Development and Preclinical Characterization of a Humanized Antibody Targeting CXCL12

Abstract

Purpose: Our goal was to develop a potent humanized antibody against mouse/human CXCL12. This report summarized its in vitro and in vivo activities.Experimental Design: Cell surface binding and cell migration assays were used to select neutralizing hamster antibodies, followed by testing in several animal models. Monoclonal antibody (mAb) 30D8 was selected for humanization based on its in vitro and in vivo activities.Results: 30D8, a hamster antibody against mouse and human CXCL12a, CXCL12b, and CXCL12g, was shown to dose-dependently block CXCL12a binding to CXCR4 and CXCR7, and CXCL12a-induced Jurkat cell migration in vitro. Inhibition of primary tumor growth and/or metastasis was observed in several models. 30D8 alone significantly ameliorated arthritis in a mouse collagen-induced arthritis model (CIA). Combination with a TNF-a antagonist was additive. In addition, 30D8 inhibited 50% of laser-induced choroidal neovascularization (CNV) in mice. Humanized 30D8 (hu30D8) showed similar in vitro and in vivo activities as the parental hamster antibody. A crystal structure of the hu30D8 Fab/CXCL12a complex in combination with mutational analysis revealed a "hot spot" around residues Asn 44 /Asn 45 of CXCL12a and part of the RFFESH region required for CXCL12a binding to CXCR4 and CXCR7. Finally, hu30D8 exhibited fast clearance in cynomolgus monkeys but not in rats. Conclusion: CXCL12 is an attractive target for treatment of cancer and inflammation-related diseases; hu30D8 is suitable for testing this hypothesis in humans.

The Intricate Role of CXCR4 in Cancer

“…For instance, CVX15 binding induces structural reconfiguration in the base of the receptor N-terminus as well as the extracellular regions of the helices. The binding interaction of CXCL12 to CXCR4 takes place with the RFFESH loop of CXCL12α interacting with the N-terminus of CXCR4, while the N-terminus of CXCL12 simultaneously interacts with the binding pocket comprising the transmembrane helices and the extracellular loops (Zhong et al, 2013). While this study has shed more light on CXCR4–ligand interactions, the observed variability in binding modes has further complicated the rationale design and predictability of essential structural backbones necessary for production of high-affinity CXCR4 binding ligands.…”

What Do Structures Tell Us About Chemokine Receptor Function and Antagonism?

“…Mogamulizumab, a monoclonal antibody targeting CCR4, recently became the first biologic to be approved for cutaneous T-cell lymphoma (33). In a complementary effort, antibodies (7, 148) and therapeutic nucleotides (35, 53, 95) are pursued as agents targeting chemokines. With all of these agents, oral availability is out of question; however, various approaches to improving metabolic stability have been successful (18, 95).…”

CXCL12 modulation of CXCR4 and CXCR7 activity in human glioblastoma stem-like cells and regulation of the tumor microenvironment

“…Several CK-directed (CCL2, CCL5, and CXCL10) antibodies have been generated and tested in phase I/II clinical trials (Klarenbeek et al, 2012) in both cancer and inflammatory diseases. 30D8, a humanized antibody against mouse/human CXCL12, inhibits tumor growth and/or metastasis and improve arthritis in experimental in vitro and in vivo models (Zhong et al, 2013). However, the majority of antibodies that successfully entered clinical trials, targets CK receptors rather than ligands.…”