Table of Contents

Volume 4, Issue 4

  July/August 2012

  Pages 419 - 552

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 Issue Catalogue (MARC XML)

EDITORIAL

Open Access Article

Marketing approval of mogamulizumab: A triumph for glyco-engineering

Alain Beck and Janice M. Reichert
Pages 419 - 425
http://dx.doi.org/10.4161/mabs.20996
Abstract | Full Text | PDF

Therapeutic properties of antibodies strongly depend on the composition of their glycans. Most of the currently approved antibodies are produced in mammalian cell lines, which yield mixtures of different glycoforms that are close to those of humans, but not fully identical. Glyco-engineering is being developed as a method to control the composition of carbohydrates and to enhance the pharmacological properties of mAbs. The recent approval in Japan of mogamulizumab (POTELIGEO^®), the first glyco-engineered antibody to reach the market, is a landmark in the field of therapeutic antibodies. Mogamulizumab is a humanized mAb derived from Kyowa Hakko Kirin’s POTELLIGENT^® technology, which produces antibodies with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity. The approval was granted April 30, 2012 by the Japanese Ministry of Health, Labour and Welfare for patients with relapsed or refractory CCR4-positive adult T-cell leukemia-lymphoma.




MEETING REPORTS

Open Access Article

1st French-Israeli International Conference on B Cells and therapeutic antibodies: October 23–25, 2011 Jerusalem, Israel

Claude-Agnès Reynaud, Sandrine Moutel, Marie-Caroline Dieu-Nosjean, Reuven Laskov and Jean-Luc Teillaud
Pages 426 - 433
http://dx.doi.org/10.4161/mabs.20289
Full Text | PDF

Open Access Article

7th Cancer Scientific Forum of the Cancéropôle Lyon Auvergne Rhône-Alpes: March 20–21, 2012, Lyon, France

Petrus J. Pauwels, Charles Dumontet, Janice M. Reichert, Alain Beck, Liliane Goetsch, Nathalie Corvaïa, Christian Klein, Bertrand Coiffier and Beverly A. Teicher
Pages 434 - 444
http://dx.doi.org/10.4161/mabs.20869
Abstract | Full Text | PDF

The Innovative Approaches in Anti-Cancer Monoclonal Antibodies meeting, held on March 20, 2012 in Lyon, was organized by Cancéropôle Lyon Auvergne-Rhône-Alps in partnership with the French competitiveness cluster Lyonbiopôle. CLARA is one of the seven cancer research clusters within France in charge of facilitating Translational Oncology Research by taking into account the objectives of the French National Cancer Plans I and II and, in coordination with the French National Cancer Institute and local authorities (mainly Grand Lyon, Rhône County and Rhône-Alpes Region), to perform economic development of research findings. The contribution of lectures by outstanding speakers as described in this report, the organization of two-round tables: “Antibody treatment in cancer: Unmet needs in solid tumors and hematological malignancies,” and “From chimeric to more than human antibodies,” together with face-to-face meetings, was shared by over 230 participants. The lectures provided an overview of the commercial pipeline of monoclonal antibody (mAb) therapeutics for cancer; discussion of the distinction between biosimilar, biobetter and next generation therapeutic antibodies for cancer; updates on obinutuzumab and the use of mAbs in lymphoma; and discussion of antibody-drug conjugates.




REVIEWS

Molecular engineering of antibodies for therapeutic and diagnostic purposes

Frédéric Ducancel and Bruno H. Muller
Pages 445 - 457
http://dx.doi.org/10.4161/mabs.20776
Abstract | Full Text | PDF

During the past ten years, monoclonal antibodies (mAbs) have taken center stage in the field of targeted therapy and diagnosis. This increased interest in mAbs is due to their binding accuracy (affinity and specificity) together with the original molecular and structural rules that govern interactions with their cognate antigen. In addition, the effector properties of antibodies constitute a second major advantage associated with their clinical use. The development of molecular and structural engineering and more recently of in vitro evolution of antibodies has opened up new perspectives in the de novo design of antibodies more adapted to clinical and diagnostic use. Thus, efforts are regularly made by researchers to improve or modulate antibody recognition properties, to adapt their pharmacokinetics, engineer their stability, and control their immunogenicity. This review presents the latest molecular engineering results on mAbs with therapeutic and diagnostic applications.

Brentuximab vedotin

Niels W. C.J. van de Donk and Eugen Dhimolea
Pages 458 - 465
http://dx.doi.org/10.4161/mabs.20230
Abstract | Full Text | PDF

Brentuximab vedotin (SGN-35; Adcetris®) is an anti-CD30 antibody conjugated via a protease-cleavable linker to the potent anti-microtubule agent monomethyl auristatin E (MMAE). Following binding to CD30, brentuximab vedotin is rapidly internalized and transported to lysosomes where MMAE is released and binds to tubulin, leading to cell cycle arrest and apoptosis.

