A humanized anti-DLL4 antibody promotes dysfunctional angiogenesis and inhibits breast tumor growth.

Xuelian Jia,Wenyi Wang,Min Wu,Tong Wang,Zhuobin Xu,Shijing Wang,Min Wang

doi:10.1038/srep27985

Xuelian Jia, Wenyi Wang + Show 5 more

Open Access

https://doi.org/10.1038/srep27985

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Abstract

Blockage of Delta-like 4 (DLL4)-directed Notch signaling induces excessive tip cell formation and endothelial proliferation resulting in dysfunctional angiogenesis in tumors. MMGZ01, as a murine anti-human DLL4 monoclonal antibody, specifically binds to human DLL4 and blocks Notch pathway. Here, the structure of MMGZ01 variable fragment (Fv) was established and framework region (FR) residues which supported complementarily determining region (CDR) loop conformation were identified. Important residues interactions were also identified through docking MMGZ01 Fv with antigen epitope in DLL4. To humanize the murine antibody, we modified MMGZ01 Fv through CDR grafting and the reconstructed antibody (H3L2) maintained similar structure and binding affinity to parental MMGZ01 after back mutation of 12 canonical murine residues in the FRs. Meanwhile, H3L2 promoted human umbilical vein endothelial cell (HUVEC) proliferation through inhibiting DLL4-directed Notch pathway. Moreover, in MDA-MB-231-bearing nude mice, H3L2 induced dysfunctional angiogenesis and tumor cell apoptosis and showed superior anti-tumor activity. In conclusion, H3L2 is an ideal humanized antibody that inhibits tumor growth through targeting DLL4-Notch pathway and has attracting potentials for clinical applications.

Highlights

Parental mouse antibodies, many CDR grafted antibodies present lower antigen binding affinities due to the lack of framework regions (FRs) canonical residues which supported CDR loop conformation[16]
The amino acid sequences of MMGZ01 VH, VL were loaded to Molecular Operating Environment (MOE) software, version 2013. 08, and FRs or CDRs in VH and VL were identified by Kabat numbering scheme[17]
Antibody structures in Protein Data Bank (PDB)[18] were searched and scored based on sequence similarity as well as structural fitness, in particular the backbone integrity, which was evaluated based on a structural scoring scheme developed by MOE

Summary

Introduction

Parental mouse antibodies, many CDR grafted antibodies present lower antigen binding affinities due to the lack of framework regions (FRs) canonical residues which supported CDR loop conformation[16]. We firstly chose suitable human V and J donors for CDR grafting based on a precisely modeled MMGZ01 variable fragment (Fv) structure. Through back mutation of 7 canonical residues in VH and 5 canonical residues in VL to the original murine one, the further designed version (H3L2) achieved comparable DLL4 binding affinity and specificity with the murine-human chimeric antibody (HCLC). Comprehensive in vivo and in vitro evaluation revealed that H3L2 was able to inhibit tumor growth and disturb tumor angiogenesis

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