The ability of a monoclonal antibody to bind to a target molecule with high affinity and specificity has led to their widespread use in cancer research and for therapeutic and diagnostic applications. Antibody-based biotherapeutics target specific cancer cell antigens to induce cell death and anti-tumor immune responses. Immunoassays utilize antibodies for specific and sensitive detection of cancer-related biomarkers for diagnosis and monitoring treatment response and disease recurrence. The utility of antibody-based biotherapeutics and diagnostics are limited by the propensity of antibodies to aggregate (Wang et al., 2009), which reduces their solubility and stability.

CA 72–4 was originally described as an antigenic determinant recognized by monoclonal antibody (mAb) B72.3; CA 72–4 was subsequently identified as a 1 MDa mucin-like, 48-kDa tumor-associated glycoprotein complex termed TAG-72 (Johnson et al., 1986). TAG-72 is overexpressed in cancers of the colon (Xu et al., 1989; Guadagni et al., 1993; Cho et al., 2019), pancreas (Pasquali et al., 1994), stomach (Filella et al., 1992; Guadagni et al., 1992), ovary (Ponnusamy et al., 2007), breast (Pizzi et al., 1999), and lung (Filella et al., 1992). TAG-72 has been investigated as a potential target for antibody-based immunotherapies and can be detected in serum as a marker of gastric and ovarian cancer by a newly developed CA 72–4 automated chemiluminescent immunoassay (Yanagihara et al., 2023).

TAG-72 was initially identified and characterized using two different monoclonal antibodies, CC49 and B72.3; CC49 is used in the CA 72–4 immunoassay, and reacts with a unique Sialyl-Tn antigen expressed on TAG-72 (Muraro et al., 1988). Previous studies have revealed aggregation-prone structural elements in the CC49 mAb that limit its utility as an immunotherapy or in immunoassays (Abergel et al., 1993). Here, we employed antibody engineering principles (Perchiacca and Tessier, 2012) to design CC49 antibodies with mutations to disrupt aggregation, and examined their stability, solubility, and purity compared to wild type CC49.