Shortly after the invention of mAb technology in laboratories all around the world a huge variety of reagents were raised against white blood cells and normal as well as malignant cells from numerous tissues. This led to a Babylonian confusion with regard to antibody names and designation of detected antigens. The problem was approached by the organization of the well-known Workshops and Conferences on Human Leukocyte Differentiation Antigens (HLDA), the first of which took place 1982 in Paris, France, and the most recent 8th meeting was held 2004 in Adelaide, Australia (Bernard et al. 1984; Zola et al. 2005). Antibody samples submitted to the Workshop were grouped into panels and simultaneously analyzed by a couple of reference laboratories with expertise for particular methods such as flow cytometr y, immunohistology, biochemistr y, or molecular genetics. The results were subsequently compared and statistically evaluated. This allowed the identification of distinct “clusters of differentiation” that became the basis of CD antigen nomenclature. To date, 339 CD antigens have been defined and characterized in depth by approaches taking advantage of immunology, cell biology, biochemistry, and molecular biology.
Monoclonal antibodies have proven to be unique reagents for the analysis of surface antigens on lymphocytes that are expressed on certain stages of lymphocyte differentiation and maturation. Using a whole panel of such antibodies has facilitated the phenotyping of functional subpopulations of normal lymphocytes. Likewise, the malignant counterparts derived from the respective stages of differentiation can be classified. Figure 2.7 illustrates how individual B cell antigens show up and vanish during B cell development. The identification and diagnosis of distinct entities among malignant lymphomas is essentially based on immunohistological staining with a set of antibodies recognizing lymphocyte differentiation antigens (Harris et al. 2001). More recently, classical immunophenotyping of lymphomas was complemented by gene expression profiling technology (Staudt and Dave 2005).
Only a small proportion of the 339 CD antigens have yet evolved as valuable targets for antibody therapy not only of malignant lymphomas but also of certain autoimmune diseases and for the prevention of allograft rejection. In Table 2.1 the characteristics of six antigens that serve as targets for therapeutic antibodies already on the market are listed. These antigens differ with respect to their tissue distribution and show different traits concerning stability of surface expression and internalization. The CD22 antigen, for example, has a high internalization rate, making it an exquisite candidate for manufacturing an immunotoxin that has to reach the cytosol of a target cell to become effective (Messmann et al. 2000; Kreitman et al. 2001).
Monoclonal antibodies have proven to be unique reagents for the analysis of surface antigens on lymphocytes that are expressed on certain stages of lymphocyte differentiation and maturation. Using a whole panel of such antibodies has facilitated the phenotyping of functional subpopulations of normal lymphocytes. Likewise, the malignant counterparts derived from the respective stages of differentiation can be classified. Figure 2.7 illustrates how individual B cell antigens show up and vanish during B cell development. The identification and diagnosis of distinct entities among malignant lymphomas is essentially based on immunohistological staining with a set of antibodies recognizing lymphocyte differentiation antigens (Harris et al. 2001). More recently, classical immunophenotyping of lymphomas was complemented by gene expression profiling technology (Staudt and Dave 2005).
Only a small proportion of the 339 CD antigens have yet evolved as valuable targets for antibody therapy not only of malignant lymphomas but also of certain autoimmune diseases and for the prevention of allograft rejection. In Table 2.1 the characteristics of six antigens that serve as targets for therapeutic antibodies already on the market are listed. These antigens differ with respect to their tissue distribution and show different traits concerning stability of surface expression and internalization. The CD22 antigen, for example, has a high internalization rate, making it an exquisite candidate for manufacturing an immunotoxin that has to reach the cytosol of a target cell to become effective (Messmann et al. 2000; Kreitman et al. 2001).
2.6.2 Epithelial Differentiation Antigens
The most frequent tumor type in humans, carcinoma, is derived from epithelial cells. Therefore, tremendous efforts have been made to identify tumor-associated or even tumor-specific membrane antigens on epithelial tumors by means of mAbs. With time it has emerged that all antigens initially regarded as tumorspecific were actually differentiation antigens and are also expressed on certain normal cells. Today it is clear that tumor-specific antigens recognized by antibodies most likely do not exist. The same experience emerged from studies focusing on other tumor types such as melanoma and brain tumors. We have learned, however, that differentiation antigens, although not tumor-specifi c, are valuable targets for antibody-based tumor therapy.
