TAG HIV Basic Science, Vaccines, and Cure Project Blog

Developing Multi-Pronged Antibodies to Target the HIV Reservoir

In an example of publication kismet, three recent open access articles all converge in describing a new strategy for depleting the HIV reservoir. The research involves engineering antibodies or antibody-like molecules capable of simultaneously binding two targets: the CD3 receptor, a human protein expressed on T cells, and parts of the HIV envelope (Env) protein, which are typically displayed on the outside of infected CD4 T cells when the virus is active. The rationale is that the region of the antibody that targets HIV Env binds to infected cells, while the CD3-targeting region binds to passing T cells and activates them to kill the cell. The idea for this two-pronged—termed bispecific—antibody attack originated in cancer research, and one candidate that targets CD19 (a protein expressed by B cells) and CD3 is already FDA-approved as a second-line treatment for certain forms of acute lymphoblastic leukemia. 

In the Journal of Clinical Investigation, Julia Sung and colleagues report results obtained in vitro with “Dual-Affinity Re-Targeting” (DART) proteins designed to bind CD3 and HIV Env epitopes targeted by the broadly binding (but non-neutralizing) monoclonal antibodies A32 or 7B2. These DART proteins were able to significantly reduce HIV levels in cultured, HIV-infected CD4 T cells and also showed activity against latently infected CD4 T cells isolated from individuals on ART and exposed to the latency-reversing agent vorinostat.

In PLoS Pathogens, a research team from Gilead Sciences and MacroGenics, Inc. describe similar studies conducted with a larger array of candidate DART proteins targeting CD3 and one of several different HIV Env epitopes, including those bound by A32, 7B2 and the broadly neutralizing antibodies PGT121, PGT145, VRC01 and 10E8.  The most active DART proteins were those derived from PGT121, PGT145, A32 and 7B2, confirming and extending the results of Sung et al. Of potential importance for clinical development, modified versions of the DART proteins designed to allow for relatively infrequent dosing in humans maintained activity. 

The third study, by Amarendra Pegu and colleagues from the Vaccine Research Center at the National Institutes of Health, employed a bispecific antibody that attaches to CD3 and the HIV Env epitope recognized by the broadly neutralizing antibody VRC07. In experiments using latently infected CD4 T cells isolated from individuals on ART, the antibody was found to both activate HIV gene expression and reduce HIV DNA levels in most donor samples. 

An important, overarching concern with all these bispecific antibodies is the extent to which the CD3-binding region might cause generalized T cell activation. Many years ago, anti-CD3 antibodies were tested in people with HIV with the aim of broadly activating CD4 T cells as means to reverse latency and deplete the HIV reservoir, but the results were disastrous: the treatment led to a massive depletion of T cells, inflammatory cytokine release (leading to transient renal failure in one case) and no evident HIV reservoir reduction (see Prins et al,1999).

All three research groups conducted laboratory assessments of T cell activation, and found little or no evidence that it occurred in the absence of HIV Env expression; in other words, binding of the bispecific antibodies to both targets appeared necessary for inducing activation. However, Pegu et al also performed a safety assessment in SHIV-infected macaques receiving antiretroviral therapy and this revealed a sharp but short-lived drop in peripheral blood CD3+ T cells and a sizable, albeit transient, increase in levels of several inflammation-related cytokines (TNF-α, MIP-1β and IL-10). These immunological perturbations resolved within 24 hours and there were no increases in SHIV viral load or clinically evident adverse events, leading the researchers to conclude that: “overall, these short-term toxicity studies indicate that the treatment was well tolerated.”

Taken together, the three papers suggest that bispecific antibodies may have promise as an anti-reservoir strategy. A particular appeal of the approach is the capacity to recruit T cells to destroy HIV-infected cells without regard to their antigen specificity—this could be important because HIV-specific T cells are typically functionally compromised in HIV-positive people. Tempering enthusiasm somewhat are the macaque data showing transient T cell depletion and inflammation, a phenomenon that will need to be evaluated in further animal studies. Barring additional safety concerns arising, human trials appear likely given the involvement of Gilead Sciences (a company with a large HIV cure research program). 

J Clin Invest. 2015 Sep 28. pii: 82314. doi: 10.1172/JCI82314. [Epub ahead of print]

Dual-Affinity Re-Targeting proteins direct T cell-mediated cytolysis of latently HIV-infected cells.

Sung JA, Pickeral J, Liu L, Stanfield-Oakley SA, Lam CK, Garrido C, Pollara J, LaBranche C, Bonsignori M, Moody MA, Yang Y, Parks R, Archin N, Allard B, Kirchherr J, Kuruc JD, Gay CL, Cohen MS, Ochsenbauer C, Soderberg K, Liao HX, Montefiori D, Moore P, Johnson S, Koenig S, Haynes BF, Nordstrom JL, Margolis DM, Ferrari G.

