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PEPTICHIP: Streamlined identification of tumour neoantigens for personalised anti-cancer immunotherapy

Periodic Reporting for period 1 - PeptiCHIP (PEPTICHIP: Streamlined identification of tumour neoantigens for personalised anti-cancer immunotherapy)

Periodo di rendicontazione: 2019-10-01 al 2021-09-30

Identification of HLA class I ligands from the tumour surface is essential for designing T-cell mediated anti-cancer therapies. However, the immunopurification process for isolating MHC-I-restricted tumour-specific peptides has been the major limiting factor for reliable tumour antigen characterisation. Other challenges to address include the scarcity of biological sample material, high costs, long and laborious protocols, and the need for extensive sample handling by highly trained personnel. In the ERC Proof-of-Concept project “PeptiCHIP” (2019-2021) awarded to Prof. Cerullo’s IVT Lab, we developed a microfluidic-based chip to identify and characterize tumour-specific ligands on clinically relevant human samples (Feola et al. PeptiCHIP: A Microfluidic Platform for Tumor Antigen Landscape Identification. ACS Nano, 15, 10, 15992-16010, available Open Access at: https://pubs.acs.org/doi/10.1021/acsnano.1c04371). In addition, we have developed HEX, an in silico platform that combines existing methods with a novel scoring algorithm, where the resemblance of the tumor peptide sequence to viral peptides not only expedites ligand selection but engages our pre-existing anti-viral immunity to improve the outcome of cancer immunotherapies. The results demonstrated that the HEX-prioritised ligands enabled the recruitment of cross-reactive cytotoxic T cells with a clear impact on both tumour growth and clearance (Chiaro et al. Viral Molecular Mimicry Influences the Antitumor Immune Response in Murine and Human Melanoma, Cancer Immunology Research 2021;9:981–93, available Open Access at: https://cancerimmunolres.aacrjournals.org/content/9/8/981).

The technical and scientific feasibility assessment of PeptiCHIP, as detailed in Feola et al., successfully:
1) assessed the potential of immobilizing a pan-HLA antibody on solid surfaces via well-characterized streptavidin–biotin chemistry, thus overcoming the limitations of the cross-linking chemistry used to prepare the affinity matrix and attach the desired antibodies in the immunopeptidomics workflow;
2) addressed the limitations with current state-of-the-art related to the handling and the limited availability of tumour samples by implementing a novel microfluidic system, where the biotinylated pan-HLA antibody is immobilized on streptavidin-functionalized surfaces, and immune-affinity purification is carried out on customized thiol–ene polymer microfluidic pillar arrays;
3) demonstrated that in comparison to current state-of-the-art, our methodology significantly reduced the needed amount of antibody, sample material, and time required for the immunopurification process. Together these also translate to significant cost savings. Importantly, we showed that the microchip could isolate the HLA-peptides from only one million (1 × 10^6) cells, whereas a typical ligandome experiment requires hundreds of millions (5 × 10^8) cells (Purcell et al., Nat. Protoc. 2019, 14 (6), 1687– 707). Following elution with mild acid, the HLA-I-presented peptides were identified by tandem mass spectrometry using a custom but robust immunopeptidomics workflow, and further investigated using in vitro methods. The microchip-based immunopurification protocol together with the immunopeptidomics workflow could be completed in less than 24 hours, instead of several days required by traditional ligandome analysis; and finally,
4) confirmed that using our technology, the ligandome can be determined also from small needle biopsy samples (typically comprised of 1.65 × 10^6 to 6 × 10^6 cells (Rajer et al., Radiol. Oncol. 2005, 39, 269– 272)), which opens new clinical avenues for both diagnostics and therapeutics' development.
5) In addition to technical characterisation and method validation, PeptiCHIP was applied to the antigen discovery process of clinical samples (PDOs) demonstrating that our microchip-based protocol was able to isolate HLA-relevant ligands from as few as 6 × 10^6 cells, instead of the 3.85 × 10^7 to 1 × 10^8 cells reported recently using a state-of-the-art method in a comparative setting (Newey et al., J. Immunother. Cancer 2019, 7, 309).
6) Our results highlight the potential to exploit microfluidics-based strategies in immunopeptidomics platforms and in personalized immunopeptidome analysis from cells isolated from individual small needle tumour 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.

In summary, PeptiCHIP enables user-friendly, accurate, and standardized tumour neoantigen identification from small tumour biopsies. Combining PeptiCHIP with the HEX in silico platform further increases the efficiency of neoantigen selection and introduces “viral or molecular mimicry” as an additional peptide selection criterion with potential to improve the outcomes of cancer immunotherapies. It should be noted that most competing technologies merely predict neoantigen signatures instead of identifying them, using protocols that are time-consuming, cumbersome, expensive, and requiring very high amounts of tumour sample. By addressing several major hurdles in the clinical translation of cancer immunotherapies, the PeptiCHIP platform helps unleash their full potential and pave the way for truly personalized immunotherapies. It will therefore offer more meaningful neoantigen identification for immunotherapy companies, clinicians, and researchers. The technology platform is particularly valuable for antigen validation in the clinical development of DC-based therapies, TCR designs, monoclonal antibody creation, and for coating oncolytic viruses, such as the PeptiCRAd technology developed in Prof. Cerullo’s ERC CoG project “PEPTICRAD” (2016-2021). We have filed a PCT application in May 2021 and are currently updating the detailed business plan together with Helsinki Innovation Services Ltd (technology transfer office) based on the selected commercialisation paths to lay the foundation for a high-potential spin-off company and support discussions with interested commercialisation partners and investors.