Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen-binding fragments (Fabs) of an mAb allow for high-avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fcγ receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half-life. To augment the effector functions or half-life of an IgG1 mAb, we constructed a novel “2Fc” mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral-like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors FcγRI, FcγRIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6- to 130-fold, resulting in apparent affinity increases of 7- to 42-fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of FcγRI or four molecules of FcRn, which is double the stoichiometry of a wild-type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.     

Isolation of B subunit‐specific monoclonal antibody clones that strongly neutralize the toxicity of Shiga toxin 2

Shiga toxin 2 (Stx2)‐specific mAb‐producing hybridoma clones were generated from mice. Because mice tend to produce small amounts of B subunit (Stx2B)‐specific antibodies at the polyclonal antibody level after immunization via the parenteral route, mice were immunized intranasally with Stx2 toxoids with a mutant heat‐labile enterotoxin as a mucosal adjuvant; 11 different hybridoma clones were obtained in two trials. Six of them were A subunit (Stx2A)‐specific whereas five were Stx2B‐specific antibody‐producing clones. The in vitro neutralization activity of Stx2B‐specific mAbs against Stx2 was greater than that of Stx2A‐specific mAbs on HeLa229 cells. Furthermore, even at low concentrations two of the Stx2B‐specific mAbs (45 and 75D9) completely inhibited receptor binding and showed in vivo neutralization activity against a fivefold median lethal dose of Stx2 in mice. In western blot analysis, these Stx2B‐specific neutralization antibodies did not react to three different mutant forms of Stx2, each amino acid residue of which was associated with receptor binding. Additionally, the nucleotide sequences of the V_H and V_L regions of clones 45 and 75D9 were determined. Our Stx2B‐specific mAbs may be new candidates for the development of mouse‐human chimeric Stx2‐neutralizing antibodies which have fewer adverse effects than animal antibodies for enterohemorrhagic Escherichia coli infection.     

Efficient bispecific monoclonal antibody purification using gradient thiophilic affinity chromatography

Bispecific monoclonal antibodies (bsMAbs), due their unique design, have a wide range of potential applications in immunodiagnostics and immunotherapy. One of the major limitations for the use of bsMAbs produced by hybrid–hybridomas is the concomitant production of parental monospecific antibodies. The relative amount of bsMAb secreted may vary between different hybrid–hybridomas. Hence, the purification of the desired bispecific molecule from other forms is crucial. Current purification methods include anion-exchange, HPLC on different matrices, and dual affinity methods. Most of those methods include multiple steps and have limitations on the purity or yield of the desired species. We report here a simple single-step purification method, using inexpensive thiophilic chromatography. This new method can potentially be scaled up, for industrial proposes. Finally, based on the amino acid sequences and assembly of the two heavy chains we attempt to explain the possible mechanism by which thiophilic chromatography was able to resolve the bsMAbs from the monospecific species.     

A monoclonal antibody identifying a T-cell marker in the horse

A cell surface molecule of equine T lymphocytes was identified and characterized using a mouse monoclonal antibody, HT23A. The molecule was detected on all T cells but not on other cells in peripheral blood, with the possible exception of a small subpopulation (about 5%) of B cells, as assessed by indirect immunofluorescence and flow cytometry. HT23A labelled T cell areas of horse lymph nodes and spleen when used in an indirect immunoperoxidase assay on frozen sections. Macrophages and neutrophils were not labelled by the antibody nor were frozen sections of horse liver, kidney, or brain. HT23A precipitated a molecule of approximately 69 kDa from 125Iodine labelled horse lymphocytes.     

再循环抗体的研究进展

单克隆抗体因其与抗原结合具有高度特异性与强亲和力,已成为抗体药物研发的主要类型。但随着天然单克隆抗体的深入研究,它的诸多缺陷也浮出水面,如与抗原结合次数有限、带来非预期的抗体清除效应和抗原累积效应。人们不再局限于天然抗体的筛选,而是想通过改造提升抗体药物的药效。近年来,一类新型再循环抗体的问世,很好地解决了天然单克隆抗体发展的瓶颈。再循环抗体可以在胞外结合抗原,在细胞内与抗原解离,使抗体结合抗原次数最大化,减少抗原介导的抗体清除效应和抗体介导的抗原累积效应,并且再循环抗体可以通过进一步的Fc改造来加强与Fc受体的亲和力。文中综述了再循环抗体的研究进展,包括其特点、改造方法及展望。     

