The embryonic antigen Receptor Tyrosine Kinase Receptor 1 (ROR1) is expressed by cells of various B-cell malignancies, but is absent in almost all healthy tissues, including B-cells. Whilst such an expression pattern suggests it could represent an attractive target for Chimeric Antigen Receptor (CAR) T-cell immunotherapy, it is present at much lower levels than other antigens that are commonly targeted on malignant B-cells. Consequently, we determined whether the level of antigen density affects the ability of CAR-expressing T-cells to recognise and destroy tumour cells, using Chronic Lymphocytic Leukaemia (CLL) as the model disease.
A CAR specific for ROR1 was generated and specificity and optimisation of targeting was determined using matched positive and negative cell lines. The level of ROR1 expression on the surface of primary CLL cells isolated from 19 patients was determined by flow cytometry. Co-cultures between CLL cells and ROR1_CAR+ T-cells were undertaken at a 1:1 ratio, with CLL survival determined by flow cytometry at defined timepoints.
A range of ROR1 expression levels was noted across our patient cohort, but this did not correlate with any known prognostic markers. Interestingly, whilst T-cells expressing a CD19-specific CAR showed substantial anti-tumour activity against autologous CLL cells from all patients, efficacy of ROR1_CAR T-cells was not observed uniformly across the cohort. Importantly, efficacy of the ROR1_CAR T-cells appeared to correlate with ROR1 expression as antigen density was significantly lower in patient samples that were not lysed, compared to those that were. To investigate this further, we are currently determining whether CLL cells isolated from patients that failed in vitro ROR1_CAR T-cell therapy and manipulated via electroporation to express enhanced ROR1 levels are subsequently susceptible to ROR1_CAR T-cell destruction. A similar phenomenon has been observed in a panel of matched cell lines, with those engineered to express a high level of ROR1 significantly more susceptible to in vitro ROR1_CAR-mediated destruction. Importantly, equivalent destruction by a CD19-specific CAR demonstrated that this increased susceptibility was not an intrinsic property of the engineered tumour cells.
The data presented here suggest that antigen density may play a key role in the efficacy of CAR-mediated T-cell retargeting. Tumour cells bearing low levels of ROR1 were significantly more resistant to CAR-mediated destruction than equivalent cells expressing higher ROR1 antigen levels. We are currently confirming whether this observation is recapitulated with other targets.
Citation Format: David Marc Davies, Solange Paredes-Moscosso, Satyen Gohil, Marco Della-Peruta, Vania Coelho, Amit Nathwani. Antigen density determines the efficacy of chimeric antigen receptor (CAR) re-targeted T-cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B125.
- ©2016 American Association for Cancer Research.