J. Datta1, S. Xu1, J. H. Terhune1, L. Lowenfeld1, C. Rosemblit1, E. Berk1, E. Fitzpatrick1, R. E. Roses1, B. J. Czerniecki1 1University Of Pennsylvania,Surgery,Philadelphia, PA, USA
Introduction: HER3 overexpression is associated with resistance to targeted therapies (e.g. BRAF inhibitor in melanoma, trastuzumab in breast cancer) and a poor oncologic prognosis. Since existing anti-HER3 strategies – antibodies inhibiting HER2-HER3 dimerization – are static but not lytic, cellular therapies targeting HER3 are needed. CD4 T-helper type 1 (Th1) cells secrete IFN-γ/TNF-α, inducing tumor apoptosis. Using the extracellular domain (ECD) of HER3 as a candidate tumor antigen, we sought to identify immunogenic Class II-promiscuous HER3 CD4 peptides that generate anti-HER3 Th1 immunity for inclusion in cancer vaccines.
Method: A library comprising 123 overlapping 15 aa-long peptides was generated from HER3-ECD. Autologous monocyte-derived DCs from patients were matured to a type 1-polarized phenotype using IFN-γ/LPS (IL-12 secreting DC1), pulsed with HER3-ECD, and co-cultured with purified CD4 T-cells. After 10 days, sensitized CD4 T-cells were restimulated against immature DCs (iDC) pulsed with HER3 peptide clusters or irrelevant CD4 control. Th1 responses, measured by IFN-γ ELISA, were considered antigen-specific if IFN-γ production was at least twice that of control groups.
Results: Th1 sensitization was performed in 6 breast cancer patients with known anti-HER3 reactivity in order to identify single immunogenic HER3 CD4 epitopes. HER3 ECD-specific CD4 Th1 were sequentially restimulated against 10-peptide clusters, narrowed to 3-peptide clusters, and subsequently to single immunogenic HER3 peptides. Four immunogenic peptides (HER351-75, HER3402-417, HER3417-432, HER3451-465) were reproducibly identified based on two-fold increase in IFN-γ production (Fig). These peptides were promiscuous across HLA-DR, DP, and DQ subtypes. When Th1 cells from 4 non-HER3 reactive donors were sensitized using DC1s pulsed with the four identified HER3 peptides, and subsequently challenged to recognize HER3 ECD-pulsed iDCs, all donors demonstrated successful sensitization not only to individual immunogenic HER3 peptides, but also recognized native HER3-ECD.
Conclusion: DC1 pulsed with an overlapping HER3-derived peptide library identified promiscuous class II peptides for CD4 Th1 vaccine development. Immunogenic HER3 CD4 peptides abrogated anti-HER3 immune self-tolerance. Utilizing these peptides in vaccine construction warrants investigation in patients harboring HER3-overexpressing cancers, and would represent the first cellular therapy targeting IFN-γ-secreting CD4 Th1 cells against HER3-ECD. These data also reveal a novel strategy to rapidly and reproducibly identify class II-promiscuous immunogenic CD4 epitopes from any self-tumor antigen for immunotherapy via a DC1-Th1 platform.
