Antibody Synthesis in Vitro

Antibody Synthesis in Vitro

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Paz Martıńez, Universitat Auto`noma de Barcelona, Barcelona, Spain . Introduction

Antoni Iborra, Universitat Auto`noma de Barcelona, Barcelona, Spain

. Generation of B-cell Response in Vivo

Several strategies are used to provide immortalized monoclonal antibody-secreting B cells, and antibodies by recombinant deoxyribonucleic acid (DNA) technology. Monoclonal antibodies are good specific reagents to recognize any chemical structure. High concentrations of monoclonal antibodies can be obtained and used in diagnostics and human therapy.

. Monoclonal Antibody Technology . Mouse Monoclonal Antibodies . Human Monoclonal Antibodies . Production of Monoclonal Antibodies . Applications of Monoclonal Antibodies . Future Prospects

Introduction

doi: 10.1038/npg.els.0001115

Karl Landsteiner demonstrated that animals could make antibodies to almost anything, including small synthetic molecules that had never previously existed. Taking advantage of this fact, it has been widely demonstrated that almost any given molecule taken as an antigen, in a suitable form for immunization, induces specific antibodies (polyclonal antibodies) in several animals. Therefore, antibodies are good specific reagents to recognize any chemical structure. The required properties for a good selective biochemical reagent for any needed application are high specificity for the antigen and availability for long time and high concentrations. Monospecific antibodies (monoclonal antibodies) that may be produced in large quantities have a wide range of applications in medicine, cell analysis and separation or human immunotherapy. Depending on their use, in laboratory techniques or in human therapy, the source for antibody may be animal or human B lymphocytes, respectively. Futher monospecific antibodies can also be made using recombinant deoxyribonucleic acid (DNA) technology. Bearing in mind how nature selects high-quality antibodies to any given antigen or structure, we will describe the requirements for the generation of these antibodies by diverse in vitro methods.

Generation of B-cell Response in Vivo Antibodies may be expressed as membrane receptors in B cells (B-cell receptors, (BCR)), or as secreted proteins. With these receptors, B lymphocytes are able to recognize the shapes or conformations of practically any native macromolecule including proteins, lipids, carbohydrates and nucleic acids, as well as external parts of more complex macromolecules (glycoproteins, lipoproteins, glycolipids). Lymphocyte receptors are remarkably diverse, and developing B cells use a complex mechanism to achieve this diversity. In each lymphocyte, functional genes are assembled by somatic recombination from sets of separate gene segments that encode the variable region of the immunoglobulin. This diversity is mostly concentrated in the DNA

encoding the complementarity determining regions (CDRs), or hypervariable regions, that conform the antigen-binding structure. In addition, mature B cells that are activated by antigens initiate a second process of maturation that includes isotype switching and somatic point mutations (hypermutation) of the variable region. This creates numerous variants of the original assembled immunoglobulin genes. Antibodies bind to antigens by reversible, noncovalent interactions, including hydrogen bonds and charge interactions. The parts of antigens that are recognized by antibodies are called epitopes or antigenic determinants. The strength with which one antigen-binding surface of antibody binds to one epitope is called affinity of the interaction. The process by which high-affinity B cells are positively selected is called affinity maturation. This affinity maturation occurs as a result of somatic hypermutation of immunoglobulin genes in dividing B cells. This is followed by the selection of high-affinity B cells by antigen displayed on the surface of follicular dendritic cells in germinal centres of the lymphoid follicles (see Figure 1a). Mutations that result in a B-cell receptor of low affinity for the antigen prevent the B cell from being activated and this B cell will die by apoptosis. Mutations that improve affinity of a B cell for the antigen are efficiently selected and expanded. During cell division, mutations and selection are repeated, and affinity and specificity of selected B cells are continually refined during the germinal centre response. Antibodies are produced by mature B lymphocytes expressing membrane-bound immunoglobulins IgM and IgD with a single antigenic specificity. Naive B lymphocytes recognize antigens but do not secrete antibodies. When naive B cells are activated by antigens, these antigen-specific cells proliferate and some differentiate into antibody-secreting effector B cells or plasma cells. The secreted antibodies have the same specificity of the naive B-cell membrane receptors. The processes of affinity maturation and isotype switching are responsible for the higher affinity exhibited in a secondary response. Thus, during a primary humoral response,

