Monoclonal antibodies as therapeutics in oncology

The specificity of antibodies has been harnessed to target cancer cells and the first therapeutic antibodies for use in oncology are now finding application in the clinic. Studies are currently under way to develop new and improved antibodies. Recent developments have been made in the identification of novel targets, including the use of genomic and proteomic technologies. Several methods are also being developed to enhance antibody efficacy.     

A Method for the Parallel and Sequential Generation of Single-Domain Antibodies for the Proteomic Analysis of Human Blood Plasma

A new efficient method for the parallel and sequential stepwise generation of single-domain antibodies to various high-abundance human-plasma proteins has been described. Single-domain antibodies have a number of features that favorably distinguish them from classical antibodies. In particular, they are able to recognize unusual unique conformational epitopes of native target proteins, small in size, and relatively easily produced and modified; have enhanced stability; and rapidly renature after denaturation. As a consequence, the immunosorbents that utilize these antibodies can be reused without any significant loss of activity. The principal novelty and universality of the described method is that it enables the sequential generation of antibodies to a number of high-abundance and yet unknown antigens of a complex protein mixture without the need for purified antigens. The effectiveness of the method is demonstrated by the example of generation of single-domain antibodies to a number of high-abundance proteins of the human blood plasma. The produced antibodies are promising biotechnological tools that can be used to develop prototypes for new diagnostic and therapeutic agents, as well as appropriate immunoaffinity-based methods for removal, enrichment, analysis, and/or targeting of specified proteins and their complexes from (in) the human blood. As we show, the generated single-domain antibodies can be efficiently used in designing new immunosorbents. As a rule, commercially available analogous immunosorbents that utilize classical antibodies remove many major proteins from the blood plasma immediately, while immunosorbents for many individual proteins are difficult to find and rather expensive. Single-domain antibodies generated by our method are unique new materials that allow for the development of more efficient and delicate approaches to pretreatment of plasma and the analysis of various blood plasma biomarkers.     

Monoclonal antibodies: methods and clinical laboratory applications

Kohler and Milstein have shown that individual clones of normal antibody-secreting lymphocytes could be immortalized by fusion with myeloma cells. These investigators initiated a new era of technology with the successful in vitro production of monoclonal antibodies via somatic cell hybridization. With the use of monoclonal antibodies, many major problems arising from the limited specificity and reproducibility of conventional antisera can be solved. Some of the commonly employed methods for the production of monoclonal antibody are: (1) fusion of sensitized lymphocytes and myelomas from different sources to produce continuous antibody-producing cell lines; (2) in vitro viral transformation of sensitized lymphocytes to form continuous antibody-producing cells; (3) hybrid fusion of sensitized lymphocytes and continuous B lymphocyte cell lines. During the past few years, monoclonal antibody methodology has been used in almost every area of biological research. Monoclonal antibodies have been used as structural probes for proteins and hormones, and as highly specific agents for histocompatibility testing, tumor localization, immunotherapy, purification of molecules, identification of new surface antigens on lymphocytes and tumor cells, and detection of drug levels and microbial and parasitic diseases. In addition, several investigators have developed alternative methods for the production of human as well as mouse and rat monoclonal antibodies. The new technology of in vitro production of animal and human monoclonal antibodies will have many future applications in diagnosis and therapy in laboratory and clinical medicine.     

Colorectal carcinoma antigens detected by hybridoma antibodies.

Hybridoma cells which secrete colorectal carcinoma-specific antibodies have been produced and used to study the antigenic structure of these tumor cells. Nineteen antibodies have been studied in detail, and 15 of these are colorectal carcinoma specific. Only two antibodies reactive with carcinoembryonic antigen (CEA) have been discovered and five other antibodies that react with distinct epitopes on the cell surface have been defined. Several antigens with distinct molecular characteristics have been shown to exist by use of hybridoma antibodies. Six hybridoma antibodies have been shown to mediate antibody-dependent cell-mediated cytotoxicity (ADCC).     

Preparation of monoclonal antibodies against glycoprotein IIIa of human platelets. Their effect on platelet aggregation

Monoclonal antibodies against purified glycoprotein IIIa (GPIIIa) of human platelet membranes have been obtained. These antibodies, except one, are able to bind to intact platelets; the exception is M108/p98 antibody which recognizes a new epitope, unmasked after proteolysis of GPIIIa in vitro. Several antigenic areas can be delineated on the molecule, by testing the ability of different antibodies to compete in their simultaneous binding to GPIIIa. One of the monoclonal antibodies inhibits ADP-induced platelet aggregation while others do not have an effect or induce agglutination of platelets independent of ADP. Conventional antiserum raised against purified GPIIIa also blocks the aggregation induced by ADP. These results favour the hypothesis that GPIIIa plays a direct role in the mechanism of platelet aggregation.     

