Abstract
Identification of HLA class I ligands from the tumor surface (ligandome or immunopeptidome) is essential for designing T-cell mediated cancer therapeutic approaches. However, the sensitivity of the process for isolating MHC-I restricted tumor-specific peptides has been the major limiting factor for reliable tumor antigen characterization, making clear the need for technical improvement. Here, we describe our work from the fabrication and development of a microfluidic-based chip (PeptiCHIP) and its use to identify and characterize tumor-specific ligands on clinically relevant human samples. Specifically, 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 immunopeptidomics workflow. Furthermore, to address the restrictions related to the handling and the limited availability of tumor samples, we further developed the concept toward 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 reduces 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 platform by immunopurifying HLA-I complexes from 1 × 106 cells both in a widely studied B-cell line and in patients-derived ex vivo cell cultures, instead of 5 × 108 cells as required in the current technology. 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 immunopeptidomics platforms and in personalized immunopeptidome analysis from cells isolated from individual tumor biopsies to design tailored cancer therapeutic vaccines. Moreover, the possibility to integrate multiple identical units on a single chip further improves the throughput and multiplexing of these assays with a view to clinical needs.
Highlights
Cancer immunotherapy relies on the priming of T cells, the generation and stimulation of cytotoxic CD8 T lymphocytes within the tumor microenvironment, and the establishment of an efficient and durable antitumor immune response.[1]
We envisioned that all the immune-purification steps could be carried out within a single microfluidic chip by adding a biotinylated pan-Human Leucocyte Antigen (HLA) antibody to a streptavidin-prefunctionalized solid support structure and eventually immobilizing the HLA-I complexes onto the pan-HLA antibody coated solid surface
1.74 × 1014 molecules of antibody could be immobilized and technically 4.5 × 106 cells could be investigated. These results clearly demonstrate the feasibility of the chip-based protocol for immobilizing the pan-HLA antibody, via quick biotin−streptavidin chemistry, which theoretically enables the identification of the HLA
Summary
Cancer immunotherapy relies on the priming of T cells, the generation and stimulation of cytotoxic CD8 T lymphocytes within the tumor microenvironment, and the establishment of an efficient and durable antitumor immune response.[1]. Presented peptide pool is the only reliable approach to identify the naturally presented HLA-I landscape in human cell lines,[3,4] tumor tissues,[5−7] and bodily fluids such as plasma.[8] This methodology is based on immunoaffinity purification (IP) of HLA-I complexes from mild detergent-solubilized lysates, followed by extraction of HLA-I peptides. Several techniques originating from an immunoaffinity purification approach are suitable for immunopeptidomics analysis.[9,10] significant technological advances in chromatography, MS, and bioinformatics tools have facilitated the analysis of thousands of HLA-I peptides and enabled a greater understanding of the dynamic nature of the entire HLA-I landscape in tumor cells.
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