A phase I/II trial of CRISPR-Cas9-mediated PD-1 knockout Epstein-Barr Virus cytotoxic lymphocytes (EBV-CTLs) for advanced stage EBV associated malignancies

Abstract

Background: EBV associated malignancies exhibits high amplification of PD-L1 as distinguished from EBV non-associated malignancies (Kim et al. Gastroenterology 2015; Chen et al. Clinical Cancer Research 2013). The up-regulation of PD-L1 restricts antitumor effect of EBV-CTLs by immune tolerance and results in poor prognosis of patients. Our previous work has generated PD-1-disrupted CTLs by CRISPR-Cas9 system which could upregulate IFN-gamma production and enhance cytotoxicity in tumor cell lines and mouse model (Su et al. Oncoimmunology 2016). Trial design: This phase I/II prospective single center clinical study (clinicaltrials.gov NCT03044743) was designed to evaluate the safety of PD-1 knockout EBV-CTLs in treating EBV positive advanced stage malignancies. Patients included should be pathologically verified EBV positive stage IV gastric carcinoma, nasopharyngeal carcinoma or lymphoma progressed after standard treatment with measurable lesions. Patients will be divided into three groups and receive 2 to 4 cycles of cell therapy according to their tolerance. PD-1 knockout EBV-CTLs from autologous origin will be generated and 2 x 10^ /kg of specific T cells will be infused in one cycle. Each cycle is divided into three administrations, with 20%, 30% and 50% respectively. To modify immune microenvironment, Fludarabine at 30mg/m and cyclophosphamide at 300mg/m will be administered 3 days (intravenous injection, i.v.) before cell infusion. Interleukin-2 will be given daily (i.v.) from the first day of the cell infusion for 5 consecutive days at the dose of 4000,000 international unit (IU)/day to sustain the survival of infused T cells. The adverse events will be evaluated after each cycle by Common Terminology Criteria for Adverse Events (CTCAE v4.0) as primary endpoint. Progression-free survival (PFS), the duration of the normalization of tumor marker and immunological markers will be evaluated as the secondary endpoints. Immunological markers will continuously be examined every two cycles.