Members of the epidermal growth factor receptor (EGFR) family, in particular, such as HER2/neu and EGFR, hold great promise as therapeutic targets since they are overexpressed in a variety of solid tumors (Hynes and Lane 2005). The induction of antitumor responses using the antibodies trastuzumab and cetuximab are discussed in Volume III in Chapters 14 and 4. Further good candidates for antibody therapy of solid tumors are the epithelial cell adhesion molecule, Ep-CAM, and the carcinoembryonic antigen, CEA. Ep-CAM represents a very stable marker even in highly de-differentiated adenocarcinomas and its overexpression is associated with poor prognosis in breast cancer (Gastl et al. 2000) whereas CEA appears to be a suitable target for radioimmunotherapy of colorectal and medullary thyroid cancer (Mayer et al. 2000; Sharkey et al. 2005).
2.6.3 Mechanisms of Action of Monoclonal Antibodies
The antitumor effects of antibodies can be induced by direct and indirect mechanisms (Table 2.2). In some instances antibody binding per se will lead to cell death. For instance, if a surface receptor is crosslinked that transmits an apoptosis signal, programmed cell suicide is started. Likewise binding to growth receptors or their ligands might abrogate vital signals required for cell proliferation. Antibodies against EGFR family members are prominent examples of this mode of action, as already mentioned. Recently, reagents interfering with angiogenesis have become increasingly attractive (Ferrara et al. 2003). Antibodies specific for VEGF or its receptors can prevent tumor vessel formation and thus deprive the tumor of nutrients. A rather special case is represented by anti-idiotype antibodies in B cell lymphoma. By mechanisms that are poorly understood, these antibodies are able to facilitate long lasting growth control of tumor cells (Davis et al. 1998).
The classical effector functions of antibody are CDC and ADCC. Depending on the isotype of the therapeutic antibody, complement component C1q is activated and triggers a cascade of enzymatic reactions resulting in recruitment of phagocytes and formation of a membrane-attack complex that finally leads to the lysis of tumor cells (Gelderman et al. 2004). In case of ADCC tumor cell-bound antibodies interact via their Fc portion with Fc receptors expressed at high density on NK cells, neutrophils, and monocytes (Ravetch and Bolland 2001). Upon activation, these effector cells release cytotoxic granules from the cytosol delivering a kiss of death to the tumor target. Unfortunately, many antibodies elicit neither direct nor indirect effects, this holds especially true for murine antibodies. However, these reagents can be successfully used as carriers for toxins, radionuclides, or chemotherapeutic substances. There has been much debate on the issue whether anti-idiotypic networks, forming an internal image of tumor antigens, really contribute to tumor regression. Finally, bispecific antibodies are synthetic molecules that carry two different antigen binding sites. By virtue of their dual specificity they can trigger effector cells via a membrane receptor and at the same time link them to a tumor cell. This interaction leads to the subsequent destruction of the tumor cell.
2.6.4 Human Monoclonal Antibodies
Great efforts have been made to take human myeloma cells in culture suitable for cell fusion in order to raise human mAbs. These attempts were largely hampered by the fact that most of the laboriously established lines later turned out to be Epstein–Barr virus (EBV) transformed lymphoblastoid B cell cultures. Although some human cell lines capable of producing human hybridomas have been described, for instance SK-007 (Olsson and Kaplan 1980), GM1500 (Croce et al. 1980) LICR-LON-Hmy2 (Edwards et al. 1982) and Karpas 707 (Karpas et al. 1982), the overall experience remains disappointing. In addition, for ethical reasons it is not possible to immunize a human volunteer with an experimental antigen. As already mentioned, in vitroimmunization was not able to solve the problem due to predominant IgM responses.
In an alternative attempt, antigen-specific B lymphocytes were isolated from the peripheral blood of human donors and immortalized by EBV to establish permanent cell lines (Steinitz et al. 1977). Unfortunately, the production rate of the lines was low and decreased with time. It further turned out that the EBVtransformed lines were extremely difficult to clone. To circumvent those problems, the EBV hybridoma technique was developed, which combined EBV-induced immortalization of human antibody-secreting cells with fusion of a variant of the human myeloma line GM1500 to obtain human–human hybrids (Kozbor and Roder 1981). This method, however, is complex and often leads to instable hybridomas that require repeated recloning. Lacking a human non-secretor myeloma cell line with high fusion frequency the production of human mAbs by the hybridoma technique was no longer pursued for many years. Recently, the EBV method has been improved to immortalize memory B cells from a patient with severe acute respiratory syndrome (SARS) coronavirus infection. Neutralizing mAbs of high affinity against the virus, conferring protection in a mouse model, were successfully isolated (Traggiai et al. 2004).
