Identification of Human Anti-HIV gp160 Monoclonal Antibodies That Make Effective Immunotoxins

Abstract

The envelope (Env) glycoprotein of HIV is the only intact viral protein expressed on the surface of both virions and infected cells. Env is the target of neutralizing antibodies (Abs) and has been the subject of intense study in efforts to produce HIV vaccines. Therapeutic anti-Env Abs can also exert antiviral effects via Fcmediated effector mechanisms or as cytotoxic immunoconjugates, such as immunotoxins (ITs). In the course of screening monoclonal antibodies (MAbs) for their ability to deliver cytotoxic agents to infected or Env-transfected cells, we noted disparities in their functional activities. Different MAbs showed diverse functions that did not correlate with each other. For example, MAbs against the external loop region of gp41 made the most effective ITs against infected cells but did not neutralize virus and bound only moderately to the same cells that they killed so effectively when they were used in ITs. There were also differences in IT-mediated killing among transfected and infected cell lines that were unrelated to the binding of the MAb to the target cells. Our studies of a well-characterized antigen demonstrate that MAbs against different epitopes have different functional activities and that the binding of one MAb can influence the interaction of other MAbs that bind elsewhere on the antigen. These results have implications for the use of MAbs and ITs to kill HIVinfected cells and eradicate persistent reservoirs of HIV infection.IMPORTANCE There is increased interest in using antibodies to treat and cure HIV infection. Antibodies can neutralize free virus and kill cells already carrying the virus. The virus envelope (Env) is the only HIV protein expressed on the surfaces of virions and infected cells. In this study, we examined a panel of human anti-Env antibodies for their ability to deliver cell-killing toxins to HIV-infected cells and to perform other antiviral functions. The ability of an antibody to make an effective immunotoxin could not be predicted from its other functional characteristics, such as its neutralizing activity. Anti-HIV immunotoxins could be used to eliminate virus reservoirs that persist despite effective antiretroviral therapy.

Design andIn VivoCharacterization of Immunoconjugates Targeting HIV gp160

“…Others propose targeting the CD4-binding site (CD4bs) with either MAbs or sCD4 itself (1, 5-7, 9-11, 13). In a companion publication, we again confirmed the efficacy of MAb 7B2 plus sCD4 in killing infected cells and cells stably expressing transfected Env compared to that of highly potent broadly neutralizing MAbs, which killed only cells stably expressing Env and not infected cells (19). We also demonstrated that size limited the ability of ITs targeted against the CD4bs to kill HIV-infected cells.…”

“…We found that scFv and other monomeric constructs targeted to the CD4bs functioned well in vitro as ITs, whereas the same V regions in the context of intact Ig were 10 to 100 times less cytotoxic to infected cells (19). In contrast, intact IgG MAbs against the V3 loop of gp120 (MAb 924) and to the gp41 helix-loop region (MAb 7B2) were highly lethal to H9/NL4-3 cells.…”

“…Our efforts have primarily focused on comparing different anti-Env MAbs for their ability to target ITs to infected cells. We have consistently found that those targeted to the gp41 external helix-loop region (for example, MAb 7B2), when used with sCD4, were the most efficacious MAbs for delivering anti-HIV ITs to HIV-infected cells (4,8,18,19,41). To extend this observation, we tested ITs and ADCs based upon MAb 7B2 for safety and efficacy in both mice and SHIV-infected macaques.…”

“…The ADCs did not kill either nontransfected 293T cells or H9/NL4-3 cells. At these very high concentrations, 7B2-RAC was toxic to nontransfected 293T, but the IT retained specific toxicity even at nanogram concentrations on cells stably expressing gp160 (19).…”

“…If bars are not marked, the results were not statistically significantly different. (19). The lack of efficacy of IgG-based ITs to the CD4bs was not an artifact of the conjugation process, because the binding of the ITs to HIV-infected cells measured by flow cytometry was not affected by conjugation to ricin, and RAC-based ITs directed against other gp160 epitopes conjugated side-by-side, using the same linker and batch of ricin, were highly effective.…”