Establishing a link between amino acid sequences and self-associating and viscoelastic behavior of two closely related monoclonal antibodies.

PURPOSE: To investigate the underlying cause for the observed differences in self-associating and viscoelastic behavior between two monoclonal antibodies, MAb1, and MAb2. METHODS: Several mutants were designed by swapping charged residues in MAb1 with those present in MAb2 at their respective positions and vice versa. Rheological analysis was done at low and high shear rates. Dynamic light scattering quantified intermolecular interactions in dilute solutions; sedimentation equilibrium analysis determined the corrected weight average molecular weight (M (wc)) to assess the self-associating behavior in high concentration. The molecular charge was estimated from electrophoretic mobility measurements. RESULTS: Replacing the charged residues in the CDR of MAb1 resulted in a lower M (wc) and solution viscosity. The corresponding changes in either just the variable light (VL) or variable heavy (VH) chain showed only a partial decrease in viscosity, whereas changes in both VL and VH chains resulted in a dramatic reduction in viscosity. The converse case where the VL and VH chains of MAb2 were made to look like MAb1 did not self-associate or show increased viscosity. CONCLUSIONS: Exposed charged residues in the CDR of MAb1 are critical in determining the self-associating and highly viscous behavior observed at high concentrations.

A therapeutic anti-VEGF antibody with increased potency independent of pharmacokinetic half-life.

Bevacizumab [Avastin; anti-vascular endothelial growth factor (VEGF) antibody] is an antiangiogenic IgG approved for treating patients with certain types of colon, breast, and lung cancer. In these indications, bevacizumab is administered every 2 to 3 weeks, prompting us to study ways to reduce the frequency of administration. Increasing affinity to neonatal Fc receptor (FcRn) may extend the pharmacokinetic half-life of an antibody, but the quantitative effect of FcRn affinity on clearance has not been clearly elucidated. To gain further insight into this relationship, we engineered a series of anti-VEGF antibody variants with minimal amino acid substitutions and showed a range of half-life improvements in primates. These results suggest that, if proven clinically safe and effective, a modified version of bevacizumab could potentially provide clinical benefit to patients on long-term anti-VEGF therapy through less-frequent dosing and improved compliance with drug therapy. Moreover, despite having half-life similar to that of wild-type in mice due to the species-specific FcRn binding effects, the variant T307Q/N434A exhibited superior in vivo potency in slowing the growth of certain human tumor lines in mouse xenograft models. These results further suggest that FcRn variants may achieve increased potency through unidentified mechanisms in addition to increased systemic exposure.

Pharmacokinetics of humanized monoclonal anti-tumor necrosis factor-{alpha} antibody and its neonatal Fc receptor variants in mice and cynomolgus monkeys.

The neonatal Fc receptor (FcRn) plays a critical role in maintaining homeostasis of IgG antibodies. Recent studies have shown that the FcRn-IgG interaction can be modulated to alter the pharmacokinetics of the antibody. This has been achieved by altering amino acid residues in the FcRn-binding domain of the antibody, resulting in a change in the pH-dependent binding affinity of the antibody to FcRn. The purpose of this study was to examine the impact of the pH-dependent FcRn binding affinity on the pharmacokinetics of the antibody with changes in the Asn434 residue. Two anti-tumor necrosis factor-alpha monoclonal antibody (mAb) FcRn variants (N434A and N434H) were engineered, and pharmacokinetic studies of the two FcRn variants together with the wild type (WT) were conducted in mice and cynomolgus monkeys. N434A, which had binding properties to murine FcRn similar to those of the WT, had the same pharmacokinetic profile as the WT in mice. N434H, with the highest binding affinity to murine FcRn at pH 7.4, had a faster clearance (16.1 ml/day/kg) and a lower bioavailability (61.3%) compared with the WT (5.07 ml/day/kg, 73.2%) and N434A (5.90 ml/day/kg, 72.4%) in mice. N434A and N434H, which had higher binding affinity at pH 6.0 to monkey FcRn with comparable affinity at pH 7.4, had significantly higher areas under the serum concentration-time curve from time 0 to day 7 than the WT (749 +/- 71.9 and 819 +/- 81.5 versus 592 +/- 56.8 microg/ml . day) in monkeys. Thus, increasing the binding affinity of mAbs to FcRn at pH 6.0 while keeping a low binding affinity at pH 7.4 improves the pharmacokinetics of these molecules.

