Stochastic assembly of biomacromolecular complexes: impact and implications on charge interpretation in native mass spectrometry.

Native mass spectrometry is a potent method for characterizing biomacromolecular assemblies. A critical aspect to extracting accurate mass information is the correct inference of the ion ensemble charge states. While a variety of experimental strategies and algorithms have been developed to facilitate this, virtually all approaches rely on the implicit assumption that any peaks in a native mass spectrum can be directly attributed to an underlying charge state distribution. Here, we demonstrate that this paradigm breaks down for several types of macromolecular protein complexes due to the intrinsic heterogeneity induced by the stochastic nature of their assembly. Utilizing several protein assemblies of adeno-associated virus capsids and ferritin, we demonstrate that these particles can produce a variety of unexpected spectral appearances, some of which appear superficially similar to a resolved charge state distribution. When interpreted using conventional charge inference strategies, these distorted spectra can lead to substantial errors in the calculated mass (up to ∼5%). We provide a novel analytical framework to interpret and extract mass information from these spectra by combining high-resolution native mass spectrometry, single particle Orbitrap-based charge detection mass spectrometry, and sophisticated spectral simulations based on a stochastic assembly model. We uncover that these mass spectra are extremely sensitive to not only mass heterogeneity within the subunits, but also to the magnitude and width of their charge state distributions. As we postulate that many protein complexes assemble stochastically, this framework provides a generalizable solution, further extending the usability of native mass spectrometry in the characterization of biomacromolecular assemblies.

Chemically Defined, High-Density Insect Cell-Based Expression System for Scalable AAV Vector Production.

The recombinant adeno-associated virus (AAV) vector is one of the most utilized viral vectors in gene therapy due to its robust, long-term in vivo transgene expression and low toxicity. One major hurdle for clinical AAV applications is large-scale manufacturing. In this regard, the baculovirus-based AAV production system is highly attractive due to its scalability and predictable biosafety. Here, we describe a simple method to improve the baculovirus-based AAV production using the ExpiSf Baculovirus Expression System with a chemically defined medium for suspension culture of high-density ExpiSf9 cells. Baculovirus-infected ExpiSf9 cells produced up to 5 × 1011 genome copies of highly purified AAV vectors per 1 mL of suspension culture, which is up to a 19-fold higher yield than the titers we obtained from the conventional Sf9 cell-based system. When mice were administered the same dose of AAV vectors, we saw comparable transduction efficiency and biodistributions between the vectors made in ExpiSf9 and Sf9 cells. Thus, the ExpiSf Baculovirus Expression System would support facile and scalable AAV manufacturing amenable for preclinical and clinical applications.

A direct role for SNX9 in the biogenesis of filopodia.

Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia.

Antibodies binding the head domain of P2X4 inhibit channel function and reverse neuropathic pain.

P2X4 is a ligand-gated ion channel implicated in neuropathic pain. Drug discovery efforts targeting P2X4 have been unsuccessful largely because of the difficulty in engineering specificity and selectivity. Here, we describe for the first time the generation of a panel of diverse monoclonal antibodies (mAbs) to human and mouse P2X4, capable of both positive and negative modulation of channel function. The affinity-optimised anti-P2X4 mAb IgG#151-LO showed exquisite selectivity for human P2X4 and induced potent and complete block of P2X4 currents. Site-directed mutagenesis of P2X4 revealed the head domain as a key interaction site for inhibitory mAbs. Inhibition of spinal P2X4 either by intrathecal delivery of an anti-P2X4 mAb or by systemic delivery of an anti-P2X4 bispecific mAb with enhanced blood-spinal cord barrier permeability produced long-lasting (>7 days) analgesia in a mouse model of neuropathic pain. We therefore propose that inhibitory mAbs binding the head domain of P2X4 have therapeutic potential for the treatment of neuropathic pain.

Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo.

Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins.

Isolation of high-affinity, neutralizing anti-idiotype antibodies by phage and ribosome display for application in immunogenicity and pharmacokinetic analyses.

Anti-idiotype antibodies against a therapeutic antibody are key reagents for the development of immunogenicity and pharmacokinetic (PK) assays during pre-clinical and clinical development. Here we have used a combination of phage and ribosome display to isolate a panel of monoclonal anti-idiotype antibodies with sub-nanomolar affinity and high specificity to a human anti-IgE monoclonal antibody. Anti-idiotype antibodies were enriched from scFv libraries using phage display, and a biochemical epitope competition assay was used to identify anti-idiotypes which neutralized IgE binding, which was essential for the intended use of the anti-idiotypes as positive controls in neutralizing anti-drug antibody (Nab) assays. The phage display-derived anti-idiotype antibodies were rapidly affinity-matured using a random point mutagenesis approach in ribosome display. Ten anti-idiotype antibodies with improved neutralizing activity relative to the parent antibodies displayed sub-nanomolar affinity for the anti-IgE antibody, representing up to 20-fold improvements in affinity from just two rounds of affinity-based selection. The optimized anti-idiotype antibodies retained the specificity of the parent antibodies, and importantly, were fit for purpose for use in PK and anti-drug antibody (ADA) assays. The approach we describe here for generation of anti-idiotype antibodies to an anti-IgE antibody is generically applicable for the rapid isolation and affinity maturation of anti-idiotype antibodies to any antibody-based drug candidate.

