Associate Professor of Urology University of California, San Diego (UCSD)
Introduction: Prostate cancer (PCa) is a heterogeneous tumor, therefore, novel treatments need to target combinations of pathways. We produced a new cell-based immunotherapy - a dual targeting CAR T-cell - for the treatment of advanced prostate cancer. We first developed a mono-specific CAR T-cell using the inhibitory antibody therapeutic, Zilovertamab, specific for the fetal oncoprotein, ROR1, a non-canonical WNT5A receptor. The Wnt ligand, WNT5A, is increased in bone metastatic, castration resistant PCa (CRPC). ROR1, is not expressed in adult tissue but is up regulated in several cancers including metastatic PCa making it a good immunotherapy target. Zilovertamab, is safe and in clinical trials for chronic lymphocytic leukemia (CLL) and metastatic breast cancer. We investigated Zilovertamab-based anti-ROR1 CAR T-cells for mCRPC and developed dual specificity anti-ROR1 CAR T-cells which additionally target Trop2 or PSMA. Methods: We generated a series of 2nd-generation T-cell CAR constructs using lentiviral vectors which were transduced into normal human T cells. Trop2 and PSMA have clinically available antibody therapeutics we used in CARs. We used ROR1+ PCa cell lines, PC3 and DU145, and our ROR1+ patient-derived xenograft (PDX), PCSD13, a small cell bone metastatic prostate cancer to test the efficacy of mono-specific Zilovertamab-CART cells and dual-specific CAR T-cells in vitro using the real time cell viability, proliferation, and cell cycle tracking assays, in an Incucyte S3 along with LDH- and 51Cr-release cytotoxic T cell assays. We used in vivo bioluminescence and tumor caliper measurements to monitor effects in vivo. Tumor tissues were collected for analyses of ROR1 RNA, protein expression and signaling. Results: We showed that ROR1 was expressed at high levels in PC3, DU145, and in PCSD13 using RNASeq, qRT-PCR, FACS and Western blotting. CRISPR-Cas9 Knock out of ROR1 in PC3 and DU145 cells showed increased sensitivity to docetaxel inhibition of proliferation in vitro. Likewise, treatment of PCSD13 PDX in vivo with Zilovertamab increased docetaxel-mediated tumor growth inhibition in vivo. Mice bearing PC3 xenografts injected intravenously with Zilovertamab anti-ROR1 CAR-T cells showed durable, long term tumor ablation. We have now produced a series of 2nd generation T-cell CAR constructs that when transduced into human T-cells, demonstrated highly potent and specific anti-tumoral activity and specificity in vitro and in vivo. Conclusions: These studies supported the recently launched Phase 1b clinical trial of Zilovertamab plus docetaxel in metastatic CRPC patients. Clinical development of GMP Zilovertamab CAR T- cells for a clinical trial in CLL is in progress and may lead to rapid progression to a clinical trial for metastatic CRPC. The highly potent, prolonged, and specific activities we observed with the Zilovertamab-based anti-ROR1 CAR T-cells was further enhanced with the anti-ROR1 dual-targeting CAR T-cell products. SOURCE OF Funding: DOD CDMRP PCRP IMPACT Award, JM Foundation, Leo and Anne Albert Charitable Trust, Padres Pedal the Cause Translation Award, UCSD Moores Cancer Center Seed Grant.
