Synergistic In Vitro Antiretroviral Activity of a Humanized Monoclonal Anti-CD4 Antibody (TNX-355) and Enfuvirtide (T-20)

Abstract

Recently, antiretroviral agents directed at several steps involved in viral entry have been shown to reduce viral replication in vitro and in vivo. We have demonstrated a high level of in vitro synergistic antiretroviral activity for two entry inhibitors that are directed at sequential steps in the entry process.Combination chemotherapy has had a profound effect on human immunodeficiency virus type 1 (HIV-1)-associated morbidity and mortality (8). Although there are now more than 20 approved antiretroviral chemotherapeutic agents, there is significant overlap in resistance patterns among these agents, effectively limiting the number of sequential antiretroviral regimens available to a given patient. Recent clinical trials have demonstrated that agents that either block the binding of HIV-1 to its chemokine coreceptors or prevent reconfiguration of the gp41 molecule significantly reduce the replication of HIV-1 in vivo (5,6,13,14). These in vivo observations have been complemented by a series of in vitro observations that demonstrate that antiviral agents acting at several steps of the entry cascade exhibit substantial antiviral synergism (15, 16). TNX-355 (formerly hu5A8) is a humanized monoclonal antibody that binds to a unique epitope in domain 2 of the CD4 molecule that is involved in the conformational change required for entry into target cells following binding of the virus to the CD4 molecule (2, 7). We report here in vitro studies that demonstrate the synergistic activity of TNX-355 and enfuvirtide against HIV-1.Peripheral blood mononuclear cells were isolated from HIV-1-uninfected donors by Ficoll-Hypaque density gradient centrifugation and grown in RPMI 1640 medium supplemented with 20% fetal calf serum, 5% interleukin-2, and 5 g/ml phytohemagglutinin (R-3 medium). Three-day phytohemagglutinin blasts (2 ϫ 10 6 ) were incubated with HIV-1 (100 50% tissue culture infective doses) and TNX-355, enfuvirtide, or both agents in 2.0 ml of medium overnight in 24-well tissue culture plates at 37°C in a humidified 5% CO 2 atmosphere. Peripheral blood mononuclear cells were washed three times in phosphate-buffered saline and resuspended in 2 ml of R-3 medium with replacement of the antiviral compound(s) being tested. HIV-1 p24 antigen production was assessed on days 4 and 7 of culture in cell-free supernatant fluid from each well by enzymelinked immunosorbent assay (Beckman Coulter, Miami, FL). Fresh R-3 medium with appropriate antiviral compound concentrations replaced the 0.5 ml of supernatant removed for p24 assay on day 4. Primary HIV-1 isolates (302076, 302077, 302143, 302054, and 301714) were provided by the National Institutes of Health AIDS Reference Reagent Repository. Human T-cell lymphotropic virus strain IIIB (HTLV-IIIB) was provided by Robert Gallo (Institute of Human Virology, Baltimore, MD). TNX-355 (Tanox, Inc., Houston, TX) was used at concentrations of 2.0, 0.4, 0.08, 0.016, 0.0032, and 0.00064 g/ml. Enfuvirtide (T-20; Trimeris, Inc., Durham, NC) was used at concentrations of 1.0, 0.2, 0.04, 0...

In VitroPre- and Post-Exposure Prophylaxis Using HIV Inhibitors as Microbicides Against Cell-Free or Cell-Associated HIV-1 Infection

“…MOI, multiplicity of infection. Previous studies using the HIV-1 inhibitors continuously present in peripheral blood mononuclear cell (PBMC) cultures against infection with ‡ SHIV-162P3 virus (Veazey et al, 2003); § Ba-L virus (Maeda et al, 2004); ¶ HIV IIIB virus (Zhang et al, 2006); # Ba-L virus (Van Herrewege et al, 2002); EC 50 , 50% effective compound concentration; MOI, multiplicity of infection; NF, not found. be used after a possible risk exposure.…”

“…Nevertheless, this class of compounds was previously shown to prevent cell-free virus infection in cervical tissue (Hu et al, 2004) and macaques (Veazey et al, 2003a(Veazey et al, , 2003b(Veazey et al, , 2005 and some binding inhibitors were active against cell-associated virus at high concentrations (Lu et al, 2006). Therefore, although binding/fusion inhibitors alone may be insufficient as single microbicides, they could make a valuable contribution to a combination microbicide strategy to protect non-infected target cells (Liu et al, 2005;Veazey et al, 2005;Sterjovski et al, 2006;Zhang et al, 2006).…”

Loss of Asparagine-Linked Glycosylation Sites in Variable Region 5 of Human Immunodeficiency Virus Type 1 Envelope Is Associated with Resistance to CD4 Antibody Ibalizumab

“…Consistent with the observed lack of cross-resistance, critical determinants of maraviroc and enfuvirtide resistance have been mapped to the V3 region and gp41, respectively, and do not appear to overlap with the determinants of ibalizumab resistance reported in this study. In vitro drug combination studies have demonstrated synergistic antiviral activities for ibalizumab and enfuvirtide (44) and additive-to-synergistic activity for ibalizumab and maraviroc (S. P. Weinheimer et al, unpublished data), providing further support for ibalizumab as a potentially valuable addition to antiretroviral drug regimens. However, more stud- 6.…”

Epitope Mapping of Ibalizumab, a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity in Infected Patients

“…Ibalizumab (formerly known as TNX-355) is a humanized IgG4 monoclonal antibody that blocks HIV-1 entry by binding to human CD4 (8,12,14,33). It was engineered from its mouse progenitor (5A8) by grafting the mouse complementary-determining region (CDR) onto a human IgG4 construct (4,5).…”