Safety, pharmacokinetics, and biological activity of CD4-mimetic BNM-III-170 in SHIV-infected rhesus macaques

Abstract

Anti-HIV-1 antibodies capable of mediating ADCC are elicited by the majority of people with HIV-1 and preferentially target the "open," CD4-bound conformation of HIV-1 envelope glycoproteins (Env). However, due to the "closed" conformation sampled by unliganded HIV-1-Envs, these antibodies are ineffective at eliminating infected cells. BNM-III-170 is a small-molecule CD4-mimetic compound that binds the Phe43 cavity of the gp120 subunit of Env, forcing Env to "open up," thus exposing epitopes targeted by CD4-induced (CD4i), ADCC-mediating antibodies. Here, we assessed the safety, pharmacokinetics, and biological activity of BNM-III-170 in uninfected and SHIV-AD8-EO-infected rhesus macaques (RMs). In uninfected RMs, single subcutaneous administrations of 3-36 mg/kg BNM-III-170 were well-tolerated, with serum half-lives ranging from 3 to 6 h. In SHIV-infected RMs, four different regimens were evaluated: 2 × 36 mg/kg daily, 1 × 24 mg/kg, 3 × 36 mg/kg every 7 days, and 3 × 36 mg/kg every 3 days. While toxicity was observed with daily doses, all other regimens demonstrated reasonable safety profiles. No changes in plasma viral loads were observed in SHIV-infected RMs following any of the evaluated BNM-III-170 dosing regimens. However, plasma collected following BNM-III-170 administration was shown to have increased binding to infected cells and to sensitize SHIV AD8-EO virions to neutralization by otherwise non-neutralizing antibodies. In addition, the plasma of treated animals mediated ADCC in the presence of BNM-III-170. These results establish a well-tolerated BNM-III-170 dosing regimen in SHIV-infected RMs and serve as proof of concept for its biological activity in promoting the targeting of infected cells by CD4i ADCC-mediating antibodies. Thus, they inform future studies evaluating CD4mc treatment in ART-treated animals.IMPORTANCEA therapeutic regimen able to eradicate or functionally cure HIV-1 remains elusive and may require a "shock-and-kill" approach to reactivate and then purge the latent HIV-1 reservoir. The small-molecule CD4-mimetic compound BNM-III-170 has previously been shown to (i) sensitize HIV-1-infected cells to ADCC mediated by plasma from people with HIV-1 (PWH) in vitro and (ii) significantly delay the time to viral rebound following ART interruption when combined with anti-CoRBS + anti-cluster A Abs or plasma from PWH in humanized mice. To evaluate the use of BNM-III-170 as part of a kill approach, we characterized the safety, pharmacokinetics, and biological activity of BNM-III-170 in uninfected and SHIV-infected RMs. Our study identifies a tolerable BNM-III-170 dosing regimen in SHIV-infected RMs and provides insights into its antiviral activities; as such, it informs future studies evaluating the efficacy of BNM-III-170 in reducing the viral reservoir.

Keywords: ADCC; CD4 mimetic; human immunodeficiency virus; in vivo therapeutic strategies; nonhuman primate.

Fig 1

BNM-III-170 pharmacokinetics in uninfected RMs and functional activity of bioavailable BNM-III-170. (A) Plasma levels of BNM-III-170 following intravenous administration of 3 mg/kg BNM-III-170 and subcutaneous administration of 3–36 mg/kg BNM-III-170 in uninfected RMs. (B) Experimental setup of the assay to measure binding of anti-CoRBS mAb 17b to Env expressed on CH058TF-infected cells following incubation with RM plasma containing bioavailable BNM-III-170. (C) Binding of 17b to CH058TF-infected cells following incubation with plasma collected from uninfected RMs at selected timepoints after subcutaneous 3–36 mg/kg BNM-III-170 administration. (D) 17b binding and bioavailable BNM-III-170 in plasma from select timepoints following subcutaneous administration of 36 mg/kg BNM-III-170. (E) Association between plasma levels of BNM-III-170 in RMs that received one SQ 36 mg/kg dose of BNM-III-170 and 17b binding to CH058TF-infected cells following incubation with plasma collected after SQ 36 mg/kg BNM-III-170 administration (Spearman correlation, n = 22).

