Luis Sanchez-Perez

Several different self-assembling peptide systems that form nanofibers have been investigated as vaccine platforms, but design principles for adjusting the character of the immune responses they raise have yet to be well articulated. Here we compared the immune responses raised by two structurally dissimilar peptide nanofibers, one a β-sheet fibrillar system (Q11), and one an α-helical nanofiber system (Coil29), hypothesizing that integrated T-cell epitopes within the latter would promote T follicular helper (Tfh) cell engagement and lead to improved antibody titers and quality. Despite significantly different internal structures, nanofibers of the two peptides exhibited surprisingly similar nanoscale morphologies, and both were capable of raising strong antibody responses to conjugated peptide epitopes in mice without adjuvant. Both were minimally inflammatory, but as hypothesized Coil29 nanofibers elicited antibody responses with higher titers and avidities against a conjugated model epitope (OVA323-339) and a candidate peptide epitope for vaccination against S. aureus. Subsequent investigation indicated that Coil29 nanofibers possessed internal CD4+ T cell epitopes: whereas Q11 nanofibers required co-assembly of additional CD4+ T cell epitopes to be immunogenic, Coil29 nanofibers did not. Coil29 nanofibers also raised stronger germinal center B cell responses and follicular helper T cell (Tfh) responses relative to Q11 nanofibers, likely facilitating the improvement of the antibody response. These findings illustrate design strategies for improving humoral responses raised by self-assembled peptide nanofibers.

BACKGROUND: First-in-human (FIH) clinical trials require careful selection of a safe yet biologically relevant starting dose. Typically, such starting doses are selected based on toxicity studies in a pharmacologically relevant animal model. However, with the advent of target-specific and highly active immunotherapeutics, both the Food and Drug Administration and the European Medicines Agency have provided guidance that recommend determining a safe starting dose based on a minimum anticipated biological effect level (MABEL) approach. METHODS: We recently developed a T cell activating bispecific antibody that effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma in mice (hEGFRvIII:CD3 bi-scFv). hEGFRvIII:CD3 bi-scFv is comprized of two single chain antibody fragments (bi-scFvs) that bind mutant epidermal growth factor receptor variant III (EGFRvIII), a mutation frequently seen in malignant glioma, and human CD3ε on T cells, respectively. In order to establish a FIH dose, we used a MABEL approach to select a safe starting dose for hEGFRvIII:CD3 bi-scFv, based on a combination of in vitro data, in vivo animal studies, and theoretical human receptor occupancy modeling. RESULTS: Using the most conservative approach to the MABEL assessment, a dose of 57.4 ng hEGFRvIII:CD3 bi-scFv/kg body weight was selected as a safe starting dose for a FIH clinical study. CONCLUSIONS: The comparison of our MABEL-based starting dose to our in vivo efficacious dose and the theoretical human receptor occupancy strongly supports that our human starting dose of 57.4 ng hEGFRvIII:CD3 bi-scFv/patient kg will be safe.

PURPOSE: The success of checkpoint blockade against glioblastoma (GBM) has been disappointing. Anti-PD-1 strategies may be hampered by severe T-cell exhaustion. We sought to develop a strategy that might license new efficacy for checkpoint blockade in GBM. EXPERIMENTAL DESIGN: We characterized 4-1BB expression in tumor-infiltrating lymphocytes (TIL) from human GBM. We implanted murine tumor models including glioma (CT2A), melanoma (B16), breast (E0771), and lung carcinomas intracranially and subcutaneously, characterized 4-1BB expression, and tested checkpoint blockade strategies in vivo. RESULTS: Our data reveal that 4-1BB is frequently present on nonexhausted CD8+ TILs in human and murine GBM. In murine gliomas, 4-1BB agonism and PD-1 blockade demonstrate a synergistic survival benefit in a CD8+ T-cell-dependent manner. The combination decreases TIL exhaustion and improves TIL functionality. This strategy proves most successful against intracranial CT2A gliomas. Efficacy in all instances correlates with the levels of 4-1BB expression on CD8+ TILs, rather than with histology or with intracranial versus subcutaneous tumor location. Proffering 4-1BB expression to T cells licenses combination 4-1BB agonism and PD-1 blockade in models where TIL 4-1BB levels had previously been low and the treatment ineffective. CONCLUSIONS: Although poor T-cell activation and severe T-cell exhaustion appear to be limiting factors for checkpoint blockade in GBM, 4-1BB agonism obviates these limitations and produces long-term survival when combined with anti-PD-1 therapy. Furthermore, this combination therapy is limited by TIL 4-1BB expression, but not by the intracranial compartment, and therefore may be particularly well-suited to GBM.