Autocrine glucocorticoid signaling in hormonally active endocrine and non-endocrine cancers induces antigen expression for immunotherapy

Abstract

The present invention relates to glucocorticoid (GC)-resistant chimeric antigen receptor (CAR) T-cell for use in the treatment of a HSD11B1-expressing cancer. The invention also relates to glucocorticoid (GC)-resistant chimeric antigen receptor (CAR) T-cell for use in the treatment of a non-endocrine cancer. The invention further encompasses related treatment methods and kits.

Description

[0001] Autocrine glucocorticoid signaling in hormonally active endocrine and non-endocrine cancers induces antigen expression for immunotherapy

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to glucocorticoid (GC)-resistant chimeric antigen receptor (CAR) T-cell for use in the treatment of a HSD11 B1 -expressing cancer. The invention also relates to glucocorticoid (GC)-resistant chimeric antigen receptor (CAR) T-cell for use in the treatment of a non-endocrine cancer. The invention further encompasses related treatment methods and kits.

[0004] BACKGROUND OF THE INVENTION

[0005] Glucocorticoids (GC) confer a profound immunosuppressive effect and are extensively used in autoimmune diseases and cancer therapy for their anti-inflammatory properties. However, this mechanism is also exploited by multiple endocrine and non-endocrine cancers that secrete GCs either by active production or metabolite recycling to modulate immune-recognition and -therapy. In this study, the inventors used adrenocortical carcinoma (ACC) as an exemplary endocrine cancer that produces high amounts of glucocorticoids (GCs) and studied cancer antigen expression and recognition. It was revealed that the oncofetal antigen R0R1 is induced by autocrine GC-signaling and that its transcription is regulated by GC-activated human glucocorticoid receptor (hGR) in complex with STAT3. The inventors also show that non-endocrine cancers are capable of recycling inactive into active GCs, which induces R0R1 expression through the same transcription complex of hGR and STAT3. After establishing R0R1 as biomarker for intratumoral GC secretion in adrenocortical, breast and pancreatic cancer models, the inventors engineered hGR-knockout ROR1-specific chimeric antigen receptor (CAR)-T cells, to exploit the exalted R0R1 expression in GC secreting cancers. GC-resistant R0R1 CAR-T cells outperformed conventional CAR-T cells and conferred complete and durable remission of GC-secreting solid tumors in vivo. Taken together, the inventors identified autocrine GC signaling as a mechanism that induces antigen expression in hormonally active endocrine and non-endocrine cancers, which can be leveraged for effective immunotherapy with genetically-engineered CAR-T cells.

[0006] Glucocorticoids (GCs) are steroid hormones that are physiologically produced in the adrenal gland from its precursor cholesterol through steroid hydroxylases. However, many tissues have a recycling pathway that enables the regeneration of circulating inactive GC metabolites by the enzyme 11 p-hydroxysteroid dehydrogenase type 1 (HSD11B1) (53). GCs confer a profound immune-suppressive effect through interference of the human glucocorticoid receptor (hGR) with transcription factors that drive proinflammatory responses, as well as transactivation of immune-suppressive genes (1,2). hGR signaling has been shown to alter the activation, differentiation and function of endogenous tumor-infiltrating lymphocytes (3), and to decrease the function of adoptively transferred T cells, including T cells that have been redirected with achimeric antigen receptor (CAR) (4-6). In cancer cells, hGR signaling has been shown to increase resistance to cellular immunotherapies through downregulation of antigen processing and presentation, adhesion molecules and costimulatory ligands, and upregulation of immune checkpoint molecules (7). In triple-negative breast cancer (TNBC), the hGR synergizes with signal transducer and activator of transcription 3 (STAT3) to instruct the transcriptional program that leads to a more aggressive basal-like tumor phenotype (8). In addition to synthetic GCs that are administered to mitigate adverse effects of chemo- and radiotherapy or severe immune-related adverse events after immunotherapy, endogenous GCs are increased in many cancer patients as part of the chronic stress response associated with the malignancy (9). Endogenous GC production has also been reported from myeloid derived cells in the tumor microenvironment to promote cancer progression and immune escape (1),and from several types of non-endocrine cancers including pancreatic (10), colorectal, gastric and esophageal cancer cells (11-14). Thus, endogenous GC production and signaling can also be coopted by tumor cells to escape immune detection or immunotherapeutic destruction.

[0007] DESCRIPTION OF THE INVENTION:

[0008] The inventors used adrenocortical carcinoma (ACC) that actively produces GCs to investigate the impact of endogenous GCs on the expression of cancer antigens that can be targeted with CAR-T cells. ACC is a devastating endocrine cancer that can only be cured by complete surgical resection if diagnosed at an early stage. The adrenolytic agent mitotane is the only approved drug for treating advanced ACC and achieves an objective response rate of 25% in combination with chemotherapy, with a 5-year overall survival rate of < 20% (15, 16). The majority of ACC lesions is lymphocyte-depleted with negligible antitumor reactivity from endogenous adaptive and innate immune cells, which has been attributed to the immune-suppressive effect of ACC-derived GCs (17, 18). Moreover, the inventors used breast and pancreatic cancer models to confirm mechanistic and therapeutic transferability of autocrine GC signaling to a broad range of GC secreting cancers. After identifying ROR1 as GC induced cancer antigen, they reasoned that additional genetic engineering may be warranted in order to neutralize the immune-suppressive effects of GCs in ACC and confirmed cross entity application potential of GC-resistant ROR1 CAR-T cells in other ROR1 + GC secreting cancers.

[0009] Accordingly, the present invention provides the following preferred embodiments:

[0010] 1. A recombinant glucocorticoid (GC)-resistant immune cell, for use in the treatment of cancer in a human cancer patient, wherein the immune cell expresses a recombinant immune receptor having an extracellular antigen-binding domain capable of binding to a cancer antigen of the cancer, and wherein the cancer is a cancer expressing at least one enzyme of a synthesis or regeneration pathway for active glucocorticoids selected from the group consisting of HSD11B1, CYP11B1, CYP11A1, CYP17A1, and CYP21A2.

[0011] 2. The recombinant immune cell for use according to item 1, wherein the cancer is a HSD11B1- expressing cancer.The recombinant immune cell for use according to any one of the preceding items, wherein the immune cell is a T cell, an NKT cell, a B lymphocyte, a natural killer cell, a monocyte, a macrophage, a dendritic cell, or a granulocyte.

[0012] The recombinant immune cell for use according to any one of the preceding items, wherein the immune cell is a T cell.

[0013] The recombinant immune cell for use according to any one of the preceding items, wherein the recombinant immune receptor is a recombinant T-cell receptor.

[0014] The recombinant immune cell for use according to any one of the preceding items, wherein the cancer antigen is an antigenic peptide of an intracellular cancer antigen and is presented on an MHC molecule.

[0015] The recombinant immune cell for use according to item 6, wherein the intracellular cancer antigen is an intracellular cancer antigen whose expression is upregulated by cortisol.

[0016] The recombinant immune cell for use according to any one of items 1-6, wherein the cancer antigen is a cell-surface cancer antigen.

[0017] The recombinant immune cell for use according to item 8, wherein the cell-surface cancer antigen is a cell-surface cancer antigen whose expression is upregulated by cortisol.

[0018] The recombinant immune cell for use according to any one of the preceding items, wherein the recombinant immune receptor a chimeric antigen receptor (CAR).

[0019] A recombinant glucocorticoid (GC)-resistant chimeric antigen receptor (CAR) T-cell for use in the treatment of a HSD11 B1-expressing cancer in a human cancer patient, wherein the CAR T-cell expresses a chimeric antigen receptor (CAR) having an extracellular antigen-binding domain capable of binding to a cell-surface cancer antigen of the cancer.

[0020] The recombinant immune cell or CAR T-cell according for use according to any one of the preceding items, wherein the cancer is a non-endocrine cancer.

[0021] The recombinant immune cell or CAR T-cell according for use according to any one of items 1-11, wherein the cancer is an endocrine cancer.

[0022] A recombinant glucocorticoid (GC)-resistant chimeric antigen receptor (CAR) T-cell for use in the treatment of a non-endocrine cancer in a human cancer patient, wherein the CAR T-cell expresses a chimeric antigen receptor (CAR) having an extracellular antigen-binding domain capable of binding to a cell-surface cancer antigen of the cancer.

[0023] The recombinant immune cell or CAR T-cell for use according to any one of the preceding items, wherein the cancer is a glucocorticoid-secreting cancer.

[0024] The recombinant immune cell or CAR T-cell for use according to item 15, wherein the glucocorticoid-secreting cancer is a cortisol-secreting cancer.

[0025] The recombinant immune cell or CAR T-cell for use according to any one of the preceding items, wherein the cancer is selected from the group consisting of acute myeloid leukemia (LAML), adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), brain lower grade glioma(LGG), breast invasive carcinoma (BRCA) such as Triple-negative breast cancer (TNBC), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), cholangiocarcinoma (CHOL), chronic myelogenous leukemia (LCML), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), lymphoid neoplasm diffuse large B-cell lymphoma (DLBC), mesothelioma (MESO), miscellaneous (MISO), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD) such as pancreatic ductal adenocarcinoma (PDAC), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), sarcoma (SARC), skin cutaneous melanoma (SKCM), stomach adenocarcinoma (STAD), testicular germ cell tumors (TGCT), thymoma (THYM), thyroid carcinoma (THCA), uterine carcinosarcoma (DCS), uterine corpus endometrial carcinoma (UCEC), multiple myeloma (MM) and uveal melanoma (UVM).

[0026] The recombinant immune cell or CAR T-cell for use according to item 17, wherein the cancer is breast invasive carcinoma (BRCA) such as Triple-negative breast cancer (TNBC) or pancreatic adenocarcinoma (PAAD) such as pancreatic ductal adenocarcinoma (PDAC).

[0027] The recombinant immune cell or CAR T-cell for use according to any one of the preceding items, wherein the cancer contains a HSD11 B1 gain-of-function mutation in the HSD11 B1 gene.

[0028] The recombinant immune cell or CAR T-cell for use according to any one of the preceding items, wherein the cancer overexpresses HSD 11 B 1.

[0029] The recombinant immune cell or CAR T-cell for use according to any one of the preceding items, wherein the cancer expresses ROR1.

[0030] The recombinant immune cell or CAR T-cell for use according to any one of the preceding items, wherein the cancer overexpresses ROR1.

[0031] The recombinant immune cell or CAR T-cell for use according to any one of the preceding items, wherein the cell-surface cancer antigen is ROR1.

[0032] The recombinant immune cell or CAR T-cell for use according to any one of the preceding items, wherein the extracellular antigen-binding domain of the CAR contains an scFv selected from: (I) an anti-ROR1 scFv comprising, preferably in an N- to C- terminal order, an antibody heavy chain variable domain amino acid sequence according to SEQ ID NO: 4, an amino acid linker sequence which is preferably the amino acid sequence according to SEQ ID NO: 6, and an antibody light chain variable domain amino acid sequence according to SEQ ID NO: 8,

[0033] (II) an anti-ROR1 scFv having the same antibody heavy chain variable domain CDRs and the same antibody light chain variable domain CDRs as the anti-ROR1 scFv according to (I), and(iii) an anti-R0R1 scFv which competes with the anti-ROR1 scFv according to (i) for binding to R0R1.

[0034] The recombinant immune cell or CAR T-cell for use according to any one of the preceding items, wherein the extracellular antigen-binding domain of the CAR contains an anti-ROR1 scFv comprising, in an N- to C- terminal order, an antibody heavy chain variable domain amino acid sequence according to SEQ ID NO: 4, an amino acid linker sequence which is preferably the amino acid sequence according to SEQ ID NO: 6, and an antibody light chain variable domain amino acid sequence according to SEQ ID NO: 8.

[0035] The recombinant immune cell or CAR T-cell for use according to any one of the preceding items, wherein the immune cell or CAR T-cell is a human glucocorticoid receptor (hGR) knockout cell. The recombinant immune cell or CAR T-cell for use according to item 26, wherein the hGR knockout is a knockout by CRISPR / Cas9-mediated gene editing.

[0036] The recombinant immune cell or CAR T-cell for use according to any one of items 1-27, wherein the immune cell or CAR T-cell is a human glucocorticoid receptor (hGR) knockdown cell.

[0037] The recombinant immune cell or CAR T-cell for use according to item 28, wherein immune cell or CAR T-cell contains an siRNA against hGR mRNA.

[0038] The recombinant immune cell or CAR T-cell for use according to item 28 or 29, wherein immune cell or CAR T-cell expresses an shRNA against hGR mRNA, and wherein the immune cell or CAR T-cell preferably contains a recombinant genomic expression cassette expressing the shRNA. The recombinant immune cell or CAR T-cell for use according to any one of the preceding items, wherein the cancer patient is not a glioblastoma patient.