Sowing the seeds of success: pharmaceutical proteins from plants

Among the many plant-based production systems that have been developed for pharmaceutical proteins, seeds have the useful advantage of accumulating proteins in a relatively small volume and in a stable environment in which they are protected from degradation. Several seed crops, including cereals, grain legumes and oilseeds, have been explored as production platforms, and the first commercial products -- all technical proteins and enzymes -- have already reached the market. Recent studies have explored the use of seeds for the production of pharmaceutical proteins, particularly replacement human proteins, recombinant antibodies and (oral) vaccines.     

The fluorescent probe Bodipy-FL-verapamil is a substrate for both P-glycoprotein and multidrug resistance-related protein (MRP)-1.

Several fluorescent probes have been used in functional studies to analyze drug transport in multidrug-resistant cells by fluorescent microscopy. Because many of these molecules have some drawbacks, such as toxicity, nonspecific background, or accumulation in mitochondria, new fluorescent compounds have been proposed as more useful tools. Among these substances, Bodipy-FL-Verapamil, a fluorescent conjugate of the drug efflux blocker verapamil, has been used to study P-glycoprotein activity in different cell types. In this study we tested by fluorescent microscopy the accumulation of Bodipy-FL-Verapamil in cell lines that overexpress either P-glycoprotein (P-gp) or multidrug resistance-related protein 1 (MRP1). Expression of P-gp and MRP1 was evaluated at the mRNA level by RT-PCR technique and at the protein level by flow cytometric analysis using C219 and MRP-m6 monoclonal antibodies. Results indicate that Bodipy-FL-Verapamil is actually a substrate for both proteins. As a consequence, any conclusion about P-gp activity obtained by the use of Bodipy-FL-Verapamil as fluorescent tracer should be interpreted with caution.     

Intracellular antibodies as specific reagents for functional ablation: future therapeutic molecules

The use of antibodies in medicine and research depends on their specificity and affinity in the recogniton and binding of individual molecules. However, these applications are limited to the extracellular targets. Advances in antibody engineering has allowed the manipulation of the antibody segments containing the antigen-binding regions and generation of small fragments that can be stably expressed in cells. These entities are called intracellular antibodies or intrabodies and have being successfully applied, mainly in the scFv format, to inhibit the function of intracellular target proteins in specific cellular compartments. As new techniques to select and isolate intrabody fragments have been developed, intrabodies are beginning to be used to interfere with the function of a greater number of relevant disease targets. Just as monoclonal antibodies are opening a new era in human therapeutics, intrabodies promise a new prospective for antibody tools for therapy and research. Their varied mode of action gives intrabodies great potential in different approaches in the treatment of human diseases, as well as in the area of functional genomics for characterisation of novel gene products and subsequent validation as potential drug targets. While techniques for identifying functional intrabodies have improved, there are still many significant problems to be overcome before intrabodies can actually be used in treatment of diseases such as cancer, AIDS or neuro-degenerative disorders.     

Beyond natural antibodies: the power of in vitro display technologies

In vitro display technologies, best exemplified by phage and yeast display, were first described for the selection of antibodies some 20 years ago. Since then, many antibodies have been selected and improved upon using these methods. Although it is not widely recognized, many of the antibodies derived using in vitro display methods have properties that would be extremely difficult, if not impossible, to obtain by immunizing animals. The first antibodies derived using in vitro display methods are now in the clinic, with many more waiting in the wings. Unlike immunization, in vitro display permits the use of defined selection conditions and provides immediate availability of the sequence encoding the antibody. The amenability of in vitro display to high-throughput applications broadens the prospects for their wider use in basic and applied research.     

Epidermal growth factor receptor: a promising therapeutic target for colorectal cancer

The epidermal growth factor receptor is a 170,000-kd transmembrane glycoprotein involved in signaling pathways affecting cellular growth, differentiation, and proliferation. An abnormal expression of the epidermal growth factor receptor (EGFR) has been described in many human tumors and implicated in the development and prognosis of malignancies, thus representing not only a possible prognostic marker, but primarily a rational molecular target for a new class of anticancer agents. The aim of this analysis is to review the available data about the biology of the EGFR and its use as a target for a new class of anticancer agents for colorectal cancer. Several clinical trials have been reported with the use of EGFR-targeted monoclonal antibodies and tyrosine kinase inhibitors, mainly in combination with chemotherapy for advanced colorectal cancer patients. Results available so far demonstrated a manageable and acceptable toxicity profile and a promising level of activity. Many critical issues are yet unresolved, such as the optimal chemotherapy regimen to combine with anti-EGFR treatment and the most adequate patient setting. Moreover, the biological selection of colorectal tumors more likely to benefit from this treatment approach is still to be defined.     

