The Antibody Society

the official website of the antibody society

The Antibody Society

An international non-profit supporting antibody-related research and development.

You are here: Home / Archives for antibody engineering

Most read from mAbs

by

The Antibody Society is pleased and proud to be affiliated with mAbs, a multi-disciplinary journal dedicated to advancing the art and science of antibody research and development. We hope you enjoy these summaries based on the abstracts of the most read papers published in a recent issue. All the articles are open access; PDFs can be downloaded by following the links below.

Issue 10.8 (November/December 2018)

Rapid, automated characterization of disulfide bond scrambling and IgG2 isoform determination. In this new report, Resemann et al. discuss a rapid LC-MALDI-TOF/TOF workflow that can both identify the IgG2 disulfide linkages and provide a semi-quantitative assessment of the distribution of the disulfide isoforms. They established signature disulfide-bonded IgG2 hinge peptides that correspond to the A, A/B, and B disulfide isoforms, and can be applied to the fast classification of IgG2 isoforms in heterogeneous mixtures.

Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis. Bailey et al. describe charge variant native mass spectrometry (CVMS), an integrated native ion exchange mass spectrometry-based charge variant analytical approach that delivers detailed molecular information in a single, semi-automated analysis. They used pure volatile salt mobile phases over a pH gradient that effectively separated variants based on minimal differences in isoelectric point. Characterization of variants such as deamidation, which are traditionally unattainable by intact mass due to their minimal molecular weight differences, were measured unambiguously by mass and retention time to allow confident MS1 identification. The authors demonstrated that efficient chromatographic separation allows introduction of the purified forms of the charge variant isoforms into the Orbitrap mass spectrometer. Based on their results, they conclude that the CVMS method allows confident assignment of intact monoclonal antibody isoforms of similar mass and relative abundance measurements across three orders of magnitude dynamic range.

A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture. In this new report, Wang et al. discuss a systematic approach for analysis and characterization of mispairing in asymmetric bispecific antibodies. This approach consists of three orthogonal components, the first of which is a liquid chromatography (LC)-mass spectrometry (MS)–based method to measure the mass of intact antibodies. This method is used for fast analysis of mispairing and requires minimal method development, which makes it an ideal choice for early-stage development. The second component is a hydrophobic interaction chromatography (HIC)–based mispairing method that is suitable for lot release testing. The HIC method is robust and quality control friendly, and offers great linearity, precision, and accuracy. The third component is a two-dimensional LC-MS method for on-line chromatographic peak identification, which not only expedites this task but also reduces the risk of undesirable modifications during conventional fraction collection. These three methods dovetail to form the foundation of a complementary toolbox for analysis and characterization of mispairing in asymmetric bispecific antibodies and provide guidance and support for process development throughout the drug development life cycle.

Characterization and analysis of scFv-IgG bispecific antibody size variants. Cao et al. report size variants that were observed for an appended scFv-IgG bispecific antibody. Structural characterization studies showed that the size variants resulted from the engineered disulfide bond on the scFv, whereby the engineered disulfide was found to be either open or unable to form an intrachain disulfide bond due to cysteinylation or glutathionylation of the cysteines. Furthermore, the scFv engineered cysteines also formed intermolecular disulfide bonds, leading to the formation of highly stable dimers and aggregates. Because both the monomer variants and dimers showed lower bioactivity, they were considered to be product-related impurities that must be monitored and controlled. To this end, the authors developed and optimized a robust, precise, and accurate high-resolution size-exclusion chromatographic method, using a statistical design-of-experiments methodology.

Like this post but not a member? Please join!

Most read from mAbs issue 10.7

by

The Antibody Society is pleased and proud to be affiliated with mAbs, a multi-disciplinary journal dedicated to advancing the art and science of antibody research and development. We hope you enjoy these summaries based on the abstracts of the most read papers published in a recent issue. All the articles are open access; PDFs can be downloaded by following the links below.

MAbs Issue 10.7 (October 2018)

Platform development for expression and purification of stable isotope labeled monoclonal antibodies in Escherichia coli.  In this report, Reddy et al. present the expression and purification of a stable isotope labeled mAb from a genetically engineered E. coli strain capable of forming disulfide bonds in its cytoplasm. Using 2D NMR spectral fingerprinting, they show that the unlabeled mAb and the mAb singly or triply labeled with 13C, 15N, 2H are well folded, with only minor structural differences relative to the mammalian cell-produced mAb that are attributed to the lack of glycosylation in the Fc domain.