[0039] The recombinant immune cell or CAR T-cell for use according to any one of the preceding items, wherein the cancer patient is not a patient receiving treatment with steroids.

[0040] Use of a recombinant glucocorticoid (GC)-resistant immune cell for the manufacture of a medicament for the treatment of a cancer in a human cancer patient, wherein the cancer is as defined in item 1, and wherein the immune cell expresses a recombinant immune receptor having an extracellular antigen-binding domain capable of binding to a cancer antigen of the cancer. A method for the treatment of a cancer in a human cancer patient, the method comprising: identifying from a group of cancer patients a patient who has a cancer expressing an enzyme as defined in item 1 by determining whether the cancers of the group of cancer patients express the enzyme; and treating the patient who has a cancer expressing the enzyme with a recombinant glucocorticoid (GC)-resistant immune cell, wherein the immune cell expresses a recombinant immune receptor having an extracellular antigen-binding domain capable of binding to a cancer antigen of the cancer.

[0041] The use or method of item 33 or 34, wherein:

[0042] (I) the cancer is as defined in any one of the preceding items;(II) the recombinant immune receptor is a recombinant immune receptor or CAR as defined in any one of the preceding items;

[0043] (III) the recombinant immune cell is a recombinant immune cell or CAR T-cell as defined in any one of the preceding items; and / or

[0044] (IV) the patient is as defined in any one of the preceding items.

[0045] 36. A kit, comprising:

[0046] (a) reagents for determining whether the cancer of a cancer patient expresses an enzyme as defined in item 1, wherein the reagents optionally comprise nucleotide primers capable of binding to the mRNA encoding the enzyme and or antibodies capable of binding to the enzyme; and (b) a recombinant glucocorticoid (GC)-resistant immune cell, wherein the immune cell expresses a recombinant immune receptor having an extracellular antigen-binding domain capable of binding to a cancer antigen of the cancer.

[0047] 37. The kit of item 36, wherein:

[0048] (I) the recombinant immune receptor is a recombinant immune receptor or CAR as defined in any one of the preceding items;

[0049] (II) the enzyme is HSD11B1, and / or

[0050] (III) the recombinant immune cell is a recombinant immune cell or CAR T-cell as defined in any one of the preceding items.

[0051] BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1: R0R1 is highly expressed in ACC with GC excess.

[0053] a, Volcano plot of mRNA gene expression assessed by RNA sequencing analysis comparing normal adrenal glands (nAG) with adrenocortical carcinoma (ACC) samples. R0R1 gene expression and other known CAR / TCR targets are annotated. The horizontal line is at an adjusted p value of 0.05. b, Comparison of ROR1 mRNA expression (normalized to a-tubulin) in normal adrenal glands (nAG) compared to ACC samples assessed by quantitative real-time PCR (qRT-PCR). c, Comparison of ROR1 mRNA expression in ACC patients with and without disease recurrence, d, Association between ROR1 mRNA expression in ACC patients with low (ENSAT 1 &2) and high (ENSAT 3&4) clinical ENSAT staging, e, Association of ROR1 mRNA expression with Ki67 proliferation index and (f) histopathological Weiss-score. g, Immunohistochemical ROR1 protein analysis comparing H-scores of nAG, primary ACC tumors, local recurrences and metastases. Representative pictures are shown alongside, h, ROR1 protein expression comparing steroid hormone inactive (Steroid-) with steroid hormone producing (Steroid*) ACC tumors, i, Waterfall plot comparing ROR1 protein expression in ACC samples with glucocorticoid (GC+) excess, androgen excess (andro.*), inactive (GC-) ACC tumors and nAGs. Mean value of ROR1 expression in inactive ACC tumors (blue line) and the highest ROR1 expression in nAGs (red line) are annotated, j, ROR1 mRNA expression of all four human ACC cell lines assessed by quantitative real-time PCR (qRT-PCR) k, ROR1 molecules per cell (x103) of all fourACC cell lines assessed by quantitative flow cytometry (n=3). Statistical analysis were performed using unpaired t-test (two-tailed) (b, c, d, h), Pearson correlation (e, f), repeated measures one-way ANOVAwith Dunnett's test for multiple comparisons (g) and were considered significant if p value was *p<0.05,**p<0.01,***p<0.001, ****p<0.0001; ns, not significant.

[0054] Figure 2: GC inhibitors diminish R0R1 expression in ACC.

[0055] a, Specific lysis of R0R1 CAR-T cells targeting the steroidogenic NCI-H295R and non-steroidogenic JIL-2266 ACC cell line in 3D cell culture after 48 hours of co-culture with glucocorticoid (GC) inhibitors. Specific lysis of CAR-T cells in combination with the individual drugs in presence was normalized for cell lysis with the individual drugs without CAR-T cells and compared to lysis of UTD T cells, n=3). b, R0R1 downregulation on NCI-H295R cells after 48 hours of co-culture with pharmaceutical inhibitors of GC effector functions. R0R1 mRNA expression was assessed using qRT-PCR (n=3) and surface density (clusters / pim2) and percentage of R0R1+cells were assessed by single molecule high resolution microscopy (dSTORM, n=30). Representative pictures are shown alongside, c, R0R1 mRNA expression after treatment with mitotane for 48 hours with different dose-dependencies (n=3). d, R0R1 mRNA expression after abolishing exposure to mitotane at different time points (n=3). e, R0R1 mRNA expression of NCI-H295R cells after 48 hours of coculture with 1 piM of dexamethasone (n=3). f, R0R1 mRNA and protein expression of ACC patients' tissue samples comparing untreated samples with those that received GC inhibitors prior to surgical removal of the relapsed tumor lesion. R0R1 protein expression in matched ACC tumor samples before g, and after h, receiving GC inhibitors. Statistical analysis were performed using repeated measures one-way ANOVAwith Dunnett's test for multiple comparisons (a, b, c), unpaired t-test (two-tailed) (f), paired t-test (e, g, h), and were considered significant if p value was *p<0.05,**p<0.01,***p<0.001, ****p<0.0001; ns, not significant; hGR, human glucocorticoid receptor.

[0056] Figure 3: R0R1 transcription in ACC is regulated by hGR and STAT3.

[0057] a, RNA gene expression of key genes between the Wnt / ROR and the h G R / Stat3 pathway (n=3) in NCI-H295R cells, b, Western Blot analysis of Wnt5A, Stat3 and the hGR in NCI-H295R cells, c, Expression of key genes between the Wnt / ROR and the hGR / Stat3 pathway (n=3) in primary R0R1 expressing ACC patient cells (R0R1+) that have been shown GC excess (GC+) or have been hormonally inactive (GC-) and either treated with GC inhibitors (treated) or left untreated (untreated) prior to surgery, d, Western Blot analysis of Wnt5A, Stat3 and the hGR in primary R0R1 expressing ACC patient cells (R0R1+) that have been shown GC excess (GC+) or have been hormonally inactive (GC~) and either treated with GC inhibitors (treated) or left untreated (untreated) prior to surgery, e, Scheme of the working model molecular pathway including the Wnt / ROR pathway and the investigated hGR / Stat3 interplay. Representative pictures of Duolink proximity ligation analysis (PLA) assessing hGR / Stat3 protein complex (PLA in red and DAPI in blue) in f, positive NCI-H295R cells in 3D cell culture before (untreated) and after 48 hours of treatment with different glucocorticoid (GC) inhibitors and g, primary ACC patient samples with and without GC excess before and after GC inhibitor treatment. Comparison of hGR / Stat3 complex positive tumor cells (per area (%) before and after treatment inh, NCI-H295R and i, primary ACC cells as well as the association between hGR / Stat3 complexes and ROR1 expression, j, k ROR1 mRNA and protein expression before and after hGR gene silencing in NCI-H295R ACC cells. Statistical analysis were performed using repeated measures one-way ANOVA with Dunnett's test for multiple comparisons (h, i), Pearson correlation (h,i), paired t-test (j) and were considered significant if p value was *p<0.05,**p<0.01,***p<0.001, ****p<0.0001; ns, not significant; hGR, human glucocorticoid receptor. Figure 4: R0R1 CAR-T cells with hGR gene-edit are resistant to ACC-derived GCs.

[0058] a, Schematic design of R0R1 CAR-T cells (R12.BBz) with human glucocorticoid receptor knockout (hGR-KO) (R0R1 CAR construct and R12.BBz+hGR-KO). Untransduced T cells (UTD) have been used for normalization, b, hGR mRNA (qRT-PCR) and c, protein expression (Western Blot) before and after CRISPR / Cas9-mediated hGR knockout, d, T cell viability after 100 nM dexamethasone treatment for 48 hours, e, ROR1 CAR-T cell subset analysis before and after CRISPR / Cas9-mediated glucocorticoid receptor knockout, f, Heatmap of dysregulated genes in T cells with and without hGR-KO. g, Specific lysis of four ROR1+ACC cell lines with different effector-to-target ratios (E-T) after 24 hours, h, IL-2 and IFN-y cytokine secretion of ROR1 CAR-T cells with and without hGR knockout after antigen contact for 24 hours was assessed by ELISA in the supernatant. I, Proliferation and expansion of ROR1 CAR-T cells upon antigen contact in all four ACC cell lines after 72 hours assessed by flow cytometry. Statistical analysis were performed using repeated measures one-way ANOVA with Dunnett's test for multiple comparisons (b, e), unpaired f-test (two-tailed) (d), paired f-test (g,h,i) and were considered significant if p-value was *p<0.05,**p<0.01,***p<0.001, ****p<0.0001; ns, not significant; ELISA, enzyme-linked immunosorbent assay.

[0059] Figure 5: GC-resistant ROR1 CAR-T cells induce durable remission of ACCGC+ / n vivo.

[0060] a, Characterization of ACC xenografts for ACC specific origin and protein markers, b, Association between glucocorticoid serum levels as determined by LC-MS / MS and tumor volume of all mice, c, Treatment scheme used for the in vivo experiment. NSG mice bearing steroidogenic NCI-H295R ACC xenografts were treated intravenously on day 14 after tumor inoculation with 1 x106CAR-T cells, d, Quantification of tumor burden for each treatment group measured by digital caliper measuring (n=7 mice per group), e, Survival curve for all tumor bearing mice treated with different CAR-T / T cell modifications, f, Quantification of tumor burden for each individual mouse and in their different treatment groups measured by digital caliper measuring (n=7 mice per group), g, Median fluorescence intensity of activated and persistent CAR-T cells that are positive for CD25 and CD69 positive for tEGFR transduction marker, h, T cell subset phenotypes at all different endpoints from all four treatment groups, i, Numbers and frequencies of CAR-T cells that have been positive for canonically expressed exhaustion markers, j, Numbers and frequencies of activated CAR-T cells that are positive for CD25 and CD69 at their different endpoints, k, Immune infiltration of CD3+T cells from all different treatment groups assessed by immunofluorescence. Representative pictures for each group are shown in I, alongside. Statistical analysis were performed using Pearson correlation (b), repeated measures one-way ANOVA with Dunnett's test for multiple comparisons (g-j), mixed-effects analysis (d,f), Log-rank (Mantel-Cox) test (e),unpaired f-test (k) and were considered significant if p value was *p<0.05,**p<0.01 ,***p<0.001 , ****p<0.0001 ; ns, not significant; hGR, human glucocorticoid receptor; LC-MS, liquid-chromatography-mass-spectrometry.

[0061] Figure 6: GC-resistant R0R1 CAR-T cells are effective in GC-secreting breast cancer

[0062] a, Cortisone-to-cortisol conversion and saturation curve as detected by LC-MS measure-ment. b, T cell viability after 72 hours of exposure to actively converted cortisol in superna-tants that are shown in a), c, Normalized R0R1 and HSD11B1 mRNA expression after treatment with different doses of cortisone, d, Correlation between R0R1 and HSD11B1 mRNA expression levels with the amounts of actively converted cortisol, e, GC concentra-tions in cell culture supernatants after 24 hours of 300nM cortisone with different treatment groups (UTD = untreated, Cort = cortisone, Met = metyrapone, Cort+Met = cortisone after preincubation with metyrapone). f, HSD11B1 and R0R1 mRNA and g, protein expression after 250nM of cortisone or metyrapone treatment, h, Specific lysis of MDA-MB231 breast cancer cells with different effector-to-target ratios (E-T) after 24 hours. I, IFN-y cytokine secretion of R0R1 CAR-T cells with and without hGR knockout after antigen contact for 24 hours was assessed by ELISA in the supernatant.

[0063] Figure 7: Glucocorticoid producing ACCs show worse prognosis than inactive tumors and ACC cell lines show similar R0R1 expression patterns.

[0064] a, Survival curve comparing recurrence free survival of ACC patients with and without glucocorticoid excess. b, Waterfall plot comparing R0R1 mRNA expression in human ACC cell lines with ACC patients' samples.

