The previous chapters dealt with the reasoning of how and why the mammalian immune system can be manipulated such that monoclonal, antibody-secreting hybridomas can be produced. The various biological, chemical and physical requirements for the production and culture of hybridomas were presented, as was a detailed description of the various assays used for detecting and characterising the hybridoma antibodies. The present chapter will be used to demonstrate, in conclusion, that monoclonal antibodies have found a wide range of uses, being applied in many different areas of the clinical and veterinary fields. The areas will be divided into research, diagnostics, preparative protein chemistry and immunochemistry, prophylaxis and therapeutics, and anti-idiotype antibodies. These categories will be discussed in this order, since this is the approximate chronological order of the development and application of monoclonal antibodies. Because the application of monoclonal antibodies in basic research has received most attention, particularly with respect to publication, this section will be subdivided into the different areas of microbiology, immunobiology and cell biology.

It is, however, impossible to cover all of these areas adequately in a single chapter, particularly a concluding chapter, nor can they be covered in a single volume. In the individual sections and subsections of this chapter, extensive reference will be made to recent reviews, monographs and books on each area of monoclonal antibody application, followed by selected examples to show how the MAb have been used and what the benefit of that application has been. An idea of how extensive this field is can be seen from the number of reviews already written (Table 6.1).

Introduction

In order to develop a clear appreciation of the problems and processes associated with the preparation and use of monoclonal antibodies (MAb), it is essential that the structure and function of the immune system be understood. The initial evidence that the body possessed a defensive system against microbial attack first came to light in the nineteenth and early twentieth centuries. This knowledge has expanded during the past 20–30 years, to give a detailed biochemical and immunochemical insight into the structure and function of the antibody molecule, the bifurcation of the immune response against foreign material (antigen) into non-specific and specific mechanisms (which has been further subdivided into cellular and humoral responses), and the genetic control of antibody diversity.

An understanding of the structure and function of the immune system is necessary for a clear appreciation of the problems and processes associated with the preparation and use of monoclonal antibodies. The stimulation of antibody production in vivo(the humoral response) and the other components of the immune response will then be introduced, and the structure of the antibody molecule and its differentiation into classes and subclasses described and related, where appropriate, to immunological functions. This will assist with the understanding of the properties and functions of monoclonal antibodies, and of both how and why they can be produced. Although the immune system is a complex network of reactions and interactions, the antibody response will be described in the greatest detail, since it is the antibody response which is of direct relevance to the production of monoclonal antibodies.

It is now clear that the immunological activities of lymphocytes are profoundly affected by the presence of various products of arachidonic acid metabolism; however, many fundamental issues concerning this interaction remain unresolved and are, at times, hotly debated.

EICOSANOID PRODUCTION AND INTERACTION WITH LYMPHOCYTES

It has not been proven conclusively that normal lymphocytes possess either cyclooxygenase or lipoxygenase enzyme systems. This is stated with caution, since studies in vitro by Parker et al. (1) suggested that purified lymphocytes produced 5-HETE, 12-HETE, and TXB2, and Goetzl reported that T lymphocytes possess a 5-lipoxygenase system (2). Furthermore, Bauminger reported that mouse thymocytes could be differentiated on the basis of their PG synthetase activities (3). Mature thymocytes appeared to contain higher concentrations of PGE and greater PG synthetase activity than did immature cells. Webb and Nowowiejski reported that both nonadherent and glass-adherent T cells release PGE into the culture medium after phytohemagglutinin (PHA) stimularion (4).

Using thin layer chromatography (TLC) and/or radioimmunoassay (RIA) procedures, Aussel et al. recently demonstrated that Jurkat cells (a human leukemic T cell line) are able to convert arachidonic acid to 6-keto-PGF1α, PGE2, PGA2, and TXB2 (5). The addition of lectins or anti-CD3 monoclonal antibody reduced the amount of PG released by the cells, as did the addition of the cyclooxygenase inhibitors indomethacin and niflumic acid.

