Abstract
Cluster of differentiation 24 (CD24) is a specific surface marker involved in the tumorigenesis and progression of hepatocellular carcinoma (HCC). However, all reported anti-CD24 antibodies are murine ones with inevitable immunogenicity. To address this, a method using both molecular structure and docking-based complementarity determining region (CDR) grafting was employed for humanization. After xenogeneic CDR grafting into a human antibody, three types of canonical residues (in the VL/VH interface core, in the loop foundation, and interaction with loop residues) that support loop conformation and residues involved in the antigen-binding interface were back-mutated. Four engineered antibodies were produced, among which hG7-BM3 has virtually identical 3-D structure and affinity parameters with the parental chimeric antibody cG7. In vitro, hG7-BM3 demonstrated superior immunogenicity and serum stability to cG7. Antibody-dependent cellular cytotoxicity (ADCC), tumor cell internalization and in vivo targeting assays indicate that hG7-BM3 has the potential for development as an antibody-drug conjugate (ADC). We therefore generated the hG7-BM3-VcMMAE conjugate, which was shown to induce tumor cell apoptosis and effectively suppress nude mice bearing HCC xenografts. In conclusion, our study provides new inspiration for antibody humanization and an ADC candidate for laboratory study and clinical applications.
Highlights
According to global cancer statistics, hepatocellular carcinoma (HCC) is the second leading cause of cancer mortality in developing countries and the sixth leading cause among males in developed countries [1]
The amino acid sequences of G7mAb Fv were loaded into the Molecular Operating Environment (MOE), and framework regions (FR) or complementarity determining region (CDR) were identified by the Kabat numbering scheme
From the Ramachandran plot (Figure 1B), we determined that all residues were distributed in the allowed regions. (The green dots indicate the residues were in the core region, yellow indicates they were in the allowed region and red indicates they were in the outlier region.) Three types of canonical residues in FRs were identified by precise structural simulation of G7mAb Fv (Figure 1C)
Summary
According to global cancer statistics, hepatocellular carcinoma (HCC) is the second leading cause of cancer mortality in developing countries and the sixth leading cause among males in developed countries [1]. Global precision medicine strategies call for new biomarkers in targeted therapies. Few treatable molecular targets in HCC have been defined [2, 3]. It is a receptor that mediates antibody internalization [8, 9]. We developed an anti-CD24 antibody series that targeted HCC both in vitro and in vivo [11, 12]. The immunogenicity of these murine-based antibodies will be an obstacle in future clinical use, especially in oncotherapy, where large doses and repeated administration are necessary to achieve significant efficacy [13, 14]
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