Viso, Carole

Abstract Although tumors from melanoma patients can often provide a source of therapeutically active T cells for adoptive therapy, it has historically proved challenging to employ peripheral blood as the T cell source for any type of cancer. We have observed nonetheless that HER2-specific T cells can be Ag-driven and enriched in culture when PBMCs from HER2-vaccinated breast cancer patients are treated with exogenous IL12. We hypothesized that stimulating innate immunity could further improve PBMC cultures by activating the DC subpopulation more effectively than adding exogenous rIL12. Bulk PBMC exposure to optimized TLR agonists produced vast quantities of IL12 and IL23, and upregulated HLA-DR, B7.1 and CD40 in the DC subpopulation, effects not produced by exogenous IL12. Subsequent IL7 exposure emulated homeostatic proliferation, selectively causing the Ag-driven T cell subset both to proliferate faster and to strongly resist apoptosis. Ag-specificity within two weeks approached 80-100% of both CD4+ and CD8+ T cells for recall Ags and 1-15% for a wide array of MUC1- and HER2-derived peptides, including the ability to distinguish glycoforms. Culture-expanded T cells retained a young CD28+/CD56- phenotype, uniformly expressed ROR-γ during culture, variably produced IL-2 and/or IL-17, and uniformly expressed T-bet and secreted IFN-γ when reexposed to Ag. Such T1/T17 bipotency may be ideal for anti-tumor adoptive therapy and highly efficient for use in Ag discovery.

Abstract The invasion of malignant glioma cells into the surrounding normal brain precludes effective clinical treatment. In this report, we investigated the role of the NH2-terminal FERM domain in the regulation of the promigratory function of Pyk2. We report that the substitution of residues that constitute a small cleft on the surface of the F3 module of the FERM domain do not significantly alter Pyk2 expression but result in the loss of Pyk2 phosphorylation. A monoclonal antibody, designated 12A10, specifically targeting the Pyk2 FERM domain was generated and recognizes an epitope located on the β5C-α1C surface of the F3 module of the FERM domain. Amino acid substitutions in the F3 module that resulted in the loss of Pyk2 phosphorylation also inhibited the binding of 12A10, suggesting that the 12A10 epitope overlaps a site that plays a role in Pyk2 activity. Conjugation of 12A10 to a membrane transport peptide led to intracellular accumulation and inhibition of glioma cell migration in a concentration-dependent manner. A single chain Fv fragment of 12A10 was stable when expressed in the intracellular environment, interacted directly with Pyk2, reduced Pyk2 phosphorylation, and inhibited glioma cell migration in vitro. Stable intracellular expression of the 12A10 scFv significantly extended survival in a glioma xenograft model. Together, these data substantiate a central role for the FERM domain in regulation of Pyk2 activity and identify the F3 module as a novel target to inhibit Pyk2 activity and inhibit glioma progression. [Mol Cancer Ther 2009;8(6):1505–14]