44P - HPV16-specific CD4 and CD8 T-cell activation and functionality in patients receiving combination PDS0101 immunotherapy

Background

The novel, investigational HPV-targeted immunotherapy PDS0101 is being studied in combination with pembrolizumab in a phase 2 clinical trial (NCT04260126) in patients with HPV16-positive head and neck cancer. Measurement of antigen-specific activation of endogenous T-cells is critical to understanding drug-induced T-cell based immunity and its association with observed clinical outcomes. In this pilot study, we sought to establish optimal stimulation conditions for in vitro activation with selected HPV16 peptide pools that would enable downstream single cell analysis of functional cytokine profiles of HPV-specific CD4 and CD8 T-cell subpopulations.

Methods

The IsoPlexis CodePlex platform was used to track cytokine profiles generated over time by peptide-activated CD4 and CD8 T-cells. Cryopreserved PBMCs from 2 study subjects collected at 3 timepoints (pre-treatment, and 12 and 36 weeks following 4 and 5 cycles of combination therapy, respectively) were thawed and recovered overnight in the presence of IL-2, before activation with overlapping HPV16 E6 and E7 peptide pools for 1hr, 6hr, 16hrs or 24hrs. Recovered stimulated cells were enriched for CD4 and CD8 populations using magnetic bead separation and the cells plated overnight. The supernatants from each of these populations were recovered and frozen at -80°C until all supernatants were available for analysis. Supernatants were loaded on to CodePlex chips and analyzed using the IsoLight instrument.

Results

Strong CD4 and CD8 T-cell responses were documented after only 1hr of peptide stimulation and at all time course timepoints. Detection of multiple cytokines (background subtracted) was captured by concentration and demonstrated post-treatment increases in granzyme B, IFN-g, TNF-a/b, MCP-1, MIP-1a/1b and perforin reflecting development of HPV-specific CD8 and CD4 T cell reactivity and immune memory. Stimulation between 6-16hrs provided the most multiparametric and robust cytokine signals.

Conclusions

PDS0101 treatment induces polyfunctional CD4 and CD8 T-cell responses across multiple timepoints. Additional studies of cell-specific functional profiles in larger numbers of subjects and correlation with clinical outcomes are planned.

Clinical trial identification

NCT04260126.

Editorial acknowledgement

Legal entity responsible for the study

PDS Biotechnology.

Funding

PDS Biotechnology.

Disclosure

L.V. Wood, D. Schaaf, N. Riebel, S. Jones: Financial Interests, Personal, Full or part-time Employment: PDS Biotechnology. All other authors have declared no conflicts of interest.