Widespread monoclonal IgE antibody convergence to an immunodominant, proanaphylactic Ara h 2 epitope in peanut allergy.

BACKGROUND: Despite their central role in peanut allergy, human monoclonal IgE antibodies have eluded characterization. OBJECTIVE: We sought to define the sequences, affinities, clonality, and functional properties of human monoclonal IgE antibodies in peanut allergy. METHODS: We applied our single-cell RNA sequencing-based SEQ SIFTER discovery platform to samples from allergic individuals who varied by age, sex, ethnicity, and geographic location in order to understand commonalities in the human IgE response to peanut allergens. Select antibodies were then recombinantly expressed and characterized for their allergen and epitope specificity, affinity, and functional properties. RESULTS: We found striking convergent evolution of IgE monoclonal antibodies (mAbs) from several clonal families comprising both memory B cells and plasmablasts. These antibodies bound with subnanomolar affinity to the immunodominant peanut allergen Ara h 2, specifically a linear, repetitive motif. Further characterization of these mAbs revealed their ability to single-handedly cause affinity-dependent degranulation of human mast cells and systemic anaphylaxis on peanut allergen challenge in humanized mice. Finally, we demonstrated that these mAbs, reengineered as IgGs, inhibit significant, but variable, amounts of Ara h 2- and peanut-mediated degranulation of mast cells sensitized with allergic plasma. CONCLUSIONS: Convergent evolution of IgE mAbs in peanut allergy is a common phenomenon that can reveal immunodominant epitopes on major allergenic proteins. Understanding the functional properties of these molecules is key to developing therapeutics, such as competitive IgG inhibitors, that are able to stoichiometrically outcompete endogenous IgE for allergen and thereby prevent allergic cascade in cases of accidental allergen exposure.

Nanoparticle Formulation Derived from Carboxymethyl Cellulose, Polyethylene Glycol, and Cabazitaxel for Chemotherapy Delivery to the Brain.

Nanoparticles provide a unique opportunity to explore the benefits of selective distribution and release of cancer therapeutics at sites of disease through varying particle sizes and compositions that exploit the enhanced permeability of tumor-associated blood vessels. Though delivery of larger as opposed to smaller and/or actively transported molecules to the brain is prima facie a challenging endeavor, we wondered whether nanoparticles could improve the therapeutic index of existing drugs for use in treating brain tumors via these vascular effects. We therefore selected a family of nanoparticles composed of cabazitaxel-carboxymethyl cellulose amphiphilic polymers to investigate the potential for delivering a brain-penetrant taxane to intracranial brain tumors in mice. Among a small set of nanoparticle formulations, we found evidence for nanoparticle accumulation in the brain, and one such formulation demonstrated activity in an orthotopic model of glioma, suggesting that such nanoparticles could be useful for the treatment of glioma and brain metastases of other tumor types.

Evaluation of IgE Antibodies to Omalizumab (Xolair®) and Their Potential Correlation to Anaphylaxis.

Omalizumab (Xolair®) is a recombinant humanized monoclonal antibody that selectively binds to human immunoglobulin E (IgE). Omalizumab is used to treat IgE-mediated diseases such as chronic idiopathic urticaria (CIU) and moderate to severe allergic asthma. In pre-marketing clinical trials in patients with asthma, anaphylaxis was reported in 3 of 3,507 (0.1%) patients. In post-marketing spontaneous reports, the frequency of anaphylaxis attributed to omalizumab use was estimated to be at least 0.2% of patients based on an estimated exposure of about 57,300 patients from June 2003 through December 2006. To better understand the risk of anaphylaxis in patients with allergic asthma receiving omalizumab, a post-marketing pharmacosurveillance study was initiated in 2009. As part of this study, an assay was developed to detect antibodies of IgE isotype to omalizumab. Serum samples from patients in the study were evaluated using this assay. Our results indicated that there was no observable correlation between either anaphylaxis or skin test reactivity and the presence of antibodies of IgE isotype to omalizumab. Here, we discuss the development of this assay as well as the results of the immunogenicity assessment.

Probody therapeutics for targeting antibodies to diseased tissue.

