ORIGINAL ARTICLE Gabriela Pavlinkova á David Colcher Barbara J. M. Booth á Apollina Goel á Surinder K. Batra Pharmacokinetics and biodistribution of a light-chain-shuf¯ed CC49 single-chain Fv antibody construct Received: 29 December 1999 / Accepted: 4 February 2000 Abstract Murine monoclonal antibodies to tumor-as- sociated glycoprotein 72 (anti-TAG-72 mAb B72.3 and CC49) are among the most extensively studied mAb for immunotherapy of adenocarcinomas. They have been used clinically to localize primary and metastatic tumor sites; however, murine mAb generally induce potent human anti-(mouse antibody) responses. The immuno- genicity of murine mAb can be minimized by genetic humanization of these antibodies, where non-human regions are replaced by the corresponding human sequences or complementary determining regions are grafted into the human framework regions. We have developed a humanized CC49 single-chain antibody construct (hu/muCC49 scFv) by replacing the murine CC49 variable light chain with the human subgroup IV germline variable light chain (Hum4 V L ). The major advantages of scFv molecules are their excellent pene- tration into the tumor tissue, rapid clearance rate, and much lower exposure to normal organs, especially bone marrow, than occur with intact antibody. The bio- chemical properties of hu/muCC49 scFv were compared to those of the murine CC49 scFv (muCC49 scFv). The association constants (K a ) for hu/muCC49 and muCC49 constructs were 1.1 ´ 10 6 M )1 and 1.4 ´ 10 6 M )1 re- spectively. Pharmacokinetic studies in mice showed similar rapid blood and whole-body clearance with a half-life of 6 min for both scFv. The biodistribution studies demonstrated equivalent tumor targeting to hu- man colon carcinoma xenografts for muCC49 and hu/ muCC49 scFv. These results indicate that the human variable light-chain subgroup IV can be used for the development of humanized or human immunoglobulin molecules potentially useful in both diagnostic and therapeutic applications with TAG-72-positive tumors. Key words Monoclonal antibodies á Single-chain antibodies á Antibody engineering á Recombinant antibody á Human colon cancer xenografts Introduction The clinical utilization of murine monoclonal antibodies (mAb) is limited by the induction of a human anti- mouse antibody (HAMA) response [29, 37]. The HAMA response may elicit allergic reactions in patients [3], and it can alter the rate of antitumor antibody clearance from serum [11, 32]. Various attempts have been made to produce human-derived monoclonal antibodies using human hybridomas [23]. Unfortunately, the yields of human mAb from such hybridomas are low and transient compared to antibody production by mouse hybridomas. Another approach to reduce the immuno- genicity of murine mAb is genetic humanization of these antibodies, in which non-human constant regions are replaced by corresponding human ones (chimerization) or/and complementary determining regions are grafted into human framework regions. However, these hu- manized antibodies retain various portions of the vari- able domains of light- and heavy-chain variable regions of non±human origin that can evoke an immunogenic reaction in patients [18]. Therefore, it is most desirable Cancer Immunol Immunother (2000) 49:267±275 Ó Springer-Verlag 2000 These studies were supported by a grant from the United States Department of Energy (DE-FG02-95ER62024) and were con- ducted in the J. Bruce Henrikson Cancer Research Laboratories G. Pavlinkova á B. J. M. Booth á A. Goel University of Nebraska Medical Center, Department of Pathology and Microbiology, 983135 Nebraska Medical Center, Omaha, NE 68198-3135, USA D. Colcher Coulter Pharmaceutical Inc., 600 Gateway Blvd., San Francisco, CA 94080, USA S. K. Batra (&) Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center; 984525 Nebraska Medical Center Omaha, NE 68198-4525, USA e-mail: sbatra@unmc.edu Tel.: +1-402-559-5455 Fax: +1-402-559-6650