Several trials have shown durable antitumor activity with a manageable safety profile in patients with relapsed/refractory Hodgkin lymphoma, systemic anaplastic large cell lymphoma, or primary cutaneous CD30-positive lymphoproliferative disorders. Peripheral sensory neuropathy is a significant adverse event associated with brentuximab vedotin administration. Neuropathy symptoms are cumulative and dose-related. Multiple ongoing trials are currently evaluating brentuximab vedotin alone or in combination with other agents in relapsed/refractory patients, as well as patients with newly diagnosed disease.




REPORTS

Open Access Article

Pharmacokinetics of engineered human monomeric and dimeric CH2 domains

Kurt R. Gehlsen, Rui Gong, Dave Bramhill, David A. Wiersma, Shaun A. Kirkpatrick, Yangping Wang, Yang Feng and Dimiter S. Dimitrov
Pages 466 - 474
http://dx.doi.org/10.4161/mabs.20652
Abstract | Full Text | PDF

Therapeutic monoclonal antibodies have several advantages over small molecule drugs and small proteins and peptides, including a long serum half-life. The long serum half-life of IgG is due, in part, to its molecular weight (150kDa) and its ability to bind FcRn. Both the CH2 and CH3 domains of Fc are involved in FcRn binding. Antibody fragments and antibody-like scaffolds have improved penetration into tissues due to their small size, yet suffer from a short serum half-life of less than one hour. The human CH2 domain (CH2D) of IgG1 retains a portion of the FcRn binding site, is amenable to modification for target binding, and may represent the smallest antibody-like scaffold retaining a relatively long serum half-life. Here we describe the generation of a dimeric CH2D (dCH2D) and determination of its pharmacokinetics (PK), as well as the PK of wild-type monomeric CH2D (mCH2D) and a short stabilized CH2D variant (ssCH2D) in normal B6 mice, human FcRn transgenic mice and cynomolgus macaques. The elimination half-life of dCH2D was 9.9, 10.4 and 11.2 hours, and that of ssCH2D was 13.1, 9.9 and 11.4 hours, in B6 mice, hFcRn mice and cynomolgus macaques, respectively. These half-lives were slightly longer than that of mCH2D (6.9 and 8.8 hours) in B6 and hFcRn mice, respectively. These data demonstrate that engineered CH2D-based variants have relatively long serum half-lives, making them a unique scaffold suitable for development of targeted therapeutics.

Production, characterization and pharmacokinetic properties of antibodies with N-linked Mannose-5 glycans

Marcella Yu, Darren Brown, Chae Reed, Shan Chung, Jeff Lutman, Eric Stefanich, Anne Wong, Jean-Philippe Stephan and Robert Bayer
Pages 475 - 487
http://dx.doi.org/10.4161/mabs.20737
Abstract | Full Text | PDF

The effector functions of therapeutic antibodies are strongly affected by the specific glycans added to the Fc domain during post-translational processing. Antibodies bearing high levels of N-linked mannose-5 glycan (Man5) have been reported to exhibit enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared with antibodies with fucosylated complex or hybrid glycans. To better understand the relationship between antibodies with high levels of Man5 and their biological activity in vivo, we developed an approach to generate substantially homogeneous antibodies bearing the Man5 glycoform. A mannosidase inhibitor, kifunensine, was first incorporated in the cell culture process to generate antibodies with a distribution of high mannose glycoforms. Antibodies were then purified and treated with a mannosidase for trimming to Man5 in vitro. This 2-step approach can consistently generate antibodies with > 99% Man5 glycan. Antibodies bearing varying levels of Man5 were studied to compare ADCC and Fcγ receptor binding, and they showed enhanced ADCC activity and increased binding affinity to the FcγRIIIA. In addition, the clearance rate of antibodies bearing Man8/9 and Man5 glycans was determined in a pharmacokinetics study in mice. When compared with historical data, the antibodies bearing the high mannose glycoform exhibited faster clearance rate compared with antibodies bearing the fucosylated complex glycoform, while the pharmacokinetic properties of antibodies with Man8/9 and Man5 glycoforms appeared similar. In addition, we identified the presence of a mannosidase in mouse serum that converted most Man8/9 to Man6 after 24 h.

Expression and biological characterization of an anti-CD20 biosimilar candidate antibody: A case study

Denise Dorvignit, Julio L. Palacios, Maylin Merino, Tays Hernández, Katya Sosa, Angel Casacó, Alejandro López-Requena and Cristina Mateo de Acosta
Pages 488 - 496
http://dx.doi.org/10.4161/mabs.20761
Abstract | Full Text | PDF

The CD20 molecule is a non-glycosylated protein expressed mainly on the surface of B lymphocytes. In some pathogenic B cells, it shows an increased expression, thus becoming an attractive target for diagnosis and therapy. Rituximab is a chimeric antibody that specifically recognizes the human CD20 molecule. This antibody is indicated for the treatment of non-Hodgkin lymphomas and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. In this work, we describe the stable expression and biological evaluation of an anti-CD20 biosimilar antibody. While rituximab is produced in fed-batch culture of recombinant Chinese hamster ovary (CHO) cells, our biosimilar antibody expression process consists of continuous culture of recombinant murine NS0 myeloma cells. The ability of the purified biosimilar antibody to recognize the CD20 molecule on human tumor cell lines, as well as on peripheral blood mononuclear cells from humans and primates, was demonstrated by flow cytometry. The biosimilar antibody induced complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and apoptosis on human cell lines with high expression of CD20. In addition, this antibody depleted CD20-positive B lymphocytes from peripheral blood in monkeys. These results indicate that the biological properties of the biosimilar antibody compare favorably with those of the innovator product, and that it should be evaluated in future clinical trials.

Open Access Article

Generation, characterization and structural data of chymase binding proteins based on the human Fyn kinase SH3 domain

Daniel Schlatter, Simon Brack, David W. Banner, Sarah Batey, Jörg Benz, Julian Bertschinger, Walter Huber, Catherine Joseph, Arne C. Rufer, Anita van der Klooster, Martin Weber, Dragan Grabulovski and Michael Hennig
Pages 497 - 508
http://dx.doi.org/10.4161/mabs.20452
Abstract | Full Text | PDF

The serine protease chymase (EC = 3.4.21.39) is expressed in the secretory granules of mast cells, which are important in allergic reactions. Fynomers, which are binding proteins derived from the Fyn SH3 domain, were generated against human chymase to produce binding partners to facilitate crystallization, structure determination and structure-based drug discovery, and to provide inhibitors of chymase for therapeutic applications. The best Fynomer was found to bind chymase with a K_D of 0.9 nM and k_off of 6.6x10⁻⁴ s⁻¹, and to selectively inhibit chymase activity with an IC₅₀ value of 2 nM. Three different Fynomers were co-crystallized with chymase in 6 different crystal forms overall, with diffraction quality in the range of 2.25 to 1.4 Å resolution, which is suitable for drug design efforts. The X-ray structures show that all Fynomers bind to the active site of chymase. The conserved residues Arg15-Trp16-Thr17 in the RT-loop of the chymase binding Fynomers provide a tight interaction, with Trp16 pointing deep into the S1 pocket of chymase. These results confirm the suitability of Fynomers as research tools to facilitate protein crystallization, as well as for the development of assays to investigate the biological mechanism of targets. Finally, their highly specific inhibitory activity and favorable molecular properties support the use of Fynomers as potential therapeutic agents.

Open Access Article

Increased serum clearance of oligomannose species present on a human IgG1 molecule

Leslie Alessandri, David Ouellette, Aima Acquah, Mathew Rieser, David LeBlond, Mary Saltarelli, Czeslaw Radziejewski, Taro Fujimori and Ivan Correia
Pages 509 - 520
http://dx.doi.org/10.4161/mabs.20450
Abstract | Full Text | PDF

The role of Fc glycans on clearance of IgG molecule has been examined by various groups in experiments where specific glycans have been enriched or the entire spectrum of glycans was studied after administration in pre-clinical or clinical pharmacokinetic (PK) studies. The overall conclusions from these studies are inconsistent, which may result from differences in antibody structure or experimental design. In the present study a well-characterized recombinant monoclonal IgG1 molecule (mAb-1) was analyzed from serum samples obtained from a human PK study. mAb-1 was recovered from serum using its ligand cross-linked to Sepharose beads. The overall purity and recovery of all isoforms were carefully evaluated using a variety of methods. Glycans were then enzymatically cleaved, labeled using 2-aminobenzamide and analyzed by normal phase high performance liquid chromatography. The assays for recovering mAb-1 from serum and subsequent glycan analysis were rigorously qualified at a lower limit of quantitation of 15 μg/mL, thus permitting analysis to day 14 of the clinical PK study. Eight glycans were monitored and classified into two groups: (1) the oligomannose type structures (M5, M6 and M7) and (2) fucosylated biantennary oligosaccharides (FBO) structures (NGA2F, NA1F, NA2F, NA1F-GlcNAc and NGA2F-GlcNAc). We observed that the oligomannose species were cleared at a much faster rate (40%) than FBOs and conclude that high mannose species should be carefully monitored and controlled as they may affect PK of the therapeutic; they should thus be considered an important quality attribute. These observations were only possible through the application of rigorous analytical methods that we believe will need to be employed when comparing innovator and biosimilar molecules.

A high throughput capillary electrophoresis method to obtain pharmacokinetics and quality attributes of a therapeutic molecule in circulation

Reema Piparia, David Ouellette, W. Blaine Stine, Christine Grinnell, Edit Tarcsa, Czeslaw Radziejewski and Ivan Correia
Pages 521 - 531
http://dx.doi.org/10.4161/mabs.20099
Abstract | Full Text | PDF

Therapeutic proteins circulating in blood are in a highly crowded, redox environment at high temperatures of ~37°C. These molecules circulate in the presence of enzymes and other serum proteins making it difficult to predict from in vitro studies the stability, aggregation or pharmacokinetics of a therapeutic protein in vivo. Here, we describe use of a high throughput capillary electrophoresis based microfluidic device (LabChip GXII) to obtain pharmacokinetics (PK) of a fluorescently labeled human mAb directly from serum. The non-labeled and labeled mAbs were evaluated in single dose rat PK studies using a traditional ELISA method or LabChip GXII, respectively. The fluorescent dye did not significantly alter clearance of this particular mAb, and PK parameters were comparable for labeled and unlabeled molecules. Further, from the CE profile we concluded that the mAb was resistant to fragmentation or aggregation during circulation. In a follow-up experiment, dimers were generated from the mAb using photo-induced cross-linking of unmodified proteins (PICUP) and labeled with the same fluorophore. The extent of dimerization was incomplete and some monomer and higher molecular weight species were found in the preparation. In rat PK studies, the serum concentration-time profile of the three entities present in the dimer preparation could be followed simultaneously with the GXII technology. While further studies are warranted, we believe this method could be adapted to obtain PK of different forms of antibodies (oxidized, deamidated or various glycosylated species) and other proteins.

Correlation of ADCC activity with cytokine release induced by the stably expressed, glyco-engineered humanized Lewis Y-specific monoclonal antibody MB314

Ralf Kircheis, Nicole Halanek, Iris Koller, Wolfgang Jost, Manfred Schuster, Gilbert Gorr, Klaus Hajszan and Andreas Nechansky
Pages 532 - 541
http://dx.doi.org/10.4161/mabs.20577
Abstract | Full Text | PDF

A major limitation to the application of therapeutic monoclonal antibodies (mAbs) is their reduced in vivo efficacy compared with the high efficacy measured in vitro. Effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) are dramatically reduced in vivo by the presence of high amounts of endogenous IgG in the serum. Recent studies have shown that modification of the glycosylation moieties attached to the Fc part of the mAb can enhance binding affinity to FcγRIIIα receptors on natural killer cells and thus may counteract the reduced in vivo efficacy. In the present study, a humanized IgG1/κ monoclonal antibody recognizing the tumor-associated carbohydrate antigen Lewis Y was stably produced in a moss expression system that allows glyco-engineering. The glyco-modified mAb (designated MB314) showed a highly homogeneous N-glycosylation pattern lacking core-fucose. A side-by-side comparison to its parental counterpart produced in conventional mammalian cell-culture (MB311, formerly known as IGN311) by fluorescence-activated cell sorting analysis confirmed that the target specificity of MB314 is similar to that of MB311. In contrast, ADCC effector function of MB314 was increased up to 40-fold whereas complement dependent cytotoxicity activity was decreased 5-fold. Notably, a release of immunostimulatory cytokines, including interferon γ, monocyte chemotactic protein-1 (MCP-1), interleukin-6 and tumor necrosis factor (TNF) was particularly induced with the glyco-modified antibody. TNF release was associated with CD14⁺ cells, indicating activation of monocytes.

An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification

Pablo H. Sotelo, Noberto Collazo, Roberto Zuñiga, Matías Gutiérrez-González, Diego Catalán, Carolina Hager Ribeiro, Juan Carlos Aguillón and María Carmen Molina
Pages 542 - 550
http://dx.doi.org/10.4161/mabs.20653
Abstract | Full Text | PDF

Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene.

In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles.




EDITOR'S CORNER

Open Access Article

2012 Upcoming Meetings

Janice M. Reichert
Pages 551 - 552
http://dx.doi.org/10.4161/mabs.20772
Abstract | Full Text | PDF

The Summer/Fall 2012 conference season provides ample opportunities to attend meetings that feature topics relevant to antibody research and development (R&D). Meetings such as these serve critical informational and educational functions and are key networking opportunities. Locations are spread throughout the world, reflecting the global nature of antibody R&D.