Potent Killing of Pseudomonas aeruginosa by an Antibody-Antibiotic Conjugate.

Pseudomonas aeruginosa causes life-threatening infections that are associated with antibiotic failure. Previously, we identified the antibiotic G2637, an analog of arylomycin, targeting bacterial type I signal peptidase, which has moderate potency against P. aeruginosa. We hypothesized that an antibody-antibiotic conjugate (AAC) could increase its activity by colocalizing P. aeruginosa bacteria with high local concentrations of G2637 antibiotic in the intracellular environment of phagocytes. Using a novel technology of screening for hybridomas recognizing intact bacteria, we identified monoclonal antibody 26F8, which binds to lipopolysaccharide O antigen on the surface of P. aeruginosa bacteria. This antibody was engineered to contain 6 cysteines and was conjugated to the G2637 antibiotic via a lysosomal cathepsin-cleavable linker, yielding a drug-to-antibody ratio of approximately 6. The resulting AAC delivered a high intracellular concentration of free G2637 upon phagocytosis of AAC-bound P. aeruginosa by macrophages, and potently cleared viable P. aeruginosa bacteria intracellularly. The molar concentration of AAC-associated G2637 antibiotic that resulted in elimination of bacteria inside macrophages was approximately 2 orders of magnitude lower than the concentration of free G2637 required to eliminate extracellular bacteria. This study demonstrates that an anti-P. aeruginosa AAC can locally concentrate antibiotic and kill P. aeruginosa inside phagocytes, providing additional therapeutic options for antibiotics that are moderately active or have an unfavorable pharmacokinetics or toxicity profile. IMPORTANCE Antibiotic treatment of life-threatening P. aeruginosa infections is associated with low clinical success, despite the availability of antibiotics that are active in standard microbiological in vitro assays, affirming the need for new therapeutic approaches. Antibiotics often fail in the preclinical stage due to insufficient efficacy against P. aeruginosa. One potential strategy is to enhance the local concentration of antibiotics with limited inherent anti-P. aeruginosa activity. This study presents proof of concept for an antibody-antibiotic conjugate, which releases a high local antibiotic concentration inside macrophages upon phagocytosis, resulting in potent intracellular killing of phagocytosed P. aeruginosa bacteria. This approach may provide new therapeutic options for antibiotics that are dose limited.

Structure of the human secretory immunoglobulin M core.

Immunoglobulins (Ig) A and M are the only human antibodies that form oligomers and undergo transcytosis to mucosal secretions via the polymeric Ig receptor (pIgR). When complexed with the J-chain (JC) and the secretory component (SC) of pIgR, secretory IgA and IgM (sIgA and sIgM) play critical roles in host-pathogen defense. Recently, we determined the structure of sIgA-Fc which elucidated the mechanism of polymeric IgA assembly and revealed an extensive binding interface between IgA-Fc, JC, and SC. Despite low sequence identity shared with IgA-Fc, IgM-Fc also undergoes JC-mediated assembly and binds pIgR. Here, we report the structure of sIgM-Fc and carryout a systematic comparison to sIgA-Fc. Our structural analysis reveals a remarkably conserved mechanism of JC-templated oligomerization and SC recognition of both IgM and IgA through a highly conserved network of interactions. These studies reveal the structurally conserved features of sIgM and sIgA required for function in mucosal immunity.

Targeted IgMs agonize ocular targets with extended vitreal exposure.

Treatment of ocular disease is hindered by the presence of the blood-retinal barrier, which restricts access of systemic drugs to the eye. Intravitreal injections bypass this barrier, delivering high concentrations of drug to the targeted tissue. However, the recommended dosing interval for approved biologics is typically 6-12 weeks, and frequent travel to the physician's office poses a substantial burden for elderly patients with poor vision. Real-world data suggest that many patients are under-treated. Here, we investigate IgMs as a novel platform for treating ocular disease. We show that IgMs are well-suited to ocular administration due to moderate viscosity, long ocular exposure, and rapid systemic clearance. The complement-dependent cytotoxicity of IgMs can be readily removed with a P436G mutation, reducing safety liabilities. Furthermore, dodecavalent binding of IgM hexamers can potently activate pathways implicated in the treatment of progressive blindness, including the Tie2 receptor tyrosine kinase signaling pathway for the treatment of diabetic macular edema, or the death receptor 4 tumor necrosis family receptor pathway for the treatment of wet age-related macular degeneration. Collectively, these data demonstrate the promise of IgMs as therapeutic agonists for treating progressive blindness.

Structure of the secretory immunoglobulin A core.

Secretory immunoglobulin A (sIgA) represents the immune system's first line of defense against mucosal pathogens. IgAs are transported across the epithelium, as dimers and higher-order polymers, by the polymeric immunoglobulin receptor (pIgR). Upon reaching the luminal side, sIgAs mediate host protection and pathogen neutralization. In recent years, an increasing amount of attention has been given to IgA as a novel therapeutic antibody. However, despite extensive studies, sIgA structures have remained elusive. Here, we determine the atomic resolution structures of dimeric, tetrameric, and pentameric IgA-Fc linked by the joining chain (JC) and in complex with the secretory component of the pIgR. We suggest a mechanism in which the JC templates IgA oligomerization and imparts asymmetry for pIgR binding and transcytosis. This framework will inform the design of future IgA-based therapeutics.

