Structure-function relationships of the soluble form of the antiaging protein Klotho have therapeutic implications for managing kidney disease.

The fortuitously discovered antiaging membrane protein αKlotho (Klotho) is highly expressed in the kidney, and deletion of the Klotho gene in mice causes a phenotype strikingly similar to that of chronic kidney disease (CKD). Klotho functions as a co-receptor for fibroblast growth factor 23 (FGF23) signaling, whereas its shed extracellular domain, soluble Klotho (sKlotho), carrying glycosidase activity, is a humoral factor that regulates renal health. Low sKlotho in CKD is associated with disease progression, and sKlotho supplementation has emerged as a potential therapeutic strategy for managing CKD. Here, we explored the structure-function relationship and post-translational modifications of sKlotho variants to guide the future design of sKlotho-based therapeutics. Chinese hamster ovary (CHO)- and human embryonic kidney (HEK)-derived WT sKlotho proteins had varied activities in FGF23 co-receptor and ß-glucuronidase assays in vitro and distinct properties in vivo Sialidase treatment of heavily sialylated CHO-sKlotho increased its co-receptor activity 3-fold, yet it remained less active than hyposialylated HEK-sKlotho. MS and glycopeptide-mapping analyses revealed that HEK-sKlotho is uniquely modified with an unusual N-glycan structure consisting of N,N'-di-N-acetyllactose diamine at multiple N-linked sites, one of which at Asn-126 was adjacent to a putative GalNAc transfer motif. Site-directed mutagenesis and structural modeling analyses directly implicated N-glycans in Klotho's protein folding and function. Moreover, the introduction of two catalytic glutamate residues conserved across glycosidases into sKlotho enhanced its glucuronidase activity but decreased its FGF23 co-receptor activity, suggesting that these two functions might be structurally divergent. These findings open up opportunities for rational engineering of pharmacologically enhanced sKlotho therapeutics for managing kidney disease.

Humanized staphylococcal enterotoxin B (SEB)-specific monoclonal antibodies protect from SEB intoxication and Staphylococcus aureus infections alone or as adjunctive therapy with vancomycin.

BACKGROUND: Staphylococcal enterotoxin B (SEB), a potential biological warfare agent, is a potent superantigen that contributes to the virulence of methicillin-resistant Staphylococcus aureus (MRSA), which is a major health threat in the United States. Efforts to develop toxin-neutralizing antibodies as adjunctive therapies are justified, given the high mortality and frequent failure of therapy despite available antibiotics. METHODS: Murine SEB-specific mAb 20B1 was humanized, and treatment benefits of Hu-1.6/1.1 and Hu-1.4/1.1 variants were investigated in mice in an SEB intoxication model, as well as in sepsis and deep-tissue infection models. RESULTS: Hu-1.6/1.1 and Hu-1.4/1.1 protected mice against SEB-induced lethal shock. Hu-1.6/1.1 also enhanced survival of mice that developed fatal sepsis after challenge with a SEB-producing MRSA strain. Combined treatment of Hu-1.6/1.1 with vancomycin further increased survival and altered cytokine responses, compared with monotherapy with either monoclonal antibody or vancomycin alone. Efficacy was also demonstrated in the deep-tissue infection model, where Hu-1.4/1.1 bound to SEB in vivo and decreased abscess formation, as well as proinflammatory cytokine levels. CONCLUSIONS: SEB-neutralizing mAb 20B1 was successfully humanized. The mAb affects outcome by modulating the proinflammatory host response in both the sepsis and the intoxication models, which justifies further development.