[0065] Figure 8: R0R1 expression in human ACC cell lines.

[0066] a, R0R1 mRNA molecules per cell of all four ACC cell lines assessed by RNAscope single cell analysis. Representative pictures are shown alongside, b, R0R1 clusters / pim2per basal plasma cell membrane of all four ACC cell lines assessed by single molecule super high-resolution microscopy (dSTORM) analysis (n=30). Representative pictures are shown alongside, c, Glucocorticoid secretion patterns of all four ACC cell lines assessed by LC-MS analysis.

[0067] Figure 9: R0R1 expression in JeKo-1 and K562 reference cell lines.

[0068] a, R0R1 mRNA expression of all four human ACC cell lines assessed by quantitative real-time PCR (qRT-PCR) b, R0R1 molecules per cell (x103) of all four ACC cell lines assessed by quantitative flow cytometry (n=3). c, R0R1 mRNA molecules per cell of JeKo-1 and K562 control cell lines assessed by RNAscope single cell analysis. Representative pictures are shown alongside, d, R0R1 clusters / pim2per basal plasma cell membrane of JeKo-1 and K562 control cell lines assessed by single molecule super high-resolution microscopy (dSTORM) analysis (n=30).

[0069] Figure 10: Glucocorticoid inhibition diminishes ROR1 CAR-T cell efficacy.

[0070] a, Cell viability of T cells after 72 hours of dexamethasone treatment. Cytokine secretion from ROR1 CAR-T cells in 3D cell culture models of b, NCI-H295R and c, JIL-2266 ACC cells after 48 hours of pretreatment with glucocorticoid inhibitors assessed by ELISA measurement, d, Cell viability of CAR-T cells after 48 hours with different doses of the applied combination treatments (normalized to DMSO, n=3). Representative dot plotsare shown alongside. Statistical analyses were considered significant if p-value was *p<0.05,**p<0.01,***p<0.001, ****p<0.0001; ns, not significant; hGR, human glucocorticoid receptor.

[0071] Figure 11: ROR1 downregulation after glucocorticoid inhibitor treatment in ACC cell lines.

[0072] ROR1 mRNA and protein expression after glucocorticoid (GC) inhibitor treatment using metyrapone, ketoconazole, relacorilant and mitotane in a, JIL-2266, b, CU-ACC1 and c, CU-ACC2 cell line assessed by qRT-PCR and dSTORM analysis, d, ROR1 mRNA and protein expression comparing untreated matched primary tumor samples with metastatic lesions of the same patient. Statistical analyses were considered significant if p-value was *p<0.05,**p<0.01,***p<0.001, ****p<0.0001; ns, not significant.

[0073] Figure 12: hGR gene silencing shows hGR related ROR1 expression.

[0074] a, RNA gene expression of ROR1 , Wnt5A, Stat3 and hGR before and after treatment with glucocorticoid (GC) inhibitors in JIL-2266 cell line, b, hGR, Stat3 and Wnt5A expression after hGR gene silencing in NCI-H295R cells. Statistical analyses were considered significant if p-value was *p<0.05,**p<0.01,***p<0.001, ****p<0.0001; ns, not significant; hGR, human glucocorticoid receptor.

[0075] Figure 13: Functional ROR1 CAR expression.

[0076] a-b, CAR positive CD8 and CD4 T cells assessed by FACS analysis. Representative dot plots of ROR1 (R12.BBz) CAR expressing T cells and CD4+ and CD8+T cell transduction efficacy are shown alongside.

[0077] Figure 14: ROR1 CAR-T cell functionality in vitro.

[0078] a, Specific lysis of ROR1+JeKo-1 mantle cell lymphoma and ROR1- K562 leukemia cell line with different effector-to-target ratios (E-T) after 24 hours, b, IL-2 and IFN-y cytokine secretion of ROR1 CAR-T cells with and without hGR knockout after antigen contact for 24 hours was assessed by ELISA in the supernatant, c, Proliferation and expansion of ROR1 CAR-T cells upon antigen contact in all four ACC cell lines after 72 hours assessed by FACS-analysis. Statistical analyses were considered significant if p-value was *p<0.05,**p<0.01,***p<0.001, ****p<0.0001; ns, not significant; hGR, human glucocorticoid receptor; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence activated cell sorting.

[0079] Figure 15: Correlation between ROR1 expression andhGR KOROR1 CAR-T cell cytokine production. a, IL-2 and b, IFN-y cytokine secretion ofhGR-KOROR1 CAR-T cells correlate with ROR1 antigen levels (RNA, density and molecule count) in ACC cell lines.

[0080] Figure 16: Histopathological staining of ACC tumors.

[0081] Immunohistochemical staining of Stat3, hGR, p53 and I nhi bin-a of ACC tumors from the steroidogenic ACC mouse model used in this study.

[0082] Figure 17: HSD11B1 expression in human cancers

[0083] Relative mRNA expression of HSD11 B1 in a, hematological and b, solid cancer entities as compared to their respective normal tissues, c, Correlation between expression of HSD11B1 with bulk RNA-Seqestimated frequency of tumor-infiltrating TCOnvs and Tregs. d, Correlation between expression of HSD11B1 and glucocorticoid response genes (TSC22D3, DUSP1, FKBP5), T cell dysfunction genes (PDCD1, LAG3, HAVCR2, TIGIT), and Treg marker genes (CCR8, CTLA4, ICOS, IL1R2) in human cancers.

[0084] Figure 18: HSD11B1 and hGR expression is linked to R0R1 expression in a variety of R0R1 expressing cancers.

[0085] Correlation between HSD11B1 and hGR mRNA expression with R0R1 mRNA expression in a, breast and b, pancreatic cancer, c, Pearson-correlation between hGR mRNA expression and R0R1 mRNA expression in a variety of R0R1+solid tumor entities. All cancer entity abbrevations are in line with TCGA study abbreviations, d, Percentage of cases that carry an intrinsic HSD11B1 CNV gain of function mutation in different TCGA tumor entities, e, Normalized R0R1 mRNA expression after treatment with different doses of cortisone, f, Representative pictures of Duolink proximity ligation analysis (PLA) assessing hGR / Stat3 protein complex (PLA in red and DAPI in blue) in primary breast cancer patient samples. Statistical analyses were considered significant if p-value was *p<0.05,**p<0.01,***p<0.001, ****p<0.0001; ns, not significant.

[0086] Figure 19: Tumoral GC metabolite recycling impairs R0R1 CAR-T cell efficacy in GO secreting pancreatic cancer

[0087] a, GC concentrations in cell culture supernatants after 24 hours of 300nM cortisone with different treatment groups (UTD = untreated, Cort = cortisone, Met = metyrapone, Cort+Met = cortisone after preincubation with metyrapone). b, HSD11B1 and R0R1 mRNA and c, protein expression after 250nM of cortisone or metyrapone treatment, d, Specific lysis of PANC-1 pancreatic cancer cells with different effector-to-target ratios (E-T) after 24 hours, e, IFN-y cytokine secretion of R0R1 CAR-T cells with and without hGR knockout after antigen contact for 24 hours was assessed by ELISA in the supernatant. Statistical analyses were considered significant if p-value was *p<0.05,**p<0.01,***p<0.001, ****p<0.0001; ns, not significant.

[0088] Figure 20: Tumors secrete GCs either by active synthesis or metabolite recycling.

[0089] Schematic picture of the proposed mechanism where tumors either secrete GCs through CYP11 B1 -mediated active GC production or through HSD11B1-mediated metabolite regeneration. Both GC secretion pathways enable hGR activation and STAT3 protein transcription factor complex coupling which in turn upregulates ROR1 expression.

[0090] Figure 21: GC-resistant ROR1 CAR-T cells are effective against non-endocrine cancers in vivo. (a) Treatment scheme used for the in vivo experiment. NSG mice bearing HSD11B1 expressing PANC-1 (PDAC) and MDA-MB231 (TNBC) xenografts were treated intravenously on day 7 after tumour inoculation with 2x106CAR-T cells, (b) Quantification of tumour burden for each treatment group measured by bioluminescence imaging (BLI) (n=6 mice per group), (c) Survival curves for all PDAC and TNBC tumour bearing mice treated with different CAR-T / T cell modifications. Statistical analysis were performed usingmixed-effects analysis (b) and log-rank (Mantel-Cox) test (c), and were considered significant if p-value was *p<0.05,**p<0.01,***p<0.001, ****p<0.0001; ns, not significant; ELISA, enzyme-linked immunosorbent assay.

[0091] Figure 22: GC-resistant R0R1 CAR-T cells are effective against non-endocrine cancers in vivo. (a) Treatment scheme used for the in vivo experiment. NSG mice bearing HSD11B1 expressing PANC-1 (PDAC) and MDA-MB231 (TNBC) xenografts were treated intravenously on day 7 after tumour inoculation with 2x106CAR-T cells, (b) Tumor volume, (c) Survival curves. Statistical analysis were performed using mixed-effects analysis (b) and log-rank (Mantel-Cox) test (c), and were considered significant if p-value was *p<0.05,**p<0.01,***p<0.001, ****p<0.0001; ns, not significant; ELISA, enzyme-linked immunosorbent assay.

[0092] Figure 23: ROR1 and HSD11B1 expression.

[0093] (a) and (b) ROR1 and HSD11 B1 mRNA and protein expression at in vivo endpoint analyses from PDAC and TNBC tumours, (c) Association between ROR1 and HSD11B1 mRNA expression in PDAC tumours, (d) HSD11B1 mRNA expression after treatment with increasing effector-to-target ratios of CAR-T cells that secrete TNFa and IFNy in PANC-1 cells. Statistical analysis were performed using one-way ANOVA with Dunnett's test for multiple comparisons (a, b,d), and Pearson-correlation (c) and were considered significant if p-value was *p<0.05,**p<0.01,***p<0.001, ****p<0.0001; ns, not significant; ELISA, enzyme-linked immunosorbent assay.

[0094] Figure 24

[0095] (a) Serum corticosterone levels in PDAC and TNBC bearing mice in all three different treatment groups, (b, c) Association between R0R1 and HSD11B1 mRNA expression in TNBC tumours; association between HSD11B1 mRNA expression and serum corticosterone levels in mice, (d) HSD11B1 mRNA expression in PANC-1 cells after increasing doses of TNFa. Statistical analysis were performed using one-way ANOVA with Dunnett's test for multiple comparisons (a,d) and Pearson correlation (b,c) and were considered significant if p-value was *p<0.05,**p<0.01,***p<0.001, ****p<0.0001; ns, not significant.

[0096] Figure 25

[0097] (a) and (b) show representative immunohistochemistry pictures of R0R1 and HSD11B1 protein expression at in vivo endpoint analyses from PDAC and TNBC tumours.

[0098] DETAILED DESCRIPTION OF THE INVENTION

[0099] Unless specifically defined below, the terms used in the present invention shall be understood with their common meaning known to the person skilled in the art.Literature references referred to herein may be cited by using the full reference, or by using an abbreviation such as a number, for instance, "(76)”, and by specifying the corresponding full reference in the "References” section. All literature reference referred to herein are incorporated by reference for all purposes in their entitrety.

[0100] As used in accordance with the invention, terms such as "active glucocorticoids” and "active GCs” refer to glucocorticoids which are capable of binding to and activating the human glucocorticoid receptor. A preferred active glucocorticoid is cortisol.

[0101] As used herein, each occurrence of terms such as "comprising” or "comprises” may optionally be substituted with "consisting of or "consists of.

[0102] Preferably the recombinant glucocorticoid (GC)-resistant chimeric antigen receptor (CAR) T-cells as used in the invention are capable of causing a decrease in cancer cell number of the cancer cells expressing the cellsurface cancer antigen. Preferably, this can be caused by cytotoxicity through necrosis or apoptosis, or this can be caused by inhibiting or stopping proliferation i.e., inhibiting growth. This can be measured by various common methods and assays known in the art, e.g., by measuring the response to the treatment in accordance with known clinical guidelines.

[0103] In one embodiment, the chimeric antigen receptor expressed by the recombinant glucocorticoid (GC)-resistant chimeric antigen receptor (CAR) T-cells as used in the invention is capable of binding to R0R1. It is understood that the chimeric antigen receptor is capable of binding to the extracellular domain of R0R1. The treatment of cancer in accordance with the invention is preferably a treatment by cancer immunotherapy. "Immunotherapy” as described herein refers to the transfer of immune cells into a patient for targeted treatment of cancer. The cells may have originated from the patient or from another individual. In immunotherapy, immune cells, preferably T cells, are typically extraced from an invdivdiual, preferably from the patient, genetically modified and cultured in vitro and administered to the patient. Immunotherapy is advantageous in that it allows growth inhibiting, preferably cytotoxic, treatment of tumor cells without the non-targeted toxicity to non-tumor cells that occurs with conventional treatments.