Probodies are proteolytically activated antibodies engineered to remain inert until activated locally in diseased tissue. In principle, any therapeutic antibody can be converted into Probody™ form. In this perspective, we highlight the emerging therapeutic potential of the Probody approach in the form of conventional IgG-based Probodies as well as in the form of 'empowered Probody' formats such as Probody-drug conjugates.

Tumor-specific activation of an EGFR-targeting probody enhances therapeutic index.

Target-mediated toxicity constitutes a major limitation for the development of therapeutic antibodies. To redirect the activity of antibodies recognizing widely distributed targets to the site of disease, we have applied a prodrug strategy to create an epidermal growth factor receptor (EGFR)-directed Probody therapeutic-an antibody that remains masked against antigen binding until activated locally by proteases commonly active in the tumor microenvironment. In vitro, the masked Probody showed diminished antigen binding and cell-based activities, but when activated by appropriate proteases, it regained full activity compared to the parental anti-EGFR antibody cetuximab. In vivo, the Probody was largely inert in the systemic circulation of mice, but was activated within tumor tissue and showed antitumor efficacy that was similar to that of cetuximab. The Probody demonstrated markedly improved safety and increased half-life in nonhuman primates, enabling it to be dosed safely at much higher levels than cetuximab. In addition, we found that both Probody-responsive xenograft tumors and primary tumor samples from patients were capable of activating the Probody ex vivo. Probodies may therefore improve the safety profile of therapeutic antibodies without compromising efficacy of the parental antibody and may enable the wider use of empowered antibody formats such as antibody-drug conjugates and bispecifics.

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.

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.

Technology comparisons for anti-therapeutic antibody and neutralizing antibody assays in the context of an anti-TNF pharmacokinetic study.

A single-dose cynomolgus monkey pharmacokinetic study was performed comparing two monoclonal anti-TNF antibodies (mAbs), GNExTNFvF and Humira. Normal pharmacokinetic profiles were observed over the first week of the study, followed by a rapid drop in serum mAb levels after day 8. In order to determine whether an anti-therapeutic antibody (ATA) response led to the abnormal clearance of antibody in this study, ATA assays were developed using two electrochemiluminescent technologies, BioVeris and Meso Scale Discovery (MSD). Characterization of the assays demonstrated that the two platforms gave similar sensitivities and tolerance to the presence of therapeutic antibody. Analysis of the cynomolgus monkey serum samples revealed that all animals developed significant ATA titers with log titer values of 2-4, with the BioVeris and MSD technologies giving very similar results. Immunodepletion studies confirmed the CDR-specificity of the ATA response for the GNExTNFvF-dosed cynos, although the Humira-dosed cynos showed both CDR-specific and human IgG1 framework-specific ATAs. To further characterize the ATA response, neutralizing antibody (NAb) assays were developed using two different approaches, flow cytometry and MSD. Flow cytometry and MSD cell-binding assays used Jurkat cells transfected with noncleavable TNF (huTNF(NC)). Neutralizing activity was assessed by the ability of ATA-positive serum samples to block the binding of biotinylated anti-TNF to huTNF(NC) Jurkat cells, showing that all but one animal developed neutralizing antibodies. Although both technologies displayed similar trends, the MSD approach showed greater differentiation between samples and could detect a broader range of neutralizing activities.

Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index.

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.

Rapid identification of reactive cysteine residues for site-specific labeling of antibody-Fabs.

Cysteines with reactive thiol groups are attractive tools for site-specific labeling of proteins. Engineering a reactive cysteine residue into proteins with multiple disulfide bonds is often a challenging task as it may interfere with structural and functional properties of the protein. Here we developed a phage display-based biochemical assay, PHESELECTOR (Phage ELISA for Selection of Reactive Thiols) to rapidly screen reactive thiol groups on antibody fragments without interfering with their antigen binding, using trastuzumab-Fab (hu4D5Fab) as a model system. The solvent accessibility values for all the amino acid residues in the hu4D5Fab were calculated using available crystal structure information. Serine, alanine and valine residues with highest solvent accessibility values were selected and tested to compare structure-based design with the PHESELECTOR biochemical method. Cysteine substitutions at partially solvent-accessible alanine or valine residues exhibited better thiol reactivity values than substitutions at serine residues. The poor correlation between fractional solvent accessibility and thiol reactivity of the engineered hu4D5Fab variants indicated the value of PHESELECTOR biochemical assay to identify reactive thiol groups on the antibody-Fab surface. Mass spectrometric analysis of biotinylated ThioFab (Fab with engineered cysteine) variants confirmed that conjugation occurred only at the engineered cysteine thiols of either light or heavy chains. ThioFabs with engineered cysteine residues in the constant domains (CL and CH(1)) should allow universal application for site-specific conjugation of antibody-Fabs.

