Development of Anti-CEA CH2 Domain-Deleted Antibody (M5A∆CH2) for the PET Imaging of Colorectal Cancer

TL;DR

Researchers developed a modified antibody for PET imaging of colorectal cancer, showing it targets tumors quickly and clears from the blood fast.

Contribution

A novel CH2 domain-deleted anti-CEA antibody was developed for improved immunoPET imaging of colorectal cancer.

Findings

The M5A∆CH2-C5 antibody showed rapid and high tumor localization in mouse models.

The antibody demonstrated fast blood clearance, which is ideal for immunoPET imaging.

The M5A∆CH2 antibodies were stable and could be purified using a new CH3 domain affinity resin.

Abstract

Recombinant antibody fragments represent a novel class of in vivo biological immunoPET imaging agents. This study developed a series of anti-carcinoembryonic antigen (CEA) CH2 domain-deleted antibodies to evaluate their rapid, high-level tumor targeting combined with fast blood clearance for immunoPET imaging in two colorectal cancer mouse models.

A series of humanized anti-CEA M5A∆CH2 recombinant antibody fragments were synthesized via transient mammalian expression and purified using a two-step process. The M5A∆CH2 antibody series was characterized by HPLC-SEC, SDS-PAGE and binding affinities. The M5A∆CH2-C5 antibody, which has five disulfide bridges in the modified IgG1/IgG3 hinge domain, was selected for positron emission tomography (PET) imaging. Site-specific thiol conjugation of the reduced hinge disulfides with the 1,4,7,10 tetraazacyclododecane-1,4,7-triacetic acid trisodium salt-vinyl sulfone (DO3A-VS) chelate was performed, followed by labeling with [64Cu-CuCl2]. The [64Cu]Cu-DO3A-M5A∆CH2-C5 was evaluated for CEA-positive tumor PET imaging at serial time points, pharmacokinetics and a terminal biodistribution study conducted in two CEA-positive colorectal cancer mouse models.

The anti-CEA M5A∆CH2 antibodies had high expression, were purified using a new CH3 domain affinity resin and were stable up to one year. ImmunoPET imaging and biodistribution studies were performed in athymic mice bearing human colorectal cancer LS174T tumors and immunocompetent transgenic-CEA (Tg-CEA) mice bearing MC-38 tumors transfected with the human CEA gene. The [64Cu]Cu-DO3A-M5A∆CH2-C5 showed rapid, high tumor localization and the expected fast blood clearance. Conclusions: A series of humanized anti-CEA M5A∆CH2 antibodies were designed for immunoPET imaging of colorectal cancer, and the [64Cu]Cu-DO3A-M5A∆CH2-C5 showed high tumor targeting and fast blood clearance supporting its potential for clinical trials.

The online version contains supplementary material available at 10.1007/s11307-025-01997-3.

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