Antitumor efficacy of CD137 ligation is maximized by the use of a CD137 single-chain Fv–expressing whole-cell tumor vaccine compared with CD137-specific monoclonal antibody infusion Hongtao Zhang, 1 Keith L. Knutson, 1 Karl Erik Hellstrom, 2 Mary L. Disis, 2 and Ingegerd Hellstrom 1 1 Department of Pathology and 2 Tumor Vaccine Group, Center for Translational Medicine in Women’s Health, University of Washington, Seattle, Washington Abstract Tumor-destructive immune responses can be generated by engaging CD137 (4-1BB) via infusing a monoclonal antibody specific for CD137 or vaccinating with a single- chain Fv (scFv) CD137-expressing whole-cell tumor vaccine. We assessed whether such a vaccine can induce tumor rejection in the neu -transgenic (neu -Tg) mouse breast cancer model and compared the antitumor efficacy of vaccination with the infusion of a CD137-specific antibody. Mammary carcinoma cells (MMC) from a neu - Tg mouse were transfected to stably express surface scFv derived from the anti-CD137 rat hybridoma 1D8 or 3H3. The anti-CD137 scFv-expressing cells were rejected when transplanted into neu -Tg mice by a mechanism that involved both CD4 + and CD8 + T cells, and vaccination with such cells delayed the outgrowth of MMC cells transplanted 3 days previously. T cells from neu -Tg mice that had been vaccinated proliferated and produced IFN-; when stimulated by MMC but not by antigen-negative variant breast cancer cells that did not express the neu tumor antigen. In addition, antibodies binding to the MMC but not to antigen-negative variant cells were detected in sera from some but not all of the immunized mice. Complete regression of s.c. transplanted MMC tumors was observed in mice repeatedly immunized against MMC-1D8 starting on the day the MMC cells were transplanted. In contrast, repeated administration of either of two different anti-CD137 monoclonal antibodies did not induce complete tumor regression, although tumor growth was delayed. [Mol Cancer Ther 2006;5(1):149 – 55] Introduction Many tumor antigens, including antigens encoded by HER- 2/neu , are self-proteins and induce peripheral tolerance that prevents the generation of a tumor-destructive immune response. One approach to circumvent tolerance is to activate tumor-specific T cells via certain costimulatory receptors. Much attention has been given to CD137 (4-1BB), which was first detected on activated T lymphocytes (1, 2) and subsequently on natural killer cells (3), monocytes (4, 5), and dendritic cells (6). Engagement of CD137 can up-regulate T-cell responses, increase natural killer cell reactivity, and depress the formation of T-cell-dependent antibodies (2, 3, 7–9). Administration of anti-CD137 monoclonal antibodies (mAb) and vaccination with tumor cells transfected to express the CD137 ligand or with a combination of cDNAs encoding tumor antigen together with costimulatory signals by CD137 ligand and CD80 or CD86 can break tolerance and induce a tumor-destructive immune response in some mouse models (10 – 16). The antitumor response by injecting anti-CD137 mAb is often greater than that by vaccinating with tumor cells transfected to express CD137 ligand. For example, mice with established sarcoma Ag104 can be treated by admin- istration of anti-CD137 mAb (10) but not by vaccination with tumor cells expressing CD137 ligand (11, 15). To construct a vaccine that engages the immune system similar to an agonistic mAb, tumor cells can be transfected to stably express, at their surface, single-chain Fv fragments (scFv) from the given mAb (17, 18). Such a vaccine may be therapeutically more effective than a systemically given mAb by delivering tumor antigens together with signals that engage CD137 and may avoid the inhibitory effect of the mAb on endogenous antibody formation. Based on these premises, Ye et al. transfected cells from the M2 clone of the K1735 mouse melanoma to express anti- CD137 scFv and showed therapeutic efficacy against small M2 tumors growing s.c. or in the lung (19). Furthermore, cells from sarcoma Ag104 became immunogenic when transfected to express anti-CD137 scFv 3 but not when transfected to express CD137 ligand (11). Received 6/20/05; revised 10/31/05; accepted 11/15/05. Grant support: NIH Specialized Program of Research Excellence grant CA-98-008 and NIH RO1 grants CA79490, CA85780, and 112073 (I. Hellstrom and K.E. Hellstrom), grant K24 CA85218 (M.L. Disis), and grant K01-CA100764 (K.L. Knutson). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: M.L. Disis and I. Hellstrom contributed equally to this work. Requests for reprints: Ingegerd Hellstrom, Box 35 99 39 Harborview Medical Center, 325 Ninth Avenue, Seattle, WA 98104-2499. Phone: 206-341-4707; Fax: 206-726-0302. E-mail: ihellstr@u.washington.edu Copyright C 2006 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-05-0206 3 Yang et al., unpublished findings. 149 Mol Cancer Ther 2006;5(1). January 2006