Directed Evolution of PD-L1-Targeted Affibodies by mRNA Display.

Therapeutic monoclonal antibodies directed against PD-L1 (e.g., atezolizumab) disrupt PD-L1:PD-1 signaling and reactivate exhausted cytotoxic T-cells in the tumor compartment. Although anti-PD-L1 antibodies are successful as immune checkpoint inhibitor (ICI) therapeutics, there is still a pressing need to develop high-affinity, low-molecular-weight ligands for molecular imaging and diagnostic applications. Affibodies are small polypeptides (∼60 amino acids) that provide a stable molecular scaffold from which to evolve high-affinity ligands. Despite its proven utility in the development of imaging probes, this scaffold has never been optimized for use in mRNA display, a powerful in vitro selection platform incorporating high library diversity, unnatural amino acids, and chemical modification. In this manuscript, we describe the selection of a PD-L1-binding affibody by mRNA display. Following randomization of the 13 amino acids that define the binding interface of the well-described Her2 affibody, the resulting library was selected against recombinant human PD-L1 (hPD-L1). After four rounds, the enriched library was split and selected against either hPD-L1 or the mouse ortholog (mPD-L1). The dual target selection resulted in the identification of a human/mouse cross-reactive PD-L1 affibody (M1) with low nanomolar affinity for both targets. The M1 affibody bound with similar affinity to mPD-L1 and hPD-L1 expressed on the cell surface and inhibited signaling through the PD-L1:PD-1 axis at low micromolar concentrations in a cell-based functional assay. In vivo optical imaging with M1-Cy5 in an immune-competent mouse model of lymphoma revealed significant tumor uptake relative to a Cy5-conjugated Her2 affibody.

Directing evolution of novel ligands by mRNA display.

mRNA display is a powerful biological display platform for the directed evolution of proteins and peptides. mRNA display libraries covalently link the displayed peptide or protein (phenotype) with the encoding genetic information (genotype) through the biochemical activity of the small molecule puromycin. Selection for peptide/protein function is followed by amplification of the linked genetic material and generation of a library enriched in functional sequences. Iterative selection cycles are then performed until the desired level of function is achieved, at which time the identity of candidate peptides can be obtained by sequencing the genetic material. The purpose of this review is to discuss the development of mRNA display technology since its inception in 1997 and to comprehensively review its use in the selection of novel peptides and proteins. We begin with an overview of the biochemical mechanism of mRNA display and its variants with a particular focus on its advantages and disadvantages relative to other biological display technologies. We then discuss the importance of scaffold choice in mRNA display selections and review the results of selection experiments with biological (e.g., fibronectin) and linear peptide library architectures. We then explore recent progress in the development of "drug-like" peptides by mRNA display through the post-translational covalent macrocyclization and incorporation of non-proteogenic functionalities. We conclude with an examination of enabling technologies that increase the speed of selection experiments, enhance the information obtained in post-selection sequence analysis, and facilitate high-throughput characterization of lead compounds. We hope to provide the reader with a comprehensive view of current state and future trajectory of mRNA display and its broad utility as a peptide and protein design tool.

mRNA Display Discovery of a Novel Programmed Death Ligand 1 (PD-L1) Binding Peptide (a Peptide Ligand for PD-L1).

Programmed death ligand 1 (PD-L1) is a critical immune checkpoint ligand whose overexpression on tumor cells provides a mechanism of escape from immune surveillance. The interaction between PD-L1 and PD-1 on T cell lymphocytes suppresses both T cell activation and effector function and is engaged by cancers to dampen antitumor immunity. Here, we used mRNA display to engineer an 18-residue linear peptide that binds to human PD-L1. This peptide, which we term SPAM (signal peptide-based affinity maturated ligand), is nonhomologous to known PD-L1 binding peptides and mAbs, with dissociation constants (KD) of 119 and 67 nM for unglycosylated and glycosylated human PD-L1, respectively. The SPAM peptide is highly selective for human PD-L1 and shows no significant binding to either mouse PD-L1 or human PD-L2. Competition binding assays indicate that the SPAM peptide binding site overlaps with the binding site of PD-1 as well as therapeutic anti-PD-L1 antibodies. Taken together, these results suggest that the SPAM peptide specifically binds to human PD-L1 and could potentially serve as a PD-L1 affinity agent and PD-L1/PD-1 pathway modulator.

Identification, characterization and application of a new peptide against anterior gradient homolog 2 (AGR2).