[0104] The present invention may use a chimeric antigen receptor (CAR) having an extracellular antigen-binding domain capable of binding to R0R1 , further comprising a transmembrane domain and at least one intracellular signalling domain, wherein said extracellular antigen binding domain comprises a R0R1 -binding element and a hinge which is preferably an lgG4 hinge, and wherein said transmembrane domain comprises a CD28 transmembrane domain, and wherein said intracellular signalling domain comprises a 4-1 BB costimulatory domain and a CD3( domain.

[0105] In a preferred embodiment of the invention, the R0R1 -binding element is an anti-ROR1 scFv which comprises, preferably in an N- to C- terminal order, the following sequences: an antibody heavy chain variable domain amino acid sequence according to SEQ ID NO: 4 or an amino acid sequence at least 90% identicalthereto and an antibody light chain variable domain amino acid sequence according to SEQ ID NO: 8 or an amino acid sequence at least 90% identical thereto.

[0106] In a preferred embodiment of the invention, the R0R1 -binding element is an anti-ROR1 scFv which comprises, preferably in an N- to C- terminal order, the following sequences: an antibody heavy chain variable domain amino acid sequence according to SEQ ID NO: 4 or an amino acid sequence at least 90% identical thereto, an amino acid linker sequence which is preferably the amino acid sequence according to SEQ ID NO: 6, and the antibody light chain variable domain amino acid sequence according to SEQ ID NO: 8 or an amino acid sequence at least 90% identical thereto.

[0107] In a preferred embodiment of the invention, the I g G4-h i nge domain is represented by the amino acid sequence according to SEQ ID NO: 10. Moreover, the IgG-hinge domain is preferably encoded by a nucleotide sequence according to SEQ ID NO: 9. The hinge connects the extracellular targeting and the transmembrane domain. In a preferred embodiment of the invention, the CD28 transmembrane domain is represented by an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 97 % or most preferably at least 99% sequence identity with an amino acid sequence according to SEQ ID NO: 12. Preferably, the CD28 transmembrane domain is represented by an amino acid sequence according to SEQ ID NO: 12. Moreover, the CD28 transmembrane domain is preferably encoded by an nucleotide sequence having at least 90%, preferably at least 95%, more preferably at least 97 % or most preferably at least 99% sequence identity with a nucleotide sequence according to SEQ ID NO: 11. Preferably, the CD28 transmembrane domain is encoded by a nucleotide sequence according to SEQ ID NO: 11. The CD28 transmembrane domain consists of a hydrophobic alpha helix, traverses the membrane of the cell and anchors the CAR to the cell surface. In a preferred embodiment of the invention, the intracellular signalling domain comprises a 4-1 BB costimulatory domain and a CD3 domain.

[0108] In a preferred embodiment of the invention, the 4-1 BB costimulatory domain is represented by an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 97 % or most preferably at least 99% sequence identity with an amino acid sequence according to SEQ ID NO: 14. Preferably, the 4-1BB costimulatory domain is represented by an amino acid sequence according to SEQ ID NO: 14. Moreover, the 4-1 BB costimulatory domain is preferably encoded by an nucleotide sequence having at least 90%, preferably at least 95%, more preferably at least 97 % or most preferably at least 99% sequence identity with an nucleotide sequence according to SEQ ID NO: 13. Preferably, the 4-1 BB costimulatory domain is encoded by a nucleotide sequence according to SEQ ID NO: 13.

[0109] In a preferred embodiment of the invention, the CD3 domain is represented by an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 97 % or most preferably at least 99% sequence identity with an amino acid sequence according to SEQ ID NO: 16. Preferably, the CD3 domain is represented by an amino acid sequence according to SEQ ID NO: 16. Moreover, the CD3 domain is preferably encoded by a nucleotide sequence having at least 90%, preferably at least 95%, more preferablyat least 97 % or most preferably at least 99% sequence identity with a nucleotide sequence according to SEQ ID NO: 15. Preferably, the CD3 domain is encoded by a nucleotide sequence according to SEQ ID NO: 15.The CD3 domain mediates downstream signaling during the T cell activation. It is derived from the intracellular signaling domain of the T cell receptor and contains ITAMs (immunoreceptor tyrosine based activation motifs).

[0110] In a preferred embodiment of the invention, the chimeric antigen receptor (CAR) having an extracellular antigen-binding domain capable of binding to R0R1 comprises an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 97 % or most preferably at least 99% sequence identity to an amino acid sequence according to SEQ ID NO: 24 or 26. Preferably, the chimeric antigen receptor (CAR) having an extracellular antigen-binding domain capable of binding to ROR1 comprises an amino acid sequence according to SEQ ID NO: 24 or 26. More preferably, the chimeric antigen receptor (CAR) having an extracellular antigen-binding domain capable of binding to ROR1 consists of an amino acid sequence according to SEQ ID NO: 24 or 26.

[0111] Moreover, in a preferred embodiment of the invention, the chimeric antigen receptor (CAR) having an extracellular antigen-binding domain capable of binding to ROR1 is encoded by a nucleotide sequence having at least 90%, preferably at least 95%, more preferably at least 97 % or most preferably at least 99% sequence identity to a nucleotide sequence according to SEQ ID NO: 23 or 25. Preferably, the chimeric antigen receptor (CAR) having an extracellular antigen-binding domain capable of binding to ROR1 is represented by a nucleotide sequence according to SEQ ID NO: 23 or 25. More preferably, the chimeric antigen receptor (CAR) having an extracellular antigen-binding domain capable of binding to ROR1 is encoded by a nucleotide sequence according to SEQ ID NO: 23 or 25.

[0112] Recombinant immune cell

[0113] In an preferred embodiment of the invention in accordance with all other embodiments, the recombinant glucocorticoid (GC)-resistant chimeric antigen receptor (CAR) T-cell is a human ecombinant glucocorticoid (GC)-resistant chimeric antigen receptor (CAR) T-cell.

[0114] Human glucocorticoid receptor (hGR) knockout CAR T-cells can be generated by methods known in the art, e.g., by CRISPR / Cas9-mediated gene editing.

[0115] As used in connection with all embodiments of the invention, the terms "glucocorticoid (GC)-resistant immune cell” and "glucocorticoid (GC)-resistant chimeric antigen receptor (CAR) T-cell” mean that the respective immune cell or chimeric antigen receptor (CAR) T-cell is partly or completely resistant to active glucocorticoids such as cortisol. When treated with active glucocorticoids such as cortisol, the respective glucocorticoid (GC)-resistant immune cell or chimeric antigen receptor (CAR) T-cell displays reduced activation or no activation of the human glucocorticoid receptor when compared to a corresponding cell of the same cell type which has not been made glucocorticoid (GC)-resistant. Activation of the human glucocorticoid receptor can be assessedby assays known in the art, for instance, measurements of the expression known target genes of the human glucocorticoid receptor.

[0116] Pharmaceutical composition

[0117] The recombinant glucocorticoid (GC)-resistant chimeric antigen receptor (CAR) T-cell used in the present invention can be used in form of a pharmaceutical composition comprising the (CAR) T-cell. Typical additional ingredients of pharmaceutical compositions such as excipients, stabilizers etc. are known in the art.

[0118] Pharmaceutical composition for use as a medicament

[0119] The present invention also relates to a pharmaceutical composition as described above for use as a medicament.

[0120] In an embodiment of the invention, the pharmaceutical composition as described above is used in a method of treating cancer, wherein in said method the pharmaceutical composition of the present is to be administered to a subject.

[0121] HSD11B1 mutations and HSD11B1 overexpression

[0122] HSD11B1 gain-of-function mutation in the HSD11B1 gene are known in the art and have been described in Li et al. (55), which is incorporated herein by reference in its entirety for all purposes.

[0123] It is understood that the phrase "the cancer overexpresses HSD11B1” means that the cancer expresses higher amounts of HSD 11 B1 than the respective normal (non-cancerous) cells of the same tissue or, in the case of non-solid cancers, the same cell type. Expression of HSD11 B1 can be determined by methods known in the art, e.g., by quantifying HSD11 B1 mRNA levels using primers capable of binding to HSD11 B1 mRNA and / or by quantifying HSD11B1 protein levels using antibodies capable of binding to HSD11B1.

[0124] It is also understood that the phrase "the cancer overexpresses R0R1” means that the cancer expresses higher amounts of R0R1 than the respective normal (non-cancerous) cells of the same tissue or, in the case of non-solid cancers, the same cell type. Expression of R0R1 can be determined by methods known in the art, e.g., by quantifying R0R1 mRNA levels using primers capable of binding to R0R1 mRNA and / or by quantifying R0R1 protein levels using antibodies capable of binding to R0R1.

[0125] The nomenclature of cancers as referred to in connection with the present invention is the nomenclature of The Cancer Genome Atlas (TCGA) and is known in the art.

[0126] Sequences

[0127] The sequences corresponding to the SEQ IDs referred to herein are indicated Table 1 below (asterisks (***) denote the stop signal / end of amino acid sequence):

[0128] Table 1:

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139] EXAMPLES

[0140] Additional aspects and details of the invention are exemplified by the following non-limiting examples.Methods

[0141] Patient samples

[0142] Tumor samples and normal adrenal glands (nAG) were obtained from the European Network for the Study of Adrenal Tumors (ENSAT) ACC biobank curated by the Division of Endocrinology & Diabetes at the University Hospital Wuerzburg, Germany. All patients provided prior written informed consent as part of the ENSAT ACC registry (30). The study followed the principles of the declaration of Helsinki, the good clinical practice guidelines and was approved by the ethics committee of the University of Wuerzburg (#88 / 11). In total 197 tumor samples (n=149 primary tumors, n=14 local recurrences, n=34 ACC metastases) from 149 individual patients with histologically confirmed ACC and 22 nAGs were obtained from the biobank. The assessed clinical and histopathological characteristics included sex, age at diagnosis, tumor size, resection status, Ki67 proliferation index, Weiss score, ENSAT classification tumor staging, hormone secretion profile, steroid inhibitor treatment (metyrapone, ketoconazole, and mitotane) prior to surgery and the presence of local recurrences and distant metastases. Single cell transcriptomic data were analysed from the RNA sequencing data set from Giordano et al. (29) Overexpression of ROR1 as compared to nAGs was confirmed by qRT-PCR in a second, independent patient cohort consisting of 62 ACC samples from 51 individual patients (n=47 primary tumors and n=15 metastases) and 13 nAGs. ROR1 expression at protein level was finally confirmed by chromogenic immunohistochemistry in 135 ACC FFPE tissue samples (n=102 primary tumors, n=14 local recurrences and n=19 metastases) and 9 nAGs. Endocrine workup confirmed autonomous glucocorticoid excess (GC) in 61.7% of all patients diagnosed by means of pathological 1 mg dexamethasone test (cortisol >5 pig / dL) in the presence of suppressed adrenocorticotropic hormone (ACTH) (30). Completeness of surgical resection of the primary tumor was based on negative surgical, pathological and imaging reports (RO) for any residual malignant tissue. At the time of diagnosis and during follow up examinations at an interval of 3-6 months, the presence of local recurrences or metastases was evaluated by computed tomography of chest and abdomen.

[0143] Cell lines, cell culture and 3D spheroid models

[0144] The ACC cells - NCI-H295R (from ATCC) (32), JIL-2266 (in house) (33),CU-ACC1 and CU-ACC2 (by Katja Kiseljak-Vassiliades)(31) - were cultured as previously described. Non-adrenal cell lines were cultured in RPMI-medium (Gibco) supplemented with 9% FCS and 1% penicillin-streptomycin (pen-strep). Besides dexamethasone (Sigma-Aldrich) treatment (72h), cells were treated with different drugs dissolved in DMSO (metyrapone, ketoconazole, relacorilant & mitotane) for 48 hours. 3D cell culture was performed with 15% methylcellulose for 3 weeks.

[0145] RNA extraction, silencing and RNA-nanostring nCounter analysis

[0146] RNA was extracted by using the Maxwell® RSC simply RNA Tissue Kit (Promega) according to the instructions of the manufacturer. RNA quality and quantity were determined using a Nanodrop 1000 (Thermo Fisher). hGR gene silencing was achieved using ACCELL siRNA. Digital multiplexed NanoString nCounteranalysis system (NanoString Technologies)-based gene expression profiling was performed on 40 ng total RNA from each sample according to the manufacturer's instructions. Nanostring RNA analysis of 785 immune cell and exhaustion-related human genes was performed using the nCounter® Immune Exhaustion Panel on the nCounter® Analysis System. Analysis and normalization of the raw Nanostring data were performed using nSolver Analysis Software v1.1 (Nanostring Technologies).