Humoral immunity to HIV-1: neutralisation and antibody effector functions

Several features of HIV have frustrated efforts to develop a vaccine able to induce broadly neutralising antibodies. The enormous genetic diversity of HIV is a major factor, accompanied by the camouflaged nature of the envelope spike, upon which HIV depends for cellular entry and to which antibodies must bind to neutralise. The picture is further complicated by the presence of nonfunctional envelope glycoproteins on the surface of HIV that are immunogenic. Consequently, HIV attracts antibodies that do not directly neutralise the virus but still activate complement and engage Fc receptors, which can both enhance and inhibit infection. The various effects that anti-envelope antibodies have on HIV infection will be reviewed here. Further research is needed to determine if these in vitro-characterised activities have relevance in vivo, and if some of the undesirable effects of non-neutralising antibodies can be avoided or the beneficial effects harnessed.     

Antibodies,viruses and vaccines

Neutralizing antibodies are crucial for vaccine-mediated protection against viral diseases. They probably act, in most cases, by blunting the infection, which is then resolved by cellular immunity. The protective effects of neutralizing antibodies can be achieved not only by neutralization of free virus particles, but also by several activities directed against infected cells. In certain instances, non-neutralizing antibodies contribute to protection. Several viruses, such as HIV, have evolved mechanisms to evade neutralizing-antibody responses, and these viruses present special challenges for vaccine design that are now being tackled.     

Principles of antibody therapy.

The success of monoclonal antibodies in clinical practice is dependent on good design. Finding a suitable target is the most important part as other properties of the antibody can be altered by genetic engineering. Antibodies that target lymphocyte antigens offer less toxic immunosuppressive treatment than currently available drugs and the first monoclonal antibody approved for human use is an immunosuppressive agent for treating rejection of renal transplants. Human trails of monoclonal antibodies to treat septic shock have been done and antibodies are also being developed to target common pathogens such as herpes simplex virus. Although monoclonal antibodies against cancer have been much heralded, their success has been limited by the poor access to the inside of tumours. Treatment of blood cancers has been more successful and a human antibody against B cell malignancies is being clinically tested. As knowledge about natural immune responses and antibody engineering increases many more monoclonals are likely to feature in clinical practice.     

Next-Generation Techniques for Discovering Human Monoclonal Antibodies

Monoclonal antibodies have found wide applications in the treatment of cancer, as well as of autoimmune, infectious, and other diseases. Several dozen new antibodies are currently undergoing different stages of clinical trials, and some of them will soon be added to the list of immunotherapeutic drugs. Most of these antibodies have been generated using hybridoma technology or a phage display. In recent years, new methods of obtaining human monoclonal antibodies have been actively developing. These methods rely on sequencing immunoglobulin genes from B lymphocytes, as well as on the creation of antibody-secreting stable B-cell lines. The term next-generation antibody-discovery platforms has already been established in the literature to refer to these approaches. Our review focuses on describing the results obtained by these methods.     

Antibody-Based Resistance to Plant Pathogens

Plant diseases are a major threat to the world food supply, as up to 15% of production is lost to pathogens. In the past, disease control and the generation of resistant plant lines protected against viral, bacterial or fungal pathogens, was achieved using conventional breeding based on crossings, mutant screenings and backcrossing. Many approaches in this field have failed or the resistance obtained has been rapidly broken by the pathogens. Recent advances in molecular biotechnology have made it possible to obtain and to modify genes that are useful for generating disease resistant crops. Several strategies, including expression of pathogen-derived sequences or anti-pathogenic agents, have been developed to engineer improved pathogen resistance in transgenic plants. Antibody-based resistance is a novel strategy for generating transgenic plants resistant to pathogens. Decades ago it was shown that polyclonal and monoclonal antibodies can neutralize viruses, bacteria and selected fungi. This approach has been improved recently by the development of recombinant antibodies (rAbs). Crop resistance can be engineered by the expression of pathogen-specific antibodies, antibody fragments or antibody fusion proteins. The advantages of this approach are that rAbs can be engineered against almost any target molecule, and it has been demonstrated that expression of functional pathogen-specific rAbs in plants confers effective pathogen protection. The efficacy of antibody-based resistance was first shown for plant viruses and its application to other plant pathogens is becoming more established. However, successful use of antibodies to generate plant pathogen resistance relies on appropriate target selection, careful antibody design, efficient antibody expression, stability and targeting to appropriate cellular compartments.     

