A5053116560- Monoclonal and Polyclonal Antibodies Research
- RNA and protein synthesis mechanisms
- Advanced Biosensing Techniques and Applications
- Chemical Synthesis and Analysis
- Glycosylation and Glycoproteins Research
- Advanced biosensing and bioanalysis techniques
- Immunotherapy and Immune Responses
- Peptidase Inhibition and Analysis
- Bacteriophages and microbial interactions
- Radiopharmaceutical Chemistry and Applications
- Medical Imaging Techniques and Applications
- Antimicrobial Peptides and Activities
- Galectins and Cancer Biology
- Click Chemistry and Applications
- Endoplasmic Reticulum Stress and Disease
- RNA Interference and Gene Delivery
- CAR-T cell therapy research
- Bacterial Genetics and Biotechnology
- Protein purification and stability
- Analytical Chemistry and Chromatography
- Lung Cancer Research Studies
- Microfluidic and Capillary Electrophoresis Applications
- Melanoma and MAPK Pathways
- Heat shock proteins research
- HER2/EGFR in Cancer Research
University of Southern California
2013-2022
California Institute of Technology
2001-2006
Scripps College
2000
Cyclic peptides are attractive scaffolds for the design of conformationally constrained molecular therapeutics. Previously, biological display libraries could only be cyclized via disulfide bonds, which labile and can reduced in an intracellular environment. In this paper, we construct high diversity, covalently mRNA (>1013 sequences) analyze cyclization reaction using MALDI-TOF MS unnatural amino acid incorporation. Our route allows extent to evaluated quantitatively is broadly applicable a...
Peptides constructed with the 20 natural amino acids are generally considered to have little therapeutic potential because they unstable in presence of proteases and peptidases. However, proteolysis cleavage can be idiosyncratic it is possible that analogues functional sequences exist highly resistant cleavage. Here, we explored this idea context peptides bind signaling protein Gαi1. To do this, used a two-step vitro selection process simultaneously select for protease resistance while...
Arginine-rich peptide motifs (ARMs) capable of binding unique RNA structures play critical roles in transcription, translation, trafficking, and packaging. Bacteriophage ARMs necessary for transcription antitermination bind to distinct boxB hairpin sequences with a characteristic induced α-helical structure. Characterization from lambdoid phages reveals that the dissociation constant P22 bacteriophage model−antitermination complex (P22N21−P22boxB) is 200 ± 56 pM free solution at physiologic...
Abstract There is great demand for high‐throughput methods to characterize ligand affinity. By combining mRNA display with next‐generation sequencing, we determined the kinetic on‐ and off‐rates over twenty thousand ligands without need synthesis or purification of individual members. Our results are reproducible as accurate those obtained other affinity measurement.
In a single round: By combining the high-efficiency enrichment through continuous-flow magnetic separation (CFMS) technique with analytical power of next-generation sequencing, generation antibody mimetics round mRNA display is made possible. This approach eliminates iterative selection cycles and provides path to fully automated ligand (see picture).
Abstract Peptides typically have poor biostabilities, and natural sequences cannot easily be converted into drug‐like molecules without extensive medicinal chemistry. We 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 library that was subjected proteolysis prior selection for function. The resulting “SUPR (scanning protease resistant) peptide” showed ≈500‐fold improvement...
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 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 diagnostic applications. Affibodies small polypeptides (∼60 amino acids) that provide stable scaffold from which evolve...
RNA loops that adopt a characteristic GNRA "tetraloop" fold are common in natural RNAs. Here, we have used vitro selection by means of mRNA-peptide fusions to select peptides bind an example this loop motif. Starting with the recognition domain from lambda N protein, constructed libraries containing 150, 1,600, and 9 trillion different peptide sequences as isolated those capable high-affinity binding. These selections resulted more than 80 same loop. The highest affinity exhibit low...
N proteins from bacteriophages λ, P22, and φ21 modulate transcription elongation by binding nascent "boxB" mRNA hairpins. This RNA recognition is mediated N-terminal arginine-rich peptide sequences capable of interacting with their cognate boxB targets. Here, we have analyzed the affinity specificity peptide−RNA interactions that this transcriptional switch. To do this, constructed a series peptides based on wild-type protein domains ranging 11 to 22 residues leftward rightward hairpin...
Even flow: Microfluidics technology can rapidly generate affinity reagents that bind to protein targets using mRNA display. The continuous-flow magnetic separation (CFMS) method (see scheme; steps a–h) offers reproducible means impose stringent selection conditions without the need for a specialized device. yields approximately 30-fold greater partition efficiency over conventional methods when interleukin-6 as model target. Since invention of hybridoma technology, generating specific target...
Programmed death ligand 1 (PD-L1) is a critical immune checkpoint whose overexpression on tumor cells provides mechanism of escape from surveillance. The interaction between PD-L1 and PD-1 T cell lymphocytes suppresses both activation effector function engaged by cancers to dampen antitumor immunity. Here, we used mRNA display engineer an 18-residue linear peptide that binds human PD-L1. This peptide, which term SPAM (signal peptide-based affinity maturated ligand), nonhomologous known...
The cancer-associated protein Anterior Gradient 2 (AGR2) has been described, predominantly in adenocarcinomas. Increased levels of extracellular AGR2 (eAGR2) have correlated with poor prognosis cancer patients, making it a potential biomarker. Additionally, neutralizing antibodies showed preclinical effectiveness murine models suggesting eAGR2 may be therapeutic target. We set out to identify peptide by mRNA display that would serve as theranostic tool targeting AGR2. This method enables the...
Dendritic cell (DC)-based vaccines have shown promise as an immunotherapeutic modality for cancer and infectious diseases in many preclinical studies clinical trials. Provenge (sipuleucel-T), a DC-based vaccine based on ex vivo-generated autologous DCs loaded with antigens, has recently received FDA approval prostate treatment, further validating the potential of modalities. However, direct antigen delivery to vivo via DC-specific surface receptors would enable more less laborious approach...
Radiolabeling of substrates with 2-[18F]fluoroethylazide exploits the rapid kinetics, chemical selectivity, and mild conditions copper-catalyzed azide-alkyne cycloaddition reaction. While this methodology has proven to result in near-quantitative labeling alkyne-tagged precursors, relatively small size fluoroethylazide group makes separation 18F-labeled radiotracer unreacted precursor challenging, particularly precursors >500 Da (e.g., peptides). We have developed an inexpensive...
Modern genomic sequencing efforts are identifying potential diagnostic and therapeutic targets more rapidly than existing methods can generate the peptide- protein-based ligands required to study them. To address this problem, we have developed a microfluidic enrichment device (MFED) enabling kinetic off-rate selection without use of exogenous competitor. We tuned conditions (bed volume, flow rate, immobilized target) such that modest, readily achievable changes in rates favor formation or...