Diastereomeric phosphonate ester adducts of chymotrypsin: 31P-NMR measurements

Generation of diastereomeric phosphonate ester adducts of chymotrypsin was evidenced for the first time by ³¹P NMR and spectrophotometric kinetic measurements. ³¹P NMR signals were recorded for 4-nitrophenyl 2-propyl methylphosphonate (IMN) at 32.2 ppm and for its hydrolysis product at 26.3 ppm downfield from phosphoric acid. The inhibition of α-chymotrypsin at pH > 8.0 by the faster reacting enantiomer of IMN or 2-propyl methylphosphonochloridate (IMCl), or other phosphonate ester analogs of these compounds, all caused a ～6.0 ppm downfield shift of the ³¹P signal to the 39–40 ppm region. IMN, when applied below the stoichiometric amount of chymotrypsin, under the same conditions, generated two signals, at 39.0 and at 37.4 ppm. Scans accumulated in hourly intervals showed the decomposition of both diastereomers, with approximate half-lives of 12 h at pH 8.0 and 22°C, into a species with a resonance at 35.5 ppm. The most likely reaction to account for the appearance of this new peak is the enzymic dealkylation of the isopropyl group from the covalently bound phosphonate ester. We base this conclusion mostly on the similarity of the upfield shift to the hydrolysis of phosphonate esters. Contrary to experience with phosphate ester adducts of serine proteases, no signal was detected higher than 25.0 ppm downfield from phosphoric acid for several phosphonate ester adducts of chymotrypsin and in no case did the resonance for the adduct shift further downfield in the course of the experiments. © 1993 Wiley-Liss, Inc.     

Analytical comparability study of recombinant monoclonal antibody therapeutics

Process changes are inevitable in the life cycle of recombinant monoclonal antibody therapeutics. Products made using pre- and post-change processes are required to be comparable as demonstrated by comparability studies to qualify for continuous development and commercial supply. Establishment of comparability is a systematic process of gathering and evaluating data based on scientific understanding and clinical experience of the relationship between product quality attributes and their impact on safety and efficacy. This review summarizes the current understanding of various modifications of recombinant monoclonal antibodies. It further outlines the critical steps in designing and executing successful comparability studies to support process changes at different stages of a product's lifecycle.     

Impact of cell culture on recombinant monoclonal antibody product heterogeneity

Recombinant monoclonal antibodies are commonly expressed in mammalian cell culture and purified by several steps of filtration and chromatography. The resulting high purity bulk drug substance still contains product variants differing in properties such as charge and size. Posttranslational modifications and degradations occurring during cell culture are the major sources of heterogeneity in bulk drug substance of recombinant monoclonal antibodies. The focus of the current review is the impact of cell culture conditions on the types and levels of various modifications and degradations of recombinant monoclonal antibodies. Understanding the relationship between cell culture and product variants can help to make consistently safe and efficacious products. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1103–1112, 2016     

Monoclonal antibody capture and viral clearance by cation exchange chromatography

Traditionally, post-production culture harvest capture of therapeutic monoclonal antibodies (mAbs) is performed using Protein A chromatography. We investigated the efficiency and robustness of cation exchange chromatography (CEX) in an effort to evaluate alternative capture methodologies. Up to five commercially available CEX resins were systematically evaluated using an experimentally optimized buffer platform and a design-of-experiment (DoE) approach for their ability to (a) capture a model mAb with a neutral isoelectric point, (b) clear three model viruses (porcine parvovirus, CHO type-C particles, and a bacteriophage). This approach identified a narrow operating space where yield, purity, and viral clearance were optimal under a CEX capture platform, and revealed trends between viral clearance of PPV and product purity (but not yield). Our results suggest that after unit operation optimization, CEX can serve as a suitable capture step.     

Generation and characterization of a unique reagent that recognizes a panel of recombinant human monoclonal antibody therapeutics in the presence of endogenous human IgG

