Intro and objectives
Bladder cancer is one of the cancer types where treatments targeting the immune system have proven the most effective. Indeed, bladder cancer bears a high number of mutations, which is crucial for developing T cell mediated immune responses. To decipher the exact role of these tumor infiltrating lymphocytes, it is important to identify their targets. Antigen discovery has always been challenging, given the very high number of peptide – MHC (pMHC) combinations and the current impossibility to predict the binding of a particular pMHC complex to a given T cell receptor (TCR).
We aim at developing a workflow to facilitate antigen discovery, this to ultimately improve precision immunotherapy in bladder cancer.
Materials and methods
We use fresh human bladder tumor samples originating from patients operated in our university hospital (UCL – Saint Luc), as well as blood and urine. From these samples, we extract T cells and sort them in 384-well plates. Single-cell RNA sequencing following the Smartseq2 protocol is then performed on these cells. This allows use to highlight interesting transcriptomic features of these cells and identify the clonotypes enriched in the tumor and their TCR sequences.
We plan to use a novel tool, designed by the Baltimore lab at Caltech, called SABR (signaling and antigen-presenting bifunctional receptors). SABRs are chimeric receptors that allow for identification of antigen-presenting cells and the discovery of the cognate peptides of selected TCRs.
For peptide presentation, we plan to transduce the SABR-carrying cell line with a tumor cDNA library.
Results
We already have successfully constructed a lentiviral cDNA library. We will analyze its representativeness of tumoral transcriptome and potential biases (eg over- or underrepresentation of certain transcripts) by performing high throughput sequencing.
We are currently adapting the published SABR protocol, which is based on mutated peptide prediction, to implement it in our lab using a cDNA library. This will allow us to broaden the spectrum of potential antigen discovery beyond classical neoantigens.
Conclusion
This approach is still under construction. It is very promising and will allow for a high throughput identification of antigens.
