[Show abstract][Hide abstract] ABSTRACT: The effector functions of therapeutic antibodies are strongly affected by the specific glycans added to the Fc domain during post-translational processing. Antibodies bearing high levels of N-linked mannose-5 glycan (Man5) have been reported to exhibit enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared with antibodies with fucosylated complex or hybrid glycans. To better understand the relationship between antibodies with high levels of Man5 and their biological activity in vivo, we developed an approach to generate substantially homogeneous antibodies bearing the Man5 glycoform. A mannosidase inhibitor, kifunensine, was first incorporated in the cell culture process to generate antibodies with a distribution of high mannose glycoforms. Antibodies were then purified and treated with a mannosidase for trimming to Man5 in vitro. This 2-step approach can consistently generate antibodies with > 99% Man5 glycan. Antibodies bearing varying levels of Man5 were studied to compare ADCC and Fcγ receptor binding, and they showed enhanced ADCC activity and increased binding affinity to the FcγRIIIA. In addition, the clearance rate of antibodies bearing Man8/9 and Man5 glycans was determined in a pharmacokinetics study in mice. When compared with historical data, the antibodies bearing the high mannose glycoform exhibited faster clearance rate compared with antibodies bearing the fucosylated complex glycoform, while the pharmacokinetic properties of antibodies with Man8/9 and Man5 glycoforms appeared similar. In addition, we identified the presence of a mannosidase in mouse serum that converted most Man8/9 to Man6 after 24 h.
[Show abstract][Hide abstract] ABSTRACT: MEHD7945A is a novel dual-action monoclonal antibody in which each of the two antigen-binding fragments is capable of binding to EGFR and HER3 with high affinity. It is being evaluated as a potential therapy for human cancer. The purpose of these studies was to characterize the pharmacokinetics (PK) of MEHD7945A in mouse and monkey and predict its human PK and efficacious dose.
PK of MEHD7945A was determined in SCID beige mice and cynomolgus monkeys after administration of single intravenous doses. Human PK profiles were projected from monkey PK profiles using a species-invariant time method, and human population PK parameters were estimated using a nonlinear, two-compartment model comprising specific (target-mediated) and nonspecific clearance pathways. The antitumor efficacy in mice bearing human tumor xenografts was used in conjunction with human PK projections to estimate human efficacious doses.
The total clearance of MEHD7945A decreased with increase in dose in both mouse and monkey. The nonspecific clearance in monkey was estimated to be 14 mL/day/kg. The predicted nonspecific clearance range in humans was 6-10 mL/day/kg. Doses of 8-12 mg/kg administered every 2 weeks in humans were predicted to achieve exposure of 300 day μg/mL per week to match the efficacious exposure observed in xenograft models.
The PK of MEHD7945A was nonlinear in mouse and monkey in the dose range tested. The nonspecific clearance in monkey was approximately twofold higher than typical humanized IgG1 antibodies. The projected human efficacious dose and dose regimen appear to be achievable in patients.
No preview · Article · Dec 2011 · Cancer Chemotherapy and Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Upper hinge is vulnerable to radical attacks that result in breakage of the heavy-light chain linkage and cleavage of the
hinge of an IgG1. To further explore mechanisms responsible for the radical induced hinge degradation, nine mutants were designed
to determine the roles that the upper hinge Asp and His play in the radical reactions. The observation that none of these
substitutions could inhibit the breakage of the heavy-light chain linkage suggests that the breakage may result from electron
transfer from Cys231 directly to the heavy-light chain linkage upon radical attacks, and implies a pathway separate from His229-mediated hinge cleavage. On the other hand, the substitution of His229 with Tyr showed promising advantages over the native antibody and other substitutions in improving the stability and function
of the IgG1. This substitution inhibited the hinge cleavage by 98% and suggests that the redox active nature of Tyr did not
enable it to replicate the ability of His to facilitate radical induced degradation. We propose that the lower redox potential
of Tyr, a residue that may be the ultimate sink for oxidizing equivalents in proteins, is responsible for the inhibition.
More importantly, the substitution increased the antibody's binding to FcγRIII receptors by 2–3-fold, and improved ADCC activity
by 2-fold, while maintaining a similar pharmacokinetic profile with respect to the wild type. Implications of these observations
for antibody engineering and development are discussed.
Full-text · Article · Dec 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Clinical use of recombinant fibroblast growth factor 21 (FGF21) for the treatment of type 2 diabetes and other disorders linked to obesity has been proposed; however, its clinical development has been challenging owing to its poor pharmacokinetics. Here, we describe an alternative antidiabetic strategy using agonistic anti-FGFR1 (FGF receptor 1) antibodies (R1MAbs) that mimic the metabolic effects of FGF21. A single injection of R1MAb into obese diabetic mice induced acute and sustained amelioration of hyperglycemia, along with marked improvement in hyperinsulinemia, hyperlipidemia, and hepatosteatosis. R1MAb activated the mitogen-activated protein kinase pathway in adipose tissues, but not in liver, and neither FGF21 nor R1MAb improved glucose clearance in lipoatrophic mice, which suggests that adipose tissues played a central role in the observed metabolic effects. In brown adipose tissues, both FGF21 and R1MAb induced phosphorylation of CREB (cyclic adenosine 5'-monophosphate response element-binding protein), and mRNA expression of PGC-1α (peroxisome proliferator-activated receptor-γ coactivator 1α) and the downstream genes associated with oxidative metabolism. Collectively, we propose FGFR1 in adipose tissues as a major functional receptor for FGF21, as an upstream regulator of PGC-1α, and as a compelling target for antibody-based therapy for type 2 diabetes and other obesity-associated disorders.