Massive parallel screening of phage libraries for the generation of repertoires of human immunomodulatory monoclonal antibodies. Sasso et al discuss a novel approach for the generation of several novel human immunomodulatory antibodies capable of binding their targets in their native conformation and useful for therapeutic applications. They performed a massive parallel screening of phage libraries by using activated human lymphocytes to generate large collections of scFvs against 10 immune checkpoints: LAG-3, PD-L1, PD-1, TIM3, BTLA, TIGIT, OX40, 4-1BB, CD27 and ICOS. By next-generation sequencing and bioinformatics analysis, they ranked individual scFvs in each collection and identified those with the highest level of enrichment. Human IgGs from three of these collections (i.e., PD-1, PD-L1 and LAG-3) were generated and shown to have comparable or better binding affinity and biological activity than clinically validated mAbs. The repertoires generated in this work represent a convenient source of agonistic or antagonistic antibodies against the ‘Checkpoint Immunome’ for preclinical screening and clinical implementation of optimized treatments.

High-throughput screening of antibody variants for chemical stability: identification of deamidation-resistant mutants. In this report, DiCara et al describe a high-throughput method for antibody stability screening during the early stages of antibody discovery and highlight the value of broad searches of antibody sequence space. They developed a high-throughput assay to characterize asparagine deamidation and used it to identify a mutation that unexpectedly stabilizes a critical asparagine. Ninety antibody variants were incubated under thermal stress in order to induce deamidation and screened for both affinity and total binding capacity. Surprisingly, a mutation five residues downstream from the unstable asparagine greatly reduced deamidation.

Extending human IgG half-life using structure-guided design. Booth et al. report on the development of a structure- and network-based framework to interrogate the engagement of IgG with multiple Fc receptors (FcRn, C1q, TRIM21, FcγRI, FcγRIIa/b, FcγRIIIa) simultaneously. They identified features that govern Fc-FcRn interactions and identified multiple distinct pathways for enhancing FcRn binding in a pH-specific manner. Network analysis provided a novel lens to study the allosteric impact of half-life-enhancing Fc mutations on FcγR engagement, which occurs distal to the FcRn binding site. Applying these principles, they engineered a panel of unique Fc variants that enhance FcRn binding while maintaining robust biophysical properties and wild type-like binding to activating receptors.

Like this post but not a member? Please join!

We encourage you to join the Society to take advantage of the substantial benefits of membership, including discounts on fees for selected KNect365, CHI, and Hanson Wade meetings, discounted subscriptions to Society-affiliated journals PEDS and mAbs (special subscription rate of US $84 online only access for Antibody Society members)  and access to information in the Members Only section of the website. In particular, we encourage members to take advantage of the discount on registration for Antibody Engineering & Therapeutics, which is the annual meeting of The Antibody Society traditionally held in San Diego in December. Membership is free for students, post-docs and employees of our corporate sponsors!

Post-translational Modification in Antibody Function

by

The Antibody Society is pleased to invite you to attend its annual Meeting, Antibody Engineering & Therapeutics, which will be held December 12-15, 2017, in San Diego, CA. We will be celebrating the 10th anniversary of the Society at the Society’s Special Session on Thursday December 14, 2017. In this session summary, Dennis R. Burton (The Scripps Research Institute) and Paul W.H.I. Parren (Leiden University Medical Center) discuss what you will learn in their session on post-translational modification in antibody function.

The critical importance of sequence variation in antibodies is well recognized. Sequence diversity in antibody variable domains is essential for specific antigen recognition while linkage to different constant domains leads to distinct Fc-mediated effector activities. Post-translational modifications (PTMs) of these domains provide an additional immune mechanism by which the binding and activity of antibodies can be modulated. PTMs vary from chain additions, such as N- and O-linked glycosylation, glycation, cysteinylation and sulfation; chain trimming, such as C-terminal lysine clipping; amino acid modifications such as cyclization (into a N-terminal pyroglutamic acid), deamidation, oxidation, isomerization and carbamylation; to disulfide scrambling of hinge region interchain disulfide bonds. Each antibody can therefore give rise to a myriad of distinct antibody molecules with large activity and potency differences. Although post-translational modifications of antibodies have been observed and studied for decades, we only now start to understand the full impact of this incredible microheterogeneity. PTMs have moved from being viewed as a mere nuisance to antibody manufacturing that requires controlling to a potential handle to modify and improve specific antibody functions.

