Monoclonal Antibodies Find Utility In Cell Biology

In its short lifetime, monoclonal antibody (MAb) technology has experienced more ups and downs than a yo-yo. As this brilliant invention has evolved into a difficult technology, numerous suppliers have emerged to facilitate researchers' access to the new antibodies. Today, MAbs are partners in such cell-biology staples as fluorescence microscopy, flow cytometry, and affinity chromatography. Investigators use these antibodies to track cells as they communicate and interact, respond to various stresses, turn cancerous, and die. But, just as antibodies are finding increasing utility in cell biology, a new Food and Drug Administration classification for those products with clinical utility may affect researchers' access to the important technology (see accompanying story). Monoclonal History MAbs were born in 1975, when Georges Kohler and Cesar Milstein at the Medical Research Council Laboratories in Cambridge, England, fused two types of cells to form a hybridoma. Part antibody-secreting B cell, part myeloma cell, a hybridoma combines the talents of each constituent, continuously releasing a single type of antibody when cultured in ascites fluid in a mouse's abdomen. This secretion of a monoclonal (single-type) antibody is in contrast to the response of an animal's challenged immune system, which is termed polyclonal because several types of antibodies are produced against a single type of foreign antigen, each zeroing in on a different portion of the target. Hybridoma technology, the research community thought, would finally provide a way to isolate and at the same time mass-produce a single antibody type, harnessing its exquisite specificity. But capturing that quality did not come easily. "When Kohler and Milstein made the first hybridoma, everyone started making them. But by the mid-1980s, hybridoma technology fell out of favor," says Krishna Balakrishnan, vice president of research and development at Richmond, Calif.-based Berkeley Antibody Company (BAbCO). Researchers found it exceedingly difficult to create hybridomas, select a youthful one, and culture it successfully enough to provide ample antibodies, he explains. But as MAb research nears its 20th anniversary, it is experiencing a resurgence. "In the early 1990s, hybridomas came back into fashion because many people realized that there are a lot of specialized applications that only a MAb can do," notes Balakrishnan, referring to MAbs that zero in on chemical groups small enough to be specific to one molecule. Finding A Niche Many researchers agree that nurturing a hybridoma to secrete a desired antibody is technically challenging. First, the animal providing the B-cell part of the hybridoma must be immunized against the appropriate antigen, then B cells isolated and fused with myeloma cells to provide them unlimited cell division, or immortality. Next is the onerous task of selecting hybridomas whose antibodies suit researchers' needs. Obstacles are everywhere in the long process--cells won't fuse, hybridomas die or won't propagate. Antibodies may be produced in too low a yield, may bind to their targets with poor affinity, or may bind to target regions (epitopes) that different molecules have in common, drastically reducing specificity and making cross-reactivity possible. These technological hurdles have opened a wide niche for dozens of companies that MAbs have overcome these obstacles and nurture for researchers. Such vendors offer extensive lists of monoclonal and polyclonal antibodies, partial antibodies, and antibodies conjugated to various marker molecules. Firms that grow hybridomas and scale up production of desired antibodies report that such contract services are extremely popular. "Lately we have been providing mostly customized products. Some people want special forms of antibodies, such as without preservatives or with a certain conjugate, or just a FAb fragment [the portion of an antibody that binds antigen]," says Bret G. Wien, president of Research Diagnostics Inc. in Flanders, N.J. Richard Plante, associate scientist at the Robert Wood Johnson Pharmaceutical Research Institute in Don Mills, Ontario, works in drug discovery and doesn't have time to create monoclonals. "We work with the glucocorticoid receptor, for which there is no good clone available," he explains. A colleague had been supplying MAbs, but the hybridoma died. "So Affinity BioReagents [Inc. of Neshanic Station, N.J.] has been a good source of the antibodies I'm interested in." Sharon Devereaux, a research scientist at Abbott Laboratories Inc. in Abbott Park, Ill., is another satisfied hybridoma customer. BAbCO grew her MAbs. "They were very cooperative, everything was documented well, and they gave timely results and feedback," she says. Backbone Of Biological Research MAbs are powerful tools in cell, molecular, and developmental biology. They have helped researchers dissect the complex pathways of signal transduction, cell adhesion, the stress response, carcinogenesis, and cell death. In short, MAbs reveal life at