Staphylococcal Enterotoxin B-specific monoclonal antibody 20B1 successfully treats diverse Staphylococcus aureus infections.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) has become a major health threat in the United States. Staphylococcal enterotoxin B (SEB) is a potent superantigen that contributes to its virulence. High mortality and frequent failure of therapy despite available antibiotics have stimulated research efforts to develop adjunctive therapies. METHODS: Treatment benefits of SEB-specific monoclonal antibody (mAb) 20B1 were investigated in mice in sepsis, superficial skin, and deep-tissue infection models. RESULTS: Mice challenged with a SEB-producing MRSA strain developed fatal sepsis, extensive tissue skin infection, and abscess-forming deep-seeded thigh muscle infection. Animals preimmunized against SEB or treated passively with mAb 20B1 exhibited enhanced survival in the sepsis model, whereas decrease of bacterial burden was observed in the superficial skin and deep-tissue models. mAb 20B1 bound to SEB in the infected tissue and decreased abscess formation and proinflammatory cytokine levels, lymphocyte proliferation, and neutrophil recruitment. CONCLUSIONS: mAb 20B1, an SEB-neutralizing mAb, is effective against MRSA infection. mAb 20B1 protects against lethal sepsis and reduces skin tissue invasion and deep-abscess formation. The mAb penetrates well into the abscess and binds to SEB. It affects the outcome of S. aureus infection by modulating the host's proinflammatory immune response.

Distinct in vitro binding properties of the anti-CD20 small modular immunopharmaceutical 2LM20-4 result in profound and sustained in vivo potency in cynomolgus monkeys.

OBJECTIVES: To characterize the in vitro binding and effector function properties of CD20-directed small modular immunopharmaceutical (SMIP) 2LM20-4, and to compare its in vivo B-cell depletion activity with the mutated 2LM20-4 P331S [no in vitro complement-dependent cytotoxicity (CDC)] and rituximab in cynomolgus monkeys. METHODS: Direct binding is examined in flow cytometry, confocal microscopy, scatchard and lipid raft assays. Effector function assays include CDC and Fc-mediated cellular toxicity. In the 6-month-long in vivo B-cell depletion study, single i.v. dosages of 1 or 10 mg/kg of anti-CD20 proteins were administered to monkeys and B-cell counts were monitored in peripheral blood, bone marrow and lymph nodes. RESULTS: 2LM20-4 has lower saturation binding to human primary B cells and recruits fewer CD20 molecules into lipid rafts compared with rituximab; however, it induces higher in vitro CDC. In competitive binding, 2LM20-4 only partially displaces rituximab, suggesting that it binds to a fraction of CD20 molecules within certain locations of the plasma membrane as compared with rituximab. In monkeys, 2LM20-4 had more sustained B-cell depletion activity than rituximab in peripheral blood and had significantly more profound and sustained activity than 2LM20-4 P331S and rituximab in the lymph nodes. CONCLUSIONS: SMIP 2LM20-4, which binds to a fraction of CD20 molecules as compared with rituximab, has more potent in vitro CDC, and more potent and sustained B-cell depletion activity in cynomolgus monkeys. Our work has considerable clinical relevance since it provides novel insights related to the emerging B-cell depletion therapies in autoimmune diseases.

Transient CHO expression platform for robust antibody production and its enhanced N-glycan sialylation on therapeutic glycoproteins.

Large-scale transient expression in mammalian cells is a rapid protein production technology often used to shorten overall timelines for biotherapeutics drug discovery. In this study we demonstrate transient expression in a Chinese hamster ovary (CHO) host (ExpiCHO-S™) cell line capable of achieving high recombinant antibody expression titers, comparable to levels obtained using human embryonic kidney (HEK) 293 cells. For some antibodies, ExpiCHO-S™ cells generated protein materials with better titers and improved protein quality characteristics (i.e., less aggregation) than those from HEK293. Green fluorescent protein imaging data indicated that ExpiCHO-S™ displayed a delayed but prolonged transient protein expression process compared to HEK293. When therapeutic glycoproteins containing non-Fc N-linked glycans were expressed in transient ExpiCHO-S™, the glycan pattern was unexpectedly found to have few sialylated N-glycans, in contrast to glycans produced within a stable CHO expression system. To improve N-glycan sialylation in transient ExpiCHO-S™, we co-transfected galactosyltransferase and sialyltransferase genes along with the target genes, as well as supplemented the culture medium with glycan precursors. The authors have demonstrated that co-transfection of glycosyltransferases combined with medium addition of galactose and uridine led to increased sialylation content of N-glycans during transient ExpiCHO-S™ expression. These results have provided a scientific basis for developing a future transient CHO system with N-glycan compositions that are similar to those profiles obtained from stable CHO protein production systems. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2724, 2019.

