Page 14 - Tyrosine-Based Bioconjugations - Jorick Bruins
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Chapter 1
in two 25 kDa fragments (light chains) and two 50 kDa fragments (heavy chains).12 This is done primarily for analytical purposes, as it can simplify both quantitative and qualitative analysis by SDS-PAGE, HPLC and LC-MS, as well as generate handles for conjugation strategies. Enzymatic digestion can be done by a variety of methods, most of which is done for analysis of antibody purity and integrity.12 Whereas chemical cleavage relies on the reduction of disulfide bridges, enzymatic cleavage relies on the hydrolysis of an amino acid sequence-specific peptide bond. These include, but are not limited to, the enzymes IdeS, papain, pepsin, and trypsin (Figure 1B).12, 13 The unique sites of cleavage by these enzymes, called proteases, allows for analysis of different fragments of the antibody, enabling rapid and detailed analysis of the structure and modifications of the antibody.
Antibody fragments can be generated genetically, by expressing only certain monomeric parts of the antibody and, optionally, fusing multiple fragments together. The generation of antibody fragments via genetic modification is mainly done for pharmaceutical applications, and extensive reviews of these antibody fragments and their functions have been reported.14, 15 A well-known example of antibody fragments is the single chain variable fragment (scFv), where the VH and VL are expressed fused together via the N-terminus of one to the C-terminus of the other via a non- specificpeptidesequenceconsistingof15–20aminoacids(Figure2B).16 ScFvsretainthebinding exhibited by their parent antibodies, but lack interaction with the Fc receptors. Bare scFvs have many uses, such as preparation of immunotoxins, therapeutic gene delivery, and as anticancer intrabodies.16 Furthermore, scFvs can also be linked to an antibody to present a second, unique antigen-binding site via protein fusion techniques, creating so-called bispecific antibodies.15, 17 The advantage of binding multiple different targets are, amongst others, to increase binding affinity,18 improve internalization of antibody-based drugs,19, 20 or redirect cytotoxic T-cells for increased immune response near the target cell.21, 22
1.1.2. Bispecific antibodies
As mentioned above, bispecific antibodies can be made in a variety of ways, such as expressing two scFvs fused in tandem (Figure 2C) or fusing scFvs to the termini of antibodies (Figure 2D).17 The latter would result in a bispecific antibody with two binding sites for each epitope, i.e. a [2]:2 format.i Another way to obtain bispecific antibodies is by using asymmetrical antibodies with two distinct antigen-binding sites. An example of this are the knob-in-hole antibodies; by changing only a few key amino acids in the CH3 domain that are pivotal to the pairing of the heavy chains, i.e. replacing a small side-chain with a large side-chain on one heavy chain (knob) and vice versa for the other heavy chain (hole), heterodimers with different epitopes or other mutations be promoted.17, 23 This method would result in a bispecific antibody with a single
i Regarding bi- and trispecific antibody notation: Numbers represent the quantity of a particular binding site or functionality, the number(s) in square brackets are the antibody’s binding sites. Numbers after the square brackets are functionalities fused and/or conjugated to the light or heavy chains.
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