ABSTRACT
Antibodies possess many desired properties for validating protein function, and early evidence indicates that antibodies can be used to block the function of intracellular targets by injecting them into individual cells [1]. Antibodies, however, cannot fold correctly or form stable structures within the reducing environment of cells [2], and they are unable to penetrate the cell membrane, which hinders their ability to
CONTENTS
19.1 Introduction .................................................................................................. 521 19.2 Intrabody Stability ........................................................................................ 523
19.2.1 Intradomain Stability of Variable Domains ..................................... 523 19.2.2 Framework Mutations to Enhance Intradomain Stability ................ 524 19.2.3 Inuence of CDR Loops on Variable Domain Stability ................... 525 19.2.4 Engineering Disulde Bonds to Enhance Variable Domain
Stability ............................................................................................. 527 19.2.5 Stabilization of VL Domains ............................................................. 527 19.2.6 VH/VL Interface Stability .................................................................. 528 19.2.7 Inuence of Variable Domain Charge on Stability .......................... 529
19.3 Selection Methods for Isolating Intrabodies ................................................. 529 19.3.1 In Vitro Stability and Afnity Selection Using Phage Display ........ 530 19.3.2 Selection of Functional Intrabodies within Cells ............................. 530
19.4 Applications .................................................................................................. 534 19.5 Summary ...................................................................................................... 536 References .............................................................................................................. 536
inhibit intracellular targets. Further, their large size and tetrameric structure limit their use as intracellular afnity reagents [2] and have driven the development of smaller antibody fragments (called “intrabodies”) for intracellular applications (Figure 19.1). Intrabodies are designed to retain the antigen afnity of antibodies without the effector function normally associated with the antibody constant region, such as antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity. The single-chain variable fragment (scFv) and the variable heavy (VH) domain are the most commonly used intrabodies. The scFv consists of a VH domain covalently linked to a variable light (VL) domain via a short peptide linker, generally consisting of glycine-serine repeats. ScFvs have advantages over smaller variable domain fragments as the binding afnity/specicity of full-length immunoglobulins (IgGs) and antigen-binding fragments (Fabs) can be easily engineered into the scFv format. Many scFvs have been developed in recent years as reagents for studying and inhibiting cancer and other diseases, but only a relatively small number have been reported for intracellular applications [3,4].
