Germlining of the HIV-1 broadly neutralizing antibody domain m36

Abstract

Engineered antibody domains (eAds) have emerged as a novel class of HIV-1 inhibitors and are currently under preclinical testing as promising drug candidates for prevention and therapy of HIV-1 infection. Reverse mutation of antibodies to germline sequences (germlining) could not only identify less mutated variants with lower probability of immunogenicity and other improved properties but also help elucidate their mechanisms of action. In this study, we sequentially reverted the framework (FRs) and complementary determining regions (CDRs) of m36, a human antibody heavy chain variable domain-based eAd targeting the coreceptor binding site of the viral envelope glycoprotein gp120, back to germline sequences. Two types of amino acid mutations and one region in the antibody V segment were identified that are critical for HIV-1 neutralization. These include four mutations to acidic acid residues distributed in the CDR1 and CDR2, two mutations to hydrophobic residues in the FR3 and CDR3, and partial FR2 and FR3 sequences flanking the CDR2 that are derived from a different gene family. An m36 variant with all five mutations in the FRs reverted back to germline showed slightly increased neutralizing activity against two HIV-1 isolates tested. Another variant with seven of twelve mutations in the V segment reverted retained potency within threefold of that of the mature antibody. These results, together with an analysis of m36-gp120-CD4 docking structures, could have implications for the further development of m36 and elucidation of its mechanism of potent and broad HIV-1 neutralization.

Keywords: Antibody domain; Germlining; HIV-1; Mutation; Neutralization.

Fig. 1

Design and generation of germlinized m36 variants. Antibody amino acid sequences are numbered and FRs and CDRs are defined according to the IMGT numbering system (http://www.imgt.org). Mutations in FRs and CDRs are highlighted with gray shading. In m36 variants, reverse mutations are indicated with single-letter amino acid code while unmutated sequences are represented by dots. The DNA fragments of m36m1 (I66Y), m36m2, m36m3 and m36m4 were synthesized commercially, digested with SfiI and cloned into pComb3X. M36m1 and its variants were generated by using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) with m36m1 (I66Y) and m36m1, respectively, as a template according to the manufacturer’s instructions. All m36 variants, which contain both the hexahistidine and FLAG tags at their C termini, were expressed in Escherichia coli HB2151 and purified from the soluble periplasmic fraction by using the Ni-NTA resin as described previously (Chen et al., 2008a).

Fig. 2

Binding and neutralizing activity of m36 variants with back mutated FRs. (A) ELISA binding to gp120_Bal-CD4 and SPA. ELISA was performed as described previously (Chen et al., 2008a). Antibodies binding to gp120_Bal-CD4 coated on 96-well plates at a concentration of 2 μg ml⁻¹ were detected by using the horseradish peroxidase (HRP)-conjugated mouse anti-FLAG tag antibody. HPR-conjugated SPA was directly used to detect binding to antibodies coated on 96-well plates at a concentration of 2 μg ml⁻¹. EC₅₀s were calculated by fitting the data to the Langmuir adsorption isotherm. (B) Pseudovirus neutralization assay. Bal and JRFL are two R5-tropic HIV-1 primary isolates from clade B. Viruses pseudotyped with HIV-1 Envs were produced in 293T cells and the assay was performed in duplicate with HOS-CD4-CCR5 cells as target cells according to previously published protocols (Chen et al., 2008a).

Fig. 3

Neutralizing activity of m36 variants with back mutated CDRs. Bal and JRFL are two R5-tropic HIV-1 primary isolates from clade B. Viruses pseudotyped with HIV-1 Envs were produced in 293T cells and the assay was performed in duplicate with HOS-CD4-CCR5 cells as target cells according to previously published protocols (Chen et al., 2008a).

Fig. 4

Molecular docking of m36 onto a gp120-CD4 crystal structure. The crystal structures of gp120_JRFL and CD4 and the model of m36 are in magenta, blue and green, respectively. The bridging sheet and V3 loop of gp120 and the CDRs of m36 are designated. Side chains of the Q44 residue in m36 FR2 and the R315 residue in gp120 V3 loop are shown as overlapping spheres. Residues predicted to be important for antibody-antigen interaction are highlighted in the squares with their side chains represented by stick and sphere models. The black dashed lines in the upper right square indicate possible formation of salt bridges or hydrogen bonds between the residues. The Z-DOCK program implanted into the Accelrys Discovery studio (http://accelrys.com) was used to dock m36 onto the surface of gp120_JRFL-CD4. Before docking, the m36 and gp120_JRFL-CD4 structures were processed as the following: Water molecules were deleted and hydrogen atoms were added at pH7.4 and an ionic strength of 0.145, and in a dielectric environment of 10; energy was minimized based on CHARMM with a cutoff of 0.9; loop regions were rebuilt according to SEQRES data; energy was used to judge the efficacy of the geometry optimization; and cysteine bridges in these proteins were defined as blocked regions. Initially produced docked poses of m36 were filtered and re-ranked to obtain top 200 poses based on ZRank scores using electrostatic and desolvation energy and non-deterministic FFT optimization, the latter were then visually scrutinized.