Effective immunoconjugate therapy in cancer models targeting a serine protease of tumor fibroblasts.

PURPOSE: Invasion and metastasis of malignant epithelial cells into normal tissues is accompanied by adaptive changes in the mesenchyme-derived supporting stroma of the target organs. Altered gene expression in these nontransformed stromal cells provides potential targets for therapy. The present study was undertaken to determine the antitumor effects of an antibody-conjugate against fibroblast activation protein-alpha, a cell surface protease of activated tumor fibroblasts. EXPERIMENTAL DESIGN: A novel antibody-maytansinoid conjugate, monoclonal antibody (mAb) FAP5-DM1, was developed to target a shared epitope of human, mouse, and cynomolgus monkey fibroblast activation protein-alpha, enabling preclinical efficacy and tolerability assessments. We have used stroma-rich models in immunodeficient mice, which recapitulate the histotypic arrangement found in human epithelial cancers. RESULTS: Treatment with mAb FAP5-DM1 induced long-lasting inhibition of tumor growth and complete regressions in xenograft models of lung, pancreas, and head and neck cancers with no signs of intolerability. Analysis of chemically distinct conjugates, resistance models, and biomarkers implicates a unique mode of action, with mitotic arrest and apoptosis of malignant epithelial cells coupled to disruption of fibroblastic and vascular structures. CONCLUSIONS: We show that mAb FAP5-DM1 combines excellent efficacy and tolerability and provides a first assessment of the mode of action of a novel drug candidate for tumor stroma targeting, thus encouraging further development toward clinical testing of this treatment paradigm.

In vivo plasmid electroporation induces tumor antigen-specific CD8+ T-cell responses and delays tumor growth in a syngeneic mouse melanoma model.

Plasmid DNA-based molecular cancer vaccines generally suffer from suboptimal immunogenicity. One of the key limitations is insufficient level of gene expression, which was surmounted in our approach by using the novel technique of in vivo plasmid electroporation-enhanced vaccination (electrovaccination). Electrovaccination with plasmids encoding the full-length autologous melanocyte antigen tyrosinase-related protein-2 induced limited melanocyte destruction in a subset of mice. Despite examples of vitiligo, vaccinated mice were not protected from a subsequent challenge of B16F10M melanoma cells. Novel constructs were then designed and submitted to a functional screen. Best performance was obtained when the relevant H-2K(b)-restricted epitope SVYDFFVWL was placed into a context of sequences of the HLA-Cw3 molecule. After animals were electrovaccinated using this construct, direct enzyme-linked immunospot analysis of peripheral blood mononuclear cells indicated that very high numbers of T cells recognizing the specific tyrosinase-related protein-2 epitope were generated. CD8+ T cells isolated from the spleen also displayed a high degree of antigen-specific reactivity and vigorously reacted toward unmodified B16F10M cells. In vivo protective effects of this construct were demonstrated in mice using two different models; outgrowth of s.c. implanted B16F10M tumor cells was significantly delayed, and vaccinated mice developed no or only very few tumor nodules in an i.v. lung metastasis model. Thus, improved antigen vectors delivered by highly effective gene transfer methods may form the basis for future human applications.

Structural elements of a protein antigen determine immunogenicity of the embedded MHC class I-restricted T cell epitope.

Substantial effort has been invested into optimization of vector structure, DNA formulation, or delivery methods to increase the effectiveness of DNA vaccines. In contrast, it has been only insufficiently explored how the higher order structure of an antigenic protein influences immunogenicity of embedded epitopes in vivo. Potent CD8+ T cell responses specific for a single immunogenic epitope are induced upon electrovaccination with plasmid DNA encoding the full-length heavy chain of the human HLA-Cw3 molecule. Contrary to expectations, a minimal construct, which provoked a substantial release of IFN-gamma from specific CTLs in vitro, did not induce a significant response in vivo. Systematically altered variants of the Cw3 molecule were thus tested both in vivo and in vitro to determine which structural parts are responsible for this discrepancy. In complementation experiments the participation of trans-acting helper epitopes was ruled out. Successive C-terminal truncations, human/mouse domain swap variants, and subdomain modifications defined the alpha3 region of the HLA heavy chain and membrane anchoring as critical elements. Based on these data, refined minimal constructs were engineered that triggered very high in vivo responses. The most advanced variant consisted only of an adenoviral leader, antigenic epitope, alpha3 domain, and 16 aa of the transmembrane domain. When a tumor Ag epitope was incorporated into one of these high performer minimal constructs, protection against melanoma metastases was attained upon vaccination. Thus, structural elements of the Ag can dominantly influence immunogenicity in vivo. These elements can also markedly improve the immunogenicity of unrelated Ags and may form the basis of a new generation of DNA vaccines.