A novel IgE-neutralizing antibody for the treatment of severe uncontrolled asthma.

The critical role played by IgE in allergic asthma is well-documented and clinically precedented, but some patients in whom IgE neutralization may still offer clinical benefit are excluded from treatment with the existing anti-IgE therapy, omalizumab, due to high total IgE levels or body mass. In this study, we sought to generate a novel high affinity anti-IgE antibody (MEDI4212) with potential to treat a broad severe asthma patient population. Analysis of body mass, total and allergen-specific IgE levels in a cohort of severe asthmatics was used to support the rationale for development of a high affinity IgE-targeted antibody therapeutic. Phage display technology was used to generate a human IgG1 lead antibody, MEDI4212, which was characterized in vitro using binding, signaling and functional assay systems. Protein crystallography was used to determine the details of the interaction between MEDI4212 and IgE. MEDI4212 bound human IgE with an affinity of 1.95 pM and was shown to target critical residues in the IgE Cε3 domain critical for interaction with FcεRI. MEDI4212 potently inhibited responses through FcεRI and also prevented the binding of IgE to CD23. When used ex vivo at identical concentration, MEDI4212 depleted free-IgE from human sera to levels ~1 log lower than omalizumab. Our results thus indicate that MEDI4212 is a novel, high affinity antibody that binds specifically to IgE and prevents IgE binding to its receptors. MEDI4212 effectively depleted free-IgE from human sera ex vivo to a level (1 IU/mL) anticipated to provide optimal IgE suppression in severe asthma patients.

Human monomeric antibody fragments to TRAIL-R1 and TRAIL-R2 that display potent in vitro agonism.

Apoptosis through the TRAIL receptor pathway can be induced via agonistic IgG to either TRAIL-R1 or TRAIL-R2. Here we describe the use of phage display to isolate a substantive panel of fully human anti-TRAIL receptor single chain Fv fragments (scFvs); 234 and 269 different scFvs specific for TRAIL-R1 and TRAIL-R2 respectively. In addition, 134 different scFvs that were cross-reactive for both receptors were isolated. To facilitate screening of all 637 scFvs for potential agonistic activity in vitro, a novel high-throughput surrogate apoptosis assay was developed. Ten TRAIL-R1 specific scFv and 6 TRAIL-R2 specific scFv were shown to inhibit growth of tumor cells in vitro in the absence of any cross-linking agents. These scFv were all highly specific for either TRAIL-R1 or TRAIL-R2, potently inhibited tumor cell proliferation, and were antagonists of TRAIL binding. Moreover, further characterization of TRAIL-R1 agonistic scFv demonstrated significant anti-tumor activity when expressed and purified as a monomeric Fab fragment. Thus, scFv and Fab fragments, in addition to whole IgG, can be agonistic and induce tumor cell death through specific binding to either TRAIL-R1 or TRAIL-R2. These potent agonistic scFv were all isolated directly from the starting phage antibody library and demonstrated significant tumor cell killing properties without any requirement for affinity maturation. Some of these selected scFv have been converted to IgG format and are being studied extensively in clinical trials to investigate their potential utility as human monoclonal antibody therapeutics for the treatment of human cancer.

Generation and characterization of LymphoStat-B, a human monoclonal antibody that antagonizes the bioactivities of B lymphocyte stimulator.

OBJECTIVE: To identify and characterize a fully human antibody directed against B lymphocyte stimulator (BLyS), a tumor necrosis factor-related cytokine that plays a critical role in the regulation of B cell maturation and development. Elevated levels of BLyS have been implicated in the pathogenesis of autoimmune diseases. METHODS: A human phage display library was screened for antibodies against human BLyS. A human monoclonal antibody, LymphoStat-B, specific for human BLyS was obtained from the library screening and subsequent affinity optimization mutagenesis. The antibody was tested for inhibition of human BLyS in vitro and in an in vivo murine model. Additionally, the consequences of BLyS inhibition were tested in vivo by administration of LymphoStat-B to cynomolgus monkeys. RESULTS: LymphoStat-B bound with high affinity to human BLyS and inhibited the binding of BLyS to its 3 receptors, TACI, BCMA, and BLyS receptor 3/BAFF-R. LymphoStat-B potently inhibited BLyS-induced proliferation of B cells in vitro, and administration of LymphoStat-B to mice prevented human BLyS-induced increases in splenic B cell numbers and IgA titers. In cynomolgus monkeys, administration of LymphoStat-B resulted in decreased B cell representation in both spleen and mesenteric lymph nodes. CONCLUSION: A fully human monoclonal antibody has been isolated that binds to BLyS with high affinity and neutralizes human BLyS bioactivity in vitro and in vivo. Administration of this antibody to cynomolgus monkeys resulted in B cell depletion in spleen and lymph node. This antibody may prove therapeutically useful in the treatment of autoimmune diseases in humans.