Facile method for the site-specific, covalent attachment of full-length IgG onto nanoparticles

Abstract

Antibodies, most commonly IgGs, have been widely used as targeting ligands in research and therapeutic applications due to their wide array of targets, high specificity and proven efficacy. Many of these applications require antibodies to be conjugated onto surfaces (e.g. nanoparticles and microplates); however, most conventional bioconjugation techniques exhibit low crosslinking efficiencies, reduced functionality due to non-site-specific labeling and random surface orientation, and/or require protein engineering (e.g. cysteine handles), which can be technically challenging. To overcome these limitations, we have recombinantly expressed Protein Z, which binds the Fc region of IgG, with an UV active non-natural amino acid benzoylphenyalanine (BPA) within its binding domain. Upon exposure to long wavelength UV light, the BPA is activated and forms a covalent link between the Protein Z and the bound Fc region of IgG. This technology was combined with expressed protein ligation (EPL), which allowed for the introduction of a fluorophore and click chemistry-compatible azide group onto the C-terminus of Protein Z during the recombinant protein purification step. This enabled the crosslinked-Protein Z-IgG complexes to be efficiently and site-specifically attached to aza-dibenzocyclooctyne-modified nanoparticles, via copper-free click chemistry.

Keywords: antibody; click chemistry; conjugation; nanoparticle; site-specific.

Figure 1. Schematic describing the production and surface conjugation of Protein Z-IgG complexes

(A) A fusion protein containing an unnatural amino acid benzoylphenylalanine (BPA) in the Protein Z domain is expressed in frame with an intein and a chitin binding domain. During the affinity purification process, the intein is used to drive expressed protein ligation between the Protein Z and an azido fluorescent peptide (AzFP) containing an N-terminal cysteine, a “clickable” azide group and a 5-FAM fluorophore. (B) After the protein Z-AzFP conjugate is mixed with IgG, long-wavelength UV irradiation is used to create a site-specific covalent bond between the BPA and Fc region of IgG. (C) The crosslinked IgG, now containing both a fluorophore and an azide moiety, can be used for site-specific conjugation with any substrate containing a free alkyne via click chemistry. A strained alkyne, aza-dibenzocyclooctyne (ADIBO), capable of copper-free click reactions is shown.

Figure 2. SDS-PAGE with Coomassie staining confirming the in vivo incorporation of BPA into expressed Protein Z

T7 competent E. coli were co-transformed with the pEVOL-pBpf plasmid containing the amber suppressor tRNA/aminoacyl transferase pair and the pTXB1 plasmid, which codes for Protein Z with an amber codon mutation (ProZ F13BPA). Following induction of protein expression, cell lysates, with or without BPA in the media, were evaluated by SDS-PAGE stained with Coomassie (lanes 1 and 2, respectively). Analogous studies were performed with E. coli that express wild-type Protein Z (lane 3) and unmodified T7 competent cells (lane 4).

Figure 3. SDS-PAGE and HPLC analysis of Protein Z following ligation with cysteine or an azido fluorescent peptide

(A) Tricine SDS-PAGE analysis of Protein Z following intein-mediated expressed protein ligation with either a cysteine (Pz-Cys) or an azido-fluorescent peptide (Pz-AzFP). Formation of the conjugate was evaluated via a white light image of the gel (left) and further confirmed by fluorescent imaging of the gel (right), which showed that only the Protein Z-AzFP conjugate was fluorescent. (B) Pz-Cys and Pz-AzFP ligation products were also analyzed by HPLC.

Figure 4. Evaluation of UV crosslinked Protein Z-rituximab conjugates via SDS-PAGE

Samples containing photoreactive Protein Z and/or rituximab (Ritux) were UV or mock irradiated and analyzed via SDS-PAGE under reducing and non-reducing conditions. (A) The reducing gel shows that the exposure of samples containing photoreactive Protein Z and rituximab to UV irradiation results in an additional band above the heavy chain (lane 5), corresponding to Protein Z crosslinked heavy chains. No additional bands were observed above the light chain. (B) The non-reducing gel clearly shows the appearance of two additional bands when rituximab is crosslinked with Protein Z (lane 5), corresponding to 1 or 2 crosslinked Protein Z per IgG. The formation of the additional bands is dependent on both the presence of rituximab and Protein Z, as well as on exposure to UV.

Figure 5. Crosslinking of photoreactive Protein Z and IgG in ascites fluid

Photoreactive F13BPA Protein Z-AzFP conjugates that were incubated with rituximab or monoclonal anti-BSA IgG in ascites fluid were either mock or UV irradiated and analyzed via a reducing SDS-PAGE gel. Gels were imaged under white light and via fluorescence.

Figure 6. Evaluation of B cell-targeted SPIO nanoparticles with site-specifically conjugated rituximab

(A) Photoreactive F13BPA Protein Z-AzFP was crosslinked to rituximab and conjugated onto ADIBO-functionalized SPIO (SPIO-Ritux). The SPIO-Ritux and rituximab alone were analyzed via a reducing SDS-PAGE gel. (B) Rituximab-conjugated SPIOs were used to label B cells in the presence and absence of excess free rituximab and the results analyzed using flow cytometry. (C) B cells were incubated with unmodified SPIO or SPIO-ritux in the presence or absence of excess free rituximab and T2*-weighted MR images of the respective cell pellets were acquired.