[CANCER RESEARCH 60, 6964 – 6971, December 15, 2000] Genetically Engineered Tetravalent Single-Chain Fv of the Pancarcinoma Monoclonal Antibody CC49: Improved Biodistribution and Potential for Therapeutic Application 1 Apollina Goel, David Colcher, Janina Baranowska-Kortylewicz, Sam Augustine, Barbara J. M. Booth, Gabriela Pavlinkova, and Surinder K. Batra 2 Departments of Biochemistry and Molecular Biology [A. G., S. K. B.], Radiation Oncology [J. B-K.], and Pathology and Microbiology [S. A., B. J. M. B., G. P., S. K. B.], College of Pharmacy [S. A.], Eppley Institute of Research in Cancer and Allied Diseases [S. K. B.], University of Nebraska Medical Center, Omaha, Nebraska 68198; and Coulter Pharmaceutical Incorporated, San Francisco, California 94080 [D. C.] ABSTRACT Failure of radiolabeled monoclonal antibodies (MAbs) in the treatment of solid tumors, for the most part, is a result of undesirable pharmacoki- netics that lead to significant radiation exposure of normal tissues and an inadequate delivery of radiation doses to tumors. Using genetic engineer- ing, antitumor MAbs can be optimized for desirable clinical applications. In the present study, we report the generation of a tetravalent single-chain Fv {[sc(Fv) 2 ] 2 } of the murine MAb CC49 that recognizes the tumor- associated glycoprotein, TAG-72. [Sc(Fv) 2 ] 2 was expressed as a secreted soluble protein in Pichia pastoris under the regulation of alcohol oxidase 1 promoter. The in vitro binding properties of the tetravalent construct were analyzed by solid-phase RIA and surface plasmon resonance studies using BIAcore. The binding affinity constant (K A ) for the [sc(Fv) 2 ] 2 and CC49 IgG were similar, i.e., 1.02  10 8 M 1 and 1.14  10 8 M 1 , respectively, and were 4-fold higher than its divalent scFv [sc(Fv) 2 ; 2.75  10 7 M 1 ]. At 6 h postadministration, the percentage of injected dose accumulated/g of LS-174T colon carcinoma xenografts was 21.3  1.3, 9.8  1.3, and 17.3  1.1 for radioiodinated [sc(Fv) 2 ] 2 , sc(Fv) 2 , and IgG, respectively. Pharmacokinetic analysis of blood clearance studies showed the elimina- tion half-life for [sc(Fv) 2 ] 2 , sc(Fv) 2 , and IgG as 170, 80, and 330 min, respectively. The gain in avidity resulting from multivalency along with an improved biological half-life makes the tetravalent construct an important reagent for cancer therapy and diagnosis in MAb-based radiopharmaceu- ticals. INTRODUCTION MAbs 3 used to be referred to as magic bullets because of their ability to target malignant cells for delivery of cytocidal agents like radionuclides, enzymes, genes, drugs, and toxins (1, 2). Currently, some MAbs are in clinical trials for both diagnostic and therapeutic purposes (3– 6). The major limitation of using intact MAbs for cancer radioimmunotherapy is because of the long persistence in circulation, thereby exhibiting side effects that are significantly deleterious to the normal tissues with only limited quantities delivered to tumors (7, 8). Furthermore, intact MAbs show poor diffusion from the vasculature into and through the tumor (9). Immunoglobulins have been engineered to retain only the domains that are required for antigen binding and/or effector functions and have also been rebuilt into multivalent, high-affinity reagents (10, 11). In scFvs, the variable regions of heavy and light chains are joined covalently by either a polypeptide linker or a disulfide bond (12, 13). As monomers (M r 30,000), these reagents are ideal for diagnostic applications because of their excellent tumor penetration, high RI (ratio of the %ID/g in the tumor:%ID/g in normal tissue), and low backgrounds (14 –18). However, the absolute amounts of scFv uptake by the tumors remain low, mainly because of their monovalent nature and fast elimination (14, 15, 17–20). The lower tumor:normal tissue ratio makes monovalent scFvs inefficient for radioimmunotherapeutic applications (8, 18, 21). Many of the therapeutically important tumor-associated antigens are either glycolipids or glycoproteins with highly repetitive structures (22). Antibodies and antibody fragments with multiple valencies therefore represent an enormous gain in the functional affinity attrib- utable to multiple interactions within a single antigen-antibody com- plex (10). Indeed, various divalent scFvs revealed improved antigen affinity in vitro when compared with the monovalent forms. Also, in animal models, divalent scFvs exhibited a significant improvement in tumor targeting over monovalent species like scFv and Fab because of their higher avidity and slower clearance properties rendered by their larger size (17, 18, 23–25). To improve further the in vitro and, ultimately, the in vivo performance of scFvs, the valency of scFv has been increased by designing trivalent and tetravalent scFvs (26 –35). However, the utility of scFv multimers in vivo for tumor targeting has not been adequately investigated. There are only a few studies where biodistribution studies were performed with trivalent antigen-binding constructs F(ab') 3 generated by chemical linkages (36 –38). In the present study, we report for the first time genetic engineering and in vivo evaluation of a tetravalent scFv construct of MAb CC49. The tetravalent scFv was formed by a noncovalent association of the covalent dimer sc(Fv) 2 . [Sc(Fv) 2 ] 2 with four potentially active anti- gen-binding sites showed improved in vitro binding properties as compared with sc(Fv) 2 and CC49 IgG. [sc(Fv) 2 ] 2 exhibited 2-fold increase in the absolute tumor uptake (from 4 h after injection on- wards). The larger molecular size of [sc(Fv) 2 ] 2 , which exceeds the renal threshold for the first pass elimination, translated into an im- proved biological half-life of [sc(Fv) 2 ] 2 . We believe that the tetrava- lent scFv, with higher avidity and prolonged pharmacokinetics in blood, meets the prerequisites of an optimum tumor-targeting reagent in radionuclide-mediated therapy and diagnosis. MATERIALS AND METHODS Vector Construction. The divalent CC49 scFv gene (V L -linker-V H -linker- V L -linker-V H ) was constructed using the linker designated as 205C (39) and was cloned into the Pichia pastoris expression vector, pPICZA (Invitrogen, Carlsbad, CA) as described earlier (25, 40). Briefly, competent P. pastoris KM71 cells (his4arg4aox1::ARG4) were transformed with 1–3 g of plas- mid DNA linearized with SacI and selected on yeast extract peptone dextrose plates with 100 g/ml Zeocin. The clones were screened for the secreted recombinant protein by solid-phase competition ELISA using biotinylated CC49 IgG. Received 4/12/00; accepted 10/17/00. 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 by a Grant from the United States Department of Energy (DE-FG02- 95ER62024). 2 To whom requests for reprints should be addressed, at Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 984525 Nebraska Med- ical Center, Omaha, NE 68198-4525. Phone: (402) 559-5455; Fax: (402) 559-6650; E-mail: sbatra@unmc.edu. 3 The abbreviations used are: MAbs, monoclonal antibodies; scFv, single chain Fv; sc(Fv) 2 , covalent dimeric scFv; [sc(Fv) 2 ] 2 , noncovalent tetrameric scFv; HPLC, high performance liquid chromatography; SPR, surface plasmon resonance; RI, radiolocaliza- tion index; %ID/g, % of injected dose/g; BSM, bovine submaxillary gland mucin; V L , variable light; V H , variable heavy. 6964 Research. on November 6, 2015. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from