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