There has been increasing concern for the immunological depression, which is unavoidably acquired as a result of major accidental or operative injury in an otherwise normal host. Immunological changes following injury are precipitous and leave the host vulnerable to life-threatening sepsis. Fortunately, injury-induced immune depression is completely reversed with patient recovery. The critical period, therefore, occurs during acute care where controlling immunity in favor of the host is the goal of both clinical care and research. Most of the lessons, to date, have been learned from thermal injuries. The immunological changes that occur as a result of burns appear to have a great deal in common with the immune consequences of other types of tissue injury.

IMMUNE DEPRESSION IN BURN PATIENTS

It is clear that major thermal injuries often precipitate a profound, multicentric immunological depression, which is thought to predispose patients to sepsis. Impairment of immune function is almost universal in patients with burns over greater than 40% of the body surface area, and in very young or very old patients with far smaller burns.

1. total loss of skin-test reactivity and recall-antigen responses;

2. the release of endotoxin, tissue-degradation products, hormones, cytokines and lymphokines with immunosuppressive properties into the general circulation;

3. activation of the complement system (both classical and alternate pathways) with the production of complement-split products with immunoregulatory capabilities;

The prostaglandins and the leukotrienes are families of oxygenated fatty acids, which have been detected in virtually every mammalian tissue thus far examined. These families include some of the most potent natural substances known. Prostaglandins/leukotrienes are not stored in tissues, however, as are biogenic amines. Rather, they are formed via the activity of specific enzymes immediately prior to their release. Synthesis takes place subsequent to any cell membrane disruption, which causes the release of free fatty acids from esterified lipid sources. The precise mechanism by which the precursors of prostaglandins/leukotrienes are released is not known; however, it is agreed that they originate from the phospholipid reserves in the cell membranes. Phospholipase A is recognized as an important enzyme in this release, and recent studies with platelets have shown that at least one additional enzyme, phosphatidylinositolspecific phospholipase C, will also release membrane diacylglycerides, which can subsequently be acted upon by various Upases to produce arachidonic acid (1).

Three primary, substrate fatty acids are currently recognized as prostaglandin/ leukotriene precursors. These are cis-5,8,11,14-eicosatetraenoic acid (arachidonic acid), and two related compounds, cis-8,11,14- eicosatrienoic acid, and cis-5,8,11,14,17-eicosapentaenoic acid. Arachidonic acid is considered to be the most important of the three, so much so that the biosynthetic pathway of prostaglandin/leukotriene production is generally referred to as the arachidonic acid cascade (Figure 2.1).

Atopic or allergic diseases affect 10–20% of the population of the United States. Genetic factors play an important role in the susceptibility to these diseases, and association with specific HLA haplotypes has been demonstrated. Allergic responses are immunological reactions mediated through the activity of IgE. As early as 1921, Prausnitz and Kustner recognized that serum from allergic individuals contained a humoral sensitizing factor, which they called reagin (1). With the advances made in immunochemistry during the 1950s and 1960s came the characterization of reaginic antibody as a fifth distinct immunoglobulin class, IgE (2). While the serum of normal individuals contains only nanogram quantities of IgE, concentrations in allergic individuals can be quite significant.

A variety of cells synthesize membrane glycoproteins that can bind the Fc portion of specific immunoglobulins, and such Fc receptor interactions initiate specific cellular functions (3). Fc receptors on mast cells and basophils have a high affinity for monomeric IgE. Human basophils normally have 103–106 of these receptors per cell; however, basophils from individuals with high levels of serum IgE have an increased number of receptors (4). As shown in Figure 10.1, the interaction of IgE with surface receptors on basophils and mast cells (as well as some other nonspecific stimuli) results in the release of biologically active mediators such as histamine (5).

Single-author books are becoming somewhat of a rarity in scientific literature. Far more common are the multiauthored loosely edited works focused on a single topic. Such publications have their uses, especially in giving the reader first-hand accounts of relevant research findings. On the other hand, multiauthored works frequently fail when it comes to providing a synthesis, an overall picture of a complex field, wherein seemingly unrelated findings are placed in the “big picture”. This is an important goal for any scientific book, especially one devoted to a field as rife with seemingly contradictory data as is the area of eicosanoids and immune function. Dr. Ninnemann succeeds admirably in meeting this goal. He has managed to integrate into a readable text an enormous amount of data, results obtained in vitro and in vivo in animals and in humans.

