Development and preclinical characterization of a humanized antibody targeting CXCL12.

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 CXCL12α, CXCL12ß, and CXCL12γ, was shown to dose-dependently block CXCL12α binding to CXCR4 and CXCR7, and CXCL12α-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-α 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/CXCL12α complex in combination with mutational analysis revealed a "hot spot" around residues Asn(44)/Asn(45) of CXCL12α and part of the RFFESH region required for CXCL12α 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.

Quantification of cortical bone loss and repair for therapeutic evaluation in collagen-induced arthritis, by micro-computed tomography and automated image analysis.

OBJECTIVE: Ex vivo and in vivo micro-computed tomography (micro-CT) combined with a novel image analysis algorithm were used to quantify cortical bone loss and periosteal new bone formation for therapeutic evaluation in a murine model of collagen-induced arthritis. METHODS: An automated algorithm was created to locate 5 metatarsophalangeal and 3 metacarpophalangeal joints in 3-dimensional micro-CT images of mouse paws for evaluation of joint cortical bone volume (JCBV) within close proximity of the joints as well as cortical bone mineral density and periosteal new bone formation within the paws. For validation, automated estimates of JCBV were compared with radiographic visual scores (RVS) in 4 treatment groups (n = 9 per group): rat anti-mouse CD11a monoclonal antibody, methotrexate (MTX), anti-CD11a plus MTX, and saline only. In a separate study, serial images of hind limbs were evaluated in 2 treatment groups: murine tumor necrosis factor receptor II-Fc fusion protein (mTNFRII; n = 10) and control antibody (n = 7). RESULTS: Automated estimates of the JCBV were significantly correlated with the RVS (hind paws R = -0.94, front paws R = -0.81, combined R = -0.87). The anti-CD11a group had significantly higher JCBV compared with controls. In the serial study, the automated estimate of JCBV detected significant treatment effects in the mTNFRII-Fc group compared with controls. Cortical bone mineral density was significantly higher in all treatment groups compared with controls. CONCLUSION: Micro-CT combined with a novel image analysis technique (estimation of JCBV) provides a fully automated means to quantify bone destruction in a mouse model of rheumatoid arthritis.