Two steps forward, one step back for targeting NKG2D in cancer treatment

When it comes to engineering proteins, Dr. Roland Strong and his lab in the Division of Basic Sciences, are probably the people you want to talk to. As Dr. Strong described it, “We’ll often take bits of human proteins and we’ll simply ‘Mr. Potatohead’ them together in some novel way.” He explained how, to do this, his lab utilizes a system that they had developed about 10 years ago called Daedalus, which allows quick, cheap and remarkably effective protein production. More recently, the lab used this system to produce NKG2D ‘decoys’, which they hoped could be used to interfere with a signaling axis that that is used by tumorigenic cells to their advantage.

Before we get ahead of ourselves, though, it is important to understand why the research team wanted to target this signaling pathway in the first place. If you know a lot about anti-tumor responses you may have heard of NKG2D, which is expressed on a variety of cells-most notably on Natural Killer or NK cells- and which binds to its ligands as a homodimer. The wide variety of ligands for NKG2D (NKG2D-L) are expressed only on tumorigenic or infected cells, which allows for a very specific response by immune cells like NK cells to kill these unwanted harbingers of disease. As such, there has been considerable interest in enhancing this signaling pathway in cancer patients to reduce tumor burden. The caveat to this, however, is that occasionally tumor cells can start producing NKG2D themselves, taking advantage of the interaction with the ligands to drive pro-tumorigenic effects. This effect has been seen in a number of cancers including breast, ovarian, colon and prostate, and higher levels of NKG2D ligand (NKG2D-L) can indicate poor prognosis. The use of some biologic to disrupt this signaling pathway, then, could represent a novel method in the treatment of cancer for some patients. A common approach to interfering with signaling pathways would be to utilize antibodies that block the receptor from interacting with its ligands, but in the case of NKG2D this comes with the risk of antibody-mediated NKG2D crosslinking and signaling. So, this is where the engineered NKG2D decoy produced in the Strong lab comes in, the structure and functionality of which were tested in the lab’s recently published paper in Heliyon, the final results of which produced mixed feelings by the research team.

Dr. Strong explained that despite the final experiments being negative data, he emphasized that “the study itself is a really cool study,” which should hopefully inform other clinical uses for their NKG2D construct. This current study built off of previous work done with former Fred Hutch investigators Thomas Spies and Veronica Groh, where the engineered NKG2D proteins were initially produced. When designing the engineered protein, they noted that one of the unique challenges with NKG2D was that, while the receptor is a homodimer, meaning both components of the receptor are identical (i.e. symmetrical), they recognize asymmetrical ligands, and a lot of them at that. The team had to devise a strategy that would keep the engineered design simple enough not to interfere with these interactions. They chose to link both halves through one of the ‘arms’ of NKG2D, as a single chain dimer (SCD), which ended up being remarkably successful. The original full construct included seven of these NKG2D_SCD domains linked as a heptamer, assuming the increase in avidity would be necessary. However, they had discovered that the linking, alone, increased binding affinity by as much as 100-fold. In the team’s recent Heliyon paper, they confirmed that they had not drastically altered the structure by linking the dimers, and further supported their hypothesis that the increase in binding affinity was likely improved due to small conformational changes. They also confirmed that the proteins were properly secreted from transduced cell lines and were functionally capable of recognizing their ligands. When summarizing this portion of their data, Dr. Strong remarked enthusiastically that, “We didn’t just succeed, it’s perfect; this is what perfection looks like.”

Further adding to the teams’ excitement for their data were the results from their pharmaco-kinetic and -dynamic experiments. They utilized four different versions of their construct for this, one monomer, two dimers—either attached to human Fc or mouse Fc—and one heptamer. They injected radiolabeled proteins into mice xenografted with an NKG2D-expressing tumor and assessed their half-life and tissue distribution. Their monomer construct was observed to have the best accumulation in the tumor of the four reagents. Additionally, they found that the longest serum retention was from their dimer construct attached to human Fc. For both measurements, however, the heptamer landed in last place. Dr. Strong explained that this unexpected poor performance could likely be due to the larger size and higher avidity making it a challenge for the protein to fully penetrate the tumor. These results did, however, give high hopes that the monomer and dimer constructs could be promising for further efficacy studies.

This is, unfortunately, where the fun ended, and the frustration began for the research team. They tested in vivo efficacy using a xenograft model in NOD SCID gamma (NSG) immunodeficient mice implanted with ovarian tumor cell lines expressing NKG2D-L and each treated with one of the four NKG2D reagents. When finally assessing all their results for this experiment, Dr. Strong lamented, “We had zero efficacy,” both in preventing tumor engraftment and growth. He noted an unexpected result, the tumor cells in their model rapidly lost NKG2D-L, limiting the functional capacity for their reagents. “We would need a new model,” Dr. Strong explained, however, the ability to develop a more biologically relevant mouse model is limited at this time. They were not discouraged from finding another way to incorporate NKG2D_SCD into other biologics that could be used in cancer treatment. Dr. Strong added that it’s “still a fantastic study. It continues to validate the NKG2D_SCD engineered binding block.” And so, despite the study being “a steaming pile of negative data,” in Dr. Strong’s words, it seems as if the use of their NKG2D_SCD in therapeutics is far from written off.

This spotlight work was funded by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, FHCC Evergreen Fund, the Washington Research Foundation, and the CCSG.

Fred Hutch/University of Washington/Seattle Children’s Cancer Center Consortium members Drs. Ted Gooley and Roland Strong contributed to this work.

Rupert PB, Buerger M, Girard EJ, Frutoso M, Parrilla D, Ng K, Gooley T, Groh V, Strong RK. 2024. Preclinical characterization of Pan-NKG2D ligand-binding NKG2D receptor decoys. Heliyon. 10(7):e28583.