Development of smart anti-glycan reagents using immunized lampreys

Tanya R Mckitrick,Hirotomo Nakahara,Max D Cooper,Brantley R Herrin,Charles S Rosenberg,Nicholas J Rivers,Richard D Cummings,Jamie Heimburg-Molinaro,Christoffer K Goth,Rosalia Falco,Alyssa M Mcquillan

doi:10.1038/s42003-020-0819-2

Abstract

Studies on the expression of cellular glycans are limited by a lack of sensitive tools that can discriminate specific structural features. Here we describe the development of a robust platform using immunized lampreys (Petromyzon marinus), which secrete variable lymphocyte receptors called VLRBs as antibodies, for generating libraries of anti-glycan reagents. We identified a wide variety of glycan-specific VLRBs detectable in lamprey plasma after immunization with whole fixed cells, tissue homogenates, and human milk. The cDNAs from lamprey lymphocytes were cloned into yeast surface display (YSD) libraries for enrichment by multiple methods. We generated VLRB-Ig chimeras, termed smart anti-glycan reagents (SAGRs), whose specificities were defined by microarray analysis and immunohistochemistry. 15 VLRB antibodies were discovered that discriminated between linkages, functional groups and unique presentations of the terminal glycan motif. The development of SAGRs will enhance future studies on glycan expression by providing sequenced, defined antibodies for a variety of research applications.

Highlights

Studies on the expression of cellular glycans are limited by a lack of sensitive tools that can discriminate specific structural features
Two weeks post-final injection, plasma from immunized and naive animals was collected and screened on the Consortium for Functional Glycomics (CFG) glycan microarray, which contains over 600 unique glycan structures
We considered that some glycan antigens might be present in the material that are below the typical detection by glycomic analyses, yet sufficient to induce antibody responses

Summary

Introduction

Studies on the expression of cellular glycans are limited by a lack of sensitive tools that can discriminate specific structural features. We describe the development of a robust platform using immunized lampreys (Petromyzon marinus), which secrete variable lymphocyte receptors called VLRBs as antibodies, for generating libraries of anti-glycan reagents. VLRB cells clonally expand and differentiate into plasma cells that secrete circulating antigenspecific VLRB proteins Such VLRBs are generated by recombinatorial assembly of hundreds of partial LRR gene segments, and this gene conversion-like assembly mechanism is capable of generating >1014 distinct receptors, comparable to the human Ab repertoire[17]. The resulting VLRB repertoire was expressed as a stable YSD library where we pursued different enrichment strategies, including glycan microarrays, labeled whole cells and lysates, to isolate distinct populations of anti-glycan antibodies. We defined a panel of mAbs prepared as Ig chimeras termed smart anti-glycan reagents (SAGRs), which have unique specificity and provide unprecedented possibilities to accurately dissect the expression and localization of distinct glycan epitopes. The stable genetic library can be screened by glycan microarray technologies and other outlined approaches to rapidly identify, enrich, and produce recombinant SAGRs that recognize specific glycan determinants

Objectives

Methods

Results

Conclusion