Abstract
<h3>Background</h3> While effective, CAR-T therapies are limited by a lack of scalability for patient-derived starting material. Alternatively, allogeneic CAR-NK cell therapies have the potential to overcome these limitations by providing an off-the-shelf product capable of delivering clinical benefits without the safety and manufacturing challenges associated with CAR-Ts. CAR-NK cell therapies are attractive in treating AML as the inherent graft-versus-leukemia activity of NK cells can be effectively augmented by a CAR directed to an AML-expressed antigen. CD70 is an attractive target for CAR therapy in AML since it is highly expressed on leukemic stem cells and blasts and is not detectable on normal bone marrow hematopoietic stem cells.<sup>1</sup> Additionally, aberrant CD70 expression is associated with several solid tumors and hematological malignancies, including AML and renal cell carcinoma (RCC) while expression in normal tissue is restricted to immune cells including T, B, DC, and NK cells.<sup>2</sup> <h3>Methods</h3> Here we demonstrate that CD70 is not expressed in resting peripheral blood NK cells but is strongly upregulated in response to NK cell activation by engineered feeder cells. As such, integration of a CD70-targeting CAR into activated NK cells leads to substantial reduction of NK cell expansion due to fratricide. Knockout (KO) of CD70 by CRISPR/Cas9 editing does not inhibit NK cell expansion nor impair cytotoxicity against various types of tumor cells, therefore a successful engineering strategy where CAR integration and self-expression of the CAR ligand is knocked out via CRISPR/Cas9 would allow for successful propagation of such a CAR-NK therapy. Using the non-viral <i>TC Buster</i> transposon system, we were able to deliver transposons containing a CD70 CAR or CD70 CAR/IL15 expression cassette while simultaneously knocking out CD70 by CRISPR/Cas9 in primary human peripheral blood NK cells. <h3>Results</h3> This single-step process resulted in >75% CD70 CAR integration/expression and >80% knockout of endogenous CD70. The resulting CD70 knockout CAR-NK cells were resistant to fratricide and expanded comparably to mock-engineered NK cells following feeder cell activation. The IL15 expression cassette enabled enhanced persistence of CAR-NK cells <i>in vitro</i> and <i>in vivo</i> without exogenous cytokine support. In functional assays, CD70 knockout CAR-NK cells mediated cytotoxicity against multiple CD70-positive tumor cell lines including AML and RCC both <i>in vitro</i> and <i>in vivo</i>. <h3>Conclusions</h3> Overall, the results demonstrate the potential for targeting CD70 with CAR-NK cell therapy for the treatment of AML, RCC, and other CD70-positive malignancies while overcoming fratricide issues by engineering with a non-viral transposon delivery system in combination with CRISPR/Cas9 editing. <h3>References</h3> Perna <i>et al</i>. 2017, <i>Cancer Cell.</i><b> 32</b>:506-519. McEarchern <i>et al</i>. 2008, <i>Clin Cancer Res</i>. <b>14</b>(23):7763-7772.
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