Pharmacokinetic (PK) and immunohistochemistry (IHC) assays are essential to the evaluation of the safety and efficacy of therapeutic monoclonal antibodies (mAb) during drug development. These methods require reagents with a high degree of specificity because low concentrations of therapeutic antibody need to be detected in samples containing high concentrations of endogenous human immunoglobulins. Current assay reagent generation practices are labor-intensive and time-consuming. Moreover, these practices are molecule-specific and so only support one assay for one program at a time. Here, we describe a strategy to generate a unique assay reagent, 10C4, that preferentially recognizes a panel of recombinant human mAbs over endogenous human immunoglobulins. This “panel-specific” feature enables the reagent to be used in PK and IHC assays for multiple structurally-related therapeutic mAbs. Characterization revealed that the 10C4 epitope is conformational, extensive and mainly composed of non-CDR residues. Most key contact residues were conserved among structurally-related therapeutic mAbs, but the combination of these residues exists at low prevalence in endogenous human immunoglobulins. Interestingly, an indirect contact residue on the heavy chain of the therapeutic appears to play a critical role in determining whether or not it can bind to 10C4, but has no affect on target binding. This may allow us to improve the binding of therapeutic mAbs to 10C4 for assay development in the future. Here, for the first time, we present a strategy to develop a panel-specific reagent that can expedite the development of multiple clinical assays for structurally-related therapeutic mAbs.     

Monoclonal antibody against a lactose epitope of glycosphingolipids binds to melanoma tumour cells

Mice were immunized with a neoglycoprotein consisting of a chemically modified carbohydrate moiety (reductively aminated 3-sialyllactose) linked to human serum albumin. By this procedure an antibody response to the normally non-immunogenic carbohydrate structure was obtained. Hybridomas were established, and monoclonal antibodies were selected in ELISA based on their binding to the saccharide hapten, or to a lactosylceramide-mimicking neoglycolipid, lactose-bis-sulfone. One of the selected antibodies, 2H4, was of particular interest, since it also bound to glycolipids present on melanoma cells. FACS analysis of a panel of 14 melanoma cell lines showed that the 2H4 antibody bound to the majority of these. In frozen, non-fixed sections or paraffin sections of biopsies the monoclonal antibody 2H4 stained melanoma cells, but not tumour infiltrating lymphocytes or normal skin. Detailed immunochemical analysis of 2H4, using thin layer chromatography revealed that it recognized an internal lactose epitope in several glycosphingolipids.Abbreviations BSA bovine serum albumin - FACS fluorescence activated cell sorter - ELISA enzyme-linked immunosorbent assay - HSA human serum albumin - LacCer lactosylceramide - MAb monoclonal antibody - PBS phosphate buffered saline     

A polymorphic monoclonal anti-BOLA class I antibody

Cytotoxic monoclonal antibody IVA 44 was generated after the intraperitoneal immunization with peripheral blood mononuclear (PBM) cells and the boost by the intrasplenic inoculation of skin graft. The detected membrane antigen isolated by immunoprecipitation appears to be composed of two subunits characteristic for the MHC class I molecules. The antibody IVA 44 exhibited a different reactivity: it recognized the BoLA A14 (A8) specificity in animals typed in the Fifth BoLA workshop, while it reacted with all A8 positive animals including subtypes A14 and A15 in Czech and Slovak cattle. It is concluded that mAb IVA 44 might detect the broad subtype of A8 covering A14 and certain A15 split(s). The diverse A15 reactivity of this mAb in the workshop and our population could be explained by the different occurrence of A15 splits in both populations.     

Preferential precipitation of acidic variants from monoclonal antibody pools

Microheterogeneity of monoclonal antibodies (mAbs) can impact their activity and stability. Formation of charge variants is considered as the most important source of the microheterogeneity. In particular, controlling the content of the acidic species is often of major importance for the production process and regulatory approval of therapeutic proteins. In this study, the preferential precipitation process was developed for reducing the content of acidic variants in mAb downstream pools. The process design was preceded by the determination of phase behavior of mAb variants in the presence of different precipitants. It was shown that the presence of polyethylene glycol (PEG) in protein solutions favored precipitation of acidic variants of mAbs. Precipitation yield was influenced by the variant composition in the mAb feed solutions, the concentration of the precipitant and the protein, and the ionic strength of the solutions. To improve yield, multistage precipitation was employed, where the precipitate was recycled to the precipitation process. The final product was a mixture of supernatants pooled together from the recycling steps. Such an approach can be potentially used either instead or in a combination with chromatography for adjusting the acidic variant content of mAbs, which can benefit in improvement in throughput and reduction in manufacturing costs.     

