Abstract
The biophysical properties of therapeutic antibodies influence their manufacturability, efficacy, and safety. To develop an anti-cancer antibody, we previously generated a human monoclonal antibody (Ab417) that specifically binds to L1 cell adhesion molecule with a high affinity, and we validated its anti-tumor activity and mechanism of action in human cholangiocarcinoma xenograft models. In the present study, we aimed to improve the biophysical properties of Ab417. We designed 20 variants of Ab417 with reduced aggregation propensity, less potential post-translational modification (PTM) motifs, and the lowest predicted immunogenicity using computational methods. Next, we constructed these variants to analyze their expression levels and antigen-binding activities. One variant (Ab612)—which contains six substitutions for reduced surface hydrophobicity, removal of PTM, and change to the germline residue—exhibited an increased expression level and antigen-binding activity compared to Ab417. In further studies, compared to Ab417, Ab612 showed improved biophysical properties, including reduced aggregation propensity, increased stability, higher purification yield, lower pI, higher affinity, and greater in vivo anti-tumor efficacy. Additionally, we generated a highly productive and stable research cell bank (RCB) and scaled up the production process to 50 L, yielding 6.6 g/L of Ab612. The RCB will be used for preclinical development of Ab612.
Highlights
Therapeutic monoclonal antibodies are the leading class of drugs on the biopharmaceutical market, largely due to their high specificity, affinity, potency, and their long in vivo half-life
Taken gether with the analyses of expression and purification processing, these results demonstrate that compared to Ab417, Ab612 showed improved biophysical properties, including increased productivity,reduced reducedaggregation aggregation propensity fragmentation, and inincreased productivity, propensity and and fragmentation, and increased creased thermal stability
These results indicated that Ab612 exhibited enhanced anti-tumor efficacy compared to Ab417 in the cholangiocarcinoma model
Summary
Therapeutic monoclonal antibodies (mAb) are the leading class of drugs on the biopharmaceutical market, largely due to their high specificity, affinity, potency, and their long in vivo half-life. Since approval of the first mAb (OKT3) in 1983, 79 therapeutic antibodies have been approved by FDA and are currently on the market, including 30 mAbs for cancer treatment [1]. Therapeutic antibody discovery and development starts with early candidates (hits), followed by selection of advanced candidates (leads). During the manufacturing process, including cell culture and downstream processing, therapeutic antibodies are at risk of physical and chemical degradation through multiple pathways [13]. Degradation may affect antigen binding, decrease antibody efficacy, or even lead to immunogenic products [14,15,16]. Protein aggregation is the most common and substantial type of physical degradation associated with therapeutic antibodies [13,17,18]
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