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Animal immunization Antigen Activated B cell

Cell division Mouse lymphocytes

Mouse myeloma cells

Somatic hypermutation Affinity

Affinity Affinity Antigen

Apoptosis Cell fusion by PEG L

Affinity

Hybrid L−L

Follicular dendritic cell

Selective medium HAT/HT M

Hybrid M−M Macrophage

Memory cell

Plasma cell

Hybridomas L−M Memory cell

Plasma cell

(a) Affinity maturation in germinal centres Screening and cloning

Screening

Monoclonal antibody

Stationary culture

–

Suspension culture

-Cell requirement -Time and economical cost -Technical experience -Antibody production

Perfusion culture

+

(b) In vitro monoclonal antibody

Figure 1 Generation of monoclonal antibody secretory hybridomas by B-cell fusion. (a) In vivo selection of high-affinity B cells in germinal centres of lymph nodes. Differentiation in plasma B cells secreting high-affinity antibodies. (b) In vitro production of large concentrations of monoclonal antibodies.

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IgM is secreted initially and its affinity is low, and this is followed by a switch to an increasing proportion of immunoglobulin (IgG, IgA or IgE) with higher affinity. Single and homogeneous molecular species of antibodies may be secreted by plasma cell tumours, called myeloma. The clones of malignant plasma cells are called plasmacytomas and secrete single reactive monoclonal antibodies.

Monoclonal Antibody Technology Animals immunized with an antigen raise a pool of polyclonal antibodies that consist of diverse antibodies of a variety of isotypes binding to different antigenic epitopes, with a diverse range of affinities. Antisera from immunized animals are variable in their specificity and of limited amounts. In 1975 Georges Ko¨hler (1946–1995) and Cesar Milstein (1927–2002) made a discovery trying to find out how antigen specificity is generated. They fused B cells with specificity for a known antigen with myelomas to make them immortal, so that they could grow in culture indefinitely and secrete antibodies of desired specificity. These hybrid cells, called B-cell hybridomas, carry the antibodysecreting ability from B cells and the immortality from myeloma cells or plasmacytomas (Ko¨hler and Milstein, 1975). In 1984 Jerne, Milstein and Ko¨hler shared the Nobel Prize of Physiology or Medicine ‘for theories concerning the specificity in development and control of the immune system and discovery of the principle for production of monoclonal antibodies’ (Jerne and Nordin, 1963). By being directed towards single epitopes on the antigen, monoclonal antibodies frequently show high specificity in terms of their low cross-reactivity with other antigens (Alkan, 2004).

Mouse Monoclonal Antibodies Screening strategies For a successful development of a monoclonal antibody the screening assay is critical and has to be developed beforehand. This selective analysis must discriminate between positive and negative antibody-secreting hybridomas in culture. Just after hybridoma supernatant testing, selected hybridomas must be cloned, grown in selective medium and recloned to ensure their monoclonality. The future application of monoclonal antibodies will often prefer one screening assay. Some antibodies will work very well in one assay but not in another. For example, antibodies that react to a fixed tissue will not necessarily react with fresh tissue. Any chosen screening protocol requires purified antigen to select the antibody specificity and to discriminate between the desired epitope and the contaminating material used in the immunization. The assay must be specific, sensitive and capable of screening large num-

bers of samples quickly. Appropriate positive and negative controls must be used. The most frequent screening techniques are immunofluorescence, enzyme-linked immunosorbent assay (ELISA) or immunoblot.

Immunization procedures Activated B lymphocytes are obtained after induction of an immune response by different methods that include: diverse immunization protocols (using different routes, adjuvants, booster injections), use of B lymphocytes of a nonimmunized healthy individual, as a naive source of specificities, or lymphocytes from disease-related sites. The most common way to obtain antigen-specific B lymphocytes is by active immunization, normally for long periods of time. In a long-term immunization schedule with one or more injections, immune responses mature in vivo and the specificity spectrum of antibodies change from broad specificity and low affinity to narrow specificity and high affinity. Alternatively, a short-term antibody synthesis procedure may be attained by a single intrasplenic injection of a DNA plasmid coding for a foreign antigen. This genetic immunization has proved to be a good method for polyclonal antisera as well as for the generation of monoclonal antibodies (Velikovsky et al., 2000).