The cancer-associated protein Anterior Gradient 2 (AGR2) has been described, predominantly in adenocarcinomas. Increased levels of extracellular AGR2 (eAGR2) have been correlated with poor prognosis in cancer patients, making it a potential biomarker. Additionally, neutralizing AGR2 antibodies showed preclinical effectiveness in murine cancer models suggesting eAGR2 may be a therapeutic target. We set out to identify a peptide by mRNA display that would serve as a theranostic tool targeting AGR2. This method enables the selection of peptides from a complex (>1011) library and incorporates a protease incubation step that filters the selection for serum stable peptides. We performed six successive rounds of enrichment using a 10-amino acid mRNA display library and identified several AGR2 binding peptides. One of these peptides (H10), demonstrated high affinity binding to AGR2 with a binding constant (KD) of 6.4 nM. We developed an AGR2 ELISA with the H10 peptide as the capture reagent. Our H10-based ELISA detected eAGR2 from cancer cell spent media with a detection limit of (20-50 ng/ml). Furthermore, we investigated the therapeutic utility of H10 and discovered that it inhibited cell viability at IC50 (9-12 µmoles/L) in cancer cell lines. We also determined that 10 µg/ml of H10 was sufficient to inhibit cancer cell migration in breast and prostate cancer cell lines. A control peptide did not show any appreciable activity in these cells. The H10 peptide showed promise as both a novel diagnostic and a potential therapeutic peptide.

Automated, Resin-Based Method to Enhance the Specific Activity of Fluorine-18 Clicked PET Radiotracers.

Radiolabeling of substrates with 2-[18F]fluoroethylazide exploits the rapid kinetics, chemical selectivity, and mild conditions of the copper-catalyzed azide-alkyne cycloaddition reaction. While this methodology has proven to result in near-quantitative labeling of alkyne-tagged precursors, the relatively small size of the fluoroethylazide group makes separation of the 18F-labeled radiotracer and the unreacted precursor challenging, particularly with precursors >500 Da (e.g., peptides). We have developed an inexpensive azide-functionalized resin to rapidly remove unreacted alkyne precursor following the fluoroethylazide labeling reaction and integrated it into a fully automated radiosynthesis platform. We have carried out 2-[18F]fluoroethylazide labeling of four different alkynes ranging from <300 Da to >1700 Da and found that >98% of the unreacted alkyne was removed in less than 20 min at room temperature to afford the final radiotracers at >99% radiochemical purity with specific activities up to >200 GBq/µmol. We have applied this technique to label a novel cyclic peptide previously evolved to bind the Her2 receptor with high affinity, and demonstrated tumor-specific uptake and low nonspecific background by PET/CT. This resin-based methodology is automated, rapid, mild, and general allowing peptide-based fluorine-18 radiotracers to be obtained with clinically relevant specific activities without chromatographic separation and with only a minimal increase in total synthesis time.

RasIns: Genetically Encoded Intrabodies of Activated Ras Proteins.

K- and H-Ras are the most commonly mutated genes in human tumors and are critical for conferring and maintaining the oncogenic phenotype in tumors with poor prognoses. Here, we design genetically encoded antibody-like ligands (intrabodies) that recognize active, GTP-bound K- and H-Ras. These ligands, which use the 10th domain of human fibronectin as their scaffold, are stable inside the cells and when fused with a fluorescent protein label, the constitutively active G12V mutant H-Ras. Primary selection of ligands against Ras with mRNA display resulted in an intrabody (termed RasIn1) that binds with a KD of 2.1µM to H-Ras(G12V) (GTP), excellent state selectivity, and remarkable specificity for K- and H-Ras. RasIn1 recognizes residues in the Switch I region of Ras, similar to Raf-RBD, and competes with Raf-RBD for binding. An affinity maturation selection based on RasIn1 resulted in RasIn2, which binds with a KD of 120nM and also retains excellent state selectivity. Both of these intrabodies colocalize with H-Ras, K-Ras, and G12V mutants inside the cells, providing new potential tools to monitor and modulate Ras-mediated signaling. Finally, RasIn1 and Rasin2 both display selectivity for the G12V mutants as compared with wild-type Ras providing a potential route for mutant selective recognition of Ras.

Directed Evolution of Scanning Unnatural-Protease-Resistant (SUPR) Peptides for in Vivo Applications.

Peptides typically have poor biostabilities, and natural sequences cannot easily be converted into drug-like molecules without extensive medicinal chemistry. We have adapted mRNA display to drive the evolution of highly stable cyclic peptides while preserving target affinity. To do this, we incorporated an unnatural amino acid in an mRNA display library that was subjected to proteolysis prior to selection for function. The resulting "SUPR (scanning unnatural protease resistant) peptide" showed ≈500-fold improvement in serum stability (t1/2 =160 h) and up to 3700-fold improvement in protease resistance versus the parent sequence. We extended this approach by carrying out SUPR peptide selections against Her2-positive cells in culture. The resulting SUPR4 peptide showed low-nanomolar affinity toward Her2, excellent specificity, and selective tumor uptake in vivo. These results argue that this is a general method to design potent and stable peptides for in vivo imaging and therapy.