Crystal structures of the two major aggrecan degrading enzymes, ADAMTS4 and ADAMTS5.

Aggrecanases are now believed to be the principal proteinases responsible for aggrecan degradation in osteoarthritis. Given their potential as a drug target, we solved crystal structures of the two most active human aggrecanase isoforms, ADAMTS4 and ADAMTS5, each in complex with bound inhibitor and one wherein the enzyme is in apo form. These structures show that the unliganded and inhibitor-bound enzymes exhibit two essentially different catalytic-site configurations: an autoinhibited, nonbinding, closed form and an open, binding form. On this basis, we propose that mature aggrecanases exist as an ensemble of at least two isomers, only one of which is proteolytically active.

Parallel loading and complete automation of a 3-step mAb purification process for multiple samples using a customized preparative chromatography instrument with networked pumps.

Advancement in high-throughput screening methods of novel therapeutic proteins for early stage research and development, specifically mAbs, have given mid-scale (milligram to gram scale) purification groups access to more of these molecules. The available purification technologies built to support mid-scale production was not efficient or versatile enough to keep up with this surge. To remedy this problem, we have designed and built a custom instrument using an ÄKTA Pure. This system enables parallel processing up to 5 samples and has the versatility to perform 1- to 3-step purification processes in a single queue. Furthermore, a unique purification scheme can be selected for each of the five samples in the queue. Overall processing time has reduced by 83% compared to manual, non-parallel load methods. Here, we describe our novel approach and demonstrate the flexibility, speed and efficiency of the instrument.

Binding and localization of recombinant lubricin to articular cartilage surfaces.

Lubricin is a secreted, cytoprotective glycoprotein that contributes to the essential boundary lubrication mechanisms necessary for maintaining low friction levels at articular cartilage surfaces. Diminishment of lubricin function is thereby implicated as an adverse contributing factor in degenerative joint diseases such as osteoarthritis. Lubricin occurs as a soluble component of synovial fluid, and is synthesized and localized in the superficial layer of articular cartilage (and thus has also been described as "superficial zone protein", or SZP); however, defined interactions responsible for lubricin retention at this site are not well characterized. In the current studies, we identified molecular determinants that enable lubricin to effectively bind to articular cartilage surfaces. Efficient and specific binding to the superficial zone was observed for synovial lubricin, as well as for recombinant full-length lubricin and a protein construct comprising the lubricin C-terminal (hemopexin-like) domain (LUB-C, encoded by exons 7-12). A construct representing the N-terminal region of lubricin (LUB-N, encoded by exons 2-5) exhibited no appreciable cartilage-binding ability, but displayed the capacity to dimerize, and thus potentially influence lubricin aggregation. Disulfide bond disruption significantly attenuated recombinant lubricin and LUB-C binding to cartilage surfaces, demonstrating a requirement for protein secondary structure in facilitating the appropriate localization of lubricin at relevant tissue interfaces. These findings help identify additional key attributes contributing to lubricin functionality, which would be expected to be instrumental in maintaining joint homeostasis.

Mechanistic understanding of the cysteine capping modifications of antibodies enables selective chemical engineering in live mammalian cells.

Protein modifications by intricate cellular machineries often redesign the structure and function of existing proteins to impact biological networks. Disulfide bond formation between cysteine (Cys) pairs is one of the most common modifications found in extracellularly-destined proteins, key to maintaining protein structure. Unpaired surface cysteines on secreted mammalian proteins are also frequently found disulfide-bonded with free Cys or glutathione (GSH) in circulation or culture, the mechanism for which remains unknown. Here we report that these so-called Cys-capping modifications take place outside mammalian cells, not in the endoplasmic reticulum (ER) where oxidoreductase-mediated protein disulfide formation occurs. Unpaired surface cysteines of extracellularly-arrived proteins such as antibodies are uncapped upon secretion before undergoing disulfide exchange with cystine or oxidized GSH in culture medium. This observation has led to a feasible way to selectively modify the nucleophilic thiol side-chain of cell-surface or extracellular proteins in live mammalian cells, by applying electrophiles with a chemical handle directly into culture medium. These findings provide potentially an effective approach for improving therapeutic conjugates and probing biological systems.

