Table of Contents

Volume 4, Issue 5

  September/October 2012

  Pages 553 - 633

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 Issue Catalogue (MARC XML)

GUEST EDITORIAL

Open Access Article

Mechanism of resistance to cetuximab therapy in colorectal cancer: Possible role of antibodies to immunoglobulin allotypes

Janardan P. Pandey
Pages 553 - 554
http://dx.doi.org/10.4161/mabs.21311
Full Text | PDF




MEETING REPORTS

Open Access Article

The new ParaDIgm: IgM from bench to clinic: November 15–16, 2011, Frankfurt, Germany

Sherif Hanala
Pages 555 - 561
http://dx.doi.org/10.4161/mabs.21449
Abstract | Full Text | PDF

The inaugural IgM event entitled “The new ParaDIgm: IgM from bench to clinic” brought together the increasingly active and growing IgM antibody community to discuss recent advances and challenges facing the discovery and development of IgM antibody therapies and technologies. Researchers, clinicians and biomanufacturing experts delivered 21 talks on the basic science and isolation of IgM, upstream and downstream development, and formulation and clinical development of the molecules. Participants networked around topics aimed at exploring the full potential of IgM antibodies. The meeting was held at DECHEMA Gesellschaft für Chemische Technik und Biotechnologie e. V. (Society for Chemical Engineering and Biotechnology), a non-profit scientific and technical society based in Frankfurt am Main, Germany. The meeting was sponsored by Patrys, Laureate Biopharma, Bio-Rad Laboratories, BIA Separations, Percivia and the Bio Affinity Company (BAC). The second New ParaDIgm: IgM from bench to clinic meeting, will be held on April 23–24, 2013 in Frankfurt, Germany.

Open Access Article

2^nd PEGS Annual Symposium on Antibodies for Cancer Therapy: April 30–May 1, 2012, Boston, USA

Mitchell Ho, Ivor Royston and Alain Beck
Pages 562 - 570
http://dx.doi.org/10.4161/mabs.21521
Abstract | Full Text | PDF

The 2nd Annual Antibodies for Cancer Therapy symposium, organized again by Cambridge Healthtech Institute as part of the Protein Engineering Summit, was held in Boston, USA from April 30^th to May 1^st, 2012. Since the approval of the first cancer antibody therapeutic, rituximab, fifteen years ago, eleven have been approved for cancer therapy, although one, gemtuzumab ozogamicin, was withdrawn from the market.  The first day of the symposium started with a historical review of early work for lymphomas and leukemias and the evolution from murine to human antibodies. The symposium discussed the current status and future perspectives of therapeutic antibodies in the biology of immunoglobulin, emerging research on biosimilars and biobetters, and engineering bispecific antibodies and antibody-drug conjugates. The tumor penetration session was focused on the understanding of antibody therapy using ex vivo tumor spheroids and the development of novel agents targeting epithelial junctions in solid tumors. The second day of the symposium discussed the development of new generation recombinant immunotoxins with low immunogenicity, construction of chimeric antigen receptors, and the proof-of-concept of ‘photoimmunotherapy’. The preclinical and clinical session presented antibodies targeting Notch signaling and chemokine receptors.  Finally, the symposium discussed emerging technologies and platforms for therapeutic antibody discovery.




REVIEWS

CD19 as an attractive target for antibody-based therapy

Ohad Hammer
Pages 571 - 577
http://dx.doi.org/10.4161/mabs.21338
Abstract | Full Text | PDF

Despite progress in the treatment of B cell disorders, novel treatment approaches are still highly needed. CD19 is a pan-B cell marker that is recognized as a potential immunotherapy target for B cell disorders, including blood-borne malignancies and autoimmune diseases. Although initial attempts to target CD19 were unsuccessful, a new wave of investigational agents is currently in development. These agents are based on novel antibody-based technologies and formats that appear to better exploit CD19's therapeutic potential, and some promising clinical study data has already been reported. This review provides an overview and the rationale for the most advanced CD19-targeting programs in development.

Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies

Yi Du, Alison Walsh, Robin Ehrick, Wei Xu, Kimberly May and Hongcheng Liu
Pages 578 - 585
http://dx.doi.org/10.4161/mabs.21328
Abstract | Full Text | PDF

The existence of multiple variants with differences in either charge, molecular weight or other properties is a common feature of monoclonal antibodies. These charge variants are generally referred to as acidic or basic compared with the main species. The chemical nature of the main species is usually well-understood, but understanding the chemical nature of acidic and basic species, and the differences between all three species, is critical for process development and formulation design. Complete understanding of acidic and basic species, however, is challenging because both species are known to contain multiple modifications, and it is likely that more modifications may be discovered. This review focuses on the current understanding of the modifications that can result in the generation of acidic and basic species and their affect on antibody structure, stability and biological functions. Chromatography elution profiles and several critical aspects regarding fraction collection and sample preparations necessary for detailed characterization are also discussed.