Multiple novel classes of APRIL-specific receptor-blocking peptides isolated by phage display.

A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) ligand superfamily and has a proliferative effect on both normal and tumor cells. The TNF family receptors (B-cell maturation antigen (BCMA), transmembrane activator and CAML-interactor (TACI), and BAFF receptor-3 (BR3)) for APRIL and the closely related ligand, B-cell activating factor of the TNF family (BAFF), bind these ligands through a highly conserved six residue DXL motif ((F/Y/W)-D-X-L-(V/T)-(R/G)). Panning peptide phage display libraries led to the identification of several novel classes of APRIL-binding peptides, which could be grouped by their common sequence motifs. Interestingly, only one of these ten classes consisted of peptides containing the DXL motif. Nevertheless, all classes of peptides prevented APRIL, but not BAFF, from binding BCMA, their shared receptor. Synthetic peptides based on selected sequences inhibited APRIL binding to BCMA with IC(50) values of 0.49-27 microM. An X-ray crystallographic structure of APRIL bound to one of the phage-derived peptides showed that the peptide, lacking the DXL motif, was nevertheless bound in the DXL pocket on APRIL. Our results demonstrate that even though a focused, highly conserved motif is required for APRIL-receptor interaction, remarkably, many novel and distinct classes of peptides are also capable of binding APRIL at the ligand receptor interface.

Engineering human IgG1 affinity to human neonatal Fc receptor: impact of affinity improvement on pharmacokinetics in primates.

The pH-dependent binding of Igs to the neonatal FcR (FcRn) plays a critical role in the in vivo homeostasis of IgGs. Modulating the interaction between Fc and FcRn through protein engineering is one method for improving the pharmacokinetics of therapeutic Abs. Recent studies disputed the direct relationship between increasing FcRn affinity and improved pharmacokinetic properties. In this work, we studied the pharmacokinetics of two human IgG1 Fc variants in cynomolgus monkey to further clarify the affinity-pharmacokinetic relationship. First, we report a number of novel Fc point mutations and combination variants, including some with primate-specific FcRn-binding improvements. By studying these variants along with some previously described variants across a wide range of affinities, we discovered a direct correlation of pH 6 affinity improvements with neutral pH improvements, suggesting that all of the tested variants exhibit similar pH dependency in FcRn binding. We then evaluated the pharmacokinetics of variants N434A and N434W, which, respectively, gave approximately 4- and 80-fold improvements in pH 6-binding affinity to both human and nonhuman primate FcRn. Surprisingly, clearance of N434W was similar to that of wild type. N434W is the first variant studied in primates that exhibits significant binding to FcRn at pH 7.4, and its clearance substantiates the principle that too much affinity improvement, i.e., beyond that of N434W, does not yield improved pharmacokinetics. In contrast, N434A exhibited a approximately 2-fold decrease in clearance in cynomolgus monkey, supporting the notion that modest increases in pH 6 FcRn affinity can result in improved pharmacokinetics in primates.

Ranibizumab inhibits multiple forms of biologically active vascular endothelial growth factor in vitro and in vivo.

Neovascular age-related macular degeneration (AMD) is the leading cause of blindness in older adults in the Western world. Ranibizumab (Lucentis), a humanized antibody fragment directed against vascular endothelial growth factor (VEGF-A), was recently approved by the US Food and Drug Administration (FDA) for the treatment of neovascular AMD. The objective of this study was to characterize the binding affinity and pharmacological activity of ranibizumab for 3 biologically active forms of VEGF-A: VEGF165, VEGF121, and VEGF110. The apparent equilibrium binding affinity of ranibizumab for VEGF-A molecules was determined by Biacore analysis; the biological activity of VEGF-A was demonstrated in a human umbilical vein endothelial cell (HUVEC) proliferation-inhibition assay. Inhibition of VEGF-A-induced vascular permeability by ranibizumab was assessed in vivo using hairless guinea pigs and a modified Miles assay. Ranibizumab was capable of binding to recombinant human VEGF165, VEGF121, and VEGF110 (KD < or = 192 pM), inhibiting VEGF-A-induced HUVEC proliferation in a concentration-dependent manner. Ranibizumab also exerted potent dose-dependent inhibition (IC(50) of 0.4-1.2 nM) of the vascular permeability-enhancing activity of VEGF165, VEGF121, and VEGF110 in the Miles assay. In conclusion, these results show that ranibizumab is capable of binding to and specifically inhibiting the activities of 3 biologically active forms of VEGF-A. As VEGF-A plays a pivotal role in the pathogenesis of neovascular AMD, ranibizumab activity, as demonstrated in this study, supports its clinical utility in the treatment of this disease.