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.

Convergent recognition of the IgE binding site on the high-affinity IgE receptor.

Two structurally distinct classes of peptides were recently identified by phage display that bind the high-affinity IgE receptor, FcepsilonRI, and block IgE binding and subsequent receptor activation. Both classes adopt highly stable structures in solution, one forming a beta hairpin, with the other forming a helical "zeta" structure. Despite these differences, the two classes bind competitively to the same site on the receptor. Structural analyses of both peptide-receptor complexes by NMR spectroscopy and/or X-ray crystallography reveal that the unrelated peptide scaffolds have nevertheless converged to present a similar three-dimensional surface to interact with FcepsilonRI and that their modes of interaction share a key feature of the IgE-FcepsilonRI complex, the proline/tryptophan sandwich.

Redesigning an antibody fragment for faster association with its antigen.

Traditional approaches for increasing the affinity of protein-protein complexes focus on constructing highly complementary binding surfaces. Recent theoretical simulations and experimental results suggest that electrostatic steering forces can also be manipulated to increase association rates while leaving dissociation rates unchanged, thus increasing affinity. Here we demonstrate that electrostatic attraction can be enhanced between an antibody fragment and its cognate antigen through application of a few simple rules to identify potential on-rate amplification sites that lie at the periphery of the antigen-antibody interface.

Sequence plasticity in the antigen-binding site of a therapeutic anti-HER2 antibody.

We have examined the plasticity of the antigen-combining site of a high-affinity antibody. In phage-displayed Fab libraries, selected CDR positions and one FR position of the humanized anti-Her2 antibody hu4D5 were substituted with all 20 amino acids. Antigen-binding selections were used to enrich for high-affinity variants, and a large number of sequences were obtained prior to convergence of the selected pool to a small set of clones. As expected, sequence variability of the antigen-binding site is overall diminished compared to known IgG sequences; however, certain positions retain much higher variability than others. The sequence variability map of the hu4D5 binding site is compared with a map derived from previous alanine-scanning of the antibody. Affinities of soluble Fab fragments for antigen confirm that multiple variants were selected with high affinity for antigen, including one variant with a single point mutation that was about threefold improved in affinity compared to the parental hu4D5. Interestingly, this mutation is one of the most radical in terms of changing side-chain chemistry (Trp for Asp) and occurs at the most plastic site as calculated by the Wu-Kabat variability coefficient. Thus variability mapping yields information about the antibody-antigen interaction that is useful and complementary to that obtained by alanine scanning mutagenesis.

Selective alpha4beta7 integrin antagonists and their potential as antiinflammatory agents.

The accumulation of leukocytes in various tissues contributes to the pathogenesis of numerous human autoimmune diseases. The integrin alpha4beta7, expressed on the surface of B and T lymphocytes, plays an essential role in lymphocyte trafficking throughout the gastrointestinal (GI) tract via interaction with its primary ligand, mucosal addressin cell adhesion molecule (MAdCAM). Elevated MAdCAM expression in the intestines and liver has been linked to GI-associated autoimmune disorders, including Crohn's disease, ulcerative colitis, and hepatitis C. Monoclonal antibodies that block the interaction of alpha4beta7 with MAdCAM inhibit lymphocyte homing to murine intestines without effecting migration to peripheral organs; this suggests that alpha4beta7-selective antagonists might be useful as GI specific antiinflammatory agents. Here, we report the discovery of highly potent and selective alpha4beta7 antagonists affinity selected from a random peptide-phage library. Subsequent optimization of initial peptide leads afforded alpha4beta7-selective heptapeptide inhibitors that competitively inhibit binding to MAdCAM in vitro and inhibit lymphocyte homing to murine intestines in vivo. Substitution of a single carboxylate moiety alters selectivity for alpha4beta7 by more than 500-fold to afford a potent and selective alpha4beta1 antagonist. The antagonists described here are the first peptides to demonstrate potency and selectivity for alpha4beta7 compared to other integrins.

