Date Published: April , 2009
Publisher: A.I. Gordeyev
Author(s): S. M. Deyev, E. N. Lebedenko.
The modular structure and versatility of antibodies enables one to modify natural immunoglobulins in different ways for various clinical applications. Rational design and molecular engineering make it possible to directionally modify the molecular size, affinity, specificity, and immunogenicity and effector functions of an antibody, as well as to combine them with other functional agents. This review focuses on up-to-date methods of antibody engineering for diagnosing and treating various diseases, particularly on new technologies meant to refine the effector functions of therapeutic antibodies.
A century ago, antibodies were regarded a “magic bullet” that would selectively strike disease hotbeds in the human organism (see Paul Ehrlich’s Nobel Lecture of 1908 ). However, Paul Ehrlich’s idea was put into practice only in 1975, when Köhler and Milstein’s study initiated the development of monoclonal antibody technology. This technology makes it possible to produce not just a set of diverse immunoglobulin molecules (natural antibodies), but also a monospecific antibody focused on one specific antigen (monoclonal antibody, Mab) in response to antigen-driven immunization. This method is still the cornerstone of antibody reshaping. Unfortunately, the first attempts to use mouse Mab for clinical purposes were not successful and revealed the following, virtually insurmountable disadvantages of Mab: in some cases, its antibody affinity is lower than that of polyclonal antiserum; it has a high immunogenicity to humans and, as a consequence, is rapidly eliminated from the body; and it is unable to activate the complement system and cellular mechanisms of the immune response in a foreign environment. Nevertheless, after three decades of battles and defeats, hopes and PR blitzes, Mab proved to be a successful medicinal product from both the clinical and commercial standpoints (Table 1). The unique potential of immunoglobulins characterized by modular structures and functions related to other structural modules was realized, and the antibodies were modified for variable clinical applications thanks to the technologies of genetic engineering and transgenic animals. Depending on the practical task, researchers can directionally modify the molecular size, specificity, affinity, and valency; they can decrease immunogenicity and refine pharmacokinetic properties and effector functions. Moreover, antibodies are obtained as recombinant-fused proteins which include other specific antibodies, cytokines, protein toxins, radioisotopes, ferments, and fluorescent proteins. Currently, about 30 antibody medicines are approved for clinical application, 89% of which are used in treating oncological and immunological diseases. antibodies are also used in treating cardiovascular, autoimmune, and infectious diseases (Table 2). On the pharmaceutical market, antibodies come in second after vaccines in production volume. By 2011, the sales volume of antibody medicines is predicted to increase to $21 billion (Table 1). More than 85% of antibodies approved for clinical application are products of antibody reshaping. The approved antibodies include chimeric, humanized, and human Mab; antibodies obtained using phage display; and genetically engineered antibody conjugates with cytokines and toxins. Hundreds of antibody derivatives are still subject to clinical testing, including synthetic antibodies produced by gene engineering: bispecific antibodies; single-chain full-sized antibodies; different variants of truncated antibodies, including dimers and monomers of Fab fragments, scFv-fragments (single-chain mini-antibodies), single-domain antibodies (nanoantibodies), etc. Different technologies that make it possible to modify immunoglobulin molecules for certain clinical purposes are considered. This review is focused on antibody reshaping for the treatment and detection of oncological diseases, because this sphere is in particular need of these medicines.
Antibodies, also known as immunoglobulins (IgG), are high-avid molecules which can detect and eliminate foreign antigens from the human organism. Most mammal antibodies have similar structures and are bivalent multidomain proteins with two antigen-binding sites (see, for example, human G immunoglobulin (IgG), Fig. 1). This compound Y-shaped molecular complex consists of two identical heavy (~50 kDa) and two identical light (~25 kDa) chains. Four inter-domain disulfide bonds provide stable conditions for the whole molecular complex. Globular structural immunoglobulin domains with a characteristic β-folded structure are stabilized by intradomain disulfide bonds and are subdivided into variable (V) and constant (C) domains. N-tail domains of light and heavy chains (VL and VH variable domains) vary significantly for different antibodies, while the remaining part of the polypeptide chains (CL and CH constant domains) varies for different antibody classes (and within one antibody class, depending on the species). The structural domains of immunoglobulin are isolated in space and perform different functions in the immune response process.