Abstract

Enhancement of HIV-specific immunity is likely required to eliminate latent HIV infection. Here, we have developed an immunotherapeutic modality aimed to improve T cell-mediated clearance of HIV-1-infected cells. Specifically, we employed Dual-Affinity Re-Targeting (DART) proteins, which are bispecific, antibody-based molecules that can bind 2 distinct cell-surface molecules simultaneously. We designed DARTs with a monovalent HIV-1 envelope-binding (Env-binding) arm that was derived from broadly binding, antibody-dependent cellular cytotoxicity-mediating antibodies known to bind to HIV-infected target cells coupled to a monovalent CD3 binding arm designed to engage cytolytic effector T cells (referred to as HIVxCD3 DARTs). Thus, these DARTs redirected polyclonal T cells to specifically engage with and kill Env-expressing cells, including CD4+ T cells infected with different HIV-1 subtypes, thereby obviating the requirement for HIV-specific immunity. Using lymphocytes from patients on suppressive antiretroviral therapy (ART), we demonstrated that DARTs mediate CD8+ T cell clearance of CD4+ T cells that are superinfected with the HIV-1 strain JR-CSF or infected with autologous reservoir viruses isolated from HIV-infected-patient resting CD4+ T cells. Moreover, DARTs mediated CD8+ T cell clearance of HIV from resting CD4+ T cell cultures following induction of latent virus expression. Combined with HIV latency reversing agents, HIVxCD3 DARTs have the potential to be effective immunotherapeutic agents to clear latent HIV-1 reservoirs in HIV-infected individuals. 

Nat Commun. 2015 Oct 20;6:8447. doi: 10.1038/ncomms9447.

Activation and lysis of human CD4 cells latently infected with HIV-1.

Pegu A, Asokan M, Wu L, Wang K, Hataye J, Casazza JP, Guo X, Shi W, Georgiev I, Zhou T, Chen X, O'Dell S, Todd JP, Kwong PD, Rao SS, Yang ZY, Koup RA, Mascola JR, Nabel GJ.

Abstract

The treatment of AIDS with combination antiretroviral therapy (cART) remains lifelong largely because the virus persists in latent reservoirs. Elimination of latently infected cells could therefore reduce treatment duration and facilitate immune reconstitution. Here we report an approach to reduce the viral reservoir by activating dormant viral gene expression and directing T lymphocytes to lyse previously latent, HIV-1-infected cells. An immunomodulatory protein was created that combines the specificity of a HIV-1 broadly neutralizing antibody with that of an antibody to the CD3 component of the T-cell receptor. CD3 engagement by the protein can stimulate T-cell activation that induces proviral gene expression in latently infected T cells. It further stimulates CD8 T-cell effector function and redirects T cells to lyse these previously latent-infected cells through recognition of newly expressed Env. This immunomodulatory protein could potentially help to eliminate latently infected cells and deplete the viral reservoir in HIV-1-infected individuals. 

PLoS Pathogens

Published: November 5, 2015DOI: 10.1371/journal.ppat.1005233 

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

Derek D. Sloan, Chia-Ying Kao Lam, Alivelu Irrinki, Liqin Liu, Angela Tsai, Craig S. Pace, Jasmine Kaur, Jeffrey P. Murry, Mini Balakrishnan, Paul A. Moore, Syd Johnson, Jeffrey L. Nordstrom, Tomas Cihlar, Scott Koenig

Abstract

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. 

Author Summary

Current HIV therapies prevent AIDS by dramatically reducing, but not eliminating, HIV infection. A reservoir of HIV-infected cells persists during long-term antiviral therapy, and individuals are at increased risk to develop non-AIDS illnesses, e.g., accelerated heart, bone, or kidney disease. Novel strategies are thus needed to safely kill HIV-infected cells and reduce or eliminate the HIV reservoir. An emerging strategy to kill HIV-infected cells involves antibodies (Abs) that bind the HIV envelope protein (Env). Env can distinguish HIV-infected cells from uninfected cells, and some Env-specific Abs can kill HIV-infected cells by recruiting immune cells, e.g., NK cells and macrophages. Here, we developed a strategy to kill HIV-infected cells that is complementary to Env-specific Abs. We designed and evaluated Dual-Affinity Re-Targeting (DART) molecules that incorporate Env-binding specificities with a CD3-binding specificity to recruit and activate cytotoxic T cells. We report that HIVxCD3 DARTs potently and selectively kill HIV-infected cells. Furthermore, HIV DARTs perturb resting and activated viral reservoirs in cells isolated from individuals on antiviral therapy. This novel strategy may be an important element of future antiviral therapies that target the HIV reservoir.

