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 NH 2 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 Magnetically purified SPIO are fluorescently labeled Cancer cells labeled with Her2-SPIO exhibit a a lower T2 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 C H 2-C H 3 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
Affinity Assays Bispecific Antibodies - 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 Full- Length IgGs Site-specific antibody conjugation methods can be used to make bispecific antibodies rapidly (<1 day) and efficiently
Cetux Cetux x Her Her Ritux Ritux x OKT3 OKT3 Her Her x OKT3 OKT3 Cetux Cetux x OKT3 OKT3 Rapid Production of Bispecific Antibodies 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
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 Acknowledgements 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