Over the past decade, immunotherapy has recruited the patients' own immune system in the fight against thetumor. In particular, the successful blocking of the interaction between programmed cell death-1 (PD-1) and itsligand PD-L1 has paved the way for the development of new treatments for several of the most devastatingcancers. In cancers such as non-small cell lung cancer (NSCLC), upregulation of PD-L1, both in the tumor cellsand in macrophages, reduces the host's immune response, thereby enhancing tumor aggressiveness. WhilePD-1/PD-L1 checkpoint-blockade immunotherapy has become a real "game-changer' for many cancer patients,response rates remain relatively low, ranging from 15 to 40%, depending on the type of cancer and the stage ofthe disease.The current assessment of whether a patient should be treated with PD-1/PD-L1 blockade therapy is based onexpression levels of PD-L1 and degree of tumor-infiltrating immune cells using immunohistochemistry (IHC).This approach has significant downsides: 1) samples are surgically obtained, posing additional risk, andpotentially triggering enhanced metastasis as a result of primary tumor perforation; and 2) these measures arerarely sensitive or specific enough in predicting response efficacy, being prone to generating false-positiveresults. In addition, IHC lacks a clean and quantitative definition of what threshold defines a PD-L1 positivetumor. It was shown that subsets of patients do not benefit from checkpoint-blockade immunotherapy despitetesting positive for PD-L1 expression using IHC, and conversely, a significant number of patients responded wellto it, despite testing negative for PD-1/PD-L1 in IHC-based tests. To spare patients from ineffective therapy andlimit the number of those exposed to potential autoimmune side effects, the search for a reliable predictivebiomarker allowing patient selection of those who would benefit from anti-PD-1/PD-L1 therapy has becomemore urgent than ever.In this proposal, we will use our ExoView technology to create an assay able to identify and quantify circulatingexosomes that carry PD-L1. Exosomes have been shown to play a critical role in many pathologies and morerecently in anti-tumor immunity. Furthermore, the vesicular nature of exosomes allows the identification of proteinsignatures which in turn may reveal valuable information about the parent cell type. Aim 1 will establish anantibody able to immobilize exosomes containing PD-L1 and validate the specificity and sensitivity of suchantibodies in human plasma. Aim 2 will quantify PD-L1 positive exosomes originating from immune cellsvs. tumor cells. Using various cellular markers will enable identifying the origin of PD-L1-expressing exosomes.Aim 3 will focus on Quantification of PD-L1 in vesicles from immune vs tumor cells in NSCLC plasmasamples and correlate to PD-L1 IHC score and outcome of anti-PD-1/PD-L1 therapy. NARRATIVE
We propose to develop a quantifiable method for identification of exosomal PD-L1 as an indicator for tumor-
derived PD-L1, in lung cancer. To date, PD1/PDL-1 blockade therapy provides limited success due to poor IHC
based prognostic testing, often leading to both false positive and negative results, which lead to ineffective
treatment. We propose a method in which our ExoView specifically captures and quantifies exosomal PD-L1
from plasma samples, thus offering lower patient risk and heightened accuracy in predicting efficacy of
PD1/PD-L1 blockade treatment. 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