Single amino acid substitution in the Fc region of chimeric TNT-3 antibody accelerates clearance and improves immunoscintigraphy of solid tumors.
Recent studies in antibody catabolism have identified residues at the CH2-CH3 interface of the IgG heavy chain critical for serum persistence of immunoglobulins. Amino acid substitutions in the Fc region of murine IgG1 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 CH2 domain of the human gamma1 constant region was made by polymerase 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 beta-phase half-life of 33.8 h, compared to 134.2 h for chTNT-3. Moreover, biodistribution 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.


CONCLUSIONS
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.
Hornick
J L
JL
Department of Pathology, University of Southern California School of Medicine, Los Angeles 90033, USA.
Sharifi
J
J
Khawli
L A
LA
Hu
P
P
Bai
W G
WG
Alauddin
M M
MM
Mizokami
M M
MM
Epstein
A L
AL
D000230:Adenocarcinoma / Q000000981:diagnostic imaging; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D003110:Colonic Neoplasms / Q000000981:diagnostic imaging; D005260:Female; D006801:Humans; D007457:Iodine Radioisotopes; D008297:Male; D051379:Mice; D008819:Mice, Nude; D009154:Mutation; D009368:Neoplasm Transplantation; D016719:Radioimmunodetection; D013997:Time Factors; D014018:Tissue Distribution; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D007457:Iodine Radioisotopes