Proteomics and antigen discovery: a keynote presentation by Prof Jim Wells at University of California
A fascinating presentation about neo-epitopes and proteolytic landscape in cancer was delivered by Prof Jim Wells from the University of California San Francisco.
In cancer, it is pivotal to understand how cell surface proteins change in response to different oncogenes.
Prof Wells addressed this by creating a proteomics platform to find oncogene-specific antigens and antibodies (Leung et al., 2020, PNAS). The platform enables antigen discovery and specificity testing of final antibodies to find potential therapeutics.
In addition to understanding expression levels of cell surface proteins during different stages of cancer, Prof Wells' lab is interested in neo-epitopes and their correlation with cell surface epitopes.
Some examples of such neo-epitopes include MHC-peptide complexes or extracellular post-translational modifications (PTMs; Douglass and Zhou et al., 2021).
Prof Wells is particularly interested in proteolysis, a prominent extracellular PTM, playing role in a number of cell processes, such as angiogenesis, growth, survival, or metastasis. Prof Wells' lab focused on the discovery of proteolytic cleavage sites and their targeting for potential therapeutics.
The group developed a method for the identification of proteolytic cleavage sites, using a caspase digestion strategy (Mahrus et al., 2008 Cell). The results from the project were released via a database of caspase cleavage points (wellslab.ucsf.edu/degrabase).
Applying subtiligase at the cell surfaces strategy (Weeks et al., 2021, PNAS), Dr Weeks from Prof Wells' lab succeeded in labeling cell-surface proteins. Dr Weeks engineered the subtiligase to make it more successful in labeling the N-terminus of cell surface proteins.
Using an improved technology coupled with LC-MS mass spectrometry, Dr Shaefer labeled proteolytic neo-epitopes, allowing Prof Wells' lab to examine ectodomain cleavage events in neo-epitopes. The lab team identified numerous ectodomain cleavage events in the N-termini.
Comparing the cell surface proteolysis to expression levels of cell surface proteins in cancer, Prof Wells claimed to find that oncogenes change proteolysis rate.
Comparing common and unique proteolytic events between KRAS G12V mutation and Her2, some of the events were shared but several were unique to the individual oncogenes. One of the proteins identified through the analysis of proteolytic events was the protein CDCP1, found highly upregulated in pancreatic cancer cells.
Dr Zhou and Dr Lim from Prof Wells' lab next looked into CDCP1 cleavage in pancreatic cancer cell model lines and found an abundance of cleaved CDCP1 in some pancreatic cancer cell lines.
Prof Wells highlighted that the cleaved form of CDCP1 is only present in cancerous cells, and therefore his lab focused on developing antibodies against CDCP1, cleaved and full-length forms, as potential cancer therapeutic.
The development of antibodies was done using a differential selection strategy following affinity maturation, raising antibodies against cleaved and full-length CDCP1.
Prof Wells’ lab then raised mouse-specific antibodies against CDCP1 forms to study the toxicity of antibodies in mice. Mice treated with cleaved form anti-CDCP1 antibodies tolerated the antibody well, but those mice treated with full-length anti-CDCP1 antibodies lost ~10% body weight following a few days of treatment.
Using mice with Fc1245 tumors, the anti-CDCP1 antibody treatment seemed promising in slowing down tumor volume growth.
The results from the anti-CDCP1 antibody experiments were so encouraging, that Prof Wells' lab next looked into raising antibodies against targets that are both upregulated in cancer cells and have proteolytic events in them. This work is currently ongoing.
"The cleaved form of CDCP1 is only present in cancerous cells, and therefore Wells' lab focused on developing antibodies against CDCP1 as potential cancer therapeutic"
In the second part of his talk, Prof Wells outlined his work on MHC-peptide targets for engineering BiTes and CAR-T cells. MHC-peptides are important for recognition by cytotoxic T cells, enabling engineering for cytotoxic T-cell recruitment in cancer.
The work in Prof Wells' lab (Dr Rettko and Dr Kirkemo) focused on engineering peptide-HLA complexes that would be secreted by cells, enabling targeting by cytotoxic T cells.
Dr Rettko and Dr Kirkemo from Prof Wells' lab identified specific HLA.A:02.01 peptides using an HLA Fc-fusion workflow and investigated these in hypoxic PDAC cells. Hypoxia is a typical tumor state, whereas normoxia refers to normal levels of oxygen.
By comparing immunopeptidome of hypoxic and normoxic pancreatic cells (MiaPaCa2), Prof Wells' lab identified 99 peptides over-represented in hypoxia and is now raising antibodies to 4 of these hypoxic pMHC targets. Dr Rettko next used a differential screening strategy to identify antibodies selective to MHC-peptide complexes.
Secreted MHC-peptide immunopeptidomics allows profiling of specific MHC-1 alleles, and the approach used by Prof Wells' lab allows to pair peptides with specific MHC.
They also built a platform for building and characterizing antibodies to specific MHC-peptide complexes, which has been applied to state-specific cell conditions such as hypoxia, MEKi, and specific oncogenes.
