Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1-directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti-PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm 3 ) and large tumors (150 mm 3 ), whereas the activity of anti-PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1-positive and PD-L1-negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.protein engineering | cancer immunotherapy | PET imaging | PD-1 | PD-L1

There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI-II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin-binding agents. We generated yeast-displayed libraries of EETI-II by substituting its 6-amino acid trypsin binding loop with 11-amino acid loops containing the Arg-Gly-Asp (RGD) integrin binding motif and randomized flanking residues. These libraries were screened in a high-throughput manner by fluorescence-activated cell sorting to identify mutants that bound to αvβ3 integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half-maximal inhibitory concentration (IC50) values of 10-30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both αvβ3 and αvβ5 integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to αvβ3, αvβ5, and α5β1 integrins. This finding has important clinical implications since all three of these receptors can be co-expressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI-II as a scaffold for protein engineering, and highlight the development of unique integrin-binding peptides with potential for translational applications in cancer.

There is a critical need for molecular imaging agents to detect cell surface integrin receptors that are present in human cancers. Previously, we used directed evolution to engineer knottin peptides that bind with high affinity (f10 to 30 nmol/L) to integrin receptors that are overexpressed on the surface of tumor cells and the tumor neovasculature. To evaluate these peptides as molecular imaging agents, we sitespecifically conjugated Cy5.5 or 64 Cu-1,4,7,10-tetra-azacyclododecane-N,N ¶,N 00

Perfect circle. We report the biosynthesis of a natively folded cyclotide, MCoTI‐II, in E. coli by intracellular backbone cyclization of a linear cyclotide–intein fusion precursor. The cyclized peptide then spontaneously folds into its native conformation. Biosynthetic access to correctly folded cyclotides allows the possibility of generating cell‐based combinatorial libraries that can be screened, inside living cells, for their ability to modulate or inhibit cellular processes.