At present, there are at least three alternative core technologies available allowing for the creation of human mAbs. The variable regions or only the CDRs from mouse heavy and light chains can be grafted onto a human IgG scaffold giving rise to chimeric or humanized antibodies, respectively (Carter 2001). Screening of large recombinant antibody libraries is exploited to build human antibodies with high specificity and affinity (Hoogenboom 2005). Transgenic mice carrying human immunoglobulin genes will respond to immunization with the production of entirely human antibodies. After fusion with mouse myeloma cells, these human antibodies are secreted by resulting hybridomas (Lonberg 2005). In addition, recombinant antibodies containing minimal binding fragments can be reconstructed to multivalent high-affinity reagents (Holliger and Hudson 2005)
2.7 Outlook
Monoclonal antibodies secreted by hybidoma cells have led to a revolution in biology, medicine, and many applied sciences due to their excellent specificity. After a first wave of innovation based on mouse monoclonals, molecular biology has provided tools for reshaping the antibody molecule to obtain chimeric, humanized, and fully human antibodies as well as recombinant antibody fragments. Therapeutic antibodies have evolved as effective pharmaceutical compounds not only for the treatment of malignant tumors but also of autoimmune diseases and infections. Currently we are encountering a third wave of scientific advancement by subtle antibody engineering, making it possible to tune the molecule in a way that it can meet special therapeutic demands (Weiner and Carter 2005). In the end there is no doubt that antibody-based therapeutics will play an outstanding role in several fields of modern medicine
The most frequent tumor type in humans, carcinoma, is derived from epithelial cells. Therefore, tremendous efforts have been made to identify tumor-associated or even tumor-specific membrane antigens on epithelial tumors by means of mAbs. With time it has emerged that all antigens initially regarded as tumorspecific were actually differentiation antigens and are also expressed on certain normal cells. Today it is clear that tumor-specific antigens recognized by antibodies most likely do not exist. The same experience emerged from studies focusing on other tumor types such as melanoma and brain tumors. We have learned, however, that differentiation antigens, although not tumor-specifi c, are valuable targets for antibody-based tumor therapy.
Members of the epidermal growth factor receptor (EGFR) family, in particular, such as HER2/neu and EGFR, hold great promise as therapeutic targets since they are overexpressed in a variety of solid tumors (Hynes and Lane 2005). The induction of antitumor responses using the antibodies trastuzumab and cetuximab are discussed in Volume III in Chapters 14 and 4. Further good candidates for antibody therapy of solid tumors are the epithelial cell adhesion molecule, Ep-CAM, and the carcinoembryonic antigen, CEA. Ep-CAM represents a very stable marker even in highly de-differentiated adenocarcinomas and its overexpression is associated with poor prognosis in breast cancer (Gastl et al. 2000) whereas CEA appears to be a suitable target for radioimmunotherapy of colorectal and medullary thyroid cancer (Mayer et al. 2000; Sharkey et al. 2005).
2.6.3 Mechanisms of Action of Monoclonal Antibodies
The antitumor effects of antibodies can be induced by direct and indirect mechanisms (Table 2.2). In some instances antibody binding per se will lead to cell death. For instance, if a surface receptor is crosslinked that transmits an apoptosis signal, programmed cell suicide is started. Likewise binding to growth receptors or their ligands might abrogate vital signals required for cell proliferation. Antibodies against EGFR family members are prominent examples of this mode of action, as already mentioned. Recently, reagents interfering with angiogenesis have become increasingly attractive (Ferrara et al. 2003). Antibodies specific for VEGF or its receptors can prevent tumor vessel formation and thus deprive the tumor of nutrients. A rather special case is represented by anti-idiotype antibodies in B cell lymphoma. By mechanisms that are poorly understood, these antibodies are able to facilitate long lasting growth control of tumor cells (Davis et al. 1998).