A novel approach to identifying beta-secretase inhibitors: bis-statine peptide mimetics discovered using structure and spot synthesis.

Beta-secretase 1 (BACE1) is an aspartic protease believed to play a critical role in Alzheimer's disease. Inhibitors of this enzyme have been designed by incorporating the non-cleavable hydroxyethylene and statine isosteres into peptides corresponding to BACE1 substrate sequences. We sought to develop new methods to quickly characterize and optimize inhibitors based on the statine core. Minimal sequence requirements for binding were first established using both crystallography and peptide spot synthesis. These shortened peptide inhibitors were then optimized by using spot synthesis to perform iterative cycles of substitution and deletion. The present study resulted in the identification of novel "bis-statine" inhibitors shown by crystallography to have a unique binding mode. Our results demonstrate the application of peptide spot synthesis as an effective method for enhancing peptidomimetic drug discovery.

Optimization of experimental conditions for functional in vitro characterization of humanized antibodies specific for staphylococcal enterotoxin B.

Staphylococcus aureus is a common nosocomial infection and its resistance to penicillin and methicillin antibiotics is a growing clinical problem. We previously described the development of a humanized anti-Staphylococcus enterotoxin B (SEB) antibody derived from the mouse antibody made by the 20B1 hybridoma. This antibody was humanized and characterized kinetically by surface plasmon resonance demonstrating that the humanized clones retained binding to SEB. Clones were then functionally characterized in an in vitro assay demonstrating that the murine 20B1, chimeric and humanized antibodies potently inhibited SEB-induced murine splenocyte proliferation assay. Here, we describe a human cell-based screening assay, optimized by varying multiple experimental parameters that resulted in an assay that was used to demonstrate full and potent neutralization by the parental, chimeric and humanized antibodies. The replacement of fetal bovine serum (FBS) with normal human serum (NHS) was found to be a crucial factor in the performance of the human cell based screening assay that enabled the calculation of mAb efficacy and potency. In addition, we found that anti-SEB antibodies showed similar efficacy and potency with a triple mutant Fc region (designed to be effector function null) or a wild-type Fc region, which is in contrast to previously described studies.

Temporal associations between interleukin 22 and the extracellular domains of IL-22R and IL-10R2.

Interleukin 22 (IL-22) is a cytokine induced during both innate and adaptive immune responses. It can effect an acute phase response, implicating a role for IL-22 in mechanisms of inflammation. IL-22 requires the presence of the IL-22 receptor (IL-22R) and IL-10 receptor 2 (IL-10R2) chains, two members of the class II cytokine receptor family (CRF2), to effect signal transduction within a cell. We studied the interaction between human IL-22 and the extracellular domains (ECD) of its receptor chains in an enzyme-linked immunoabsorbant assay (ELISA)-based format, using biotinylated IL-22 (bio-IL-22) and receptor-fusions containing the ECD of a receptor fused to the Fc of hIgG1 (IL-22R-Fc and IL-10R2-Fc). IL-22 has measurable affinity for IL-22R-Fc homodimer and undetectable affinity for IL-10R2. IL-22 has substantially greater affinity for IL-22R/IL-10R2-Fc heterodimers. Further analyses involving sequential additions of receptor homodimers and cytokine indicates that the IL-10R2(ECD) binds to a surface created by the interaction between IL-22 and the IL-22R(ECD), and thereby further stabilizes the association of IL-22 within this cytokine-receptor-Fc complex. Both a neutralizing rat monoclonal antibody, specific for human IL-22, and human IL-22BP-Fc, an Fc-fusion of the secreted IL-22 binding-protein and proposed natural antagonist for IL-22, bind to similar cytokine epitopes that may overlap the binding site for IL-22R(ECD). Another rat monoclonal antibody, specific for IL-22, binds to an epitope that may overlap a separate binding site for IL-10R2(ECD). We propose, based on this data, a temporal model for the development of a functional IL-22 cytokine-receptor complex.

Disposition of human recombinant lubricin in naive rats and in a rat model of post-traumatic arthritis after intra-articular or intravenous administration.