[0147] Quantitative real-time-PCR (qRT-PCR)

[0148] Gene mRNA expression analysis was investigated by quantitative real-time polymerase chain reaction (qRT-PCR). RNA was isolated as described in the previous paragraph. Reverse transcription of mRNA into cDNA was performed by using the QuantiTect Reverse Transcription Kit (Qiagen) according to manufacturer's recommendations. mRNA expression was analyzed in duplicates and normalized to o-Tubulin. Gene amplification during qRT-PCR was performed on a CFX96 real-time thermocycler (Bio-Rad) and Bio-Rad CFX Manager 2.0 software in a 50 l reaction consisting of 5.0 ng cDNA, 300 nM gene-specific forward and reverse primers (Table 2) and 25 l Power SYBR Green PCR Master Mix (Applied Biosystems). Cycling conditions were 95°C for 10 minutes (min) followed by 40 cycles of 95°C for 15 s, 60°C for 1 min. The cycle threshold (Ct) was determined using SDS software v2.2.2 (Applied Biosystems) and the level of gene expression calculated by using the ACt method (2-(ACt)).

[0149] Table 2

[0150]

[0151] RNAscope single cell analysis

[0152] RNAScope is a custom RNA in situ hybridization solution (Advanced Cell Diagnostics) that allows the staining of RNA fragments on FFPE tissue slides. Staining has been performed according to manufacturer's protocol and extensively elucidated priorly (44). Three pictures of representative areas of each slide were taken with the Leica Aperio Versa brightfield scan-ning microscope (Leica) at 40x magnification. For scoring the slides, optional image analysis algorithm ‘RNA ISH vT of the Aperio ImageScope software v.12.x (Leica) has been used on the entirety of the pictures. This algorithm automatically detects and counts the number of RNA molecules (each brown stained spot is one molecule of RNA) and the number of cells (by detecting the hematoxylin-stained nuclei in a defined area. Thresholds for the detection were manually adjusted for a high-fidelity assessment of the signal. We used the ratio of RNA molecules per cells for each slide to quantify R0R1 gene expression (Hs-R0R1, #402831, Advanced Cell Diagnostics) .

[0153] Chromogenic and immunofluorescence immunohistochemistry (IHC) and H-scores Chromogenic and fluorescence immunohistochemical staining on human formalin-fixed, paraffin-embedded (FFPE) tumor slides has been performed as already reported (17,44). The primary antibodies that have been used were the following: ROR1 (4A5, BD Pharmingen), CYP11 B1 and CYP11 B2 (provided by Celso Gomez-Sanchez) (45),SF-1 (N1665, R&D Systems), KI67 (MIB-1, Dako), CD31 (orb10314, Biobyrt), CD3 (ab699, abeam), p53 (DO-7, Bio-Rad), hGR (D4X9S, Cell Signaling), Stat3 (D1B2J, Cell Signaling), pStat3 (D3A7, Cell Signaling), Inhibin-o (R1, Bio-Rad). Microscopic assessment and H-score evaluation was performed using Aperio Leica scanner and Aperio Leica ImageScope software analysis (Leica).

[0154] Direct stochastic optical reconstruction microscopy (dSTORM)

[0155] Purified anti-human ROR1 antibody was purchased from Biolegend (Cat. 357802). Buffer exchange was carried out using Zeba Spin Desalting Columns and antibodies were conjugated with Alexa Fluor 647 using the AF647-NHS ester kit (Thermo Scientific) according to the manufacturer's protocols. The concentration of the final conjugated antibody solution was determined on a Tecan Spark. 8-well chambered cover glass chambers (Cellvis) were coated with 0.02 mg / ml PLL or PDL. Tumor cells were harvested with a cell scrapper, resuspended in FACS buffer. Fc-Receptor blockade was carried out using 1:9 diluted TruStain FcX, followed by ROR1 staining using 10 pig / ml ROR1-AF647 antibody for 30 min at 4°C in the dark. Background signal from unspecific antibody binding was removed by sequen-tial wash steps. Cells were plated in coated cover glass chambers and allowed to adhere for 15 min on ice and fixated 3% formaldehyde and 0.25% glutaraldehyde in PBS for 15 min. Fixation solution was removed and washed 3x with PBS. For direct stochastic optical reconstruction microscopy (dSTORM), an ONI Nanoimager S was used. For dSTORM measurements, imaging buffer (100 mM cysteamine hydrochloride in PBS, pH 7.4) was added to cover slides. Images were taken in total inner reflection fluorescence (TIRF) mode. Pixel size was 117 nm. Typically, 15000 frames at 10 ms exposure time were taken per movie with a laser power at 640 nm wavelength of ~3.5 kW / cm2. dSTORM image reconstruction was performed with rapidSTORM 3.3 (46). Drift correction was performed using the linear drift correction tool of rapidSTORM 3.3. Selection of cell region of interests (ROIs) was performed with Napari and cross checked with widefield images. For cluster analysis, a custom localization analysis tool LOGAN was used, which employs a density-based spatial clustering of applications with noise (DBSCAN) algorithm (47). Cluster parameters of DBSCAN were minPoints = 3 and epsilon = 20 nm.

[0156] Western Blot analysis

[0157] For Western Blot (WB) analysis, proteins were extracted from fresh-frozen ACC tissues and cells as previously described (48). Protein concentration was quantified as previously described (49). The primary antibodies that have been used were the following: ROR1 (D6T8C, Cell Signaling, #16540S), Wnt5A (2392S, Cell Signalling,#2392S), hGR (D4X9S, Cell Signalling, #47411S), Stat3 (D1B2J, Cell Signalling, #30835S), pStat3 (D3A7, Cell Signaling, #9145S).

[0158] Duolink® Proximity Ligation Assay (PLA®)

[0159] ACC tumor samples and 3D cell culture spheroids have been formalin-fixed, paraffine-embedded and applied on a microscopic slide. Duolink® Proximity Ligation Assay (PLA®) has been performed according to the instructions of the manufacturer. In brief, tissue slides have been initially blocked for 1 h at 37°C in a heated chamber with the Duolink® blocking solution (Merck). After multiple washing steps, cells were incubated successively with PLA probes, ligation solution and amplification solution at 37 °C (Duolink© In Situ PLA® Probe Anti-Mouse MINUS & Anti-Rabbit PLUS). Antibodies have been the same from IHC. Samples have been covered with DAPI, cover slips and sealed with transparent nail polisher. Images were examined using Aperio Leica scanner (Leica).

[0160] (Quantitative) flow cytometry (FACS) analysis and cell phenotyping

[0161] Cells were washed with FACS buffer before staining. Staining was performed in FACS buffer for 20 min at 4°C. Cells were then washed once with FACS buffer and analyzed on a BD FACS Canto II. FACSDiva software (BD) was used for data collection and FlowJo software (BD) was used for data analysis. T cells were detected for CAR expression using an tEGFR antibody. The following antibodies were used for cell staining: ROR1 (AF647, clone 2A2, BioLegend), CD45 (VioBlue, clone REA747, Miltenyi), CD4 (PeVio770, clone REA, Miltenyi), CD8 (APC-Cy7, clone SK1, BioLegend), tEGFR (APC, clone AY13, Bio-Legend), PD-1 (PE, clone PD1.3.1., Miltenyi), Lag3 (PerCP-Cy5.5, clone 11C3C65, Bio-Legend), CTLA-4 (PeCy7, clone L3D10, BioLegend), Tim-3 (CD366), APC-Cy7, clone F382E2, BioLegend), CD45RO (FITC, clone UCHL1, BioLegend), CD45RA (PE, clone T6D11, Miltenyi), CD62L (PerCP-Cy5.5, clone DREG-56, BioLegend), CD25, PeCy7, clone BC96, BioLegend), CD69 (APC-Cy7, clone FN50, BioLegend). Quantitative flow cytometry was performed using PE-labeled antibodies and the BD-Quantibrite PE-Bead Assay (BD Biosciences).

[0162] Liquid chromatography-tandem mass spectrometry (LC-MS) analysis

[0163] Steroid hormones in cell culture supernatants of ACC cell lines and mouse blood were quantified with using a liquid chromatography-tandem mass spectrometry system (QTRAP 6500+.SCIEX®) including an Agilent 1290 UHPLC (G4226A autosampler, infinityBinPump, G1316C column-oven, G1330B thermostat) and the MassChrom-Steroids in Serum / Plasma® IVDR conform kit (Chromsystems®) was used for steroid measurements. 15 steroid hormones in the positive MRM-Mode (aldosterone and DHEAS in the negative mode) were quantified via corresponding isotope-labeled standards according to the manufacturer's instruction. 500 pl serum / plasma / supernatants were utilized for offline solid phase extraction (SPE) and finally 15 pl were used for LC-MS / MS. Raw data were processed by Analyst® Software (1.6.3) via 6 point calibration curve and 1 / x weighting. Periodic participation in ring trails and commercial quality controls ensured the correctness of measurements for the steroids.T cell isolation, activation, expansion and culture

[0164] T cells were isolated from PBMCs by negative selection using CD4+and CD8+T cell isolation kits (Miltenyi), and have been expanded and activated using anti-CD3 / anti-CD28 bead stimulation (Invitrogen) prior to nucleofection. They were cultured in T cell medium (RPMI-1640 supplemented with 9% human serum, 1% pen-strep, 0.1% GlutaMax) and 50 U / mL recombinant human interleukin (IL)-2 (Proleukine). Patient-derived and healthy donor CD4+and CD8+T cells were isolated from peripheral blood by negative selection. CAR-transduced T cells were enriched using biotinylated anti-EGFR mAb and anti-biotin beads (Miltenyi), prior to expansion using a rapid expansion protocol (50,51).

[0165] CAR-T cell generation and CRISPR / Cas9-mediated glucocorticoid receptor (hGR) knockout Two days after isolation and stimulation, 1.2x106CD4+and CD8+T cells were used for nucleofection adding 1 pig / reaction of the sleeping beauty vector containing the R12.BBz CAR construct and 0.6 pig minicircle DNA encoding the SB100X transposase (Plasmid factory) to the transfection medium. HGR molecule expression was disrupted by CRISPR / Cas9-mediated gene editing. Single guide RNA for hGR was purchased from Integrated DNA Technologies and Cas9 protein from PNA Bio. Electroporation was performed according to the manufacturer's protocol using the 4D-NucleofectorTM (Lonza, program EO-115). The electroporated cells (WOpil) were then transferred to a 48-well plate with 0.9 ml pre-warmed CTL medium and a half medium change with CTL supplemented with 100 U / ml recombinant human IL-2 was performed after 4h. CD3 / CD28 Dynabeads were removed magnetically 4 days after transfection. Transfection efficiency was assessed the day after bead removal by flow cytometry via staining of the tEGFR transfection marker with biotinylated anti-tEGFR mAb (in-house).

[0166] CAR-T cell proliferation assay

[0167] T cells were labeled with 0.2 mmol / L carboxyfluorescein succinimidyl ester (cell trace CFSE; life technologies), washed, and plated in triplicates with inactivated tumor cells at E:T ratio of 1:1 in cell culture medium without exogenous cytokines. After a 72 hours incubation, cells were labeled with CD8, CD4, tEGFR antibody. 7AAD (BD Biosciences) staining was conducted for live / dead cell discrimination. Samples were acquired by flow cytometry (FACS) to assess cell division by measuring CFSE dilution in living CAR+ T cells.

[0168] CAR-T cell cytotoxicity and cytokine analysis

[0169] CAR-T cells were co-cultured with 50,000 tumor cells (2D cell culture) at the specified E:T ratios in RPMI medium on 96-well flat-bottom plates. For 3D cell culture, an E:T ratio of 0.25:1 was applied using 200,000 tumor cells per spheroid and well. Co-cultures were incubated at 37°C and analyzed after 24 h. The basic analytic feature was used to quantify ATP to evaluate cell viability using the CellTiter Gio Assay (Promega) according to the manufacturer's protocol. Specific lysis was calculated by subtracting T cell viability from the total viability of untreated tumors and normalizing it to UTD T cell reference. After 24 h of co-culture, supernatants were collected and cytokine analysis was quantified by enzyme-linked immunosorbent assay (ELISA). Interferon (IFN)-y and Interleukin (IL)-2 ELISA Kits were obtained from BioLegend. The analyseswere performed according to the instructions of the manufacturer and absorbance was measured using a NanoQuant Infinite M200 Pro plate reader (TECAN).

[0170] In vivo experiments and tumor engraftment in ACC xenograft mouse models

[0171] All animal studies were carried out according to the animal experiment license that has been granted by the competent authorities and to the guidelines of the institutional animal care and use committee (University of Wuerzburg). Immunodeficient NOD.Cg-PrkdcSCID Il2rgtm1 Wjl / SzJ (NSG) mice were purchased from Charles River. 8-week-old female mice were inoculated subcutaneously with 1 x107NCI-H295R cells 14 days before T cell injection in 200pil sterile PBS with 1%sterile FCS. Mice were randomized and 1x106T cells were injected intravenously one time, 5 days after activation. Mice were monitored two to three times a week and tumor burden was quantified weekly using digital caliper measurement. Tumor area was calculated by multiplication of 3D tumor measurements using hemi ellipsoid formula (2 / 3 x TT X length x width x depth). Mice were humanely euthanized when tumor burden reached the dropout criteria or showed signs of morbidity that were not in line with animal welfare or reached a body condition score exceeding 15.

