Covalent disulfide-linked anti-CEA diabody allows site-specific conjugation and radiolabeling for tumor targeting applications

Abstract

An engineered anti-carcinoembryonic antigen (CEA) diabody (scFv dimer, 55 kDa) was previously constructed from the murine anti-CEA T84.66 antibody. Tumor targeting, imaging and biodistribution studies in nude mice bearing LS174T xenografts with radiolabeled anti-CEA diabody demonstrated rapid tumor uptake and fast blood clearance, which are favorable properties for an imaging agent. Current radiolabeling approaches result in random modification of the protein surface, which may impair immunoreactivity especially for smaller antibody fragments. Site-specific conjugation approaches can direct modifications to reactive groups located away from the binding site. Here, cysteine residues were introduced into the anti-CEA diabody at three different locations, to provide specific thiol groups for chemical modification. One version (with a C-terminal Gly-Gly-Cys) existed exclusively as a disulfide-bonded dimer. This cysteine-modified diabody (Cys-diabody) retained high binding to CEA and demonstrated tumor targeting and biodistribution properties identical to the non-covalent diabody. Furthermore, following reduction of the disulfide bond, the Cys-diabody could be chemically modified using a thiol-specific bifunctional chelating agent, for radiometal labeling. Thus, the Cys-diabody provides a covalently linked alternative to conventional diabodies, which can be reduced and modified site-specifically. This format will provide a versatile platform for targeting a variety of agents to CEA-positive tumors.

An engineered anti-CA19-9 cys-diabody for positron emission tomography imaging of pancreatic cancer and targeting of polymerized liposomal nanoparticles

“…To this end, we engineered, produced, and characterized the anti-CA19-9 cys-diabody. We show that our cys-diabody exhibits similar biochemical properties to other engineered cys-diabodies, displaying a molecular weight of 55 kDa and forming a covalent dimer [7,13]. In addition, we demonstrate specific binding of CA19-9 with the cys-diabody by flow cytometry and immunofluorescence with similar binding affinity compared with the parental anti-CA19-9 diabody.…”

“…Micro-PET was performed at 4 and 20 h after injection. These time points for imaging were chosen based on previously measured half-lives of diabody fragments and corresponded to approximately one half-life and five half-lives, respectively [7,20,21]. Also, to appropriately compare the in vivo targeting ability of the anti-CA19-9 cys-diabody to the anti-CA19-9 diabody, we performed the imaging experiments at the same time points as previously published for the anti-CA19-9 diabody [12].…”

“…The diabody format has been shown to exhibit superior binding affinity and avidity compared with the scFv, likely because of its bivalency for its antigen [6]. Moreover, a covalent dimer of two scFv fragments (cys-diabody) can be created by engineering C-terminal cysteine residues into the DNA construct of the scFv [7]. The cys-diabody has the ability to be site specifically conjugated with fluorophores or radioisotopes through the free sulfhydryl groups of the cysteine residues after reduction of the disulfide bond [7].…”

“…Moreover, a covalent dimer of two scFv fragments (cys-diabody) can be created by engineering C-terminal cysteine residues into the DNA construct of the scFv [7]. The cys-diabody has the ability to be site specifically conjugated with fluorophores or radioisotopes through the free sulfhydryl groups of the cysteine residues after reduction of the disulfide bond [7]. The work by Carmichael et al [8] elucidated the crystal structure of the anti-Carcinoembyonic antigen diabody and showed that the C-termini are at the opposite end of the protein's antigen-binding pocket, making it an appealing site to engineer the cysteine residues.…”

Positive Progress in ImmunoPET—Not Just a Coincidence

“…Cu-labeled anti-CEA minibody 24 and cysteine-modified diabody 25 have been evaluated preclinically, and the list has now been extended to include a 64 Cu-labeled intact antibody. In a study by Li et al, 26 a humanized anti-CEA intact antibody, hT84.66-M5A (M5A), was labeled with 64 Cu using the chelator 1,4,7-tris(carboxymethyl)-10-(vinylsulfone)-1,4,7,10-tetraazacy clododecane (DO3A-VS).…”