# New protein engineering-based tools and technologies for characterizing cell surface proteolysis in cancer cells for novel neo-epitope biomarkers and drug targets

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2021 · $363,351

## Abstract

Project Summary
Extracellular proteolysis is a hallmark of cancer. Proteolysis has major functional consequences for
remodeling cell surface proteins and matrix including receptor activation and shedding of receptor
extracellular domains. Of the more than 600 human proteases, over half are on the cell surface or
secreted reflecting their significance for processing the extracellular space. Yet we understand very
little about the targets for cell surface proteolysis (CSP), the proteases responsible, and resulting neo-
cleaved products that could serve as new biomarkers or drug targets.
 What has been missing are robust technologies for unbiased identification of the CSP targets,
their specific sites of cleavage, the proteases responsible for promoting disease, and the generation of
neo-epitope specific antibodies. My group has engineered a novel peptide ligase, called subtiligase,
and shown it robustly labels sites of proteolysis of soluble proteins. Our preliminary data show that this
powerful tool can be used to determine the targets and precise sites of cleavage for CSP. We propose
this novel CSP technology can be used to determine CSP signatures in cancer. We will initially focus
on KRAS, the most dominant oncogene, especially in pancreatic cancer. We will determine KRAS-
induced proteolysis in isogenic pancreatic cell lines, with and without mutant KRAS, grown in hypoxic
and normoxic conditions in the context of two- and three-dimensional culture. We will identify the
surface proteases up-regulated using cell surface capture (CSC) proteomics and confirm their activities
using CRISPRi or small molecule inhibitors. We will characterize their specificities using new substrate
phage technology coupled with NGS and match these to neo-epitopes we discover. Lastly, we will
further exploit differential antibody phage display selection technology, and generate antibodies specific
to the cleaved forms of various identified targets, including CDCP1. We will develop important
technologies for new neo-epitope biomarkers and potential immune-oncology reagents for the
characterization of these important cleavage events.
 Impact: Our studies will provide robust protein engineering-based technologies for unbiased
insights into how CSP remodels cells, the proteases responsible, and actionable oncogene specific
neo-epitopes to generate antibodies as potential biomarkers and therapeutics. We believe these
approaches will be generally applicable to characterizing proteolysis in other solid tumor cancers.

## Key facts

- **NIH application ID:** 10102220
- **Project number:** 5R01CA248323-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** JAMES A WELLS
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $363,351
- **Award type:** 5
- **Project period:** 2020-04-01 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10102220

## Citation

> US National Institutes of Health, RePORTER application 10102220, New protein engineering-based tools and technologies for characterizing cell surface proteolysis in cancer cells for novel neo-epitope biomarkers and drug targets (5R01CA248323-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10102220. Licensed CC0.

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