AIDS Res Hum Retroviruses 6:465C479. infected cell lines that were Idasanutlin (RG7388) 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 conversation of other MAbs that bind elsewhere around the antigen. These Mouse monoclonal to KDM3A results have implications for the use of MAbs and ITs to kill HIV-infected cells and eradicate persistent reservoirs of HIV contamination. IMPORTANCE There is increased interest in using antibodies to treat and remedy HIV infection. Antibodies can neutralize free computer virus and kill cells already carrying the computer virus. The computer 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 computer virus reservoirs that persist despite effective antiretroviral therapy. values were decided using Pearson’s method, and the values, determined by Pearson’s correlation, are not corrected for multiple comparisons. values in strong are those that remained statistically significant after correction for multiple hypothesis testing via the use of both stringent (Bonferroni correction) and more lenient (Benjamini-Hochberg false discovery rate) criteria. It has been established that for an IT to effectively kill a target cell, some portion of the IT-antigen must be internalized and routed to the endosome-Golgi apparatus (33). Those immune complexes that are routed to lysosomes do not make effective ITs, unless brokers that disable lysosomal function are used (34). It has also been exhibited that ITs that Idasanutlin (RG7388) bind to epitopes more proximal to the plasma membrane are more effective than those that bind to epitopes on the same antigen more distal to the plasma membrane, and it has been suggested that this facilitates the conversation of the toxic moiety with the membrane (35). Unless Idasanutlin (RG7388) it is known whether and how an antigen-MAb complex is usually internalized and routed intracellularly, it is not possible to predict which MAbs will make effective ITs. To facilitate the screening of MAbs for their efficacy as ITs, we used an indirect assay in which cells were first incubated with a given MAb and then incubated with anti-human IgG conjugated to ricin A chain (RAC). This assay has been shown to be highly predictive in determining whether a MAb will make an effective IT when it is conjugated to RAC (36,C38) (Fig. 1A). The results highlight three target regions on Env where the cytotoxicity of a MAb was observed: the CD4-binding site (CD4bs), the V3 loop of gp120, and the gp41 HR/loop region. Not surprisingly, the addition of sCD4 inhibited the cytotoxic efficacy of MAbs against the CD4bs. As previously observed (13), sCD4 greatly enhanced the killing by anti-gp41 MAbs. However, not all anti-gp41 MAbs would necessarily make effective Idasanutlin (RG7388) ITs, since Idasanutlin (RG7388) they bind to different epitopes. For example, the neutralizing anti-membrane proximal external region (MPER) MAbs 2F5, 4E10, and 10E8 showed almost no cytotoxic activity. The anti-V3 MAbs fell into three groups: (i) MAbs 447-52D and 2191, which bind to the tip of the V3 loop, which killed the best, and which showed improvement in binding in the presence of sCD4; (ii) MAbs 2219, PGT121, and PGT126, which were directed against complex epitopes around the sides or base of the loop and which killed the best in the absence of CD4, and (iii) the remainder of the MAbs, which showed marginal evidence of killing..