How important are arachidonic acid metabolites in the normal immune response? We assume that cyclooxygenase metabolites are not critical for normal functioning, because both humans and animals seem to do quite well on long-term treatment with cyclooxygenase inhibitors. On the other hand, overproduction of cyclooxygenase metabolites and/or changes in sensitivity to these metabolites would appear to play a role in the disturbances in immunoregulation associated with several diseases. Less is known about the role of the lipoxygenase metabolites of arachidonic acid, but it is clear that one or more of these lipoxygenase metabolites must play an important role in normal immune function, because all the lipoxygenase inhibitors recently developed are powerful immunosuppressants.

Rheumatoid arthritis is a chronic inflammatory disease involving the joints and connective tissues throughout the body. The disease is most common in females between the ages of 30 and 40 years, and has a generally unpredictable course. The etiology of rheumatoid arthritis is still unknown; however, there is evidence that immune complexes are involved in the pathogenesis of the synovial lesions characteristic of the disease. These antigen–antibody complexes can activate complement, a process which releases chemotactic factors and attracts granulocytes. The granulocytes release lysosomal enzymes and vasoactive amines and cause inflammatory tissue destruction. Complexes consisting of cell nuclear fragments and anti-DNA antibodies also contribute to joint inflammation. Antinuclear factors are often present in the joint fluid but absent in the serum. It appears that other diseases, such as gout, spondylitis, psoriatic arthritis, colitic arthritis, and Reiter's syndrome share some of these characteristics (1).

Another hallmark of rheumatoid arthritis is the development and circulation of rheumatoid factor, which is antibody to IgG. Rheumatoid factor includes both 7S and 19S immunoglobulins with specificity to certain determinants on the IgG molecule, determinants thought to be exposed by physical aggregation of IgG or immune-complex formation (2). Rheumatoid factor can be detected in the serum of approximately 80% of patients with rheumatoid arthritis (3) and can also be seen in many nonrheumatoid diseases including viral and parasitic infections, tuberculosis, leprosy, and subacute bacterial endocarditis (4).

In his revolutionary book The Structure of Scientific Revolutions, Thomas Kuhn describes the nature of scientific progress, and how new, better answers to scientific problems become validated and accepted. Progress, he suggests, comes from the construction, and frequent revision, of models or paradigms that explain sets of information. These paradigms are the underlying assumptions of any scientific field. It is significant that Dr. Kuhn's philosophical constructions continue to attract attention and have helped organize thinking in a number of complex fields, including immunology.

It has become clear to me in the preparation of this monograph that a new immunological paradigm is emerging that relates to the participation of the products of arachidonic acid metabolism in the immune response. It is now clear that the prostaglandins (particularly PGE2), the leukotrienes (particularly LTB4), and probably the lipoxins act as primary and/or secondary mediators in a wide range of immune functions and disease states. Synthesis of this paradigm began with the observations of many individuals, then progressed to reviews on mediator involvement in specific diseases or cell types, and finally to recognition of the ubiquity of the prostaglandins and leukotrienes in normal and abnormal immune responses.

Our understanding of the exact nature and importance of the participation of these mediators in immune function is in its infancy, however, and some important basic questions have not yet been answered to everyone's satisfaction. Before any system can be manipulated to clinical advantage, it must be understood.

The products of arachidonic acid metabolism, including the prostaglandins and the leukotrienes, are surprisingly versatile compounds, which participate in an extraordinary variety of normal physiological processes, such as maintaining blood pressure and body temperature, protecting organs from damage caused by disease, traumatic injury and stress, and regulating parturition. In addition, an imbalance in these same metabolites has been implicated in shock and a wide variety of disease states including arthritis, malignancy, and allergic disorders.