In this session, we will hear about current state-of-the-art in PTM detection and novel insights into the role and modulation of PTMs in our immune system as well as the way in which we can exploit PTMs to make better (therapeutic) antibodies. The first and the second (after the break) part of our session will be initiated with lectures by renowned experts in their fields. Professor Albert Heck (Utrecht University) is a world-expert on the structural analysis of proteins by mass spectrometry. He received the Frank H. Field and Joe L. Franklin Award for outstanding achievement in mass spectrometry from the American Chemical Society and in 2017 he was received the NWO Spinoza Prize, which is the highest award in Dutch Science. Prof Heck will discuss how innovative and advanced mass spectrometry methods can be used to map antibody heterogeneity due to PTMs. Leendert Trouw (Leiden University Medical Center) will discuss the role of two amino acid modifications (citrullination and carbamylation) in the autoimmune disease rheumatoid arthritis (RA). On the one hand, the presence of antibodies against citrullinated or carbamylated proteins represents a prognostic marker for the disease. How antibodies recognize diverse antigens carrying these modifications is therefore an important area of study. Carbamylation of antibodies furthermore may also have functional consequences for antibody effector functions which will be highlighted. Professor Gerhard Krönke (University of Erlangen) will discuss how the PTM profile and inflammatory activity of autoantibodies in RA is regulated by TH17 helper T cells. His work gives us a novel insight into a mechanism by which the cellular immune system regulates the activity of antibodies and how its derailment may lead to the initiation of (autoimmune) disease.

After the break, Taia Wang (Stanford University School of Medicine) will discuss the diverse downstream proinflammatory, anti-inflammatory and immunomodulatory consequences of the engagement of type I and type II Fc receptors, which are influenced by the Fc’s amino acid sequence and the complex, biantennary Fc-associated N-linked glycan, in the context of infectious, autoimmune, and neoplastic disorders. Yingda Xu (Adimab) will bring us back to the importance of PTMS in manufacturing and control of therapeutic antibody production. He will show novel data on the identification of chemically labile sites in antibodies and how this information may be used in therapeutic antibody lead selection. Finally, Raiees Andrabi (The Scripps Research Institute) will discuss how sulfation of residues in the antibody binding site is critical for certain broadly neutralizing anti-HIV-1 antibodies targeting the envelope glycoprotein.

We hope that this session will convey the current interest and high excitement in antibody PTMs and will serve to promote further research into the importance and impact of PTM microheterogeneity for polyclonal antibody responses as well as for monoclonal antibody therapeutics.

 

Interested in attending the meeting? Learn more from this PDF, which includes all session summaries written by the chairpersons.

Society members can save 15% on the registration fee! Not a member? Please join!

Membership is free for students and employees of the Society’s corporate sponsors.

 

 

Novel engineering strategies to enhance antibody functions

by

Please join us at the Society’s annual meeting, Antibody Engineering & Therapeutics, on December 11-15, 2017 at the Manchester Grand Hyatt, San Diego, CA! Here, Paul J. Carter, Ph.D., Senior Director and Staff Scientist, Antibody Engineering, Genentech, discusses what you will learn in his session on novel engineering strategies to enhance antibody functions, which will be held on Friday December 15, 2017.

A recurring theme with this conference since it began in the early 1990s has been the presentation of new antibody engineering technologies and their application to the design of antibodies for specific clinical applications. True to this tradition, this session will showcase a broad range of different antibody platform technologies for improving existing antibody properties or engineering antibodies with brand new capabilities. Antibody technologies to be highlighted include bispecific antibodies in many different formats, PEGylation, glyco-engineering and engineering of isoelectric point (pI).

Greg Lazar (Genentech) will present on agonizing the TNFR superfamily independent of FcgR-mediated cross-linking using multiple antibody technology platforms. This talk will highlight in vitro and in vivo proof-of-concept data. Taichi Kuramochi (Chugai Pharmabody Research) will provide a case study on pI engineering of an antibody variable domains and constant regions to enhance the potency of pH-dependent antigen binding antibody. Gestur Vidarsson (Sanquin Research) will describe how the natural glycans of antibodies can be tailored to increase ADCC and/or CDC activities. For example, combining hypergalactosylation with afucosylation increases the potency of ADCC activity beyond that achieved by afucosylation alone.

After the networking break, Martin Steegmaier (Roche Innovation Center Munich) will discuss applications of CrossMAbs in bivalent (1+1), trivalent (2+1) and tetravalent (2+2) formats for cancer immunotherapy and the use of novel targeting approaches for neurological diseases. Additionally, bispecific antibodies in DutaFab format for ophthalmologic diseases will be described.  Qing Li (Medimmune) will demonstrate tumor uptake of a PEGylated antibody fragment. Specifically, this presentation will focus on investigation of the correlation between hydrodynamic size, pharmacokinetic parameters and tumor uptake of a PEGylated diabody. Kartik Chandran (Albert Einstein College of Medicine) will close the session by presenting a bispecific antibody “Trojan horse” approach for broad protection against ebolaviruses. One arm of the bispecific antibody binds a conserved epitope on ebolavirus glycoprotein. After internalization of the virus/antibody complex into cells and trafficking to late endosomes, the second arm of the bispecific blocks infection by binding to the viral entry receptor, NPC1.

Interested in attending the meeting? Society members can save 15% on the registration fee! Contact us at membership@antibodysociety.org for the code.

Not a member? Please join!

Membership is free for students and employees of the Society’s corporate sponsors.