QnAs with Kenneth W. Kinzler

Kenneth W. Kinzler has spent a long and distinguished career studying the genetics of human cancer. He has identified several cancer driver genes and has also helped develop multiple techniques that have improved the analysis of gene expression and mutations in human cancer. Continuing his nearly four decades of cancer research at The Johns Hopkins University, Kinzler is currently a professor of oncology and was elected to the National Academy of Sciences in 2016. In his Inaugural Article (1), Kinzler and his colleagues describe an immunotherapeutic approach to specifically target loss of heterogeneity, a widespread genetic alteration present in cancer cells.

Kenneth W. Kinzler. Image credit: Johns Hopkins Kimmel Cancer Center.

PNAS: What makes targeting tumor cells challenging?

Kinzler: The holy grail of oncology is specificity: How to kill a tumor cell without killing a normal cell. Being able to exploit the differences between a normal and tumor cell is critical. It sounds like it should be easy, but you have to consider that a tumor cell, as deadly as it is, genetically looks greater than 99.99% like a normal cell. The vast majority of genes are exactly the same and, typically, there are tens to hundreds of genes that are different in only one amino acid due to somatic mutations. As we learned more about the cancer genome, we expected to find lots of altered genes that could be therapeutically targeted, and the fact was there weren’t as many as we hoped. So, we had to refocus on alternative ways of targeting the genetic alterations in cancer.

PNAS: What is loss of heterozygosity, and can it be targeted?

Kinzler: Basically, we all have two copies—or alleles—of each autosomal gene, one from our mother and one from our father. Tumors often lose one of these copies for large numbers of genes due to chromosomal or subchromosomal loss events. This loss of one copy, called loss of heterozygosity or LOH, is one of the most common genetic events in cancers. It occurs in about 90% of all cancers, so this was an attractive target. However, therapeutically targeting loss events is relatively challenging. Fortunately, the ability to engineer T cells to be activated with chimeric antigen receptors (CARs) and suppressed by inhibitory CARs (iCARs) made it possible to think about using T cells to target LOH.

PNAS: How did you and your colleagues at The Johns Hopkins University develop the specific approach called neoplasm-targeting allele-sensing CAR (NASCAR) that you describe in the article (1)?

Kinzler: Three lines of observations stimulated this approach. The first was based on the recent advances in immunotherapy for cancer. People have suspected for a long time that the immune system is really a powerful opponent of cancers. The ability of immune checkpoint blockades to produce dramatic results in human cancer really highlighted to us the power of immune cells to make a clinically meaningful therapeutic response. Second was the expanding ability to engineer T cells to recognize and differentially respond to new targets. The third was based on our work developing antibodies to target mutation-associated neoantigens (2). This was a separate line of work in which we showed that you could make antibodies that recognize a peptide from a mutated oncogene, but not the wild-type peptide, when it was presented on the cell surface. This piece of the puzzle showed that we could readily make immunotargeting reagents that recognized single amino acid differences, which opened up all the missense polymorphisms of our genome for making reagents that could recognize one allele versus the other allele. So, to summarize, the ability to make artificial T cells that recognize an antigen, to engineer those same T cells to be inhibited by an antigen, and the ability to make reagents that can distinguish between polymorphic versions of a protein all came together. It’s taken a long time to assemble all of the components, and now we’ve been working on demonstrating systematically our ability to put this into practice.

PNAS: How did you test this approach, and what did you find?

Kinzler: There are several ways this approach could be used to target LOH. We felt it was particularly powerful to demonstrate in the case where we used CARs and iCARs targeting to two common alleles of the same gene. That’s advantageous because, when the target is expressed in any given cell, both alleles should typically be expressed excluding genes that undergo imprinting. So, the allele that promotes killing should only be expressed in cells that are also expressing the allele that suppresses and prevents killing. The major deviation from this, in practice, should be in cancer cells because of loss of heterozygosity. There are going to be occasional cells that don’t express two alleles because of some sort of aberrant methylation or isolated mutational event, but killing one in a million cells here or there is not an issue. The only clonal deviation from this biallelic expression should be in the tumor cells. And we showed that we can actually engineer T cells to recognize and kill cancer cells with LOH in vitro in three independent cell lines and also in vivo in mice. It’s a solid foundation for why we think it could be a selective killer, and we hope it translates to humans. But there’s still some significant development work that needs to be done.

PNAS: Where do you see this research going in the future?

Kinzler: We presented this with HLA (human leukocyte antigen) as an example, but you can imagine expanding this to many different polymorphic proteins subject to LOH. We could also make better next-generation versions of the engineered T cells, using more sophisticated cellular engineering approaches to more precisely regulate the CAR and iCAR activity. We’re very excited, and we want to share the story so more and more people can start working on this because there’s tons of work to be done and there’s tons of opportunity.