410. The Development of HLAnull K562 Cells Expressing CD1d and Co-Stimulatory Molecules as Artificial Antigen-Presenting Cells for Clinical Scale Expansion of NKT Cells With Superior In Vivo Persistence and Anti-Tumor Potential

Abstract

CD1d-restricted type-I Natural Killer T cells (NKTs) have been shown to mediate antitumor responses in mouse models and are associated with improved outcome in several types of cancer. However, the therapeutic application of NKTs has been limited by low numbers and functional defects of these cells in patients with cancer. Herein we present an effective method for ex-vivo NKT-cell numeric expansion for adoptive immunotherapy. The method is based on engineered properties of K562 cells to function as artificial antigen-presenting cells (aAPC). K562 cells express a single endogenous HLA allele, HLA-Cw3, an NK-cell activating ligand which favors the expansion of NK cells that compete with NKTs in culture. We rendered K562 cells HLAnull by eliminating HLA-C gene from K562 cell genome using a HLA-C-specific zinc finger nuclease. We then transduced parental and HLAnull K562 cells with CD1d cDNA followed by single cell sorting and clonal expansion. The clones were pulsed with αGalactosylceramide and tested as irradiated aAPC for NKTs using CFSE proliferation assay. We found that in contrast to HLA-Cw3+ K562/CD1d, HLAnullK562/CD1d clones selectively expanded NKTs when added to primary PBMC, and clones with an intermediate level of CD1d expression induced the highest rate of NKT-cell proliferation. Next, a selected HLAnullCD1dmed clone was further modified to express CD86 alone or in combination with 4-1BBL and/or OX40L followed by single cell sorting and clonal expansion, producing 250 clones. One clone with the phenotype HLAnullCD1dmedCD86high4-1BBmedOX40Lmed (designated B-8-2) consistently induced the highest rate of NKT-cell expansion. B-8-2 aAPC were able to efficiently expand both primary NKTs as well as NKTs modified to express a chimeric antigen receptor (CAR) specific for CD19 antigen, a clinically validated therapeutic target for B-cell malignancies. CAR expression in NKTs rendered them highly cytotoxic against CD19+ Raji leukemia cells. Compared with CAR-NKTs expanded with autologous PBMC, those expanded with B-8-2 cells exhibited a Th-1-skewed cytokine profile, prolonged in vivo persistence, and superior therapeutic activity in Raji leukemia model. Gene expression analysis of NKTs expanded with B-8-2 cells vs. those expanded with autologous PBMC revealed a significant up-regulation of genes associated with T- and NK-cell memory (CCR7, CD45RA, KLRG1, KLRD1, IL-21R), a mixture of Th17- and Th-1-associated molecules (IL-17A, IL-17F, STAT1, IL12RB1, GZMA, GZMB GNLY) and downregulation of a particular pro-apoptotic gene from the BCL2 family, BCL2L11. Therefore, the engineered K562-derived B-8-2 cells can be used as a highly efficient aAPC for ex vivo propagation of fully functional primary as well as gene-modified NKTs amenable for cancer immunotherapy.