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Refractory solid tumors remain an ongoing clinical problem with significant morbidity and mortality. CAR T cell therapy has the potential to address this issue. However, in contrast to their success in B cell malignancies, CAR T cell therapy targeting solid cancer antigens has had limited success so far due to several reasons including tumor antigen heterogeneity. To address this issue, we have previously generated a second-generation CAR recognizing the carbohydrate antigen Lewis Y (LeY). Given that LeY is highly expressed in various epithelial cancers but appears to have only limited expression on the surface of normal tissues making this antigen an ideal target for cancer immunotherapy. In a Phase I clinical trial involving transfer of anti-LeY CAR T cells in AML patients the detection and monitoring of CAR T cells in peripheral blood was challenging. To overcome this problem we have generated a retroviral vector containing the anti-LeY CAR, and a truncated CD34 gene. We successfully generated a stably expressing packaging cell line for this anti-LeY-CAR-CD34 containing retroviral vector. Retroviral supernatant derived from these packaging cells was then used to successfully transduce primary human T cells with the anti-LeY CAR and positive cells were sorted by flow cytometry using the CD34 marker. After sorting by flow cytometry, the majority of the T cells (>90%) were double positive for the anti-LeY CAR and CD34 marker. Antigen-specific function of the gene-modified T cells was then examined in cytokine and cytotoxicity assays to examine whether the presence of the CD34 marker may have compromised CAR function. The anti-LeY-CD34 CAR-T cells were found to secrete high levels of interferon γ, TNFα and IL-2 following direct stimulation through the CAR using immobilized plate bound anti-idiotype antibody or LeY+ tumor targets. The CAR T cells also specifically killed LeY+ tumor cells but not LeY− targets cells indicating that functional responses were not affected by the presence of the CD34 marker. Our results suggest that these new hu-CD34 CARs against LeY tumor antigen could offer new-targeted therapy with acceptable toxicity for patients with solid cancers given the low expression of this antigen on normal tissues. Furthermore this innovative tracking system with the CD34 marker will allow better characterisation and monitoring of the infused CAR-T cells in vivo in preclinical mouse models and in patients in future trials. [ABSTRACT FROM AUTHOR]