Fig 2

Biological activity of BNM-III-170 against SHIV variants. (A) Infectivity of SHIV variants when incubated with the indicated concentrations of BNM-III-170. (B) Infectivity of SHIV variants when incubated with non-neutralizing CD4i antibody 19b in the presence or absence of 2 µM BNM-III-170. (C) 17b binding to CEM.NKr-CCR5-sLTR-Luc cells infected with SHIV variants in the absence (DMSO) or presence of the indicated concentrations of BNM-III-170 from three independent experiments. Statistical analyses were performed using Kruskal-Wallis with Dunnett’s multiple comparisons tests. (D) Binding of plasma from PWH (n = 9) to CEM.NKr-CCR5-sLTR-Luc cells infected with SHIV variants in the absence (DMSO) or presence of the indicated concentrations of BNM-III-170. Statistical analyses were performed using repeated-measures one-way ANOVA with Tukey’s multiple comparisons tests. Percent ADCC-mediated killing of CEM.NKr or primary CD4 T cells infected with SHIV variants in the absence (DMSO) or presence of 50 µM BNM-III-170 as measured by (E) luciferase-based and (F) FACS-based ADCC assays, respectively. Statistical analyses were performed using ordinary ANOVA with Dunnett’s multiple comparisons tests in C-D and Wilcoxon matched-pairs signed rank test in E-F. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001, ****P-value < 0.0001.

Fig 3

Summary of study design of subcutaneous BNM-III-170 treatment in SHIV AD8-EO-infected RMs. 36 mg/kg and 24 mg/kg doses of BNM-III-170 are represented by blue and red arrows, respectively. Two animals underwent necropsy (NX) due to an adverse event of unknown cause (animal 14C013) or weight loss due to diarrhea that started prior to BNM-III-170 dosing (animal 34901).

Fig 4

BNM-III-170 administration in viremic, SHIV AD8-EO-infected RMs results in increased neutrophils and monocytes and a non-specific reduction in lymphocytes. (A) WBC counts and neutrophil, lymphocyte, and monocyte percentages and counts in peripheral blood as determined by CBC panels. (B) Frequencies and absolute counts of CD4 T cells, CD8 T cells, B cells, and NK cells in PBMCs as determined by multi-color flow cytometry. CD4 T-cell and CD8 T-cell frequencies were reported as percentages of CD3+ lymphocytes and B-cell and NK cell frequencies as percentages of CD3- lymphocytes. Absolute counts were reported as cells per μL blood. Statistical analyses were performed using Wilcoxon matched-pairs signed rank tests comparing counts/frequencies from 0 h of the first dose and 24 h post-terminal dose for each treatment cycle for all animals. *P-value < 0.05, **P-value < 0.01.

Fig 5

Bioavailable BNM-III-170 in plasma of treated, SHIV AD8-EO-infected RMs is correlated with increased plasma binding to HIV-1-infected cells. (A) Plasma binding and bioavailable BNM-III-170 in plasma during each BNM-III-170 treatment cycle. Plasma binding to mock-infected (gray) or CH058TF-infected (black) CEM.NKr cells is plotted on the left y-axis. Plasma levels of BNM-III-170 (red) are plotted on the right y-axis. (B) Association between plasma levels of BNM-III-170 and plasma binding to CH058TF-infected CEM.NKr cells for timepoints from each respective BNM-III-170 treatment cycle (Spearman correlation).

Fig 6

Plasma viral loads for each BNM-III-170 treatment cycle. Plasma viral loads (SHIV RNA copies/mL) were measured by RT-qPCR at multiple timepoints during each BNM-III-170 dosing regimen. The dashed horizontal line represents the assay’s limit of detection (LOD; 60 copies/mL). The red and blue vertical lines represent 36 mg/kg and 24 mg/kg doses of BNM-III-170, respectively.

Fig 7

SHIV AD8-EO-infected RMs generate CD4i antibodies capable of binding to infected cells, mediating ADCC, and neutralizing virus following BNM-III-170 treatment. (A) Longitudinal CD4i antibody titers were measured via CD4-bound gp120core ELISA for all four animals. (B) Percent reduction in plasma binding to CD4 bound gp120core following 17b Fab blocking compared to no blocking. (C) Longitudinal anti-cluster A antibody titers were measured via stabilized gp120 inner domain protein (ID2) ELISA for all four animals. (D) Capacity of plasma from each animal collected at 0 h of each dose of BNM-III-170 to bind CH058TF-infected CEM.NKr cells in the presence or absence of 50 µM added BNM-III-170. (E) Capacity of plasma from each animal collected at 0 h of each dose of BNM-III-170 to mediate ADCC responses against CH58TF-infected CEM.NKr cells in the presence or the absence of 50 µM added BNM-III-170. Statistical analyses for D and E were performed using Wilcoxon matched-pairs signed rank tests. *P-value < 0.05, ** P-value < 0.01, ***P-value < 0.001, ****P-value < 0.0001. (F) Percent infectivity of SHIV AD8-EO following incubation with indicated dilutions of plasma collected from pre- and post-treatment timepoints during the 2 × 36 mg/kg BNM-III-170 treatment cycle with 1 day dosing interval.