Derrick R. Witcher

Monoclonal Antibody Clearance IMPACT OF MODULATING THE INTERACTION OF IgG WITH THE NEONATAL Fc RECEPTOR

The neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. There are mixed reports on whether modification of the interaction with FcRn can be used as an engineering strategy to improve the pharmacokinetic and pharmacodynamic properties of monoclonal antibodies. We tested whether the T250Q/M428L mutations, which improved the pharmacokinetics of humanized IgGs in the rhesus monkey, would translate to a pharmacokinetic benefit in both cynomolgus monkeys and mice when constructed on a different humanized IgG framework (anti-tumor necrosis factor-α (TNFα)). The T250Q/M428L anti-TNFα variant displayed an ∼40-fold increase in binding affinity to cynomolgus monkey FcRn (C-FcRn) at pH 6.0, with maintenance of the pH binding dependence. We also constructed another anti-TNFα variant (P257I/Q311I) whose binding kinetics with the C-FcRn was similar to that of the T250Q/M428L variant. The binding affinity of the T250Q/M428L variant for murine FcRn was increased ∼500-fold, with maintenance of pH dependence. In contrast to the interaction with C-FcRn, this interaction was driven mainly by a decrease in the rate of dissociation. Despite the improved in vitro binding properties of the anti-TNFα T250Q/M428L and P257I/Q311I variants to C-FcRn, the pharmacokinetic profiles of these molecules were not differentiated from the wild-type antibody in cynomolgus monkeys after intravenous administration. When administered intravenously to mice, the T250Q/M428L anti-TNFα variant displayed improved pharmacokinetics, characterized by an ∼2-fold slower clearance than the wild-type antibody. The discrepancy between these data and previously reported benefits in rhesus monkeys and the inability of these mutations to translate to improved kinetics across species may be related to a number of factors. We propose extending consideration to differences in the absolute IgG-FcRn affinity, the kinetics of the IgG/FcRn interaction, and differences in the relative involvement of this pathway in the context of other factors influencing the disposition or elimination of monoclonal antibodies.

Behavioral, biochemical, and genetic analysis of iron metabolism in high‐intensity blood donors

BACKGROUND: Individuals donating whole blood 13 times in a 2‐year period without development of iron deficiency anemia (superdonors) are a self‐selected population that is deferred for low hematocrit (Hct) level less frequently than other donors.

A Dual-Monoclonal Sandwich ELISA Specific for Hepcidin-25

BACKGROUND Hepcidin, a key regulator of iron metabolism, binds to the iron transporter ferroportin to cause its degradation. In humans, hepcidin deficiency has been linked to hemochromatosis and iron overload, whereas increased concentrations have been reported in anemia of cancer and chronic disease. There is currently an unmet clinical need for a specific immunoassay with a low limit of quantification to measure serum concentrations of hepcidin-25, the active form of the protein. METHODS We generated 2 antihepcidin-25 monoclonal antibodies and used them to build a sandwich ELISA. We correlated ELISA results to hepcidin-25 measurements by LC-MS and used ELISA to measure serum hepcidin-25 concentrations in normal individuals, cancer patients, and patients with rheumatoid arthritis. RESULTS The sandwich ELISA was highly specific for hepcidin-25, having a limit of quantification of 0.01 μg/L (10 pg/mL). Serum concentrations of hepcidin-25 measured by ELISA correlated with hepcidin-25 concentrations measured by using an independent LC-MS assay (r = 0.98, P < 0.001). Hepcidin-25 concentrations were increased in patients with cancer (median 54.8 μg/L, 25%-75% range 23.2-93.5 μg/L, n = 34) and rheumatoid arthritis (median 10.6 μg/L, 25%-75% range 5.9-18.4 μg/L, n = 76) compared with healthy individuals (median 1.20 μg/L, 25%-75% range 0.42-3.07 μg/L, n = 100). CONCLUSIONS The use of 2 monoclonal antibodies in a sandwich ELISA format provides a robust and convenient method for measuring concentrations of the active form of hepcidin. This ELISA should help to improve our understanding of the role of hepcidin in regulating iron metabolism.

Growth differentiation factor 15 in anaemia of chronic disease, iron deficiency anaemia and mixed type anaemia.

