Pluripotent stem cell-derived NK cells with high-affinity noncleavable CD16a mediate improved antitumor activity.

Antibody-dependent cellular cytotoxicity (ADCC) is a key effector mechanism of natural killer (NK) cells that is mediated by therapeutic monoclonal antibodies (mAbs). This process is facilitated by the Fc receptor CD16a on human NK cells. CD16a appears to be the only activating receptor on NK cells that is cleaved by the metalloprotease a disintegrin and metalloproteinase-17 upon stimulation. We previously demonstrated that a point mutation of CD16a prevents this activation-induced surface cleavage. This noncleavable CD16a variant is now further modified to include the high-affinity noncleavable variant of CD16a (hnCD16) and was engineered into human induced pluripotent stem cells (iPSCs) to create a renewable source for human induced pluripotent stem cell-derived NK (hnCD16-iNK) cells. Compared with unmodified iNK cells and peripheral blood-derived NK (PB-NK) cells, hnCD16-iNK cells proved to be highly resistant to activation-induced cleavage of CD16a. We found that hnCD16-iNK cells were functionally mature and exhibited enhanced ADCC against multiple tumor targets. In vivo xenograft studies using a human B-cell lymphoma demonstrated that treatment with hnCD16-iNK cells and anti-CD20 mAb led to significantly improved regression of B-cell lymphoma compared with treatment utilizing anti-CD20 mAb with PB-NK cells or unmodified iNK cells. hnCD16-iNK cells, combined with anti-HER2 mAb, also mediated improved survival in an ovarian cancer xenograft model. Together, these findings show that hnCD16-iNK cells combined with mAbs are highly effective against hematologic malignancies and solid tumors that are typically resistant to NK cell-mediated killing, demonstrating the feasibility of producing a standardized off-the-shelf engineered NK cell therapy with improved ADCC properties to treat malignancies that are otherwise refractory.

An evolutionarily conserved TNF-alpha-responsive enhancer in the far upstream region of human CCL2 locus influences its gene expression.

Comparative cross-species genomic analysis has served as a powerful tool to discover novel noncoding regulatory regions that influence gene expression in several cytokine loci. In this study, we have identified several evolutionarily conserved regions (ECRs) that are shared between human, rhesus monkey, dog, and horse and that are upstream of the promoter regions that have been previously shown to play a role in regulating CCL2 gene expression. Of these, an ECR that was ~16.5 kb (-16.5 ECR) upstream of its coding sequence contained a highly conserved NF-κB site. The region encompassing the -16.5 ECR conferred TNF-α responsiveness to homologous and heterologous promoters. In vivo footprinting demonstrated that specific nucleotide residues in the -16.5 ECR were protected or became hypersensitive after TNF-α treatment. The footprinted regions were found to bind NF-κB subunits in vitro and in vivo. Mutation/deletion of the conserved NF-κB binding site in the -16.5 ECR led to loss of TNF-α responsiveness. After TNF-α stimulation, the -16.5 ECR showed increased sensitivity to nuclease digestion and loss of histone signatures that are characteristic of a repressive chromatin. Chromosome conformation capture assays indicated that -16.5 ECR physically interacts with the CCL2 proximal promoter after TNF-α stimulation. Taken together, these results suggest that the -16.5 ECR may play a critical role in the regulation of CCL2.

The rs1024611 regulatory region polymorphism is associated with CCL2 allelic expression imbalance.

CC chemokine ligand 2 (CCL2) is the most potent monocyte chemoattractant and inter-individual differences in its expression level have been associated with genetic variants mapping to the cis-regulatory regions of the gene. An A to G polymorphism in the CCL2 enhancer region at position -2578 (rs1024611; A>G), was found in most studies to be associated with higher serum CCL2 levels and increased susceptibility to a variety of diseases such as HIV-1 associated neurological disorders, tuberculosis, and atherosclerosis. However, the precise mechanism by which rs1024611influences CCL2 expression is not known. To address this knowledge gap, we tested the hypothesis that rs1024611G polymorphism is associated with allelic expression imbalance (AEI) of CCL2. We used haplotype analysis and identified a transcribed SNP in the 3'UTR (rs13900; C>T) can serve as a proxy for the rs1024611 and demonstrated that the rs1024611G allele displayed a perfect linkage disequilibrium with rs13900T allele. Allele-specific transcript quantification in lipopolysaccharide treated PBMCs obtained from heterozygous donors showed that rs13900T allele were expressed at higher levels when compared to rs13900C allele in all the donors examined suggesting that CCL2 is subjected to AEI and that that the allele containing rs1024611G is preferentially transcribed. We also found that AEI of CCL2 is a stable trait and could be detected in newly synthesized RNA. In contrast to these in vivo findings, in vitro assays with haplotype-specific reporter constructs indicated that the haplotype bearing rs1024611G had a lower or similar transcriptional activity when compared to the haplotype containing rs1024611A. This discordance between the in vivo and in vitro expression studies suggests that the CCL2 regulatory region polymorphisms may be functioning in a complex and context-dependent manner. In summary, our studies provide strong functional evidence and a rational explanation for the phenotypic effects of the CCL2 rs1024611G allele.