The classical effector functions of antibody are CDC and ADCC. Depending on the isotype of the therapeutic antibody, complement component C1q is activated and triggers a cascade of enzymatic reactions resulting in recruitment of phagocytes and formation of a membrane-attack complex that finally leads to the lysis of tumor cells (Gelderman et al. 2004). In case of ADCC tumor cell-bound antibodies interact via their Fc portion with Fc receptors expressed at high density on NK cells, neutrophils, and monocytes (Ravetch and Bolland 2001). Upon activation, these effector cells release cytotoxic granules from the cytosol delivering a kiss of death to the tumor target. Unfortunately, many antibodies elicit neither direct nor indirect effects, this holds especially true for murine antibodies. However, these reagents can be successfully used as carriers for toxins, radionuclides, or chemotherapeutic substances. There has been much debate on the issue whether anti-idiotypic networks, forming an internal image of tumor antigens, really contribute to tumor regression. Finally, bispecific antibodies are synthetic molecules that carry two different antigen binding sites. By virtue of their dual specificity they can trigger effector cells via a membrane receptor and at the same time link them to a tumor cell. This interaction leads to the subsequent destruction of the tumor cell.
2.6.4 Human Monoclonal Antibodies
Great efforts have been made to take human myeloma cells in culture suitable for cell fusion in order to raise human mAbs. These attempts were largely hampered by the fact that most of the laboriously established lines later turned out to be Epstein–Barr virus (EBV) transformed lymphoblastoid B cell cultures. Although some human cell lines capable of producing human hybridomas have been described, for instance SK-007 (Olsson and Kaplan 1980), GM1500 (Croce et al. 1980) LICR-LON-Hmy2 (Edwards et al. 1982) and Karpas 707 (Karpas et al. 1982), the overall experience remains disappointing. In addition, for ethical reasons it is not possible to immunize a human volunteer with an experimental antigen. As already mentioned, in vitroimmunization was not able to solve the problem due to predominant IgM responses.
In an alternative attempt, antigen-specific B lymphocytes were isolated from the peripheral blood of human donors and immortalized by EBV to establish permanent cell lines (Steinitz et al. 1977). Unfortunately, the production rate of the lines was low and decreased with time. It further turned out that the EBVtransformed lines were extremely difficult to clone. To circumvent those problems, the EBV hybridoma technique was developed, which combined EBV-induced immortalization of human antibody-secreting cells with fusion of a variant of the human myeloma line GM1500 to obtain human–human hybrids (Kozbor and Roder 1981). This method, however, is complex and often leads to instable hybridomas that require repeated recloning. Lacking a human non-secretor myeloma cell line with high fusion frequency the production of human mAbs by the hybridoma technique was no longer pursued for many years. Recently, the EBV method has been improved to immortalize memory B cells from a patient with severe acute respiratory syndrome (SARS) coronavirus infection. Neutralizing mAbs of high affinity against the virus, conferring protection in a mouse model, were successfully isolated (Traggiai et al. 2004).
At present, there are at least three alternative core technologies available allowing for the creation of human mAbs. The variable regions or only the CDRs from mouse heavy and light chains can be grafted onto a human IgG scaffold giving rise to chimeric or humanized antibodies, respectively (Carter 2001). Screening of large recombinant antibody libraries is exploited to build human antibodies with high specificity and affinity (Hoogenboom 2005). Transgenic mice carrying human immunoglobulin genes will respond to immunization with the production of entirely human antibodies. After fusion with mouse myeloma cells, these human antibodies are secreted by resulting hybridomas (Lonberg 2005). In addition, recombinant antibodies containing minimal binding fragments can be reconstructed to multivalent high-affinity reagents (Holliger and Hudson 2005)
2.7 Outlook
Monoclonal antibodies secreted by hybidoma cells have led to a revolution in biology, medicine, and many applied sciences due to their excellent specificity. After a first wave of innovation based on mouse monoclonals, molecular biology has provided tools for reshaping the antibody molecule to obtain chimeric, humanized, and fully human antibodies as well as recombinant antibody fragments. Therapeutic antibodies have evolved as effective pharmaceutical compounds not only for the treatment of malignant tumors but also of autoimmune diseases and infections. Currently we are encountering a third wave of scientific advancement by subtle antibody engineering, making it possible to tune the molecule in a way that it can meet special therapeutic demands (Weiner and Carter 2005). In the end there is no doubt that antibody-based therapeutics will play an outstanding role in several fields of modern medicine
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