Right on target: eradicating leukemic stem cells

Less than a third of adults with acute myeloid leukemia (AML) are cured by current treatments, emphasizing the need for new approaches to therapy. The discovery over a decade ago that myeloid leukemias originate from rare stem-like cells that can transfer the disease to immunodeficient mice suggested that these 'leukemia stem cells' (LSCs) are responsible for relapse of leukemia following conventional or targeted cancer therapy and that eradication of LSCs might be necessary to cure the disease permanently. Several recent studies have provided insight into the signaling pathways underlying the LSC phenotype and have also described approaches to eliminate LSCs with antibodies. Here, we review recent advances in LSC research and discuss novel therapeutic strategies to specifically target LSCs.     

Cationization, a process for the delivery of antibodies to the central nervous system. Problems encountered in its application for immunotherapy strategies such as those for clostridial poisoning

The central nervous system is separated from the rest of the body by the blood-brain barrier. This barrier prevents many substances, such as the antibodies, to penetrate into the brain making it difficult to use them for the treatment of brain diseases, such as tetanus and botulism. These two diseases are caused by the development of bacilli of the genus Clostridium which release neurotropic toxins. Specific antibodies can neutralize toxin activity when the toxin is in the blood but are ineffective when it is transported into nerve cells. Various invasive strategies have been used to deliver antibodies to the brain. However, they can induce seizures and transient neurologic deficits and may be applicable only for diseases restricted to the brain surface. Physiologically based strategies utilizing transport systems naturally present at the blood-brain barrier appear to be a more promising approach to brain delivery of antibodies. Cationization is a chemical treatment that causes the conversion of superficial carboxyl groups on a protein into extended primary amino groups. This is used to increase interactions of this protein with the negative charges at the luminal plasma membrane of the brain endothelial cells. The cationized protein can then undergo adsorptive mediated transcytosis through the blood-brain barrier. There are many problems yet to be solved in successfully carrying out in vivo applications of cationized antibodies. One of these problems is that cationization can cause damage to an antibody molecule and, thus, can compromise its binding affinity. Depending on the radiolabelling of the cationized antibodies, a serum inhibition phenomenon can possibly alter the pharmacokinetics and the organ distribution of these molecules. The antibodies can be cationized using various, synthetic (hexamethylenediamine) or naturally occuring (e.g., putrescine) polyamines. Hexamethylenediamine-induced and putrescine-induced brain uptakes of various antibodies and proteins have been shown, but the results obtained suggest that cationization with putrescine may be a more efficient approach to blood-brain barrier delivery. The development of animal or cellular models to check for therapeutic efficacy of cationized antibodies is necessary. In spite of the difficulties, the studies described in this paper indicate that cationization can be a realistic delivery strategy for carrying antibodies across the blood-brain barrier. The advances made in antibody technologies help generate more appropriate immunological structures for brain transfer with better effector functions and decreased immunogenicity or toxicity. Taken together, these two aspects can lead to further developments in treatment of intoxications caused by the clostridial neurotoxins.     

Detection of catalytic monoclonal antibodies.

Several laboratories have now shown that monoclonal antibodies having enzyme-like properties can be generated. The generation of catalytic antibodies makes use of the same basic procedures that have been used for the generation of binding monoclonal antibodies, yet the process involves an additional crucial step: screening for catalytic activity. In this paper we address the unique problems involved in the detection of inefficient catalytic activity that is accompanied by uncatalyzed background reaction. An analysis that allows optimization of assay conditions and estimation of the minimal antibody concentration required to observe catalysis is presented. The results indicate that the structure of the substrate should be optimized to increase its affinity (i.e., decrease its Km) and reduce its concentration to pseudo-first-order conditions (S(O) much less than Km) so that the signal observed in the presence of a catalytic antibody (delta Pcat) is significantly higher than that of the background (delta P(uncat)). Other factors involved in the screening procedures, e.g., sensitivity of the assay, solubility and reactivity of the substrate, and purity of the antibody preparation, are also discussed. The effect of these assay parameters on the ability to detect catalytic activity is demonstrated with p-nitrophenyl ester-hydrolyzing antibodies.     

Investigating the Endogenous Component of Human Circadian Rhythms: A Review of Some Simple Alternatives to Constant Routines

Several types of constant routine are accepted as an important means by which the endogenous component of circadian rhythms can be studied. Nevertheless, they are impracticable to perform and unsuitable for routine use in many individuals. We describe a group of simple methods with which rhythms measured in normal circumstances can be dissociated into the components due to masking and the internal clock. Each method is best suited to a particular type of experimental condition. Results from a variety of protocols are analysed by these and conventional methods to assess the validity of the new methods.