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Molecular Constructor on the Basis of Barnase–Barstar Module

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Molecular Constructor on the Basis of Barnase–Barstar Module ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2009, Vol. 35, No. 6, pp. 685–701. © Pleiades Publishing, Ltd., 2009. Original Russian Text © S.M. Deev, E.N. Lebedenko, 2009, published in Bioorganicheskaya Khimiya, 2009, Vol. 35, No. 6, pp. 761–778. Antibody Engineering: Molecular Constructor on the basis of Barnase–Barstar Module S. M. Deev1 and E. N. Lebedenko Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia Received May 19, 2009; in final form, May 29, 2009 Abstract—Today, antibody engineering for clinical applications is a rapidly progressing field of science and a big business. The basic functions of an antibody can be spatially differentiated and attributed to various struc- tural domains of a molecule. Therefore, each of them may be an object for engineering with the aim of using a definite antibody function. In this sense, the potential of antibodies is unique. In this article, recent achieve- ments and current problems of antibody engineering are briefly reviewed. The main attention is focused on a molecular constructor that allows for obtaining, with the help of a versatile barnase–barstar module, mono- and multivalent miniantibodies and their derivatives with outlined properties. Key words: single chain antibodies, multivalency, bispecificity, targeted delivery, barnase–barstar module DOI: 10.1134/S1068162009060041 INTRODUCTION mAbs), active or diagnostic agents, for instance, cytok- ines, protein toxins and radioisotopes, enzymes, fluo- Currently, antibodies have the second largest pro- rescent proteins, etc. duction value in the pharmaceutical market after vac- cines. More than 85% of antibodies permitted for clin- The current review will focus on a molecular con- ical use are products of antibody engineering. They structor developed in our laboratory that allows for the include chimeric, humanized and fully human mono- production of mono- and multivalent miniantibodies clonal antibodies (mAbs), phage-display derived anti- and their derivatives with previously defined properties bodies, and recombinant conjugates of antibodies with using a multipurpose barnase–barstar module. cytokines and toxins. Genetic and cell engineering techniques have allowed for the implementation of the unique potential of immunoglobulins that have a mod- 1. STRUCTURAL DOMAINS OF ANTIBODIES ular structure and a set of functions connected with sep- AND THEIR FUNCTIONS arate structural modules, modifying them for very dif- ferent clinical applications. Hundreds of antibody Natural antibodies or immunoglobulins (Ig) are derivatives are currently in clinical trials, including components of the immune system intended to identify recombinant antibodies of artificial formats: bispecific and remove xenogenic antigens. They are complex pro- antibodies; single-chain full-sized antibodies; different teins with an evident domain structure and a number of variants of shortened antibodies, specifically, dimers different functions. Figure 1 shows the structure of and monomers of Fab fragments; scFv fragments (sin- immunoglobulin by the example of IgG, which makes gle-chain miniantibodies); one-domain antibodies up more than 65% of the total amount of antibodies in (nanoantibodies); and others. Meanwhile, the develop- human blood serum. The great diversity of antibodies ment of one specific and highly effective antibody for (the theoretically available number of combinations is clinical use remains a complex and difficult task. Sub- 109–1011) lies in the mosaic structure of genes of light ject to given application, it is necessary to vary the anti- and heavy chains and emerges due to DNA rearrange- body’s size, its specificity, affinity, valency, to decrease ments and subsequent somatic mutations during the the immunogenicity, and to optimize the pharmacoki- process of the maturation of immune cells [1]. We netics and effector properties. In addition, to increase defined more precisely a detailed mechanism of gene treatment efficiency, recombinant antibodies may be fit rearrangement in the κ locus of human immunoglobu- up with antibodies of another specificity (bispecific lin light chains [2] that has been confirmed in leading 1 Corresponding author; phone: +7 (495) 429-8810; e-mail: world laboratories and appears to be universally recog- [email protected]. nized [3, 4]. 