From Whole Monoclonal Antibody to Single Domain Antibody (sdAb)
From Mouse Antibody to Human Antibody
The development of therapeutic monoclonal antibodies over the past 35 years has led to the emergence of a new class of useful therapeutic molecules, and mostly whole IgG molecules have been developed for therapeutic use in humans so far. In the early 1980s, the vast majority of mAbs were of rodent origin. However, when mAbs of murine origin were infused to patients, this became a significant concern quickly as the detection of human anti-mouse antibodies (HAMAs) with high titers, which lower or block the therapeutic efficacy of mAbs and provoke side effects.
Therefore, cellular engineering and powerful molecular engineering techniques are used to humanize antibodies, which can reduce the immunogenicity of the murine mAbs by grafting complementarity determining regions (CDRs) derived from murine antibodies with the desired specificity onto carefully chosen human frameworks (FRs). The powerful technique for generating fully human antibodies is the phage display technology that allows the rapid selection of specific antibodies and is primarily used to format a whole recombinant human mAb. Furthermore, humanized mice can mount an antigen-specific human antibody response following immunization, and human antigen-specific mAbs can be generated. About a part of the mAbs currently in clinical development is human antibodies derived either from phage display libraries or from transgenic mice.
Many Formats of Antibody Fragments
IgG can be cleaved into three well-defined fragments: two antigen-binding fragments (Fabs) and one fragment crystallizable (Fc). Although both Fab and Fc fragments exert critical biological functions, mostly antigen-binding fragments have been developed for therapeutic purposes. Moreover, single-chain variable fragment (scFv) is made of one heavy chain variable domain (VH) linked through a flexible spacer (usually a repeated motif of 3 × GGGGS) to one light chain variable domain (VL).
Furthermore, antibodies composed of heavy-chain dimers and devoid of light chains were found in the serum of camelids, which exhibiting extensive antigen-binding repertoires. These lead to the cloning and engineering of single domain antibodies with remarkable biophysical properties (dAbs or sdAbs) by phage display. Their unique characteristics make them particularly attractive as useful diagnostic and/or therapeutic tools for research diagnosis, as well as clinical use. Studies of such single domain antibodies provide insight into the design of recombinant antibody fragments.
Fig. 1 Various antibody formats.
Antibody fragments of varied structures and lacking an Fc domain, are examples of how antibody-based biotherapeutic agents are expanding in class and scope. These antibody fragments are especially amenable to genetic manipulations or covalent modifications aimed at improving their affinity, stability, and function. Due to their specific structural, physiological, and pharmacological properties, new forms of antibody fragments continue to emerge and offer new biopharmaceutical opportunities. The structures and characteristics of these different antibody fragments are listed in the table below.
Table.1 Features of different antibody fragments.
| Antibody | Molecular Weight | Structural Feature | CDR3 Length | Disulfide Bond |
|---|---|---|---|---|
| sdAb | 15 kDa | Only VH, VHH or VNAR | 7-24 |
FR1~FR3 CDR1/FR1~CDR3 (some germlines) |
| scFv | 28 kDa | Connection with VH and VL | 7-12 | FR1~FR3 |
| Fab | 55 kDa | Complete light chain, VH and CH1 domain of heavy chain | ||
| IgG | 160 kDa | Complete light chains and heavy chains |
Case Study
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