Hwang et al. engineered T cells with NOT-gate logic to target loss of heterozygosity (LOH) of HLA alleles. scFvs specifically binding present or missing HLA alleles were identified, linked with either activating (CD28 intracellular domain) or inhibitory (PD-1 intracellular domain) CAR constructs, respectively, and expressed on human T cells (called NASCARs). Compared to cells expressing the CAR domain alone, NASCARs did not respond to “healthy” target cells expressing both alleles. When confronted with LOH tumor cells expressing only one allele, NASCARs secreted IL-2 and IFNγ and displayed cytotoxicity in vitro, and eliminated xenograft tumors in vivo.
Contributed by Alex Najibi
ABSTRACT: Developing therapeutic agents with potent antitumor activity that spare normal tissues remains a significant challenge. Clonal loss of heterozygosity (LOH) is a widespread and irreversible genetic alteration that is exquisitely specific to cancer cells. We hypothesized that LOH events can be therapeutically targeted by "inverting" the loss of an allele in cancer cells into an activating signal. Here we describe a proof-of-concept approach utilizing engineered T cells approximating NOT-gate Boolean logic to target counterexpressed antigens resulting from LOH events in cancer. The NOT gate comprises a chimeric antigen receptor (CAR) targeting the allele of human leukocyte antigen (HLA) that is retained in the cancer cells and an inhibitory CAR (iCAR) targeting the HLA allele that is lost in the cancer cells. We demonstrate that engineered T cells incorporating such NOT-gate logic can be activated in a genetically predictable manner in vitro and in mice to kill relevant cancer cells. This therapeutic approach, termed NASCAR (Neoplasm-targeting Allele-Sensing CAR), could, in theory, be extended to LOH of other polymorphic genes that result in altered cell surface antigens in cancers.