Abstract 1578: MTBhsp70 fusion protein targeting mesothelin augments tumor specific T cell responses and prolongs survival in a murine model of ovarian cancer

Abstract

Abstract Background and Purpose: There is a clear logic to adjuvant immunotherapy to enhance conventional treatment of ovarian cancer. Mesothelin (MSLN) is a surface glycoprotein expressed at high levels by ovarian cancers and is already a clinical target for immunotherapy. We have created a novel immunotherapeutic agent that links a single-chain antibody variable fragment (scFv) targeting MSLN to Mycobacterium tuberculosis (MTB) heat shock protein 70 (Hsp70), which is known as a potent immune activator. This fusion protein binds MSLN on tumor cells and activates antigen presenting cells (APCs) through the interaction of MTBhsp70 with CD40 on APCs, thus promoting tumor antigen presentation and cross presentation and adjuvanting antitumor specific CD4 and CD8 T cell responses. We tested the hypothesis that the MSLN targeted MTBhsp70 fusion protein would improve survival and increase antitumor immune responses. Experimental Procedures: The efficacy of the MSLN targeted fusion protein was evaluated in a syngeneic mouse model of papillary ovarian cancer in immune competent FVB/NJ mice. Mice received 4 intraperitoneal (ip) treatments with experimental or control proteins from 7 days post ip injection of BR5FVB ovarian cancer cells. Survival time was compared from the day of tumor inoculation to that of the onset of disease (clinically evident ascites). In immunological studies, mice were sacrificed 26 days after tumor cell inoculation. Splenocytes were stained for CD3, CD4, CD8, CD25 and Foxp3, and examined by flow cytometry. Splenocytes were stimulated with MSLN or Her2/neu peptide, or mitomycin C-treated BR5FVB cells. IFNα-generated CD3+CD8+ T cells and CD3+CD8+ T Cell-surface exposure of CD107a/b were detected by flow cytometry. To examine the role of CD8+ T cells in the antitumor effect, we performed in vivo antibody depletion experiments using standard methodologies. Results: MSLN targeted MTBhsp70 fusion protein significantly delayed the onset of disease compared to saline (p=0.0025) or an unfused mixture of the component proteins, MTBhsp70 and scFv(MSLN) (p=0.0149). The survival advantage of the fusion protein treatment was abrogated by CD8 T cell depletion in tumor bearing mice. The fusion protein also reduced the proportion of CD4+CD25+Foxp3 Treg cells (p=0.0159) and increased the proportion of CD3+CD8+ T cells (p=0.0079) in the spleen compared to controls. In preliminary studies, significantly greater CD8 T cell responses were demonstrated in the spleen from the majority of fusion protein-treated mice compared to controls. The MSLN targeted fusion protein was also shown to evoke dendritic cell activation as well as antigen presentation and cross presentation in vitro. Conclusion: MSLN targeted MTBhsp70 fusion protein treatment of ovarian cancer significantly enhances survival and slows tumor growth while augmenting anti-tumor antigen responses in mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1578. doi:1538-7445.AM2012-1578