Site-specific modification of antibody fragments by combining chemoenzymatic bioconjugation and click chemistry


Site-specific modification of antibody fragments by combining chemoenzymatic bioconjugation and click chemistry

Zarschler, K.; Singh, G.; Schlein, E.; Pufe, J.; Comba, P.; Pietzsch, J.; Bachmann, M.; Stephan, H.

Introduction
A wealth of preclinical experiments has clearly demonstrated that site-specific protein modification results in a more homogenous product population with defined and tunable properties compared to traditional bioconjugation techniques (1, 2). This is of particular importance for antibodies and their fragments as random conjugation strategies can compromise their functionality and interfere with or abolish their immunoreactivity (3). That specifically applies to small-sized antibody formats such as single chain variable fragments and single-domain antibody-fragments due to their compact structure and limited number of available functional reactive residues outside the antigen-binding site (4-6). Here, we describe a versatile two-step approach for site-specific introduction of bifunctional radiometal-chelating agents into antibody-derived fragments for the generation of highly defined radioimmunotracers.

Methods
The first step of this approach uses enzyme-mediated bioconjugation for regioselective incorporation of an azide-containing linker into an antibody fragment. The second step of this modular approach involves the modification of the azide-tagged antibody fragment with dibenzocyclooctyne (DBCO)-containing fluorescent dyes or DBCO-modified bispidine via Cu-free strain-promoted alkyne-azide cycloaddition.

Results
In an exemplary way, we demonstrate the versatility of this two-step approach by the site-specific incorporation of fluorescent dyes or radiometal chelators for fluorescence or positron emission tomographic imaging at physiological conditions. In vitro binding studies on different human cancer cell lines using dye- or 64Cu-labeled antibody fragments showed high specificity, co-localization and a receptor-mediated cellular uptake of the EGFR-specific probes. Noteworthy, we did not observe any detrimental effect to the functionality of the antibody fragments.

Discussion and Conclusion
The site-specific insertion of a bioorthogonal handle into the targeting moiety allows the subsequent convenient and straightforward incorporation of a variety of complementary probes. In addition, the heterogeneity of the conjugate population is substantially reduced as the conjugation site is defined and maximally one probe is attached per antibody fragment.

References
1. Agarwal P, Bertozzi CR. Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development. Bioconjug Chem. 2015;26(2):176-92.
2. Sletten EM, Bertozzi CR. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew Chem Int Ed Engl. 2009;48(38):6974-98.
3. Schumacher D, Hackenberger CP, Leonhardt H, Helma J. Current Status: Site-Specific Antibody Drug Conjugates. J Clin Immunol. 2016;36 Suppl 1:100-7.
4. Meyer JP, Adumeau P, Lewis JS, Zeglis BM. Click Chemistry and Radiochemistry: The First 10 Years. Bioconjug Chem. 2016;27(12):2791-807.
5. Massa S, Xavier C, Muyldermans S, Devoogdt N. Emerging site-specific bioconjugation strategies for radioimmunotracer development. Expert Opin Drug Deliv. 2016;13(8):1149-63.
6. Schumacher D, Helma J, Schneider AFL, Leonhardt H, Hackenberger CPR. Nanobodies: Chemical Functionalization Strategies and Intracellular Applications. Angew Chem Int Ed Engl. 2018;57(9):2314-33.

  • Lecture (Conference)
    Annual congress of the European Association of Nuclear Medicine (EANM'18), 13.-17.10.2018, Düsseldorf, Deutschland

Permalink: https://www.hzdr.de/publications/Publ-27375
Publ.-Id: 27375