# Uniquely constrained peptides for modulating TNF receptor activity

> **NIH NIH F32** · UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN · 2022 · $67,582

## Abstract

PROJECT SUMMARY
There is a steady rise in the clinical use of biologics, particularly monoclonal antibodies (mAbs). We present
lasso peptides as a potential drug modality within the underleveraged region of chemical space between mAbs
and small molecules. Lasso peptides represent a class of ribosomally-synthesized and post-translationally
modified peptide (RiPP). Biosynthesis of lasso peptides involves a genetically encoded precursor peptide and
maturation enzymes that catalyze a macrolactam ring and configure the peptide into a unique lariat knot. Lasso
peptides recapitulate the desired properties of synthetic cyclic peptide but with key advantages: (i) lasso peptides
are uniquely constrained, highly stable globular structures with the correct shape to engage peptide-binding
targets, (ii) lasso peptides are entirely genetically encoded, straightforward generation of large and sequence-
diverse libraries, (iii) enzymatic tolerance supports modification of the entire solvent-exposed surface, non-
natural amino acids, and post-translational modifications, and (iv) the C-terminus provides a facile conjugation
site for peptide display, and high-throughput screening. Due to their unique topology, lasso peptides cannot be
chemical synthesized, thus we rely on the biosynthetic enzymes to thread the peptide. This proposal will
design a biosynthesis-informed library of lasso peptides and as a proof-of-concept, use this library to
select for lasso peptides that bind OX40. OX40 is an immunostimulatory member of the tumor necrosis factor
receptors (TNFR), studies have found that the native OX40 ligand, OX40L, and agonist antibodies can activate
OX40 to stimulate the proliferation and activation of T cells. There is a compelling need to validate immune
targets so that therapeutics can be developed, complementing the success of anti-PD1 (ie Keytruda) and anti-
TNFα (ie Humira) biologics. Our group has developed a high-throughput method with cell-free biosynthesis
(CFB) to produce lasso peptide libraries. In Aim I we will combine CFB with mRNA display to identify library
members that are successfully cyclized. Aim II will select for lasso peptides that bind OX40. We will assess
structure-activity relationships by validating the three-dimensional structure of the OX40-binding variants and
conduct binding affinity studies and in vitro signaling assays to evaluate their activity. Motifs related to OX40
binding and activity will be identified through structural and mutagenesis data. Improved understanding of
receptor binding-activity relationship will significantly inform pharmaceutical efforts. This fellowship will provide
the necessary training to prepare me for an academic career in mentoring researchers and teaching students.
My goal is to develop my abilities to become a more comprehensive scientist through improving communication
in both oral and written form, a strong collaborative work ethic, and mentorship of undergraduate and graduate
students. Working wi...

## Key facts

- **NIH application ID:** 10387498
- **Project number:** 1F32GM145122-01
- **Recipient organization:** UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
- **Principal Investigator:** Ashley M. Kretsch
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $67,582
- **Award type:** 1
- **Project period:** 2022-02-16 → 2023-02-15

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10387498

## Citation

> US National Institutes of Health, RePORTER application 10387498, Uniquely constrained peptides for modulating TNF receptor activity (1F32GM145122-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10387498. Licensed CC0.

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