T-Cell Exhaustion Biomarkers Linked to Time to Viral Load Rebound in the SPARTAC Trial

A study published in Nature Communications has drawn a fair amount of press coverage—and some hyperbolic headlines—by reporting that certain immunological biomarkers may be able to predict the likelihood of maintaining a low viral load after an antiretroviral therapy (ART) interruption.

The research involves a subset of participants from the SPARTAC trial, which randomized individuals with primary HIV infection to one of three arms: no treatment (the standard of care at the time the trial was conducted) or 48 or 12 weeks of ART followed by an interruption (the primary results were published in NEJM in 2013). Drawing on samples from a subset of participants who received 48 weeks of ART, analyses were conducted to assess if there were associations between a multitude of immunological biomarkers and the time it took for viral load to rebound to detectable levels after ART interruption (these analyses were not planned in the original SPARTAC protocol and are thus considered exploratory). Senior author John Frater from Oxford University originally described the work at CROI earlier this year (the webcast of his talk is available online). 

The crux of the results is that baseline (pre-ART) levels of three biomarkers linked to T cell exhaustion—PD-1, Tim-3 and Lag-3—were statistically associated with time to viral load rebound to over 400 copies after ART interruption. Participants with baseline levels of these exhaustion biomarkers on CD4 and CD8 T cells that were below the median for the cohort took longer to rebound, on average, compared to those with levels above the median. However, no such association was seen when the biomarkers were measured in samples taken immediately prior to the ART interruption (the only predictor of time to viral load rebound at this timepoint was total HIV DNA, as reported previously).

The researchers suggest that the study represents a step toward the development of biomarker algorithms capable of identifying ideal candidates for ART interruptions, based on the likelihood of post-treatment control of viral load. However, it is a very early step, as is stressed by the UK website NHS Choices, which published a commentary in response to a wildly overstated Daily Mail headline about the paper (“HIV breakthrough could lead to a CURE: Scientists identify markers on immune cells that 'predict who can stop drug therapy and stay well'”). There are also uncertainties as to whether post-treatment control necessarily equates to a state of health equivalent to that obtained by ART-mediated HIV suppression (see “The Challenge of Defining HIV Remission” in the most recent TAGline). 

In the discussion section of the paper, the authors note that anti-PD-1 antibodies have shown success as cancer immunotherapies and that there is interest in studying their therapeutic effects in HIV. In laboratory studies, anti-PD-1 antibodies have been shown to stimulate HIV production by latently infected CD4 T cells, and restore functionality to exhausted HIV-specific T cells. There are concerns, however, about the potential for adverse effects, particularly the induction of autoimmune responses (which has been observed in some cancer trials).

The uncertainty about the risk/benefit of anti-PD-1 antibodies in otherwise healthy HIV-positive people has prompted a research team led by Thomas Uldrick at the National Cancer Institute to design a trial that will enroll HIV-positive individuals with relapsed, refractory, or disseminated cancers. The main aims are to evaluate safety and anticancer effects; assessments of the HIV reservoir and HIV-specific T cell immunity are included as tertiary endpoints. The anti-PD-1 antibody that will be administered is pembrolizumab (trade name Keytruda), which recently gained FDA approval for the treatment of certain forms of melanoma and non-small cell lung cancer. With helpfully coincidental timing, the trial was recently entered in clinicaltrials.gov and is due to launch in January.

Nat Commun. 2015 Oct 9;6:8495. doi: 10.1038/ncomms9495.

Immunological biomarkers predict HIV-1 viral rebound after treatment interruption.

Hurst J, Hoffmann M, Pace M, Williams JP, Thornhill J, Hamlyn E, Meyerowitz J, Willberg C, Koelsch KK, Robinson N, Brown H, Fisher M, Kinloch S, Cooper DA, Schechter M, Tambussi G, Fidler S, Babiker A, Weber J, Kelleher AD, Phillips RE, Frater J.

Abstract

Treatment of HIV-1 infection with antiretroviral therapy (ART) in the weeks following transmission may induce a state of 'post-treatment control' (PTC) in some patients, in whom viraemia remains undetectable when ART is stopped. Explaining PTC could help our understanding of the processes that maintain viral persistence. Here we show that immunological biomarkers can predict time to viral rebound after stopping ART by analysing data from a randomized study of primary HIV-1 infection incorporating a treatment interruption (TI) after 48 weeks of ART (the SPARTAC trial). T-cell exhaustion markers PD-1, Tim-3 and Lag-3 measured prior to ART strongly predict time to the return of viraemia. These data indicate that T-cell exhaustion markers may identify those latently infected cells with a higher proclivity to viral transcription. Our results may open new avenues for understanding the mechanisms underlying PTC, and eventually HIV-1 eradication.