Comparison and critical analysis of robotized technology for monoclonal antibody high-throughput production

We have previously demonstrated how to transform the conventional method of hybridoma production and screening into a fast, high-throughput technology. Nevertheless, there were still open questions related to automated procedures and immunization protocols that we address now by comparing the hybridoma production work-flow in automated and manually executed processes. In addition, since the animals' antibody responses to single or multiple antigen challenge affect monoclonal antibody throughput, different immunization and fusion strategies were tested. Specifically, the results obtained with multiplexing (multiple target antigens injected into a single animal) and single antigen immunization followed by splenocyte pooling immediately before fusion were compared with conventional methods. The results presented here demonstrate that the optimal protocol consists of automated somatic-cell fusion and hybridoma dilution followed by manual plating of hybridoma cells. Additionally, more specific and productive hybridoma clones were obtained with multiplexed immunization in a single animal with respect to the splenocyte pooling from single antigen immunized animals. However, in terms of overall antibody yield, the conventional method consisting of single immunization for each single animal assured ten times more specific hybridoma cell lines than the strategy based on the multiple antigen immunization followed by separate fusion step. In conclusion, the most productive approach for recovering a large number of suitable antibodies relies on single antigen immunization followed by automated fusion and dilution steps and manual plating.     

Urinary biomarkers suggest that estrogen-DNA adducts may play a role in the aetiology of non-Hodgkin lymphoma

A variety of evidence suggests that estrogens may induce non-Hodgkin lymphoma (NHL). The reaction of catechol estrogen quinones with DNA to form depurinating estrogen-DNA adducts is hypothesized to initiate this process. These adducts are released from DNA, shed from cells into the bloodstream and excreted in urine. The aim of this study was to determine whether or not the depurinating estrogen-DNA adducts might be involved in the aetiology of human NHL. Estrogen metabolites, conjugates and depurinating DNA adducts were identified and quantified in spot urine samples from 15 men with NHL and 30 healthy control men by using ultraperformance liquid chromatography/tandem mass spectrometry. The levels of estrogen-DNA adducts were significantly higher in the men with NHL than in the healthy control men. Thus, formation of estrogen-DNA adducts may play a critical role in the aetiology of NHL, and these adducts could be potential biomarkers of NHL risk.     

Identification of SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization

1. Download : Download high-res image (195KB)
2. Download : Download full-size image

     

A rat monoclonal antibody that catalyses the hydrolysis of a nitrophenyl-beta-lactam

We report the first example of a monoclonal antibody-catalysed hydrolysis of a beta-lactam where the antibodies were generated by a simple transition-state analogue. A rat monoclonal antibody (1/91c/4d/26) generated by using an acyclic 4-nitrophenylphosphate immunogen catalysed the hydrolysis of corresponding 4-nitrophenyl carbonates but, more importantly, also catalysed the hydrolysis of N-(4-nitrophenyl)-azetidinone at pH 8 with k(cat)=8.7 x 10(-6)s(-1) and K(M)=35 microM. This is the first example of a rat monoclonal catalytic antibody.     

Synthesis and analysis of DNA adducts of arylamines

Atylamines and nitroarenes are very important environmental and occupational pollutants. Genotoxic effects of arylamines are believed to be initiated by the formation of DNA adducts. DNA adducts of arylamines have been found in experimental animals and in exposed humans, and are predominantly formed with the carbon 8 of 2'-deoxyguanosine. Reference standards are necessary to develop methods for the quantification of DNA-adducts. Therefore, we have synthesized the 2'-deoxyguanosin-8-yI adducts of 2-methylaniline, 2-chloroaniline, 4-chloroaniline, 2,4dimethylaniline, and 2,6-dimethylaniline. The products were characterized by ¹H-NMR, ¹³C-NMR, MS and UV. The corresponding 2'-deoxyguanosine-3' -monophosphate adducts were synthesized for the quantification of DNA adducts by the ³²P-postlabelling technique. A GC-MS method was developed for the analysis of the new adducts as an alternative to the ³²P-postlabelling. DNA was spiked with the synthesized adducts and treated with 0.3 m NaOH overnight at 110 °C in the presence of a deuterated internal standard. We observed up to 80% recovery from about 1 adduct in 10⁸ to 1 in 10⁵ nucleotides.     

Potential antioxidant activity of Morita-Baylis-Hillman adducts

The wide variety of potent biological activities of Morita-Baylis-Hillman adducts (MBH) encouraged us to synthesize new series of products belonging to this class of compounds, possessing different functionalities and exhibiting potential antioxidant activity.As part of our on-going program on targeting molecules with antioxidant activity, we describe herein different DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activities of MBH alcohols and their derivatives including acetates, phosphonates and hydrazonophosphonates. The obtained results showed that the strongest DPPH radical scavenging activity was observed in the case of hydrazonophosphonates in comparison to the other MBH derivatives.