Abstract
Conjugation of various reagents to antibodies has long been an elegant way to combine the superior binding features of the antibody with other desired but non-natural functions. Applications range from labels for detection in different analytical assays to the creation of new drugs by conjugation to molecules which improves the pharmaceutical effect. In many of these applications, it has been proven advantageous to control both the site and the stoichiometry of the conjugation to achieve a homogeneous product with predictable, and often also improved, characteristics. For this purpose, many research groups have, during the latest decade, reported novel methods and techniques, based on small molecules, peptides, and proteins with inherent affinity for the antibody, for site-specific conjugation of antibodies. This review provides a comprehensive overview of these methods and their applications and also describes a historical perspective of the field.
Highlights
Antibodies have a long-standing reputation as excellent tools in many medical and biological applications because of their capacity to selectively bind to specific target molecules with high affinity
Further improving the cross-linking abilities of the Z domain, Hui et al reported up to 80% cross-linking of mouse IgG3 heavy chains by introducing BPA at position 17 in the protein domain and 47% conjugation efficiency for human IgG1 with BPA at position 35.27 For this particular labeling domain, BPA was introduced in the backbone of the Z domain using the technique described in section 2, where an orthogonal tRNA/aminoacyl tRNA synthetase pair incorporates the noncanonical amino acids (ncAAs) in response to the amber stop codon.[48]
DeLano et al isolated two peptides, FcI and FcII, from a phage library of 4 × 109 different disulfide-induced cyclic peptides of the form XiCXjCXk. These peptides were further matured by construction of new libraries that resulted in the 13-mer peptide FcIII (DCAWHLGELVWCT), binding to the fragment crystallizable (Fc) region with a strength of approximately half of Protein A and G, which both have equilibrium binding constants (KD) around 10 nM
Summary
Antibodies have a long-standing reputation as excellent tools in many medical and biological applications because of their capacity to selectively bind to specific target molecules with high affinity. By introducing a lysine residue in position 8 of this peptide and thereafter cross-linking it to the nearby Lys[248] of an IgG-Fc through the cross-linker DSG, this peptide has been used to prepare both an ADC by conjugation of the maytansine derivative DM1 and a bispecific antibody by conjugation to a nanobody.[62] This same peptide, together with other previously known peptides such as the original FcIII and the minimized Z domain described above, forms the basis of the AJICAP technology, described by Yamada et al This technology is based on the functionalization of native antibodies with thiol groups through the use of these affinity reagents, which allows for further conjugation of cytotoxic payloads.[63] The same group later describes the use of the AJICAP technology for gram-scale synthesis of stable and homogeneous ADCs.[64] Taken together, these examples show how nonmodified antibodies can be efficiently site- conjugated by utilizing a peptide as small as 13 amino acids. Since the peptide is so small, it can be produced by a chemical synthesis approach, which in turn entails a simple way of introducing many other non-natural functional groups
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