An ultrapotent RBD-targeted biparatopic nanobody neutralizes broad SARS-CoV-2 variants.

The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, convalescent patients derived natural antibodies are vulnerable to SARS-CoV-2 Spike mutation. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb, named Nb1-Nb2, with tight affinity and super-wide neutralization breadth against multiple SARS-CoV-2 variants of concern. Deep-mutational scanning experiments identify the potential binding epitopes of the Nbs on the RBD and demonstrate that biparatopic Nb1-Nb2 has a strong escape-resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that the Nb1-Nb2 broadly neutralizes multiple SARS-CoV-2 variants at sub-nanomolar levels, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1), and Mu (B.1.621). Furthermore, a heavy-chain antibody is constructed by fusing the human IgG1 Fc to Nb1-Nb2 (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability, and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0 × 10-12 M) and strong neutralizing activity (IC50 = 1.46 nM for authentic Omicron virus). Together, we developed a tetravalent biparatopic human heavy-chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.

Reteplase Fc-fusions produced in N. benthamiana are able to dissolve blood clots ex vivo.

Thrombolytic and fibrinolytic therapies are effective treatments to dissolve blood clots in stroke therapy. Thrombolytic drugs activate plasminogen to its cleaved form plasmin, a proteolytic enzyme that breaks the crosslinks between fibrin molecules. The FDA-approved human tissue plasminogen activator Reteplase (rPA) is a non-glycosylated protein produced in E. coli. rPA is a deletion mutant of the wild-type Alteplase that benefits from an extended plasma half-life, reduced fibrin specificity and the ability to better penetrate into blood clots. Different methods have been proposed to improve the production of rPA. Here we show for the first time the transient expression in Nicotiana benthamiana of rPA fused to the immunoglobulin fragment crystallizable (Fc) domain on an IgG1, a strategy commonly used to improve the stability of therapeutic proteins. Despite our success on the expression and purification of dimeric rPA-Fc fusions, protein instability results in high amounts of Fc-derived degradation products. We hypothesize that the "Y"- shape of dimeric Fc fusions cause steric hindrance between protein domains and leads to physical instability. Indeed, mutations of critical residues in the Fc dimerization interface allowed the expression of fully stable rPA monomeric Fc-fusions. The ability of rPA-Fc to convert plasminogen into plasmin was demonstrated by plasminogen zymography and clot lysis assay shows that rPA-Fc is able to dissolve blood clots ex vivo. Finally, we addressed concerns with the plant-specific glycosylation by modulating rPA-Fc glycosylation towards serum-like structures including α2,6-sialylated and α1,6-core fucosylated N-glycans completely devoid of plant core fucose and xylose residues.

Increasing Transfection Efficiency of Lipoplexes by Modulating Complexation Solution for Transient Gene Expression.

Transient gene expression is a suitable tool for the production of biopharmaceutical candidates in the early stage of development and provides a simple and rapid alternative to the generation of stable cell line. In this study, an efficient transient gene expression methodology using DC-Chol/DOPE cationic liposomes and pDNA in Chinese hamster ovary suspension cells was established through screening of diverse lipoplex formation conditions. We modulated properties of both the liposome formation and pDNA solution, together called complexation solutions. Protein expression and cellular cytotoxicity were evaluated following transfection over the cell cultivation period to select the optimal complexation solution. Changes in hydrodynamic size, polydispersity index, and ζ potential of the liposomes and lipoplexes were analyzed depending on the various pH ranges of the complexation solutions using dynamic light scattering. The transfer of lipoplexes to the cytosol and their conformation were traced using fluorescence analysis until the early period of transfection. As a result, up to 1785 mg/L and 191 mg/L of human Fc protein and immunoglobulin G (bevacizumab), respectively, were successfully produced using acidic liposome formation and alkaline pDNA solutions. We expect that this lipoplex formation in acidic and alkaline complexation solutions could be an effective methodology for a promising gene delivery strategy.

