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Leukocytes are the cells of the immune system and are centrally involved in defense against infection, in autoimmune disease, allergy, inflammation, and in organ graft rejection. Lymphomas and leukemias are malignancies of leukocytes, and the immune system is almost certainly involved in most other cancers. Each leukocyte expresses a selection of cell surface glycoproteins and glycolipids which mediate its interaction with antigen, with other components of the immune system, and with other tissues. It is therefore not surprising that the leukocyte surface molecules (CD molecules) have provided targets for diagnosis and therapy. Among the “celebrities” are CD20, a target for lymphoma therapeutic antibodies which earns $2 billion annually (and makes a significant difference to lymphoma patients), and CD4, the molecule used by the human immunodeficiency virus (HIV) as an entry portal into cells of the immune system. This short review provides a background to the CD molecules and antibodies against them, and summarizes research, diagnostic, and therapeutic applications of antibodies against these molecules.
The immune system evolved (presumably) because it protects complex organisms from being overwhelmed by infection. In mammals, the immune system is complex, multi-layered, and tightly controlled. Immune responses are directed against foreign but not self targets, and are controlled by feedback inhibition so as to minimize damage to tissue. The immune system consists of a network of organs, cells, and soluble mediators. Inevitably, the system can malfunction, leading to disease.
The cells of the immune system are the white blood cells, the leucocytes. These include a number of major distinguishable populations, such as the dendritic cells which first capture antigen, process it to a form that can be recognized by T lymphocytes, and present it to the T cells. Lymphocytes are a morphologically distinct population, but are functionally heterogeneous. Lymphocytes are divided into B cells, which make antibodies, and T cells, which control B cells and many other aspects of the immune response. T cells can be sub-divided into multiple functional subsets which interact with each other and with other components of the immune system. Immunological memory, which allows rapid recovery from a second or subsequent infection with an organism experienced previously, resides in T cells and B cells.
Gross abnormalities of lymphocytes are associated with certain diseases, such as chronic lymphocytic leukemia, which is a malignant proliferation of a single clone of B cells, or HIV infection, which leads to the depletion of the CD4+ “helper T cell” population. More subtle abnormalities of lymphocytes are associated with many other diseases, including the autoimmune and allergic diseases.
There are a number of excellent Immunology texts available for the reader who wants to delve deeper, for example Mak and Saunders (1).
The interactions of leucocytes with their universe—other cells, tissue matrix, and antigen—occur through the cell membrane, and specifically through membrane proteins, glycoproteins, and glycolipids. Specialized cell function is reflected in specialized cell surface composition. For example a B lymphocyte binds antigen through membrane immunoglobulin (Ig), which is characteristic of B cells and is absent from other leucocytes. Furthermore, when antigen binds Ig, complex molecular machinery involving several other membrane proteins (including CD79, CD19, CD81, and CD21) comes into play. This complex transduces activation signals to the inside of the cell, and regulates activation. Some of these molecules are also restricted to B cells, while CD81 mediates a similar function in T cells.
The characterization and naming of leukocyte surface molecules has been the responsibility of an organization formerly called Human Leukocyte Differentiation Antigens (HLDA) and more recently re-named Human Cell Differentiation Molecules (HCDM). This organization devised the CD nomenclature and publishes periodic reports on human cell surface molecules (2–9). There are currently some 500 characterized leukocyte cell surface molecules, many of them with CD names. It has been estimated that there may be 2,500 leukocyte cell surface molecules in total (10). Although most of these have yet to be characterized and named, the complete set of leukocyte surface molecules will be referred to in this article as CD molecules.
Comprehensive databases of CD molecules include the HCDM web site (www.hcdm.org). The reports of the HLDA Workshops provide detailed information on the molecules as they are characterized, and a comprehensive directory of CD molecules is in press (11). Efforts are under way to identify the “missing” CD molecules by proteomic analysis (12).
While a number of techniques can be used in the study of CD molecules, antibodies are particularly specific, versatile, and powerful reagents (Table 1). Antibodies can be used analytically to label the molecules and hence cells bearing them, allowing, in turn, measurement of the amount of a CD molecule, the number of cells bearing it, as well as the localization of the molecule and cells bearing it in tissue. Antibodies can be used preparatively to purify (or remove) the molecule from serum or a tissue extract, or to purify (or remove) cells bearing it from cell suspensions.
The analytical applications of antibodies lead to diagnostic assays, while the preparative applications have therapeutic counterparts. Finally, antibody against a CD molecule can be used to probe, simulate, or inhibit the function of the molecule, and this also suggests therapeutic applications.
Lists of antibodies against CD molecules are available from a variety of web sites (including www.hcdm.org) and from suppliers of CD antibodies.
