An engineered high-affinity PD-1 small protein that can bind to PD-L1 on tumours was found to be a more effective anticancer immunotherapeutic than conventional anti- PD-L1 antibodies, and this small protein was more effective in synergising with other immunotherapies compared with conventional anti-PD-L1 antibodies, according to preclinical data presented at the CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference, held Sept. 16–19.

“We now have many antibody drugs [such as pembrolizumab and ipilimumab] that target immune checkpoints such as PD-1, PD-L1, CTLA4, and other immune-regulatory pathways.

However, these types of drugs have some overlooked drawbacks that can limit their effectiveness,” said Sydney Gordon, a graduate student in the laboratory of Irving Weissman, MD, at the Ludwig Center at Stanford University School of Medicine in California.

“First, it would be ideal if drugs that target PD-L1 could get inside tumours.

However, antibodies, because of their large size, cannot get there effectively, which prevents them from working to their full potential,” Gordon said. “Second, antibodies against PD-1 and PD-L1 can result in counterproductive collateral damage, depleting some of the very anti-tumour immune cells they are supposed to activate.”

Gordon; Aaron Ring, who is an MD/PhD student from Weissman’s lab and will shortly be assistant professor in the Department of Immunobiology at Yale University School of Medicine; and colleagues hypothesised that a small engineered protein that targets PD-L1 and is capable of blocking PD-1/PD-L1 signalling could potentially overcome these setbacks and function as a superior immunotherapy.

The researchers designed a small-protein antagonist of PD-L1 by using a process called directed evolution.

“In the end, our best PD-1 variants bound to PD-L1 about 50,000 times more strongly than the natural PD-1 protein,” Ring said.

The small-protein, high-affinity PD-1 variant they developed was one-tenth the size of an antibody.

First, the researchers tested whether the high-affinity PD-1 protein could penetrate solid tissues more effectively than anti-PD-L1 antibodies.

To do this, they labelled the high-affinity PD-1 protein and an anti-PD-L1 antibody with two differently coloured fluorescent dyes and injected tumour-bearing mice with both fluorescent proteins at the same time.

When they surgically removed the tumours and looked at them under a microscope, they found that the antibody was mostly found near blood vessels (outside the tumour), whereas the high-affinity PD-1 protein was able to distribute much more deeply into the tumours.

Next, the researchers tested what impact the high-affinity PD-1 protein had on immune cells.

For this, they took blood samples and lymph nodes from mice treated with the PD-1 protein and those treated with the PD-L1 antibody, and estimated the number of T cells.

They found that the high-affinity PD-1 protein did not affect T-cell numbers in the blood or lymph nodes, but the anti-PD-L1 antibody caused significant depletion of T cells.

Further, they found that this depletion was worsened when they combined the antibody with other immunotherapies. “This may have important implications when considering how to optimise immunotherapeutic combinations for patients,” Gordon said.

Finally, they tested the anti-tumour activity of the high-affinity PD-1 protein and found that when mice had tumours about the size of a grain of rice (50 mm3), both PD-1 protein and anti-PD-L1 antibodies were effective in shrinking the tumours.

However, when the mice had tumours about the size of a pea (150 mm3), the anti-PD-L1 antibodies were entirely ineffective, even when they were combined with anti-CTLA4 antibodies.

The high-affinity PD-1 protein was able to slow the growth of big tumours, and it was more effective when combined with anti-CTLA4 antibodies.

“Given how well anti-PD-1 and anti-PD-L1 antibodies are working for cancer patients, it may seem like a reasonable assumption that we have adequately targeted the pathway at this point.

However, our results show that significantly more anti-tumour activity is possible using a small- protein therapeutic compared with a conventional antibody,” Ring said.

“The broader implication is that small-protein therapeutics could present the same advantages in additional immunotherapeutic pathways outside of PD-1 and PD-L1.”

Because of their small size, the high-affinity PD-1 proteins may get excreted from the body more quickly, which means that they would have to be administered more frequently than antibodies.

Further, they may be “immunogenic,” meaning patients could develop an immune response to the drug itself.

“Both issues would need to be addressed for high-affinity PD-1 or a similar small-protein therapeutic to be developed for clinical use,” Gordon said.

Source: AACR