Construction and optimization of a CC49-Based scFv-β-lactamase fusion protein for ADEPT

Abstract

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is over-expressed and exposed on a large fraction of solid malignancies. We constructed a single chain fragment (scFv) based on CC49 and fused it to beta-lactamase. The first generation fusion protein, TAB2.4, was expressed at low levels in Escherichia coli and significant degradation was observed during production. We optimized the scFv domain of TAB2.4 by Combinatorial Consensus Mutagenesis (CCM). An improved variant TAB2.5 was identified that resulted in an almost 4-fold improved expression and 2.5 degrees higher thermostability relative to its parent molecule. Soluble TAB2.5 can be manufactured in low-density E.coli cultures at 120 mg/l. Our studies suggest that CCM is a rapid and efficient method to generate antibody fragments with improved stability and expression. The fusion protein TAB2.5 can be used for antibody directed enzyme prodrug therapy (ADEPT).

The Design and Characterization of Oligospecific Antibodies for Simultaneous Targeting of Multiple Disease Mediators

“…Generally, scFv have lower denaturation temperatures than the other antibody domains; their unfolding is characterized by a single transition event. [43][44][45] Of note, the scFv linked to the N-terminus of the light chain in Bs1Ab does not destabilize the overall stability of the antibody scaffold. In fact, the T m of 73°C for the denaturation of the Fab domain of Bs1Ab is not significantly different from the T m observed for the parental Fab, which is 75°C ( Fig.…”

The Cosmc connection to the Tn antigen in cancer

“…Human tumor-associated glycoprotein-72 (TAG-72) is a carcinoma mucin molecule expressed in colon [184,185], breast [186], pancreatic [187], ovarian [188], lung, and gastric cancers [189]. Epitopes of TAG-72 recognized by the mAbs B72.3 and CC49 are STn and Sialyl T antigens, respectively [190–192]. CC49 has been analyzed and a humanized antibody [193–195] is under clinical trial for use in radioimmunoguided surgery (RIGS).…”

Update: Recombinant Antibodies: From the Laboratory to the Clinic

“…Less immunogenic variants or other enzymes, preferably of human origin, are needed to fulfill the therapeutic potential of ADEPT. 45,73 Immunonanoparticles Targeting of synthetic carrier and delivery systems can be achieved by conjugating an antibody or antibody fragment to PEG moieties present on the surface of the vehicle for increased solubility and circulation-time adjustments. Nanoparticles are highly promising and versatile vectors.…”

“…127 Drug and toxin immunoconjugates with higher antitumor activity are being explored; for the BL22 anti-CD22 immunotoxin (RFB4(dsFv)-PE38), this was achieved by mutating an arginine, located in the toxin catalytic domain, to an alanine. 128 In the ADEPT approach for targeted therapy, that has shown promise in clinical trials, less immunogenic drug-cleaving enzymes are being assessed in vitro, 73,129 as well as targeting Ab format. 18 Immunonanoparticles were used for selective toxicity delivery to tumor cells.…”