Side chain requirements for affinity and specificity in D5, an HIV-1 antibody derived from the VH1-69 germline segment

Joseph S Harrison,Lauren K Regula,Jonathan R Lai,Alex Stewart

doi:10.1186/1471-2091-14-9

Abstract

BackgroundAnalysis of factors contributing to high affinity antibody-protein interactions provides insight into natural antibody evolution, and guides the design of antibodies with new or enhanced function. We previously studied the interaction between antibody D5 and its target, a designed protein based on HIV-1 gp41 known as 5-Helix, as a model system [Da Silva, G. F.; Harrison, J. S.; Lai, J. R., Biochemistry, 2010, 49, 5464–5472]. Antibody D5 represents an interesting case study because it is derived from the VH1-69 germline segment; this germline segment is characterized by a hydrophobic second heavy chain complementarity determining region (HCDR2) that constitutes the major functional paratope in D5 and several antibodies derived from the same progenitor.ResultsHere we explore side chain requirements for affinity and specificity in D5 using phage display. Two D5-based libraries were prepared that contained diversity in all three light chain complementarity determining regions (LCDRs 1–3), and in the third HCDR (HCDR3). The first library allowed residues to vary among a restricted set of six amino acids (Tyr/Ala/Asp/Ser/His/Pro; D5-Lib-I). The second library was designed based on a survey of existing VH1-69 antibody structures (D5-Lib-II). Both libraries were subjected to multiple rounds of selection against 5-Helix, and individual clones characterized. We found that selectants from D5-Lib-I generally had moderate affinity and specificity, while many clones from D5-Lib-II exhibited D5-like properties. Additional analysis of the D5-Lib-II functional population revealed position-specific biases for particular amino acids, many that differed from the identity of those side chains in D5.ConclusionsTogether these results suggest that there is some permissiveness for alternative side chains in the LCDRs and HCDR3 of D5, but that replacement with a minimal set of residues is not tolerated in this scaffold for 5-Helix recognition. This work provides novel information about this high-affinity interaction involving an antibody from the VH1-69 germline segment.

Highlights

Analysis of factors contributing to high affinity antibody-protein interactions provides insight into natural antibody evolution, and guides the design of antibodies with new or enhanced function
While the HCDR1 regions are highly similar between both antibodies, an S30R mutation in CR6261 was shown to be a specificity determinant in its interaction with HA [14]. These results suggest that, while the hydrophobic HCDR2 may serve as a critical anchor point to engage in antigen recognition, other regions could play an important role in specificity determination
Specificity Profiles of D5 and CR6261 Given the similarity of HCDR1 and HCDR2 among D5, CR6261 and the common VH1-69 germline segment [6,11,12,13,14,15], we sought to explore the degree of specificity of these two antibodies toward their native antigens

Summary

Introduction

Analysis of factors contributing to high affinity antibody-protein interactions provides insight into natural antibody evolution, and guides the design of antibodies with new or enhanced function. We previously studied the interaction between antibody D5 and its target, a designed protein based on HIV-1 gp known as 5-Helix, as a model system [Da Silva, G. We previously studied the interaction between the HIV-1 antibody D5 and its target (a protein mimic of HIV-1 gp known as ‘5-Helix’) as a model system for antibody-protein recognition (Figure 1a) [5,6,7]. Antibodies that bind proteins with high affinity contain extensively mutated (i.e., evolved) complementarity determining regions (CDRs); the lower mutation rate of D5 suggests that some naïve antibodies may (a)

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