Antibody Drug Development: On Target

Courtesy of Abbott Laboratories BETTER LIVING THROUGH IMMUNOLOGY: Though the exact cause of rheumatoid arthritis (RA) is unknown, people suffering from the disease have an excess of tumor necrosis factor alpha (TNF-a) that accumulates in their joints. Abbott Laboratories' Humira, a humanized monoclonal antibody that targets TNF-a, helps prevent the inflammation characteristic of RA and inhibits the progression of structural joint damage. As soon as Köhler and Milstein described hybridoma technology for generating monoclonal antibodies in 1975,1 the biomedical community began working to convert these tailor-made reagents into pharmacological magic bullets. Success didn't take long: In 1986, Orthoclone OKT®-3 from Bridgewater, NJ-based Ortho Biotech became the first therapeutic monoclonal antibody (mAb) to gain Food and Drug Administration approval for use as an immunosuppressant to reverse transplant rejection. But therapeutic mAbs did not take off as expected because the nonhuman molecules (usually murine), generated using conventional hybridoma technology, do not fare well in human patients. Mouse mAbs have a short serum half-life in humans, limiting their efficacy, and they don't interact optimally with the human effector system, an important component of some treatment scenarios. Perhaps most discouraging: They can actually trigger an immune response. As a result, the once-considerable hoopla surrounding therapeutic mAbs died down for some time. However, as advances in genetic engineering enabled the development of more humanized mAbs, the arsenal of FDA-approved therapeutic mAbs slowly and steadily expanded.2-5 A handful of therapeutic mAbs have found their way into the veins of patients with cancers, autoimmune disorders, or newly transplanted organs, among other things. More weaponry is in the pipeline as scientists work to see just how far therapeutic mAbs can go; presently, there are more than 200 drugs in clinical trials. These new antibodies are steadily diverging from the textbook image of an immunoglobulin, with researchers tweaking both sequence and structure to yield constructs that can, for example, recruit a more robust immune response or penetrate cells more easily. Some tote radioactive or cytotoxic payloads, too. Far from magical in nature, today's therapeutic mAbs might better be described as "smart" molecular ammunition. And given the intensity and creativity of researchers working in this field, the future may witness even more sophisticated mAb-based armaments. THE HUMAN TOUCH Advances in molecular engineering techniques have enabled drug developers to design antibodies that are more human-like in sequence. First on the scene were chimeric antibodies, which pair the variable or hypervariable regions of a mouse mAb with human sequences for the constant and/or variable framework regions. A number of these antibodies were FDA-approved during the mid-to-late 1990s, including Simulect from Basel, Switzerland-based Novartis, which is used to reverse kidney transplant rejection; and Remicade from Malvern, Pa.-based Centocor, which is approved for treating Crohn disease and rheumatoid arthritis. Progress continues with more fully humanized antibodies in which only the mAb's antigen-binding portions are murine in origin. In these constructs, the complementarity determining regions responsible for antigen binding are grafted into the human variable domain framework. Several humanized mAbs have been FDA-approved, including South San Francisco, Calif.-based Genentech's Herceptin. This IgG1 mAb binds epidermal growth factor receptor 2 (HER2/ErbB2), thereby inhibiting tumor cell proliferation and migration. Compared with conventional chemotherapy alone, treatment with Herceptin plus other cytotoxic drugs improves clinical outcomes for women with metastatic breast cancer overexpressing HER2/ErbB2. Herceptin was FDA-approved for this use in 1998. Other humanized mAbs include Zenapax, Campath, and Xolair. Specific for the interleukin-2 receptor alpha chain (CD25), Zenapax is an immunosuppressive drug used to ward off kidney transplant rejection. It was developed by Freemont, Calif.-based Protein Labs, FDA-approved in 1997, and is now licensed to the Roche Group in Basel, Switzerland. Campath is specific for the CD52 antigen, and was FDA-approved in 2001 for treating refractory B-cell chronic lymphocytic leukemia. Developed by Cambridge, Mass.-based Millennium Pharmaceuticals and San Antonio, Texas-based ILEX Oncology, this humanized mAb is also available under the trade name MabCamath in Europe. And FDA-approved just this June, Xolair is an IgE-specific humanized mAb,developed by Genentech, Novartis, and Houston-based Tanox, used for treating allergic asthma. Drug manufacturers have also devised techniques for generating antibodies that are fully humanized in sequence. Transgenic mice genetically engineered to express human antibodies can be used for the production of such molecules via hybridoma technology. But other researchers use any of a variety of display technologies to screen libraries representing human, preimmune antibody-producing repertoires in vitro.2 Companies such as Affitech of Oslo, Norway, and Cambridge Antibody Technology in the UK, screen phage libraries that express single-chain Fv (scFv) or Fab fragments fused to viral coat proteins. Such fragments can be directly