Recently, the iron and erythropoiesis‐controlled growth differentiation factor 15 (GDF15) has been shown to inhibit the expression of hepcidin in β‐thalassaemia patients, thereby increasing iron absorption despite iron overload. To access the diagnostic and pathogenic impact of GDF15 in inflammatory anaemia the association of GDF15 expression with serum iron parameters and hepcidin was studied in patients suffering from iron deficiency anaemia (IDA), anaemia of chronic disease (ACD) and ACD subjects with true iron deficiency (ACD/IDA). GDF15 was significantly increased in both ACD and ACD/IDA, but not in IDA subjects as compared to controls. In contrast, hepcidin levels were significantly lower in IDA and ACD/IDA subjects than in ACD patients. IDA and ACD/IDA, but not ACD, showed an association between GDF15 and soluble transferrin receptor, an indicator of iron requirement for erythropoiesis. However, GDF15 did not correlate to hepcidin in either patient group. While GDF15 levels were linked to the needs for erythropoiesis and iron homeostasis in IDA, the immunity‐driven increase of GDF15 may not primarily affect iron homeostasis and hepcidin expression. This indicates that other ACD‐related factors may overcome the regulatory effects of GDF15 on hepcidin expression during inflammation.

Characterization of the purified N-type Ca2+ channel and the cation sensitivity of ω-conotoxin GVIA binding

A functional N-type Ca2+ channel (omega-conotoxin GVIA receptor) has been purified from rabbit brain and shown to be composed of four subunits of molecular weights 230 K (alpha 1B), 160 K (alpha 2 delta), 95 K and 57 K (beta 3) [Witcher D. R., De Waard M., Sakamoto J., Franzini-Armstrong C., Pragnell M., Kahl S.D. and Campbell K. D. (1993) Science 261: 486-489]. These four subunits migrate on sucrose density gradients as a single complex and are identified by subunit specific polyclonal antibodies. Polyclonal antibodies against the purified receptor complex immunoprecipitate greater than 90% of the [125I]omega-conotoxin GVIA (omega-CgTx) binding sites in solubilized crude rabbit brain membranes. Furthermore, polyclonal antibodies affinity-purified against unique GST fusion proteins from two of the cloned subunits in the complex (alpha 1B and beta 3) specifically immunoprecipitated [125I]omega-CgTx binding sites and not [3H]PN200-110 binding sites. Analysis of [125I]omega-CgTx binding to the purified N-type Ca2+ channel demonstrated that the equilibrium binding was sensitive to increasing cation concentration. The IC50 for calcium and barium was 2.5 and 5 mM, respectively. [125I]omega-CgTx binding was not significantly reduced within 15 min after the addition of 50 mM barium. However, single channel analysis of the purified N-type Ca2+ channel preincubated with 10 microM omega-CgTx demonstrated that in the presence of 50 mM barium and 0.5 microM omega-CgTx, channel activity was detected but at a low open state probability (P < 0.10). These data suggest that the Ca2+ binding site(s) allosterically regulates the omega-CgTx binding site. Since the channel gating persisted in the presence of omega-CgTx, the omega-CgTx binding site may not be located within the pore of the channel and may be different from intra-pore Ca2+ binding sites.

Regulation of iron metabolism through GDF15 and hepcidin in pyruvate kinase deficiency.

Iron absorption is inadequately increased in patients with chronic haemolytic anaemia, which is commonly complicated by iron overload. Growth differentiation factor 15 (GDF15) has been identified as a bone marrow‐derived factor that abrogates hepcidin‐mediated protection from iron overload under conditions of increased erythropoiesis. Increased concentrations of GDF15 have been reported in β‐thalassaemia patients and GDF15 has been found to suppress hepcidin expression in vitro. To further study the interdependencies of iron metabolism and erythropoiesis in vivo, the concentrations of hepcidin and GDF15 were determined in sera from 22 patients with pyruvate kinase deficiency (PKD) and 21 healthy control subjects. In PKD patients, serum hepcidin levels were 13‐fold lower than in controls (2·0 ng/ml vs. 26·2 ng/ml) and GDF15 was significantly higher (859 pg/ml vs. 528 pg/ml). Serum hepcidin concentrations correlated positively with haemoglobin and negatively with serum GDF15. These results suggest that GDF15 contributes to low hepcidin expression and iron loading in PKD.

