Evolved Molecules that Destroy Cancer Relevant Proteins

ABSTRACT
Cancer research is often driven by hypotheses that postulate that a specific protein is important for the
disease, hypotheses often tested by adding an antibody to that system. The technology to be developed here will
create new reagents (AEGISbodies and AEGISCleavers) that not only bind a target protein (like an antibody), but
also cleave it, at a cost 10 to 100 fold lower than a service-provided antibody, in weeks rather than months, and
across a range of affinities that support quantitative research. These will be obtained by applying laboratory in
vitro evolution (LIVE) to libraries built from an artificially expanded genetic information system (AEGIS), and
decorated with precisely selected chemical groups. AEGIS is a biopolymer like DNA, but with up to 12 building
blocks, enhanced folding, greater stability, and functional groups that assist in the cleavage reaction, which
gives products where an AEGISCleaver is covalently attached to a fragment of the target peptide.
AEGIS-LIVE has high level of technical readiness because of preliminary studies that created (a) platforms to
manufacture AEGIS DNA, (b) a molecular biology to support AEGIS-LIVE, and (c) sequencing, fold prediction,
and other tools to analyze the products of AEGIS-LIVE. The team has proven the cancer-relevance of AEGIS-LIVE,
creating AEGISBodies that bind specifically and selectively to breast and liver cancer cells, to cancer-relevant
proteins heterologously expressed on mammalian cell surfaces, and to isolated proteins from anthrax. The first
catalytic AEGISzymes are in hand. To further this technology development, we will:
Aim 1. Apply AEGIS-LIVE to create AEGISbodies that bind Programmed Death-Ligand 1 (PD-L1), a recognized
target for cancer therapy. We will also target extracellular peptide loops in the PD-L1 fold, made from L- and D-
amino acids. The second are mirror images of the natural amino acids, and allow production of mirror image L-
AEGISbodies that are stable against nuclease digestion. We will metric these for binding affinities and stability,
and validate their efficacies compared to current technologies.
Aim 2. Apply AEGIS-LIVE to create L- and D-AEGISCleavers that cleave peptides from PD-L1, and the analogous
segments when embedded in the complete protein. We will metric and benchmark these for affinity, cleavage
rates, and stability against nucleases.
The new reagents will be transformative since they do things that antibodies cannot: covalently tag and destroy
target proteins. Further, the reagents are expected to be stable in biological fluids, including cell culture, blood,
and living animals. Longer term, ultra-stable AEGISCleavers may even come to be diagnostic and therapeutic
tools.

Grant Number: 5R61CA291117-02
NIH Institute/Center: NIH
Principal Investigator: Elisa Biondi