used as therapeutic agents, or if needed, can be converted to full-length mAbs of the desired isotype. Humira, the first such therapeutic to gain FDA acceptance, binds TNF-a, a cytokine involved in the inflammatory response. Discovered through collaboration between Abbott Laboratories, Abbott Park, Ill., and Cambridge Antibody Technology, this fully humanized, recombinant IgG1 monoclonal was approved for the treatment of rheumatoid arthritis in the United States in December 2002, and in Europe as of September 2003. Other display technologies also may prove useful for screening human variable region sequences in vitro. Cambridge Antibody Technology, for example, is currently refining a ribosome display platform for the development of therapeutic mAbs. According to the company's Web site, this system offers the "potential to build even larger libraries than the phage display method allows." Some companies forego display technologies altogether. The Celltech Group in Berkshire, UK, uses a version of the selected lymphocyte antibody method originally licensed from Abgenix in 2001. In this approach, specific B cells, usually obtained from peripheral blood of an antigen-challenged animal, are selected using hemolytic plaque assays for the specific antibodies of interest. The light- and heavy-chain variable regions are then amplified by reverse transcriptase PCR, ligated into an appropriate vector, and reconstituted in an expression system of choice. POTENT PAYLOAD Therapeutic mAbs and mAb-based constructs can be engineered to deliver any of a variety of payloads, including toxins, radioisotopes, cytokines, or even enzymes or DNA, to their targets. For example, Mylotarg, which was codeveloped by Celltech Group and Madison, NJ-based Wyeth, is used for the treatment of acute myeloid leukemia (AML). FDA-approved in 2000, this conjugate consists of a humanized IgG4 mAb linked to the bacterial toxin calicheamicin, which induces DNA strand breaks and apoptosis. Mylotarg targets CD33, which is expressed in roughly 90% of AML cases. Then there's Zevalin, the first radio-immunotherapeutic mAb to gain FDA approval. The drug is used in conjunction with Rituxan (known as MabThera in Europe), which isn't radioactive at all. Developed by San Diego-based IDEC Pharmaceuticals and Genentech, Rituxan is a chimeric IgG1 mAb that targets CD20, an antigen found on the surface of mature B cells. Used to treat relapsed or refractory, low-grade or follicular B-cell non-Hodgkin's lymphoma (NHL), Rituxan recruits the body's natural defenses to attack and kill the marked B cells. Also developed by IDEC Pharmaceuticals, Zevalin shares Rituxan's clinical indications and its antigen-binding specificity, but packs an additional wallop. Unlike its unlabeled, chimeric predecessor, Zevalin carries the radioisotope Yttrium-90, whose beta emission induces cellular damage in the target and neighboring cells. Another difference: Zevalin is a mouse antibody, whose rapid clearance minimizes the patient's radiation exposure. In June of this year, the FDA approved another CD20-specific antibody for radioimmunotherapy. The mouse mAb Bexxar is indicated for patients with CD20-positive, follicular, NHL that has relapsed following chemotherapy with Rituxan. Discovered and comarketed by Seattle-based Corixa and Middlesex, UK-based GlaxoSmithKline, Bexxar delivers a one-two punch, pairing the cytotoxic action of the unlabeled, nonradioactive mAb with the targeted destruction afforded by patient-specific doses of its Iodine-131 labeled counterpart. PLANS OF ATTACK With the technologies for making humanized antibodies now well established, researchers have turned their attention to other aspects of mAb design. "Therapeutic mAbs are now being designed to function in a more sophisticated manner," comments Neil Weir, head of antibody therapeutics at Celltech Group, noting the growing trend towards first identifying the therapeutic mechanisms of choice, "then going on to engineer the mAb or related molecule best suited to carry out the mission." Several mission profiles exist. Some antibodies carry toxic payloads; others block soluble mediators, such as growth factors, cytokines, and toxins from reaching their target receptors or binding partners. Specific cell populations can be targeted by mAbs that bind appropriate cell-surface molecules. Some antibodies that bind signaling receptors perturb their target's biological functions. These effects can be stimulatory or inhibitory, which is more often but not always the case for therapeutic mAbs; mechanisms range from straightforward competition for ligand binding to receptor downregulation or Fc-mediated effector functions. In some cases, the binding of the antibody or derived molecule alone mediates the desired therapeutic effects, and side effects associated with stimulating the immune response are not necessarily wanted. In other scenarios, effective treatment requires a more general immune response, and antibody designers may try to boost effector functions such as complement-dependent cytotoxicity (CDC), or antibody-dependent cellular cytotoxicity (ADCC). Although lgG-Fc-receptor crosslinking generally leads to ADCC or phagocytosis of the target cell, it also can directly induce the target cell to undergo apoptosis in some cases, as when the mAb binds certain signaling receptors. Effector functions can be moderated, abolished, or ramped