FcRn Affinity-Pharmacokinetic Relationship Of Five Human IgG4 Antibodies Engineered For Improved In Vitro FcRn Binding Properties In Cynomolgus Monkeys

The pH-dependent binding of IgGs to the neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. Enhancing interactions between Fc and FcRn via protein engineering has been successfully used as an approach for improving the pharmacokinetics of monoclonal antibodies (mAbs). Although the quantitative translatability of the in vitro FcRn affinity enhancement to an in vivo pharmacokinetic benefit has been supported by several studies, there are also published reports indicating a disconnect in this relation. The body of literature suggests there are likely additional biochemical and biophysical properties of the mAbs along with their FcRn affinity that influence the in vivo pharmacokinetics. Herein, we more broadly evaluate the in vitro Fc-FcRn interactions and biochemical properties of five humanized IgG4 antibodies each with two Fc variant sequences (T250Q/M428L and V308P) and their corresponding pharmacokinetics in cynomolgus monkeys. Our findings indicate that the FcRn affinity-pharmacokinetic relationship does not show a direct correlation either across different IgGs or between the two variant sequences within a platform. Other parameters that have been suggested to contribute to mAb pharmacokinetic properties, such as the pH-dependent dissociation of the FcRn-IgG complexes, mAb biophysical properties, and nonspecific/charge binding characteristics of the mAbs, also did not independently explain the differing pharmacokinetic behaviors. Our results suggest that there is likely not a single in vitro parameter that readily predicts in vivo pharmacokinetics, but that the relative contribution and interplay of several factors along with the FcRn binding affinity are important determinants of mAb pharmacokinetic properties.

Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor.

B-cell activating factor (BAFF) is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect.

Contact-dependent regulation of N-type calcium channel subunits during synaptogenesis

The developmental regulation of the N-type calcium channel during synaptogenesis was studied using cultured rat hippocampal neurons to elucidate the roles of extrinsic versus intrinsic cues in the expression and distribution of this channel. Prior to synapse formation, alpha1B and beta3 subunits of the N-type calcium channel were distributed diffusely throughout neurites, growth cones, and somata. As synaptogenesis proceeded, the subunit distributions became punctate and colocalized with the synaptic vesicle protein synaptotagmin. Isolated neurons were also examined to test for the requirement of extrinsic cues that control N-type calcium channel expression and distribution. These neurons expressed N-type calcium channel subunits, but their distributions remained diffuse. Functional omega-conotoxin GVIA-sensitive channels were expressed in isolated neurons, although the distribution of alpha1B subunits was diffuse. The distribution of the alpha1B subunit and synaptotagmin only became punctate when neuron-neuron contact was allowed. Thus, the expression of functional N-type calcium channels is the result of an intrinsic program while extrinsic regulatory cues mediated by neuron-neuron contact are required to control their distribution during synaptogenesis.

Engineered FcRn Binding Fusion Peptides Significantly Enhance the Half-Life of a Fab Domain in Cynomolgus Monkeys

There is a rapidly growing reinvigoration of the investigation of small proteins, cyclic peptides, and mAb derived domains as biotherapies. The drugability of these structures are challenged by fast peripheral clearance properties that can reduce their potential to be realized as medicines. Engineering strategies have been of limited value because mechanistically the half-life benefit is manifested by increasing the molecular weight and/or the hydrodyanimc radius which slows the molecules renal elimination, but can result in the inherent loss of activity and target accessibility. The present work evaluated an alternative approach using smaller peptide sequences which bind to the neonatal Fc receptor (FcRn). Results revealed, small linear and cyclic FcRn binding peptides (FcRnBPs) fused to a combination of the N- and C-termini of a Fab can significantly improve the pharmacokinetics of the protein in cynomolgus monkeys relative to the parental Fab. The linear and cyclic conformations, as well as, the number of FcRnBPs fused to the Fab both influence the clearance and the extent of pharmacokinetic benefit. FcRnBP fusion protein kinetics were also affected by a combination of post-translation modifications and non-specific binding properties. The results in this report lay some foundation in fostering the advent of newer technologies toward successfully improving the pharmacokinetics of proteins, peptides, and mAb-derived domains. Additional work in the integration of a variety of factors including the intended site of action, tissue disposition, metabolism, toxicity and pharmacokinetic, and pharmacodynamics relationship of the intended therapeutic modality are key areas for advancement of these approaches.