the biochemical level. And investigators are finding that their small, individual glimpses into the lives of cells are connected, through signaling pathways. "The whole cell biology area has taken off, and signal transduction is at the core of that. A lot of the signal-transduction work is antibody-related," comments Wesley Russ, immunodetection product manager and senior scientist in research and development at Life Technologies in Gaithersburg, Md. Geneticists are using small MAbs to identify proteins. "We're finding more and more molecular biologists working with recombinant DNA turning to monoclonals as tools to isolate molecules. For example, a researcher with a partial gene sequence reconstructs the corresponding peptide, then uses a monoclonal to fish out the gene product," notes BAbCO's Balakrishnan. "You couldn't do that with polyclonal antibodies--you need a highly specific signal." Researchers can use generic antibodies to obtain gene products via an approach called epitope tagging. To do this, a scientist attaches a synthetic oligonucleotide encoding six to 10 amino acids to a fragment of the gene of interest. The oligonucleotide specifies a "peptide tag," an extra bit of protein attached to the gene's product. BAbCO supplies MAbs that are attracted to such tags. "Then you express the gene product as a fusion protein, and use the universal antibody to detect the fusion protein. The epitope tag antibody purifies the gene product from a mixture," Balakrishnan explains. "It is very powerful because of its universal use." An antibody's specificity allows developmental biologists to isolate and highlight individual steps of differentiation. This is the case for anti-agrin MAbs, a product of StressGen Biotechnologies Corp. of Victoria, British Columbia in Canada. Agrin is a protein found in the basal lamina (the connective tissue boundary between tissues) that, early in development, stimulates receptors for the neurotransmitter acetylcholine to cluster at the tip of a muscle fiber--a prelude to formation of a neuromuscular junction. "Agrin is released into the space [between a nerve and muscle cell] and it clusters the receptors to increase the signal. We offer a group of five monoclonals to different regions [epitopes] of agrin," notes Henry Rodriguez, director of StressGen's biochemical division. The anti-agrin antibodies became available last month. Many companies offering MAbs also supply polyclonal antibodies, which, when purified by affinity chromatography, are specific enough for many research needs. "Affinity-purified peptide polyclonals have specificities similar to those of monoclonals, with the advantage of multiple clones to the same epitope, which provides higher affinity. A lot of people are going this way, because it's easier than going through the process of screening for monoclonals," according to James Stiehr, president of Affinity BioReagents. Big sellers are polyclonal antibodies to nitric-oxide synthase, the enzyme that catalyzes formation of nitric oxide, a messenger molecule that takes part in vasodilation, neurotransmission, and cytotoxicity. Antibodies In Biotech Monoclonal antibodies are key components of several biotechnologies. They are conjugated to other molecules so that they can be detected, such as to a fluorescent dye for fluorescence microscopy, or to an electron-dense marker for electron microscopy. Antibody bound to an inert matrix forms an affinity column, with the antibody pulling out its target molecule from an extract passed over the column. An antibody's signal can be amplified by binding it (at a region other than the antigen binding site) to a second antibody, which is in turn bound to a label. In another popular approach, an antibody is bound to the vitamin biotin, which then binds a labeled form of the bacterial protein streptavidin. Labeled MAbs are also used in Western blots to detect peptides or proteins separated on a polyacrylamide gel by electrophoresis. In an enzyme-linked immuno-sorbent assay (ELISA), a primary antibody binds to its target antigen as well as to a secondary antibody. The secondary antibody is bound to an enzyme, such as alkaline phosphatase, that catalyzes a reaction whose products are easily detectable. Because one enzyme molecule is used over and over, catalyzing thousands of reactions, a single primary antibody, via its linkage to a single enzyme, gives off a very powerful signal. Jaspreet Sidhu, a senior fellow in the Department of Environmental Health at the University of Washington, Seattle, is evaluating a new ELISA kit for Life Technologies. "We're field-testing an ELISA to measure levels of the second-messenger cyclic AMP [cAMP], looking at stimulation of intracellular cAMP in various cells, such as human smooth muscle and rat hepatocytes," he reports. Fluorescently labeled MAbs are also used in flow cytometry, a method of separating cells or their components in a device called a fluorescence-activated cell sorter. In the technique, up to four groups of cells are labeled with MAbs coupled to different fluorescent dyes. The