No preview · Article · Dec 2011 · Science translational medicine
[Show abstract][Hide abstract] ABSTRACT: Extensive crosstalk among ErbB/HER receptors suggests that blocking signaling from more than one family member may be essential to effectively treat cancer and limit drug resistance. We generated a conventional IgG molecule MEHD7945A with dual HER3/EGFR specificity by phage display engineering and used structural and mutational studies to understand how a single antigen recognition surface binds two epitopes with high affinity. As a human IgG1, MEHD7945A exhibited dual action by inhibiting EGFR- and HER3-mediated signaling in vitro and in vivo and the ability to engage immune effector functions. Compared with monospecific anti-HER antibodies, MEHD7945A was more broadly efficacious in multiple tumor models, showing that combined inhibition of EGFR and HER3 with a single antibody is beneficial.
[Show abstract][Hide abstract] ABSTRACT: Extensive crosstalk among ErbB/HER receptors suggests that blocking signaling from more than one family member may be essential to effectively treat cancer and limit drug resistance. We generated a conventional IgG molecule MEHD7945A with dual HER3/EGFR specificity by phage display engineering and used struc-tural and mutational studies to understand how a single antigen recognition surface binds two epitopes with high affinity. As a human IgG1, MEHD7945A exhibited dual action by inhibiting EGFR-and HER3-medi-ated signaling in vitro and in vivo and the ability to engage immune effector functions. Compared with mono-specific anti-HER antibodies, MEHD7945A was more broadly efficacious in multiple tumor models, showing that combined inhibition of EGFR and HER3 with a single antibody is beneficial. INTRODUCTION
[Show abstract][Hide abstract] ABSTRACT: Off-target binding can significantly affect the pharmacokinetics (PK), tissue distribution, efficacy and toxicity of a therapeutic antibody. Herein we describe the development of a humanized anti- fibroblast growth factor receptor 4 (FGFR4) antibody as a potential therapeutic for hepatocellular carcinoma (HCC). A chimeric anti FGFR4 monoclonal antibody (chLD1) was previously shown to block ligand binding and to inhibit FGFR4 mediated signaling as well as tumor growth in vivo. A humanized version of chLD1, hLD1.vB, had similar binding affinity and in vitro blocking activity, but it exhibited rapid clearance, poor target tissue biodistribution and limited efficacy when compared to chLD1 in a HUH7 human HCC xenograft mouse model. These problems were traced to instability of the molecule in rodent serum. Size exclusion high performance liquid chromatography, immunoprecipitation and mass spectral sequencing identified a specific interaction between hLD1.vB and mouse complement component 3 (C3). A PK study in C3 knock-out mice further confirmed this specific interaction. Subsequently, an affinity-matured variant derived from hLD1.vB (hLD1.v22), specifically selected for its lack of binding to mouse C3 was demonstrated to have a PK profile and in vivo efficacy similar to that of chLD1 in mice. Although reports of non-specific off-target binding have been observed for other antibodies, this represents the first report identifying a specific off-target interaction that affected disposition and biological activity. Screens developed to identify general non-specific interactions are likely to miss the rare and highly specific cross-reactivity identified in this study, thus highlighting the importance of animal models as a proxy for avoiding unexpected clinical outcomes.
[Show abstract][Hide abstract] ABSTRACT: Fibroblast growth factor 19 (FGF19) is a hormone-like protein that regulates carbohydrate, lipid and bile acid metabolism. At supra-physiological doses, FGF19 also increases hepatocyte proliferation and induces hepatocellular carcinogenesis in mice. Much of FGF19 activity is attributed to the activation of the liver enriched FGF Receptor 4 (FGFR4), although FGF19 can activate other FGFRs in vitro in the presence of the coreceptor βKlotho (KLB). In this report, we investigate the role of FGFR4 in mediating FGF19 activity by using Fgfr4 deficient mice as well as a variant of FGF19 protein (FGF19v) which is specifically impaired in activating FGFR4. Our results demonstrate that FGFR4 activation mediates the induction of hepatocyte proliferation and the suppression of bile acid biosynthesis by FGF19, but is not essential for FGF19 to improve glucose and lipid metabolism in high fat diet fed mice as well as in leptin-deficient ob/ob mice. Thus, FGF19 acts through multiple receptor pathways to elicit pleiotropic effects in regulating nutrient metabolism and cell proliferation.