Engineering CD70-Directed CAR-NK Cells for the Treatment of Hematological and Solid Malignancies
Authors: Choi Eugene , Chang Jae-Woong , Krueger Joshua , Lahr Walker S , Pomeroy Emily , Walsh Meghan , Khamhoung Annie , Johnson Jennifer , Franco Charlotte , Swiech Lukasz , Pabla Simarjot , Richardson Celeste , Suri Vipin , Webber Beau R , Moriarity Branden S 
Published: November 2021

Abstract
Advances in cellular immunotherapy have led to multiple FDA approvals for autologous CAR-T cell therapies in acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphomas (NHL), and multiple myeloma (MM). While effective, autologous CAR-T therapies are limited by safety concerns, lack of scalability for patient derived starting material, and long vein-to-vein timelines. Allogeneic CAR-NK cell therapies have the potential to overcome these limitations by providing an off-the-shelf product capable of delivering clinical benefit without the safety and manufacturing challenges associated with CAR-T therapy. CAR-NK cell therapies are particularly attractive in 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 expression is associated with several indications, including AML, NHL, and renal cell carcinoma (RCC), and it is an attractive target for CAR therapy in AML since it is highly expressed on leukemic stem cells and blasts and is absent in normal bone marrow hematopoietic stem cells. 1 While aberrant expression of CD70 is associated with several solid and hematological indications, its expression in normal tissue is restricted to immune cells including T, B, DC, and NK cells. 2 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. Introduction of CARs targeting CD70 into activated NK cells leads to substantial reduction of NK cell expansion due to fratricide. While CD70 is expressed in activated NK cells, knockout (KO) of CD70 by CRISPR/Cas9 editing does not inhibit NK cell expansion nor impair endogenous cytotoxicity against K562 target cells. Using the non-viral TcBuster™ Transposon System (Bio-Techne), we were able to deliver transposons containing a CD70 CAR and an IL15 expression cassette while simultaneously knocking out CD70 by CRISPR/Cas9 in primary human peripheral blood NK cells. This single-step process resulted in &gt;70% CAR integration/expression and &gt;80% knockout of 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 in vitro without exogenous cytokine support. In functional assays, CD70 KO NK cells engineered with the CD70 CAR and IL15 expression cassette mediated cytotoxicity against multiple CD70-positive tumor cell lines, expressed the degranulation marker CD107a (LAMP1), and expressed the cytokines IFNγ and TNFα. 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 the risk posed by fratricide by engineering with a non-viral transposon delivery system in combination with CRISPR/Cas9 editing.
1 Perna et al. 2017, Cancer Cell. 32:506-519.
2 McEarchern et al. 2008, Clin Cancer Res. 14(23):7763-7772.

Disclosures
Choi: Unum Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Walsh: Obsidian Therapeutics: Ended employment in the past 24 months. Khamhoung: Rubius Therapeutics, Inc.: Ended employment in the past 24 months. Johnson: Celsius Therapeutics: Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Franco: KSQ Therapeutics: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months. Swiech: Agenus: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months; Unum Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Richardson: Novartis Pharma: Current equity holder in publicly-traded company; Obsidian Therapeutics: Current holder of stock options in a privately-held company, Ended employment in the past 24 months.