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Volume 3 Supplement 2

30th Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2015)

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Minimizing leukemia escape: implementing a dual anti-CD20- and CD19-scFv-based chimeric antigen receptor (CAR)

Adoptive immunotherapy with chimeric antigen receptor (CAR) transduced T lymphocytes has shown promising results in both pediatric and adult B cell malignancies. Nevertheless, both CAR-based and antibody-based anti-CD19 therapies, e.g. blinatumomab, have seen treatment failures attributed to the loss of CD19 or an epitope of CD19 on the surface of the malignant B cell. It may be possible to overcome antigen escape by targeting two tumor antigens simultaneously, i.e. CD19 and CD20 using a tandem construct with two scFv-based CAR binding domains. Lentiviral vectors encoding chimeric antigen receptors comprised of anti-CD19 and anti-CD20 targeting domains expressed alone or in tandem were transduced into T cells from healthy donors to generate the corresponding CAR19, CAR20, CAR19_20 (CD19 scFv more distal to the T cell plasma membrane) and CAR20_19 T cells (CD20 scFv distal). The transduced T cells were 50-70% CAR positive as determined by protein L flow cytometric analysis. Expression of CAR proteins of the expected molecular weight was confirmed by Western blot analysis of transduced T cells. When CAR-transduced T cells were combined with CD19+CD20+ Raji target cells, but not CD19-CD20- K562 cells, all four CAR T cell types demonstrated comparable efficient killing of leukemia targets (E:T ratio >2), and target-dependent induction of IFN-γ, as measured in co-culture supernatants by ELISA.

We then began a series of in vitro co-culture experiments where we used very low E:T ratios to examine the potential for CAR-induced antigen loss on surviving leukemia cells. All CAR-T cells expressing an anti-CD19 scFv induced rapid loss of CD19 surface expression. In contrast, the CD20 surface marker was less prone to down-regulation by CAR-T cells expressing anti-CD20 scFv. Upon flow cytometric analysis of surviving leukemia cells on day 5, CD19 expression was reduced to 3%, 48%, 73%, 90% and 93% of control when co-cultured with CAR T cells expressing CAR19_20, CAR20_19, CAR19, CAR20, and control T cells, respectively. Similar results were seen when experiments were of longer, 7 days, or shorter, 1 day, duration. In conclusion, tandem CAR T cells are as effective as single CAR19 or CAR20 T cells in leukemia cell killing. Importantly, dual scFv-expressing CARs are more potent in preventing tumor antigen escape via target antigen down-regulation.

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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

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Schneider, D., Xiong, Y., Roy, A. et al. Minimizing leukemia escape: implementing a dual anti-CD20- and CD19-scFv-based chimeric antigen receptor (CAR). j. immunotherapy cancer 3 (Suppl 2), P122 (2015).

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