Purpose: Engineering T cells with receptors to redirect the immune system against cancer has most recently been described as a scientific breakthrough. However, a main challenge remains the GMP-grade purification of immune cells selectively expressing the introduced receptor in order to reduce potential side effects due to poorly or nonengineered cells.

Experimental Design: In order to test a novel purification strategy, we took advantage of a model γδT cell receptor (TCR), naturally interfering with endogenous TCR expression and designed the optimal retroviral expression cassette to achieve maximal interference with endogenous TCR chains. Following retroviral transduction, nonengineered and poorly engineered immune cells characterized by a high endogenous αβTCR expression were efficiently depleted with GMP-grade anti-αβTCR beads. Next, the engineered immune cells were validated for TCR expression, function against a panel of tumor cell lines and primary tumors and potential allo-reactivity. Engineered immune cells were further validated in two humanized mouse tumor models.

Results: The untouched enrichment of engineered immune cells translated into highly purified receptor-engineered cells with strong antitumor reactivity both in vitro and in vivo. Importantly, this approach eliminated residual allo-reactivity of engineered immune cells. Our data demonstrate that even with long-term suboptimal interference with endogenous TCR chains such as in resting cells, allo-reactivity remained absent and tumor control preserved.

Conclusions: We present a novel enrichment method for the production of untouched engineered immune cells, ready to be translated into a GMP-grade method and potentially applicable to all receptor-modified cells even if interference with endogenous TCR chains is far from complete. Clin Cancer Res; 21(17); 3957–68. ©2015 AACR.