General, Label-Free Method for Determining K(d) and Ligand Concentration Simultaneously.

Some of the most commonly used affinity reagents (e.g., antibodies) are often developed and used in conditions where their input concentrations ([L]0) and affinities (K(d)) are not known. Here, we have developed a general approach to determine both [L]0 and K(d) values simultaneously for affinity reagents (small molecules, proteins, and antibodies). To do this, we perform quantitative equilibrium exclusion immunoassays with two different concentrations of target and fit the data simultaneously to determine K(d) and [L]0. The results give accurate and reproducible measures of both values compared to established methods. By performing detailed error analysis, we demonstrate that our fitting gives unique solutions and indicates where K(d) and [L]0 measures are reliable. Furthermore, we found that a divalent model of antibody binding gives accurate K(d) and [L]0 values in both the forward (antibody immobilized) and the reverse (target immobilized) assays-addressing the long-term problem of obtaining quantitative data from reverse assays.

Serum stable natural peptides designed by mRNA display.

Peptides constructed with the 20 natural amino acids are generally considered to have little therapeutic potential because they are unstable in the presence of proteases and peptidases. However, proteolysis cleavage can be idiosyncratic, and it is possible that natural analogues of functional sequences exist that are highly resistant to cleavage. Here, we explored this idea in the context of peptides that bind to the signaling protein Gαi1. To do this, we used a two-step in vitro selection process to simultaneously select for protease resistance while retaining function-first by degrading the starting library with protease (chymotrypsin), followed by positive selection for binding via mRNA display. Starting from a pool of functional sequences, these experiments revealed peptides with 100-400 fold increases in protease resistance compared to the parental library. Surprisingly, selection for chymotrypsin resistance also resulted in similarly improved stability in human serum (~100 fold). Mechanistically, the decreases in cleavage results from both a lower rate of cleavage (kcat) and a weaker interaction with the protease (Km). Overall, our results demonstrate that the hydrolytic stability of functional, natural peptide sequences can be improved by two orders of magnitude simply by optimizing the primary sequence.

Robust, quantitative analysis of proteins using peptide immunoreagents, in vitro translation, and an ultrasensitive acoustic resonant sensor.

A major benefit of proteomic and genomic data is the potential for developing thousands of novel diagnostic and analytical tests of cells, tissues, and clinical samples. Monoclonal antibody technologies, phage display and mRNA display, are methods that could be used to generate affinity ligands against each member of the proteome. Increasingly, the challenge is not ligand generation, rather the analysis and affinity rank-ordering of the many ligands generated by these methods. Here, we developed a quantitative method to analyze protein interactions using in vitro translated ligands. In this assay, in vitro translated ligands generate a signal by simultaneously binding to a target immobilized on a magnetic bead and to a sensor surface in a commercial acoustic sensing device. We then normalize the binding of each ligand with its relative translation efficiency in order to rank-order the different ligands. We demonstrate the method with peptides directed against the cancer marker Bcl-xL. Our method has 4- to 10-fold higher sensitivity, using 100-fold less protein and 5-fold less antibody per sample, as compared directly with ELISA. Additionally, all analysis can be conducted in complex mixtures at physiological ionic strength. Lastly, we demonstrate the ability to use peptides as ultrahigh affinity reagents that function in complex matrices, as would be needed in diagnostic applications.

Antibody-mimetic ligand selected by mRNA display targets DC-SIGN for dendritic cell-directed antigen delivery.

Dendritic cell (DC)-based vaccines have shown promise as an immunotherapeutic modality for cancer and infectious diseases in many preclinical studies and clinical trials. Provenge (sipuleucel-T), a DC-based vaccine based on ex vivo-generated autologous DCs loaded with antigens, has recently received FDA approval for prostate cancer treatment, further validating the potential of DC-based vaccine modalities. However, direct antigen delivery to DCs in vivo via DC-specific surface receptors would enable a more direct and less laborious approach to immunization. In this study, the recombinant extracellular domains (ECD) of human and mouse DC-SIGN (hDC-SIGN and mDC-SIGN) were generated as DC-specific targets for mRNA display. Accordingly, an antibody-mimetic library was constructed by randomizing two exposed binding loops of an expression-enhanced 10th human fibronectin type III domain (e10Fn3). After three rounds of selection against mDC-SIGN, followed by four rounds of selection against hDC-SIGN, we were able to evolve several dual-specific ligands, which could bind to both soluble ECD of human and mouse DC-SIGNs. Using a cell-binding assay, one ligand, eFn-DC6, was found to have high affinity to hDC-SIGN and moderate affinity to mDC-SIGN. When fused with an antigenic peptide, eFn-DC6 could direct the antigen delivery and presentation by human peripheral blood mononuclear cell (PBMC)-derived DCs and stimulate antigen-specific CD8(+) T cells to secrete inflammatory cytokines. Taken together, these results demonstrate the utility of mRNA display to select protein carriers for DC-based vaccination and offer in vitro evidence that the antibody-mimetic ligand eFn-DC6 represents a promising candidate for the development of an in vivo DC-based vaccine in humans.

