Niv Papo
Krill Prize 2014
Ben Gurion University
Dr. Niv Papo (ד”ר ניב פפו)
Research Interests:
Multifunctional Cancer Therapeutics
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.
These desired functions could for example be improvements in protein physico-chemical
properties (i.e. expression, solubility and stability) or modifications in substrate affinity and specificity. These optimized functions can be used for a variety of applications. For
medical diagnostics, high binding affinities, selectivity, and exceptional thermal, chemical, and protease stability play a major role. In vivo pharmacokinetic characteristics like serum stability, tissue penetration, blood clearance, and target retention are mostly important for therapeutic applications. All these parameters can now be optimized by modern evolution strategies. The main topics in my lab include:
i. Analysing proein 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. For that, 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.
ii. 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 use 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 not only for cancer detection but also to follow treatment, which for
example is necessary in case of radiotherapy. We are also using the 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.
iii. 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.