Mouse–mouse hybridomas Antibody-secreting hybridomas are derived from the fusion of a murine myeloma cell line that can grow indefinitely and B splenocytes from an immunized mouse. A large number of mouse plasmacytoma immortalized cell lines, secreting different immunoglobulin classes, are available in the American Type-Culture Collection (ATCC). Nonsecretory myeloma cell lines deficient for the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT) have been established as fusion partners, because they are not able to use the salvage pathway for DNA synthesis in the presence of aminopterin. Cell fusion is facilitated by polyethylene glycol, by a viral infection, or by electroporation. To select viable hybridomas, the cells are grown in selective medium with hypoxanthine, aminopterin and thymidine (HAT medium), in which only the hybridomas proliferate. The myeloma cells that fuse with another myeloma cell or do not fuse at all die in HAT medium, since they are HGPRT-negative. The B cells and B-cell hybrids also die because they are not able to grow indefinitely. Hybridomas resulting from cell fusion are a heterogenous population, with a broad range of antibody specificities. After screening for specificity, hybridomas are cloned by limiting dilution, normally, to obtain single secreting clones. Figure 1 summarizes the procedure for murine monoclonal antibody production from a hyperimmunized mouse. 3


Human immunization

Disease related sites

Naive repertoire

Animal immunization

Mouse lymphocyte Phage display Mouse myeloma Human lymphocyte Fv scFv Mouse hybridoma

Infected with EBV

Human myeloma

Heterohybridoma

Immortalizaton Human hybridoma

Chimeric and humanized antibodies Antibody fragments and constructs

Antibody expression in transfected cell lines

Figure 2 Strategies for human monoclonal antibody production.

The application of murine monoclonal antibodies is widespread in many areas from biological and medical research to clinical diagnostics. Monoclonal antibodies also have potential in human therapeutic applications, but their usefulness is limited due to negative effects including the immune response to the murine antibodies (human antimouse antibody response (HAMA). Special interest of monoclonal antibodies suitable for their use in human immunotherapy has led to the development of human monoclonal antibodies.

tissues, such as spleen, tonsil, lymph nodes and peripheral blood mononuclear cells (PBMC). As compared with other sources, PBMC are readily accessible and can be sampled repeatedly. Several strategies for human monoclonal antibody production have been applied, such as the immortalization of B cells by virus, human B lymphocyte fusion with a heterologous myeloma cell line or the use of recombinant DNA technology. All these strategies are described in Figure 2.

Immortalization of B lymphocytes

Human Monoclonal Antibodies Human monoclonal antibodies, and monoclonal antibodies from several species other than the mouse or the rat, are difficult to generate because of the lack of myeloma cell lines. B cells may be derived from different lymphoid 4

Epstein–Barr virus (EBV) is double-stranded DNA virus belonging to the herpes virus family. The main targets of EBV are B and epithelial cells. B-cell infection with EBV involves virus attachment to the B cell-specific surface protein CR2 (CD21) through the viral glycoprotein gp350/ 220. After infection of B cells, the virus DNA forms a circle


and persists as an episome in the nuclei of infected cells, establishing a latent infection and cell immortalization. Immortalized EBV transformed B cell lines have been used as a source of human monoclonal antibodies. However, data suggest that EBV transformation favour nonimmunoglobulin-producing B cells. Therefore, EBVtransformed B cells often produce relatively low-affinity IgM antibodies, although some useful higher affinity antibodies can occasionally be obtained. Futhermore, the cell lines frequently lose their ability to secrete antibody if cultured for long periods of time, although they can sometimes be rescued by fusion with a myeloma cell line to produce hybridomas. Recent developments have allowed interesting new approaches. Splenocytes derived from transgenic mice H-2Kb-tsA58 (Immortomice) harbouring a mutant temperature-sensitive simian virus (SV-40) tumour antigen, under the control of a mouse major histocompatibility promoter, are conditionally immortal at permissive temperatures (338C) and may be used to produce monoclonal antibodies. If H-2Kb-tsA58 mice are immunized, the splenocytes obtained grow and can be selected and cloned for their positivity by some screening methods. This cell culture system ensures a reliable and reproducible source of monoclonal antibodies and eliminates the need for hybridoma generation. Antibody-synthesizing cells grow slowly at 338C and are genetically stable for at least several months in culture, and survive freeze–thaw without loss or inactivation of antibody secretion. In vitro immunization is enhanced through the presence of other spleenderived cells, such as macrophages, dendritic cells and fibroblasts that facilitate antibody production. In the future, crossing H-2Kb-tsA58 mice with mice expressing human immunoglobulin-cloned genes will eventually enable production of human monoclonal antibodies by this very efficient method (Pasqualini and Arap, 2004).

