Karen Billeci

Variants of the antibody herceptin that interact with HER2 and VEGF at the antigen binding site

The interface between antibody and antigen is often depicted as a lock and key, suggesting that an antibody surface can accommodate only one antigen. Here, we describe an antibody with an antigen binding site that binds two distinct proteins with high affinity. We isolated a variant of Herceptin, a therapeutic monoclonal antibody that binds the human epidermal growth factor receptor 2 (HER2), on the basis of its ability to simultaneously interact with vascular endothelial growth factor (VEGF). Crystallographic and mutagenesis studies revealed that distinct amino acids of this antibody, called bH1, engage HER2 and VEGF energetically, but there is extensive overlap between the antibody surface areas contacting the two antigens. An affinity-improved version of bH1 inhibits both HER2- and VEGF-mediated cell proliferation in vitro and tumor progression in mouse models. Such “two-in-one” antibodies challenge the monoclonal antibody paradigm of one binding site, one antigen. They could also provide new opportunities for antibody-based therapy.

Hepsin activates pro-hepatocyte growth factor and is inhibited by hepatocyte growth factor activator inhibitor-1B (HAI-1B) and HAI-2

Hepsin, a type II transmembrane serine protease, is highly upregulated in prostate cancer and promotes tumor progression and metastasis. We generated a soluble form of hepsin comprising the entire extracellular domain to show that it efficiently converts single‐chain hepatocyte growth factor (pro‐HGF) into biologically active two‐chain HGF. Hepsin activity was potently inhibited by soluble forms of the bi‐Kunitz domain inhibitors HAI‐1B (IC50 21.1 ± 2.7 nM) and HAI‐2 (IC50 1.3 ± 0.3 nM). Enzymatic assays with HAI‐1B Kunitz domain mutants (R260A and K401A) further demonstrated that inhibition was due to Kunitz domain‐1. The results suggest a functional link between hepsin and the HGF/Met pathway, which may contribute to tumor progression.

Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent

Significance Therapeutic antibodies have revolutionized the treatment of human disease. Despite these advances, antibody bivalency limits their utility against some targets. Here, we describe the development of a one-armed (monovalent) antibody, onartuzumab, targeting the receptor tyrosine kinase MET. While initial screening of bivalent antibodies produced agonists of MET, engineering them into monovalent antibodies produced antagonists instead. We explain the structural basis of the mechanism of action with the crystal structure of onartuzumab antigen-binding fragment in complex with MET and HGF-β. These discoveries have led to an additional antibody-based therapeutic option and shed light on the underpinnings of HGF/MET signaling. Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coli-derived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF binding and receptor phosphorylation and signaling and has antibody-like pharmacokinetics and antitumor activity. Biochemical data and a crystal structure of a ternary complex of onartuzumab antigen-binding fragment bound to a MET extracellular domain fragment, consisting of the MET Sema domain fused to the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF β-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain (but not β-chain) binding to MET. These data suggest a likely binding site of the HGF α-chain on MET, which when dimerized leads to MET signaling. Onartuzumab, therefore, represents the founding member of a class of therapeutic monovalent antibodies that overcomes limitations of antibody bivalency for targets impacted by antibody crosslinking.

Utilizing the activation mechanism of serine proteases to engineer hepatocyte growth factor into a Met antagonist

Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase Met, is secreted as single chain pro-HGF that lacks signaling activity. Pro-HGF acquires functional competence upon cleavage between R494 and V495, generating a disulfide-linked α/β-heterodimer, where the β-chain of HGF (HGF β) has a serine protease fold that lacks enzymatic activity. We show that, like serine proteases, insertion of the newly formed N terminus in the β-chain is critical for activity, here by allosterically stabilizing interactions with Met. The HGF β crystal structure shows that V495 inserts into the “activation pocket” near the Met binding site where the positively charged N terminus forms a salt bridge with the negatively charged D672, and the V495 side chain has hydrophobic interactions with main- and side-chain residues. Full-length two-chain HGF mutants designed to interrupt these interactions (D672N, V495G, V495A, G498I, and G498V) displayed <10% activity in Met receptor phosphorylation, cell migration, and proliferation assays. Impaired signaling of full-length mutants correlated with >50-fold decreases in Met binding of the low-affinity HGF β domain alone bearing the same mutations and further correlated with impaired N-terminal insertion. Because high-affinity binding resides in the HGF α-chain, full-length mutants maintained normal Met binding and efficiently inhibited HGF-mediated Met activation. Conversion of HGF from agonist to antagonist was achieved by as little as removal of two methyl groups (V495A) or a single charge (D672N). Thus, although serine proteases and HGF have quite distinct functions in proteolysis and Met signal transduction, respectively, they share a similar activation mechanism.

Effect of IFN-γ on the killing of S. aureus in human whole blood: Assessment of bacterial viability by CFU determination and by a new method using alamarBlue

Given the increasing incidence of methicillin resistant Staphylococcus aureus (MRSA) and the recent emergence of MRSA with a reduced susceptibility to vancomycin, alternative approaches to the treatment of infection are of increasing relevance. The purpose of these studies was to evaluate the effect of IFN-gamma on the ability of white blood cells to kill S. aureus and to develop a simpler, higher throughput bacterial killing assay. Using a methicillin sensitive clinical isolate of S. aureus, a clinical isolate of MRSA, and a commercially available strain of MRSA, studies were conducted using a killing assay in which the bacteria were added directly into whole blood. The viability of the bacteria in samples harvested at various time points was then evaluated both by the classic CFU assay and by a new assay using alamarBlue. In the latter method, serially diluted samples and a standard curve containing known concentrations of bacteria were placed on 96-well plates, and alamarBlue was added. Fluorescence readings were taken, and the viability of the bacteria in the samples was calculated using the standard curve. The results of these studies demonstrated that the CFU and alamarBlue methods yielded equivalent detection of bacteria diluted in buffer. For samples incubated in whole blood, however, the alamarBlue method tended to yield lower viabilities than the CFU method due to the emergence of a slower growing subpopulation of S. aureus upon incubation in the blood matrix. A significant increase in bacterial killing was observed upon pretreatment of whole blood for 24 h with 5 or 25 ng/ml IFN-gamma. This increase in killing was detected equivalently by the CFU and alamarBlue methods. In summary, these studies describe a method that allows for the higher throughput analysis of the effects of immunomodulators on bacterial killing.

Characteristics of rhVEGF Release from Topical Hydrogel Formulations

PurposeTo study recombinant human vascular endothelial growth factor (rhVEGF), the release characteristics from topical gel formulations, and its interaction with the gelling agents.MethodsThe release kinetics were followed by quantifying rhVEGF that diffused into the receptor chamber of Franz cells. Analytical ultracentrifuge (AUC) was used to characterize the sedimentation velocity of rhVEGF experienced in the gel. The interactions were characterized by isothermal calorimetry (ITC), and rhVEGF conformation was assessed by circular dichroism (CD).ResultsThe fraction of protein released was linear with the square root of time. The release rate constants did not show significant change within a wide range of bulk viscosities created by different concentrations of hydroxypropyl methylcellulose (HPMC) or MC gels. Sedimentation velocity determined by AUC generated comparable sedimentation coefficients of protein in these gels. AUC and ITC revealed no significant interaction between rhVEGF and HPMC and some change on secondary structure of the protein by Far UV CD, which was not the case with carboxymethyl cellulose (CMC).ConclusionsMicroviscosity, not bulk viscosity, was the key factor for the release of rhVEGF from cellulosic gels such as HPMC. Interaction between rhVEGF and CMC resulted in slower, and reduced amount of, release from the gel.

