241 TUMOR ANTIGENS CAN BE COVALENTLY CROSSLINKED TO ENHANCE ANTITUMOR IMMUNITY

Abstract

INTRODUCTION AND OBJECTIVES: Cancer vaccines are promising therapies for immunoresponsive solid tumors such as renal cell carcinoma (RCC) and melanoma. A simple and novel strategy is described for enhancing the immunostimulatory properties of cancer vaccines. Covalently crosslinking a tumor antigen in vitro produces a tumor vaccine that can generate antitumor immunity even when an adjuvant is not used. METHODS: Antitumor immunity was assessed using mouse models for RCC (RENCA) and melanoma (B16). CA9 was the target antigen for RENCA tumors implanted in Balb/c mice. Gp100 was the target antigen for B16 tumors implanted in C57/Bl mice. Cancer vaccines consisted of recombinant CA9 or gp100 covalently crosslinked (CL) by incubating with 3,3’dithiobis(sulfosuccinimidylpropionate), which was then dialyzed off. Dendritic cells (DCs) were freshly generated from mouse bone marrow. The specificity of immune stimulation was studied using T-cell receptor transgenic mouse models (OT-1 and Pmel-1). In these transgenic mice, nearly all the CD8 T cells recognize a single, well-characterized epitope. RESULTS: In a murine RCC model, vaccination with CA9 alone produced little immune-mediated antitumor response; however, CLCA9 was highly effective in inhibiting tumor growth. Crosslinking enhanced antitumor immunity even when CA9 was substituted by an immunodominant peptide of CA9. Similarly, in a murine melanoma model, CL-gp100 inhibited tumor growth, demonstrating that crosslinking enhances vaccines in multiple animal models. Crosslinking of tumor antigen enhanced the cellular immune response as measured using an ELISPOT assay or a Cr release cytotoxicity assay. Potential mechanisms were explored. CL-antigens had enhanced binding to fresh DCs when FITC-labeled antigens were monitored by fluorescence microscopy, flow cytometry or immunoblotting. Crosslinking did not enhance markers of DC activation such as MHC I, MHC II, CD80 and CD86; however, crosslinking preferentially enhanced antigen processing through the proteosomal pathway in DCs, which is expected to result in crosspresentation of antigens. Finally, DCs stimulated with CL-gp100 or CL-ovalbumin were capable of stimulating antigen-specific lymphocytes derived from Pmel-1 or OT-1 mice, respectively. CONCLUSIONS: Crosslinking of tumor antigens is a simple and effective strategy for enhancing tumor vaccines, which results in greater capacity to bind DCs and stimulate antigen processing. DCs stimulated with crosslinked antigens are highly effective in activating antigen-specific lymphocytes.