Abstract 3805: A novel anti-CD24 monoclonal antibody, humanized and affinity maturated for targeting gastrointestinal cancers
Authors: Shapira Shiran , Kazanov Dina , Padler Karavani Vered , Benhar Itai , Arber Nadir 
Published: July 2016

Abstract
Background: CD24 is a cell-surface heavily glycosylated GPI-anchored protein. We had previously shown that CD24 in an important player in the multistep process of GI carcinogenesis (Gastro 2006, Clin Can Res 2007, Can Res 2008). The creation of chimeric, humanized or fully human antibodies was a major breakthrough and led to a wave of US FDA-approved antibodies.
Aim: To further improve the efficacy of the humanized anti-CD24 mAb by increasing its binding strength and thereby generating a novel therapy tool for GI malignancy
Methods: From murine to humanized, unarmed and conjugated, small derivatives and full IgG antibodies were recombinantly engineered. The antibody genes were recovered, amplified and cloned into appropriate vectors. Then the vectors were introduced into a host (mammalian and E.coli) and adequate amounts of functional antibody were achieved. Sequence analysis of the CDR loops was the base for library designing. Affinity maturation was performed in two-steps selection (CDR walking) and by using phage display technique. The binding of the different derivatives were evaluated on full Glycan array in which more than 70 sugar moieties were printed.
Results: In vivo antibody targeting and accumulation within a CD24 positive tumor and its excess clearance was clearly demonstrated using live imaging device (Maestro Cri device). High-affinity antibodies were selected and created from combinatorial phage-displayed antibody libraries that contain varying degrees of diversity at randomized positions. A chosen matured clone was isolated and showed higher binding strength (1.8×10-8), compared to the parental murine and humanized Abs. The matured antibody showed selective recognition and binding to the CD24 antigen which proves that the genetic manipulations carried out did not affect its properties. Its stability was enhanced following the maturation process,as well as its pharmacokinetics parameters which showed a long serum half-life.The matured antibody mediates ADCC (antibody-dependent cell cytotoxicity), 75% of target cell lysis was demonstrated. Combined treatment with standard chemotherapy and natural products, such as monoterpenes (terpinen-4-ol), showed significant reduction in cell viability (90% cell death). Binding of anti-CD24 Ab to glycan microarray could not be detected while high binding intensities were observed where the whole CD24 protein was printed, indicating that the antibodies bind to the core peptide and not to its sugar residues.
Conclusion: Targeting CD24 may be a promising treatment for GI malignancies in combination with chemotherapy and natural agents. The resulted matured humanized anti-CD24 mAb proved to be more effective than the murine parental Ab. The long serum half-life is desirable as it would decrease the need for repetitive injections.
Citation Format: Shiran Shapira, Dina Kazanov, Vered Padler Karavani, Itai Benhar, Nadir Arber. A novel anti-CD24 monoclonal antibody, humanized and affinity maturated for targeting gastrointestinal cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3805.