Julianna LeMieux Ph.D. Senior Science Writer GEN

Immune-Evasion Mechanism May Help Explain the Differences in Responses to Immunotherapy

A team of researchers from the University of Pennsylvania (UPenn) School of Medicine revealed that the exosomes (extracellular vesicles) that contain programmed death-ligand 1 (PD-L1), released by melanoma cells, are found far beyond the tumor microenvironment and could be purified from the plasma of patients.

James L. Gulley, M.D., Ph.D., a senior investigator and the chief at the genitourinary malignancies branch of the National Cancer Institute, tells GEN “we have been so used to thinking about the PD-1/PD-L1 interaction in the tumor microenvironment, this work shifts it into a different light…we are going to start thinking about this differently now.”

The paper, entitled “Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response” and published in the journal Nature, is a collaboration between Wei Guo, Ph.D., a professor of biology in the School of Arts and Sciences at UPenn, and Xiaowei Xu, M.D., Ph.D., a professor of pathology and laboratory medicine in the Perelman School of Medicine at UPenn.

The researchers purified exosomes from a panel of human primary and metastatic melanoma cells lines by differential centrifugation, and analyzed the proteins associated with the exosomes by reverse phase protein array (RPPA) a large-scale antibody-based quantitative proteomics technology.

PD-1, found on the surface of T cells, regulates the immune system’s response to cells of the human body acting to inhibit T cells from attacking other cells in the body. However, when PD-1 is bound to PD-L1, the T-cell killing is inhibited. Tumor cells evade immune surveillance by upregulating the surface expression of PD-L1—which, when bound to PD-1 on T cells, initiates the anticancer response (or immune checkpoint response). Checkpoint inhibitors block PD-1, reinvigorating the T cells, and opening up the attack on the cancer cells by the immune system.

To look at the secretion of exosomal PD-L1 by melanoma cells in vivo, the researchers established human melanoma xenografts in nude mice. Human PD-L1 was identified on the circulating exosomes from mice bearing human melanoma xenografts, but not the control mice. Not only did the level of circulating PD-L1+ exosomes correlate with tumor size, but adding PD-L1+ exosomes accelerated the progression of melanoma tumors. In addition, the researchers found that the PD-L1+ exosomes inhibit the T cells.

In essence, the tumors are releasing the exosomes to circulate in the body via the bloodstream, to inhibit T cells before they reach the site of the tumor. Dr. Guo describes it by saying, “essentially, exosomes secreted by melanoma cells are immunosuppressive,” and that they “propose a model in which these exosomes act like drones to fight against T cells in circulation, even before the T cells get near to the tumor.”

Indeed, PD-L1 has been found in blood samples derived from melanoma patients. While primarily focused on metastatic melanoma, the team found that breast and lung cancer also release the PD-L1-carrying exosomes.

Currently, tumor PD-L1 levels are used as a predictive biomarker for clinical responses to anti-PD-1 therapy. The higher the percentage of cells expression PD-L1, in general, the more likely the patient is to respond to immune checkpoint inhibition.

Dr. Gulley tells GEN that “this work opens the door to an important question as to whether circulating PD-L1+ exosomes could be a clinical predictor if they are correlated to patient outcomes.” He adds that it raises the attractive possibility of developing “a liquid biopsy that could may tell you the patient’s PD-L1 level in a blood-based test.” Dr. Gulley says that this work is very promising, but “needs to be looked at in large, clinically annotated datasets to understand the true impact of this.”

Tasuku Honjo’s, M.D., Ph.D., lab at Kyoto University first brought programmed cell death protein 1 (PD-1) into focus in 1992, with the paper “Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death” in EMBO J. Since then, PD-1 has been at the heart of cancer research as a primary target for one of the most successful innovations in cancer therapy—the checkpoint inhibitor drugs.

Currently, there are two FDA-approved PD-1 inhibitors (Pembrolizumab [Keytruda; Merck & Co.] and Nivolumab [Opdivo; Bristol-Myers Squibb]) and three FDA approved PD-L1 inhibitors (Atezolizumab [Tecentriq; Genentech], Avelumab [Bavencio; EMD Serono], and Durvalumab [Imfinzi; AstraZeneca]). A sixth FDA approval is expected in the next few months from Regeneron and their partners at Sanofi.

These drugs are designed to block the PD-1/PD-L1 interaction and have shown great promise in treating tumors. However, the patient response rate is low. Dr. Guo says, “Immunotherapies are life-saving for many patients with metastatic melanoma, but about 70% of these patients don’t respond.” In addition, “these treatments are costly and have toxic side effects so it would be very helpful to know which patients are going to respond.” He adds that this is why “identification of a biomarker in the bloodstream could potentially help make early predictions about which patients will respond.”

The UPenn researchers found that the exosomal PD-L1 has the same membrane topology as cell surface PD-L1 and that exosomal PD-L1 binds to PD-1 in a concentration-dependent manner, an interaction that can be disrupted by PD-L1 blocking antibodies. This research offers a paradigm-shifting picture of how cancers take a systemic approach to suppressing the immune system.

According to Dr. Gulley, this work will “change how many people think about PD-L1 only being important in the tumor microenvironment. [It] offers a lot of opportunities to interrogate clinical datasets.” He adds that we know a lot of information about some patients and that both academic and pharma companies will probably act quickly to figure out how clinically relevant this is. To erase any doubts, Dr. Gulley says with enthusiasm that he will be sharing this work with his team of researchers as soon as possible. He hopes that this will lead to rapid insights.



































































































































































