Design of bioactive peptides based on antibody hypervariable region structures. Development of conformationally constrained and dimeric peptides with enhanced affinity.

W V Williams,J Vonfeldt,M I Greene,T Kieber-Emmons,D B Weiner

doi:10.1016/s0021-9258(19)67772-0

W V Williams, J Vonfeldt + Show 3 more

Open Access

https://doi.org/10.1016/s0021-9258(19)67772-0

Copy DOI

Abstract

The variable regions of antibody molecules bind antigens with high affinity and specificity. This binding is imparted largely by the hypervariable portions of the variable region. Hypervariable regions typically fold into reverse turn or loop structures. Peptides derived from antibody hypervariable region sequences can bind antigens with similar specificity, albeit with markedly lower affinity. In this study, cyclic and dimeric peptide analogs of an anti-idiotypic/antireceptor antibody hypervariable region were developed. This antibody (87.92.6) binds to reovirus type 3 receptors on cells as well as to a neutralizing anti-reovirus type 3 monoclonal antibody (9B.G5). The cyclic peptides were utilized to probe the optimal conformation for binding to both the receptor and 9B.G5. By dimerizing or constraining the conformation of these peptides, higher affinity binding was produced. By utilizing several different cyclic peptides, the optimal conformation for binding was established. The conformationally optimized cyclic peptide possessed greater than 40-fold higher affinity for the receptor and the idiotype than the linear analog. This study suggests that conformationally constrained and dimeric peptides derived from antibody hypervariable loop sequences can bind antigens (including receptors) with reasonable affinity. hypervariable loop sequences can bind antigens (including

Highlights

Peptide Cyclization-One way to evaluate the optimal folding conformation of the VL peptide is reflected by the ability of cysteine-containing variants to cyclize
If the cysteine residues are placed in various positions on one or the other side of a predicted reverse turn, the residues placed in the most energetically favorable locations for assuming a reverse turn structure should form disulfide bridges most rapidly
These peptides were subjected to oxidation by agitating a solution (2 mg/ml in 0.1 M NaHC03)at 37 "C for varying periods of time exposed to air

Summary

Design pe Reo

- 3 dinated by the chloramine-T method [10].Murine L cells (fibroblasts) were grown in Joklik's modified minimal essential medium with 10%. A 100 X 1-cm column (Bio-Rad) was constructed with Sephadex G-25 (superfine; Pharmacia LKB Biotechnologies, Inc) and equilibrated s), with distilled/deionized water This was utilized along with an inflow peristaltic pump(2232Microperpex ultraviolet monitor (2138 interaction between the unmodified VL peptide and both Uvicord S),fraction collector (2212Helirac), and chartrecorder To assess cyclization versus oligomerization, peptides were run following reduction with jj"mercaptoethano These aspects lower the net free energy of binding, making a X molar excess of 0-mercaptoethanol added to peptides diluted to 1 high affinity interactiondifficult to obtain. To decrease these mg/ml in distilled/deionized HZ0 and sealed under N2 for 1 h) and energy considerations, we havedevelopedconformationally were run on the Sephadex G-25 column followingequilibration constrained analogs otfhe VLpeptide.

MATERIALS AND METHODS

RESULTS

Design

DISCUSSION