Allosteric Peptide Activators of Pro-Hepatocyte Growth Factor Stimulate Met Signaling

Hepatocyte growth factor (HGF) binds to its target receptor tyrosine kinase, Met, as a single-chain form (pro-HGF) or as a cleaved two-chain disulfide-linked α/β-heterodimer. However, only two-chain HGF stimulates Met signaling. Proteolytic cleavage of the Arg494-Val495 peptide bond in the zymogen-like pro-HGF results in allosteric activation of the serine protease-like β-chain (HGF β), which binds Met to initiate signaling. We use insights from the canonical trypsin-like serine protease activation mechanism to show that isolated peptides corresponding to the first 7–10 residues of the cleaved N terminus of the β-chain stimulate Met phosphorylation by pro-HGF to levels that are ∼25% of those stimulated by two-chain HGF. Biolayer interferometry data demonstrate that peptide VVNGIPTR (peptide V8) allosterically enhances pro-HGF β binding to Met, resulting in a KDapp of 1.6 μm, only 8-fold weaker than the Met/HGF β-chain affinity. Most notably, in vitro cell stimulation with peptide V8 in the presence of pro-HGF leads to Akt phosphorylation, enhances cell survival, and facilitates cell migration between 75 and 100% of that found with two-chain HGF, thus revealing a novel approach for activation of Met signaling that bypasses proteolytic processing of pro-HGF. Peptide V8 is unable to enhance Met binding or signaling with HGF proteins having a mutated activation pocket (D672N). Furthermore, Gly substitution of the N-terminal Val residue in peptide V8 results in loss of all activity. Overall, these findings identify the activation pocket of the serine protease-like β-chain as a “hot spot” for allosteric regulation of pro-HGF and have broad implications for developing selective allosteric activators of serine proteases and pseudoproteases.

Substrate capacity considerations in developing kinase assays.

In developing a screening assay for a serine/threonine kinase, we evaluated various formats of an in-plate enzyme-linked immunosorbent assay (ELISA), as well as solution-phase kinase assays using either ELISA or AlphaScreen detection. Substrate was available both as a biotinylated 15-residue peptide and as a 25-residue peptide containing the same sequence expressed as a glutathione S-transferase fusion protein. When increasing concentrations of either of these substrates were coated directly onto ELISA plates, the rates of the kinase reactions progressively increased. In contrast, when the biotin-peptide was captured onto NeutrAvidin-coated plates, the finite peptide binding capacity of the plates limited the amount of substrate that could be incorporated into the assay system and thereby limited the rate of the reaction at a given kinase concentration. Solution-phase kinase reactions can tolerate high substrate concentrations; however, analysis of kinase reaction samples containing biotin-peptide concentrations higher than the binding capacity of NeutrAvidin-coated plates resulted in an inability to detect differences between reactions run at different substrate concentrations. For AlphaScreen detection following solution-phase kinase reactions, limitations in the binding capacity of the donor and acceptor beads caused loss of signal for substrate concentrations above the maximum binding capacity. Overall, the solution-phase assays required significantly more kinase than the in-plate assays (1-4 microg/ml versus <100 ng/ml, respectively). These studies demonstrate that the amount of substrate that can be incorporated into an assay system substantially affects the rate of the kinase reaction and therefore the amount of kinase required for the assay.

Implementation of an Automated High-Throughput Plasmid DNA Production Pipeline.

Biologics sample management facilities are often responsible for a diversity of large-molecule reagent types, such as DNA, RNAi, and protein libraries. Historically, the management of large molecules was dispersed into multiple laboratories. As methodologies to support pathway discovery, antibody discovery, and protein production have become high throughput, the implementation of automation and centralized inventory management tools has become important. To this end, to improve sample tracking, throughput, and accuracy, we have implemented a module-based automation system integrated into inventory management software using multiple platforms (Hamilton, Hudson, Dynamic Devices, and Brooks). Here we describe the implementation of these systems with a focus on high-throughput plasmid DNA production management.