AML-396: Development of a Relevant Cell Model for NKG2D CAR-T Efficiency Assessment

Abstract

Context AML is a severe blood pathology with a complicated therapy strategy and high risk of relapse. Objective Different types of cancer cells exhibit various expression rates of NKG2D ligands. An effective cell model is a crucial stage for profound NKG2D CAR-T functional cell estimation. Design We have designed two NKG2D plasmid constructs for two types of CAR-T cells' production and consequent modification with costimulatory signals (4-1BB, CD 28, TIGIT, ICOS). By means of qPCR, we analyzed expression of main NKG2D receptor ligands: MICA, MICB, ULBP 1–6 in K562 (myelogenous leukemia), NB-4 (acute myeloid leukemia), and HeLa (cervical cancer) tumor cell lines. Prominent ligand expression within HeLa cells makes it a sensitive model for NKG2D CAR-T efficiency verification. For cell model creation, we designed shRNA constructs for knockdown of the mentioned NKG2D ligands in HeLa cells. Setting The first designed NKG2D vector includes a suicide iCasp9 sequence. The second vector contains EGFR with a signal sequence. For cloning these sequences and plasmid assembly, the lentiviral pLVT1 vector was used in both cases. The NKG2D-CAR gene sequence was adopted from the Baumeister et al. study and contains human cytoplasmic CD3ζ fused to the full-length human NKG2D gene (KLRK1). NKG2D-CD3ζ fusion was obtained after gene synthesis (Evrogen, Russia). Both newly designed plasmids contain DAP10 sequence for NKG2D signal transduction enhancement. shRNA sequences were designed for MICA, MICB, and ULBP 1–6 ligands and cloned into pLKO.1 vector (Addgene, Plasmid #10878). After lentiviral assembly, all designed shRNA was transduced into the HeLa cell line. Results We observed the levels of MICA, MICB, and ULBP3–6 to be the highest and upregulated in HeLa cells, compared to K562 and NB-4 (p Conclusions Due to patient-specific patterns of ligand expression and its influence on CAR-T cell therapy efficiency, we developed tumor cell line models allowing the assessment of NKG2D CAR-T efficiency in the case of different levels of ligands on tumor cell membranes.