Mass Spectrometry-Based Multi-Protein Panel Assay for Detecting Immune Checkpoint Proteins in Immune Cells of Oral Cancer Patients

Cancer immunotherapy represents a transformative approach
to cancer
treatment, paving the way for personalized and precision medicine
strategies. Despite the significant advances in immunotherapy, the
treatment response rate remains a major clinical challenge. The stratification
of patients into responders and nonresponders is paramount for immunotherapy
treatment response, which reduces the likelihood of treatment challenges,
expenses, and potentially severe adverse effects in cases where patients
are not likely to respond. Therefore, in the present study, we optimized
a mass spectrometry-based analytical assay deploying data-independent
acquisition analysis (DIA) to simultaneously detect a panel of six
well-known immune checkpoint proteins. Further, the optimized assay
was analytically validated to assess the technical performance, and
preliminary clinical feasibility testing was conducted using the clinical
samples of immune cells from oral cancer patients. Altogether, we
tested the method for the detection of the candidate proteins in samples
from 20 oral cancer patients and healthy individuals. A proof-of-concept
method using DIA-MS was optimized with a shorter turnaround time,
facilitating a multiplexed detection and quantitation of protein targets
within the panel. This DIA-MS method could be applied to detect potential
immune checkpoint proteins without additional enrichment techniques.
Furthermore, this optimized method can be used in clinical settings
to guide appropriate drug selection based on the abundance of target
protein(s) in each patient. The assay may also be adapted for other
cancer types using the same multiprotein panel, or expanded in the
future to include additional proteins validated as potential immunotherapeutic
targets.