Abstract 1897: PeptiCHIP: A novel microfluidic-based chip platform for tumor antigen landscape identification
Authors: Feola Sara , Happala Markus , Peltonen Karita , Capasso Cristian , Antignani Gabriella , Federico Antonio , Pietiäinen Vilja , Chiaro Jacopo , Feodoroff Michaela , Ranniko Antti , Fusciello Manlio , Koskela Satu , Partanen Jukka , Hamdan Firas , Tähkä Sari , Ylösmäki Erkko , Greco Dario , Grönholm Mikaela , Branca Rui M M , Lehtiö Janne , Siikanen Tiina , Cerullo Vincenzo 
Published: July 2021

Knowledge about naturally presented HLA class I ligands from the tumor surface (the ligandome or immunopeptidome) is essential for designing T-cell mediated cancer therapeutic approaches. Indeed, the generation of specific anti-tumor CD8+ T cells relies on the recognition of tumor-antigens in the HLA-I complex. However, the sensitivity of the process for isolating the tumor antigens have been the major limiting factor for reliable HLA-I peptide characterization, making clear the need for technical improvement.Here, we assessed the potential of immobilizing a pan-HLA antibody on solid surfaces via well-characterized streptavidin-biotin chemistry, overcoming the limitations of the cross-linking chemistry used to prepare the affinity matrix with the desired antibodies in the immunopeptidomic workflow. Furthermore, to address the restrictions related to the handling and the limited availability of tumor samples, we further developed the concept towards the implementation of a microfluidic through-flow system.Thus, the biotinylated pan-HLA antibody was immobilized on streptavidin-functionalized surfaces, and immune-affinity purification (IP) was carried out on customized microfluidic pillar arrays made of thiol-ene polymer. Compared to the standard methods reported in the field, our methodology drastically reduces the handling, the amount of antibody and the time required for peptide isolation.In this work, we carefully examined the specificity and robustness of our customized technology for immunopeptidomics workflows. We tested this novel platform by immunopurifying HLA-I complexes from as few as 106 cells both in a widely studied B-cell line and in patients-derived ex vivo cell cultures. After the final elution in mild acid, HLA-I-presented peptides were identified by tandem mass spectrometry and further investigated by in vitro methods. These results highlight the potential to exploit microfluidics-based strategies in immunopeptidomic platforms and in personalized immunopeptidome analysis from cells isolated from individual tumor biopsies to design tailored cancer therapeutic vaccines.

Citation Format: Sara Feola, Markus Happala, Karita Peltonen, Cristian Capasso, Gabriella Antignani, Antonio Federico, Vilja Pietiäinen, Jacopo Chiaro, Michaela Feodoroff, Antti Ranniko, Manlio Fusciello, Satu Koskela, Jukka Partanen, Firas Hamdan, Sari Tähkä, Erkko Ylösmäki, Dario Greco, Mikaela Grönholm, Rui M M Branca, Janne Lehtiö, Tiina Siikanen, Vincenzo Cerullo. PeptiCHIP: A novel microfluidic-based chip platform for tumor antigen landscape identification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1897.