A preclinical study of allogeneic CD19 chimeric antigen receptor double-negative T cells as an off-the-shelf immunotherapy drug against B-cell malignancies.

OBJECTIVES: To evaluate the manufacturability, efficacy and safety of allogeneic CD19 chimeric antigen receptor double-negative T cells (CD19-CAR-DNTs) as an off-the-shelf therapeutic cell product. METHODS: A membrane proteome array was used to assess the off-target binding of CD19-CAR. DNTs derived from healthy donors were transduced with lentiviral vectors encoding the CD19-CAR. The manufacture of the CD19-CAR-DNTs was under GMP conditions, and their surface molecule expression patterns were characterised using flow cytometry. We investigated the off-the-shelf potential of CD19-CAR-DNTs by evaluating the cryopreserved CD19-CAR-DNTs in terms of cell viability as well as their cytotoxicity against various CD19+ target cell lines and primary patient blasts in vitro. We evaluated the persistence and safety of the cryopreserved CD19-CAR-DNTs in xenograft models in vivo. RESULTS: GMP-grade CD19-CAR-DNTs were manufactured and cryopreserved for use in advance. The cryopreserved CD19-CAR-DNTs maintain their viability and antitumor activity against various CD19+ target cell lines and primary patient blasts. These cells significantly prolonged the survival in Raji-Luc-xenografted NOG mice. Multiple infusions of the cells can further augment their efficacy. Remarkably, following a single infusion in mice, CD19-CAR-DNTs rapidly got distributed among well-perfused organs initially, and progressively spread to most tissues, peaking at Day 43. In toxicity studies, CD19-CAR-DNTs significantly reduced tumor burden and ameliorated tissue damage in tumor-bearing NOG mice. Critically, no immunotoxicity or graft versus host disease was observed in non-tumor-bearing NOG mice. CONCLUSIONS: The allogeneic CD19-CAR-DNTs fulfil the requirements of an off-the-shelf therapeutic cell product, offering a promising new approach to the treatment of haematological malignancies.