Binding and Cytotoxicity of HPMA Copolymer Conjugates to Lymphocytes Mediated by Receptor-Binding Epitopes Aijun Tang, 1 Pavla Kopec ˇkova ´, 1,2 and Jindr ˇich Kopec ˇek 1,2,3 Received September 17, 2002; accepted December 4, 2002 Purpose. Studies on the recognition of epitopes presented on a tem- plate peptide showed the potential of nonapeptide-related sequences to act as biorecognition sites for the B-cell CD21 receptor. This study was intended to evaluate the capability of three epitope sequences to mediate specific cell binding and to enhance the cytotoxicity of HPMA copolymer conjugates. Methods. HPMA copolymer conjugates were synthesized containing three different epitopes at various contents and either a fluorescent marker or doxorubicin (DOX). The binding and cytotoxicity of the conjugates to CD21 + Raji B cells and CD21 - HSB-2 T cells were evaluated. Results. The epitope-containing conjugates were found to bind to Raji cells at different apparent affinities depending on epitope struc- ture and content. The conjugates generally possessed higher affinities for Raji cells than for HSB-2 cells. Targeted HPMA copolymer– DOX conjugates exhibited higher cytotoxicities than the nontargeted conjugate, likely indicative of enhanced internalization by receptor- mediated endocytosis. HSB-2 cells were more sensitive to both free and polymer-bound DOX than Raji cells; however, the enhancement of cytotoxicity of the conjugates by incorporation of epitopes was more pronounced for Raji cells. Conclusions. The results verified the concept of using receptor- binding epitopes as targeting moieties in HPMA copolymer conju- gates for the delivery of anticancer drugs to lymphoma cells. KEY WORDS: HPMA copolymer conjugates; epitope; CD21 recep- tor; binding affinity; cytotoxicity; lymphocytes. INTRODUCTION Leukemia, lymphoma, and myeloma are related cancers characterized by the uncontrolled growth of cells with similar functions and origins. These blood-related cancers accounted for about 8.6% of 1,268,000 new cancer cases diagnosed in the United States in 2001 and were responsible for nearly 11% of the total deaths from cancers (1). “Lymphoma” is a broad term encompassing a group of cancers that originate in the lymphatic system. Current treatments for these blood-related cancers include chemotherapy, radiation therapy, a combina- tion of chemo- and radiotherapy, immunotherapy, or, in spe- cific cases, high-dose chemotherapy followed by bone marrow or stem cell transplantation. Chemotherapy is largely respon- sible for the dramatic improvement in the treatment of these cancers. However, it is often accompanied by unwanted side effects. The development of new chemotherapy drugs has greatly increased the cure rates and remission period and has reduced the side effects of some malignancies; however, im- provement of the therapeutic index of existing anticancer drugs is highly desirable. One promising approach to improv- ing chemotherapy is to use drug delivery systems. The water-soluble polymer system N-(2-hydroxy- propyl)methacrylamide (HPMA) copolymers has been stud- ied for nearly two decades as a carrier for low-molecular- weight drugs to improve cancer chemotherapy (2,3). Covalent binding of anticancer drugs to HPMA copolymers can simul- taneously improve the solubility of the drugs and prolong their blood circulation time. More importantly, polymer- bound drugs are believed to have increased accumulation in solid tumors as a result of enhanced permeability and a re- tention (EPR) effect (4). In addition, HPMA copolymer- bound doxorubicin (DOX) has been shown to overcome P- glycoprotein-associated multidrug resistance (5) and to have greater efficacy than the free drug (6,7). Incorporation of targeting moieties in the conjugate system often results in an improvement in the therapeutic efficacy of polymer-bound drugs by facilitating binding of the conjugates to target cells and promoting conjugate internalization. A concomitant ben- efit with the use of targeting moieties is a lowered nonspecific toxicity of the drug. Examples of targeting moieties that have been used in HPMA copolymer conjugate system include car- bohydrates (8) and antibodies and antibody fragments (9,10). In addition to the well-established targeting strategies, a rather new approach is to use receptor-binding epitopes as the biorecognition sites in HPMA copolymer conjugates that mediate specific interactions of the conjugates with receptor- bearing cells. Advantageous properties of small epitopes for targeting moieties include the possibility of multivalent inter- actions if multiple epitope molecules are linked to each mac- romolecular chain, and probably easier transcompartmental transport of the epitope-containing conjugates because of their small size relative to the large antibody-containing con- jugates. The CD21 receptor is a 145-kDa integral membrane gly- coprotein found mainly on mature B cells (11) and on certain T cells (12). The CD21 or CD21-like receptor was shown to overexpress on a subset of cancerous cells relative to normal cells (13,14). This observation indicated that CD21 might be a useful target for the delivery of anticancer drugs to CD21 + malignant cells. Epstein–Barr virus (EBV) was found to bind to the CD21/EBV receptor on B cells through its main enve- lope glycoprotein gp350/220 (15). Specifically, a nonapeptide (NP) sequence (EDPGFFNVE) close to the N-terminus of gp350/220 seemed to play an important role (16). Previous work using NP-containing HPMA copolymer conjugates showed that the NP sequence induced specific binding of the conjugates to B and T cells and that the binding affinities seemed to increase with increasing peptide content in the conjugates (17). Further studies investigated the recognition of NP-related epitopes presented on coiled coil stem loop peptides by purified soluble CD21 receptor and CD21 + Raji cells (18). The results indicated that two other epitope sequences, peptides “D” (EDPGFFNVEIPEF) and “F” (EFGLDPGNFVEGF), might also act as biorecognition sites 1 Department of Pharmaceutics and Pharmaceutical Chemistry, Uni- versity of Utah, Salt Lake City, Utah 84112. 2 Department of Bioengineering, University of Utah, Salt Lake City, Utah 84112. 3 To whom correspondence should be addressed. (e-mail: Jindrich.Kopecek@m.cc.utah.edu) Pharmaceutical Research, Vol. 20, No. 3, March 2003 (© 2003) Research Paper 360 0724-8741/03/0300-0360/0 © 2003 Plenum Publishing Corporation