Cancer is a target-rich environment, but the targets can be hard to see. Even when cancer cells display antigens that are recognized by immune cells—as is evident when an immune cell receptor infiltrates a cancer cell—the antigens remain obscure. But antigen identification, which amounts to the identification of matched antigen–receptor pairs, may become more practical, say scientists based at Stanford University.

These scientists have developed a new kind of screen. It builds on years of work led by Christopher Garcia, Ph.D., a professor of molecular and cellular physiology and of structural biology, to better understand how the immune system “sees” antigens.

“This screen is a completely unbiased way of taking a random T-cell receptor (TCR) that's infiltrated a tumor and interrogating it to find out exactly what antigen it is actually seeing,” asserted Dr. Garcia, who is also a Howard Hughes Medical Institute investigator.

The screen he and his colleagues devised pulls data from two resources—first, TCRs found on colon cancer tumors that recognized unidentified antigens and thus qualified as “orphan receptors,” and second, a hefty repository of antigen sequences of white blood cells.

Using yeast as a vehicle, the team scanned some 400 million of these antigen sequences, all possible matches to 20 orphan receptors derived from colon cancer tissue samples. Four of the 20 receptors found matches.

This work has implications for cancer immunotherapy, which depends on immune cells recognizing specific antigens and killing the tumor cells that display them. “That's the basis of the actual killing event—where the rubber hits the road,” said Dr. Garcia. “But currently we know very few tumor antigens, and there's just been no good way of discovering them.” The new screening approach could also serve to identify potential antigens relevant to other immunotherapies, such as those that combat autoimmune or infectious diseases.

To show how the antigen identification process might be expedited, Dr. Garcia and colleagues presented their screening approach in an article (“Antigen Identification for Orphan T Cell Receptors Expressed on Tumor-Infiltrating Lymphocytes”) that appeared December 21 in the journal Cell. The article describes how Dr. Garcia’s team used yeast-display libraries of peptide-human leukocyte antigen (pHLA) to screen for antigens of “orphan” TCRs expressed on tumor-infiltrating lymphocytes (TILs) from human colorectal adenocarcinoma.

“Four TIL-derived TCRs exhibited strong selection for peptides presented in a highly diverse pHLA-A02:01 library,” the article’s authors wrote. “Three of the TIL TCRs were specific for non-mutated self-antigens, two of which were present in separate patient tumors, and shared specificity for a non-mutated self-antigen derived from U2AF2.”

These results, the authors suggested, show that the exposed recognition surface of major histocompatibility complex (MHC)-bound peptides accessible to the TCR contains “sufficient structural information to enable the reconstruction of sequences of peptide targets for pathogenic TCRs of unknown specificity.”

What's in the screen, and what's getting screened, Dr. Garcia explained, are mimics of the original receptors and antigens—accurate, but not 100% identical. So, after the initial screen, the four receptors that bound antigens were then sequenced and run through an algorithm, which ultimately figured out the correct corresponding identity of the human antigen. With this technique, the team unambiguously identified two human antigens of the four receptors that found matches in the yeast-based library, and they're currently in the process of identifying a third one.

The somewhat modest ratio is a product of chance. The receptors are restricted by genotype and will only bind to antigens of a matching genotype. Between that and the enormous variability of possible antigens, pegging a receptor–antigen match is “a bit like winning the lottery,” Dr. Garcia noted. “The key to increasing the odds is to increase the throughput of the experiments, kind of like putting more coins into the slot machine.”

There's an ongoing debate, Dr. Garcia commented, about the most important types of antigens that T cells “see” and attack in tumors. One currently popular notion is that TCRs react with neoantigens, or antigens that are mutated or uniquely part of a cancer, rather than self-antigens, which both cancerous and healthy cells can have in common. Unexpectedly, however, evidence from the new study suggests otherwise, as one of the two antigens was “self.” In addition, the self-antigen turned out to be shared between two patients—a key to developing immunotherapies.

“It was a huge surprise to find that one of the antigens was a nonmutated, shared self-antigen, and the implications are that if you screen many more TCRs, you'll likely find a lot of shared antigens,” Garcia said. “So in theory, you could have one immunotherapy that targets this antigen, and it'd be effective for multiple patients.” But this, he said, brings us to the million-dollar question: How do we generate antitumor immunity against an antigen that is attached to both healthy and cancerous cells?

“Right now, we don't have an answer, but there are a lot of efforts going into that problem, and it's something that I'm very interested in. It's a problem that we're going to have to solve because there are going to be a lot of tumor antigens that will be nonmutated, and so we have to figure out how to localize the attack to just the tumor, and not the rest of the host.”

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