Characterization of a CC49-Based Single-Chain Fragment−β-Lactamase Fusion Protein for Antibody-Directed Enzyme Prodrug Therapy (ADEPT)

Abstract

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is overexpressed and exposed on the cell surface in a large fraction of solid malignancies. We constructed a single-chain fragment (scFv) based on CC49 and fused it to beta-lactamase (BLA). Following optimization of the scFv domain by combinatorial consensus mutagenesis (CCM) for increased expression and stability, we characterized the protein variant for binding, in vivo pharmacokinetics (PK), and antitumor efficacy. The fusion protein TAB2.5 possessed a similar binding specificity relative to the parent antibody CC49. TAB2.5 also showed prolonged retention (T(1/2) = 36.9 h) in tumor-bearing mice with tumor/plasma ratios of up to 1000. Preliminary evaluation of TAB2.5, in combination with a novel prodrug, GC-Mel, resulted in significant efficacy in a colorectal xenograft tumor model and supports the utility of the protein as an agent for tumor-selective prodrug activation.

Update: Recombinant Antibodies: From the Laboratory to the Clinic

“…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.…”

Novel β-lactamase-random peptide fusion libraries for phage display selection of cancer cell-targeting agents suitable for enzyme prodrug therapy

“…In this type of targeted therapy, the β-lactamase enzyme protein is chemically (Meyer et al, 1993; Svensson et al, 1995) or biologically (Rodrigues et al, 1995; McDonagh et al, 2003; Cortez-Retamozo et al, 2004; Alderson et al, 2006) conjugated to a cancer-specific ligand (usually an antibody) for delivering the enzyme to the tumor site. The presence of β-lactamase enzyme predominantly at the tumor site amplifies availability of the active drug concentrations locally following a cephalosporin prodrug infusion.…”

Harnessing the immune system to improve cancer therapy

“…Antibodies or antibody fragments can be conjugated via their Fc to radioisotopes (e.g., the anti-CD20 mAb 131 I-tositumomab), cytokines [e.g., the anti-GD2/interleukin (IL)-2 fusion protein EMD 273063] and toxins (e.g., gemtuzumab ozogamicin, a fusion of a cytotoxic antibiotic to a mAb targeting CD33 on leukemic myeloblasts) (10). In Ab-directed enzyme prodrug therapy (referred to as ADEPT), an enzyme linked to the mAb Fc converts a nontoxic prodrug, given systemically, into a potent cytotoxic agent (e.g., fusion of Fc to α-lactamase that converts C-Mel into melphalan) (11). All aforementioned approaches deposit the cytotoxic agent to the vicinity of the tumor, thus minimizing adverse events.…”