Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells

<div><p>HIV reservoirs and production of viral antigens are not eliminated in chronically infected participants treated with combination antiretroviral therapy (cART). Novel therapeutic strategies aiming at viral reservoir elimination are needed to address chronic immune dysfunction and non-AIDS morbidities that exist despite effective cART. The HIV envelope protein (Env) is emerging as a highly specific viral target for therapeutic elimination of the persistent HIV-infected reservoirs via antibody-mediated cell killing. Dual-Affinity Re-Targeting (DART) molecules exhibit a distinct mechanism of action via binding the cell surface target antigen and simultaneously engaging CD3 on cytotoxic T lymphocytes (CTLs). We designed and evaluated Env-specific DARTs (HIVxCD3 DARTs) derived from known antibodies recognizing diverse Env epitopes with or without broadly neutralizing activity. HIVxCD3 DARTs derived from PGT121, PGT145, A32, and 7B2, but not VRC01 or 10E8 antibodies, mediated potent CTL-dependent killing of quiescent primary CD4 T cells infected with diverse HIV isolates. Similar killing activity was also observed with DARTs structurally modified for in vivo half-life extension. In an ex vivo model using cells isolated from HIV-infected participants on cART, combinations of the most potent HIVxCD3 DARTs reduced HIV expression both in quiescent and activated peripheral blood mononuclear cell cultures isolated from HIV-infected participants on suppressive cART. Importantly, HIVxCD3 DARTs did not induce cell-to-cell virus spread in resting or activated CD4 T cell cultures. Collectively, these results provide support for further development of HIVxCD3 DARTs as a promising therapeutic strategy for targeting HIV reservoirs.</p></div

HIVxCD3 DARTs retarget cytolytic CD3⁺ T-cells to Env-expressing HIV-infected CD4⁺ T-cells.

<p>(A) Mechanism of cytolysis. The CD3 arm (orange) of the bi-specific DART binds to CD3 at the surface of CD3⁺ T-cells and the HIV arm (blue) binds to HIV Env at the surface of HIV-infected CD4⁺ T-cells. Cell surface Env may be in the form of functional mature trimers or nonfunctional variant forms such as cleaved or uncleaved gp160 monomers or gp41 stumps [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005233#ppat.1005233.ref043" target="_blank">43</a>]. DART-mediated engagement of target and effector cells results in activation of effector cell cytolytic responses and target cell killing. (B) Variety of Env epitopes targeted by HIVxCD3 DARTs. Locations on the mature HIV-1 Env trimer surface of epitopes recognized by the anti-Env Abs used as sources of the HIV binding arms of DARTs are shown. Broadly neutralizing Abs PGT121, PGT145, VRC01 and 10E8 target epitopes located in the V3 glycan (N332; green), V2 glycan (N160K, blue), CD4 binding site (CD4bs, orange) and gp41 MPER (cyan), respectively, that are preferentially expressed on functional Env trimers, whereas non-neutralizing Abs A32 and 7B2 target epitopes located in CD4-induced sites (CD4i conformation epitopes are not visible in the depicted pre-CD4 binding Env structure) and in the gp41 stalk (cyan), respectively, that are preferentially expressed on nonfunctional forms of Env. The depicted structure of Env trimer is derived from pdb 4NCO.</p

HIV DARTs induce CD8 T cell-dependent cytolysis of CD4 T cells infected with diverse HIV-1 isolates in vitro.

<p>Unstimulated CD4 T cells were infected with HIV-1 RW, IN, or BaL isolates and co-cultured with autologous CD8 T cells in the absence or presence of active DARTs (HIVxCD3) or control DART (RSVxCD3) at concentrations ranging from 0.6 to 2,000 pM and at a CD8 T cell:CD4 T cell ratio of 2:1 for 72 hours. Cytolytic activities were determined as described in Materials and Methods. HIVxCD3 DARTs with 6 different Env-specificities were evaluated. There was no appreciable killing mediated by 10E8xCD3 or VRC01xCD3 DARTs. Representative data with cells from a single participant are depicted. Results from multiple participants are summarized in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005233#ppat.1005233.t001" target="_blank">Table 1</a>.</p

Antigen binding properties of HIVxCD3 DARTs and parental anti-HIV Env IgGs.

