Developing allogeneic double-negative T cells as a novel off-the-shelf adoptive cellular therapy for cancer

Jong Bok Lee, Hyeonjeong Kang, Linan Fang, Cheryl D'Souza, Oyedele Adeyi, Li Zhang

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29 Scopus citations


PURPOSE: To expand clinical-grade healthy donor-derived double-negative T cells (DNT) to a therapeutically relevant number and characterize their potential to be used as an "off-the-shelf" adoptive cellular therapy (ACT) against cancers.

EXPERIMENTAL DESIGN: We developed methods to expand DNTs under GMP conditions and characterized their surface molecule expression pattern using flow cytometry-based high-throughput screening. We investigated the off-the-shelf potential of clinical-grade DNTs by assessing their cytotoxicity against various cancer types and their off-tumor toxicity in vitro and in xenograft models and determining the effect of cryopreservation under GMP conditions on cell viability and cytotoxicity. Further, we determined the susceptibility of DNTs to conventional allogeneic T cells in vitro and in vivo.

RESULTS: Clinical-grade DNTs expanded 1,558 ± 795.5-fold in 17 days with >90% purity. Expanded DNTs showed potent in vitro cytotoxic activity against various cancer types in a donor-unrestricted manner. DNTs enhanced the survival of mice infused with a lethal dose of EBV-LCL and significantly reduced leukemia engraftment in xenograft models. Expanded DNTs cryopreserved using GMP-compliant reagents maintained viability and anticancer functions for at least 600 days. Live allogeneic DNTs did not induce cytotoxicity of alloreactive CD8 + T cells in vitro, and coinfusion of DNTs with peripheral blood mononuclear cells (PBMC) from a different donor into mice resulted in coengraftment of DNTs and PBMC-derived allogeneic conventional T cells in the absence of cytotoxicity toward DNTs, suggesting the lack of host-versus-graft reaction.

CONCLUSIONS: We have established a method to generate therapeutic numbers of clinical-grade DNTs that fulfill the requirements of an off-the-shelf ACT.

Original languageEnglish (US)
Pages (from-to)2241-2253
Number of pages13
JournalClinical Cancer Research
Issue number7
StatePublished - Apr 1 2019

Bibliographical note

Funding Information:
We thank Dr. Dalam Ly and Branson Chen for their critical insights and suggestions on this work. L. Zhang was supported by a Canadian Cancer Society (grant #704121) and Canadian Institutes of Health Research Proof of Principle Grant Phase II (grant #141723). L. Zhang is the Maria H. Bacardi Chair of Transplantation.

Publisher Copyright:
© 2019 American Association for Cancer Research.

PubMed: MeSH publication types

  • Journal Article
  • Research Support, Non-U.S. Gov't


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