In spite of their great importance, most of the lay world and a large portion of the scientific and medical communities, were not introduced to the prostaglandins until the announcement of the Nobel Prize in Medicine for 1982. Swedish chemist Sune Bergström, his colleague Bengt Samuelsson, both of the Karolinska Institute, and British pharmacologist John Vane of Wellcome Research Laboratories received Nobel recognition for their part in determining the structure and the biological role of the prostaglandins. Bergström's pioneering contribution was the discovery that prostaglandins are synthesized in vivo from dietary polyunsaturated fatty acids. Along with von Euler, Bergstrom was able to purify and further characterize the prostaglandins, which had been discovered 20 years earlier. The metabolic fate and disposition of the prostaglandins in the body were determined mainly by Samuelsson and his group. Vane demonstrated the ability of antiinflammatory substances such as aspirin to inhibit prostaglandin synthesis. Bergström, Samuelsson, and Vane also participated in the elucidation of many of the fatty acid derivatives, and the recognition that they often work as antagonistic pairs.

The role of the products of arachidonic acid metabolism in the immunological response to transplanted tissues has been very difficult to evaluate. This is primarily because a large volume of seemingly contradictory data has been generated by experiments in which cyclooxygenase and/or lipoxygenase inhibitors have been used, with quite mixed results, as an adjunct to transplant survival. For example, several investigators have reported that the presence of PGs, particularly PGE, significantly improved skin allograft survival in mice, while others have reported quite the opposite effect (1–4). In a recent paper, however, Foegh et al. have outlined an approach, which helps reconcile such opposing data (5). This is summarized in Figure 7.1. Arachidonic acid metabolites can be considered to belong to one of two groups: those products promoting tissue rejection, and those preventing it. It appears that most, if not all, of these metabolites are present in each transplant, and that survival or rejection of the tissue is dependent upon their balance and net cumulative activity. It is not surprising, therefore, that blanket pharmacological inhibition of cyclooxygenase activity might or might not be successful at prolonging transplant survival. A targeted manipulation of specific metabolites might lead to the generation of more interpretable data.

PGI2, PGE2, and PGD2 are recognized as antirejection metabolites.

The monocyte/macrophage has a variety of immunological functions including presentation of antigen to lymphocytes, production of immunologically active mediators such as interleukin 1(IL-1), and regulation of cell–cell cooperation and activity. This cell is essential for the development of almost all normal immunological responses. Activated macrophages can also act as cytotoxic effector cells that are especially critical to antimicrobial and antitumor defenses (1).

Monocytes, macrophages, and macrophagelike cells produce a number of mediators critical to immune responsiveness. For example, IL-1 is secreted by macrophages, macrophagelike cells (such as Langerhans cells of the skin), and by astrocytes, keratinocytes, fibroblasts, and natural killer (NK) cells (2). Collectively, these mediator-producing cells are called accessory cells (3). Accessory-cell IL-1 is directly involved in the activation of helper-T lymphocytes, and the stimulation of a variety of other cell types to produce “secondary mediators” with far-reaching influence. As summarized in Figure 3.1, these secondary mediators include prostaglandins and leukotrienes.

THE EFFECT OF PROSTAGLANDINS ON MACROPHAGE FUNCTION

The products of arachidonic acid metabolism are important regulators of macrophage activity and macrophage–lymphocyte cooperation. Many studies have been devoted to examining the effects of prostaglandin E on macrophages. In these studies, PGE has been shown to have a profound inhibitory effect on the release of lysosomal hydrolase in human macrophages, as well as on macrophage locomotion, shape change, and phagocytosis (4).

Inflammation is a response by which the body repairs tissue damage and defends itself against infection. It is not an immunological response in the traditional sense, since it is nonspecific and can be initiated by completely nonimmune pathways (1–3). Immunological cells and effector mechanisms, however, are commonly participants in the process. The purpose of inflammation is to deliver plasma and cellular components of the blood to extravascular tissues, causing dilution of toxic materials, increase in lymph flow, phagocytosis of damaged tissue and contaminating materials or organisms, and (in the late stages of inflammation) formation of a fibrotic barrier which walls off infection.

Inflammatory responses can result from a variety of immunological processes including a T-lymphocyte-mediated mechanism in allergic contact dermatitis, and a humoral-antibody-mediated process in pemphigus. The cells involved in acute inflammation include mast cells, neutrophils, platelets, and eosinophils, which appear to act in a predictable sequence. The inflammatory sequence is initiated by chemical mediators released from the mast cells, which are found adjacent to small arterioles and in submucosal tissues. These mediators, summarized in Figure 5.1, dilate vessels and initiate a cascade of important events, including participation of the neutrophil in the inflammatory response.

