Immunoprotective Activities of Multiple Chaperone Proteins Isolated from Murine B-Cell Leukemia/Lymphoma 1 Michael Graner, Amy Raymond, Davis Romney, Lin He, Luke Whitesell, and Emmanuel Katsanis 2 Department of Pediatrics, Steele Memorial Children’s Research Center, University of Arizona, Tucson, Arizona 85724-5073 ABSTRACT Although the use of tumor-derived heat shock/chaper- one proteins (HSPs) as anticancer vaccines is gaining wider study and acceptance, there have thus far been no reports concerning chaperone antitumor activities against dissemi- nated hematological malignancies. We have devised an effi- cient and effective method for purification of the chaperone proteins grp94/gp96, HSP90, HSP70, and calreticulin from harvested A20 murine leukemia/lymphoma tumor material. We have demonstrated that these purified proteins, when used as vaccines, can induce potent and specific immunity against a lethal tumor challenge. Individual chaperone pro- teins were differentially effective in their abilities to provide immune protection. The increase in survival generated by the most effective chaperone vaccine, HSP70, resulted from at least a 2-log reduction in tumor burden. Syngeneic gran- ulocyte macrophage colony-stimulating factor producing fi- broblasts were injected at the site of vaccination in an attempt to augment the immune response. Surprisingly, lo- calized granulocyte macrophage colony-stimulating factor production inhibited the protective effects of chaperone vac- cination. These studies provide evidence that chaperone pro- teins can be isolated from B-cell tumors and used effectively to immunize against disseminated lymphoid malignancies. INTRODUCTION The activity of purified tumor-derived chaperone proteins as adjuvants in cancer immunotherapy has become increasingly well-documented (1–5). It is clear from these reports that im- munization of mice with chaperones purified from a given tumor can induce specific immunoprotection against a subse- quent challenge with the same tumor, whereas chaperones pu- rified from normal murine tissues provide no such protection. The data indicate that the chaperones are not immunogenic per se, but rather serve as carriers of tumor-derived peptides (6 – 8). The molecular and cellular mechanisms behind this immuno- protection are not clear, but it appears that chaperone-peptide complexes are taken up by host professional APCs 3 and are directed toward the endogenous (MHC class I) presentation pathway (3, 9, 10). Presentation of the (formerly chaperoned) peptides to T cells may lead to the generation of tumor-specific cytotoxic T cells. A variety of tumor types growing as s.c. masses have been reported to be responsive to tumor-derived chaperone vaccina- tion (1). However, none of these studies have addressed the effectiveness of chaperone vaccines in generating antitumor immunity against disseminated hematological malignancies such as leukemias or lymphomas. This report describes our attempts to elicit antitumor immune responses via chaperones purified from the spontaneously derived murine A20 B-cell leukemia/lymphoma (11). When A20 cells are injected i.v. into BALB/c mice, they induce a disseminated disease characterized by infiltration of lymph nodes, liver, and spleen and the pres- ence of malignant cells in bone marrow and peripheral blood. This leukemia is radioresistant, with even myeloablative doses of irradiation followed by syngeneic bone marrow transplanta- tion failing to cure A20 bearing mice (data not shown; Ref. 12). A20 is poorly immunogenic; vaccination with irradiated wild- type tumor cells confers little protection to subsequent tumor challenge. Systemic T-cell immunity against A20 may be gen- erated, however, following immunization with genetically mod- ified tumor cells engineered to secrete GM-CSF and other cytokines (13). Herein we report a method for sequential purification of the major immunologically active chaperone proteins HSP70, HSP90, grp94/gp96, and calreticulin from a single A20 tumor sample and demonstrate the relative efficacy of these protein preparations in generating specific antitumor responses in this aggressive murine leukemia model. Syngeneic GM-CSF-secret- ing fibroblasts were injected at the immunization site in an attempt to stimulate antigen uptake and presentation by profes- sional APCs. Interestingly, production of GM-CSF at the vac- cination site by transduced 3T3 fibroblasts actually abrogated the protective effect. MATERIALS AND METHODS Mice. Six-to-10-week-old female BALB/c (H-2 d ) mice (Jackson Laboratories, Bar Harbor, ME) were used for the experiments. The animals were housed in a specific pathogen- Received 6/16/99; revised 12/7/99; accepted 12/8/99. 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. 1 Supported in part by the American Cancer Society Grant IM-785, the Arizona Disease Control Research Commission, the W. M. Keck Foun- dation, the Enid and Mel Zuckerman Fund, the Arizona Elks Program in Transplantation Research, and the Michael Landon Fund. 2 To whom requests for reprints should be addressed, at University of Arizona, Department of Pediatrics, 1501 North Campbell Avenue, P. O. Box 245073, Tucson, AZ 85724-5073. Phone: (520) 626-6527; Fax: (520) 626-4220; E-mail: katsanis@peds.arizona.edu. 3 The abbreviations used are: APC, antigen-presenting cell; GM-CSF, granulocyte macrophage colony-stimulating factor; ConA, concanavalin A; HSP, heat shock/chaperone protein; ns, not significant. 909 Vol. 6, 909 –915, March 2000 Clinical Cancer Research