Multivalent antibodies: when design surpasses evolution

Evolutionary pressure has selected antibodies as key immune molecules acting against foreign pathogens. The development of monoclonal antibody technology has allowed their widespread use in research, real-time diagnosis and treatment of multiple diseases, including cancer. However, compared with hematologic malignancies, solid tumors have often proven to be relatively resistant to antibody-based therapies. In an attempt to improve the tumor-targeting efficacy of antibodies, new formats with modified, multivalent properties have been generated. Initially, these formats imitated the structure of native IgG, creating mostly monospecific, bivalent antibodies. Recently, novel trivalent antibodies have been developed to maximize tumor targeting capabilities through enhanced biodistribution and functional affinity. We review recent advances in the engineering of multivalent antibodies and further discuss their promise as agents for invivo diagnostics and therapy.

Introduction

Antibodies are found in blood and other body fluids and are used by the immune system to identify and neutralize foreign pathogens, such as bacteria and viruses. The immune system has evolved unique genetic mechanisms that enable it to generate an almost unlimited number of antibody specificities. Antibodies act as molecular connectors, linking specific recognition of epitopes on their targets with nonspecific effector mechanisms, including antibody-dependent, cell-mediated cytotoxicity, complement-dependent cytotoxicity and antibody-dependent, cell-mediated phagocytosis. Intact antibody molecules are heavy (∼150 kDa), globular glycoproteins. The basic unit of each antibody is a monomer [one immunoglobulin G (IgG) unit] composed of two identical light chains (L) and two identical heavy chains (H). The four chains are connected to one another by a combination of noncovalent and covalent (disulfide) bonds, which forms a Y-shaped molecule with the antigen-binding sites located at the tip of the two ‘arms’. Each IgG is thus bivalent and able to attach to two identical antigenic sites, which increases antibody functional affinity and confers high retention times. The recruitment of effector functions is mediated by the Fc domain. Also, owing to molecular weights beyond the renal threshold limit of 70 kDa, as well as interaction with the neonatal Fc receptor (FcRn) – a major histocompatibility complex class I-related molecule known to protect IgG and albumin from intracellular catabolic degradation following pinocytic capture [1], IgG molecules show slow clearance from the circulation, thus explaining the long half-life of this antibody class in serum [2].

The advent of monoclonal antibody (mAb) technology has made it possible to raise antibodies against specific antigens, including tumor-associated antigens. MAbs are unique, versatile molecules that have found applications in research, diagnosis and in the treatment of multiple diseases, including cancer [3]. Currently, there are 26 mAbs approved by the US Food and Drug Administration (FDA) for therapeutic and diagnostic use. However, solid tumors have often proven to be relatively resistant to IgG or intact antibody-based therapies. Additionally, it has been shown that only a small amount of intravenously administered mAbs accumulates in the tumor tissue [4].

In an attempt to improve tumor penetration (see Glossary), recombinant antibodies with modified properties have been generated. Novel antibody formats, such as the single-chain Fv (scFv; 25–30 kDa), exhibit better pharmacokinetics than intact IgGs for tissue penetration, and enable binding specificity to be encoded by a single gene [2]. However, scFv antibody fragments exhibit rapid blood clearance as well as fast off-rates and poor retention times on the target due to their small size and their monovalent binding properties [2]. A wide range of strategies has therefore been developed to improve the pharmacokinetic properties of antibody fragments [5]. The covalent conjugation of small recombinant antibody molecules with poly(ethylene glycol) (PEG) – also known as ‘PEGylation’ – is a well-established procedure to improve serum half-life by increasing the molecular mass and the hydrodynamic radius [6]. Another approach is the genetic fusion of antibody fragments to albumin or albumin-binding proteins. This strategy is based on the observation that albumin has a half-life similar to that of IgG and also utilizes FcRn-mediated recycling processes [5]. Alternatively, the use of bispecific antibodies to recruit serum IgG provides the Fc-associated effector functions and prolongs residence time in serum. Multimerization is a particularly appealing approach to optimize antibody size for the desired pharmacokinetics, and to maximize tumor uptake by enhancing the functional affinity (avidity) of antibody fragments. Here, we review recent progress in the design of multimeric antibodies, and suggest that trivalent antibodies display the best combination of size and avidity for cancer diagnostics and therapy.