Histamine is the major preformed mast cell mediator, synthesized from the amino acid L-histidine by the action of the enzyme L-histidine decarboxylase. The biological effects of histamine are mediated through two distinct receptor systems, H1 and H2.

Arachidonic acid metabolites have been implicated as participants in every stage of the carcinogenic process, and as a probable cause for the lack of an effective antitumor immune response in many cancer patients. The realization that PGs might be involved in the suppression of a tumordirected immune response had its origin in the work of several groups concerned with the mechanism of cancer-associated hypercalcemia. As early as 1970, experiments by Klein and Raisz demonstrated that, along with parathyroid hormone, PGE2 was a particularly strong stimulator of bone resorption (1). Using a murine model, Tashjian et al. showed that the secretion of PGE2 by certain nonmetastatic cancers was associated with severe hypercalcemia (2). The association of PGE2 production and hypercalcemia has been reconfirmed in humans on many occasions (3). Largely because of this work, it became clear that many naturally occurring and experimentally induced malignant tumors synthesized large quantities of PGs. High concentrations of PGE and/or PGE metabolites have been found in the serum of patients with Hodgkin's and non- Hodgkin's lymphomas (Figure 6.1) (4), breast cancer (5), squamous cell carcinoma of the lung (6), renal cell carcinoma, and a large variety of other malignancies (6,7). In other studies, elevated blood levels of 6-keto- PGF1α, a degradation product of PGI2, have been found to occur in patients with various cancers, but its source has not been identified.

Process development work involves two main areas.

1. (a) The discovery and isolation of microorganisms producing new antibiotics, as a result of screening programmes, and the development of efficient fermentation processes for the production of material for testing. This usually involves the production of improved mutants and media. The object is to provide material for testing, in the laboratory and clinically. To make larger quantities of material, pilot plant fermenters are often used.

2. (b) Development associated with manufacture; this may be simple at first, but of increasing sophistication as yields increase. Such work is usually done in research units at the factory.

The various classes of work to be done are: (1) the planning of fermentation experiments and the gathering of data, (2) the organisation and interpretation of data, strain improvement, and selection, and (3) development of processes that give maximised results, materially and economically, using techniques that become more critical as yields increase. These subjects form the bases of the chapters in this part of the book.

While academic work tends to be speculative, as it is intended to explore and expand scientific knowledge, industrial research may seem rather narrowly based. Industrial research requires very reliable experimental apparatus and results. For this reason equipment of a high standard is customary. In a recent article, the equipment for a pilot laboratory was quoted as costing £1.5 million, illustrating the degree of elaboration involved in this type of work. Such work is demanding and also very interesting in its own right for those who like to see a new discovery brought into use as an aid to humankind.

Introduction

At the end of a fermentation experiment, a set of results will be obtained. This may consist of antibiotic titres and some pH values, or, in a large-scale culture trial, there may be many results covering antibiotic production, growth, and other factors. An immediate requirement is to check the general appearance and behaviour of the culture to establish its normality and reliability. The analytical data can then be collected and examined.

As a rule, antibiotic production is a key indicator, while other factors express the manner of growth and metabolism. From these data it is possible to obtain a picture of how the fermentation works, and to obtain ideas for its future development.

It is possible to visualise the fermentation mechanism as like a wire sculpture, in which wires rise from the base, intertwine, and form a network tower. Here, the wires represent the reactions or material flows described in Chapters 3 and 4. One of these wires might represent growth, others might represent respiration and the use of nutrients and the behaviour of other reactions, but the most important one would represent antibiotic synthesis, which should ascend to the maximum possible height.

If the sculpture was based on theory, there would be many wires; in reality only a few reactions are monitored, so there would be only a few wires. These would represent factors that could be measured continuously, like pH, feed, carbon dioxide, and oxygen. But with growth and antibiotic production, measured at intervals, only beads would appear representing these spot values. In plant work, the sculpture might consist only of these beads.