A substrate-phage approach for investigating caspase specificity.

We have developed a substrate-phage approach for examining the substrate specificities of an important group of proteases involved in apoptosis--the caspases. After establishing selection conditions with caspases-3 and caspase-8 vs control substrate-phage, we sorted X4 and X6 diversity libraries, identified consensus motifs that agree with previously defined caspase substrate motifs, confirmed the selection of active substrates using synthetic peptide rate assays under a range of buffer conditions, and compared kinetic parameters for selected substrates. The libraries produced some variations on the canonical motifs. From caspase-3 selections, a phage-derived synthetic peptide, DLVD, was hydrolyzed up to 170% faster than the canonical substrate DEVD. The P4 Asp residue was essential for good protease-sensitivity, but even substrates with substitutions at P4 were selected by phage and shown to be hydrolyzed. Caspase-8 selections, as expected, yielded predominantly clones containing a Glu at P3. In this case, the most frequent phage-derived peptide, LEVD, was cleaved at a rate of only 20% of the canonical caspase-8 substrate LETD. However, based on substitutions observed in the phage selectants at P4, a substrate peptide, AETD, was designed and shown to be hydrolyzed up to 160% faster than LETD. We consider factors that may contribute to differences in caspase substrate-phage selections vs synthetic peptide studies on the caspases, and suggest that the two approaches may offer complementary information.

Isolating substrates for an engineered alpha-lytic protease by phage display.

Panning of a substrate phage library with an alpha-lytic protease mutant showed that substrate phage display can be used to isolate sequences with improved protease sensitivity even for proteases of relatively broad specificity. Two panning experiments were performed with an engineered alpha-lytic protease mutant known to have a preference for cleavage after His or Met residues. Both experiments led to the isolation of protease-sensitive phage containing linker sequences in which His and Met residues were enriched compared with the initial library. Despite the relatively hydrophobic substrate binding site of the enzyme, the predominant protease-sensitive sequence isolated from the second library panning had the sequence Asp-Ser-Thr-Met. Kinetic studies showed that this sequence was cleaved up to 4.5-fold faster than rationally designed positive controls. Protease-resistant phage particles were also selected and characterized, with the finding that Gly and Pro appeared frequently at the putative P4 positions, whereas Asp dominated the putative P1 position.

The polymorphic human glutathione transferase T1-1, the most efficient glutathione transferase in the denitrosation and inactivation of the anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea.

A member of the Theta class of human glutathione transferases (GST T1-1) was found to display the greatest catalytic activity towards the cytostatic drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) of the GSTs studied. In this investigation (the most extensive to date), enzymes from four classes of the soluble human GSTs were heterologously expressed, purified, and kinetically characterized. From the 12 enzymes examined, only GST M2-2, GST M3-3 and GST T1-1 had significant activities with BCNU. This establishes that the activity is not a characteristic of a particular class of GSTs. Although GST M3-3 was previously reported to have the greatest activity with BCNU, the current investigation demonstrates that GST M2-2 is equally active and that GST T1-1 has an approximately 20-fold higher specific activity than either of the Mu class enzymes. A more rigorous kinetic analysis of GST T1-1 gave the following parameters with BCNU: a k(cat) of 0.035 +/-0.003s(-1) and a K(M) of 1.0 +/- 0.1mM. The finding that GST T1-1 has the highest activity towards BCNU is significant since GST T1-1 is expressed in the brain, a common target for BCNU treatment. Furthermore, the existence of a GST T1-1 null allele in up to 60% in some populations, may influence both the sensitivity of tumors to chemotherapy and the severity of adverse side-effects in patients treated with this agent.