Automated Production of Gene-Edited CAR-T Cells for People Living with HIV.

Adoptive transfer of gene-modified autologous T cells, for example, expressing chimeric antigen receptors (CARs), is a promising therapy option for a variety of malignant and nonmalignant diseases. Given their increased risk to develop certain cancer types, namely lymphoma, and the current progress with HIVenv-directed CARs, people living with HIV (PLWH) might particularly profit from CAR-T therapies. At the same time, clinical translation of novel advanced therapy medicinal products remains challenging, partly due to the elaborate ex vivo production under good manufacturing practice (GMP) conditions. This holds especially true for T-cell products from PLWH, which require special manufacturing conditions to suppress HIV replication. We aimed to exemplarily address this limitation in the context of CD19-CAR-T cells for HIV-positive cancer patients. To this end, we developed an integrated, GMP-compliant process for lentiviral CAR transfer and simultaneous knockout of the HIV coreceptor C-C chemokine receptor 5 (CCR5) by mRNA electroporation of our transcription activator-like effector nuclease (CCR5-Uco-hetTALEN) based on the CliniMACS Prodigy. We successfully performed three manufacturing runs to produce CCR5-edited CD19-CAR-T cells on a clinical scale, generating up to 3.9 × 109 viable T cells. Thirty-seven percent to 55% of the T cells in the final product expressed functional CD19-CARs, the overall CCR5 editing rates ranged from 42% to 54%. The third large-scale process was performed in the presence of the antiretroviral drug saquinavir to suppress potential viral replication during manufacturing of cells from HIV-positive patients. In summary, we have developed a GMP-compliant manufacturing process for gene-edited CAR-T cells under antiretroviral suppression, which might help to accelerate the translation of cellular immunotherapies, namely CAR-T cells for patients living with HIV in the future.