High-Affinity PD-1 Protein Has Potential

A small, engineered protein that selectively binds to PD-L1 was more effective in shrinking tumors and synergizing with other immunotherapies than conventional PD-L1 antibodies in a preclinical study, according to data presented at the CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference in New York, NY, in September.

The high-affinity PD-1 protein may overcome some drawbacks of existing antibody-based immune checkpoint inhibitors, says study corresponding author Aaron Ring, an MD/PhD student at California's Stanford University School of Medicine who will soon join the faculty at Yale University School of Medicine in New Haven, CT.

“It is approximately 10 times smaller than an antibody and it lacks the antibody ‘Fc’ moiety that is recognized by Fc receptors on cells like macrophages,” he says. “Consequently, the high affinity PD-1 protein penetrates deeper into tumors and, unlike antibodies, does not cause unwanted depletion of PD-L1–positive T cells that mediate antitumor immunity.”

Ring and his colleagues used directed evolution to design the small protein. Using this technique, they first created a library of over 100,000,000 different PD-1 variants that they displayed on the surface of yeast. They then used magnetic and fluorescence cell sorting techniques to select for the tightest binders to recombinant PD-L1 protein. Through increasingly more difficult binding conditions, they zeroed in on one variant that bound to PD-L1 about 50,000 times more tightly than wild-type PD-1.

To assess its effectiveness in penetrating solid tumors compared with anti–PD-L1 antibodies, the researchers labeled both proteins with fluorescent dyes and simultaneously injected them into mice. Using fluorescence microscopy, they visualized the degree of tumor penetration.

“We were able to directly see how well anti–PD-L1 antibodies and our high-affinity PD-1 protein penetrate tumors, and there was a striking difference,” says Ring. “The antibody is mostly found close to blood vessels and at the tumor periphery, whereas the smaller PD-1 protein spreads more extensively throughout the tumor.”

They also found that the small protein was more effective at treating larger tumors than PD-L1 antibodies. Both therapies shrank tumors 50 mm³ in size, but only the small protein was effective against tumors measuring 150 mm³. Adding an anti-CTLA4 antibody to anti–PD-L1 therapy in the larger tumors did not improve the efficacy of anti–PD-L1, whereas combining an anti-CTLA4 antibody with the small protein resulted in greater efficacy compared with either treatment alone.

“Our hypothesis is that as tumors grow larger, the need for effective penetration by the therapeutic agent becomes more important,” says Ring.

Several issues should be addressed before the high-affinity protein is ready for clinical testing, the researchers emphasize. For example, due to its small size, it is excreted from the body more quickly than antibodies, and therefore it may need to be administered more frequently. Furthermore, like any protein drug, there is also a risk of immunogenicity.

However, the study achieved its aim, says Ring. “Beyond the specific molecules that we made, we demonstrated the potential for small proteins to complement traditional biologics in cancer immunotherapy.”

For more news on cancer research, visit Cancer Discovery online at http://CDnews.aacrjournals.org.