Figure 1 shows some analytical data on lymphocytes in a sample of blood. The analysis shows the proportions of T cells, B cells and two major functional subsets of T cells. This is a healthy control sample, but this type of analysis has a number of diagnostic applications which are used routinely in hundreds of pathology laboratories daily. Patients with immune deficiencies may lack one or more lymphocyte types. In acquired immune deficiency due to HIV infection (AIDS), the CD4 cells are attacked, and counts of CD3 or CD4 cells are performed frequently to monitor disease, make treatment decisions, and monitor the effectiveness of therapy. Patients with B cell leukemia will have elevated numbers of B cells and a corresponding fall in the proportion of T cells.
A different example of a diagnostic test based on a CD molecule is the use of CD64. CD64 expression on neutrophils is increased within hours by inflammation or tissue damage. A kit is available from IQ Products (www.iqproducts.nl) which facilitates the analysis of neutrophil CD64 and is marketed for the diagnosis and monitoring of sepsis.
The use of additional CD antibodies allows a more detailed analysis of cells and their probable function; for the most part, these provide information that can help build a picture of disease processes but are, as yet, not well enough established to be accepted as diagnostic tests. For example, there has been a recent surge in interest in cells called regulatory T cells (Tregs), which are thought to be deficient in number or function in autoimmune disease and allergy, and over-represented or overactive in patients with malignancies that are not being controlled by the immune system. Thus Treg numbers (21) and function (22) have been described as deficient in the autoimmune disease type 1 diabetes. Treg identification is an area of active research (14), and it is likely that we do not yet have the best markers for Tregs.
We can anticipate that many new and more discriminating diagnostic assays will emerge as the full complement of CD molecules is characterized and antibodies are available to numbers of laboratories studying pathophysiology. These will go beyond answering the diagnostic question, “What disease does this patient have?” to the more useful question, “What is the best treatment for this patient at this time?”
Antibodies have been used therapeutically for many years, starting (as far as we know) with the use of horse antisera against bacterial toxins by Emil von Behring and Shibasaburo Kitasato. In the field of CD molecules, an early success was the use of OKT3, a CD3 antibody, to reverse organ graft rejection. With this exception, CD antibodies failed for many years to live up to expectations. The reasons are interesting, but the turn around is much more interesting. Monoclonal antibodies, particularly against CD molecules and related components of the immune system, are currently having a major impact on a number of diseases and perhaps a bigger impact on the biotechnology industry. The number of antibodies undergoing clinical trial and late stage preclinical evaluation is even more impressive. A recent highly-publicized adverse event (23) reminds us of the dangers, and there have been other unsuccessful trials, but the successes are impressive.
Tables 2 and and33 list some of the CD antibodies in current clinical use. About 200 antibodies are undergoing clinical evaluation (46), while an industry web site provides a list of many antibodies undergoing pre-clinical testing (PharmaProjects Database PJB Publications, available at http://www.pjbpubs.com/pharmaprojects/index.htm).
After a long period of very slow progress, the therapeutic applications of monoclonal antibodies are expanding at an explosive pace. What are the limits? First, expansion is limited by the pool of available diseases and patients in economies that can afford such relatively expensive therapeutics. We may anticipate that advances will come in the form of, for example, a better CD20 (47), which will be good for patients but will compete with the existing CD20 therapeutics, thus slowing the rate of growth. Cost is a limitation that will always be with us. Unlike small-molecule chemical “drugs,” antibodies will always be expensive to make, and cut-price “generics” will still be expensive. It is not yet clear whether fully-human antibodies will be limited by anti-idiotypic responses—these will undoubtedly occur, but may not be limiting (48).
Nevertheless, there are still many opportunities in cancer, transplantation, inflammation and autoimmune diseases, and the infectious diseases. Whereas antibody-mediated therapies once were seen as having a limited application, of interest only until a relatively cheap chemical drug was available, antibodies are now seen as having significant advantages over chemical drugs, because of their specificity. This does not mean an absence of side effects (23), but it should be possible to predict therapeutic and undesirable effects from knowledge of the biological mechanisms addressed by the antibodies.
Successful as therapeutic antibodies have been in recent years, we are still working with a small number of target molecules, and a limited set of effector mechanisms. Therapeutic effects are impressive, but far from complete. The CD20 antibody in current clinical use is effective for only a proportion of CD20-positive lymphomas (47), and the several anti-inflammatory treatments based on antibodies also address only a proportion of patients successfully. The use of immunotoxins (49) may be part of the answer, while understanding and utilizing the full range of immunological mechanisms available to an antibody (50,51) will also help. If our estimate that there are many more CD molecules to be discovered (10) is correct, we will have more targets from which to chose. When we have a better understanding of the mechanisms involved when antibodies interact with cells, we may be able to control tumors and adverse immune reactions more effectively than we can now.
The Author’s studies in the area of leukocyte cell surface molecules have been supported by the Australian National Health and Medical Research Council, by the Co-operative Research Centre for Diagnostics, and by the Human Leukocyte Differentiation Antigens Workshops. I thank Dr Alice Beare and Ms Silvia Nobbs for Figure 1.
Online address: http://www.molmed.org