To understand why cancer vaccine-induced T cells often fail to eradicate tumors, we studied immune responses in mice vaccinated with gp100 melanoma peptide in incomplete Freund’s adjuvant (IFA), commonly used in clinical cancer vaccine trials. Peptide/IFA vaccination primed tumor-specific CD8+ T cells, which accumulated not in tumors but at the persisting, antigen-rich vaccination site. Once there, primed T cells became dysfunctional and underwent antigen-driven, Interferon-γ (IFN-γ) and Fas ligand (FasL)-mediated apoptosis, resulting in hyporesponsiveness to subsequent vaccination. Provision of anti-CD40 antibody, Toll-like receptor 7 (TLR7) agonist and interleukin-2 (IL-2) reduced T cell apoptosis but did not prevent vaccination site sequestration. A non-persisting vaccine formulation shifted T cell localization towards tumors, inducing superior anti-tumor activity while reducing systemic T cell dysfunction and promoting memory formation. Persisting peptide/IFA vaccine depots can induce specific T cell sequestration, dysfunction and deletion at vaccination sites; short-lived formulations may overcome these limitations and result in greater therapeutic efficacy of peptide-based cancer vaccines.

Cancer immune evasion is an emerging hallmark of disease progression. Functional studies to understand the role of myeloid-derived suppressor cells (MDSC) in the tumor microenvironment however, are limited by the lack of available specific cell surface markers. We adapted a competitive peptide phage display platform to identify candidate peptides binding MDSC specifically and generated peptide-Fc fusion proteins (peptibody). In multiple tumor models peptibody injection iv completely depleted blood, splenic, and intratumoral MDSC in tumor-bearing mice, without affecting proinflammatory immune cell types, such as dendritic cells. While control Gr-1 antibody depleted primarily granulocytic MDSC, peptibodies depleted both granulocytic and monocytic subsets. Remarkably, peptibody treatment was associated with inhibition of tumor growth in vivo, which was superior to Gr-1. Immunoprecipitation of MDSC membrane proteins identified S100 family proteins as candidate targets. Our strategy may be useful to identify novel diagnostic and therapeutic surface targets on rare cell subtypes, including human MDSC.

Purpose: Aberrant expression of inflammatory molecules, such as inducible nitric oxide (NO) synthase (iNOS), has been linked to cancer, suggesting that their inhibition is a rational therapeutic approach. Whereas iNOS expression in melanoma and other cancers is associated with poor clinical prognosis, in vitro and in vivo studies suggest that iNOS and NO can have both protumor and antitumor effects. We tested the hypothesis that targeted iNOS inhibition would interfere with human melanoma growth and survival in vivo in a preclinical model.Experimental Design: We used an immunodeficient non-obese diabetic/severe combined immunodeficient xenograft model to test the susceptibility of two different human melanoma lines to the orallygiven iNOS-selective small molecule antagonistand without cytotoxic cisplatin chemotherapy.Results: L-nil significantly inhibited melanoma growth and extended the survival of tumor-bearing mice. L-nil treatment decreased the density of CD31+ microvessels and increased the number of apoptotic cells in tumor xenografts. Proteomic analysis of melanoma xenografts with reverse-phase protein array identified alterations in the expression of multiple cell signaling and survival genes after L-nil treatment. The canonical antiapoptotic protein Bcl-2 was downregulated in vivo and in vitro after L-nil treatment, which was associated with increased susceptibility to cisplatin-mediated tumor death. Consistent with this observation, combination therapy with L-nil plus cisplatin in vivo was more effective than either drug alone, without increased toxicity.Conclusions: These data support the hypothesis that iNOS and iNOS-derived NO support tumor growth in vivo and provide convincing preclinical validation of targeted iNOS inhibition as therapy for solid tumors. Clin Cancer Res; 16(6); 1834-44. ©2010 AACR.Upregulation of proinflammatory molecules by tumor cells is a poorly understood phenomenon, which can play a role in both the induction and maintenance of certain human cancers (1). One such molecule, inducible nitric oxide (NO) synthase (iNOS) is constitutively overexpressed in many cancers, including melanoma and gastric (2), breast (3), colon (4), and head and neck (5, 6) carcinomas. iNOS and its product NO have wide-ranging and varied effects on cellular physiology, signal transduction, and cell survival. At high levels, such as produced by activated macrophages during inflammatory responses to pathogens, NO alone or in combination with reactive oxygen species can have a direct cytotoxic effect on pathogens or tumor cells (7). At lower levels, NO can affect signal transduction pathways by interacting with metal ligands of proteins (8) or covalently modifying proteins through nitration and nitrosylation (9, 10). These protein Authors' Affiliations: