CRISPR/Cas9-mediated glucocorticoid receptor knockout effectively enhances antitumor efficacy of ROR1 specific CAR-T cells in advanced adrenocortical carcinoma

Marc Philipp Schauer; Justus Weber; Laura-Sophie Landwehr; Peter Spieler; Barbara Altieri; Tanja Maier; Matthias Kroiss; Stefan Kircher; Max Kurlbaum; Liz Tony; Silviu Sbiera; Martin Fassnacht; Michael Hudecek

doi:10.1530/endoabs.99.OC7.1

OC7.1

Prev Next

Section Contents Cite

Endocrine Abstracts (2024) 99 OC7.1 | DOI: 10.1530/endoabs.99.OC7.1

ECE2024 Oral Communications Oral Communications 7: Endocrine-related Cancer (6 abstracts)

CRISPR/Cas9-mediated glucocorticoid receptor knockout effectively enhances antitumor efficacy of ROR1 specific CAR-T cells in advanced adrenocortical carcinoma

Marc Philipp Schauer ^1,2 , Justus Weber ² , Laura-Sophie Landwehr ¹ , Peter Spieler ² , Barbara Altieri ¹ , Tanja Maier ³ , Matthias Kroiss ^1,3,4 , Stefan Kircher ⁵ , Max Kurlbaum ¹ , Liz Tony ² , Silviu Sbiera ¹ , Martin Fassnacht ^1,4 & Michael Hudecek ²

Views

Author affiliations

¹University Hospital Würzburg, Department of Internal Medicine I, Division of Endocrinology & Diabetes, Germany; ²University Hospital Würzburg, Department of Internal Medicine II, Chair for Cellular Immunotherapy, Germany; ³LMU Hospital Munich, Department of Internal Medicine IV, Division of Endocrinology & Diabetes, Germany; ⁴Comprehensive Cancer Center Mainfranken, University of Würzburg, Germany; ⁵University Hospital Würzburg, Institute of Pathology, Germany

Background: Adrenocortical carcinoma (ACC) is a rare and aggressive endocrine malignancy with poor prognosis and very limited treatment options in advanced disease. The only curative approach is complete surgical resection. Additionally, 60% of patients show endogenous glucocorticoid (GC) excess with clinical apparent hypercortisolism and low to no immune cell infiltration. To date, no therapeutically relevant surface markers are known for ACC, which is why it has not been considered for cell therapeutic interventions like CAR-T cell therapy.

Methods: In this study, we identified the receptor tyrosine kinase-like orphan receptor-1 (ROR1) as new promising target antigen for CAR-T cell therapy in a representative cohort of 197 ACC patients´ samples using RNA-sequencing, qRT-PCR (n=62) and IHC (n=135). We further evaluated ROR1 expression in five human ACC cell lines using qRT-PCR, qFACS, super high resolution single molecule microscopy (dSTORM) and RNAscope single cell analysis. Lastly, we assessed immunotherapeutic performance of different next-generation ROR1 CAR-T cell modifications in different preclinical models of ACC.

Results: We demonstrate ROR1 to be highly upregulated in in 92.7% of human ACC specimen (n=197) as compared to healthy tissue and as clinically relevant prognostic marker for GC excess and disease progression in ACC. All five ACC cell lines were expressing ROR1 in different levels. ROR1 CAR-T cells were generated and functionally tested using preclinical models of ACC. Considering the GC-enriched inhibitory tumor microenvironment, we recapitulated these modalities using steroidogenic ACC cell lines, exogenous supplementation of dexamethasone, 3D cell culture and a novel difficult-to-treat steroidogenic ACC xenograft mouse model. We observed a considerable decrease in antitumor efficacy elicited by ROR1 CAR-T cells in vitro and in vivo. Pharmaceutical inhibition of GC effector functions induced significant mitigation of CAR-T cell potency and antitumor killing due to a corticosteroid inhibitor-related downregulation of ROR1 on ACC tumor cells. This effect could be attributed to the inhibition of activated hGR/Stat3 tethered proximal transcription factor signaling. To maintain immunotherapeutic efficacy, we therefore desensitized ROR1 CAR-T cells by CRISPR/Cas9-mediated genome editing of the GC receptor (hGR) locus enabling (^hGRKO ROR1 CAR-T cells to completely eradicate all ACC xenografts while persisting in vivo, keeping periodic tumor regressions stable and inducing complete and sustained remission in 30% of all mice without toxicity.

Conclusions: Our results demonstrate ROR1 as promising target antigen for CAR-T cell therapy in ACC, while hGRKOROR1-CAR-T cells have been superior to a combined therapy using pharmaceutical blockade of GC effector functions in preclinical models of ACC.