[0172] Assessment of T cell expansion, persistence and exhaustion in vivo

[0173] To assess T cell expansion in vivo, blood samples were taken at day 10, 14, 21, 28 and when mice had to be removed from the experiment. Blood, tumor, spleen and bone marrow were collected, counted and analyzed using FACS analysis. All samples have been stained for human CD45, CD4, CD8, and CAR (tEGFR). Moreover, to assess T cell persistence, activation and exhaustion in vivo, at endpoint analyses intratumoral T cells were isolated by a tumor dissociation kit (Miltenyi) and stained for human CD45, PD-1, LAG3, CTLA-4, Tim-3 (CD366) and CAR (tEGFR). T cell subsets and memory have been evaluated by staining for human CD45, CD45RO, CD45RA, CD62L, CD25, CD69 and CAR (tEGFR).

[0174] Statistical analysis

[0175] Statistical analyses were performed with Excel v.16.64 (Microsoft) and GraphPad Prism v.9.3.1 (GraphPad). Graphical schemes were designed with Biorender.com. Licenses are available on demand. Unless otherwise noted, analyses testing for significant differences between groups were conducted with unpaired two-tailed t-tests (when comparing two un-matched groups), paired t-tests (when comparing two matched groups), a oneway ANOVAwith Dunnett's test for multiple comparisons (when comparing more than two groups). We also used linear regression analysis to assess correlations between expression data and distinct clinical parameters. Survival curves were compared with the log-rank Mantel-Cox test. In vivo tumor growth was compared with a repeated measures ANOVA with correction for multiple comparisons. In vivo T cell proliferation and persistence were compared using the two-tailed Mann-Whitney test. P-values of less than 0.05 were considered statistically significant.

[0176] Supplementary Text

[0177] The inventorsassessed ROR1 expression in a set of ACCGC+cell lines - CU-ACC1, CU-ACC2, JIL-2266 and NCI-H295R - that each have a distinct signature of GC release - but glucocorticoid positive (Fig. 8c). The inventors quantified ROR1 transcripts by qRT-PCR and found a similar range of ROR1 expression betweentheir patient cohort and the set of ACC cell lines (Fig. 7b). Further quantitative analyses by RNAscope and by flow cytometry R0R1 expression in ACC cell lines. (Fig. 1 J, K, Fig. 8a-c). The inventors employed super-high resolution single molecule microscopy (dSTORM) to analyze the distribution and density of R0R1 protein on the surface of ACC cell lines and determined CU-ACC2 to be ROR1h'sh(0.428 clusters / pim2), JIL-2266 (0.116 clusters / pim2) and NCI-H295R (0.239 clusters / pim2) to be ROR1med, and CU-ACC1 to be ROR1low(0.054 clusters / pim2), using ROR1+(Jeko-1) and RORT (K562) hematologic cancer cell lines as reference (Fig. 8b, 9a-d).

[0178] Results

[0179] Example 1: ROR1 is highly expressed in ACC with GC excess

[0180] The inventors hypothesized that the intrinsic production of GCs in ACC induces a distinct transcriptional profile compared to normal adrenal glands and analyzed RNA transcriptome array data to identify genes that encode surface antigens (29). This analysis identified R0R1 as the top candidate and cancer antigen that was markedly overexpressed in ACC (Fig. 1a). The inventors confirmed overexpression of R0R1 transcripts in their collection of 62 clinically annotated primary ACC samples (Table 3), even though they also noted a range with high and low expressers (Fig. 1b and Fig. 7b). In multivariate analyses, high ROR1 expression in primary ACC was associated with an increased risk for rapid recurrence after first-line therapy, associated with higher ACC aggressiveness based on the ENSAT clinical staging system (30),KI67 proliferation index and the histopathological Weiss score (Fig. 1 c-f and Fig. 7b). These data suggested that ROR1 was a potential target for immunotherapy in ACC. The inventors assessed 135 clinically annotated ACC tissue sections by immunohistochemistry and found that ROR1 expression was stable between ACC samples obtained at diagnosis and at relapse, and between primary and metastatic ACC lesions (Fig. 1g, Table 3). Among the seven patients with the highest ROR1 expression as determined by immunohistochemistry, all patients had clinically presented with systemic steroid excess. Therefore, the inventors grouped samples according to the presence vs. absence of clinically reported steroid excess (ACCsteroid+vs. ACCsteroid-) and found a clear segregation of the two groups, with higher ROR1 expression in ACCsteroid+(Fig. 1 h, I). Of note, in the subgroup of ACCsteroid+patients that produced androgens rather than glucocorticoids, ROR1 expression was similar to ACCsteroid’. The inventors confirmed high ROR1 expression in a set of four ACC cell lines CU-ACC1 & CU-ACC2(31), NCI-H295R(32) and JIL-2266(33) that the inventors, therefore, used in addition to primary ACC in our subsequent experiments (Fig. 1j,k and Fig. 8a-c, 9a-d). Taken together, the inventors' analyses identified ROR1 as a highly expressed cancer antigen and marker of aggressive phenotype in ACC. In primary ACC, ROR1 expression was particularly high in the subgroup of ACCsteroid+patients with glucocorticoid excess (ACCGC+).

[0181] Table 3: Adrenocortical carcinoma (ACC): patients’ and tumor characteristics.

[0182] Data represent median values with ranges or total numbers. Tumor stage at the time of diagnosis according to the European Network for the Study of Adrenal Tumors (ENSAT) classification. In patients who experiencedlocal recurrences or distant metastases during mitotane treatment, endocrine activity was classified according to the information available at primary diagnosis.

[0183] qRT-PCR cohort IHC cohort N of ACC samples 62 135

[0184] N of patients 51 123 Sex(F:M) 35:16 85:42 Age (years) 52.5 (18-80) 46.9 (18-75) Tumour type:

[0185] Primary tumour 47 (75.8%) 102 (75.6%) Local recurrence - 14 (10.4%) Metastasis 15 (24.2%) 19 (14.1%) Hormone secretion:

[0186] Glucocorticoids 25 (49.0%) 67 (54.5%) Androgens or aldosterone 15 (29.4%) 10 (8.1 %) Inactive 9 (17.6%) 20 (16.3%) Unknown 2 (3.9%) 26 (21.1%) ENSAT tumour stage:

[0187] l-ll 24 (47.1%) 55 (44.7%) III 9 (17.6%) 39 (31.7%) IV 18 (35.3%) 25 (20.3%) unknown - 4 (3.3%) Resection status:

[0188] R0-RX 30 (58.8%) 80 (65.0%) R1-R2 13 (25.5%) 36 (29.3%) unknown 8 (15.7%) 7 (5.7%) Weiss score 5 (3-9) 6 (3-9) Ki67% index 22 (0-90) 17 (0-90)

[0189] Example 2: GC inhibitors diminish R0R1 expression in ACC

[0190] The inventors reasoned that excessive release of immune-suppressive GCs would compromise recognition of ACC by CAR-T cells (Fig. 10a) and anticipated a synergistic effect of ROR1 CAR-T cells and GC inhibitors. Therefore, the inventors treated ACC cells either with an inhibitor of steroidogenesis (i.e. mitotane,metyrapone or ketoconazole) or a hGR inhibitor (relacorilant), and performed co-culture experiments with R0R1 CAR-T cells to assess antitumor function. Unexpectedly, the inventors found a decrease in cytolytic activity and cytokine release from R0R1 CAR-T cells in the presence of GC inhibitor-treated vs. non-treated ACC cells (Fig. 2a and Fig. 10b-d). Upon further analyses, the inventors found decreased expression of R0R1 transcripts and of R0R1 protein after GC inhibitor treatment that was consistent in all four ACC cell lines that were analyzed (NCI-H295R, JIL-2266, CU-ACC1, CU-ACC2) (Fig. 2b and Fig. 11a-c). Mitotane, the most commonly used GC inhibitor in the clinic, exerted the most potent effect and conferred a > 90% decrease in R0R1 transcripts and converted almost all ACC cells from being ROR1+to RORT as assessed by dSTORM super-resolution microscopy analyses (Fig. 2b and Fig. 11a). The effect of mitotane was dosedependent and persistent, but completely reversible (Fig. 2c).

[0191] After discontinuation of mitotane treatment, R0R1 transcripts recovered to baseline and subsequently, ROR1 protein expression on the surface of ACC cells also returned to baseline (Fig.2d). When dexamethasone was added to the culture medium, the time to recovery of R0R1 transcripts and ROR1 protein to baseline was substantially shorter, and even exceeded baseline expression (Fig. 2d,e). A similar effect was confirmed for metyrapone and ketoconazole, and for relacorilant, respectively (Fig.2b and Fig. 11a-c). To corroborate their data, the inventors turned to their set of primary ACC and their clinical annotation and indeed found significantly lower R0R1 transcript and ROR1 protein expression in samples obtained from ACC patients that had received mitotane or other GC inhibitors prior to sampling (Fig. 2f). The decrease in ROR1 expression was particularly evident between matched samples from primary and metastatic ACC lesions that were obtained prior to and after GC inhibitor therapy, respectively (Fig. 2g, h and Fig. 11 d). Taken together, these data show that GC inhibitors diminish ROR1 expression in ACCGC+and support the hypothesis of a direct mechanistic link between GC excess and ROR1 overexpression.

[0192] Example 3: ROR1 transcription in ACC is regulated by hGR and STAT3

[0193] The inventors were intrigued that both inhibitors of steroidogenesis and a hGR inhibitor diminished ROR1 expression, suggesting the potential that an autocrine GC pathway was active in ACC. To evaluate the underlying mechanism, the inventors evaluated WNT5A expression, which has been shown to be a ligand for ROR1 and to induce ROR1 signaling, which in turn augments ROR1 expression through upregulating STAT3.24 26The inventors scrutinized ACC cells that had been treated with GC inhibitors and detected higher amounts of hGR and STAT3 transcripts, as well as higher amounts of hGR and STAT3 protein compared to non-treated ACC cells (Fig. 3a-d and Fig. 12a). Furthermore, the inventors detected increased amounts of the ROR1 ligand WNT5A in ACC cells after GC inhibitor treatment, suggesting a positive feedback loop to sustain ROR1 signalling (Fig. 3a-d). Therefore, the inventors hypothesized that R0R1 expression in ACC was regulated by a transcription complex of GC-activated hGR and STAT3, and that the formation of this complex can be blocked by GC inhibitors (Fig. 3e). The inventors performed proximity ligation analyses to demonstrate the direct interaction of hGR and STAT3 in ACC cells, and found that treatment with GC inhibitors decreased the amount of hGR / STAT3 complexes (Fig 3f), which was associated with reduced ROR1 surface expression (Fig. 3h). The inventors used primary ACC from their clinical cohort to confirm these data and todemonstrate that hGR / STAT3 complexes were significantly more abundant in ACCGC+compared to ACCGC-. The data showed that clinical therapy with GC inhibitors essentially abrogated the formation of hGR / STAT3 complexes and expression of R0R1 in both ACCGC+and ACCGC- (Fig. 3g). In all clinical ACC samples and cell lines, the number of hGR / STAT3 complexes strongly correlated with R0R1 expression (Fig. 3h,i). To provide definitive evidence for the dependence of R0R1 expression on hGR signaling, the inventors silenced hGR in ACC cells and found a significant reduction in R0R1 expression, whereas WNT5A and STAT3 expression was strongly upregulated (Fig. 3j,k and Fig. 12b). Collectively, these data demonstrate that autocrine GC signaling induces R0R1 in ACCGC+and show that a transcription complex of hGR and STAT3 regulates R0R1 expression, which can be abrogated by GC inhibitors.