We have recently demonstrated that intra-articular (IA) administration of human recombinant lubricin, LUB:1, significantly inhibited cartilage degeneration and pain in the rat meniscal tear model of post-traumatic arthritis. In this report, we show that after a single IA injection to naïve rats and rats that underwent unilateral meniscal tear, [(125)I]LUB:1 had a tri-phasic disposition profile, with the alpha, beta, and gamma half-life estimates of 4.5 h, 1.5 days, and 2.1 weeks, respectively. We hypothesize that the terminal phase kinetics was related to [(125)I]LUB:1 binding to its ligands. [(125)I]LUB:1 was detected on articular cartilage surfaces as long as 28 days after single IA injection. Micro-autoradiography analysis suggested that [(125)I]LUB:1 tended to localize to damaged joint surfaces in rats with meniscal tear. After a single intravenous (IV) dose to rats, [(125)I]LUB:1 was eliminated rapidly from the systemic circulation, with a mean total body clearance of 154 mL/h/kg and a mean elimination half-life (t (1/2)) of 6.7 h. Overall, LUB:1 has met a desired disposition profile of a potential therapeutic intended for an IA administration: target tissue (knee) retention and fast elimination from the systemic circulation after a single IA or IV dose.

Prevention of cartilage degeneration in a rat model of osteoarthritis by intraarticular treatment with recombinant lubricin.

OBJECTIVE: Lubricin, also referred to as superficial zone protein and PRG4, is a synovial glycoprotein that supplies a friction-resistant, antiadhesive coating to the surfaces of articular cartilage, thereby protecting against arthritis-associated tissue wear and degradation. This study was undertaken to generate and characterize a novel recombinant lubricin protein construct, LUB:1, and to evaluate its therapeutic efficacy following intraarticular delivery in a rat model of osteoarthritis (OA). METHODS: Binding and localization of LUB:1 to cartilage surfaces was assessed by immunohistochemistry. The cartilage-lubricating properties of LUB:1 were determined using a custom friction testing apparatus. A cell-binding assay was performed to quantify the ability of LUB:1 to prevent cell adhesion. Efficacy studies were conducted in a rat meniscal tear model of OA. One week after the surgical induction of OA, LUB:1 or phosphate buffered saline vehicle was administered by intraarticular injection for 4 weeks, with dosing intervals of either once per week or 3 times per week. OA pathology scores were determined by histologic analysis. RESULTS: LUB:1 was shown to bind effectively to cartilage surfaces, and facilitated both cartilage boundary lubrication and inhibition of synovial cell adhesion. Treatment of rat knee joints with LUB:1 resulted in significant disease-modifying, chondroprotective effects during the progression of OA, by markedly reducing cartilage degeneration and structural damage. CONCLUSION: Our findings demonstrate the potential use of recombinant lubricin molecules in novel biotherapeutic approaches to the treatment of OA and associated cartilage abnormalities.

Oligomerization is required for the activity of recombinant soluble LOX-1.

LOX-1 is a scavenger receptor that functions as the primary receptor for oxidized low-density lipoprotein (OxLDL) in endothelial cells. The binding of OxLDL to LOX-1 is believed to lead to endothelial activation, dysfunction, and injury, which constitute early atherogenic events. Because of its potential pathological role in atherosclerosis, LOX-1 has been proposed as a therapeutic target for the treatment of this disease. In order to antagonize the ligand-binding function of cell surface LOX-1, we generated a series of recombinant human LOX-1-crystallizable fragment (Fc) fusion proteins and subsequently characterized their biochemical properties and ligand-binding activities in vitro. Consistent with the notion that oligomerization of cell surface LOX-1 is required for high-avidity binding of ligands, we found that LOX-1-Fc fusion protein containing four ligand-binding domains per Fc dimer, but not the one containing two ligand-binding domains, exhibited ligand-binding activity. Optimal ligand-binding activity could be achieved via crosslinking of LOX-1-Fc fusion proteins with a polyclonal antibody against Fc. The crosslinked LOX-1-Fc protein also effectively inhibited the binding and internalization of OxLDL by cell surface LOX-1. These findings demonstrate that functional oligomerization is required for recombinant LOX-1-Fc to function as an effective antagonist.