up by mutagenesis of the Fc domain, and designer constructs combining portions of Fc domains from different isotypes can be devised. Effector function also can be enhanced by other means, for example, by modifying the antibody with an extra dollop of glycosylation. A variety of strategies exist for enhancing the T-cell response, such as loading mAbs with pieces of antigenic material. In some cases, the activation of T-cell proliferation is the primary objective. Würzburg, Germany-based TeGenero, for example, is developing the SuperMAB (super-agonistic mAbs) which single-handedly activate T cells regardless of their T-cell receptor (TCR) specificity. According to the company, its most advanced SuperMAB, which is specific for CD28, initiates T-cell proliferation directly, with no need for antigen-mediated TCR stimulation. STRUCTURAL DIVERSITY Researchers also are evaluating a host of creative mAb-based constructs that make those that haven't been tinkered with seem almost plain vanilla in comparison. Bispecific mAbs, for example, contain arms of differing specificities. One arm of a given bispecific molecule could nab a tumor-specific antigen, while the other hails a cytotoxic T cell for a boosted effector response. When effector functions are not required, smaller antigen-binding constructs, such as scFv or Fab fragments, can be called into play. These smaller fragments penetrate tumors more efficiently than do whole antibodies, and they can be produced relatively inexpensively in bacteria. They are less stable than intact mAbs, but this can be compensated for, at least in part, by techniques such as modification with polyethylene glycol. Antibody engineers can use scFv or Fab fragments as building blocks to create more complex configurations. "Diabodies" and "triabodies," for example, correspond to dimeric and trimeric forms of scFv fragments. These oligovalent fragments offer increased avidity as well as the ability to crosslink target molecules without the Fc domain. Scientists also can design bispecific diabodies equipped with different variable domains. Other constructs under evaluation include tandem diabodies,7 coupled to achieve a greater crosslinking effect, and "pepbodies," comprised of Fab or scFv fragments linked to effector-binding peptides. In another strategy, antibody fragments conjugated to the surfaces of liposomes can be loaded with any of a variety of therapeutic agents, including small-molecule drugs, cytotoxic proteins, and antitumor DNA. These "immunoliposomes" release their contents after binding to, and becoming internalized by their targets, such as cancer cells or tumor vasculature. Others are evaluating whether antibody fragments such as scFvs can be adapted for use as "intrabodies" that bind intracellular therapeutic targets.8 Despite the progress in generating therapeutic antibodies, "getting the right antigen is sometimes more problematic," according to Björn Cochlovius, Affitech's director of discovery and development. "This is especially true in the area of cancer therapeutics, because cancer-specific antigens are often splice variants, or are the result of abnormal patterns of posttranslational modifications such as glycosylation patterns or proteolytic digestion," he explains. "Subjecting hundreds of mAbs to preclinical testing in the hopes that one will prove efficacious is not generally the best use of time, effort, or money," adds Michael Braunagel, Affitech's director of strategic alliances and licensing. "Therapeutic mAb developers are better served by finding the best antigen target early on, then using their resources to custom develop the best mAb or mAb derivative for the application at hand." Researchers respond to this need by using a variety of approaches, both genomic and proteomic, in profiling studies geared toward antigen discovery. For cases involving a general immune response, as in cancers or infectious diseases, some scientists also use various tactics for identifying antibodies already being made by patients. Deborah Fitzgerald is a freelance writer and can be reached via the contact form at www.SciWriters.com. References 1. G. Köhler, C. Milstein, "Continuous cultures of fused cells secreting antibody of predefined specificity," Nature, 256:495-7, 1975. 2. O.H. Brekke, I. Sandlie, "Therapeutic antibodies for human diseases at the dawn of the twenty-first century," Nat Rev Drug Discov, 2:52-62, 2003. 3. M. Trikha et al., "Monoclonal antibodies as therapeutics in oncology," Curr Opin Biotechnol, 13:609-14, 2002. 4. L.J. Sannes et al., "Prospects for commercialization of human monoclonal antibodies," Cambridge Healthtech Institute, 2002. 5. T.A. Waldmann, "Monoclonal antibodies in diagnosis and therapy," Science, 252:1657-62, 1991. 6. D.J. Slamon et al., "Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2," N Engl J Med, 344:783-92, 2001. 7. B. Cochlovius et al., "Cure of Burkitt's lymphoma in severe combined immunodeficiency mice by T cells, tetravalent CD3 x CD19 tandem diabody, and CD28 costimulation," Cancer Res, 60:4336-41, 2000. 8. M.N. Lobato, T.H.Rabbits, "Intracellular antibodies and challenges facing their use as therapeutic agents," Trends Mol Med, 9:390-6, September 2003. function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? Not recommended 1 2 3 4 5 Highly recommended Please register your vote

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