Current Pharmaceutical Biotechnology - Volume 9, Issue 6, 2008

Today, monoclonal antibodies (MAbs) are the fastest growing class of human pharmaceuticals. Nearly 30 antibodies and antibodyderivatives (Fab fragments, radioimmunoconjugates, immunoconjugates and Fc-fusion proteins) based on mice and human G-type immunoglobulins (IgGs) have been approved worldwide in around 20 years (see Fig. 1 and A Beck et al in this issue). Several hundreds more are investigated in clinical trials in various therapeutic indications including oncology, autoimmune and infectious diseases, organ transplantation, cardiology, rheumatoid diseases, allergy, tissue growth and repair (J. Reichert in this issue). The worldwide revenues of antibody treatments generated around 20 billion USD in 2007. Seven therapeutic antibodies reached ‘blockbuster’ status with more than 1 billion USD turnover. The sale growth forecast from 2006 to 2012 is 14 % a year, compared to 0.6 % for small molecules. Monoclonal antibodies belong to a safe and target-specific category of pharmaceuticals that have a relative high success rate from early clinical to the licensure (25-29 % for antibodies vs. 11 % for small-molecule drugs; J. Reichert in this issue). These highly promising therapeutic and commercial features translated during the past few years into several business acquisitions of biotechs companies by large pharmaceutical companies, such as Cambridge Antibody Technology and MedImmune by Astra Zennecca or Abgenix by Amgen. The physicochemical structure of recombinant humanized or human antibodies is similar to that of circulating IgGs existing in >10 g/L concentration in human serum. By themselves, these molecules are potentially less toxic than xenobiotic small drugs. Nevertheless, blocking, activating or cross-linking an antigen target can trigger adverse events like for others conventional drugs. Particularly, new targets or new antibody formats have to be carefully investigated in pre-clinical safety studies especially following the dramatic experience of TGN-1412 anti-CD28 superagonist MAb first-in-man trial (C. Schneider in this issue). Several new regulatory issues are currently under discussion by European and US Agencies, such as the application of a more stringent calculation of the ‘first dose in man’ based on the MABEL (minimal anticipated biological effect level) approach and it will likely be recommended for high-risk medicinal products (e.g., novel protein format, novel target protein). As illustrated below, a major breakthrough for therapeutic applications of monoclonal antibodies, was achieved in 1997 with the commercialization of the first chimeric and humanized Mabs (Rituxan and Zenapax, respectively). Since that time, thirteen humanized antibodies as well as two fully human antibodies generated by phage-display (Humira; H. Thies, S. Dubel et al. in this issue) or by using transgenic mice bearing an human IgG repertoire (Vectibix) and immunized with the targeted antigen have been introduced on the market. For all these different kind of antibodies, approximately ten years were needed between the first paper dealing with the new technology and the market approval of the first candidate. Currently, most of the recombinant chimeric (Ch), humanized (Hz) or human (Hu) IgGs and Fc-fusion proteins (Enbrel, Amevive, Orencia and Arcalyst) are produced in mammalian cells (CHO, NS0, SP2/0) in up to 20,000 liters bioreactors by straightforward and wellestablished bioprocesses (M. Chartrain and L. Chu in this issue). Alternatively, two of the commercial Fab-fragments, which are not glycosylated are produced in E. coli (Lucentis and Cimzia).

Monoclonal antibodies (mAbs) comprise the majority of protein candidates currently in clinical development because of their versatility as therapeutic agents. While traditionally associated with the biotechnology industry, mAb therapeutics are now being developed and marketed by most major pharmaceutical firms. A total of 21 products are approved in the US, with additional products marketed outside the US, and over 200 mAb candidates are currently undergoing clinical study. Benchmark data for mAb therapeutics, such as clinical development and US Food and Drug Administration approval times, approval success rates, and clinical phase transition probabilities, are critical for strategic planning purposes. Trends in these benchmarks for various types of mAbs, with an emphasis on those studied as anticancer and immunological therapeutics, are discussed.

The development of new monoclonal antibodies (mAbs) is a still evolving field in finding new therapeutics. Structurally, mAbs have evolved over the past years by change from fully murine molecules to chimaeric antibodies or even humanized or fully human molecules. Although being “monoclonal” in terms of specificity, mAbs can be heterogeneous with respect to molecular features like microheterogeneity and glycosylation due to their complex manufacturing processes. Small changes in these processes can have considerable consequences on the product and also clinical safety and/or efficacy. Thus, quality, non-clinical and clinical data should not be seen as separate fields, but can impact on each other. For clinical trials of mAbs, non-clinical data from relevant species are required to evaluate the potential toxicity. Demonstration of relevance can be a challenging task, and should not be restricted to comparison of amino acid sequence of the target. Non-clinical development should also be seen as a tool for proactive risk identification. For first-in-human clinical trials, recent incidences have had considerable impact on regulatory handling, and have meanwhile led to a European guideline on risk identification and mitigation. For pivotal clinical trials, the requirements for mAbs are in principle the same as for other, non-biotechnological products. However, based on their long half-life and particular mechanism of action, enhanced safety measures can become necessary for mAbs to adequately detect and characterize also unexpected adverse reactions.

This article gives an overview about the development of human therapeutic antibodies generated by phage display. After an introduction to the method, the focus is on approved antibodies and those currently in clinical trials, 14 of which are described in detail.

This article provides an overview of the upstream technologies used in the industrial production of therapeutic monoclonal antibodies (mAbs) based on the cultivation of mammalian cells. More specifically, in a first section, after a short discussion of relevant biochemical characteristics of antibodies, we review the cell lines currently employed in commercial production and the methods of constructing and isolating production clones. This is followed with a review of the most current methods of commercial scale production and their associated technologies. Selected references and short discussions pertaining to emerging and relevant technologies have been embedded throughout the text in order to give a sense of the overall direction the field is taking.

