Background

= = = = =Antibody Structure=

The following sections describe the physical and chemical properties of conventional antibodies and associated therapeutic proteins, as well as camelid heavy chain antibodies.

Conventional Antibody Structure
The human immune system structurally modifies antibodies thereby increasing their affinity for unique antigens. Consequently, antibodies are diverse proteins with variable amino acid sequences. 7 Despite this variability, antibodies exhibit several common structural characteristics. An antibody consists of four protein chains: two identical light chains and two identical heavy chains connected by disulfide bonds [Figure 3]. Each chain is subdivided into two regions: a constant domain of low amino acid variability, as well as a variable domain that displays greater diversity. The light chains consist of a single variable (VL) and constant domain (CL), while the heavy chains contain a variable domain (VH) and three constant domains (CH1, CH2 and CH3) [Figure 4]. Three hypervariable complementary determining regions (CDR1, CDR2, CDR3) comprised of 10-12 amino acids, exist within the variable region of each chain. 7 These sequences are the most variable region of a protein chain. The hypervariable segments form hydrophilic amino acid loops which interact with antigen binding sites (epitopes). The structure and binding specificity of the loop is determined by the properties of the amino acid residues. The amino acid sequences that separate the complementary determining regions display diminished variability and are termed the framework regions (FR). 7

Antibodies contain two identical antigen binding sites (paratopes) formed by pairing of the light and heavy protein chains. Each conventional paratope consists of six hypervariable loops; three light chain CDRs and three heavy chain CDRs. 5 The coupled heavy and light variable domains form a complex structure that interacts with concave or flat surficial antigen sites. 16 Epitopes recessed within molecular clefts are unrecognized by human antibodies. Exploitation of these sites by engineered antibodies displaying greater antigen affinity and stability is desirable for therapeutic applications, however many of the molecular principles that contribute to epitope recognition remain poorly understood. 5



Conventional Antibody Fragments
Variable domain antibody fragments that exhibit antigen binding specificity and enhanced tissue penetration capabilities have been isolated as therapeutic candidates. Proteins of interest include antigen binding fragments (Fab), single chain variable fragments (scFv) and single heavy variable domains of conventional antibodies (VH). 16 Smaller proteins, such as variable domains, may be synthesized in vectors whereas conventional antibody production is restricted to mammalian cells. The former cell cultures considerably reduce production expenses. The synthesis may be complicated by mispairing of light and heavy variable domains. 9 The efficiency of isolated antibody fragments is also limited by the tendency for protein aggregation and poor target retention time. In addition to these limitations, mediocre drug candidate performances have stimulated efforts to develop superior proteins.



Heavy Chain Antibodies
In camelids, antigens induce the formation of heavy chain antibodies, which deviate from the structural characteristics of conventional antibodies. The primary disparity is the absence of light protein chains. 9,16 As a result, amino acid residues associated with hydrophobic interaction between the VH and VL domains in conventional antibodies are unnecessary. In heavy chain antibodies, several hydrophobic residues are substituted with hydrophilic amino acids. This prevents aggregation of camelid antibodies in polar solutions. Another important feature of camelid antibodies is the elongation of the CDR3 amino acid sequence. Human sequences contain approximately 12 amino acid residues whereas 16-18 amino acids are frequent in heavy chain antibodies (hAds). 16 This enables heavy chain antibodies to interact with recessed or cryptic epitopes inaccessible to conventional antibodies. Further dissimilarities include absence of the CH1 domain and greater variability of the CDR1 sequence. Incorporation of these attributes in Nanobodies offers several advantages over conventional antibody fragments.

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