<p>Binding of DARTs to (A) soluble recombinant human CD3 protein, to (B) JRFL gp140 protein, or to (C) both soluble recombinant human CD3 and JRFL gp140 proteins. Binding of IgGs with CDRs exactly matching those utilized in the HIV arms of HIVxCD3 DARTs to (D) JRFL gp140 protein was measured by ELISA, as described in Materials and Methods. WNV IgG (negative control) is an antibody to the envelope protein of West Nile virus.</p

Combinations of HIVxCD3 DARTs induce CD8 T cell-dependent cytolysis of CD4 T cells infected with HIV-1 in vitro.

<p>Unstimulated CD4 T cells were infected with HIV-1 BaL or IN and co-cultured with autologous CD8 T cells at a CD8 T cell:CD4 T cell ratio of 2:1 for 72 hours in the presence of indicated individual DARTs or DART combinations. Cytolytic activities were determined as described in Materials and Methods. Representative data with cells from a single participant are depicted. Results from multiple participants are summarized in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005233#ppat.1005233.t002" target="_blank">Table 2</a>.</p

HIVxCD3 DARTs in MP3 format and basic format induce the CD8 T cell-dependent killing of HIV-Infected CD4 T cells in vitro with comparable potency.

<p>Unstimulated CD4 T cells were infected with HIV-1 BaL and co-cultured with autologous CD8 T cells at a CD8 T cell:CD4 T cell ratio of 2:1 for 72 hours in the presence of either a regular format PGT121xCD3 DART or an extended half-life MP3 format PGT121xCD3 DART. After 72 hours of co-culture, the % reduction in p24-positive CD4 T cells for each condition relative to no DART control were determined by FACS. Representative data from a single participant are depicted.</p

HIVxCD3 DARTs reduce ex vivo HIV expression in PBMCs isolated from HIV-infected participants on suppressive cART.

<p>(A-B) Resting Model: Unstimulated PBMCs from 4 HIV-infected participants on suppressive cART were cultured with 400 pM of a DART combination (200 pM each of PGT121xCD3 and 7B2xCD3) or 400 pM of RSVxCD3. Supernatant HIV RNA was quantitated on Day 8 (A) and Day 14 (B). By Day 14, statistically significant reduction in vRNA level was observed in 3 out of 4 participants treated with the HIVxCD3 DART combination, but not with control DART compared to no DART (*p<0.05, **p<0.005, 2-tailed Mann-Whitney U-Test). Horizontal bars represent the medians for each group. (C) PKC Agonist-Inducible Model. PBMCs from 4 HIV-infected participants on suppressive cART were cultured untreated or treated with 1 μM indolactam, a PKC agonist, and with or without an HIVxCD3 DART combination (200 pM PGT121xCD3 + 200 pM 7B2xCD3) or control RSVxCD3 DART (400 pM). After 7 days of incubation, total CD4 T cells were isolated from PBMCs and re-stimulated with 1 μM indolactam. After an additional 3 days of incubation, supernatant HIV RNA was quantitated. In 2 out of 4 participants, HIVxCD3 DARTs significantly reduced the indolactam-induced vRNA vs. control DART or no DART (**p<0.005, *p<0.05, 2-tailed Mann-Whitney U-Test). Horizontal bars represent the medians for each group.</p

HIVxCD3 DARTs in basic and MP3 format.

<p>(A) Schematics of DARTs in basic and MP3 (Fc-bearing) format are shown. The anti-HIV and anti-CD3 domains are colored in blue and orange, respectively, and the human IgG1 Fc domains are shaded in grey. The chains of the Fc domain are modified to contain L234A/L235A mutations that inhibit Fc binding to activating FcγRs. The chains of the Fc domain do not inhibit binding to FcRn, which prolongs serum half-life and exposure. (B) Pharmacokinetic profile in human FcRn transgenic mice. The A32xCD3 MP3 DART was administered at 5 mg/kg by iv injection and serum concentrations were measured over time (closed circles). For comparison, the serum concentration-time curve for a basic DART with different specificities is shown (open squares). The PK parameters for the A32xCD3 MP3 DART are presented in the table.</p