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Thirty years ago, César Milstein and Georges Köhler, two researchers at the Medical Research Council's (MRC) Laboratory of Molecular Biology in Cambridge, were investigating the mechanisms underlying the remarkable diversity of antibodies. As part of their research they constructed a continuously growing cell line expressing a specific, predefined antibody.1
Over the next three decades, their discovery led to invaluable medicines, insightful diagnostics, and inspirational research. Since 1975, researchers have made numerous variations on Köhler and Milstein's seminal discovery. But antibody-producing hybridomas, for which Köhler and Milstein shared the Nobel prize in 1984, remain the
MAbs are exquisitely sensitive, able to pick the proverbial molecular needle from the surrounding cellular haystack. Thousands of bioscience researchers worldwide rely on MAbs to understand the structures, functions, and interactions of complex biomolecules. Like biochemical flashlights, MAbs transformed biology from a process of groping around in the dark. "It is hard to imagine any other experimental approach to investigate these problems," says Markus Enzelberger, from the Munich-based biotech, MorphoSys.
But MAbs provided more than light, allowing for much grander plans. Indeed, many biotech companies can trace their scientific origins to Milstein and Köhler's pioneering research. Currently, 18 MAbs are approved for therapeutic use in the United States, and the global therapeutic MAb market is worth $5.4 billion. MAbs also contribute to the in vitro diagnostics market expected to be worth some $34 billion this year, according to a market analysis by Theta Reports.2
MAbs aid the diagnosis of heart disease, cancer, infections, and many other conditions. In the future, MAbs could bolster our defenses against bioterrorism, and they already form a central plank in proteomics and systems biology. Nevertheless, researchers still need MAbs able to bind numerous important biological targets, including some lipids, most metabolites of small-molecule drugs, and many G protein-coupled receptors. They are searching for ways to further enhance MAbs' sensitivity, selectivity, and multiplexing ability, but production still takes time and experience.
Making Monoclonal History
The body produces a vast range of antibodies to target and destroy invading pathogens. Each B lymphocyte carries around 50,000 antibody molecules on its surface, and all are specific for a single epitope. The human immune repertoire contains hundreds of millions of B cells, each with a different specificity. Indeed, humans typically have B cells specific for 108 epitopes at any time.
Antibody-binding sites formed by regions of the heavy and light chains in the arms (variable region) of the familiar Y-shaped structure interact with the epitope. These sites account for their exquisite sensitivities. These are the complementarity-determining regions (CDRs) or hypervariable regions, which bind epitopes, such as proteins or sugars on the surfaces of bacteria, viruses, or cancer cells. The heavy chains continue into the constant region, the stem of the Y. Sites here allow the antibody to activate effectors such as the complement system or Fc receptors on macrophages, which trigger phagocytosis. Overall, the body can produce some 1010 rearrangements or recombinations of the light and heavy chains.
Antibody binding triggers clonal expansion of B cells. Some differentiate into plasma cells, which pump out more antibodies to mark pathogens for destruction. Others become memory B cells, which prime the immune response for future exposure. The CDR can recognize small areas on a sugar or protein, such as a peptide containing between five and eight amino acids. So, various parts of the same epitope's molecular structure can stimulate different B lymphocytes. This means that a typical immune reaction consists of numerous clones showing different specificities and affinities – a polyclonal response.
Polyclonal responses help the immune system protect against a plethora of potential pathogens. But researchers usually want to recognize a single molecule, or even a single epitope on a single molecule. Until 1975, this task usually proved very difficult.
Milstein and Köhler then discovered how to isolate and generate unlimited amounts of pure, specific MAbs from the polyclonal cocktail. B lymphocytes can survive for only a few days in culture. Mouse myelomas don't produce antibodies, but their lines survive indefinitely in culture. So Milstein and Köhler fused murine myelomas to B cells isolated from a mouse immunized with sheep red blood cells. The resulting hybridomas produce antibodies and divide indefinitely. Milstein and Köhler's discovery allowed researchers to select individual hybridomas that produced predefined MAbs.
Over the years, laboratories around the world developed variations on this theme. Then in 1990, a group led by Greg Winter, also working at the MRC Laboratory of Molecular Biology, developed an alternative system to produce highly specific human antibody fragments. The MRC team incorporated genes encoding human antibody variable regions (V genes) into a bacteriophage. Each phage carries a different human V gene, which directs infected bacteria to make the human antibody fragments, expressed on the viruses' surface.
The resulting phage display library contains billions of bacteriophages, each carrying a unique human antibody fragment. These libraries can show equivalent antibody diversity to the human immune system and allow researchers to rapidly isolate antibodies that recognize specific antigens.3 The fragments can be used directly for some imaging studies. Researchers can fuse the fragments to other components such as recombinant immunoglobulins using genetic engineering. Winter also pioneered techniques to humanize mouse antibodies, which made MAbs more appropriate for clinical use.
"Classical monoclonal antibodies, produced by cell fusion, are still the method of choice for analysis of complex antigen, such as a cell surface," Winter says. "Phage display, while excellent for pure antigen, has proved much less successful under those conditions, partly because the phage are sticky, and secondly because a small number of specificities quickly come to dominate in the phage population."
A MOLECULAR SCALPEL
Courtesy of Abbott Laboratories
MAbs can act as a molecular scalpel allowing researchers to dissect complex cell structures and pathways. They can selectively block proteins in signaling or regulatory pathways, and they can identify different sites on a single protein.
