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The Antibody Society

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Most read from mAbs, April 2019

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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 freely downloaded by following the links below.

Issue 11.3 (April 2019)

CH2 domain orientation of human immunoglobulin G in solution: Structural comparison of glycosylated and aglycosylated Fc regions using small-angle X-ray scattering

Yageta et al examined the small-angle X-ray scattering (SAXS) profile of the glycosylated Fc region (gFc) and aglycosylated Fc region (aFc) in solution to determine if removal of the N-linked glycan alters the CH2 domain orientation in the Fc region. For both gFc and aFc, the best-fitted SAXS profiles corresponded to ones calculated based on the crystal structure of gFc that formed a “semi-closed” CH2 domain orientation. Collectively, the data indicated that the removal of the N-linked glycan only negligibly affected the CH2 domain orientation in solution. Their findings will guide the development of methodology for the production of highly refined functional Fc variants.

Charge variants characterization and release assay development for co-formulated antibodies as a combination therapy

Characterization of co-formulated biologics can be challenging due to the high degree of similarity in the physicochemical properties of co-formulated proteins, especially at different concentrations of individual components. In this new report, Cao et al present the results of a deamidation study of one monoclonal antibody component (mAb-B) in co-formulated combination antibodies (referred to as COMBO) that contain various ratios of mAb-A and mAb-B. A single deamidation site in the complementarity-determining region of mAb-B was identified as a critical quality attribute (CQA) due to its impact on biological activity. A conventional charge-based method of monitoring mAb-B deamidation presented specificity and robustness challenges, especially when mAb-B was a minor component in the COMBO, making it unsuitable for lot release and stability testing. The authors developed and qualified a new, quality-control-friendly, single quadrupole Dalton mass detector (QDa)–based method to monitor site-specific deamidation. Their approach can be also used as a multi-attribute method for monitoring other quality attributes in COMBO. This analytical paradigm is applicable to the identification of CQAs in combination therapeutic molecules, and to the subsequent development of a highly specific, highly sensitive, and sufficiently robust method for routine monitoring CQAs for lot release test and during stability studies.

Capture and display of antibodies secreted by hybridoma cells enables fluorescent on-cell screening

Puligedda et. al describe a system in which hybridomas specifically capture and display the mAbs they secrete. Using On-Cell mAb Screening (OCMS™), monoclonal antibodies (mAbs) displayed on the cell surface can be rapidly assayed for expression level and binding specificity using fluorescent antigens with high-content (image-based) methods or flow cytometry. OCMS™ demonstrated specific mAb binding to poliovirus and rabies virus by forming a cell surface IgG “cap”, as a universal assay for anti-viral mAbs. The authors produced and characterized OCMS™-enabled hybridomas secreting mAbs that neutralize poliovirus and used fluorescence microscopy to identify and clone a human mAb specific for the human N-methyl-D-aspartate receptor. They also used OCMS™ to assess expression and antigen binding of a recombinant mAb produced in 293T cells.

Physicochemical and functional assessments demonstrating analytical similarity between rituximab biosimilar HLX01 and the MabThera®

As discussed by Xu et al, development of bio-therapeutics has exhibited exponential growth in China over the past decade. However, no biosimilar drug has been approved in China (CN) due to the lack of a national biosimilar regulatory guidance. HLX01, a rituximab biosimilar developed in China under European Medicines Agency biosimilar guidelines and requirements, was the first such drug submitted for regulatory review in China, and it is expected to receive approval there as a biosimilar product. To demonstrate the analytical similarities of HLX01, CN-rituximab (sourced in China but manufactured in Europe) and EU-rituximab (sourced and manufactured in Europe), an extensive 3-way physicochemical and functional similarity assessment using a series of orthogonal and state-of-the-art techniques was conducted, following the similarity requirement guidelines recently published by China’s Center for Drug Evaluation. The results of the similarity study showed an identical protein amino acid sequence and highly similar primary structures between HLX01 and the reference product (RP) MabThera®, along with high similarities in higher order structures, potency, integrity, purity and impurity profiles, biological and immunological binding functions, as well as degradation behaviors under stress conditions. In addition, HLX01 presented slightly lower aggregates and better photostability compared with the RP. Despite slight changes in relative abundance of glycan moieties and heavy chain C-terminal lysine modification, no differences in biological activities and immunological properties were observed between the RP and HLX01. In conclusion, HLX01 is highly similar to CN- and EU-sourced RP in terms of physicochemical properties and biological activities, suggesting similar product quality, efficacy, and safety. The regulatory requirements interpreted and applied towards the HLX01 marketing application sets a precedent for analytical similarity assessment of biosimilar products in China.

