A Supramolecular Vaccine Platform Based on α‑Helical Peptide Nanofibers

A supramolecular peptide vaccine system was designed in which epitope-bearing peptides self-assemble into elongated nanofibers composed almost entirely of α-helical structure. The nanofibers were readily internalized by antigen presenting cells and produced robust antibody, CD4+ T-cell, and CD8+ T-cell responses without supplemental adjuvants in mice. Epitopes studied included a cancer B-cell epitope from the epidermal growth factor receptor class III variant (EGFRvIII), the universal CD4+ T-cell epitope PADRE, and the model CD8+ T-cell epitope SIINFEKL, each of which could be incorporated into supramolecular multiepitope nanofibers in a modular fashion

<i>In vivo</i> effects of daclizumab on the effector T-cell to regulatory T-cell ratio.

<p>(A–B) Effector T-cells (CD4⁺ or CD8⁺) to T_Reg ratios were derived by dividing the absolute number of effector T-cells by the absolute number of T_Regs at the indicated time-points; the absolute number of cells was determined by a combination of CBC and FACS analysis. The ratios of effector T-cells to T_Regs as compared to baseline were generated by dividing the individual patient CD4⁺∶T_Reg or CD8⁺∶T_Reg ratio at every time point by the ratio at vaccine 1 (V-1 = baseline). A two sample t-test averaged over the V-2, V-3 and LP time-points was utilized to examine the difference between the daclizumab and saline groups in the CD4⁺∶T_Reg (p = 0.0757) and CD8⁺∶T_Reg (p = 0.0153) ratios.</p

The frequency of T_Regs and anti-PEPvIII humoral responses are inversely correlated.

<p>Patient sera from peripheral blood (vaccine 4) and leukapheresis samples were analyzed for levels of anti-PEPvIII antibodies and humoral responses were plotted against the frequency of T_Regs (Foxp3⁺ of CD4⁺). Assuming the assessments within individuals are independent, the Spearman correlation coefficient for both saline and daclizumab treated patients overall is (R = −0.93, p<0.0001).</p

Regulatory T-cells are significantly depleted by a single infusion of daclizumab.

<p>The frequency of CD4⁺Foxp3⁺ T_Regs was determined by FACS analysis of peripheral blood samples drawn prior to vaccination (V) or leukapheresis (LP). Percent change was calculated in comparison to baseline (vaccine 1). For each follow-up assessment, percent change from baseline (vaccine 1) was computed. For statistical comparisons of the daclizumab and saline groups, the average percent change at vaccine 2 (V-2), vaccine 3 (V-3), and leukapheresis (LP) was computed for each patient. Daclizumab showed a significantly greater reduction in CD4⁺Foxp3⁺ regulatory T-cells (p = 0.0464).</p

Daclizumab remains bound to T_Regs a month after administration.

<p>(A) CD4⁺Foxp3⁺ T_Regs from day 35±2 leukapheresis samples (saline n = 3, daclizumab n = 3) were determined by flow cytometry and were additionally stained with anti-CD25 antibodies that bind the same CD25 eptiope as daclizumab (competing clone 2A3) or bind a separate epitope (non-competing clone MA251). The ratio of the frequency of CD4⁺CD25⁺Foxp3⁺ T_Regs as determined by MA251 or 2A3 binding was used as an indirect indicator of surface daclizumab expression. The percent change in the ratio was calculated from ratios determined from baseline (vaccine 1) samples, unpaired t-test *p = 0.0353. (B) CD4⁺CD25⁺Foxp3⁺ T_Regs were determined by FACS analysis of PBMC and examined for human antibody expression as a direct indicator of daclizumab binding to the surface of T_Regs. PBMCs from a saline and a daclizumab treated patient from vaccine 1 (Pre-Daclizumab) and leukapheresis at day 35±2 (Post-Daclizumab) time-points were assessed.</p

ZAP IT Patient Characteristics.

<p>> As of the lock date of the data, the indicated patient had 12 cycles of TMZ but continued with additional cycles of TMZ treatment.</p

<i>In vitro</i> effects of IL2Rα inhibition on CD4⁺, CD8⁺ and regulatory T-cells.

<p>Normal donor peripheral blood mononuclear cells (PBMCs) were cultured for 48 hours with increasing concentrations of daclizumab followed by an additional 14 days stimulation/expansion with CMV pp65 RNA-pulsed DCs along with IL-2 or IL-15. PBMC were then isolated and stimulated for 6 hours with SEB or pp65 peptide mix in the presence of CD28/CD49d costimulation and Brefeldin A. The IFN-γ secretion of (A) CD3⁺CD4⁺CD69⁺ or (B) CD3⁺CD8⁺CD69⁺ T-cells was determined by flow cytometry.</p