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Single Amino Acid Substitution in the Fc Region of Chimeric TNT-3 Antibody Accelerates Clearance and Improves Immunoscintigraphy of Solid Tumors

Departments of Pathology and Radiology, University of Southern California School of Medicine, Los Angeles, California

Correspondence: For correspondence or reprints contact: Alan L. Epstein, MD, PhD, University of Southern California School of Medicine, Department of Pathology, 2011 Zonal Ave., HMR 210, Los Angeles, CA 90033.

ABSTRACT

Recent studies in antibody catabolism have identified residues at the C_H2-C_H3 interface of the IgG heavy chain critical for serum persistence of immunoglobulins. Amino acid substitutions in the Fc region of murine lgG₁ were shown to drastically accelerate antibody clearance in mice. Our laboratory has previously described a human—mouse chimeric TNT-3 (chTNT-3) monoclonal antibody directed against a universal nuclear antigen that has potential for the radioimmunotherapy of many solid tumors. In the current study, we engineered a chTNT-3 mutant containing a single amino acid substitution, to determine whether a more rapid clearance profile would make the antibody suitable for diagnostic imaging. Methods: A single amino acid substitution in the C_H2 domain of the human 1 constant region was made by potymerase chain reaction mutagenesis. High-level expression was achieved using the Glutamine Synthetase Gene Amplification System, and the chTNT-3 mutant was purified by protein A affinity and ion-exchange chromatography. A radioimmunoassay was performed to examine antigen binding, and in vivo studies were undertaken to evaluate clearance and tumor targeting in human tumor xenograft models. Results: The chTNT-3 mutant retained the high affinity of chTNT-3, with a binding constant of 1.5 x 10^-9 mol/L The mutant was eliminated rapidly from BALB/c mice, with a -phase half-life of 33.8 h, compared to 134.2 h for chTNT-3. Moreover, btodistribution studies in human colon tumor-bearing nude mice reflected this accelerated clearance. Tumor levels of the mutant were, respectively, 65%, 39%, and 36% of the tumor levels achieved with the parental chTNT-3 6, 12, and 24 h postinjection. The rapid clearance of the chTNT-3 mutant from the blood resulted in higher tumor-to-normal organ ratios for many normal tissues. Imaging of tumor-bearing mice with ^99mTc-labeled chTNT-3 mutant demonstrated early visualization of tumors in 3 different solid tumor xenograft models. Conclusion: The accelerated clearance produced by a single amino acid substitution in the Fc region of chTNT-3 leads to improved imaging in tumor-bearing mice. These studies suggest that a rapidly clearing antibody generated by this approach may be useful for the immunoscintigraphy of human tumors.

Key Words: chimeric monoclonal antibody • pharmacokinetics • immunoscintigraphy • tumor targeting • FcRn

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