Site-Specific Photoconjugation of Antibodies Using Chemically Synthesized IgG-Binding Domains

Abstract

Site-specific labeling of antibodies can be performed using the immunoglobulin-binding Z domain, derived from staphylococcal protein A (SpA), which has a well-characterized binding site in the Fc region of antibodies. By introducing a photoactivable probe in the Z domain, a covalent bond can be formed between the Z domain and the antibody by irradiation with UV light. The aim of this study was to improve the conjugation yield for labeling of different subclasses of IgG having different sequence composition, using a photoactivated Z domain variant. Four different variants of the Z domain (Z5BPA, Z5BBA, Z32BPA, and Z32BBA) were synthesized to investigate the influence of the position of the photoactivable probe and the presence of a flexible linker between the probe and the protein. For two of the variants, the photoreactive benzophenone group was introduced as part of an amino acid side chain by incorporation of the unnatural amino acid benzoylphenylalanine (BPA) during peptide synthesis. For the other two variants, the photoreactive benzophenone group was attached via a flexible linker by coupling of benzoylbenzoic acid (BBA) to the ε-amino group of a selectively deprotected lysine residue. Photoconjugation experiments using human IgG1, mouse IgG1, and mouse IgG2A demonstrated efficient conjugation for all antibodies. It was shown that differences in linker length had a large impact on the conjugation efficiency for labeling of mouse IgG1, whereas the positioning of the photoactivable probe in the sequence of the protein had a larger effect for mouse IgG2A. Conjugation to human IgG1 was only to a minor extent affected by position or linker length. For each subclass of antibody, the best variant tested using a standard conjugation protocol resulted in conjugation efficiencies of 41-66%, which corresponds to on average approximately one Z domain attached to each antibody. As a combination of the two best performing variants, Z5BBA and Z32BPA, a Z domain variant with two photoactivable probes (Z5BBA32BPA) was also synthesized with the aim of targeting a wider panel of antibody subclasses and species. This new reagent could efficiently couple to all antibody subclasses that were targeted by the single benzophenone-labeled Z domain variants, with conjugation efficiencies of 26-41%.

In vivo biotinylation and incorporation of a photo‐inducible unnatural amino acid to an antibody‐binding domain improve site‐specific labeling of antibodies

“…The aim of this project was to develop an IgG binding domain with improved crosslinking abilities compared to previously published domains [9][10][11][12].…”

“…Even though this type of labeling is straightforward and commonly used, the drawback is that the level, One solution to this problem is site specific labeling of antibodies using immunoglobulin (IgG) binding domains such as the B domain of Staphylococcal protein A [6,7] or the C2 domain of Streptococcal protein G [8] that all have affinity for the fragment crystallizable (Fc) and/or fragment antigen binding (Fab) portions of the antibody. Previous work by Jung et al [9], Konrad et al Accepted Article Biotechnology Journal [10], Yu et al [11] and Perols et al [12], have all demonstrated covalent labeling of antibodies using an approach where the photo inducible benzophenone group have been incorporated near the binding site of the small IgG binding domain.…”

“…The benzophenone group has been incorporated either in the backbone of the protein as the unnatural amino acid p-benzoylphenylalanine (BPA) [13] using Solid Phase Peptide Synthesis [10,12] or coupled to a maleimide group reacting with a cysteine [9,11]. Benzophenone can form a covalent bond to nearby amino acids upon UV irradiation around 350 nm [14].…”

Antibody Conjugates: From Heterogeneous Populations to Defined Reagents

“…A possible strategy to covalently link an FcBD to an antibody is to equip the domain with a photoactivable probe, e.g., benzophenone that, upon irradiation, is activated and forms a covalent bond to a closely located amino acid on the immunoglobulin surface ( Figure 7C). This methodology has been successfully applied by using various FcBDs including different variants of the monomeric Z-domain [154][155][156][157] and a minimal domain of protein G [158]. …”

LASIC: Light Activated Site-Specific Conjugation of Native IgGs

“…The yields of expression for all variants were high at around 5 mg/L, consistent with previous reports of BPA incorporation into proteins. 8,18 Next, we screened these variants for their ability to covalently label a range of IgG subclasses from various hosts upon exposure to long wavelength UV light (Supplemental Figure S2 A–C). Since each IgG is composed of two identical heavy chains, it can be labeled with up to two Protein G-based adapters, which can be deciphered using non-reducing SDS-PAGE.…”