(M1030-02-08) Simplifying Cellular Immunogenicity Monitoring for Novel Therapeutics through a sMHC-1 Immunopeptidomics Approach

Purpose: Can sHLA-1 in plasma be used to detect antigen-specific cellular immune response? Humoral immunogenicity against the drug product is routinely monitored in the cell and gene therapy products. Cellular immunogenicity is another important aspect of immunogenicity against these drug products, but implementing assays to monitor this in clinical studies has been difficult. ELISpot assay that is widely used for cellular immunogenicity readout, requires high blood volume, complex sample processing and the final readout can only demonstrate the activation of cellular immunogenicity by a peptide pool. Additional runs are required to further identify the specific peptides that are activating cellular immunogenicity. We have been exploring alternative approaches that require less blood from patients, are easier to implement and also be able to identify petides activiating cellular immunogenicity. In this poster we present data from our evaluation of a less invasive plasma-based assessment of soluble HLA associated with peptides fromAAV-5 dosed cyno monkeys to as an alternative approach for imunogenicity assessment.

Methods: The Immunopeptidomics by Biotinylated Antibodies and Streptavidin – Mass Spectrometry (IMBAS-MS) method developed by the Mann Lab¹ allows for the isolation and identification of sMHC-1 immunopeptides in plasma. We optimized these conditions to improve sensitivity and background for detection in both human and cynomolgus plasma.

Results: Optimized methodology for sMHC-1 pulldown and detection of class 1 immunopeptides in both human and cynomolgus monkey plasma samples. Using six cynomolgus subjects (3 pre-existing ADA+ and 3 pre-existing ADA- statuses) dosed with AAV5 capsids, we were able to observe time-dependent changes in plasma associated peptides involved with inflammatory and cellular stress related pathways. Predominant detection of 9-mer peptides is consistent with the size of MHC-1 immunopeptides, indicating successful MHC-1 pull down. Results demonstrate this method’s ability to utilize immunopeptides to detect, follow, and potentially characterize cellular immune responses after AAV5 infection

Conclusion: We established an immunopeptidomics method to extract and detect immunopeptides from soluble MHC-1 in human and cynomolgus plasma Preliminary results from the cynomolgus study indicate more peptides from proteins involved in inflammatory and cellular stress response pathways to be associated with soluble MHC-1 in samples collected post-AAV5 treatment. Upon further optimization (e.g. plasma enrichment for the detection of AAV5 peptides), this could serve as an alternative methodology for cellular immunogenicity characterization This indirect approach can not only provide an understanding of peptides associated with Class I MHC that could potentially drive a cellular (CTL or T effector) immune response, but additionally, the set of peptides eluted from sHLA molecules could also serve as potential biomarkers for the immune monitoring of patients. Refinement and successful implementation of this approach will be beneficial to future cell and gene therapy programs by eliminating the need to collect a large amount of blood to perform involved and more expensive cellular immunogenicity assays.

References: 1. Wahle M, Thielert M, Zwiebel M, Skowronek P, Zeng WF, Mann M. IMBAS-MS Discovers Organ-Specific HLA Peptide Patterns in Plasma. Mol Cell Proteomics. 2024