OLIGONUCLEOTIDES 15:51–59 (2005) © Mary Ann Liebert, Inc. Serum Alters the Uptake and Biologic Activity of CpG Oligodeoxynucleotides in B Cell Chronic Lymphocytic Leukemia BERND JAHRSDÖRFER, 1 SUE E. BLACKWELL, 1 JAMES E. WOOLDRIDGE, 1,2 CHRISTIANA M. TAYLOR, 1 and GEORGE J. WEINER 1,2 ABSTRACT Immunostimulatory CpG-containing oligodeoxynucleotides (CpG ODN) have a number of effects on B cells, including upregulation of immunogenic molecules, and, therefore, appear attractive as po- tential components of immunotherapy for B cell chronic lymphocytic leukemia (B-CLL). Previous in vitro studies investigating the effect of CpG ODN on B-CLL cells used serum-low conditions and did not account for the longer-half life of CpG ODN in vitro. The present study was designed to explore how the presence of serum and exposure time affect CpG ODN-mediated changes on B-CLL cells. The optimal concentration for CpG ODN-mediated effects in the presence of 100% serum or plasma was higher (10–20 g/ml) than for serum-low conditions. Maximal CpG ODN-mediated effects re- quired the presence of ODN for no longer than 3 hours. The inhibition of CpG ODN-mediated effects by serum correlated with lower uptake of ODN into B-CLL cells in the presence of serum. A thresh- old effect on biologic response was observed, with a given amount of ODN internalized, resulting in phenotypic changes. In conclusion, systemic short-term application of CpG ODN appears to be suf- ficient to induce phenotypic changes, but higher doses of CpG ODN than previously thought may be necessary because of inhibition of their uptake by serum. INTRODUCTION A LTHOUGH SEVERAL GROUPS have found evidence for the existence of chronic lymphocytic leukemia (CLL)-specific T cells in patients with B cell CLL (B- CLL) (Goolsby et al., 2000; Krackhardt et al., 2002; Rez- vany et al., 2000), B cell malignancies, including B-CLL, are thought to have a relatively poor immunogenic ca- pacity (Donepudi et al., 2003; Van den Hove et al., 1997). In recent years, different strategies have been tested to upregulate costimulatory and antigen-presenting molecules in B cell malignancies in an effort to increase their immunogenicity. There is some evidence that cyto- toxic agents already used in clinical settings, such as mel- phalan, have a positive impact on the immunogenicity of malignant and benign B cells (Donepudi et al., 2003; So- jka et al., 2000). More direct attempts at enhancing the immunogenicity of B-CLL involve CD40 ligation, the application of cytokines, gene therapy to induce en- hanced expression of immunostimulatory molecules, and the use of immunostimulatory CpG-containing oligodeoxynucleotides (CpG ODN) (Anether et al., 2002; Chu et al., 2002; Decker et al., 2000a,b; Jahrsdör- fer et al., 2001, 2002; Tretter et al., 1998; Van den Hove et al., 1997; Wendtner et al., 2002). In animal models, we and others have shown that CpG ODNs are able to facilitate the establishment of immu- nologic memory against the tumor-specific antigen and induce regression of B cell lymphomas and other tumors (Heckelsmiller et al., 2002; Liu et al., 1998; Sandler et al., 2003; Weiner et al., 1997). These responses most likely involve several immune cell subsets, including 1 The Holden Comprehensive Cancer Center, 2 Department of Internal Medicine, University of Iowa, Iowa City, IA 52242. 51