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Most read from mAbs, Feb/March 2019

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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 freely downloaded by following the links below.

Issue 11.2 (February/March 2019)

Antibodies to watch in 2019.
For the past 10 years, the annual ‘Antibodies to watch’ articles have provided updates on key events in the late-stage development of antibody therapeutics, such as first regulatory review or approval, that occurred in the year before publication or were anticipated to occur during the year of publication. To commemorate the 10th anniversary of the article series and to celebrate the 2018 Nobel Prizes in Chemistry and in Physiology or Medicine, which were given for work that is highly relevant to antibody therapeutics research and development, Kaplon and Reichert expanded the scope of the data presented to include an overview of all commercial clinical development of antibody therapeutics and approval success rates for this class of molecules. The data indicate that:
1) antibody therapeutics are entering clinical study, and being approved, in record numbers;
2) the commercial pipeline is robust, with over 570 antibody therapeutics at various clinical phases, including 62 in late-stage clinical studies; and
3) Phase 1 to approval success rates are favorable, ranging from 17–25%, depending on the therapeutic area (cancer vs. non-cancer).

Ion channels as therapeutic antibody targets.
In this review, Hutchings et al evaluate the technical challenges of raising antibodies to membrane-spanning proteins together with enabling technologies that may facilitate the discovery of antibody therapeutics to ion channels. They also discuss the potential targeting opportunities in the anti-ion channel antibody landscape, along with a number of case studies where functional antibodies that target ion channels have been reported. Antibodies currently in development and progressing towards the clinic are highlighted.

Influence of N-glycosylation on effector functions and thermal stability of glycoengineered IgG1 monoclonal antibody with homogeneous glycoforms.
The separation of various glycoforms to investigate the biological and functional relevance of glycosylation is a major challenge, and the individual contributions of each glycoform is usually not considered when evaluating mAbs with highly heterogeneous distributions. In this study, Wada et al used chemoenzymatic glycoengineering incorporating an endo-β-N-acetylglucosaminidase (ENGase) EndoS2 and its mutant with transglycosylation activity to generate mAb glycoforms with highly homogeneous and well-defined N-glycans to better understand and precisely evaluate the effect of each N-glycan structure on Fc effector functions and protein stability. They demonstrated that the core fucosylation, non-reducing terminal galactosylation, sialylation, and mannosylation of IgG1 mAb N-glycans impact not only on FcγRIIIa binding, antibody-dependent cell-mediated cytotoxicity, and C1q binding, but also FcRn binding, thermal stability and propensity for protein aggregation.

Co-engaging CD47 and CD19 with a bispecific antibody abrogates B-cell receptor/CD19 association leading to impaired B-cell proliferation.
In this report, Hatterer et al describes the generation of a CD47xCD19 bispecific antibody (biAb) to target and deplete B cells via multiple antibody-mediated mechanisms. Interestingly, the biAb, constructed of a CD19 binding arm and a CD47 binding arm, inhibited BCR-mediated B-cell proliferation with an effect even more potent than a CD19 monoclonal antibody (mAb). The inhibitory effect of the biAb was not attributable to CD47 binding because a monovalent or bivalent anti-CD47 mAb had no effect on B cell proliferation. Fluorescence resonance energy transfer analysis demonstrated that co-engaging CD19 and CD47 prevented CD19 clustering and its migration to BCR clusters, while only engaging CD19 (with a mAb) showed no impact on either CD19 clustering or migration. The lack of association between CD19 and the BCR resulted in decreased phosphorylation of CD19 upon BCR activation. Furthermore, the biAb differentially modulated BCR-induced gene expression compared to a CD19 mAb. Taken together, this unexpected role of CD47xCD19 co-ligation in inhibiting B cell proliferation illuminates a novel approach in which two B cell surface molecules can be tethered, to one another in order, which may provide a therapeutic benefit in settings of autoimmunity and B cell malignancies.

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Grad/PostDoc Poster Awards presented at Antibody Engineering &Therapeutics

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Congratulations to our 2018 Graduate Student and Post-doctoral Poster Award winners! An award ceremony was held on December 12th at the Society’s annual meeting, Antibody Engineering &Therapeutics, to recognize the recipients. The winners were:

Junpeng Qi (Postdoctoral Associate, The Scripps Research Institute). Poster title: Potent and selective antitumor activity of a T cell-engaging bispecific antibody targeting a membrane-proximal epitope of ROR1.

Pietro Sormanni (Borysiewicz Biomedical Sciences Fellow (postdoctoral), University of Cambridge). Poster title: Third generation antibody discovery: In silico rational design.

Madeleine Jennewein (Ph.D. candidate, Harvard University). Poster title: Trans-placental antibody transfer selects for highly functional antibodies.


Junpeng Qi – Scripps Research Institute (The Rader Lab)

“I would to thank The Antibody Society for giving me this award. It is a great opportunity, especially for a young scientist, to get the latest progress in antibody engineering and therapeutics, and to be connected with the excellent scientists in the antibody field. From this impressive annual meeting I learned that we can do amazing science with antibodies and develop fantastic antibody therapeutics benefiting patients as well.”

Pietro Sormanni – University of Cambridge (The Vendruscolo Lab)

“I am immensely grateful to The Antibody Society for selecting my application, and even more for organizing this award. This award has given me the opportunity to attend this terrific meeting, to learn about world-class research in both industry and academia, and more importantly to share and discuss my own work with leading scientists from across the world. I would like to stress the importance of the existence of awards such as this, and I call for the Society and the sponsors of the meeting to make available more of these awards and travel grants to early career researchers. Because at the end of the day, the growing community of postdoctoral researchers and graduate students is the engine that powers biomedical research, certainly in academia, and increasingly also in industry. And these travel grants provide us with the unique opportunity to get out of the lab, come to these meetings and greatly expand our research horizons, and generate new ideas. So I hope to see a bit more space dedicated to early career researchers in future editions of this meeting, thank you all for your attention, and again to The Antibody Society for this award.”

Madeleine F Jennewein – Harvard University (The Alter Lab)

Madeleine Jennewein was unexpectedly unable to join us at the meeting in San Diego, but we thank her for her participation and wish her the best with her work.

The Antibody Society at Antibody Engineering & Therapeutics 2018

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The Antibody Society held its 2018 annual meeting at Antibody Engineering & Therapeutics in San Diego on December 9-13. It was a a great opportunity for the board members and volunteers to meet our society members and provide updates on Society initiatives.

Informative keynote addresses were given by Prof. Andreas Plückthun (University of Zurich), Prof. David Baker (University of Washington), Prof. Rachael Clark (Harvard Medical School) and Dr. Badrul Chowdhury (Medimmune).

One of the highlights of the conference was the Antibodies to Watch in 2019 presentation by Dr. Janice Reichert (Executive Director of TAbS and Editor-in-Chief of mAbs).


The ‘Antibodies to watch in 2019’ paper is currently online in the accepted (manuscript) form. Society members will be informed when the final article, which will be open access, is available.

The Antibody Society booth at Antibody Engineering &Therapeutics

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.

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