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. 2022 Sep 7;19(1):143.
doi: 10.1186/s12985-022-01876-1.

HIV-1 bispecific antibody iMab-N6 exhibits enhanced breadth but not potency over its parental antibodies iMab and N6

Tumelo Moshoette  1 , Maria Antonia Papathanasopoulos  1 , Mark Andrew Killick  2
Affiliations

HIV-1 bispecific antibody iMab-N6 exhibits enhanced breadth but not potency over its parental antibodies iMab and N6

Tumelo Moshoette et al. Virol J. .

Abstract

The recently published AMP trial (HVTN 703/HPTN 081 and HVTN704/HPTN 085) results have validated broad neutralising antibodies (bNAbs) as potential anti-HIV-1 agents. However, single bNAb preparations are unlikely to cope with the onslaught of existing and de novo resistance mutations, thus necessitating the use of bNAb combinations to achieve clinically relevant results. Specifically engineered antibodies incorporating two bNAbs into a single antibody structure have been developed. These bispecific antibodies (bibNAbs) retain the benefits of bNAb combinations, whilst several conformations exhibit improved neutralisation potency over the parental bNAbs. Here we report on the engineering of a bibNAb comprising of an HIV-1 spike targeting bNAb N6 and a host CD4 targeting antibody ibalizumab (iMab). Antibodies were expressed in HEK293T cells and purified by protein-A affinity chromatography followed by size exclusion chromatography to achieve homogenous, monomeric, bibNAb preparations. Antibody purity was confirmed by SDS-PAGE whilst epitope specificity and binding were confirmed by ELISA. Finally, antibody breadth and potency data were generated by HIV-1 neutralisation assay (n = 21, inclusive of the global panel). iMab-N6 exhibited better neutralisation breadth (100% coverage) in comparison to its parental bNAbs iMab (90%) and N6 (95%). This is encouraging as exceptional neutralisation breadth is necessary for HIV-1 treatment or prevention. Unfortunately, iMab-N6 did not exhibit any enhancement in potency over the most potent parental antibody, iMab (p = 0.1674, median IC50 of 0.0475 µg/ml, and 0.0665 µg/ml respectively) or the parental combination, iMab + N6 (p = 0.1964, median IC50: combination 0.0457 µg/ml). This result may point to a lack of dual engagement of the bibNAb Fab moieties necessary for potency enhancement. Against the previously reported bibNAbs; iMab-CAP256, 10E08-iMab, and PG9-iMab; iMab-N6 was the lowest performing bibNAb. The re-engineering of iMab-N6 to enhance its potency, while retaining breadth, is a worthwhile endeavour due to its clinical potential.

Keywords: Bispecific antibodies; Bliss-Hill potency prediction; Broadly neutralizing antibodies; HIV-1 prevention; HIV-1 therapy.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Bispecific antibody design, purification and SDS-PAGE analysis. A Left panel: Schematic of iMab-N6 engineered using knob-in-a-hole mutations in the CH3 region of N6 and CrossMAbCH1_CL mutations in iMab. These mutations promote preferential assembly of iMab-N6 by limiting the formation of by-products. A Right panel: To show that these mutations do not affect the function of the antibody, iMab monoclonal antibody was engineered with knob-in-a-hole and CrossMAbCH1_CL mutations. B Chromatograms of size exclusion chromatography (SEC) purified antibodies with elution fractions corresponding to the correctly assembled antibody confirmation represented by the shaded rectangles. C Antibodies were resolved on a gradient SDS-PAGE post SEC under reduced and non-reduced conditions
Fig. 2
Fig. 2
Binding characterisation of purified antibodies by ELISA. A Left panel: HIV-1 Env targeting moieties (N6 and iMab-N6) were tested against gp140FVCGCN4 with iMab being used as a negative control. A Right panel: iMab-N6 was used at double the concentration of N6 to try and correct for the difference in binding affinity seen in A Left panel. B CD4 binding moieties (iMab and iMab-N6) were tested against 4dCD4 with N6 serving as an appropriate negative control
Fig. 3
Fig. 3
Comparison of antibody breadth and potency against a panel of 21 HIV-1 pseudoviruses. A IC50 heat map of the parental monoclonal antibodies alone, iMab and N6; antibody combination iMab + N6; and the bibNAb iMab-N6. The numbers in brackets next to iMab + N6 represent the fold difference in potency (red = enhancement, black = reduction) of iMab-N6 over iMab + N6. The numbers in brackets next to iMab-N6 represent the fold difference in potency (green = no enhancement) of iMab-N6 over parental N6 and iMab antibodies, respectively. Individual cells are colour-coded according to IC50 potency (number), with lower numbers and darker red representing greater potency. ND not done. B Scatter plot depicting IC50 values of dual sensitive HIV-1 pseudoviruses for each antibody with the median and IQR shown. Comparisons were done using non-parametric t-test (Wilcoxon matched-pairs signed rank test) with *p < 0.0167 and ***p < 0.001
Fig. 4
Fig. 4
Predicting the clinical utility of iMab-N6. A–F Scatter plots depicting IC80 values with median and IQR shown (A, C and E), and Breadth-Potency curves (B, D, and F) of each antibody against the full panel (n = 21) (A and B), dual sensitive viruses (n = 18) (C and D) and the expanded CATNAP virus panel (n = 97) (E and F). A–F Dotted lines represent the IC80 = 1 µg/ml threshold. A and C Statistical analysis were done using non-parametric t-test (Wilcoxon matched-pairs signed rank test) with *p < 0.0167, **p < 0.005 and ***p < 0.001. A–D bibNAb iMab-N6 exhibited median IC80 6–6.5× lower than 1 µg/ml AMP trial threshold and showed good neutralisation coverage of 90–94% at IC80 < 1 µg/ml. E and F N6 and iMab data was sourced from the CATNAP data base and used to generate iMab + N6, Bliss-Hill prediction data
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