Mouse–human heterohybridomas Heterohybridomas are created by fusing murine myeloma with human B cells. PBMC are used preferentially to generate secreting hybrids after fusion with an heteromyeloma fusion partner. The hybridomas originated are very difficult to clone, to maintain long time in culture without loss of specificity, and they normally produce very low yields of immunoglobulin. A new approach to obtain human secretory hybridoma cell lines was developed by fusing heteromyeloma cells (HNC20) with lymphocytes derived from actively immunized individuals. After heterohybridoma establishment, because these cells are unstable, DNA sequences of lightand heavy-chain variable regions were isolated, cloned and inserted into expression vectors containing human IgG constant regions. Transfected Chinese hamster ovary (CHO) cells with recombinant human IgG gene secrete human antibodies. Genetic stability of these transfected

CHO cell lines appears to be suitable for long-term culture and a source of specific monoclonal antibodies (Chin et al., 2003).

Human–human hybridomas Human myeloma cell lines have been very difficult to derive, so further alternatives are needed. A myeloma cell line from a patient has been adapted to be used as a fusion partner for the establishment of human–human hybridomas. This myeloma line has been used for the fusion with Epstein–Barr virus-transformed cells and peripheral blood lymphocytes, giving rise to stable hybridomas that secrete human monoclonal antibodies (Karpas et al., 2001).

Antibody engineering To avoid undesirable reactions in immunotherapy there is an effort to engineer monoclonal antibodies and antibodybinding sites with recombinant DNA technology. It is possible to construct genes that encode immunoglobulins in which the variable regions come from one species and the constant regions come from another. These hybrids are used for the production of mouse–human monoclonal antibodies or humanized antibodies (Jones et al., 1986). In these antibodies the antigenic specificity (variable region) is derived from the mouse DNA. The immunoglobulin isotype (constant region) is derived from the human DNA. Other forms of chimerization and CDR grafting have been developed in attempts to humanize rodent antibodies to reduce their immunogenicity. An advantage of humanized chimeric antibodies is that they are less immunogenic in humans and that they have the biological effector functions of human antibodies. These antibodies can be obtained in large amounts after expression in mammalian cells. However, glycosylation in mice-humanized antibodies is different than in human antibodies, so they are still immunogenic in some patients over repeated courses of treatment. It is possible to reduce this immunogenicity inducing T-cell tolerance to therapeutic antibodies by presenting all the potential light- and heavy-chain epitopes in a nonimmunogenic way. Alternatives have been developed that can reduce immunogenicity. Antibody fragments, such as Fab fragments, monovalent Fv fragments (variable heavy-chain (VH) domain+variable light-chain (VL) domain), monovalent scFv constructs (VH+VL-peptide linked) and multivalent Fv antibody constructs can be used instead. These Fv fragments have only half the size of Fab, and have rapid blood clearance and better penetration when used in humans.

Phage display technology A different approach to generate monoclonal antibodies employs the phage display technology first described by 5


