Background on the cancer vaccine program
A clinical-stage biotech company approached KCAS Bio to support their oncology immunotherapy program, a first-in-class cancer vaccine leveraging pathogen-derived peptide sequences sharing high homology with disease-associated protein variants expressed in cancer cells. The sponsor wanted to monitor and characterize vaccine-specific immune responses, evaluate their cross-reactivity against tumor-associated antigens (TAA), their persistence and clinical relevance across a full Phase 1/2 clinical study in oncohematology. Samples were collected from patient screening through active treatment and into long-term follow-up extending months to over a year after treatment completion.
ELISpot and flow cytometry: two platforms, one goal
To meet the sponsor’s requirements, KCAS Bio deployed two complementary cell-based assays: ELISpot IFN-g and flow cytometry-based tetramer staining. Together, they delivered a comprehensive picture of both the functional capacity and the phenotype of antigen-specific T cells, something neither platform could achieve alone.
PBMCs, frozen by clinical sites worldwide over the course of the study, were thawed and stained to track Tetramer+ CD8+ T cells among the different circulating T cell subsets and assess their capacity to secrete IFN-g across all timepoints.
Flow cytometry and tetramer staining: seeing the cells themselves
Flow cytometry was used to detect and enumerate peptide-specific T cells at the single-cell level, and to assess their differentiation status. By using multiple fluorochrome-conjugated Tetramers, each specific to a different peptide of interest restricted by a specific HLA, KCAS Bio could simultaneously detect multiple T-cell clones from a single patient sample and characterize their memory phenotype. The sensitivity of this approach allowed the team to detect individual peptide-specific T-cell clones circulating in the blood, in some patients several months after completion of treatment.
ELISpot: quantifying T-cell functional fitness
ELISpot is one of the most sensitive methods available for quantifying and characterizing the functionality of antigen-reactive T cells. It was used here to assess the ability of this novel vaccine to induce a strong immune response to the tumor in treated patients, by measuring IFN-γ-secreting antigen-reactive T cells ex vivo. In each analytical run, sample integrity was checked out by running negative controls (medium + DMSO), positive controls, consisting of peptides derived from commonly encountered pathogens (CEFSX), and polyclonal T-cell activation via anti-CD3 stimulation.
From these data, the sponsor was able to draw meaningful conclusions about the induction, persistence, and clinical relevance of the antigen-specific CD8+ T-cell populations detected across the study timepoints.
Overcoming key challenges, through K.C.A.S.
Delivering this program required navigating several significant operational and scientific challenges, which map to the four pillars of the KCAS Bio approach.
K – Knowledge. Tetramer staining requires appropriate instrument settings, careful fluorescence compensation, and a robust gating strategy to ensure the confident detection of a rare population of tetramer-positive cells and exclude any artefactual event.
C – Collaboration. Running ELISpot and flow cytometry assays in parallel, on the same PBMC samples, required intense coordination between two specialized teams. Smooth communication between analysts and scientists, combined with clearly defined priority hierarchies established, with the Sponsor, in the bioanalytical plan ahead of time, ensured no sample was wasted and valuable data were generated.
A – Accountability. PBMC yields varied across patients and visits, requiring real-time adjustments to experimental conditions. Experienced analysts adapted protocols on the fly to ensure no sample was wasted while maintaining data integrity. This accountability extended to assay transfer: the protocol was successfully transferred from a reference laboratory to KCAS Bio, with results generated on the same samples showing high concordance.
S – Services. The sponsor frequently needed results on accelerated timelines to support key program milestones. KCAS Bio was able to optimize processing, analysis, and QC timelines to meet key milestones without compromising quality.
Conclusion
This study is a strong example of how the collaboration between multiple platforms and areas of expertise under the same roof allows getting the most out of precious clinical samples. With teams working in close coordination, this integrated approach generated impactful data for the vaccine candidate, ultimately benefiting the patients. It combines the detection and longitudinal tracking of circulating antigen-specific T cells with a functional assay assessing their capacity to release IFN-γ, a powerful cytokine playing a crucial role in activating cellular immunity and subsequently stimulating anti-tumor immune responses.
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