Structural and functional integrity of antigen receptors

Abstract
Antibodies play a pivotal role in the adaptive immune system due to their ability to specifically bind to a diverse
array of antigens. There are two structurally diverse forms of antibodies that have arisen throughout evolution:
1) immunoglobulin (Ig)-based antibodies, which are found in gnathostomes (i.e., jawed vertebrates); and 2)
variable lymphocyte receptor (VLR)-based antibodies which are found in agnathans (i.e., jawless vertebrates).
Gnathostomes generate Ig-based antibodies by two diversification methods. Primary diversification of Ig-based
antibodies occurs via V(D)J recombination where the variable antigen-binding exon is assembled from various
gene segment options to generate a diversity of binding specificities in developing B lineage cells. Secondary
diversification of Ig-based antibodies occurs through somatic hypermutation (SHM) which is mediated by
activation induced cytidine deaminase (AID) in activated germinal center B cells (mammals) or B cell-rich
lymphoid structures early in life (chickens). Agnathans have an alternative form of leucine rich repeat
(LRR)-based antibodies called variable lymphocyte receptors (VLRs). While there are many convergent
properties of Ig and VLR antibodies, there has been no evidence of SHM in VLRs despite the existence of a
homolog to AID in lamprey, which opens doors to evaluate the impact of Ig- and LRR-based structures in
tolerating diversification through an SHM-mediated protein-variation mechanism. Preliminary computational
data suggest that the diverse structures of Ig- and LRR-based antibodies may vary in their capacity to maintain
structural integrity during hypermutation, a process crucial for the expression of antibody diversity. These
results are consistent with a hypothesis that structural stability constraints may play a role in real-time protein
mutability potential. This proposal will test that hypothesis that Ig-based antibodies are more mutationally
tolerant than LRR-based antibodies, with two specific aims: (i) determine the mutational tolerance of Ig-based
and LRR-based antigen receptors in vitro, and (ii) define diversity and maturation of Ig and VLR substrates in
vivo. Aim 1 will evaluate the ability for large mutation libraries of sets of three Igs and three VLRs to continue to
be expressed and bind antigen. Aim 2 will compare VLR diversification and affinity maturation to a single chain
immunoglobulin-based antibody (i.e., VHH) to immunogens in vivo. The expected outcome of this proposal will
be to (i) deliver biophysical insights into the aptitude of evolutionarily distinct but functionally convergent
adaptive immune receptors for mutation-driven diversification, and (ii) begin to inform optimal substrate design
receptive to mutation-library-driven biotechnological evolution of biomedically relevant substrates.

Grant Number: 5F31AI186232-02
NIH Institute/Center: NIH
Principal Investigator: Jessica Chen