124I-labeled engineered anti-CEA minibodies and diabodies allow high-contrast, antigen-specific small-animal PET imaging of xenografts in athymic mice

Abstract

Prolonged clearance kinetics have hampered the development of intact antibodies as imaging agents, despite their ability to effectively deliver radionuclides to tumor targets in vivo. Genetically engineered antibody fragments display rapid, high-level tumor uptake coupled with rapid clearance from the circulation in the athymic mouse/LS174T xenograft model. The anticarcinoembryonic antigen (CEA) T84.66 minibody (single-chain Fv fragment [scFv]-C(H)3 dimer, 80 kDa) and T84.66 diabody (noncovalent dimer of scFv, 55 kDa) exhibit pharmacokinetics favorable for radioimmunoimaging. The present work evaluated the minibody or diabody labeled with (124)I, for imaging tumor-bearing mice using a high-resolution small-animal PET system.

Methods: Labeling was conducted with 0.2-0.3 mg of protein and 65-98 MBq (1.7-2.6 mCi) of (124)I using an iodination reagent. Radiolabeling efficiencies ranged from 33% to 88%, and immunoreactivity was 42% (diabody) or >90% (minibody). In vivo distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA-positive) and C6 rat glioma (CEA-negative) xenografts. Mice were injected via the tail vein with 1.9-3.1 MBq (53-85 microCi) of (124)I-minibody or with 3.1 MBq (85 microCi) of (124)I-diabody and imaged at 4 and 18 h by PET. Some mice were also imaged using (18)F-FDG 2 d before imaging with (124)I-minibody.

Results: PET images using (124)I-labeled minibody or diabody showed specific localization to the CEA-positive xenografts and relatively low activity elsewhere in the mice, particularly by 18 h. Target-to-background ratios for the LS174T tumors versus soft tissues using (124)I-minibody were 3.05 at 4 h and 11.03 at 18 h. Similar values were obtained for the (124)I-diabody (3.95 at 4 h and 10.93 at 18 h). These results were confirmed by direct counting of tissues after the final imaging. Marked reduction of normal tissue activity, especially in the abdominal region, resulted in high-contrast images at 18 h for the (124)I-anti-CEA diabody. CEA-positive tumors as small as 11 mg (<3 mm in diameter) could be imaged, and (124)I-anti-CEA minibodies, compared with (18)F-FDG, demonstrated highly specific localization.

Conclusion: (124)I labeling of engineered antibody fragments provides a promising new class of tumor-specific probes for PET imaging of tumors and metastases.

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.

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.

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.

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.