Originally, the most attractive property of antibodies in terms of the “magic bullet” construction was their specific binding with the antigen, which was assumed to allow the delivery of an active agent (for instance, a radioactive isotope) to a target molecule. The governing factors which would provide the efficiency of this delivery are the affinity and specificity of the antibody targeted to the antigen, as well as its physicochemical properties, such as valency, surface charge, and size [8, 9].
a wide range of theoretical and experimental data testifies to the fact that the simplest method to overcome the imperfection of the recombinant single-chain Fv-fragments is a return to multivalency, which is a primary characteristic property of antibodies. Indeed, every effort was spent to construct multimeric formats of truncated antibodies in order to optimize their pharmacokinetic properties and to improve the biodistribution of the active agents delivered by antibodies. Currently, a wide variety of such constructions is subject to clinical testing [28, 29].
One of the first problems arising in the course of creating antibodies for clinical purposes is the high immunogenicity of Mab, which is xenogenic for a human being. The human anti-mouse antibodies response (HaMa) causes serious systematic reactions in the human organism up to allergic shock (Table 2). Using gene engineering methods allowed the partial replacement of the mouse Mab immunogenic sites with the corresponding fragments of human antibodies.
Antibodies bound to the surface of the target cell can cause its death by triggering the ADCC and CDC mechanisms (see above) (Fig. 2) . Far from all clinical applications of Mab find these mechanisms useful. For instance, the Mab effector functions are not required for the directed delivery of radioimmunoisotopes or blocking transplant rejection; moreover, they can cause dangerous complications. In other cases, the action of Mab may be intensified with the aid of killer cells. One such method was creating bispecific Mab, which attracted cytotoxic T-lymphocytes to the target cells (see Section 6). Moreover, in recent years researchers have detected a wide range of immunocyte receptors responsible for the interaction with antibodies and determining the role of IgG sites in this interaction. Recent investigations have showed that the efficiency of a therapeutic antibody depends on its affinity to FcyRIIIa (CD16) and FcyRIIa (CD32), which is characteristic for the activating receptors of most killer cells. It became apparent that the reaction of a patient to treatment by Mab medicines depends on his phenotype. Patients with FcyRIIIa-158 V/V and FcyRIIa-131 H/H phenotypes are at an advantage when being treated for follicular and non-Hodgkin lymphoma with Rituxan© (Abciximab) . analogous results were obtained when breast cancer was treated by Mab Herceptin© (Trastuzumab) . On the other hand, a therapeutic antibody can bind to a FcR receptor on non-toxic cells (thrombocytes and B-cells), which are able to inhibit the activation of effector killer cells (for instance, the FcyRIIb receptor on macrophages). Experiments on animals show that the FcyRII inhibitor isoform decreases the therapeutic effect of humanized Herceptin© (Trastuzumab) . The presented data testify to the fact that, at the current stage of gene engineering, it is essential to optimize the effector functions of the antibody depending on the sphere of application in order to increase the efficiency of the therapeutic treatment.
Natural antibodies are monospecific, i.e., they bind to antigens of only one species. antibody engineering technologies allow bispecific antibodies (Fig. 3, 2, 4, 5, 8) with a specificity to two different antigens to be created [29, 69]. The first bispecific antibodies were produced by fusing two hybridome cell lines. The quadroma created yields a random mixture of a target-oriented bispecific antibody and primary antibodies. Then, the required variant is obtained in the course of a complicated separation. a range of approaches was offered to increase the production of bispecific molecules, for instance, the creation of an interspecific mouse quadrome in the course of pairing the light and heavy chains of one species  and delicate “knob-and-holes” technology . Two mutants were injected into the Mab CH3-domains: “the knob” for replacing the small amino-acid residue (threonine) with a bulky amino-acid residue (tyrosine) and “the hole,” for reverse replacement.
