Molecular engineering and design of therapeutic antibodies

Since the first murine monoclonal antibody was approved for human therapeutic use over a decade ago, the realization that monoclonal antibody therapeutics could be engineered to improve their efficacy has inspired an astonishing array of novel antibody constructs. Early focus was on reducing the immunogenicity of rodent antibodies via humanization and generation of antibodies in transgenic mice; as those techniques were being established and then provided marketed therapeutic antibodies, the focus expanded to include engineering for enhanced effector functions, control of half-life, tumor and tissue accessibility, augmented biophysical characteristics such as stability, and more efficient (and less costly) production. Over the past two years significant progress in designing antibodies with improved pharmacokinetic properties, via modified interaction with the neonatal Fc receptor (FcRn), has been achieved. Likewise, the ability to alter the communication of a therapeutic antibody with the immune system has been advanced, using both manipulation of the immunoglobulin protein sequence and its glycosylation. Although clinical evaluation of these engineered modifications has yet to be reported, results in primates are encouraging.

Introduction

Owing to the increasingly diverse and specialized categories of antibody engineering that reflect the success of monoclonal antibodies (mAbs) as therapeutics over the past two decades, assembling a broad review on antibody engineering has become more difficult. Engineering rodent antibodies to be acceptable human therapeutics via humanization of variable domains has blossomed into engineering of the constant domains for enhanced effector function, a profusion of antibody formats (e.g. diabodies, minibodies, IgG fusion proteins, bispecific antibodies, intrabodies), new selection technologies (e.g. phage-, ribosome-, yeast-display), new production systems (e.g. mammalian cell, bacterial, yeast, plant), and new methods for increasing stability and aggregation resistance. This review will primarily focus on important developments from 2006 to 2008 in the areas of Fc engineering and selection technologies.

The Fc portion of an antibody (see Figure 1), composed of the hinge and constant domains (CH2 and CH3), performs two basic functions. First, it communicates with the immune system once the antibody has bound its target, which is especially important for cell-bound targets. Communication occurs through effector functions: antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). The first two are mediated by interaction of the Fc with specific receptors, FcγR, expressed on a variety of immune system cells such as natural killer cells, monocytes, neutrophils, and macrophages. By contrast, CDC is mediated through interaction of the Fc with a series of blood proteins that constitute the complement system, including C1q. A second function of the Fc is interaction with the neonatal Fc receptor (FcRn) expressed on a variety of cells; this interaction controls the half-life of immunoglobulins.

For some therapeutic antibodies and Fc-fusion proteins, enhancement of the effector functions may amend their therapeutic efficacy; abrogation of effector function has to some extent already been supplied by nature in the form of human IgG2 and IgG4 that exhibit decreased susceptibility to ADCC and CDC—though improvements on these could also be achieved. Similarly, for some therapeutic antibodies being able to increase or decrease the ‘normal’ IgG half-life could enhance their therapeutic efficacy. This review covers changes in the protein sequence of immunoglobulins and their affect on effector functions; another method of altering effector functions, glycosylation engineering of antibodies, will be covered in a companion review in this issue by T Shantha Raju.

The section on selection will focus on novel ways to utilize these types of technologies to generate antibodies with biophysical characteristics that lend themselves to improved production or that simplify the amino acid repertoire of the antibody complementarity-determining regions (CDRs; the loops in the antibody variable domains that interact with the target molecule).