High-Throughput, Quantitative Analysis of Peptide-Exchanged MHCI Complexes by Native Mass Spectrometry

Abstract

Immune monitoring in cancer immunotherapy involves screening CD8+ T-cell responses against neoantigens, the tumor-specific peptides presented by Major histocompatibility complex Class I (MHCI) on the cell surface. High-throughput immune monitoring requires methods to produce and characterize small quantities of thousands of MHCI−peptide complexes that may be tested for a patient's T-cell response. MHCI synthesis has been achieved using a photocleavable peptide that is exchanged by the neoantigen; however, assays that measure peptide exchange currently disassemble the complex prior to analysis�precluding direct molecular characterization. Here, we use native mass spectrometry (MS) to profile intact recombinant MHCI complexes and directly measure peptide exchange. Coupled with size-exclusion chromatography or capillary-zone electrophoresis, the assay identified all tested human leukocyte antigen (HLA)/peptide combinations in the nanomole to picomole range with minimal run time, reconciling the synthetic and analytical requirements of MHCI−peptide screening with the downstream T-cell assays. We further show that the assay can be "multiplexed" by measuring exchange of multiple peptides simultaneously and also enables calculation of Vc 50 , a measure of gas-phase stability. Additionally, MHCI complexes were fragmented by top-down sequencing, demonstrating that the intact complex, peptide sequence, and their binding affinity can be determined in a single analysis. This screening tool for MHCI−neoantigen complexes represents a step toward the application of state-of-the-art MS technology in translational settings. Not only is this assay already informing on the viability of immunotherapy in practice, the platform also holds promise to inspire novel MS readouts for increasingly complex biomolecules used in the diagnosis and treatment of disease.

Exposing the molecular heterogeneity of glycosylated biotherapeutics

“…Hence, current methods denature and de-glycosylate the complex prior to analysis, focusing only on the released peptide. 44,46 With the DIA-PTCR approach, we detected two populations of the intact, peptide-bound MHCII containing either a full-length or truncated alpha chain, each with distributions of masses consistent with extensive and heterogeneous glycosylation (Fig. 2b-d).…”

“…Figure 4d,e). 43 For a further proof-of-concept, the DIA-PTCR method was employed to pro le the glycoform distribution of the peptide-bound Major Histocompatibility Complex class II DPA1*02:02/DPB1*05:01 allele (MHCII), 44,45 a non-covalent heteromeric complex which heretofore could not be characterized intact by MS due to the molecular heterogeneity arising from 4 N-linked glycosylation sites (inset of Fig. 2a).…”

UniDec Processing Pipeline for Rapid Analysis of Biotherapeutic Mass Spectrometry Data

“…The example workflows shown here demonstrate their potential for biopharmaceutical applications, but the same framework could be readily applied to drug discovery or protein design by simply adjusting the spreadsheet columns. For example, UPP could be applied to high-throughput analysis of non-covalent MHCI complexes to screen for neoantigen candidates by quantifying the amount of successful peptide exchange . Accessible inputs and outputs make the software easy to interface with other tools.…”

“…For example, UPP could be applied to high-throughput analysis of non-covalent MHCI complexes to screen for neoantigen candidates by quantifying the amount of successful peptide exchange. 33 Accessible inputs and outputs make the software easy to interface with other tools. Finally, because it is free, cross-platform, and containerized, it can be run in individual workstations, local servers, or cloud providers without licensing restrictions or requirements to transfer data offsite or to a third-party ecosystem.…”

Human leukocyte antigen and tumor immunotherapy (Review)

“…The analysis and screening of tumor antigen peptides that efficiently bind to MHC molecules is of great significance for the rapid development of safe and effective tumor vaccines (119). Mass spectrometric analysis of tumor antigen peptides has been used to identify effective tumor neoantigen peptides from tumor cells or tissues (120). Oncolytic viruses induce tumor cells to produce new MHC class I molecular ligands to activate CD8 + T-cell responses (121).…”