Increases in free, unbound insulin-like growth factor I enhance insulin responsiveness in human hepatoma G2 cells in culture.

Insulin-like growth factor-binding protein (IGFBP)-1 binds to insulin-like growth factor (IGF)-I and -II with high affinity and has been shown to modulate IGF-I actions in vivo and in vitro. The synthesis of IGFBP-1 is suppressed by insulin, and administration of IGFBP-1 to rats results in impaired glucose metabolism. A synthetic peptide (bp1-01) has been shown to have a high affinity and specificity for human IGFBP-1 and to inhibit IGF-I binding. The current studies were undertaken to determine if, after incubation of bp1-01 with IGF-I.IGFBP-1 complexes, anabolic and insulin-like effects of IGF-I could be detected in human hepatoma (HepG2) cell cultures and to determine the receptor subtype(s) through which these effects were mediated. Incubation of HepG2 cells with bp1-01 (200 nm) increased IGF-I-stimulated protein synthesis by 44% and glycogen synthesis by 170% compared with stimulation by IGF-I alone. Incubation with bp1-01 also enhanced IGF-I-stimulated tyrosine phosphorylation of the IGF-I/insulin hybrid receptor and insulin receptor substrate 1. Exposure of the cells to bp1-01 alone enhanced glycogen synthesis and phosphorylation of IGF-I/insulin hybrid receptors. This was not a direct effect of bp1-01 because it did not bind to the receptor and did not activate tyrosine kinase activity in the presence of an anti-IGF-I receptor antibody. The addition of bp1-01 (200 nm) plus insulin to HepG2 cell culture medium resulted in increased tyrosine phosphorylation of the hybrid receptor, insulin receptor substrate 1, and the glycogen synthesis response compared with the effects of insulin alone. This enhancement of hybrid receptor phosphorylation and glycogen synthesis by bp1-01 peptide was diminished by preincubation with an inhibitory antibody for the alpha subunit of IGF-I receptor (alphaIR3). bp1-01 stimulated the hybrid receptor phosphorylation response to IGF-I, and this effect was inhibited by prior incubation of the cells with alphaIR3. In conclusion, bp1-01 competes with IGF-I for binding to IGFBP-1, which leads to release of free IGF-I from IGF-I.IGFBP-1 complexes. This released IGF-I stimulates biologic actions that are mediated predominantly through the IGF-I/insulin hybrid receptor.

Stable "zeta" peptides that act as potent antagonists of the high-affinity IgE receptor.

Recently we described a family of peptides, unrelated in sequence to IgE, that form stable beta-hairpins in solution and inhibit IgE activity in the microM range [Nakamura, G. R., Starovasnik, M. A., Reynolds, M. E. & Lowman, H. B. (2001) Biochemistry 40, 9828-9835]. Using an expanded set of peptide-phage libraries, we found a simpler motif, X(2)CPX(2)CYX, for binding to the high-affinity IgE receptor. In solution, one of these peptides spontaneously formed a covalent antiparallel dimer. We subsequently linked these monomers in a single-chain construct on phage and optimized receptor binding. Ultimately, peptides with 30 nM affinity were produced. NMR studies showed that the peptide adopts a stable fold consisting of two "zeta" (zeta)-shaped moieties. Structure-activity analyses reveal a single binding site created by the zeta-dimer, with two tyrosine residues important for structural stability and two proline residues important for Fc epsilon RI binding. The peptides inhibit histamine release from cultured cells and are extremely stable in biological fluids. The zeta peptides appear to act as competitive IgE inhibitors and suggest possibilities for design of novel IgE antagonists.