High-resolution glycosylation site-engineering method identifies MICA epitope critical for shedding inhibition activity of anti-MICA antibodies.

As an immune evasion strategy, MICA and MICB, the major histocompatibility complex class I homologs, are proteolytically cleaved from the surface of cancer cells leading to impairment of CD8 + T cell- and natural killer cell-mediated immune responses. Antibodies that inhibit MICA/B shedding from tumors have therapeutic potential, but the optimal epitopes are unknown. Therefore, we developed a high-resolution, high-throughput glycosylation-engineered epitope mapping (GEM) method, which utilizes site-specific insertion of N-linked glycans onto the antigen surface to mask local regions. We apply GEM to the discovery of epitopes important for shedding inhibition of MICA/B and validate the epitopes at the residue level by alanine scanning and X-ray crystallography (Protein Data Bank accession numbers 6DDM (1D5 Fab-MICA*008), 6DDR (13A9 Fab-MICA*008), 6DDV (6E1 Fab-MICA*008). Furthermore, we show that potent inhibition of MICA shedding can be achieved by antibodies that bind GEM epitopes adjacent to previously reported cleavage sites, and that these anti-MICA/B antibodies can prevent tumor growth in vivo.

TPL2 kinase action and control of inflammation.

Tumor progression locus 2 (TPL2, also known as COT or MAP3K8) is a mitogen-activated protein kinase kinase (MAP3K) activated downstream of TNFαR, IL1R, TLR, CD40, IL17R, and some GPCRs. TPL2 regulates the MEK1/2 and ERK1/2 pathways to regulate a cascade of inflammatory responses. In parallel to this, TPL2 also activates p38α and p38δ to drive the production of various inflammatory mediators in neutrophils. We discuss the implications of this finding in the context of various inflammatory diseases.

Assembly and Function of Heterotypic Ubiquitin Chains in Cell-Cycle and Protein Quality Control.

Posttranslational modification with ubiquitin chains controls cell fate in all eukaryotes. Depending on the connectivity between subunits, different ubiquitin chain types trigger distinct outputs, as seen with K48- and K63-linked conjugates that drive protein degradation or complex assembly, respectively. Recent biochemical analyses also suggested roles for mixed or branched ubiquitin chains, yet without a method to monitor endogenous conjugates, the physiological significance of heterotypic polymers remained poorly understood. Here, we engineered a bispecific antibody to detect K11/K48-linked chains and identified mitotic regulators, misfolded nascent polypeptides, and pathological Huntingtin variants as their endogenous substrates. We show that K11/K48-linked chains are synthesized and processed by essential ubiquitin ligases and effectors that are mutated across neurodegenerative diseases; accordingly, these conjugates promote rapid proteasomal clearance of aggregation-prone proteins. By revealing key roles of K11/K48-linked chains in cell-cycle and quality control, we establish heterotypic ubiquitin conjugates as important carriers of biological information.

In vitro Fab display: a cell-free system for IgG discovery.

Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF.

Engineering and structural characterization of a linear polyubiquitin-specific antibody.

Polyubiquitination is an essential posttranslational modification that plays critical roles in cellular signaling. PolyUb (polyubiquitin) chains are formed by linking the carboxyl-terminus of one Ub (ubiquitin) subunit to either a lysine residue or the amino-terminus of an adjacent Ub. Linkage through the amino-terminus results in linear polyubiquitination that has recently been demonstrated to be a key step in nuclear factor κB activation; however, tools to study linear chains have been lacking. We therefore engineered a linear-linkage-specific antibody that is functional in Western blot, immunoprecipitation, and immunofluorescence applications. A crystal structure of the linear-linkage-specific antibody Fab fragment in complex with linear diubiquitin provides molecular insight into the nature of linear chain specificity. We use the antibody to demonstrate that linear polyUb is up-regulated upon tumor necrosis factor α stimulation of cells, consistent with a critical role in nuclear factor κB signaling. This antibody provides an essential tool for further investigation of the function of linear chains.

Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies.

Posttranslational modification of proteins with polyubiquitin occurs in diverse signaling pathways and is tightly regulated to ensure cellular homeostasis. Studies employing ubiquitin mutants suggest that the fate of polyubiquitinated proteins is determined by which lysine within ubiquitin is linked to the C terminus of an adjacent ubiquitin. We have developed linkage-specific antibodies that recognize polyubiquitin chains joined through lysine 63 (K63) or 48 (K48). A cocrystal structure of an anti-K63 linkage Fab bound to K63-linked diubiquitin provides insight into the molecular basis for specificity. We use these antibodies to demonstrate that RIP1, which is essential for tumor necrosis factor-induced NF-kappaB activation, and IRAK1, which participates in signaling by interleukin-1beta and Toll-like receptors, both undergo polyubiquitin editing in stimulated cells. Both kinase adaptors initially acquire K63-linked polyubiquitin, while at later times K48-linked polyubiquitin targets them for proteasomal degradation. Polyubiquitin editing may therefore be a general mechanism for attenuating innate immune signaling.