124I-Labeled Engineered Anti-CEA Minibodies and Diabodies Allow High-Contrast, Antigen-Specific Small-Animal PET Imaging of Xenografts in Athymic Mice

¹²⁴I-Labeled Engineered Anti-CEA Minibodies and Diabodies Allow High-Contrast, Antigen-Specific Small-Animal PET Imaging of Xenografts in Athymic Mice

Gobalakrishnan Sundaresan, PhD¹, Paul J. Yazaki, PhD², John E. Shively, PhD²^,3, Ronald D. Finn, PhD⁴, Steven M. Larson, MD⁴, Andrew A. Raubitschek, MD²^,3, Lawrence E. Williams, PhD², Arion F. Chatziioannou, PhD¹, Sanjiv S. Gambhir, MD, PhD¹^,5^,6 and Anna M. Wu, PhD¹^,2^,3^,5

¹ Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, California
² Beckman Research Institute, City of Hope National Medical Center, Duarte, California
³ City of Hope Comprehensive Cancer Center, Duarte, California
⁴ Radiopharmaceutical Chemistry Service and Nuclear Medicine Service, Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, New York
⁵ UCLA Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, Los Angeles, California
⁶ Department of Radiology and Bio-X Program, Stanford University, Stanford, California

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FIGURE 1. Schematic drawing shows domain structure of parental intact T84.66 antibody (A), T84.66 diabody (B), and T84.66 minibody (C). Anti-CEA minibody (80 kDa) and diabody (55 kDa) were derived from T84.66, a high-affinity and highly specific antibody that recognizes an epitope on A3 domain of CEA. Minibody (C) has glycine-serine-rich 18-amino-acid linker (GS18) between variable light (V_L) and variable heavy chains (V_H) and human IgG1 hinge (H) joining them to CH3 domain, also derived from human IgG1. Gene encoding diabody (B) encodes glycine-serine-rich 8-amino-acid linker (GS8) between V_L and V_H regions. Amino acid sequence is GSTSGGGSGGGSGGGGSS for GS18 and GGGSGGGG for GS8.

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FIGURE 2. Size-exclusion HPLC analysis of ¹²⁴I-radiolabeled anti-CEA minibody (A) and diabody (B). ¹²⁴I was conjugated to T84.66 minibody and T84.66 diabody as described in Materials and Methods. Radiolabeling efficiency was determined by integrating areas on HPLC trace and determining radioactivity associated with 80-kDa protein peak for minibody or 55-kDa peak for diabody as percentage of total radioactivity eluted. Labeling efficiencies were 33% (not shown) or 46% for minibody (A) and 88% for diabody (B). Peak fractions (based on protein absorbance) were pooled for animal studies. Smaller peaks represent unincorporated label and low-molecular-weight components.

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FIGURE 3. (A) Subcutaneous LS174T (left shoulder) and C6 glioma (right shoulder) xenografts were established in nude mouse. (B) PET imaging with ¹⁸F-FDG was done 2 d before injection of ¹²⁴I (10/64 planes shown). (C) Mouse was injected with 3.1 MBq (85 µCi) ¹²⁴I-minibody and imaged at 18 h by PET (10/64 planes shown). Both these images (B and C) are on same color scale. (D) After 18-h scan, mouse was euthanized and frozen, and whole-body coronal sections were cut in cryostat and processed for DWBA. (E) DWBA confirms specific localization of ¹²⁴I minibody to CEA-positive tumor and low levels of background activity.

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FIGURE 4. Comparison of ¹²⁴I minibody and ¹²⁴I diabody by serial PET imaging at 4 and 18 h. Mice bearing LS174T (LS) and C6 rat glioma (C6) xenografts were injected via tail vein with 1.9–3.1 MBq (65–85 µCi) ¹²⁴I minibody (A and B) or diabody (C–E) and imaged at 4 and 18 h. At 18 h, background activity in central region was minimal in ¹²⁴I diabody (D and E)–injected animal, resulting in high contrast, which can be seen in these maximum a posteriori reconstructed images (10/64 planes shown). A–D are on common scale. D was rescaled to E to illustrate excellent contrast achieved with ¹²⁴I diabody at 18 h.