High-resolution microPET imaging of carcinoembryonic antigen-positive xenografts by using a copper-64-labeled engineered antibody fragment

Abstract

Rapid imaging by antitumor antibodies has been limited by the prolonged targeting kinetics and clearance of labeled whole antibodies. Genetically engineered fragments with rapid access and high retention in tumor tissue combined with rapid blood clearance are suitable for labeling with short-lived radionuclides, including positron-emitting isotopes for positron-emission tomography (PET). An engineered fragment was developed from the high-affinity anticarcinoembryonic antigen (CEA) monoclonal antibody T84.66. This single-chain variable fragment (Fv)-C(H)3, or minibody, was produced as a bivalent 80 kDa dimer. The macrocyclic chelating agent 1,4,7, 10-tetraazacyclododecane-N,N',N", N"'-tetraacetic acid (DOTA) was conjugated to the anti-CEA minibody for labeling with copper-64, a positron-emitting radionuclide (t(1/2) = 12.7 h). In vivo distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA positive) and C6 rat glioma (CEA negative) xenografts. Five hours after injection with (64)Cu-DOTA-minibody, microPET imaging showed high uptake in CEA-positive tumor (17.9% injected dose per gram +/- 3.79) compared with control tumor (6.0% injected dose per gram +/- 1.0). In addition, significant uptake was seen in liver, with low uptake in other tissues. Average target/background ratios relative to neighboring tissue were 3-4:1. Engineered antibody fragments labeled with positron-emitting isotopes such as copper-64 provide a new class of agents for PET imaging of tumors.

Figure 1

Schematic overview of anti-CEA minibody. A gene encoding the minibody is assembled in the order V_L-linker-V_H-hinge-C_H3, with the hinge and C_H3 domains derived from human IgG1. The protein self assembles into 80-kDa dimers.

Figure 2

Size-exclusion HPLC analysis of ⁶⁴Cu-radiolabeled anti-CEA minibody. (A) Analysis and purification of radiolabeled minibody; peak fractions were pooled for animal studies. (B) Upper trace, starting sample used for evaluation of immunoreactivity. Lower trace, after incubation with CEA the bulk of the ⁶⁴Cu-minibody is found in antibody/antigen complexes.

Figure 3

(A) MicroPET scan of mouse administered 200 μCi ¹⁸F-FDG and scanned 1 h after injection. The mouse carried a C6 glioma xenograft on the left shoulder and an LS174T xenograft on the right shoulder (arrows). (B) MicroPET scan of the same mouse injected with 26 μCi ⁶⁴Cu-anti-CEA minibody and imaged at 5 h with the highest retention in the LS174T tumor (arrow) and liver and lower retention in the control tumor (arrowhead). (C) Mouse was killed for whole-body autoradiography, and an anatomic photograph was taken at the time of sectioning. (D) Digital autoradiograph of adjacent section.

Figure 4

Serial microPET scans of a mouse bearing bilateral C6 (arrowhead) and LS174T (arrow) xenografts, injected with ⁶⁴Cu minibody and imaged at 2 h (A), 6 h (B), and 24 h (C). After the final scan, tumors were excised and subjected to immunohistochemical staining by using anti-CEA cT84.66 as the primary antibody. Photomicrographs of C6 (D) and LS174T (E) xenografts.