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Rapid Production of Bispecific Antibodies Using ‘Off-the-Shelf’ IgG Andrew Tsourkas, Ph.D. Professor Department of Bioengineering University of Pennsylvania Limitations/Challenges of Nanoparticle Bioconjugations • Conventional Chemistries are inefficient (typically <10%) and are not site-specific • Click-chemistries are efficient, but are not-specific & require labeling of antibody Creating Targeted Nanoparticles Targeting Group Crosslinker Contrast Agent/Nanoparticle HOOC NH2 Desirable attributes for nanoparticle-targeting ligand conjugations • Ability to work with any off-the-shelf antibody • Highly efficient • Site-specific/proper orientation on nanoparticle surface • No effect on antibody affinity Sortase-Tag Expressed Protein Ligation (STEPL) • Site-specific C-terminal modification of any single-chain recombinant protein with any desirable tag (imaging agents, drug, peg, click moieties, etc.) % Protein Ligated % Peptide Utilized Proximity-Based Sortase Ligation (PBSL) • Binding partners are used to bring Sortase and Sortase Recognition Motif into close proximity, to increase the efficiency of ligation. Proximity-Based Sortase Ligation (PBSL) • Binding partners are used to bring Sortase and Sortase Recognition Motif into close proximity, to increase the efficiency of ligation. Improving Nanoparticle Bioconjugations • Confirmation of Click conjugation • Cancer cells labeled with Her2-SPIO exhibit a a lower T2 • Magnetically purified SPIO are fluorescently labeled • Ligand density has large effect on avidity Improving Nanoparticle Bioconjugations • Her2-SPIO can be used to effectively detect Her2/neu-positive tumors Rapid Production of Modular Bispecific Targeting Ligands • Two affibody constructs were used to make 8 different bispecific targeting ligands Improved Specificity of Bispecific Targeting Ligands • Bispecific targeting ligands can be used to acquired enhanced specificity for double-positive target cell lines Site-Specific IgG-Nanoparticle Conjugations • EPL-Click can be combined with the incorporation of unnatural amino acids to allow for the site-specific attachment of IgG to nanoparticles. Site-specific Modification of IgG • Photoreactive Antibody binding domains (pAbBDs) bind at the CH2-CH3 hinge • pAbBDs efficiently crosslink a wide range of antibodies from various hosts and subclasses Optimization of IgG Bioconjugations • pAbBDs can be covalently linked to IgG very rapidly and efficiently. • A 1:1 pAbBD-to-IgG heavy chain ratio is adequate for complete labeling Modification of a Single IgG Heavy Chain • Steps can be take to modify only a single IgG heavy chain with Protein Z/G. • IgG modified with pAbBDs exhibit the same affinity for their target receptor as native IgG. IgG-Nanoparticle Bioconjugations • pAbBDs enables the covalent linkage of IgG to Nanoparticles IgG-Nanoparticle Bioconjugations • Imaging VCAM-1 in murine inflammation model Alternative IgG-Bioconjugate Applications • pAbBDs allow us to functionalize antibodies with a wide array of chemical and biological moieties for use in diverse applications One-Step Production of Bispecific Antibodies • pAbBD-scFv fusions allows IgG-scFv bispecific antibodies to be produced in a single step. pAbBDscFv • Bispecifics can be produced rapidly, in parallel, and with high purity Bispecific Antibodies • Affinity Assays - Bispecific antibodies exhibit no loss in affinity compared with the monoclonal antibody (mAb) from which they are derived. Bispecific Antibodies Exhibit High Potency • Bispecific antibodies exhibit an EC50 of ~2 ng/mL (PMBCs, E:T = 10:1) Jeko B cells: Rituximab x anti-CD3 K562 cells: Rituximab x anti-CD3 Jeko B cells: Rituximab + anti-CD3 Jeko B cells: Rituximab Rapid Production of Bispecific Antibodies from Two FullLength IgGs • Site-specific antibody conjugation methods can be used to make bispecific antibodies rapidly (<1 day) and efficiently Rapid Production of Bispecific Antibodies OKT3 Cetux x OKT3 Cetux OKT3 Her x OKT3 Her OKT3 Ritux x OKT3 Ritux Her Cetux x Her Cetux • 4 unique bispecific antibodies were generated by a one person in less than 24 hrs. Rapid Production of Bispecific Antibodies • Bispecifics exhibit similar affinity as individual wild-type IgG • FPLC can be used to improve purity Trimer Dimer Monomer Acknowledgements Graduate Students Rob Warden James Hui Elizabeth Higbee Jessica Liu Post-Docs/Research Scientists Ching-Hui Huang Xumei Zhang Kido Nwe Yang Song Lesan Yan Jayesh Thawani Joel Stein Burcin Altun Collaborators Zhiliang Cheng (Upenn) Vladimir Popik (UGa) Small Animal Imaging Facilities Weixia Liu Stephen Pickup Funding: NSF, NIH/NCI, NIH/NIBIB, American Cancer Society, CDMRP BCRP
