Ph.D.: Weizmann Institute of Science, Israel
Post-doctorate: Stanford University, CA, USA
Position: Senior Lecturer
Department of Biotechnology Engineering
Faculty of Engineering Sciences
E-mail: papo@bgu.ac.il
Utilizing combinatorial approach to target tumor neovasculature for cancer imaging and therapy
Evolution has generated the great diversity of proteins with all different functions required for the processes of life primarily by using the natural twenty amino acids. Despite all efforts to fully understand the nature of proteins, we are still far from being able to create completely new proteins with desired structure and function simply by rational design. By utilizing state-of-the-art yeast surface display (YSD) directed evolution technology, it is now possible to generate protein variants with new functions. Such desired functions could include improvements in protein physico-chemical properties (i.e. expression, solubility and stability) or modifications in substrate affinity and specificity. These optimized properties could be used for a variety of applications including medical diagnostics where issues such as high binding affinities, selectivity, and exceptional thermal, chemical, and protease stability play a major role. In vivo pharmacokinetic characteristics such as serum stability, tissue penetration, blood clearance and target retention are critically-important for therapeutic applications. All these parameters can now be optimized by modern evolution strategies.
1. Analysing protein interaction interfaces - We are developing a YSD compatible method for protein scanning to provide a high-throughput alternative to traditional alanine scanning for mapping of the binding energy contributions of residues in protein-protein interfaces. To that end, we are using a binomial mutagenesis approach (with binomial degenerate codons) that utilizes yeast-displayed protein scaffold libraries in which each site within the protein scaffold is allowed to vary as only the wild-type or a single substitution to alanine. Following YSD-based affinity selection to a specific target, a statistical analysis of the high affinity variant pool is then being used to assess the functional (binding) contributions of individual side chains in the scaffold.
2. Molecular imaging - Cancer treatment is currently shifting towards more personalized approaches which require knowledge about differences in expression patterns of cancer markers. We are using the evolved affinity proteins as in vivo imaging agents to detect and identify these markers. To evaluate these proteins as molecular imaging agents, we are site-specifically attaching a fluorescent probe or radiolabel to a free Cys or N termini and using optical and positron emission tomography (PET) imaging, respectively, to measure their tumor uptake and biodistribution in different cancer models. We are also using affinity proteins to monitor cancer treatment, which is necessary in case of radiotherapy. Furthermore, we are also investigating protein scaffolds as platforms for integrating cancer imaging and therapy by using a bi- or trifunctional scaffold coupled to effector compounds (such as small-molecule toxins and radioactive isotopes) and an optical (or radiolabeled) probe.
3. Cancer Therapy - The therapeutic effect of alternative scaffolds is obtained by blocking and antagonizing cancer-related molecular targets. In addition, fusions with cytokines or toxins, which are very difficult to produce with antibodies, provide affinity proteins with effector functions. Cytokine fusions activate the function of cytotoxic cells at a tumor site, whereas toxin fusions have a direct killing effect. Cysteine-free scaffolds thus offer the additional advantage that unique cysteines can be introduced by protein engineering, allowing convenient site-directed coupling of effector compounds, such as small-molecule toxins and radioactive isotopes. Bivalency enhances the affinity of traditional antibodies to surface-bound antigens and their Fc region increases their in vivo half-life. Bi- or oligovalency is achieved in the alternative scaffolds, either by making an oligomer genetically as a head-to-tail fusion protein, by Fc-fusions or by fusing other oligomerization domains to the protein.
Papo N.*, Braunstein A.*, Eshhar Z. and Shai Y. (2004). Suppression of Human Prostate Tumor Growth in Mice by a Cytolytic D-, L-amino Acid Peptide: Membrane Lysis, Increased Necrosis, and Inhibition of Prostate-Specific Antigen Secretion. Cancer Res. 64:5779-5786. (*equal contribution)
Papo N. and Shai Y. (2005.) A Molecular Mechanism for Lipopolysaccharide Protection of Gram-Negative Bacteria from Antimicrobial Peptides. J. Biol. Chem. 280:10378-10387.
Papo N., Seger D., Makovitzki A., Kalchenko V., Eshhar Z., Degani H. and ShaiY. (2006). Inhibition of Tumor Growth and Elimination of Multiple Metastases in Human Prostate and Breast Xenografts by Systemic Inoculation of a Host-Defense-Like Lytic Peptide. Cancer Res. 66:5371-5378.
Kipnis Y.*, Papo N.*, Haran G. and Horovitz A. (2007). Concerted ATP-induced Allosteric Transitions in GroEL Facilitate Release of Protein Substrate Domains in an All-or-None Manner. Proc. Natl. Acad. Sci. U.S.A. 104:3119-3124. (*equal contribution)
Papo N.*, Kipnis Y.*, Haran G. and Horovitz A. (2008). Concerted release of substrate domains from GroEL by ATP is demonstrated with FRET.” J. Mol. Biol. 380:717-725. (*equal contribution)
Papo N., Silverman A.P., Lahti J.L. and Cochran J.R. (2011). Antagonistic VEGF Variants Engineered to Simultaneously Bind to and Inhibit VEGFR2 and αvβ3 Integrin. Proc. Natl. Acad. Sci. U.S.A. 108:14067-14072.