Generation and characterization of a high-affinity chimeric anti-OX40 antibody with potent antitumor activity.

OX40 is a costimulatory molecule that belongs to the tumor necrosis factor receptor (TNFR) superfamily. OX40 agonist-based combinations are emerging as promising candidates for novel cancer immunotherapy. Clinical trials have shown that OX40 agonist antibodies could lead to better results in cancer patients. Using a hybridoma platform and three different types of immunization strategies, namely recombinant protein, DNA, and overexpressing cells, we identified a chimeric anti-OX40 antibody (mAb035-hIgG1 from DNA immunization) that shows excellent binding specificity, and slightly stronger activation of human memory CD4+ T cells and similar potent antitumor activity compared with BMS 986178, an anti-OX40 antibody currently being evaluated for the treatment of solid tumors. This paper further systematically investigates the antigen-specific immune response, the number of binders, epitope bins, and functional activities of antibodies among different immunization strategies. Interestingly, we found that different immunization strategies affect the biological activity of monoclonal antibodies.

High-level expression of human CH2 domain from the Fc region in Pichia pastoris and preparation of anti-CH2 antibodies.

Pichia pastoris is a popular eukaryotic system employed for the fast, simple and inexpensive production of recombinant protein including biotherapeutics such as human albumin. The CH2 domain of human Immunoglobulin G (IgG) is a promising scaffold for developing novel therapeutics. To accelerate the research of CH2 domain, we have established a procedure to highly express human CH2 domain (∼150 mg/l) as well as human Fc (∼30 mg/l) in yeast P. pastoris. The procedure yields, simultaneously, a major glycosylated (∼70%) and non-glycosylated (∼30%) fractions. They can be easily separated with high purity. Although both forms of CH2 domain have essentially the same secondary structure, the presence of the glycan increased the thermal stability of the CH2 domain by about 5°C as determined from calorimetry. The purified glycosylated CH2 domain elicited polyclonal antibodies in mouse, recognizing not only the CH2 domain, but also recombinant human Fc and the commercial IgG1 antibody Rituxan. Protein A and Protein G binding to the kink region between CH2 domain and CH3 domain of human Fc are used to purify therapeutic proteins. Therefore, these antibodies are candidates to develop a novel affinity material to purify human antibodies using their CH2 domain.

Production of a novel heterodimeric two-chain insulin-Fc fusion protein.

Insulin is a peptide hormone produced by the pancreas. The physiological role of insulin is the regulation of glucose metabolism. Under certain pathological conditions the insulin levels can be reduced leading to the metabolic disorder diabetes mellitus (DM). For type 1 DM and, dependent on the disease progression for type 2 DM, insulin substitution becomes indispensable. To relieve insulin substitution therapy for patients, novel insulin analogs with pharmacokinetic and pharmacodynamic profiles aiming for long-lasting or fast-acting insulins have been developed. The next step in the evolution of novel insulins should be insulin analogs with a time action profile beyond 1-2 days, preferable up to 1 week. Nowadays, insulin is produced in a recombinant manner. This approach facilitates the design and production of further insulin-analogs or insulin-fusion proteins. The usage of the Fc-domain from immunoglobulin as a fusion partner for therapeutic proteins and peptides is widely used to extend their plasma half-life. Insulin consists of two chains, the A- and B-chain, which are connected by two disulfide-bridges. To produce a novel kind of Fc-fusion protein we have fused the A-chain as well as the B-chain to Fc-fragments containing either 'knob' or 'hole' mutations. The 'knob-into-hole' technique is frequently used to force heterodimerization of the Fc-domain. Using this approach, we were able to produce different variants of two-chain-insulin-Fc-protein (tcI-Fc-protein) variants. The tcI-Fc-fusion variants retained activity as shown in in vitro assays. Finally, prolonged blood glucose lowering activity was demonstrated in normoglycemic rats. Overall, we describe here the production of novel insulin-Fc-fusion proteins with prolonged times of action.