Smith (Smith, 1985) and reviewed by Azzazy and Highsmith (Azazzy and Highsmith, 2002). Phage display libraries contain millions of variants that can be constructed simultaneously. First, the polymerase chain reaction (PCR) is employed to amplify the DNA that encodes antibody heavy and light chain from B cells, plasma cells or hybridomas. DNA sequences of interest are inserted into a location in the genome of a filamentous bacteriophage, such that the encoded protein is expressed or displayed on the surface of the phage as a fusion protein to one of the phage coat proteins. These libraries are introduced into Escherichia coli cells, which are then superinfected with helper phage, to facilitate the replication and assembly of viable phage particles. By this procedure an enormous diversity of antibody specificities can be generated, similar to the human in vivo repertoire. These antibodies may be obtained without any need for immunizing. However, the in vivo humoral immune response produces antibodies that increase their affinity as the immune response progresses. The great advantage of phage display is that once a library has been created, it can be used to select antibodies that bind to any target antigen of interest with the screening process being completed in only a few weeks. In the phage display system the affinity maturation, which occurs in vivo, can be simulated by an ‘in vitro affinity maturation’, using site-directed CDR mutagenesis, scFv multimer formation and by V-gene chain shuffling. By using in vitro selection, an improved affinity of 10210 or 10 –11 M is frequently obtained which is comparable to the in vivo affinity range.

Production of Monoclonal Antibodies Over many years, monoclonal antibodies have been produced in mice that have been used extensively to grow secretory hybridomas in their ascitic fluid. However, a growing number of countries have imposed severe ethical restrictions to the use of rodents to avoid distress or pain in animals, and recommend alternative methods for in vitro antibody production. We will review some basic methods of in vitro monoclonal antibody production from cultured hybridoma cells that include stationary culture methods with flasks, suspension culture methods in stirred cultures and perfusion culture methods such as several types of bioreactors. These methods are schematized in Figure 1b. In stationary low-density culture methods, culture plates and flasks are used to expand and maintain hybridomas that secrete small quantities of antibody. The antibodycontaining medium is harvested 1–2 times per week, and the yield is around 10–100 mg mL21 of antibody. In suspension-stirred cultures, the cells and medium are continuously shaken to maintain the hybridomas. Spinner flasks and roller bottles are widely used for the 6

production of high amounts of monoclonal antibodies: 10–220 mg mL21 of antibody. These methods are more expensive than stationary cultures in flasks, and require more incubator space. Large concentrations of cells are needed to prepare the growing of hybridomas by this system. In perfusion systems that are considered high-density cultures, cells are included in a capillary, with a medium recirculation system, and monitoring the metabolic supply, metabolic waste, pH, vitality and antibody concentration. Several hollow fibre systems are used to produce large amounts of antibody in high concentrations, as much as 100 mg month21. This long-term culture can be maintained for very long periods of time.

Applications of Monoclonal Antibodies Hybridoma technology and antibody engineering have contributed to the diagnostics of malignancies, tissue typing, identification and separation of individual cell types, detection of cytokines, growth factors and hormones. In recent years, an emerging and revolutionary technology has been the protein microarray for the proteomic analysis. These arrays can measure protein expression levels, protein–protein interactions or enzymatic activities. Monoclonal antibodies have proven to be excellent capture agents for protein chips developed to date. There are hundreds of companies selling antibodies against a wide range of targets that provide these molecules for their use on arrays. Monoclonal antibodies have been applied in immunotherapy of lymphomas, cancer, chronic inflammatory and autoimmune diseases, infectious pathologies and acute rejection in transplantation. In cancer patients, antibodies coupled to another molecule are used to target tumour cells and also to deliver an insult such as radiation or toxin. However, the majority of unconjugated monoclonal antibodies for clinical use find their application in autoimmunity and in immunosuppression. Some autoimmune and inflammatory diseases have been treated with anti-tumour necrosis factor a (TNF-a) chimeric or humanized monoclonal antibodies (see Table 1). Extensive pharmaceutical research based on the therapeutical use of monoclonal Table 1 Autoimmune and inflammatory diseases treated with TNF-a monoclonal antibody Crohn disease Chronic obstructive pulmonary disease Endometriosis Infectious diseases Malignant disorders Psoriasis Rheumatoid arthritis Toxic shock-virus mediated


Table 2 Use of monoclonal antibodies in human therapy Generic name

Trade name

Target antigen

Type of mAb

Indication

Abciximab Adalimumab Alemtuzumab Basilimab Daclizumab Efalizumab Etelimumab Fontolizumab Gemtuzumab Ibritumomab Infliximab Muromonab Natalizumab Palivizumab Rituximab Trastuzumab

ReoPro HUMIRA Campath Simulect Zenapax Raptiva Cat-192 HuZAF Mylotarg Y-90-Zevalin Remicade OKT3 Antregen Synagis Rituxan Herceptin

