*Article* **Improvement of Biophysical Properties and Affinity of a Human Anti-L1CAM Therapeutic Antibody through Antibody Engineering Based on Computational Methods**

**Heesu Chae 1,2, Seulki Cho 3, Munsik Jeong 1, Kiyoung Kwon 1, Dongwook Choi 4, Jaeyoung Lee 2, Woosuk Nam 2, Jisu Hong 1, Jiwoo Lee 1, Seonjoo Yoon 2,\* and Hyojeong Hong 1,3,\***


**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.

**Keywords:** therapeutic antibody; anti-cancer antibody; antibody engineering; biophysical properties; computational methods; research cell bank