[0194] Example 4: R0R1 CAR-T cells with hGR gene-edit are resistant to ACC-derived GCs

[0195] The inventors reasoned that due to the high density of R0R1, ACCGC+ought to be susceptible to recognition and elimination by R0R1 CAR-T cells, provided that these were sufficiently protected from the immune-suppressive effect of ACC-derived GCs. Therefore, the inventors performed gene-editing of the hGR locus to generate GC-resistant R0R1 CAR-T cells (hGR-K0R0R1 CAR-T cells; Fig. 4a-d and Fig. 13a,b).hGR-K0R0R1 CAR-T cells and non-edited R0R1 CAR-T cells had a similar composition with naive, effector and memory subpopulations at the end of manufacturing (Fig. 4e). The inventors performed a GC challenge with a pulse of 100 nM dexamethasone for 72 hours and found a marked drop in T cell viability in non-edited ROR1 CAR-T cells, whereashGR KOROR1 CAR-T cells maintained viability and yield (Fig. 4d). After the dexamethasone pulse, the inventors found that genes involved in T cell activation and signaling, antigen presentation, as well as immune checkpoint pathways and epigenetic modulation were differentially expressed between hGR-KO and non-edited ROR1 CAR-T cells (Fig. 4f). The inventors performed reference testing against the non-steroidogenic lymphoma and leukemia cell lines JeKol and K562 and found thathGR KOROR1 CAR-T cells exerted similarly potent effector and helper functions as non-edited ROR1 CAR-T cells (Fig. 14a). The inventors then challengedhGR KOROR1 CAR-T cells with ACC cell lines and recorded superior lysis of ACC target cells compared with non-edited ROR1 CAR-T cells, particularly evident against NCI-H295R cells that produce the highest amount of GCs (Fig. 4g and Fig. 8c).hGR-KOROR1 CAR-T cells also maintained their ability to produce and release IFN-y and IL-2, and to undergo productive proliferation after antigen-specific stimulation (Fig. 4h,i and Fig. 14b, c). Importantly,hGR-KOROR1 CAR-T cell activation and function remained firmly dependent on the presence and correlated with the amount of ROR1 antigen on ACC target cells (Fig.

[0196] 15a,b).

[0197] Example 5: GC-resistant ROR1 CAR-T cells induce durable remission of ACCGC+ / n vivo

[0198] The inventors established ACCGC+tumors in immunodeficient mice through s.c. inoculation of steroidogenic NCI-H295R cells. The inventors confirmed that ACCGC+tumors were ROR1+, were in a highly proliferative state by Ki67 staining, were fully developed as evidenced by CD31 expression on tumor vasculature, and expressed several characteristic cytochrome monooxygenases and ACC transcription factors (Fig. 5a and Fig. 17). Serum analyses confirmed GC excess, which correlated with ACCGC+tumor volume (r=0.6571, p=0.0016) (Fig. 5b). On day 14 after tumor inoculation, mice were randomized to treatment groups to receivea single i.v. dose of 1 x 106modified vs. non-modified T cells (Fig. 5c). All of the mice that were treated withhGR-KOROR1 CAR-T cells showed a drastic reduction in tumor burden (overall response rate 100%) within seven days of T cell infusion and remained in complete remission for 5 consecutive weeks (Fig. 5d-f). In the group of mice that were treated with non-edited ROR1 CAR-T cells, there were 2 / 7 mice that experienced tumor regression, which was stable for up to 2 weeks. However, at 3 weeks after T cell infusion, all of the mice presented with progressing tumors (Fig. 5d-f). In the group of mice that were treated withhGR’K0T cells (non-CAR-modified) or untransduced (UTD) T cells, all of the mice showed continuous tumor progression (Fig. 5d-f). With further follow-up, 6 / 7 mice that were treated withhGR-KOROR1 CAR-T cells remained in either partial or complete remission. There was a significant survival benefit with median overall survival not reached vs. 36 days for mice treated with hGR-KO vs. non-edited ROR1 CAR-T cells (p<0.0001) (Fig. 5d,e).hGR-KOROR1 CAR-T cells showed consistent engraftment, in vivo proliferation and persistence (Fig. 5g-l), as well as efficient migration into ACC lesions (Fig. 5k, I). There was long-term persistence ofhGR KOROR1 CAR-T cells (Fig. 5h) with a high frequency of stem central memory (Lem) and central memory (Tcm) cells, a low frequency of PD-1 / TIM-3 / LAG-3 triple-positive T cells, and elevated expression of the activation markers CD25 and CD69 (Fig. 5i,j). Taken together, these data show thathGR-KOROR1 CAR-T cells circumvent the inhibitory effects of GCs in steroidogenic ACCGC+in vivo and outperform conventional, non-edited ROR1 CAR-T cells. Treatment withhGR KOROR1 CAR-T cells induced durable complete remissions and partial remissions, suggesting the potential for therapeutic efficacy against clinical ACCGC+in humans.

[0199] Example 6: Non-endocrine cancers secrete active GCs through recycling of inactive GC metabolites The inventors sought to explore if this mechanism of autocrine GC signaling also accounts for other solid cancers and hypothesized that certain tumors might upregulate HSD11B1 as well as hGR expression for effective GC recouperation and ROR1 upregulation. The inventors found HSD11B1 to be overexpressed in a variety of hematological and solid tumors (Fig. 17a,b). Furthermore, bulk RNAseq data showed a strong correlation between HSD11B1 expression and the frequency of tumor-infiltrating conventional and regulatory T cells (Leg) (Fig. 17c) as well as glucocorticoid-response genes, T cell dysfunction and Leg marker genes in human cancers (Fig. 17d). By analyzing TCGA databases, the inventors found a strong correlation between hGR and ROR1 mRNA expression in hematologic and non-hematologic, endocrine and non-endocrine cancers (including i.a. ACC, lymphoma, breast, pancreatic, colorectal, renal, prostate cancer) (Fig. 18a-c). In all analyzed types of cancer the inventors found intrinsic HSD11B1 copy-number-variant gain of function mutations (Fig. 18d). The inventors therefore analyzed and confirmed HSD11B1 expression in MDA-MB231 breast cancer and PANC-1 pancreatic cancer cells, and analyzed the ability of these cell lines to convert inactive cortisone into active cortisol. The inventors observed efficient conversion (59-84%) of cortisone into active forms of cortisol after 24 hours (Fig 6a). The inventors confirmed the biologic activity of the converted cosrtisol and found diminished T cell viability and yield after T-cell culture in medium containing increasing concentrations of converted cortisol (Fig. 6b). The inventors next asked if cortisone treatment can stimulate HSD11B1 as well as ROR1 expression and observed a dose-dependent increase of HSD11B1 and ROR1 transcripts (Fig. 6c) that both strongly correlated with the amount of secreted GCs (Fig. 6d). Using aphysiologic concentration of cortisone the inventors checked if this conversion could be abrogated by metyrapone, which inhibits CYP11B1 and HSD11B1 (52). While cortisone was efficiently converted into cortisol in untreated cells, metyrapone inhibited regeneration and prevented R0R1 upregulation through abrogated hGR / STAT3 signaling (Fig. 6e-g and Fig. 19a-c. Lastly, the inventors confirmed the presence of hGR / STAT3 complexes in two exemplary primary breast cancer samples (Fig. 18e), thus confirming the same GC signaling mechanism in R0R1 expressing cancers.

[0200] Example 7: GC-resistant R0R1 CAR-T cells are effective in GC secreting cancers

[0201] The invnetors sought to determine the effect of GC recycling in non-endocrine cancers on the phenotype and function of R0R1 CAR-T cells. The inventors treated breast and pancreatic cancer cells for 24 hours with 250nM of cortisone to induce its conversion into cortisol, and assessed R0R1 CAR-T cell function. The data show that GC-resistant R0R1 CAR-T cells conferred superior anti-tumor functions with higher tumor cell lysis, higher cytokine secretion, proliferation and viability compared to conventional non-GC-resistant R0R1 CAR-T cells (Fig. 6h,i and Fig. 19d,e).

[0202] Taken together, the data demonstrate that autocrine GC signaling and GC secretion either through de novo production (example: ACC) or recycling from inactive GCs (example: ACC, breast cancer, pancreatic cancer) impairs the anti-tumor reactivity of CAR-T cells (Fig. 20). In both endocrine and non-endocrine cancers, expression of HSD11B1 is a marker that indicates an immune-suppressive tumor and tumor milieu and instructs the use of GC-resistant CAR-T cells in order to achieve anti-tumor efficacy.

[0203] Example 8: GC-resistant ROR1 CAR-T cells are effective against PDAC and TNBC in vivo

[0204] The inventors were encouraged by their data to assess the efficacy ofhGR KOROR1 CAR-T cells in xenograft models of PDAC (PANC-1) and TNBC (MDA-MB-231), respectively, in vivo (Fig. 21a). They confirmed that HSD11 B1 converted murine 11 -deoxycorticosterone (DOC) into active corticosterone, and also confirmed the presence of DOC in serum of NSG mice (Fig. 24a). Following tumour engraftment, mice were randomized to treatment groups to receivehGR KOROR1 CAR-T cells, non-edited ROR1 CAR-T cells, or UTD T cells, respectively. In both, the PDAC and the TNBC model,hGR-KOROR1 CAR-T cells conferred superior anti-tumour efficacy compared with non-edited ROR1 CAR-T cells, resulting in a statistically significant survival benefit compared with non-edited ROR1 CAR-T cells and UTD T cells (Fig. 21 b,c & 22b, c).

[0205] The inventors obtained progressing PDAC and TNBC tumours at the experiment endpoint in all treatment groups and found that R0R1 transcript and ROR1 protein expression were higher in mice that had receivedhGR-KOROR1 CAR-T cells, and to a lesser extent also non-edited ROR1 CAR-T cells, compared to UTD T cells (Fig. 23a, b & 25a, b). This was surprising, as antigen downregulation or even antigen loss is frequently observed in tumours that persist after CAR-T cell therapy (56, 57). Furthermore, there was a strong correlation between HSD11B1 and R0R1 expression in tumour lesions (Fig. 23c & 24b). The inventors also noted that there was a strong correlation between HSD11B1 expression in tumour lesions and DOC concentration in mouse serum (Fig. 24b, c), supporting that HSD11B1 was catalytically active and fueled the autocrinesignalling pathway that augments R0R1 expression with recycled GCs in cancer cells in both xenograft models in vivo.

[0206] Of particular interest, the inventors found that HSD11B1 transcripts in tumour cells were higher after treatment withhGR K0R0R1 CAR-T cells and non-edited R0R1 CAR-T cells compared with UTD T cells (Fig. 23a, b).

[0207] TNFo and other immune cell cytokines have been reported to induce HSD11B1 in cancer cells (56), suggesting that PDAC and TNBC may respond to immunological stress from CAR-T cells with this mechanism, resulting in augmented GO recycling and subsequent immune-suppression. Indeed, when the inventors exposed PANC-1 cells and MDA-MB-231 cells to increasing numbers ofhGR-K0R0R1 CAR-T cells and different doses of TNFo in vitro, there was a gradual increase in HSD11B1 transcripts in tumour cells that they obtained during the anti-tumour response (Fig. 23d, 24d). Taken together, these data show thathGR-K0R0R1 CAR-T cells are resistant to the immunosuppression imparted by GC recycling and release in PDAC and TNBC, and confer potent anti-tumour reactivity in xenograft models in vivo. CAR-T cell cytokines that are released during the anti-tumour response, trigger a further increase in HSD11 B1 activity in PDAC and TNBC cells that augments GC recycling and release, but also R0R1 expression due to autocrine GC signaling. Collectively, the data obtained in the setting of PDAC and TNBC provide examples of autocrine GC signaling that regulates R0R1 expression in steroidogenic non-endocrine cancers, and show that thathGR K0R0R1 CAR-T cells are resistant to the paracrine effects of recycled GCs.

[0208] Discussion

[0209] The inventors study uncovered autocrine GC signaling in hormonally active cancers as a new mechanism that induces expression of cancer-associated antigens, which can be targeted with CAR-T cell immunotherapy. Autocrine signaling in hormonally active cancers has thus far received limited attention in basic and translational cancer research. Prior studies have reported on autocrine signaling through vascular endothelial growth factor and heat shock proteins that promote proliferation and progression in gastric cancer (34, 35). The inventors show that GC-activated hGR forms a transcription complex with STAT3 to regulate R0R1 expression in ACC and that this hGR / STAT3 complex can be disrupted by GC inhibitors. STAT3 has been shown to be constitutively expressed in several types of cancer and has been positively correlated with immune cell infiltration (36). Synergy between STAT3 and hGR has been reported in basal-like TNBC (8).A recent study has reported that administration of dexamethasone promotes the formation of metastases in preclinical models of breast cancer and reported increased expression of R0R1 after dexamethasone treatment (11 ). Another group reported that GCs induce differentiation and chemoresistance in ovarian cancer, and that dexamethasone increases R0R1 expression in ovarian cancer cell lines. They postulated an indirect mechanism between hGR signaling and R0R1 expression, based on the observation that R0R1 downregulation did not affect hGR expression (37). Their study reveals that GC-activated hGR signaling is directly linked to R0R1 transcription in ACC as also demonstrated by an antagonistic effect of GC-inhibitors and an agonistic action by endogenous / exogenous GCs on R0R1 expression. The inventors confirmed this antagonism on data obtained from our clinical cohort of ACCGC+patients. The clinical data also showed thathigh ROR1 expression in ACC correlates with advanced clinical stage at first diagnosis, aggressive disease course and worse clinical outcome compared to ACCGC-. These data support the inventors' working model where autocrine GC signaling induces ROR1 as an oncogenic driver that augments and perpetuates the aggressive phenotype of ACCGC+. The inventors also detected ROR1 in ACCGC-, that produce lower amounts of GCs without systemic excess but were still susceptible to GC-inhibitor treatment, which abrogated hGR / STAT3 complex formation. Upon treatment with GC-inhibitors, the inventors detected increased WNT5A in both ACCGC+and ACCGC’. WNT5A has been shown to be a ligand for and activator of ROR1 signaling in lymphoma (24-26), supporting the inventors'r conclusion that ROR1 is active and an oncogenic driver in ACC.

