The antigen-binding entity of an antibody, reduced in size to one single domain, is referred to as a “single-domain antibody.” Various strategies have been explored with variable success to arrive at functional single-domain antibodies. The potential of single-domain antibodies, as research tools or in medicine, is reflected by the three companies – founded in Europe – with a mission to bring these molecules to the market. Domantis using human VH-derived single-domain antibodies started in 2000 and was bought by GSK for £300M in December 2007. Haptogen employing shark single-domain antibodies was acquired by Wyeth, and Ablynx focusing on llama-derived single-domain antibodies received over €70M in three rounds of venture capitalist investments and another €80M on the Euronext stock market in November 2007. Regarding therapeutic applications, Arana Therapeutics in Australia entered a Phase 2 clinical trial with its single-domain antibody derivative. In this chapter, we will review (1) the various antibodies used for generating single-domain antibodies, (2) the properties of single-domain antibodies that create an added value for use in immunotherapy, and (3) a number of therapeutic applications.

THE DEVELOPMENT OF SINGLE-DOMAIN ANTIBODIES

Antibodies comprise two identical heavy chain polypeptides (H) carrying chains of carbohydrates and two identical light chain proteins (L). Their ability to bind specifically to an antigen is dictated by the paired variable regions of the heavy (VH) and the light (VL) chain (Figure 16.1).

During evolution, antibodies have acquired several invaluable properties that are now being exploited for clinical applications. First, they can bind a wide variety of target molecules with exquisite specificity. This property can be used to block the action of ligands such as TNFα in patients with rheumatoid arthritis or the Her-2 receptor in patients with breast cancer. In contrast to this mode of action, antibodies can also imitate ligand binding and stimulate various signaling pathways. Antibodies binding to CD20, for example, can induce apoptotic signals in the malignant cells of patients with non-Hodgkin's lymphoma. Additional effector functions are provided by the Fc domains, which can induce cell lysis by binding to complement (CDC) or by binding to Fc receptors on natural killer cells and macrophages (ADCC). An additional binding domain for the neonatal receptor on endothelial cells facilitates their uptake and recycling, enabling antibody therapeutics to remain in the circulation for many weeks.

To optimize the properties of an antibody for a particular indication or for use as a diagnostic, it would be preferable to improve or even delete particular characteristics. For example, to achieve better tumor penetration or a better tumor-to-blood ratio for visualizing metastases, it would be preferable to have a relatively small antibody fragment with a fairly short half-life. On the other hand, the antibody should not be too small in order to avoid a rapid clearance immediately after its application. It would also be very advantageous for certain clinical applications to improve the effector functions.

The immune system creates binding sites of high specificity and affinity in the variable domains of antibodies by generating sequence and consequently structural diversity in the complementarity-determining region (CDR) loops, which are located at the N-terminal ends of these domains. Sequence variations in the CDR loops of an antibody generally do not have a significant influence on the overall structure of the variable domain that carries them. This feature of variable domains is actually observed in a more general sense in immunoglobulin-like domains, which are known to have a similar general shape in the core beta-barrel and high structural variability in the loops. Furthermore, overall sequence similarity of domains with an immunoglobulin fold is mainly below 25%, while their structural similarity is high, with a root mean square (rms) deviation of Cα atoms always below 3.9 Å (Halaby et al., 1999).

We therefore set out to explore whether this inherent stability and conservation of the immunoglobulin fold allows loops of immunoglobulin domains other than the CDR loops to accommodate sequence variation without negatively impacting the overall structure and stability of the protein. As shown in Figure 17.1, the candidate loops of an IgG1 for this kind of engineering are manifold, including the N- and C-terminal loops of the constant domains as well as C-terminal loops of the variable domains.

In the examples described in this chapter, we engineered the AB and the EF loops of the third constant domain (CH3) of human IgG1 by randomizing a number of residues and also by inserting random sequences in the loops, thereby generating new binding sites.

Since its reemergence following the discovery of monoclonal antibodies in the early 1980s, the field of antibody therapy in cancer has progressed in leaps and bounds. From murine to chimeric, through humanized to fully human, we are now in a situation where, with over 200 antibodies having passed through some kind of clinical testing (Reichert & Valge-Archer, 2007), the monoclonal is now an accepted form of treatment for malignancy. In fact, for some malignancies, most notably non-Hodgkin's lymphoma, monoclonals are routinely used as frontline therapy. As such, we are past the point of asking whether monoclonal therapy works and into the more expansive territory of asking how it works and how we can make it work better.

While antibodies can function to combat a tumor in a number of ways – for example, sequestration of factors essential to survival or growth and stimulation of the immune response – one of the best-studied mechanisms of action is direct tumor cell killing. Here we will begin by looking in detail at the mechanisms by which antibodies can mediate cell killing, and which of these mechanisms is likely to be most important. Subsequently, we will review briefly the possible ways that this cell killing can be increased through the process of protein engineering, several of which will be expanded upon by the authors of subsequent chapters.

With over 20 therapeutic antibodies currently approved by the Food and Drug Administration (FDA) and close to 100 leads in clinical trials, therapeutic antibodies are responsible for a considerable part of the therapeutic proteins sales worldwide. The observation of immunogenicity with the early therapeutic antibodies did not come as a surprise, as many of them were murine antibodies or chimeric variants, consisting of murine variable parts in conjunction with a human constant domain. Over time, there has been a strong evolution toward the development of humanized and fully human antibodies, thereby reducing the observed immunogenicity to a significant extent. General side effects such as anaphylaxis and allergy against protein therapeutics are also less prevalent but this is due to better manufacturing processes giving more homogeneous products.

However, some of the currently available fully human antibodies have induced significant immunogenic responses over time. This has led to the regulatory instances in Europe and the United States supporting the development of guidelines to assess the likelihood of observing immunogenicity and its potential severity and side effects.

IMMUNOGENICITY DRIVERS

Several factors contribute to the potential immunogenicity of a protein therapeutic:

• Homology to human or endogenous proteins: The degree of “foreignness” of a protein to the host is one of the major contributors to an immune response. Indeed, the likelihood to observe immunogenicity related to a bacterium-derived protein therapeutic, such as staphylokinase, is higher than against proteins that show high homology to endogenous proteins, such as erythropoietin (EPO) and insulin. The overall immunogenicity of antibody therapeutics has been severely reduced by the development of fully human and humanized therapeutic antibodies as compared to the first-generation murine and chimeric antibodies (Hwang and Foote, 2005).

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