In vitro selection of protein and peptide libraries using mRNA display.

In vitro genetic approaches are powerful solutions to the protein design problem. mRNA display is an in vitro selection technique enabling the design of peptide and protein ligands ranging from one to over 100 amino acids. Libraries containing more than 10 trillion unique sequences can be synthesized and sieved with exquisite control over binding stringency and specificity.

Interactome of ErbB4 unveiled.

MacBeath and colleagues (Kaushansky et al., 2008) use a protein array technology to find binding partners of ErbB4 in a genome-wide and quantitative fashion, shedding new light on how ErbB4 initiates cellular signaling events and why ErbB4 is not a potent oncogene.

The N-end rule pathway as a nitric oxide sensor controlling the levels of multiple regulators.

The conjugation of arginine to proteins is a part of the N-end rule pathway of protein degradation. Three amino (N)-terminal residues--aspartate, glutamate and cysteine--are arginylated by ATE1-encoded arginyl-transferases. Here we report that oxidation of N-terminal cysteine is essential for its arginylation. The in vivo oxidation of N-terminal cysteine, before its arginylation, is shown to require nitric oxide. We reconstituted this process in vitro as well. The levels of regulatory proteins bearing N-terminal cysteine, such as RGS4, RGS5 and RGS16, are greatly increased in mouse ATE1-/- embryos, which lack arginylation. Stabilization of these proteins, the first physiological substrates of mammalian N-end rule pathway, may underlie cardiovascular defects in ATE1-/- embryos. Our findings identify the N-end rule pathway as a new nitric oxide sensor that functions through its ability to destroy specific regulatory proteins bearing N-terminal cysteine, at rates controlled by nitric oxide and apparently by oxygen as well.

Context and conformation dictate function of a transcription antitermination switch.

In bacteriophage l, transcription elongation is regulated by the N protein, which binds a nascent mRNA hairpin (termed boxB) and enables RNA polymerase to read through distal terminators. We have examined the structure, energetics and in vivo function of a number of N-boxB complexes derived from in vitro protein selection. Trp18 fully stacks on the RNA loop in the wild-type structure, and can become partially or completely unstacked when the sequence context is changed three or four residues away, resulting in a recognition interface in which the best binding residues depend on the sequence context. Notably, in vivo antitermination activity correlates with the presence of a stacked aromatic residue at position 18, but not with N-boxB binding affinity. Our work demonstrates that RNA polymerase responds to subtle conformational changes in cis-acting regulatory complexes and that approximation of components is not sufficient to generate a fully functional transcription switch.

mRNA display: ligand discovery, interaction analysis and beyond.

In vitro peptide and protein selection using mRNA display enables the discovery and directed evolution of new molecules from combinatorial libraries. These selected molecules can serve as tools to control and understand biological processes, enhance our understanding of molecular interactions and potentially treat disease in therapeutic applications. In mRNA display, mRNA molecules are covalently attached to the peptide or protein they encode. These mRNA-protein fusions enable in vitro selection of peptide and protein libraries of >10(13) different sequences. mRNA display has been used to discover novel peptide and protein ligands for RNA, small molecules and proteins, as well as to define cellular interaction partners of proteins and drugs. In addition, several unique applications are possible with mRNA display, including self-assembling protein chips and library construction with unnatural amino acids and chemically modified peptides.

Designed arginine-rich RNA-binding peptides with picomolar affinity.

Arginine-rich peptide motifs (ARMs) capable of binding unique RNA structures play critical roles in transcription, translation, RNA trafficking, and RNA packaging. Bacteriophage ARMs necessary for transcription antitermination bind to distinct boxB RNA hairpin sequences with a characteristic induced alpha-helical structure. Characterization of ARMs from lambdoid phages reveals that the dissociation constant of the P22 bacteriophage model-antitermination complex (P22(N21)-P22boxB) is 200 +/- 56 pM in free solution at physiologic concentrations of monovalent cation, significantly stronger than previously determined by gel mobility shift and polyacrylamide gel coelectophoresis, and 2 orders of magnitude stronger than the tightest known native ARM-RNA interaction at physiological salt. Here, we use a reciprocal design approach to enhance the binding affinity of two separate alpha-helical ARM-RNA interactions; one derived from the native lambda phage antitermination complex and a second isolated using mRNA display selection experiments targeting boxB RNA.