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.

L1 cell adhesion molecule (L1CAM, CD171) is a transmembrane glycoprotein important for nervous system development. Conversely, L1CAM is overexpressed in many human solid tumors with poor prognosis. Nonclinical safety evaluation and pharmacokinetics (PK) of Ab612, a human anti-L1CAM monoclonal antibody (mAb), were conducted to support a first-in-human (FIH) clinical trial for the treatment of L1CAM overexpressing tumors. A tissue cross-reactivity (TCR) assessment showed that Ab612 exhibited staining in various tissues from mouse, rat, canine, cynomolgus monkey, and human, including those associated with peripheral nerves, myenteric plexus, spinal nerve roots, and certain cell types, such as mononuclear cells (lymphocytes), cardiac Purkinje fibers, and epithelial cell in the lung and kidney. These findings were consistent with the reported expression of the L1CAM by these cell types. Dose range-finding (DRF) studies were conducted in rats and cynomolgus monkeys at doses up to 500 and 400 mg/kg, respectively, by intravenous (IV) administration once weekly for 2 weeks. Results showed no treatment-related adverse findings, and the maximum tolerated dose (MTD) was the highest dose tested. Four-week repeat-dose toxicity studies were conducted in rats and cynomolgus monkeys at doses up to MTD, by IV administration once weekly for 4 weeks with a 28-day recovery period. Results showed no treatment-related adverse findings, and the no-observed-adverse-effect-level (NOAEL) was the highest dose tested. Single dose PK studies in rats and cynomolgus monkeys with doses of 5–100 and 5–50 mg/kg, respectively, confirmed the PK in the expected range for mAbs. These preclinical data indicate no safety concerns and provide adequate safety margins for the planned dose levels in the FIH trial.