[0210] The inventors further demonstrate that ROR1 is a suitable biomarker for intratumoral GC secretion and that CAR-T cells that are shielded from the immune-suppressive effects of GCs maintain their function and confer an effective antitumor response in mice. The inventors confirmed GC secretion in ROR1 expressing solid tumors and demonstrate that non-systemic intratumoral GC secretion impairs CAR-T cell function. This is particular noteworthy, as ROR1 is expressed by a variety of solid tumors and associated with aggressive disease (37, 24, 54). While, the development of GC-resistant T cells has been of interest for treating viral infections in recipients of allogeneic bone marrow transplantation that suffer from graft-versus-host disease (38), the inventors' data expand the utility of GC-resistant CAR-T cells for a broad range of hormonally active cancers. A prior study reported on the development of allogeneic off-the shelf I L 13Ra2 CAR-T cells with zinc-finger mediated hGR gene-editing for glioblastoma therapy, where steroids are administered to treat cerebral edema (39). The clinical development of CAR-T cells that are shielded from exogenous GCs may also be of interest for maintaining efficacy in cancer patients that experience cytokine release syndrome (CRS), immunecell associated neurotoxicity or hematotoxicity syndrome (ICANS / ICAHT), where dexamethasone is frequently used to control systemic inflammation (40). In this case, exogenous GCs would not compromise the efficacy of hGR-Kog R-Tce||Sj an(jworkonnon-CAR-modified adaptive and innate immune cells, and endothelial cells in vasculature and blood-brain barrier that are part of the adverse outcome pathways (41-43). The inventors challenge the paradigm that the therapeutic use of GCs ought to be generally avoided in the context of cancer immunotherapy, and illustrates the potential to exploit GC-induced antigen expression on cancer cells with GC-resistant antigen-specific CAR-T cells. The inventors also highlight the exciting potential to essentially steer ROR1 expression in ACC through administration of either GC-inhibitors or GCs and tune ROR1 antigen density to a sweet spot with optimal efficacy-to-toxicity ratio.hGR KOROR1 CAR-T cells may also be useful for treating other endocrine and non-endocrine cancers that produce GCs intrinsically or in their microenvironment (11-14). The inventors have not detected induction of ROR1 after dexamethasone treatment in non-malignant cells, likely owing to the transient and tightly regulated expression of STAT3. In summary, the inventors conclude that autocrine signaling in hormonally active cancers is a newly discovered mechanism that induces antigen expression that can be exploited for targeting cancer with immunotherapy. Considering that many endocrine and non-endocrine cancers have the ability to produce GCs, steroids and other hormones, this discovery has broad implications for cancer biology and therapy.Industrial applicability

[0211] The CARs, the CAR T-cells for use and the methods and medical uses according to the invention are industrially applicable. For example, they can be used for the production of, pharmaceutical products.

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Claims

CLAIMS1. A recombinant glucocorticoid (GC)-resistant immune cell, for use in the treatment of cancer in a human cancer patient, wherein the immune cell expresses a recombinant immune receptor having an extracellular antigen-binding domain capable of binding to a cancer antigen of the cancer, and wherein the cancer is a cancer expressing at least one enzyme of a synthesis or regeneration pathway for active glucocorticoids selected from the group consisting of HSD11B1, CYP11B1, CYP11A1, CYP17A1, and CYP21A2.

2. The recombinant immune cell for use according to claim 1, wherein the cancer is a HSD11B1- expressing cancer.

3. The recombinant immune cell for use according to any one of the preceding claims, wherein the immune cell is a T cell, an NKT cell, a B lymphocyte, a natural killer cell, a monocyte, a macrophage, a dendritic cell, or a granulocyte.

4. The recombinant immune cell for use according to any one of the preceding claims, wherein the immune cell is a T cell.

5. The recombinant immune cell for use according to any one of the preceding claims, wherein the recombinant immune receptor is a recombinant T-cell receptor.

6. The recombinant immune cell for use according to any one of the preceding claims, wherein the cancer antigen is an antigenic peptide of an intracellular cancer antigen and is presented on an MHC molecule.

7. The recombinant immune cell for use according to claim 6, wherein the intracellular cancer antigen is an intracellular cancer antigen whose expression is upregulated by cortisol.

8. The recombinant immune cell for use according to any one of claims 1-6, wherein the cancer antigen is a cell-surface cancer antigen.

9. The recombinant immune cell for use according to claim 8, wherein the cell-surface cancer antigen is a cell-surface cancer antigen whose expression is upregulated by cortisol.

10. The recombinant immune cell for use according to any one of the preceding claims, wherein the recombinant immune receptor a chimeric antigen receptor (CAR).

11. A recombinant glucocorticoid (GC)-resistant chimeric antigen receptor (CAR) T-cell for use in the treatment of a HSD11 B1-expressing cancer in a human cancer patient, wherein the CAR T-cell expresses a chimeric antigen receptor (CAR) having an extracellular antigen-binding domain capable of binding to a cell-surface cancer antigen of the cancer.

12. The recombinant immune cell or CAR T-cell according for use according to any one of the preceding claims, wherein the cancer is a non-endocrine cancer.

13. The recombinant immune cell or CAR T-cell according for use according to any one of claims 1-11, wherein the cancer is an endocrine cancer.

14. A recombinant glucocorticoid (GC)-resistant chimeric antigen receptor (CAR) T-cell for use in the treatment of a non-endocrine cancer in a human cancer patient, wherein the CAR T-cell expressesa chimeric antigen receptor (CAR) having an extracellular antigen-binding domain capable of binding to a cell-surface cancer antigen of the cancer.

15. The recombinant immune cell or CAR T-cell for use according to any one of the preceding claims, wherein the cancer is a glucocorticoid-secreting cancer.

16. The recombinant immune cell or CAR T-cell for use according to claim 15, wherein the glucocorticoid-secreting cancer is a cortisol-secreting cancer.

17. The recombinant immune cell or CAR T-cell for use according to any one of the preceding claims, wherein the cancer is selected from the group consisting of acute myeloid leukemia (LAML), adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), brain lower grade glioma (LGG), breast invasive carcinoma (BRCA) such as Triple-negative breast cancer (TNBC), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), cholangiocarcinoma (CHOL), chronic myelogenous leukemia (LCML), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), lymphoid neoplasm diffuse large B-cell lymphoma (DLBC), mesothelioma (MESO), miscellaneous (MISO), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD) such as pancreatic ductal adenocarcinoma (PDAC), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), sarcoma (SARC), skin cutaneous melanoma (SKCM), stomach adenocarcinoma (STAD), testicular germ cell tumors (TGCT), thymoma (THYM), thyroid carcinoma (THCA), uterine carcinosarcoma (DCS), uterine corpus endometrial carcinoma (UCEC), multiple myeloma (MM) and uveal melanoma (UVM).

18. The recombinant immune cell or CAR T-cell for use according to claim 17, wherein the cancer is breast invasive carcinoma (BRCA) such as Triple-negative breast cancer (TNBC) or pancreatic adenocarcinoma (PAAD) such as pancreatic ductal adenocarcinoma (PDAC).

19. The recombinant immune cell or CAR T-cell for use according to any one of the preceding claims, wherein the cancer contains a HSD11 B1 gain-of-function mutation in the HSD11 B1 gene.

20. The recombinant immune cell or CAR T-cell for use according to any one of the preceding claims, wherein the cancer overexpresses HSD 11 B 1.

21. The recombinant immune cell or CAR T-cell for use according to any one of the preceding claims, wherein the cancer expresses ROR1.

22. The recombinant immune cell or CAR T-cell for use according to any one of the preceding claims, wherein the cancer overexpresses ROR1.

23. The recombinant immune cell or CAR T-cell for use according to any one of the preceding claims, wherein the cell-surface cancer antigen is ROR1.

24. The recombinant immune cell or CAR T-cell for use according to any one of the preceding claims, wherein the extracellular antigen-binding domain of the CAR contains an scFv selected from:46(i) an anti-R0R1 scFv comprising, preferably in an N- to C- terminal order, an antibody heavy chain variable domain amino acid sequence according to SEQ ID NO: 4, an amino acid linker sequence which is preferably the amino acid sequence according to SEQ ID NO: 6, and an antibody light chain variable domain amino acid sequence according to SEQ ID NO: 8,(ii) an anti-ROR1 scFv having the same antibody heavy chain variable domain CDRs and the same antibody light chain variable domain CDRs as the anti-ROR1 scFv according to (I), and(ill) an anti-ROR1 scFv which competes with the anti-ROR1 scFv according to (I) for binding to R0R1.

25. The recombinant immune cell or CAR T-cell for use according to any one of the preceding claims, wherein the extracellular antigen-binding domain of the CAR contains an anti-ROR1 scFv comprising, in an N- to C- terminal order, an antibody heavy chain variable domain amino acid sequence according to SEQ ID NO: 4, an amino acid linker sequence which is preferably the amino acid sequence according to SEQ ID NO: 6, and an antibody light chain variable domain amino acid sequence according to SEQ ID NO: 8.

26. The recombinant immune cell or CAR T-cell for use according to any one of the preceding claims, wherein the immune cell or CAR T-cell is a human glucocorticoid receptor (hGR) knockout cell.

27. The recombinant immune cell or CAR T-cell for use according to claim 26, wherein the hGR knockout is a knockout by CRISPR / Cas9-mediated gene editing.

28. The recombinant immune cell or CAR T-cell for use according to any one of claims 1-27, wherein the immune cell or CAR T-cell is a human glucocorticoid receptor (hGR) knockdown cell.

29. The recombinant immune cell or CAR T-cell for use according to claim 28, wherein immune cell or CAR T-cell contains an siRNA against hGR mRNA.

30. The recombinant immune cell or CAR T-cell for use according to claim 28 or 29, wherein immune cell or CAR T-cell expresses an shRNA against hGR mRNA, and wherein the immune cell or CAR T-cell preferably contains a recombinant genomic expression cassette expressing the shRNA.

31. The recombinant immune cell or CAR T-cell for use according to any one of the preceding claims, wherein the cancer patient is not a glioblastoma patient.

32. The recombinant immune cell or CAR T-cell for use according to any one of the preceding claims, wherein the cancer patient is not a patient receiving treatment with steroids.

33. Use of a recombinant glucocorticoid (GC)-resistant immune cell for the manufacture of a medicament for the treatment of a cancer in a human cancer patient, wherein the cancer is as defined in claim 1, and wherein the immune cell expresses a recombinant immune receptor having an extracellular antigen-binding domain capable of binding to a cancer antigen of the cancer.

34. A method for the treatment of a cancer in a human cancer patient, the method comprising:identifying from a group of cancer patients a patient who has a cancer expressing an enzyme as47defined in claim 1 by determining whether the cancers of the group of cancer patients express the enzyme; and treating the patient who has a cancer expressing the enzyme with a recombinant glucocorticoid (GC)-resistant immune cell, wherein the immune cell expresses a recombinant immune receptor having an extracellular antigen-binding domain capable of binding to a cancer antigen of the cancer.

35. The use or method of claim 33 or 34, wherein:(I) the cancer is as defined in any one of the preceding claims;(II) the recombinant immune receptor is a recombinant immune receptor or CAR as defined in any one of the preceding claims;(III) the recombinant immune cell is a recombinant immune cell or CAR T-cell as defined in any one of the preceding claims; and / or(IV) the patient is as defined in any one of the preceding claims.

36. A kit, comprising:(a) reagents for determining whether the cancer of a cancer patient expresses an enzyme as defined in claim 1, wherein the reagents optionally comprise nucleotide primers capable of binding to the mRNA encoding the enzyme and or antibodies capable of binding to the enzyme; and (b) a recombinant glucocorticoid (GC)-resistant immune cell, wherein the immune cell expresses a recombinant immune receptor having an extracellular antigen-binding domain capable of binding to a cancer antigen of the cancer.

37. The kit of claim 36, wherein:(I) the recombinant immune receptor is a recombinant immune receptor or CAR as defined in any one of the preceding claims;(II) the enzyme is HSD11B1, and / or(III) the recombinant immune cell is a recombinant immune cell or CAR T-cell as defined in any one of the preceding claims.

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