Platelet TNFa CD52 CD25 CD25 CD11 TGFb INFg CD33 CD20 TNFa CD3 a4-Integrin RSV CD20 HER-2/neu

Chimeric Phage display CDR-grafted Chimeric CDR-grafted Chimeric Phage display Chimeric CDR-grafted Murine Chimeric Murine Chimeric CDR-gafted Chimeric CDR-grafted

Antiplatelet Rheumatoid arthritis Chronic lymphocytic leukaemia Organ rejection Organ rejection Psoriasis Scleroderma Crohn disease Chronic lymphocytic leukaemia Non-Hodgkin lymphoma Rheumatoid arthritis Crohn disease Organ rejection Multiple sclerosis Respiratory syncytial virus disease Non-Hodgkin lymphoma Breast cancer

antibodies for passive administration in the treatment of human diseases led to the commercialization of several specific antibodies that are summarized in Table 2. Several anti-cytokine monoclonal antibodies are used in human therapy to counteract cytokine-induced pathogenic effects in some diseases (Zagury and Gallo, 2004). Antibodies with a dual specificity, or bispecific antibodies have been designed in which one half of the antibody has specificity for a tumour and the other half has specificity for a surface molecule on an immune effector cell (NK cell, cytotoxic T lymphocyte). Recently, bispecific monoclonal antibodies have been introduced in immunotherapy by redirecting cytotoxicity to tumour cells, by localizing fibrinogen activators to dissolve fibrin clots, or by delivering antigen specifically to antigen-presenting cells as vaccines (Kufer et al., 2004).

microarrays that are widely accepted by researchers as fast and reliable tools for the past decade. However, the new emerging nanotechnologies allow extremely sensitive detection of biological interactions without the need of labelling, optical excitation or external probes. Nanoscale devices and nanoscale components of larger devices are simialr in size to large biological macromolecules. Small devices can interact with biomolecules on the cell surface, within the cell or with soluble molecules. Thus, with minimal sample preparation (antigen) the specific binding to a small number of monoclonal antibodies on nanoscale cantilevers produce a measurable change in the device conductivity. Nanotechnology research, based on the use of monoclonal antibodies, may have three main applications in the future: laboratory-based diagnostics, in vivo diagnostics and therapeutics.

References

Future Prospects This review summarizes several strategies for the synthesis of high-affinity monoclonal antibodies. Other important questions include the detection and the use of these antibodies. The challenge areas of the use of monoclonal antibodies involves developing micro- and nanoscale techniques for diagnostics and immunotherapy. New biomedical technologies will benefit from the combined efforts of scientists from many disciplines: physical, biological and biochemical sciences together with engineering. This may produce rapid changes in the foundations of diagnosis, treatment and prevention of several diseases. Development of new techniques for rapid and sensitive detection of relevant molecules may be used on antibody

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Ko¨hler G and Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256(5517): 495– 497. Kufer P, Lutterbu¨se R and Baeuerle PA (2004) A revival of bispecific antibodies. Trends in Biotechnology 22(5): 238–244. Pasqualini R and Arap W (2004) Hybridoma-free generation of monoclonal antibodies. Proceedings of the National Academy of Sciences of the USA 101: 257–259. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228(4705): 1315–1317. Velikovsky CA, Cassataro J, Sanchez M et al. (2000) Single-shot plasmid DNA intrasplenic immunization for the production of monoclonal antibodies. Persistent expression of DNA. Journal of Immunological Methods 224: 1–7.

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Zagury D and Gallo RC (2004) Anti-cytokine Ab immune therapy: present status and perspectives. Drug Discovery Today 9(2): 72–81.

Further Reading Goldsby RA, Kindt TJ, Osborne BA and Kuby J (2003) Immunology 10th edn. New York: W.H. Freeman and Company. Paul WE (2003) Fundamental Immunology, 5th edn. Philadelphia, PA: Lippincott, Williams & Wilkins. Roitt IM and Delves PJ (2001) Essential Immunology, 10th edn. Oxford: Blackwell Science. Special issue: 25 years of monoclonal antibodies (2000). Immunology Today 21(8): 355–412. Zola H (2000) Monoclonal Antibodies. Oxford, UK: BIOS Scientific Publishers Ltd.