A novel efficient bispecific antibody format, combining a conventional antigen-binding fragment with a single domain antibody, avoids potential heavy-light chain mis-pairing.

Due to the technical innovations in generating bispecific antibodies (BsAbs) in recent years, BsAbs have become important reagents for diagnostic and therapeutic applications. However, the difficulty of producing a heterodimer consisting of two different arms with high yield and purity constituted a major limitation for their application in academic and clinical settings. Here, we describe a novel Fc-containing BsAb format (Fab × sdAb-Fc) composed of a conventional antigen-binding fragment (Fab), and a single domain antibody (sdAb), which avoids heavy-light chain mis-pairing during antibody assembly. In this study, the Fab x sdAb-Fc BsAbs were efficiently produced by three widely used heavy-heavy chain heterodimerization methods: Knobs-into-holes (KIH), Charge-pairs (CP) and controlled Fab-arm exchange (cFAE), respectively. The novel Fab x sdAb-Fc format provided a rapid and efficient strategy to generate BsAb with high purity and a unique possibility to further purify desired BsAbs from undesired antibodies based on molecular weight (MW). Compared to conventional BsAb formats, the advantages of Fab x sdAb-Fc format may thus provide a straightforward opportunity to apply bispecific antibody principles to research and development of novel targets and pathways in diseases such as cancer and autoimmunity.

Fusion of pseudorabies virus glycoproteins to IgG Fc enhances protective immunity against pseudorabies virus.

Molecular adjuvants are vaccine delivery vehicle to increase specific antigens effectiveness. Herein, we concentrated on IgG Fc, an effective molecular adjuvant, to develop novel pseudorabies virus (PRV) subunit vaccines. Two major protective antigen genes of PRV were constructed and linked into the mouse IgG Fc fragment. The gD, gD-IgG2aFc, gB and gB-IgG2aFc proteins were expressed using a baculovirus system. Mice intranasally immunized with gD-IgG2aFc or gB-IgG2aFc subunit vaccine exhibited significantly higher PRV-specific antibodies, neutralizing antibodies and intracellular cytokines than the mice intranasally immunized with gD or gB subunit vaccine. Moreover, no histopathological lesions were observed in mice immunized with gB-IgG2aFc subunit vaccine via histopathology examination. Further, the gB-IgG2aFc subunit vaccine was efficient for PRV infection compared with live attenuated vaccine. Overall, these results suggest that IgG2a Fc fragment, as a potential molecular adjuvant, fused with PRV antigen might be a promising and efficient PRV vaccine candidate.

Characterization of intact glycopeptides reveals the impact of culture media on site-specific glycosylation of EPO-Fc fusion protein generated by CHO-GS cells.

With the increasing demand to provide more detailed quality attributes, more sophisticated glycan analysis tools are highly desirable for biopharmaceutical manufacturing. Here, we performed an intact glycopeptide analysis method to simultaneously analyze the site-specific N- and O-glycan profiles of the recombinant erythropoietin Fc (EPO-Fc) protein secreted from a Chinese hamster ovary glutamine synthetase stable cell line and compared the effects of two commercial culture media, EX-CELL (EX) and immediate advantage (IA) media, on the glycosylation profile of the target protein. EPO-Fc, containing the Fc region of immunoglobulin G1 (IgG1) fused to EPO, was harvested at Day 5 and 8 of a batch cell culture process followed by purification and N- and O-glycopeptide profiling. A mixed anion exchange chromatographic column was implemented to capture and enrich N-linked glycopeptides. Using intact glycopeptide characterization, the EPO-Fc was observed to maintain their individual EPO and Fc N-glycan characteristics in which the EPO region presented bi-, tri-, and tetra-branched N-glycan structures, while the Fc N-glycan displayed mostly biantennary glycans. EPO-Fc protein generated in EX medium produced more complex tetra-antennary N-glycans at each of the three EPO N-sites while IA medium resulted in a greater fraction of bi- and tri-antennary N-glycans at these same sites. Interestingly, the sialylation content decreased from sites 1-4 in both media while the fucosylation progressively increased with a maximum at the final IgG Fc site. Moreover, we observed that low amounts of Neu5Gc were detected and the content increased at the later sampling time in both EX and IA media. For O-glycopeptides, both media produced predominantly three structures, N1F1F0SOG0, N1H1F0S1G0, and N1H1F0S2G0, with lesser amounts of other structures. This intact glycopeptide method can decipher site-specific glycosylation profile and provide a more detailed characterization of N- and O-glycans present for enhanced understanding of the key product quality attributes such as media on recombinant proteins of biotechnology interest.

Reducing recombinant protein expression during CHO pool selection enhances frequency of high-producing cells.

Chinese hamster ovary (CHO) cells are the most widely used mammalian host for industrial-scale production of monoclonal antibodies (mAbs) and other protein biologics. Isolation of rare high-producing CHO cell lines from heterogeneous populations of stable transfectants is a daunting task and delays the process of manufacturing of novel biologics. A variety of factors that contribute to the low frequency of high-producing clones have been described; however, the impact of metabolic burden and other stresses (eg. ER stress) associated with sustained high-level expression of recombinant protein (r-protein) during selection of stable transfectants has not been fully appreciated. CHO cell line development has not traditionally received much optimization in this area because the vast majority of platforms use constitutive expression systems to produce biologics. Previously, we developed a cell line (CHOBRI/rcTA) containing a robust inducible expression system, based on the cumate gene switch, that allows r-protein expression to be down-regulated during selection. Using this switch, we generated inducible CHOBRI/rcTA pools expressing an Fc-fusion protein within two weeks of transfection with volumetric productivity of up to 1.1 g/L at 17 days post-induction in a fed-batch culture process. Herein, we show that the ability to regulate r-protein expression during pool generation confers a substantial advantage for selecting high-producing stable clones. Reducing expression levels ("off-state") during pool selection dramatically enhances high-producer frequency compared to a pool in which expression was maintained at a high level during selection ("on-state", mimicking a constitutive expression system). Overexpression of the r-protein during the pool selection process negatively affects pool recovery and is associated with subtle but significant increases in BiP expression and cell death compared to pool selection in the "off-state". Our data shows that the cumate gene switch is a valuable platform for stable clone generation and supports the wider application of inducible systems for scalable production of biologics in CHO cells.

Generation of site-distinct N-glycan variants for in vitro bioactivity testing.

Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed-batch cell culture batches yielded consistent glycoprofiles of a Fc-fusion antibody comprizing three different N-glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N-glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site-specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the FcγRIIIa receptor binding affinity and the biological activity (cell-based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N-glycan structures, a higher degree of antennarity, and a site-specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc-site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased FcγRIIIa receptor binding affinity. All N-glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity.

Expression of 5T4 extracellular domain fusion protein and preparation of anti-5T4 monoclonal antibody with high affinity and internalization efficiency.

5T4, a membrane protein, is overexpressed in many tumor tissues but rarely expressed in normal tissues. Here, CHO-5T4+ cells were generated and served as the antigen to immunize mice. Hybridoma techniques were employed to produce monoclonal antibodies (mAbs). The recombinant protein of human IgG Fc-fused extracellular domain of 5T4 (5T4 ECD-Fc) was obtained from transient expression in HEK293F cells. The fusion protein 5T4 ECD-Fc and CHO-5T4+ cells were respectively utilized to screen anti-5T4 antibodies that could bind to the native antigen. In preliminary screening, three hundred and fifty mAbs were obtained. Via surface plasmon resonance and flow cytometry screening, seven anti-5T4 mAbs stood out. Among them, H6 showed a high affinity (KD = 1.6 × 10-11 M) and internalization percentage (36% for 1 h and 80% for 4 h). The molecular weight and isoelectric point of H6 were determined by LC-MS and iCIEF. Moreover, the specific reactivity of H6 was demonstrated by western blotting, flow cytometry, and immunohistochemistry, respectively. In conclusion, we produced human recombinant protein of 5T4 extracellular domain and developed high-affinity internalizing monoclonal antibodies which may be applied in the 5T4-targeting ADC therapy and basic research.

Effects of amino acid substitutions on the biological activity of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori).

Recombinant monoclonal antibodies (mAbs) have been used in various therapeutic applications including cancer therapy. Fc-mediated effector functions play a pivotal role in the tumor-killing activities of some tumor-targeting mAbs, and Fc-engineering technologies with glyco-engineering or amino acid substitutions at the antibody Fc region have been used to enhance cytotoxic activities including antibody-dependent cellular cytotoxicity (ADCC). We previously reported that the mAbs produced using transgenic silkworms showed stronger ADCC activity and lower complement-dependent cytotoxicity (CDC) activity than mAbs derived from Chinese hamster ovary (CHO) cells due to their unique N-glycan structure (lack of core-fucose and non-reducing terminal galactose). In this study, we generated anti-CD20 mAbs with amino acid substitutions using transgenic silkworms and analyzed their biological activities to assess the effect of the combination of glyco-engineering and amino acid substitutions on the Fc-mediated function of mAbs. Three types of amino acid substitutions at the Fc region (G236A/S239D/I332E, L234A/L235A, and K326W/E333S) modified the Fc-mediated biological activities of silkworm-derived mAbs as in the case of CHO-derived mAbs, resulting in the generation of Fc-engineered mAbs with characteristic Fc-mediated functions. The combination of amino acid substitutions at the Fc region and glyco-engineering using transgenic silkworm made it possible to generate Fc-engineered mAbs with suitable Fc-mediated biological functions depending on the pharmacological mechanism of their actions. Transgenic silkworms were shown to be a promising system for the production of Fc-engineered mAbs.

Mechanisms of precipitate formation during the purification of an Fc-fusion protein.

Protein precipitates that arise during bioprocessing can cause manufacturing challenges, but they can also aid in clearance of host-cell protein (HCP) and DNA impurities. Such precipitates differ from many protein precipitates that have been studied previously in their heterogeneous composition, particularly in the presence of high concentrations of the product protein. Here, we characterize the precipitates that form after neutralization of protein A purified and viral-inactivated material of an Fc-fusion protein produced in Chinese hamster ovary cells. The physical growth of precipitate particles was observed by optical microscopy, transmission electron microscopy, dynamic light scattering, and small-angle and ultra-small-angle X-ray scattering to characterize the precipitate microstructure and growth mechanism. The precipitate microstructure is well-described as a mass fractal with fractal dimension approximately 2. The growth is governed by a diffusion-limited aggregation mechanism as indicated by a power-law dependence on time of the size of the principal precipitate particles. Optical microscopy shows that these primary particles can further aggregate into larger particles in a manner that appears to be promoted by mixing. Absorbance experiments at varying pH and salt concentrations reveal that the growth is largely driven by attractive electrostatic interactions, as growth is hindered by an increase in ionic strength. The solution conditions that resulted in the most significant particle growth are also correlated with the greatest removal of soluble impurities (DNA and HCPs). Proteomic analysis of the precipitates allows identification of O ( 100 ) unique HCP impurities, depending on the buffer species (acetate or citrate) used for the viral inactivation. Most of these proteins have pI values near the precipitation pH, supporting the likely importance of electrostatic interactions in driving precipitate formation.

Rapid Purification of Human Bispecific Antibodies via Selective Modulation of Protein A Binding.

Methods to rapidly generate high quality bispecific antibodies (BsAb) having normal half-lives are critical for therapeutic programs. Here, we identify 3 mutations (T307P, L309Q, and Q311R or "TLQ") in the Fc region of human IgG1 which disrupt interaction with protein A while enhancing interaction with FcRn. The mutations are shown to incrementally alter the pH at which a mAb elutes from protein A affinity resin. A BsAb comprised of a TLQ mutant and a wild-type IgG1 can be efficiently separated from contaminating parental mAbs by differential protein A elution starting from either a) purified parental mAbs, b) in-supernatant crossed parental mAbs, or c) co-transfected mAbs. We show that the Q311R mutation confers enhanced FcRn interaction in vitro, and Abs harboring either the Q311R or TLQ mutations have serum half-lives as long as wild-type human IgG1. The mutant Abs have normal thermal stability and Fcγ receptor interactions. Together, the results lead to a method for high-throughput generation of BsAbs suitable for in vivo studies.

Designing Fcabs: well-expressed and stable high affinity antigen-binding Fc fragments.

Fc fragment with antigen-binding (Fcab) is a novel construct which can be selected to recognize specifically a wide variety of target proteins. We describe the selection and affinity maturation of Fcab clones targeting VEGF, an important pro-angiogenesis factor. To investigate the extent of engineering permissible to Fcabs we applied targeted mutagenesis to all three C-terminal loop structures and the C-terminus of the CH3 domain to isolate high-affinity binders by directed evolution and yeast display. The matured clone, CT6, binds to VEGF with low nanomolar affinity and inhibits VEGF-stimulated proliferation of human umbilical vein endothelial cells in vitro. Molecular dynamics simulations were performed to address flexibility of the molecular structure of CT6 and to approximate a structural ensemble in aqueous solution. Significantly higher RMSF levels of CT6 in comparison to wild-type Fc were limited to the elongated CD-loop in the CH3 domain, while the overall structural integrity was retained. This allowed the Fcab to replace the Fc portion of a mAb, in which both the CH3 and Fab are capable of antigen engagement: a construct called mAb2 was assembled with CT6 and the Fab of bevacizumab. This bispecific molecule showed more potent antagonistic activity than bevacizumab in vitro. Further evaluation for the potential of the CT6 Fcab in targeted therapy is warranted due to the possibility of being combined with other therapeutically meaningful targets.

5B9, a monoclonal antiplatelet factor 4/heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies.

Essentials No humanized monoclonal antibody was available to study heparin-induced thrombocytopenia (HIT). We developed the first anti-platelet factor 4 (PF4)/heparin antibody with a human Fc fragment. This antibody (5B9) fully mimics the effects of human HIT antibodies. 5B9 binds two regions within PF4 that may be critical for the pathogenicity of HIT antibodies. SUMMARY: Background The diagnosis of heparin-induced thrombocytopenia (HIT) is based on clinical and biological criteria, but a standard is lacking for laboratory assays. Moreover, no humanized HIT antibody is available for pathophysiological studies. Objective To characterise 5B9, a chimeric monoclonal antibody, which fully mimics the effects of human HIT antibodies. Methods/Results 5B9, a chimeric anti-platelet factor 4/heparin complexes IgG1 antibody, was obtained after immunizing specific transgenic mice. 5B9 induced heparin FcγRIIA-dependent platelet aggregation and tissue factor mRNA synthesis in monocytes. It also induced significant thrombocytopenia and thrombin generation in mice expressing human PF4 and FcγRIIA receptors. The binding of 5B9 to PF4/H complexes was inhibited by 15 of 25 HIT plasma samples and only three of 25 samples containing non-pathogenic anti-PF4/H antibodies. KKO, a murine IgG2b HIT antibody, also inhibited the binding of 5B9 to PF4/H, suggesting that epitopes recognized by both antibodies are close. A docking analysis based on VH and VL sequences of 5B9 showed that binding of 5B9 Fab to PF4 involved 12 and 12 residues in B and D monomers, respectively, including seven previously identified as critical to the formation of a PF4/KKO complex. Two regions (Asp-7 to Thr-15 and Ala-32 to Thr-38) therefore appeared important for the binding of 5B9 and KKO on PF4 modified by heparin. Conclusions 5B9 is the first anti-PF4/H monoclonal antibody with a human Fc fragment, which induces similar cellular activation as HIT antibodies. Moreover, 5B9 binds epitopes within PF4 that are likely to be critical for the pathogenicity of HIT antibodies.

Human Anti-Lipopolysaccharid (LPS) antibodies against Legionella with high species specificity.

Legionella are Gram-negative bacteria that are ubiquitously present in natural and man-made water reservoirs. When humans inhale aerosolized water contaminated with Legionella, alveolar macrophages can be infected, which may lead to a life-threatening pneumonia called Legionnaires' disease. Due to the universal distribution of Legionella in water and their potential threat to human health, the Legionella concentration in water for human use must be strictly monitored, which is difficult since the standard detection still relies on lengthy cultivation and analysis of bacterial morphology. In this study, an antibody against L. pneumophila has been generated from the naïve human HAL antibody libraries by phage-display for the first time. The panning was performed on whole bacterial cells in order to select antibodies that bind specifically to the cell surface of untreated Legionella. The bacterial cell wall component lipopolysaccharide (LPS) was identified as the target structure. Specific binding to the important pathogenic L. pneumophila strains Corby, Philadelphia-1 and Knoxville was observed, while no binding was detected to seven members of the families Enterobacteriaceae, Pseudomonadaceae or Clostridiaceae. Production of this antibody in the recombinant scFv-Fc format using either a murine or a human Fc part allowed the set-up of a sandwich-ELISA for detection of Legionella cells. The scFv-Fc construct proved to be very stable, even when stored for several weeks at elevated temperatures. A sensitivity limit of 4,000 cells was achieved. The scFv-Fc antibody pair was integrated on a biosensor, demonstrating the specific and fast detection of L. pneumophila on a portable device. With this system, 10,000 Legionella cells were detected within 35 min. Combined with a water filtration/concentration system, this antibody may be developed into a promising reagent for rapid on-site Legionella monitoring.

Effective strategies for host cell protein clearance in downstream processing of monoclonal antibodies and Fc-fusion proteins.

Recombinant therapeutic proteins are typically produced through cell culture process. Host cell proteins (HCPs) are endogenous proteins derived from the host cells used for such bioproduction. HCPs form a major class of process-related impurities and even at low levels they can potentially compromise the safety and efficacy of biopharmaceuticals. Therefore, they need to be adequately removed via the downstream process. HCPs are complex mixtures with diverse physiochemical properties, and certain subpopulations can bind to the intended product. Hence reducing them to the generally accepted level can be challenging. This article reviews effective HCP removing strategies at different stages of downstream process for monoclonal antibodies and Fc-fusion proteins. When used in combination, these strategies can greatly enhance the chance of meeting the drug substance specifications for residual HCP.

The Role of Neutrophil Proteases in LPS-Induced Production of Regulatory Rheumatoid Factor that Suppresses Autoimmunity.

BACKGROUND: We recently identified a regulatory rheumatoid factor (regRF), the production of which is associated with autoimmune disease resistance and remission. In studies of regRF in the blood of healthy rats, spontaneous increases in the regRF level were noted. We suggest that in the normal state, a mechanism exists for maintaining the activity of the pool of regRF-producing lymphocytes at a level that makes it possible to control the expansion of autoreactive lymphocytes. OBJECTIVE: The purpose of this study was to test the hypothesis that the endogenous stimulator of regRF production is Fc fragments of IgG that are formed upon exposure to the proteases of neutrophils. RESULTS: Injection of Salmonella typhi LPS caused neutrophilic leukocytosis in the rats, followed by elevated level of regRF. Neutrophils were obtained from LPS-treated rats and then treated with LPS in vitro to degranulate them to form pre-split IgG that exposes antigenic determinants for regRF. A condition required for Fc fragments to be formed by neutrophils is that the pre-split IgG must be treated with a thiol reducing agent. Antigenic determinants for regRF were retained by Fc fragments of IgG. CONCLUSION: Thus, the pre-split IgG and Fc fragments of IgG formed by LPS-activated neutrophils are the potential physiological activators of regRF production.