The expanding field of monoclonal antibody-based pharmaceuticals has triggered increased interest in analytical characterization of these large proteins and in understanding of their heterogeneity and degradation pathways. As a result, a large number of enzymatic modifications as well as chemical and physical degradations have been reported in monoclonal antibodies in recent years. Most heterogeneity is related to changes in the surface charge of the antibody, either directly, as a change in the number of charged residues, or indirectly as a chemical or physical alteration that changes surface-charge distribution. This review presents an overview of the sources of charge-related heterogeneity in monoclonal antibodies and the methods used for their detection. A detailed section is dedicated to deamidation of asparagine and isomerization of aspartic acid residues, two ubiquitous degradation pathways detected in antibodies and other proteins as well. Finally, kinetic modeling of the accumulation of antibody variants is presented as a tool to determine the expected fraction of molecules that have undergone one or more degradation reactions.

Monoclonal antibodies (MAbs) are the fastest growing class of human pharmaceuticals. More than 20 MAbs have been approved and several hundreds are in clinical trials in various therapeutic indications including oncology, inflammatory diseases, organ transplantation, cardiology, viral infection, allergy, and tissue growth and repair. Most of the current therapeutic antibodies are humanized or human Immunoglobulins (IgGs) and are produced as recombinant glycoproteins in eukaryotic cells. Many alternative production systems and improved constructs are also being actively investigated. IgGs glycans represent only an average of around 3 % of the total mass of the molecule. Despite this low percentage, particular glycoforms are involved in essential immune effector functions. On the other hand, glycoforms that are not commonly biosynthesized in human may be allergenic, immunogenic and accelerate the plasmatic clearance of the linked antibody. These glyco-variants have to be identified, controlled and limited for therapeutic uses. Glycosylation depends on multiple factors like production system, selected clonal population, manufacturing process and may be genetically or chemically engineered. The present account reviews the glycosylation patterns observed for the current approved therapeutic antibodies produced in mammalian cell lines, details classical and state-of-the-art analytical methods used for the characterization of glycoforms and discusses the expected benefits of manipulating the carbohydrate components of antibodies by bio- or chemical engineering as well as the expected advantages of alternative biotechnological production systems developed for new generation of therapeutic antibodies and Fc-fusion proteins.

Recent advances in combinatorial protein engineering have made it possible to develop antibody-based and non-Ig protein scaffolds that can potentially substitute for most whole antibody-associated properties. In theory, many different natural human protein backbones are suitable to be used as recombinant templates for engineering : antibodyderived scaffolds, carrier proteins that display a single binding interface, backbones that provide a rigid core structure suitable for grafting loops or protein scaffolds allowing the incorporation of variable loops in a favorable 3D configuration. In practice however, only a few have yielded the necessary properties to be translated into ‘druggable Biologicals’. Amongst these properties, potential broad specificities towards any kind of target, ease of production, small size, good tolerability and low immunogenicity are essential and will be discussed in this review. Intellectual property is another key issue for the development of these protein scaffolds; although circumventing antibody-associated patents is often a major if not primary goal, clear advantages compared to whole antibodies must be presented to translate scaffold discovery into successful therapeutic drug candidates. In this review, a particular emphasis will be given to the most validated scaffolds that have reached the clinical development phase. Although the question of their immunogenicity is still open, preliminary clinical data do not point to any particular adverse immunogenic reactions although these are highly dependent on dosage, administration route and therapeutic indication. Finally, some of the emerging Biotechs developing protein scaffolds have been associated during the last two years with successful acquisitions by Big Pharmas and we will speak on the perspective positions of these proteins within the global Biologicals market.

This study aims to test the predictive power of gene expression data derived from NIH's database dbEST, which collects gene expression results from a large number and variety of DNA array experiments. The motivation of this study is to make comparable experimental studies, which are usually performed only for one or a few tissues or organs, with a wide variety of other tissues. Confirmation of a good predictive power of dbEST would put a number of interesting and partially surprising recent findings, solely based on data mining, on a more solid basis than available so far. The expression of nine genes (eIF4E, DDX6, HAT1, USP28, HSP90β, PKM2, PLK1, COX2 and OPN) plus two calibration genes in paired normal and cancer colon tissues of eight individual patients was investigated by quantitative RT-PCR and compared with the predictions made by the data - base. GUS and β-actin reveal only little variation among different patients, making them good internal calibration standards. In normal colon tissue, data mining correctly predicts the expression of all nine genes, which covers two orders of magnitude. In cancer, dbEST is somewhat less precise, but still valuable for the comparison with clinical results.

Apolipoprotein M (apoM) has been suggested to play a role in reverse cholesterol transport. Here we studied the influence of liver X-receptor (LXR) agonist on the transcriptional regulation of apoM. Studies were performed in murine liver and intestinal mucosal cells in vivo and in human intestinal Caco-2 cells in vitro. The expression of apoM was analyzed by quantitative real time PCR, and compared to well-established LXR target genes. Mice fed with TO901317 for six days showed a downregulation of apoM and apoAI in the liver to 40 % and 60 % respectively and an upregulation of Cyp7A1 to 280 %. In the small intestine, however, apoM and apoAI were upregulated by 30-60 % and ABCA1 by 250-430 %. In Caco-2 cells TO901317 caused a 60 % upregulation and the natural LXR agonist 22-hydroxycholesterol a 40 % upregulation of apoM. Possible causes for the differential effects in liver and intestine are discussed.