Selection, characterization, and thermal stabilization of llama single domain antibodies towards Ebola virus glycoprotein

Abstract

Background: A key advantage of recombinant antibody technology is the ability to optimize and tailor reagents. Single domain antibodies (sdAbs), the recombinantly produced variable domains derived from camelid and shark heavy chain antibodies, provide advantages of stability and solubility and can be further engineered to enhance their properties. In this study, we generated sdAbs specific for Ebola virus envelope glycoprotein (GP) and increased their stability to expand their utility for use in austere locals. Ebola virus is extremely virulent and causes fatal hemorrhagic fever in ~ 50 percent of the cases. The viral GP binds to host cell receptors to facilitate viral entry and thus plays a critical role in pathogenicity.

Results: An immune phage display library containing more than 10⁷ unique clones was developed from a llama immunized with a combination of killed Ebola virus and recombinantly produced GP. We panned the library to obtain GP binding sdAbs and isolated sdAbs from 5 distinct sequence families. Three GP binders with dissociation constants ranging from ~ 2 to 20 nM, and melting temperatures from ~ 57 to 72 °C were selected for protein engineering in order to increase their stability through a combination of consensus sequence mutagenesis and the addition of a non-canonical disulfide bond. These changes served to increase the melting temperatures of the sdAbs by 15-17 °C. In addition, fusion of a short positively charged tail to the C-terminus which provided ideal sites for the chemical modification of these sdAbs resulted in improved limits of detection of GP and Ebola virus like particles while serving as tracer antibodies.

Conclusions: SdAbs specific for Ebola GP were selected and their stability and functionality were improved utilizing protein engineering. Thermal stability of antibody reagents may be of particular importance when operating in austere locations that lack reliable refrigeration. Future efforts can evaluate the potential of these isolated sdAbs as candidates for diagnostic or therapeutic applications for Ebola.

Keywords: Antibody engineering; Ebola virus; Glycoprotein; Single domain antibodies; Virus like particles.

Fig. 1

Representative EBOV GP binding sdAbs selected after three rounds of biopannings. The sdAbs were divided into sequence families based on similarity of their CDRs. These 10 sdAbs include representatives from the different families. Each of these sdAb clones was produced and characterized

Fig. 2

Sequence modification to enhance physicochemical properties. a EBOV-GP-A8-neg+ sequence has four amino acid substitutions within FR1 (Q5V, A6E, A13V, and G17D), while EBOV-GP-G6-neg+ in b and EBOV-GP-H7-neg+ in c have only two substitutions in FR1, Q5V, A6E. All of the mutants have an insertion of a disulfide bond formed by two substituted Cys, A54C and I78C, within FR2 and FR3 indicated in a-c. EBOV-GP-A8-fneg+ sequence in a has the same sequence as EBOV-GP-A8-neg+ except with replaced C106S, which eliminates an unpaired Cys thus disrupting the potential formation of an inter-disulfide bond. d The underlined GGGGSGGGGKKK (GSKKK) sequence in bold font is fused onto the C-terminus of EBOV-GP-A8-fneg+, EBOV-GP-G6-neg+, and EBOV-GP-H7-neg+. Following each clone name the suffix “neg” indicates mutations in FR1 that result in the addition of negative charges, and the “+” indicates the addition of one disulfide bond formed by two substituted Cys in FR2 and FR3. The “f” denotes “fixing” the EBOV-GP-A8 sdAb through mutating an unpaired Cys to Ser in FR3 as indicated in a

Fig. 3

Mutations mapped onto the sdAb structure. The ribbon structure is transparent with the CDRs colored red, green and blue for CDR 1, 2, and 3 respectively. The yellow sticks are the canonical disulfide. The orange sticks are the residues that were changed to make the new disulfide. The red sticks are the VE residues near the N-terminus. The green sticks are the other changes made to EBOV-GP-A8-fneg+. Positions that have been changed by mutagenesis are indicated with the IMGT number of the amino acid that was mutated. The base structure (PDB 5LZ0) is a llama sdAb [50]

Fig. 4

Comparison of EBOV GP binding for sdAb-GSKKK fusions. Bt-sdAb-neg+-GSKKK fusions (b, d, f) and Bt-sdAb-neg+ (a, c, e) were used as tracers to detect EBOV GP captured by conjugated mAb KZ52, mAb 4F3 and EBOV-GP-A8-fneg+

Fig. 5

Binding of EBOV GP binders to EBOV VLPs. Three Bt-sdAb-neg+-GSKKK fusions were used as tracer to detect captured EBOV VLPs by conjugated mAb, KZ52, mAb 4F3, and EBOV-A8-fneg+-GSKKK. Among three fusions, Bt-G6-neg+-GSKKK b and Bt-A8-fneg+-GSKKK c have higher signals than Bt-H7-neg+-GSKKK a and both detect VLPs as low as 1.85 μg/mL

Fig. 6

Comparison of VLP binding among EBOV-sdAbs and their corresponding engineered constructs using ELISA. a Binding of captured EBOV VLPs at various concentrations is assessed using Bt-EBOV-GP-A8 and its engineered constructs as tracers. b Comparison of binding of VLPs to EBOV-sdAbs and their GSKKK fusions