Peptibodies: A flexible alternative format to antibodies

Grant Shimamoto, Colin Gegg, Tom Boone and Christophe Quéva
Pages 586 - 591
http://dx.doi.org/10.4161/mabs.21024
Abstract | Full Text | PDF

Peptibodies or peptide-Fc fusions are an attractive alternative therapeutic format to monoclonal antibodies. They consist of biologically active peptides grafted onto an Fc domain. This approach retains certain desirable features of antibodies, notably an increased apparent affinity through the avidity conferred by the dimerization of two Fcs and a long plasma residency time. Peptibodies can be made in E. coli using recombinant technology. The manufacturing process involves fermentation and downstream processing, including refolding and multiple column chromatographic steps, that result in overall yields and quality suitable for commercial development. Romiplostim, marketed under the brand name Nplate®, is the first peptibody to be approved by the United States Food and Drug Administration and the European Medicines Agency and is indicated for the treatment of immune thrombocytopenic purpura. AMG 386, a peptibody antagonist to angiopoietins 1 and 2, is being evaluated in Phase 3 clinical testing in combination with chemotherapy in women with ovarian cancer. AMG 819, a peptibody targeting nerve growth factor for pain has also progressed to clinical trials. These peptibodies illustrate the versatility of the modality.




REPORTS

High-affinity monoclonal antibodies to cell surface tumor antigen glypican-3 generated through a combination of peptide immunization and flow cytometry screening

Yen Phung, Wei Gao, Yan-Gao Man, Satoshi Nagata and Mitchell Ho
Pages 592 - 599
http://dx.doi.org/10.4161/mabs.20933
Abstract | Full Text | PDF

Isolating high-affinity antibodies against native tumor antigens on the cell surface is not straightforward using standard hybridoma procedures. Here, we describe a combination method of synthetic peptide immunization and high-throughput flow cytometry screening to efficiently isolate hybridomas for cell binding. Using this method, we identified high-affinity monoclonal antibodies specific for the native form of glypcian-3 (GPC3), a target heterogeneously expressed in hepatocellular carcinoma (HCC) and other cancers. We isolated a panel of monoclonal antibodies (YP6, YP7, YP8, YP9 and YP9.1) for cell surface binding. The antibodies were used to characterize GPC3 protein expression in human liver cancer cell lines and tissues by flow cytometry, immunoblotting and immunohistochemistry. The best antibody (YP7) bound cell surface-associated GPC3 with equilibrium dissociation constant, K_D = 0.3 nmol/L and was highly specific for HCC, not normal tissues or other forms of primary liver cancers (such as cholangiocarcinoma). Interestingly, the new antibody was highly sensitive in that it detected GPC3 in low expression ovarian clear cell carcinoma and melanoma cells. The YP7 antibody exhibited significant HCC xenograft tumor growth inhibition in nude mice. These results describe an improved method for producing high-affinity monoclonal antibodies to cell surface tumor antigens and represent a general approach to isolate therapeutic antibodies against cancer. The new high-affinity antibodies described here have significant potential for GPC3-expressing cancer diagnostics and therapy.

Open Access Article

Simultaneous targeting of TNF and Ang2 with a novel bispecific antibody enhances efficacy in an in vivo model of arthritis

Palanisamy Kanakaraj, Bridget A. Puffer, Xiao-Tao Yao, Spandana Kankanala, Ernie Boyd, Rutul R. Shah, Geping Wang, Dimki Patel, Rajesh Krishnamurthy, Shashi Kaithamana, Rodger G. Smith, David W. LaFleur, Carlos F. Barbas III, David M. Hilbert, Peter A. Kiener and Viktor V. Roschke
Pages 600 - 613
http://dx.doi.org/10.4161/mabs.21227
Abstract | Full Text | PDF

Despite the clinical success of anti-tumor necrosis factor (TNF) therapies in the treatment of inflammatory conditions such as rheumatoid arthritis, Crohn disease and psoriasis, full control of the diseases only occurs in a subset of patients and there is a need for new therapeutics with improved efficacy against broader patient populations. One possible approach is to combine biological therapeutics, but both the cost of the therapeutics and the potential for additional toxicities needs to be considered. In addition to the various mediators of immune and inflammatory pathways, angiogenesis is reported to contribute substantially to the overall pathogenesis of inflammatory diseases. The combination of an anti-angiogenic agent with anti-TNF into one molecule could be more efficacious without the risk of severe immunosuppression. To evaluate this approach with our Zybody technology, we generated bispecific antibodies that contain an Ang2 targeting peptide genetically fused to the anti-TNF antibody adalimumab (Humira®). The bispecific molecules retain the binding and functional characteristics of the anti-TNF antibody, but with additional activity that neutralizes Ang2. In a TNF transgenic mouse model of arthritis, the bispecific anti-TNF-Ang2 molecules showed a dose-dependent reduction in both clinical symptoms and histological scores that were significantly better than that achieved by adalimumab alone.

Trifunctional antibody ertumaxomab: Non-immunological effects on Her2 receptor activity and downstream signaling

Simone Diermeier-Daucher, Olaf Ortmann, Stefan Buchholz and Gero Brockhoff
Pages 614 - 622
http://dx.doi.org/10.4161/mabs.21003
Abstract | Full Text | PDF

Background: The trifunctional antibody ertumaxomab bivalently targets the human epidermal growth factor receptor 2 (Her2) on epithelial (tumor) cells and the T cell specific CD3 antigen, and its Fc region is selectively recognized by Fcγ type I/III receptor-positive immune cells. As a trifunctional immunoglobulin, ertumaxomab therefore not only targets Her2 on cancer cells, but also triggers immunological effector mechanisms mediated by T and accessory cells (e.g., macrophages, dendritic cells, natural killer cells). Whether molecular effects, however, might contribute to the cellular antitumor efficiency of ertumaxomab are largely unknown.

Methods: Potential molecular effects of ertumaxomab on Her2-overexpressing BT474 and SK-BR-3 breast cancer cells were evaluated. The dissociation constant K_d of ertumaxomab was calculated from titration curves that were recorded by flow cytometry. Treatment-induced changes in Her2 homodimerization were determined by flow cytometric fluorescence resonance energy transfer measurements on a cell-by-cell basis. Potential activation / deactivation of Her2, ERK1/2, AKT and STAT3 were analyzed by western blotting, Immunochemistry and immunofluorescent cell staining.

Results: The K_d of ertumaxomab for Her2-binding was determined at 265 nM and the ertumaxomab binding epitope was found to not overlap with that of the therapeutic anti-Her2 monoclonal antibodies trastuzumab and pertuzumab. Ertumaxomab caused an increase in Her2 phosphorylation at higher antibody concentrations, but changed neither the rate of Her2-homodimerization /-phosphorylation nor the activation state of key downstream signaling proteins analyzed.

Conclusions: The unique mode of action of ertumaxomab, which relies more on activation of immune-mediated mechanisms against tumor cells compared with currently available therapeutic antibodies for breast cancer treatment, suggests that modular or sequential treatment with the trifunctional bivalent antibody might complement the therapeutic activity of other anti-Her2/anti-ErbB receptor reagents.

Open Access Article

The assay design used for measurement of therapeutic antibody concentrations can affect pharmacokinetic parameters: Case studies

Saloumeh K. Fischer, Jihong Yang, Banmeet Anand, Kyra Cowan, Robert Hendricks, Jing Li, Gerald Nakamura and An Song
Pages 623 - 631
http://dx.doi.org/10.4161/mabs.20814
Abstract | Full Text | PDF

To interpret pharmacokinetic (PK) data of biotherapeutics, it is critical to understand which drug species is being measured by the PK assay. For therapeutic antibodies, it is generally accepted that “free” circulating antibodies are the pharmacologically active form needed to determine the PK/ pharmacodynamic (PD) relationship, safety margin calculations, and dose projections from animals to humans and the eventual characterization of the exposure in the clinic. However, “total” drug may be important in evaluating the dynamic interaction between the drug and the target, as well as the total drug exposure. In the absence of or with low amounts of soluble ligand /shed receptor, total and free drug species are often equivalent and their detection is less sensitive to assay formats or reagent choices. In contrast, in the presence of a significant amount of ligand, assay design and characterization of assay reagents are critical to understanding the PK profiles. Here, we present case studies where different assay formats affected measured PK profiles and data interpretation. The results from reagent characterizations provide a potential explanation for the observed discrepancies and highlight the importance of reagent characterization in understanding which drug species are being measured to accurately interpret PK parameters.




EDITOR'S CORNER

Open Access Article

2012 Upcoming Meetings

Janice M. Reichert
Pages 632 - 633
http://dx.doi.org/10.4161/mabs.21612
Abstract | Full Text | PDF

The Fall/Winter 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.