685 686 1 Fv V V V V L H H L Killer cell Fab × 3.0 6.5 nm C C 1C1 C Apoptos L H H L d 6.0 nm c RUSSIAN JOURNAL OFBIOORGANICCHEMISTRY JOURNAL RUSSIAN Target cell Hinge 2 åÄC DEEV, LEBEDENKO DEEV, C C1q H 2 2 C H 3 Killer cell Fc 3 C Activation 6.5 × 6.0 nm C H H 3 of killer cell Inflammation dc 5.8 nm reactions 4 Carbohydrate component of antibody FcγR Antibody specific to receptors of target cell FcRn Surface receptors Lisosome of target cell pH < 6.0 FcRn Products of complement Vol. 35 Vol. pH 7.5 reaction cascade Fig. 1. Structural units of antibodies and their functions as objects of engineering. Structural units: Fv – variable fragment consisting of variable domains of light (V ) and heavy No. 6 L (VH) chains; in every variable domain, three variable sites responsible for the complementary interaction with an antigen—CDRs (complementary determined regions)—are marked with black; Fab – antigen binding fragment of IgG; and Fc – ë-terminal part of IgG consisting of constant domains ëç2 and ëç3 of heavy chains. Functions: (1) bivalent binding with a target, for example, with pathogenic cell surface antigens; (2) binding with FcγR on the surface of immune system cells (leucocytes) and the activation of antibody- 2009 dependent cell cytotoxicity; (3) binding with protein complex C1q and the activation of the complement system cascade of reactions that lead to the formation of a membrane attack complex (MAC) and lysis of the target cell (mechanism of complement-dependent cytotoxicity); and (4) binding with neonatal receptor Fc (FcRn) and the recirculation of anti- bodies from lysosomes, and also the transport of IgG through epithelial and endothelial barriers, specifically, the transition of immunity from mother to child. ANTIBODY ENGINEERING: MOLECULAR CONSTRUCTOR 687 The structural domains of immunoglobulin are not Thus, the antibodies that are produced by an organ- only separated genetically and spatially, but also per- ism during an immune reaction may include several form different functions in the immune response pro- cellular mechanisms of xenogenic antigen removal cess (Fig. 1). An individual antibody possesses a unique simultaneously and a whole cascade of biochemical –8 –11 specificity and high affinity to its antigen (äd 10 –10 M) reactions aimed at performing this. The major functions due to the complementary nature of the antigen-binding of an antibody may be spatially differentiated and site of the antibody to a definite fragment of the antigen attributed to individual molecule domains. Therefore, molecule (epitope) (Fig. 1, function 1). Each antigen- every domain may be an object for bioengineering with the aim of using (increasing or decreasing) a definite binding site is formed by two variable domains, VH and immunoglobulin function (Fig. 2). At this point, the VL, that belong to the heavy and light chains of immu- noglobulin, respectively. The exceptions are only some bioengineering potential of immunoglobulin molecules kinds of antibodies in the Camelidae family and sharks, is absolutely unique. Today, antibody engineering for which do not have a light chain, and only the V clinical use is an emerging field of science and a big H business (see review [6]). In the last decade, a signifi- domain is responsible for antigen binding [5]. cant breakthrough in the study of the constant part and The constant part of immunoglobulin consists of carbohydrate component of immunoglobulins has been performed; new cell technologies for producing full- one domain in light chains (CL) and three or four domains (it depends on the class of antibody) for heavy sized humanized and human antibodies with a defined type of glycosylation have been created [7–13]. What is chains (ëH). Hydrophobic sites between CH1 and CH2 domains maintain their comparative mobility, forming our niche in the field of antibody engineering and what a hinge area that provides a shift of Fabs (antigen bind- is its uniqueness? ing fragments) and their rotation around the hinge. The ability of antibodies to bind two antigens simulta- 2. ANTIBODY ENGINEERING: neously is defined by their complex spatial structure, ACHIEVEMENTS AND PROBLEMS which is stabilized by disulfide bonds, and the existence of hinges that alleviate the precise adjustment when two The hybridome technology applied by Köhler and antigens are bound simultaneously (Fig. 1, function 1). Milstein in 1975 [75] initiated the emergence of current The bivalency of natural immunoglobulins substan- antibody engineering. The hybridoma cell line, which tially increases their functional affinity (avidity) and is obtained by the fusion of immune lymphocytes syn- binding time on surface cell receptors and other polyva- thesizing antibodies of a desired specificity and cells of lent antigens. an immortal myeloma line, secretes monoclonal anti- body (mAb), antibody of one type specific to antigen The constant part of IgG includes sites for binding a used for immunization. The subculturing of hybridoma compliment system of protein C1q and cell receptors of γ in vitro or its cultivation as mice ascite allows for the Fc fragments (Fc R) that mediate the effectors’ (sec- obtainment of a constant source of antibodies
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