cells are propelled through a stream of water. A vibrating crystal breaks the stream into droplets, which pass through a laser that excites the dyes linked to the MAbs marking cells. The cell sorter, programmed to recognize specific combinations of dyes, emits pulses of electricity that deflect droplets carrying different types of marked cells into separate fractions. Life Technologies offers several MAbs specific for cluster-of-differentiation (CD) cell-surface antigens, con- jugated to fluorochromes for use in flow cytometry. "A fluorochrome-labeled antibody recognizes an epitope on a cell, such as CD4 marked with fluorescein," explains the company's Wesley Russ. "A certain percentage of cells will be selected because they have that protein exposed. Then add an antibody to something on the cell surface, labeled with phytoerythrin, such as CD8, and then get the percentage of cells marked with CD4 and CD8. Add a third antibody, and get additional information." Specific Applications: Cancer: Antibodies can be used to follow the many ways that cancer cells depart from normalcy. Directed by expressed oncogenes or dampened tumor-suppressor genes, cancer cell surfaces and shapes grow more fluid, and the cells secrete biochemicals that enable them to invade and travel through healthy tissue. Antibodies that target any biochemical implicated in carcinogenesis or metastasis are valuable tools for basic research as well as in the clinic, as the basis of diagnostic tests and for evaluating response to therapy. Such antibody targets include variants of normal molecules, altered patterns of cell-surface antigens, or normal proteins in abnormal places. Consider cathepsins. Inside a cell, these enzymes are confined to lysosome sacs, where they degrade old proteins. But invading tumor cells release cathepsins instead, and they chew through the basal lamina. As a result, the cancer grows and spreads. Calbiochem-Novabiochem International Inc. of La Jolla, Calif., offers polyclonal antibodies to cathepsins, enabling researchers to detect the leading edge of a tumor. Monoclonal antibodies--from Minneapolis-based R&D Systems Inc.--to a cell-surface glycoprotein called CD44 are also being used to probe carcinogenesis. Because CD44 pokes out onto the cell's surface, extends through the cell membrane, and has a tail portion emerging on the membrane's inner face, it probably plays a major role in signal transduction--and disrupted relay of messages into a cell can lead to cancer. The link between CD44 and cancer is that certain types of cancer cells have characteristic combinations of CD44 variants--but there's only one CD44 gene. The variants arise from expression of different exons (protein-encoding sequences) of the gene. R&D Systems' MAbs to CD44 correspond to portions of the protein that different subsets of exons encode, allowing users to further follow this glycoprotein's role in carcinogenesis. Multidrug Resistance: At Immunotech Inc. in Westbrook, Maine, the cancer focus is on multidrug resistance (MDR). On a cellular level, MDR is an adaptive response to protect the cell from toxins--such as the botanical biochemicals that are the bases of many cancer drugs. The MDR response derives from a transmembrane protein called permeability-glycoprotein, or P-gp, which pumps drugs out of the cell. Immunotech sells a MAb that is specific for a portion of P-gp that protrudes from the surfaces of cancer cells resistant to the commonly used chemotherapeutic drug adriamycin. "If a patient's cells have the MDR marker, this indicates that response to adriamycin will not be great," says product manager Linda Diou, adding that the monoclonal is for research use only. Steroid hormones such as estrogen diffuse into a cell, then bind to receptors. The hormone-receptor complex then binds to DNA, activating certain genes whose action produces the hormone-associated effects. MAbs to estrogen receptors are very useful in studying breast cancer. Affinity BioReagents markets a monoclonal that targets a nuclear steroid receptor called the anti-retinoid x receptor b, or RxRb. This receptor binds to other steroid receptors as well as to DNA. "The RxRb antibody is licensed from NIH. It forms heterodimers with other steroid receptors to differentially control gene expression in different tissues," says Affinity president Stiehr. The antibody can be used to show which genes the RxRb receptor binds to in different cell types. Apoptosis: Cell death is at the forefront of cell biology research. Two general mechanisms are recognized. In necrosis, a passive process, the cell membrane deteriorates. In apoptosis, an active process, a specific sequence of events culminates in the cell synthesizing endonucleases that cut its own DNA into recognizably sized chunks. Apoptosis is important in the programmed cell death that forms the embryo; in establishing T- and B-cell populations in the immune system; and in the normal degradation of worn-out tissue. In pathology, apoptosis is linked to graft-versus-host disease, viral hepatitis, and cancer. PanVera Corp. of