MAb roles don't end with vertebrate adaptive immunity. Paul Knox at the Centre for Plant Sciences, University of Leeds, UK, uses MAbs to understand the function of plant cell-wall polymers, which he describes as "one of nature's most sophisticated biomaterials." The Leeds group raise MAbs to specific structural features of individual polymers that play important roles in cell development. "This capability has been crucial to develop our understanding of the highly complex structures in plant cell walls, such as pectic polysaccharides and arabinogalactan proteins, and their association with specific cell processes," says Knox.
Moreover, MAbs remain vital for high-throughput screening. Hervé Le Calvez, director of business development at the San Diego biotech, Abgent, comments that MAbs are still the tool of choice for detection. For example, MAbs can be used on protein chips to differentiate between peptides.
Additionally, MAbs have numerous diagnostic roles, including grouping blood types and identifying infectious agents, as well as testing for pregnancy, blood clots, heart disease, and some cancers. MorphoSys, for instance, recently produced an MAb for immunocytochemistry against CD-33, which might modulate monocyte activation, although its function isn't fully understood.4 Normal hematopoietic stem cells don't express CD-33. But in most cases of acute myeloid leukemia (AML), malignant blast cells do. MAbs against CD-33 might aid diagnosis, such as helping clinicians distinguish between AML and other forms of leukemia.
Meanwhile, Rod Capaldi, from the University of Oregon, is also chief scientific officer for MitoSciences, a startup company in Eugene, Ore., aiming to develop diagnostic tests for diseases associated with mitochondrial dysfunction. For example, Parkinson disease, Alzheimer disease, and late-onset (type II) diabetes may arise, at least in part, from accumulated oxidative damage to mitochondrial proteins. MAbs against mitochondrial proteins, such as those in the oxidative phosphorylation chain – pyruvate dehydrogenase and adenine nucleotide translocase – might aid diagnosis.5
IMPROVING ON NATURE
SHINING ITS LIGHT:
Courtesy Paul Knox
This cross section through the inflorescence stem of
Despite the tremendous advances MAbs have facilitated, they've not wholly lived up to expectations. When they were first developed, researchers hoped that MAbs would be molecular magic bullets able to inhibit specific cellular functions of proteins. "It turned out that very few MAbs were function blocking: less than 1% by some assessments," says Daniel Jay from Tufts University School of Medicine.
In 1998 Jay's group published a study that used MAbs to bind a chromophore to specific proteins in cells and tissues. Laser irradiation then targets the chromophore, destroying the specific protein without collateral damage to surrounding cellular components. Using a laser also allows researchers to target a single cell. In other words, chromophore-assisted laser inactivation allows researchers to ascribe functions to the proteins. The technique Jay pioneered spawned a family of related technologies to selectively destroy proteins.6
Efficient production poses another problem. Abgent's Le Calvez notes that MAb production remains labor intensive, and generating suitable hybridomas frequently requires considerable experience. For example, producing IgG MAbs often demands at least three immunizations given at intervals of three weeks. In some cases, alternatives exist. Animals injected with a peptide and an adjuvant generate polyclonal antibodies that can be isolated from the blood. "The immunogen is so short that a peptide-specific polyclonal antibody can mimic an MAb," he says. "If the antibody properties need to be well characterized and consistent, such as in the diagnostic and pharmaceutical fields, then a MAb is the best tool."
Enzelberger adds that epitope-specific MAbs are necessary to detect subtle differences such as splice variants or differences in protein folding. Polyclonals are more likely to detect an antigen in different presentations or glycosylation states and show pan-species reactivity. Moreover, polyclonal antibodies, when used as secondary reagents, can boost the signal in some immunostaining experiments.
A wide range of biologically important epitopes still aren't targeted by MAbs. Enzelberger notes the difficulty in raising MAbs against some targets, including sugars, lipids, and G protein-coupled receptors. Researchers also need MAbs against nonprotein antigens, such as metabolites of small-molecule drugs, which would aid clinical monitoring. Novel MAbs against bacterial-surface molecules, such as complex polysaccharides or toxins, could bolster defenses against biological warfare or bioterrorism. Finally, multiplexing different analytes in a single sample poses a particular problem. "This will require MAbs that don't cross-react, have similar affinities for their targets, and show low background in body fluids," Enzelberger says.
A GLIMPSE INTO THE FUTURE
Improvements are likely. Today, the limit of detection using gold-standard experimental techniques is around 500 pg/ml. Highly sensitive detection systems such as electrochemoluminescence may increase the dynamic ranges, says Enzelberger. Furthermore, protein engineering could, he says, substantially improve MAb affinity.
Currently, MAb production takes around three to four weeks longer than polyclonal antibody production. New immunization protocols could shorten this. For example, Le Calvez suggests that coupling the antigen to a T-cell receptor allows direct antigen presenting and boosts the immune response. This could cut MAb production by two to three weeks. "The quality of the antibody [produced in this way] still needs to be investigated," he says.
So thirty years after Milstein and Köhler's landmark discovery, MAbs remain a mainstay of basic and applied bioscience research. They're essential diagnostic and therapeutic tools in clinical medicine. They helped drive the development of the multibillion-dollar biotechnology industry. Yet, they originated in an attempt to resolve a fundamental problem in academic research. As basic research budgets come under increasing scrutiny worldwide, it's a lesson that politicians and policy makers tightening the purse strings need to recall. As the then-MRC chief executive George Radda commented in 2000: "The monoclonal antibody story shows that funding the best scientists really is the key to dramatic change in the standard and success of medical care across the UK and the rest of the world, generating wealth through industry in the process. It is also a good reminder that we should always take a long-term view of rewards and benefits."