Antibody engineering to generate anti-tumor-associated glycoprotein 72 mouse recombinant CC49 IgG with improved solubility, purity, and thermal stability

Abstract

Tumor-associated glycoprotein 72 (TAG-72) is a mucin that is overexpressed heterogeneously on the surface of cancer cells, and is a potential target for immunotherapies for various cancer types. As a tumor marker, TAG-72 is measured with the cancer antigen (CA) 72–4 immunoassay. The murine monoclonal antibody (mAb) CC49 is a second-generation IgG that targets an antigen on TAG-72; however, CC49 has an unfavorable propensity to aggregate, which results in antibody impurity, instability, and low solubility and thus low potency and efficacy for therapeutic and diagnostic applications. Sequence analysis of CC49 revealed aggregation-prone motifs in the variable domain of the light chain. Using antibody engineering approaches, we developed three aggregation-resistant CC49 mIgG2a mutants (CC49M1, CC49M2, and CC49M3). The engineered CC49 mIgG2a mutants retained compatible binding performance with a significantly higher thermal stability. The CC49 mIgG2a mutants also demonstrated an almost 15-fold improvement in solubility, with 97% purity vs 70% purity of the parent molecule at 0.3 mg/mL. The enhanced stability and improved solubility of engineered CC49 could have significant advantages for diagnostic and therapeutic applications.