Madison, Wis., offers a MAb, called anti-Fas, that induces apoptosis in vitro. "Certain cell lines have the Fas antigen. The anti-Fas antibody binds to the antigen on the cell surface, and cross-links other proteins. This disturbs the membrane, which leads to downstream events," according to Alex Vodenlich, technical services manager. The cross-linking occurs because the antibody is a pentamer, he adds. PanVera distributes antibodies developed at two Japanese companies, TaKaRa Shuzo Co. Ltd. in Kyoto and Medical and Biological Laboratories Co. Ltd. in Otsu, Shiga. Cellular Stress Response: Heat shock proteins are part of a larger group of stress proteins, which increase in abundance in response to viral infection, cancer, toxic exposure, ischemia, or oxidation, in addition to heat. "Stress proteins interact in many different systems. They are ubiquitous and conserved from one species to another, which leads people to speculate that they are essential for life," says James Stiehr of Affinity BioReagents, which recently added anti-stress protein antibodies to its product line. Interest in heat-shock proteins has mushroomed in recent years because they have been identified as molecular chaperones. They assist protein synthesis by controlling transcription factors and protein kinases, then folding peptides as they form and escorting the maturing proteins along the secretory network. The long list of antibodies so many companies offer may taper off, but MAbs continue to meet their potential as envisioned in 1975, to provide highly specific tools with which to dissect events within and between cells. Ricki Lewis, author of several college biology textbooks, is a freelance science writer based in Scotia, N.Y. (The Scientist, Vol:8, #24, p.16, December 12, 1994) (Copyright, The Scientist, Inc.) Date : December 12, 1994 Back To:Monoclonal Antibodies Find Utility In Cell Biology As cell biology and clinical medicine increasingly overlap, antibody suppliers are finding themselves in what many people in the field fear may turn into a regulatory hell. On October 21, antibody-industry representatives met with several pathology organizations and Food and Drug Administration (FDA) officials for the "Hematological and Pathological Devices Panel Meeting," at which the convened panel recommended that antibody-based products that pathologists might use be regulated as class II medical devices. Many such products have not been regulated by FDA because they are used for research purposes only, but that is changing as more and more antibodies find clinical utility. Class II designation requires the manufacturer to provide various "special controls" (such as postmarketing surveillance and restricting use to in vitro applications) and to file a premarket notification, commonly known as a 510(k). The pathologists, wishing to avoid a more restrictive class III designation but still desiring assurance of consistent safety and efficacy, pushed for class II status. But a recently formed coalition of antibody suppliers, the Joint Council of Immunohistochemistry Manufacturers, argued for less-restrictive class I status. The recommendation, if approved by the FDA commissioner following a review period, may have drastic effects on the research community, especially concerning availability of products with markets too small to justify the cost of complying with regulations. Delores Graham, manager of regulatory affairs at antibody supplier Dako Corp. in Carpinteria, Calif., and cochairwoman of the joint council, said at the October 21 meeting: "In considering the costs of getting an antibody cleared through the 510(k) process [$23,000 to $40,000], we have estimated that only 10 percent of the 300 antibodies determined to be clinically useful would have the sales to justify submission. When we evaluated our product line, 60 percent of the products fell below a liberal sales cutoff of $10,000 for submission consideration, and 20 products below this cutoff could be found on the pathologists' list of clinically useful products." Some antibody manufacturers fear that they may eventually be required to keep track of which researchers are using a particular product for basic research and which for diagnosis, says James Stiehr of Affinity BioReagents Inc., Neshanic Station, N.J. This will raise costs even more. However, Carolyn Jones, director of technology regulatory affairs at the Health Industry Manufacturers Association in Washington, D.C., does not feel that the new classification will stifle researchers' access to antibodies. Instead, she predicts, the industry will fragment somewhat, with product lines shrinking as only the larger firms remain able to afford to market low-demand research antibodies with clinical applications. FDA regulation could be in place sometime in 1995, just as monoclonal antibody technology celebrates its 20-year anniversary. Concludes Stiehr: "This new classification will have a fairly major impact on the industry." --R.L.

Monoclonal Antibodies Find Utility In Cell Biology

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