Abstract Details

G2 - Immune Targeting and Approaches with Genetically-Modified Cells and Cell Therapies (Including CAR-T, CAR-NK, TCR editing)

829: Virus-Free CRISPR Knock-In of a Chimeric Antigen Receptor into KLRC1 Generates Potent Target-Specific Natural Killer Cells

Type: Poster Session

Poster Board Number: 829

Presentation Details

Session Title: Wednesday Posters: Immune Targeting and Approaches with Genetically-Modified Cells and Cell Therapies






Natural killer (NK) cells are strong candidates for “off-the-shelf” immunotherapy products due to their high cytotoxicity profile. However, their success against solid tumors remains suboptimal when compared to hematological malignancies due to the upregulation of NK-inhibitory ligands, such as HLA-E, within the tumor microenvironment. Moreover, the manufacturing of NK cell therapies largely relies on viral vectors, risking insertional mutagenesis. Here, we describe a robust gene editing platform utilizing CRISPR-Cas9 to disrupt the KLRC1 gene, encoding for the HLA-E binding NKG2A inhibitory receptor, and virus-free insertion of large transgenes (>2.7 kb), including a GD2-targeting chimeric antigen receptor (CAR) within human primary NK cells. The use of Cas9 ribonucleoprotein (RNP) complexes yields high protein level knock-out of the KLRC1 gene with 70% efficiency and specific insertion of the CAR transgene as high as 22% (Figure A). KLRC1-CAR NK cells display robust and specific cytotoxicity and cytokine release against GD2⁺ human melanoma cells (Figure B). Moreover, deep sequencing analysis of the KLRC1-CAR NK cells shows minimal off-target activity, highlighting the specificity of our edit. Using a completely non-viral workflow, this work provides compelling evidence for the use of the CRISPR-Cas9 system to manufacture potent and effective allogeneic NK cell products from routine peripheral blood donation and collection.

Figure. CRISPR-engineered anti-GD2 CAR NK cells show improved potency against melanoma target cells. (A) Representative histogram plots showing knockout of NKG2A and knock-in of CAR one week after nucleofection. X-axis describes protein expression levels. Y-axis describes frequency after normalization to the mode. UTF samples are shown as black histograms in both plots. The percent shown for each gate represents protein levels in the KLRC1-KO (left) and KLRC1-CAR (right) samples. (B) Following FACS, NK cells were co-cultured with M21 melanoma cells at a 5:1 E:T ratio for 24 hours, after which secreted LDH was measured with the CyQuant LDH Cytotoxicity Assay kit. Data is shown as the mean + SD across three donors, with 5-6 replicates per donor (n = 3).

Plain Language Summary

Natural killer NK cells are a vital component of the immune system as they are part of the first line of defense against viruses, tumors, and infections. However, within solid tumors, NK cells can become inactivated by the cancer cells due to the tumor's immunosuppressive nature. Here, we describe a safe and efficient method to genetically engineer NK cells to overcome the suppression caused by cancer and eliminate solid tumors. The current method to modify NK cells uses viruses, which can have unintended gene editing outcomes and can complicate the safety profiles of the modified cells. In contrast, our method employs a virus-free strategy to modify NK cells specifically and precisely, to detect and eliminate melanoma tumor cells. When exposed to cancer cells, our engineered NK cells display an improved ability to kill the melanoma cells when compared to their unmodified counterparts.

Keerthana Shankar^1,2, Isabella Zingler-Hoslet¹, Lei Shi^3,4, Varun Katta⁵, Brittany Russell¹, Shengdar Q. Tsai⁵, Christian Capitini^3,4, Krishanu Saha<sup>1,2,4

1</sup>Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI,²Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI,³Department of Pediatrics, University of Wisconsin-Madison, Madison, WI,⁴Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, WI,⁵Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN"