Search

Monoclonal antibodies (mAbs) that efficiently neutralize SARS-CoV-2 have been developed at an unprecedented speed. Notwithstanding, there is a vague understanding of the various Ab functions induced beyond antigen binding by the heavy-chain constant domain. To explore the diverse roles of Abs in SARS-CoV-2 immunity, we expressed a SARS-CoV-2 spike protein (SP) binding mAb (H4) in the four IgG subclasses present in human serum (IgG1-4) using glyco-engineered Nicotiana benthamiana plants. All four subclasses, carrying the identical antigen-binding site, were fully assembled in planta and exhibited a largely homogeneous xylose- and fucose-free glycosylation profile. The Ab variants ligated to the SP with an up to fivefold increased binding activity of IgG3. Furthermore, all H4 subtypes were able to neutralize SARS-CoV-2. However, H4-IgG3 exhibited an up to 50-fold superior neutralization potency compared with the other subclasses. Our data point to a strong protective effect of IgG3 Abs in SARS-CoV-2 infection and suggest that superior neutralization might be a consequence of cross-linking the SP on the viral surface. This should be considered in therapy and vaccine development. In addition, we underscore the versatile use of plants for the rapid expression of complex proteins in emergency cases., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

While achieving rapid developments in recent years, bispecific antibodies are still difficult to design and manufacture, due to mispair of both heavy and light chains. Here we report a novel technology to make bispecific molecules. The knob-into-hole method was used to pair two distinct heavy chains as a heterodimer. IgG 4 S228P CH1-CL interface was then partially replaced by T-cell receptor α/β constant domain to increase the efficiency of cognate heavy and light chain pairing. Following expression and purification, the bispecific antibody interface exchange was confirmed by Western blotting and LC-MS/MS. To ensure its validity, we combined a monovalent bispecific antibody against PD-1 (sequence from Pembrolizumab) and LAG3 (sequence from Relatlimab). The results showed that the molecule could be assembled correctly at a ratio of 95% in cells. In vitro functional assay demonstrated that the purified bispecific antibody exhibits an enhanced agonist activity compared to that of the parental antibodies. Low immunogenicity was predicted by an open-access software and ADA test., (© 2021. The Author(s).)

There has been increasing momentum recently in the biopharmaceutical industry to transition from traditional batch processes to next-generation integrated and continuous biomanufacturing. This transition from batch to continuous is expected to offer several advantages which, taken together, could significantly improve access to biologics drugs for patients. Despite this recent momentum, there has not been a commercial implementation of a continuous bioprocess reported in the literature. In this study, we describe a successful pilot-scale proof-of-concept demonstration of an end-to-end integrated and continuous bioprocess for the production of a monoclonal antibody (mAb). This process incorporated all of the key unit operations found in a typical mAb production process, including the final steps of virus removal filtration, ultrafiltration, diafiltration, and formulation. The end-to-end integrated process was operated for a total of 25 days and produced a total of 4.9 kg (200 g/day or 2 g/L BRX/day) of the drug substance from a 100-L perfusion bioreactor (BRX) with acceptable product quality and minimal operator intervention. This successful proof-of-concept demonstrates that end-to-end integrated continuous bioprocessing is achievable with current technologies and represents an important step toward the realization of a commercial integrated and continuous bioprocessing process., (© 2021 Wiley Periodicals LLC.)

A high degree of charge heterogeneity is an unfavorable phenomenon commonly observed for therapeutic monoclonal antibodies (mAbs). Removal of these impurities during manufacturing often comes at the cost of impaired step yields. A wide spectrum of posttranslational and chemical modifications is known to modify mAb charge. However, a deeper understanding of underlying mechanisms triggering charged species would be beneficial for the control of mAb charge variants during bioprocessing. In this study, a comprehensive analytical investigation was carried out to define the root causes and mechanisms inducing acidic variants of an immunoglobulin G1-derived mAb. Characterization of differently charged species by liquid chromatography-mass spectrometry revealed the reduction of disulfide bonds in acidic variants, which is followed by cysteinylation and glutathionylation of cysteines. Importantly, biophysical stability and integrity of the mAb are not affected. By in vitro incubation of the mAb with the reducing agent cysteine, disulfide bond degradation was directly linked to an increase of numerous acidic species. Modifying the concentrations of cysteine during the fermentation of various mAbs illustrated that redox potential is a critical aspect to consider during bioprocess development with respect to charge variant control., (© 2020 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Hybridoma technology has been valuable in the development of therapeutic antibodies. More recently, antigen-specific B-cell selection and display technologies are also gaining importance. A major limitation of these approaches used for antibody discovery is the extensive process of cloning and expression involved in transitioning from antibody identification to validating the function, which compromises the throughput of antibody discovery. In this study, we describe a process to identify and rapidly re-format and express antibodies for functional characterization. We used two different approaches to isolate antibodies to five different targets: 1) flow cytometry to identify antigen-specific single B cells from the spleen of immunized human immunoglobulin transgenic mice; and 2) panning of phage libraries. PCR amplification allowed recovery of paired V H and V L sequences from 79% to 96% of antigen-specific B cells. All cognate V H and V L transcripts were formatted into transcription and translation compatible linear DNA expression cassettes (LEC) encoding whole IgG or Fab. Between 92% and 100% of paired V H and V L transcripts could be converted to LECs, and nearly 100% of them expressed as antibodies when transfected into Expi293F cells. The concentration of IgG in the cell culture supernatants ranged from 0.05 µg/ml to 145.8 µg/ml (mean = 18.4 µg/ml). Antigen-specific binding was displayed by 78-100% of antibodies. High throughput functional screening allowed the rapid identification of several functional antibodies. In summary, we describe a plasmid-free system for cloning and expressing antibodies isolated by different approaches, in any format of choice for deep functional screening that can be applied in any research setting during antibody discovery.

During preclinical studies, there is a great need to develop monoclonal antibodies (mAbs) that are specific to human immunoglobulin (IgG), without binding to monkey IgG, to detect therapeutic human mAb in non-human primates. We took advantage of the latest rabbit B cell cloning technology to develop six unique rabbit anti-human IgG mAb clones for this purpose. These clones are capable of binding to both human IgG and Fab with high affinity without nonspecific binding to cynomolgus monkey IgG. These clones have been evaluated as a generic capture reagent for the detection of human IgG and Fab, in the presence of cynomolgus monkey serum, by Gyrolab™ immunoassay. They may be used in singlet or as pairs for the detection of human IgG, in any host animal, to meet the need for therapeutic mAb development in preclinical studies.

High priority stereospecific targeting (SST) featuring selective production of conformation-specific monoclonal antibodies was directed against a native receptor, EphA2 (ephrin type-A receptor 2). A critical point for this technology is selection of sensitized B lymphocytes by antigen-expressing myeloma cells through their B-cell receptors (BCRs). The essential point is that antigens expressed on myeloma cells retain their original three dimensional structures and only these are recognized. Immunization with recombinant plasmid vectors as well as antigen-expressing CHO cells elicits enhanced sensitization of target B lymphocytes generating stereospecific antibodies. More than 24% of hybridoma-positive wells were identified to be cell-ELISA positive, confirming high efficiency. IgG-typed conformation-specific monoclonal antibodies could be also produced by the SST technique. Immunofluorescence analysis confirmed specific binding of sensitized B lymphocytes to antigen-expressing myeloma cells. Furthermore, stereospecific monoclonal antibodies to EphA2 specifically recognized EphA2-expressing cancer cells as demonstrated by Cell-ELISA. In the present study, we were able to develop priority technology for selective production of conformation-specific monoclonal antibodies against an intact receptor EphA2, known to be overexpressed by epithelial tumor cells of multiple cancer types., Competing Interests: Declaration of Competing Interest This work was supported in part by JSPS KAKENHI grant numbers JP24360339, JP25630376 and JP17H03468 as well as the Tosoh Corporation. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript other than those disclosed., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective To prepare a monoclonal antibody (mAb) against human CD33 by immunizing mice with recombinant vector and analyze its characteristics and clinical application. Methods The eukaryotic expression vector pcDNA3.1(+)/CD33 was constructed and used to immunize mice. The mouse monoclonal antibody against human CD33 was then harvested using the hybridoma technique. Its properties were evaluated and the clinical performance was validated. Results One hybridoma cell line capable of secreting mouse anti-human CD33 monoclonal antibody was successfully obtained, which was named HI33a for clone identification with a subclass of IgG2a, κ. Flow Cytometry analysis revealed that the antibody could stain myeloid cell lines but not lymphoid cell lines, and it could inhibit the binding of similar imported antibodies with HL-60 cells competitively. Western blotting verified that it could bind a M r 67 000 membrane protein extracted from HL-60 cells, which was a strong indication of the characteristics of CD33 protein molecule. Labeled with PE fluorescein, CD33-PE was tested as an antibody reagent in comparison with other similar imported products. Its overall performance including the accuracy, linearity, and precision all met the industrial standard. Further clinical evaluation of 558 bone marrow samples showed that the results were highly consistent with those by the imported reagents used as controls. Conclusion A hybridoma cell line stably secreting anti-human CD33 mAb was prepared.

In the past decades, monoclonal antibodies have made an unprecedented transformation from research tools to a rapidly growing class of therapeutics. Advancements in the yeast surface display platform enable the selection of favorable mouse or human antibody variants from large B-cell receptor (BCR) gene repertoires that are derived from immunized normal or transgenic animals. Application of high-throughput fluorescence-activated cell sorting (FACS) screening along with well-chosen selection settings can be utilized to identify variants with desired affinities and predefined epitope binding properties. In the following chapter, we describe in detail a multiparameter screening protocol for the selection of antibody variants from yeast libraries generated from BCR gene repertoires from immunized transgenic rats. The procedure provides guidance for the selection of antigen-specific, high-affinity binding, and species cross-reactive human antibodies with a broad epitope coverage. Essentially, this can accelerate target-specific antibody characterization as multiple desirable antibody features can be easily integrated into the selection procedure. In addition, we provide information on how to validate binding behavior of selected candidates after expression as soluble, full-length IgG molecules.

During the manufacturing of protein biologics, product variability during cell culture production and harvest needs to be actively controlled and monitored to maintain acceptable product quality. To a large degree, variants that have previously been described are covalent in nature and are easily analyzed by a variety of techniques. Here, we describe a noncovalent post translational modification of recombinantly expressed antibodies, containing variable domain tryptophans, that are exposed to culture media components and ambient laboratory light. The modified species, designated as conformer, can be monitored by hydrophobic interaction chromatography and often exhibits reduced potency. We studied conformer formation and identified key elements driving its accelerated growth using an IgG2 monoclonal antibody. Conformer is a result of a noncovalent interaction of the antibody with riboflavin, an essential vitamin added to many production cell culture formulations. Chemical and physical factors that influence the impact of riboflavin are identified, and methods for process control of this product quality attribute are addressed in order to prevent loss of antibody potency and potential safety issues. Identifying therapeutic antibody drug candidates with the potential to form conformers can be performed early in development to avoid this undesirable product quality propensity., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The structure of N-linked glycosylation is a very important quality attribute for therapeutic monoclonal antibodies. Different carbon sources in cell culture media, such as mannose and galactose, have been reported to have different influences on the glycosylation patterns. Accurate prediction and control of the glycosylation profile are important for the process development of mammalian cell cultures. In this study, a mathematical model, that we named Glycan Residues Balance Analysis (GReBA), was developed based on the concept of Elementary Flux Mode (EFM), and used to predict the glycosylation profile for steady state cell cultures. Experiments were carried out in pseudo-perfusion cultivation of antibody producing Chinese Hamster Ovary (CHO) cells with various concentrations and combinations of glucose, mannose and galactose. Cultivation of CHO cells with mannose or the combinations of mannose and galactose resulted in decreased lactate and ammonium production, and more matured glycosylation patterns compared to the cultures with glucose. Furthermore, the growth rate and IgG productivity were similar in all the conditions. When the cells were cultured with galactose alone, lactate was fed as well to be used as complementary carbon source, leading to cell growth rate and IgG productivity comparable to feeding the other sugars. The data of the glycoprofiles were used for training the model, and then to simulate the glycosylation changes with varying the concentrations of mannose and galactose. In this study we showed that the GReBA model had a good predictive capacity of the N-linked glycosylation. The GReBA can be used as a guidance for development of glycoprotein cultivation processes., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Single-use bioreactors have increasingly been used in recent years, for both research and development as well as industrial production, especially in mammalian cell-based processes. Among the numerous single-use bioreactors available today, wave-mixed bags and stirred systems dominate. Wave-mixed single-use bioreactors are the system of choice for inoculum production, while stirred single-use bioreactors are most often preferred for antibody expression. For this reason, the present chapter describes protocols instructing the reader to use the wave-mixed BIOSTAT ® RM 50 for cell expansion and to produce a monoclonal antibody (mAb) in Pall's Allegro™ STR 200 at pilot scale for the first time. All methods described are based on a Chinese hamster ovary (CHO) suspension cell line expressing a recombinant immunoglobulin G (IgG).

Background: Immunoglobulin (Ig) G is the most commonly used therapeutic antibodies. Recently, the interest in IgA antibodies to treat respiratory infectious diseases has been increasing. The reason for the inefficient use of IgA is recombinant antibody aggregation in cell culture, affecting the longevity and productivity of cell lines. Lactate is an important metabolite that affects the cultivation of stable cell lines producing monoclonal antibodies., Methods: In the present study, we investigated whether different combinations of succinic acid and micro-additives affect lactate production, which correlates with productivity. The effect of succinic acid substitution on productivity of cells producing IgG/IgA was analyzed using the static culture method in a six-well plate. Lactate was measured in supernatant of cell culture indirectly by using the activity of Lactate Dehydrogenase (LDH).A low lactate level was observed in cultivation medium supplemented with succinic acid or asparagine combined with some inorganic salts., Results: The results also demonstrated the effect of component supplementation on homogeneity, longevity, and productivity of cell culture. Supplementation of succinic acid eliminated cell aggregation and improved homogeneity of stable cell lines producing IgG and, especially, IgA., Conclusion: Overall, succinic acid supplementation to the culture medium has potential biotechnological applications in the production IgG and IgA., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The guinea pig serves as a useful animal model for a number of human diseases and has played an important role during development and testing of experimental vaccines and disease therapies. However, the availability of reagents to examine the immunological response in this species is very limited. Monoclonal antibodies (mAb) specific for cell surface proteins or products of immune cells have been useful tools for characterizing and quantifying immune responses in humans and in murine models of human disease, but very few similar reagents are available for characterizing and manipulating the immune response of guinea pigs. A rat IgG2a mAb specific for guinea pig CD4 has previously been described and was shown to inhibit T cell proliferation, but was inefficient at depleting CD4+ T cells in vivo. We hypothesized that the in vivo CD4+ T cell depletion function of this mAb could be improved by expression of the rat IgG2b heavy chain. We show that the purified mAb from an IgG2b class-switch variant, but not the parental IgG2a mAb, significantly depleted CD4+ T cells from secondary lymphoid tissue of guinea pigs. Further, treatment of guinea pigs with the IgG2b mAb at 2.0 mg/kg resulted in depletion of CD4+ T cells from peripheral blood and spleen. The use of this modified antibody to specifically alter the immune response of guinea pigs should prove useful in a number of guinea pig infectious disease models., (Copyright © 2019 Elsevier B.V. All rights reserved.)

To fulfil the optimization needs of current biopharmaceutical processes the knowledge how to improve cell specific productivities is of outmost importance. This requires a detailed understanding of cellular metabolism on a subcellular level inside compartments such as cytosol and mitochondrion. Using IgG1 producing Chinese hamster ovary (CHO) cells, a pioneering protocol for compartment-specific metabolome analysis was applied. Various production-like growth conditions ranging from ample glucose and amino acid supply via moderate to severe nitrogen limitation were investigated in batch cultures. The combined application of quantitative metabolite pool analysis, 13 C tracer studies and non-stationary flux calculations revealed that Pyr/H + symport (MPC1/2) bore the bulk of the mitochondrial transport under ample nutrient supply. Glutamine limitation induced the concerted adaptation of the bidirectional Mal/aKG (OGC) and the Mal/HPO 4 2- antiporter (DIC), even installing completely reversed shuttle fluxes. As a result, NADPH and ATP formation were adjusted to cellular needs unraveling the key role of cytosolic malic enzyme for NADPH production. Highest cell specific IgG1 productivities were closely correlated to a strong mitochondrial malate export according to the anabolic demands. The requirement to install proper NADPH supply for optimizing the production of monoclonal antibodies is clearly outlined. Interestingly, it was observed that mitochondrial citric acid cycle activity was always maintained enabling constant cytosolic adenylate energy charges at physiological levels, even under autophagy conditions., (Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

The cell-specific growth rate (µ) is a critical process parameter for antibody production processes performed by animal cell cultures, as it describes the cell growth and reflects the cell physiological state. When there are changes in these parameters, which are indicated by variations of µ, the synthesis and the quality of antibodies are often affected. Therefore, it is essential to monitor and control the variations of µto assure the antibody production and achieve high product quality. In this study, a novel approach for on-line estimation of µ was developed based on the process analytical technology initiative by using an in situ dielectric spectroscopy. Critical moments, such as significant µ decreases, were successfully detected by this method, in association with changes in cell physiology as well as with an accumulation of nonglycosylated antibodies. Thus, this method was used to perform medium renewals at the appropriate time points, maintaining the values of µ close to its maximum. Using this method, we demonstrated that the physiological state of cells remained stable, the quantity and the glycosylation quality of antibodies were assured at the same time, leading to better process performances compared with the reference feed-harvest cell cultures carried out by using off-line nutrient measurements., (© 2019 Wiley Periodicals, Inc.)

Metal ions can be enzyme cofactors and can directly influence the kinetics of biochemical reactions that also influence the biological production and quality attributes of therapeutic proteins, such as glycan formation and distribution. However, the concentrations of metals in commercially available chemically defined media can range from 1 to 25,000 ppb. Because such concentration changes can impact cell growth, manufacturing yield and product quality the alteration/fluctuation in media composition should be well controlled to maintain product quality. Here, we describe a platform of analytical methods to determine the composition of several metals in different sample matrices using an advanced automated Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). These methods, validated to ICH Q2R1 regulatory validation parameters, were successfully applied to- (a) screen cell culture media; (b) determine changes in the metal concentration during cell growth in spinner flasks, and, (c) determine effect on the glycosylation pattern and homogeneity of an IgG3:κ produced from a murine-hybridoma cell line in bench-top parallel bioreactors due to a spike in copper and iron concentration. Our results show that maintenance of metal content in the cell culture media is critical for product consistency of the IgG3:κ produced., (Published by Elsevier B.V.)

Acute allergic symptoms are caused by allergen-induced crosslinking of allergen-specific immunoglobulin E (IgE) bound to Fc-epsilon receptors on effector cells. Desensitization with allergen-specific immunotherapy (SIT) has been used for over a century, but the dominant protective mechanism remains unclear. One consistent observation is increased allergen-specific IgG, thought to competitively block allergen binding to IgE. Here we show that the blocking potency of the IgG response to Cat-SIT is heterogeneous. Next, using two potent, pre-selected allergen-blocking monoclonal IgG antibodies against the immunodominant cat allergen Fel d 1, we demonstrate that increasing the IgG/IgE ratio reduces the allergic response in mice and in cat-allergic patients: a single dose of blocking IgG reduces clinical symptoms in response to nasal provocation (ANCOVA, p = 0.0003), with a magnitude observed at day 8 similar to that reported with years of conventional SIT. This study suggests that simply augmenting the blocking IgG/IgE ratio may reverse allergy.

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L 1 Mab-13 (IgG 1 , kappa) was produced using a cell-based immunization and screening (CBIS) method. We investigated hPD-L1 expression in lung cancer using flow cytometry, Western blot, and immunohistochemical analyses. L 1 Mab-13 specifically reacted hPD-L1 of hPD-L1-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous hPD-L1 of KMST-6 (human fibroblast) in flow cytometry and Western blot. Furthermore, L 1 Mab-13 reacted with lung cancer cell lines (EBC-1, Lu65, and Lu99) in flow cytometry and stained lung cancer tissues in a membrane-staining pattern in immunohistochemical analysis. These results indicate that a novel anti-hPD-L1 mAb, L 1 Mab-13, is very useful for detecting hPD-L1 of lung cancers in flow cytometry, Western blot, and immunohistochemical analyses.

Immunotherapy is now at the forefront of cancer therapeutic development. Gliomas are a particularly aggressive form of brain cancer for which immunotherapy may hold promise. Pritumumab (also known in the literature as CLNH11, CLN-IgG, and ACA-11) was the first monoclonal antibody tested in cancer patients. Pritumumab is a natural human monoclonal antibody developed from a B lymphocyte isolated from a regional draining lymph node of a patient with cervical carcinoma. The antibody binds ecto-domain vimentin on the surface of cancer cells. Pritumumab was originally tested in clinical trials with brain cancer patients in Japan where it demonstrated therapeutic benefit. It was reported to be a safe and effective therapy for brain cancer patients at doses 5-10 fold less than currently approved antibodies. Phase I dose escalation clinical trials are now being planned with pritumumab for the near future. Here we review data on the development and characterization of pritumumab, and review clinical trails data assessing immunotherapeutic effects of pritumumab for glioma patients.

Monoclonal antibody production in commercial scale cell culture bioprocessing requires a thorough understanding of the engineering process and components used throughout manufacturing. It is important to identify high impact components early on during the lifecycle of a biotechnology-derived product. While cell culture media selection is of obvious importance to the health and productivity of mammalian bioreactor operations, other components such as antifoam selection can also play an important role in bioreactor cell culture. Silicone polymer-based antifoams were known to have negative impacts on cell health, production, and downstream filtration and purification operations. High throughput screening in micro-scale bioreactors provides an efficient strategy to identify initial operating parameters. Here, we utilized a micro-scale parallel bioreactor system to study an IgG1 producing CHO cell line, to screen Dynamis, ProCHO5, PowerCHO2, EX-Cell Advanced, and OptiCHO media, and 204, C, EX-Cell, SE-15, and Y-30 antifoams and their impacts on IgG1 production, cell growth, aggregation, and process control. This study found ProCHO5, EX-Cell Advanced, and PowerCHO2 media supported strong cellular growth profiles, with an IVCD of 25-35 × 10 6 cells-d/mL, while maintaining specific antibody production (Q p  > 2 pg/cell-d) for our model cell line and a monomer percentage above 94%. Antifoams C, EX-Cell, and SE-15 were capable of providing adequate control of foaming while antifoam 204 and Y-30 noticeably stunted cellular growth. This work highlights the utility of high throughput micro bioreactors and the importance of identifying both positive and negative impacts of media and antifoam selection on a model IgG1 producing CHO cell line. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:262-270, 2018., (© 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers.)

One of the key quality attributes of monoclonal antibodies is the glycan pattern and distribution. Two terminal galactose residues typically represent a small fraction of the total glycans from antibodies. However, antibodies with defined glycosylation properties including enhanced galactosylation have been shown to exhibit altered properties for these important biomedical modalities. In this study, the disruption of two α-2,3 sialyltransferases (ST3GAL4 and ST3GAL6) from Chinese Hamster Ovary (CHO) cells was combined with protein engineering of the Fc region to generate an IgG containing 80% bigalactosylated and fucosylated (G2F) glycoforms. Expression of the same single amino acid mutant (F241A) IgG in CHO cells with a triple gene knockout of fucosyltransferase (FUT8) plus ST3GAL4 and ST3GAL6 lowered the galactosylation glycoprofile to 65% bigalactosylated G2 glycans. However, overexpression of IgGs with four amino acid substitutions recovered the G2 glycoform composition approximately 80%. Combining genome and protein engineering in CHO cells will provide a new antibody production platform that enables biotechnologists to generate glycoforms standards for specific biomedical and biotechnology applications., (© 2017 Wiley Periodicals, Inc.)

In response to immunization, B-cells generate a repertoire of antigen-specific antibodies. Antibody-based immunotherapies hold great promise for treating a variety of diseases in humans. Application of antibody-based immunotherapy in cats is limited by the lack of species-specific complete sequences for mRNAs encoding rearranged heavy and light chain immunoglobulins in B cells. To address this barrier, we isolated mRNAs from feline peripheral blood mononuclear cells (PBMCs), and used available immunoglobulin sequences and 5' and 3' RACE to clone and sequence heavy and light chain immunoglobulin mRNAs. We recovered mRNA from PBMCs from two cats, cloned and sequenced the variable and constant domains of the feline heavy chains of IgG1a (IGHG1a), IgG2 (IGHG2), and IgA (IGHA), and the light chains (lambda and kappa). Using these sequences, we prepared two bicistronic vectors for mammalian expression of a representative feline heavy (IGHG1a) together with a light (lambda or kappa) chain. Here we report novel feline Ig sequences, a technique to express antigen-specific felinized monoclonal antibodies, and the initial characterization of a functional felinized monoclonal antibody against feline panleukopenia virus.

Rodent monoclonal antibodies with specificity towards important biological targets are developed for therapeutic use by a process of humanisation. This process involves the creation of molecules, which retain the specificity of the rodent antibody but contain predominantly human coding sequence. Here, we show that some humanised heavy chains (HCs) can fold, form dimers and be secreted even in the absence of a light chain (LC). Quality control of recombinant antibody assembly in vivo is thought to rely upon folding of the HC C H 1 domain. This domain acts as a switch for secretion, only folding upon interaction with the LC C L domain. We show that the secreted heavy-chain dimers contain folded C H 1 domains and contribute to the heterogeneity of antibody species secreted during the expression of therapeutic antibodies. This subversion of the normal quality control process is dependent on the HC variable domain, is prevalent with engineered antibodies and can occur when only the Fab fragments are expressed. This discovery will have an impact on the efficient production of both humanised antibodies and the design of novel antibody formats., (© 2017 The Author(s).)

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., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Industrial bioprocesses place high demands on the energy metabolism of host cells to meet biosynthetic requirements for maximal protein expression. Identifying metabolic phenotypes that promote high expression is therefore a major goal of the biotech industry. We conducted a series of 13 C flux analysis studies to examine the metabolic response to IgG expression during early stationary phase of CHO cell cultures grown in 3L fed-batch bioreactors. We examined eight clones expressing four different IgGs and compared with three non-expressing host-cell controls. Some clones were genetically manipulated to be apoptosis-resistant by expressing Bcl-2Δ, which correlated with increased IgG production and elevated glucose metabolism. The metabolic phenotypes of the non-expressing, IgG-expressing, and Bcl-2Δ/IgG-expressing clones were fully segregated by hierarchical clustering analysis. Lactate consumption and citric acid cycle fluxes were most strongly associated with specific IgG productivity. These studies indicate that enhanced oxidative metabolism is a characteristic of high-producing CHO cell lines., (Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Bispecific antibodies offer a clinically validated platform for drug discovery. In generating functionally active bispecific antibodies, it is necessary to identify a unique parental antibody pair to merge into a single molecule. However, technologies that allow high-throughput production of bispecific immunoglobulin Gs (BsIgGs) for screening purposes are limited. Here, we describe a novel bispecific antibody format termed tethered-variable CLBsIgG (tcBsIgG) that allows robust production of intact BsIgG in a single cell line, concurrently ensuring cognate light chain pairing and preserving key antibody structural and functional properties. This technology is broadly applicable in the generation of BsIgG from a variety of antibody isotypes, including human BsIgG1, BsIgG2 and BsIgG4. The practicality of the tcBsIgG platform is demonstrated by screening BsIgGs generated from FGF21-mimetic anti-Klotho-β agonistic antibodies in a combinatorial manner. This screen identified multiple biepitopic combinations with enhanced agonistic activity relative to the parental monoclonal antibodies, thereby demonstrating that biepitopic antibodies can acquire enhanced functionality compared to monospecific parental antibodies. By design, the tcBsIgG format is amenable to high-throughput production of large panels of bispecific antibodies and thus can facilitate the identification of rare BsIgG combinations to enable the discovery of molecules with improved biological function., (© The Author 2017. Published by Oxford University Press.)

Protein glycosylation is arguably the paramount post-translational modification on recombinant glycoproteins, and highly cited in the literature for affecting the physiochemical properties and the efficacy of recombinant glycoprotein therapeutics. Glycosylation of human immunoglobulins follows a reasonably well-understood metabolic pathway, which gives rise to a diverse range of asparagine-linked (N-linked), or serine/threonine-linked (O-linked) glycans. In N-linked glycans, fucose levels have been shown to have an inverse relationship with the degree of antibody-dependent cell-mediated cytotoxicity, and high mannose levels have been implicated in potentially increasing immunogenicity and contributing to less favorable pharmacokinetic profiles. Here, we demonstrate a novel approach to potentially reduce the presence of high-mannose species in recombinant human immunoglobulin preparations, as well as facilitate an approximate 100% replacement of fucosylation with arabinosylation in Chinese hamster ovary cell culture through media supplementation with D-arabinose, an uncommonly used mammalian cell culture sugar substrate. The replacement of fucose with arabinose was very effective and practical to implement, since no cell line engineering or cellular adaptation strategies were required. Arabinosylated recombinant IgGs and the accompanying reduction in high mannose glycans, facilitated a reduction in dendritic cell uptake, increased FcγRIIIa signaling, and significantly increased the levels of ADCC. These aforementioned effects were without any adverse changes to various structural or functional attributes of multiple recombinant human antibodies and a bispecific DVD-Ig. Protein arabinosylation represents an expansion of the N-glycan code in mammalian expressed glycoproteins.

IgG bispecific antibodies (BsAbs) represent one of the preferred formats for bispecific antibody therapeutics due to their native-like IgG properties and their monovalent binding to each target. Most reported studies utilized transient expression in HEK293 cells to produce BsAbs. However, the expression of biotherapeutic molecules using stable CHO cell lines is commonly used for biopharmaceutical manufacturing. Unfortunately, limited information is available in the scientific literature on the expression of BsAbs in CHO cell lines. In this study we describe an alternative approach to express the multiple components of IgG BsAbs using a single plasmid vector (quad vector). This single plasmid vector contains both heavy chain genes and both light chain genes required for the expression and assembly of the IgG BsAb, along with a selectable marker. We expressed, purified, and characterized four different IgG BsAbs or "hetero-mAbs" using transient CHO expression and stable CHO minipools. Transient CHO titers ranged from 90 to 160 mg/L. Stable CHO titers ranged from 0.4 to 2.3 g/L. Following a simple Protein A purification step, the percentage of correctly paired BsAbs ranged from 74% to 98% as determined by mass spectrometry. We also found that information generated from transient CHO expression was similar to information generated using stable CHO minipools. In conclusion, the quad vector approach represents a simple, but effective, alternative approach for the generation of IgG BsAbs in both transient CHO and stable CHO expression systems. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:469-477, 2017., (© 2017 American Institute of Chemical Engineers.)

By simultaneous binding two disease mediators, bispecific antibodies offer the opportunity to broaden the utility of antibody-based therapies. Herein, we describe the design and characterization of Bs4Ab, an innovative and generic bispecific tetravalent antibody platform. The Bs4Ab format comprises a full-length IgG1 monoclonal antibody with a scFv inserted into the hinge domain. The Bs4Ab design demonstrates robust manufacturability as evidenced by MEDI3902, which is currently in clinical development. To further demonstrate the applicability of the Bs4Ab technology, we describe the molecular engineering, biochemical, biophysical, and in vivo characterization of a bispecific tetravalent Bs4Ab that, by simultaneously binding vascular endothelial growth factor and angiopoietin-2, inhibits their function. We also demonstrate that the Bs4Ab platform allows Fc-engineering similar to that achieved with IgG1 antibodies, such as mutations to extend half-life or modulate effector functions.

Background: There is an ever-increasing need of monoclonal antibodies (mAbs) for biomedical applications and fully human binders are particularly desirable due to their reduced immunogenicity in patients. We have applied a strategy for the isolation of antigen-specific B cells using tetramerized proteins and single-cell sorting followed by reconstruction of human mAbs by RT-PCR and expression cloning., Results: This strategy, using human peripheral blood B cells, enabled the production of low affinity human mAbs against major histocompatibility complex molecules loaded with peptides (pMHC). We then implemented this technology using human immunoglobulin transgenic rats, which after immunization with an antigen of interest express high affinity-matured antibodies with human idiotypes. Using rapid immunization, followed by tetramer-based B-cell sorting and expression cloning, we generated several fully humanized mAbs with strong affinities, which could discriminate between highly homologous proteins (eg. different pMHC complexes)., Conclusions: Therefore, we describe a versatile and more effective approach as compared to hybridoma generation or phage or yeast display technologies for the generation of highly specific and discriminative fully human mAbs that could be useful both for basic research and immunotherapeutic purposes.

One direct route for the discovery of therapeutic human monoclonal antibodies (mAbs) involves the isolation of peripheral B cells from survivors/sero-positive individuals after exposure to an infectious reagent or disease etiology, followed by single-cell sequencing or hybridoma generation. Peripheral B cells, however, are not always easy to obtain and represent only a small percentage of the total B-cell population across all bodily tissues. Although it has been demonstrated that tandem mass spectrometry (MS/MS) techniques can interrogate the full polyclonal antibody (pAb) response to an antigen in vivo, all current approaches identify MS/MS spectra against databases derived from genetic sequencing of B cells from the same patient. In this proof-of-concept study, we demonstrate the feasibility of a novel MS/MS antibody discovery approach in which only serum antibodies are required without the need for sequencing of genetic material. Peripheral pAbs from a cytomegalovirus-exposed individual were purified by glycoprotein B antigen affinity and de novo sequenced from MS/MS data. Purely MS-derived mAbs were then manufactured in mammalian cells to validate potency via antigen-binding ELISA. Interestingly, we found that these mAbs accounted for 1 to 2% of total donor IgG but were not detected in parallel sequencing of memory B cells from the same patient.

This review serves to highlight approaches that may improve the access of antibody drugs to regions of the brain affected by Alzheimer's Disease. While previous antibody drugs have been unsuccessful in treating Alzheimer's disease, recent work demonstrates that Alzheimer's pathology can be modified if these drugs can penetrate the brain parenchyma with greater efficacy. Research in antibody blood-brain barrier drug delivery predominantly follows one of three distinct directions: (1) enhancing influx with reduced antibody size, addition of Trojan horse modules, or blood-brain barrier disruption; (2) modulating trancytotic equilibrium and/or kinetics of the neonatal Fc Receptor; and (3) manipulation of antibody glycan carbohydrate composition. In addition to these topics, recent studies are discussed that reveal a role of glycan sialic acid in suppressing antibody efflux from the brain.

Background: Recombinant monoclonal antibodies (mAbs) are useful in research, diagnosis, and therapy. The increased demands of recombinant mAbs require efficient production systems. A variety of expression vectors have been developed for stable or transient production of mAbs in mammalian cells. Although a few commercial expression systems of mAbs can be listed, the high expense often impedes academic research., Methods: In this study, we described the development of a transient mammalian system based on a bicistronic vector, which contained an internal ribosome entry site (IRES) and enhancer elements to express the IgG1 light chain (LC) and heavy chain (HC) in one transcript. Optimization of all components of the expression system, including gene transfer methods and regulatory elements, yielded maximal expression levels in serum-free 293 suspension cells (Freestyle 293-F)., Results: This method enabled consistent production of the anti-programmed cell death protein 1 (PD-1) mAb up to 300 mg/L in less than one week under transiently transfected conditions. Furthermore, purified anti-PD-1 IgGs showed specific affinity to the target antigen human PD-1 and PHA-stimulated human T cells., Conclusion: The simplicity of the procedure made it suitable for the fast and high-yield production of IgG antibodies in small scales, which expedited the screening of a large number of recombinant candidates., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Aim: CD44s antigens have been suggested as an efficient biomarker for cancer stem cells. Current study aimed to develop a hybridoma that producing a high affinity murine anti-human CD44 monoclonal antibody for early diagnostic laboratory tests of some cancer., Materials and Methods: To make hybridoma against CD44, mice were immunized with MDA-MB-468 cells. Resulted hybridomas using three culture media were screened by indirect ELISA, then cloned by limiting dilution, and isotype was determined after obtaining ascitic fluid and antibody purification., Results: We obtained a stable secreting clone, capable of secreting a high-affinity monoclonal antibody against CD44 protein, IgG2a kappa, with the affinity of 5.4 × 10-8 M without cross-reactivity., Conclusion: We could establish a hybridoma in a native form. This stable and high-affinity anti-CD44 mAb has a potential for diagnostic procedures and laboratory research. Thus, it could be exploited as a suitable tool for target-specific diagnosis and even treatment in several cancers.

Protein purification is often a bottleneck during protein generation for large molecule drug discovery. Therapeutic antibody campaigns typically require the purification of hundreds of monoclonal antibodies (mAbs) during the hybridoma process and lead optimization. With the increase in high-throughput cloning, faster DNA sequencing, and the use of parallel protein expression systems, a need for high-throughput purification approaches has evolved, particularly in the midsize range between 20 ml and 100 ml. To address this we modified a four channel Gilson solid phase extraction system (referred to as MG-SPE) with switching valves and sample holding loops to be able to perform standard affinity purification using commercially available columns and micro-titer format deep well blocks. By running 4 samples in parallel, the MG-SPE has the capacity to purify up to 24 samples of greater than 50 ml each using a single-step affinity purification protocol or a two-step protocol consisting of affinity chromatography followed by desalting/buffer exchange overnight (∼12 h run time). Our evaluation of affinity purification using mAbs and Fc-fusion proteins from mammalian cell supernatants demonstrates that the MG-SPE compared favorably with industry standard systems for both protein quality and yield. Overall the system is simple to operate and fills a void in purification processes where a simple, efficient, automated system is needed for affinity purification of midsize research samples., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In previous studies, hybridomas producing human immunoglobulin G, the antibodies 5E4 and 5A7 against influenza A and B virus were established using a novel human lymphocyte fusion partner, SPYMEG. In the present study, we succeeded in achieving the recombinant production and secretion of 5E4 and 5A7 in Chinese hamster ovary cells. Our N-glycan analysis by intact-mass detection and liquid chromatography mass spectrometry showed that recombinant 5E4 and 5A7 have one N-glycan and the typical mammalian-type N-glycan structures similar to those in hybridomas. However, the glycan distribution was slightly different among these antibodies. The amount of high-mannose-type structures was under 10% of the total N-glycans of recombinant 5E4 and 5A7, compared to 20% of the 5E4 and 5A7 produced in hybridomas. The amount of galactosylated N-glycans was increased in recombinants. Approximately 80% of the N-glycans of all antibodies was fucosylated, and no sialylated N-glycan was found. Recombinant 5E4 and 5A7 neutralized pandemic influenza A virus specifically, and influenza B virus broadly, quite similar to the 5E4 and 5A7 produced in hybridomas, respectively. Here we demonstrated that recombinants of antibodies identified from hybridomas fused with SPYMEG have normal N-glycans and that their neutralizing activities bear comparison with those of the original antibodies., (Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Glycan distribution has been identified as a "critical quality attribute" for many biopharmaceutical products, including monoclonal antibodies. Consequently, determining quantitatively how process variables affect glycan distribution is important during process development to control antibody glycosylation. In this work, we assess the effect of six bioreactor process variables on the glycan distribution of an IgG1 produced in CHO cells. Our analysis established that glucose and glutamine media concentration, temperature, pH, agitation rate, and dissolved oxygen (DO) had small but significant effects on the relative percentage of various glycans. In addition, we assessed glycosylation enzyme transcript levels and intracellular sugar nucleotide concentrations within the CHO cells to provide a biological explanation for the observed effects on glycan distributions. From these results we identified a robust operating region, or design space, in which the IgG1 could be produced with a consistent glycan distribution. Since our results indicate that perturbations to bioreactor process variables will cause only small (even if significant) changes to the relative percentage of various glycans (<±1.5%)-changes that are too small to affect the bioactivity and efficacy of this IgG1 significantly-it follows that the glycan distribution obtained will be consistent even with relatively large variations in bioreactor process variables. However, for therapeutic proteins where bioactivity and efficacy are affected by small changes to the relative percentage of glycans, the same analysis would identify the manipulated variables capable of changing glycan distribution, and hence can be used to implement a glycosylation control strategy. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1149-1162, 2016., (© 2016 American Institute of Chemical Engineers.)

Spermidine/spermine N1-acetyltransferase (SSAT) is a catabolic regulator of polyamines, ubiquitous molecules essential for cell proliferation and differentiation. Anti-SSAT antibodies (monoclonal antibodies [mAbs]) of high titer were prepared by immunizing BALB/c mice with multifocal intradermal injections and by fusing high-titer antibody-producing spleen cells with myeloma cells of SP2/0 origin. Four mAbs were selected for further characterization as classes and subclasses. Antibodies were produced by these three clones with high affinities ranging from 10(9) to 10(11) M(-1). These clones were found to be of the immunoglobulin IgG1 subclass with kappa light chain. They could recognize SSAT as determined by Western blot and immunohistochemistry. The specificity of one clone, 4H6, was studied by using the small interfering RNA (siRNA) on SSAT. 4H6 was also compared with the commercial antibody. The produced mAbs will be a useful tool for further investigation of SSAT functions in organisms.

We previously reported that a maraviroc (MVC)-resistant human immunodeficiency virus type 1variant, generated using in vitro selection, exhibited high sensitivity to several neutralizing monoclonal antibodies (NMAbs) and autologous plasma IgGs. The MVC-resistant variant acquired 4 sequential mutations in gp120: T297I, M434I, V200I, and K305R. In this study, we examined the mutation most responsible for conferring enhanced neutralization sensitivity of the MVC-resistant variant to several NMAbs and autologous plasma IgGs. The virus with the first resistant mutation, T297I, was sensitive to all NMAbs, whereas the passage control virus was not. The neutralization sensitivity of the variant greatly increased following its acquisition of the second mutation, M434I, in the C4 region. The M434I mutation conferred the greatest neutralizing sensitivity among the 4 MVC-resistant mutations. Additionally, the single M434I mutation was sufficient for the enhanced neutralization of the virus by NMAbs, autologous plasma IgGs, and heterologous sera relative to that of the parental virus.

Toxoplasma gondii infects animals habiting terrestrial and aquatic environments. Its oocysts and tissue cysts are important for the horizontal transmission of this parasite. The oocyst and tissue cyst walls are crucial for the ability of the parasite to persist in the environment or in animal tissues, respectively. However, the composition of these walls is not well understood. We report the generation of monoclonal antibodies directed against wall components using mice immunized with oocyst antigens of T. gondii. One monoclonal antibody (mAb) G1/19 reacted solely with T. gondii sporozoites. The respective antigen had a relative molecular weight (Mr) of 30 kDa. MAb G1/19 failed to react with sporozoites of any other coccidian parasite species tested (Hammondia hammondi, Hammondia heydorni, Cystoisospora felis, Eimeria bovis, Sarcocystis sp.). Another mAb, designated K8/15-15, recognized antigens in sporocyst walls of the parasite and in the walls of in vivo or in vitro produced tissue cysts, as demonstrated by immunofluorescence and immunoblot assays. Antigens of 80 to a high molecular weight protein of about 350 kDa Mr were recognized by this antibody using antigen extracts from sporocysts, and from in vitro or in vivo generated tissue cysts of the parasite. Tissue cyst and sporocyst walls of H. hammondi and H. heydorni, and tissue cysts of Neospora caninum were also recognized by mAb K8/15-15. Sporocyst walls of C. felis also reacted to this mAb. The cyst walls of Sarcocystis sp. and Besnoitia besnoiti were not recognized by mAb K8/15-15. Reactivity by a single mAb against T. gondii antigens in tissue cysts and sporocysts had not been reported previously. MAb K8/15-15 may be a practical tool for the identification of both cysts and sporocysts of the parasite, and may also be potentially employed in proteomic studies on the identification of new components of the cyst and sporocyst walls of T. gondii., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive). These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP) fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking antibody from mice with an affinity of 90 pM.

Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants because of beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution, and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell-mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. We also identified divergent patterns of colostrum Env-specific B-cell lineage evolution with respect to crossreactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk immunoglobulin G (IgG) repertoire. Maternal vaccine strategies to specifically target this breast milk B-cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants.

We employed top- and middle-down analyses with multiple fragmentation techniques including electron transfer dissociation (ETD), electron capture dissociation (ECD), and matrix-assisted laser desorption ionization in-source decay (MALDI-ISD) for characterization of a reference monoclonal antibody (mAb) IgG1 and a fusion IgG protein. Fourier transform ion cyclotron resonance (FT-ICR) or high performance liquid chromatography electrospray ionization (HPLC-ESI) on an Orbitrap was employed. These experiments provided a comprehensive view on the protein species; especially for different glycosylation level in these two proteins, which showed good agreement with oligosaccharide profiling. Top- and middle-down MS provided additional information regarding glycosylation sites and different combinational protein species that were not available from oligosaccharide mapping or conventional bottom-up analysis. Finally, incorporating a limited enzymatic digestion by immunoglobulin G-degrading enzyme of Streptococcus pyogene (IdeS) with MALDI-ISD analysis enabled extended sequence coverage of the internal region of protein without pre-fractionation., Biological Significance: Oligosaccharide profiling together with top- and middle-down methods enabled: 1) detection of heterogeneous glycosylated protein species and sites in intact IgG1 and fusion proteins with high mass accuracy, 2) estimation of relative abundance levels of protein species in the sample, 3) confirmation of the protein termini structural information, and 4) improved sequence coverage by MALDI-ISD analysis for the internal regions of the proteins without sample pre-fractionation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Rhizosecretion of recombinant pharmaceuticals from in vitro hydroponic transgenic plant cultures is a simple, low cost, reproducible and controllable production method. Here, we demonstrate the application and adaptation of this manufacturing platform to a human antivitronectin IgG1 monoclonal antibody (mAb) called M12. The rationale for specific growth medium additives was established by phenotypic analysis of root structure and by LC-ESI-MS/MS profiling of the total protein content profile of the hydroponic medium. Through a combination of optimization approaches, mAb yields in hydroponic medium reached 46 μg/mL in 1 week, the highest figure reported for a recombinant mAb in a plant secretion-based system to date. The rhizosecretome was determined to contain 104 proteins, with the mAb heavy and light chains the most abundant. This enabled evaluation of a simple, scalable extraction and purification protocol and demonstration that only minimal processing was necessary prior to protein A affinity chromatography. MALDI-TOF MS revealed that purified mAb contained predominantly complex-type plant N-glycans, in three major glycoforms. The binding of M12 purified from hydroponic medium to vitronectin was comparable to its Chinese hamster ovary (CHO)-derived counterpart. This study demonstrates that in vitro hydroponic cultivation coupled with recombinant protein rhizosecretion can be a practical, low-cost production platform for monoclonal antibodies., (© 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

This review describes the history of Ebola monoclonal antibody (mAb) development leading up to the recent severe Ebola outbreak in West Africa. The Ebola virus has presented numerous perplexing challenges in the long effort to develop therapeutic antibody strategies. Since the first report of a neutralizing human anti-Ebola mAb in 1999, the straightforward progression from in vitro neutralization resulting in in vivo protection and therapy has not occurred. A number of mAbs, including the first reported, failed to protect non-human primates (NHPs) in spite of protection in rodents. An appreciation of the role of effector functions to antibody efficacy has contributed significantly to understanding mechanisms of in vivo protection. However a crucial contribution, as measured by post-exposure therapy of NHPs, involved the comprehensive testing of mAb cocktails. This effort was aided by the use of plant production technology where various combinations of mAbs could be rapidly produced and tested. Introduction of appropriate modifications, such as specific glycan profiles, also improved therapeutic efficacy. The resulting cocktail, ZMapp™, consists of three mAbs that were identified from numerous mAb candidates. ZMapp™ \ is now being evaluated in human clinical trials but has already played a role in bringing awareness to the potential of antibody therapy for Ebola.

Background: Tetanus neurotoxin (TeNT) is composed of a light (LC) and heavy chain (HC) polypeptides, released by anaerobic bacterium Clostridium tetani and can cause fatal life-threatening infectious disease. Toxin HC and LC modules represents receptor binding and zinc metalloprotease activity, respectively. The passive administration of animal-derived antibodies against tetanus toxin has been considered as the mainstay therapy for years. However, this treatment is associated with several adverse effects due to the presence of anti-isotype antibodies., Objective: In the present study, we have produced the fully human single chain antibody fragments (HuScFv) from two human antibody phage display libraries., Material and Methods: Twenty-four different HuscFvs were isolated from two anti TeNT immune libraries. Our produced human ScFv (HuScFv) were converted to IgG platform and analyzed regarding their specific reactivity to TeNT., Results: All of the selected scFvs have the same VL but different VH. Three HuscFvs from the first library (TTX15, 51, 75) and two HuscFvs from the second library (TTX16, 20) were chosen to convert to IgG1 using pOptiVEC and pcDNA3.3 systems. Production of IgG1 from transfected DG44 and binding capacity of them to tetanus toxin and toxoid were measured by ELISA. ELISA results showed no detectable production of TTX16 and TTX20 IgG1. Although, TTX51 and TTX75 were converted and produced as IgG1, no reactivity to tetanus toxin and toxoid was observed. However, TTX15 was successfully produced as whole IgG1 platform with reactivity to both tetanus toxin and toxoid. The latter would be an appropriate replacement for conventional polyclonal antibodies if would meet the further characterization including specificity determination, affinity measurement and toxin neutralizing assays., Conclusion: Our results demonstrated production of functional IgG1 derived from TTX15 scFv and might be an appropriate replacement for polyclonal Tetabulin but it needs further characterization.

Podoplanin (PDPN) is a platelet aggregation-inducing factor, which is known as an endogenous ligand of C-type lectin-like receptor-2 (CLEC-2). PDPN is also expressed in several normal tissues, such as lung type I alveolar cells and kidney podocytes. Although many monoclonal antibodies (MAbs) against human PDPN (hPDPN) or mouse PDPN (mPDPN) have been established, anti-rat PDPN (rPDPN) MAbs, especially against platelet aggregation-stimulating (PLAG) domain (29-54 amino acids) of rPDPN, have not been developed. Therefore, functional analysis of rPDPN in normal tissues has been limited. Here, we immunized mice with rPDPN peptides (38-51 amino acids) and developed a novel mouse anti-rPDPN MAb, PMab-2 (IgG1, kappa), which possesses high affinity compared with anti-hPDPN or mPDPN MAbs. The KD of PMab-2 was determined to be 5.9 × 10(-10) M. PMab-2 is useful, not only in flow cytometry and Western blot analysis against endogenous rPDPN, which is expressed in rat dermal fibroblast, but also in immunohistochemistry against normal tissues. PMab-2 showed extraordinarily high sensitivity in immunohistochemistry, indicating that PMab-2 is very advantageous for functional analysis of rPDPN.