The goal of achieving a “magic bullet” and the low efficiency of “unloaded” antibodies relative to the targets induced researchers to conjugate antibodies with other effector molecules (Fig. 3): radioisotopes, toxins, interleukins, and ferments activating medical products. In these immunoconjugates, antibodies usually act as a targeting component which delivers an active (cytotoxic) or diagnostic agent to the target. Chemical conjugation methods are commonly used for the conjugation of low-molecular agents, for instance, radioisotopes or low-molecular fluorescent dyes [11, 87]. Cell-penetrating peptides (CCP), capable of penetrating the membrane and transferring other proteins inside a cell, are conjugated to antibodies in the tumor by gene engineering in order to increase the penetration degree of radioimmunoconjugates. The most efficient cell-penetrating peptides are penetratine, which represents oligopeptide (43-58 a.r.) from the homeodomain of the drosophila antp protein, and TaT, which is an oligopeptide (49-57 a.r.) from the transactivator of HIV transcription. Mini-antibody radioimmunoconjugates, which are specific to the TaG72 tumor antigen and provided with penetratine or TaT oligopeptide, are accumulated 2.5-3 times better in the tumors of xenographic mice .
Today, producing stable and high-affinity Mab in a sufficient amount for pre-clinical and clinical investigations is a “bottleneck” for the wider application of these therapeutic compounds. The rapid growth in demand for antibodies and their quality has led to the development of numerous systems for producing antibodies and their fragments on the basis of Gram-positive (Bacillus) and Gram-negative (Escherichia) bacteria, ferments, filamentous fungi, and the cell lines of insects and mammals [118-120]. High-technological systems such as bacterial and ferment producers, which favor the overgrowth of biomass in fermenters and the production of highly efficient recombinant proteins, can solve the problem of creating truncated Mab fragments, non-glycosylated Fab-fragments, and scFv provided, as a rule, with special tail peptides for fast activation on affine sorbent. Currently, antibodies are created in transgenic plants and animals which are appropriate for the production of full-size antibodies [72, 121]. The expression system is created on the surface of bacterial cells (so called “E-clonal” antibodies) used for both full-size and divalent formats  for the fast production and selection of full-size antibodies. The glycosylation of antibodies in ferments, plants, and insects differs from that in a human being: therefore, antibodies obtained in those systems are applicable only for experimental investigations. Today, therapeutic antibodies are produced in transgenic mice, whose immunoglobulic locuses are inactivated and replaced with the genes of human immunoglobulins [10, 123]. The most well-known mice transgenic lines are as follows: Xenomouse (abgenix, http://www.abgenix.com), HuMab mouse (GenPharm, http://www.genpharm.ca), and TC mouse and KM mouse (Kirin Brewery Company, http://www.medarex.com). Medicines with Mab created in these producers are characterized by low immunogenicity due to xenogenic post-translation modifications and, primarily, to glycosylation specifics. Moreover, the researchers are developing new technologies which would allow the production of Mab with a human profile of glycosylation in ferments, insects, and transgenic plants [124, 125].
In the early 21st century, one hundred years after Ilya Mechnikov and Paul Ehrlich, the founders of the current immunity theory (1908), received the Nobel Prize, knowledge about the delicate molecular mechanisms of antibody functioning and interaction with the organism’s protective systems has made the greatest step forward. This progress is provided by the innovative technologies used in the course of scientific investigations and the accumulation and systematization of large amounts of information. The quick development of bioinformatics allows us to model the compounds with preprogrammed properties, while revolutionary progress in genetic and cell engineering technologies makes it possible to create biotechnological producers of therapeutic medicines.
S.M. Deyev’s work at the antibody Engineering Laboratory was supported by the Russian Foundation for Basic Research (project no. 09-04-01201-a), the Federal Target Program for the Research and Development of Priority Directions of Scientific and Technological Complex in Russia in 2007-2012, and the “Molecular and Cell Biology” and “Fundamental Research of Nanotechnologies and Nanomaterials” programs of the Russian academy of Sciences Presidium.