Site-Specific Bioconjugation Approaches for the Preparation of Novel Bispecific Antibody Platforms

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1 Site-Specific Bioconjugation Approaches for the Preparation of Novel Bispecific Antibody Platforms Andrew Tsourkas, Ph.D. Professor Department of Bioengineering University of Pennsylvania

2 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

3 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

4 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

5 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.

6 Proximity-Based Sortase Ligation (PBSL) PBSL exhibits a significantly higher ligation efficiency than traditional sortase reactions.

7 Rapid Production of Modular Bispecific Targeting Ligands Two affibody constructs were used to make 8 different bispecific targeting ligands

8 Improved Specificity of Bispecific Targeting Ligands Bispecific targeting ligands can be used to acquired enhanced specificity for double-positive target cell lines

9 Site-Specific IgG-Nanoparticle Conjugations STEPL and PBSL can be combined with the incorporation of unnatural amino acids to allow for the site-specific attachment of IgG to nanoparticles.

10 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

11 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

12 Alternative IgG-Bioconjugate Applications pabbds allow us to functionalize antibodies with a wide array of chemical and biological moieties for use in diverse applications

13 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

14 Chemically-Conjugated Bispecific Antibodies Chemically-conjugated bispecific antibodies closely resemble genetically prepared tetravalent, bispecific antibodies Chemical conjugation approach to producing bispecific antibodies could allow libraries of bispecific antibodies to be rapidly screened for more potent compositions and lead to the creation of design rules.

15 Summary STEPL can combine protein purification and site-specific labeling of single chain proteins into a single step PBSL allows for the facile site-specific modification of difficult to express proteins and proteins expressed in yeast and mammalian cells Photoreactive-antibody binding domains can be used to rapidly, efficiently and site-specifically label any off-the-shelf antibody. Photoreactive antibody-binding domains can be used to generate libraries of bispecific antibodies in just a few hours. Photoreactive antibody-binding domains have no effect on antibody function.

16 Graduate Students Rob Warden James Hui Henry Wang Fabiana Zappala 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