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 Fc-mediated 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 HIV-infected 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.

Keywords: HIV envelope; epitope; gp160; immunotoxin; monoclonal antibody.

FIG 1

Indirect IT assays and the relationship of cytotoxicity to other MAb functions. (A) Indirect IT assays on a panel of human anti-gp160 MAbs, grouped according to target epitopes (vertical bars on the left). CD4bs, CD4 binding site; CD4i, CD4 inducible; MPER, membrane proximal external region. MAb (1 μg/ml) with or without sCD4₁₈₃ (300 ng/ml) was added to H9/NL4-3 cells, and 1 h later RAC-conjugated anti-human IgG was added at 500 ng/ml. Three days later cell viability was assayed as MTS dye reduction. The results are displayed as percent cytotoxicity (means of triplicate values; SEMs are not shown). Each MAb was tested at least 2 times, and some were tested >10 times. (B) Comparison of different functional activities of selected MAbs. Three different anti-gp120 MAbs (top) and two anti-gp41 MAbs (bottom) were tested for binding to H9/NL4-3 cells by FACS with or without sCD4 (left; gray curve, negative control; the horizontal scale is the log fluorescent intensity), cytotoxicity by indirect IT assay with or without sCD4, neutralization of cell-free virus, and neutralization of cell-cell infection. Results represent the means and SEMs from triplicate determinations. Each MAb was tested from 2 to >10 times in each assay. (C) Correlation of cytotoxicity with other functions in the panel of MAbs. We performed correlation analyses to determine whether there was a correlation between the cytotoxic effect of a MAb (in the presence of CD4) and its other activities. The P values were determined using Pearson's method, and the R² values were determined by least-squares analysis. When multiple analysis corrections were applied, none of the correlations were significant. mfi, median fluorescence intensity.

FIG 2

The size of ITs directed against the CD4-binding site limits cytotoxicity, whereas size does not affect neutralization. ITs were targeted to the CD4-combining site of Env using either the Ab HY or CD4 itself. (A) Cytotoxicity of directly conjugated ITs for H9/NL4-3 cells determined using MTS dye reduction. Small ITs targeted to the CD4-binding site effectively killed the cells (left), whereas those based on intact IgG were much less efficient (right). The small ITs were scFv-HY fused to PE and monomeric sCD4₁₈₃ also fused to PE. The larger ITs include CD4-IgG2-RAC and HY-IgG1-RAC. The results obtained with these ITs were compared to those obtained with intact IgG-based ITs targeted to other epitopes: 7B2-RAC plus CD4-IgG2 and MAb 924-RAC (targeting the gp120 V3 loop). Data are means and SEMs for triplicate samples. Each IT was tested a minimum of 5 times. We made 7 and 4 distinct preparations of HY-RAC and CD4-IgG2-RAC, respectively. Each was biochemically characterized and shown to have RAC conjugated to the MAb. (B) Size does not limit the neutralization of cell-free NL4-3 virus by CD4 derivatives. Tetrameric CD4-IgG2 and single-chain sCD4₁₈₃ were tested for their ability to neutralize cell-free NL4-3 virus in a TZM-bl cell assay. Data represent means and SEMs from triplicate determinations.

FIG 3

Direct immunofluorescence demonstrates cooperative interactions between anti-gp41 MAb 7B2 and anti-gp120 MAbs. Uninfected H9 cells (A) or H9/NL4-3 cells (A, B, and C) were incubated for 1 h with the indicated unconjugated MAb (30 μg/ml) or no MAb. Then, Alexa Fluor 488-conjugated 7B2 was added (final concentration, 3 μg/ml) and cells were incubated for 16 h at 4°C. Ten thousand cells were analyzed by flow cytometry. (A) The shaded histogram was obtained in the absence of any labeled 7B2. (Left) Fluorescent (Fl) MAb 7B2 (blue histogram) did not bind to uninfected H9 cells; (center) fluorescent-MAb 7B2 bound to H9/NL4-3 cells (blue); this binding was completely blocked by unconjugated MAb 7B2 (green) but unaffected by the isotype control Ab (Iso) (red); (right) binding of fluorescent MAb 7B2 was significantly (***, P < 0.001) enhanced in the presence of anti-gp120 MAbs VRC01 (CD4bs specific), 924 (V3 loop), and 1.7b (gp120 bridging sheet). Assays were repeated 4 to 6 times. (B) Dose-response curve of enhancement of MAb 7B2 binding by unconjugated anti-gp120 MAbs and blocking by unconjugated MAb 7B2. Statistically significant enhancement (P < 0.001) was observed at concentrations of MAb 924 of ≥4.6 ng/ml and MAb 1.7b of ≥14 ng/ml and at all concentrations of CD4-IgG2 tested (≥0.5 ng/ml). (C) Enhancement or blocking of 7B2 binding by a broad panel of unconjugated MAbs tested at a concentration of 30 μg/ml. The vertical line indicates binding in the absence of cooperating MAb. Asterisks indicate statistical significance (**, P < 0.01; ***, P < 0.001). Results are representative of those from 2 or more separate experiments.

FIG 4

Cooperative interactions among gp120 and gp41 MAbs affect binding to H9/NL4-3 cells. Six different anti-gp120 MAbs (top two rows) and three anti-gp41 MAbs (bottom row) were directly conjugated to Alexa Fluor 488. The effects of a panel of 19 unconjugated anti-Env MAbs (30 μg/ml) on binding of the Alexa Fluor-conjugated MAb (3 μg/ml) to H9/NL4-3 cells were studied. Results are indicated as the median fluorescent intensity. The vertical lines indicate binding in the absence of interacting MAb. Error bars show SEMs.

FIG 5

Enhancement of IT activity by CD4 and CD4i MAbs but not by anti-CD4bs or V3 MAbs. The cytotoxic activity of 7B2-RAC on H9/NL4-3 cells was measured at different IT concentrations in the presence of unconjugated anti-gp120 MAbs at 10 μg/ml (CD4-IgG2 was used at 1 μg/ml). Triplicate samples were run, and the results are means and SEMs. Asterisks indicate the degree of significance for MAb-mediated enhancement of IT activity, as determined by a one-tailed, paired (by IT concentration) t test (*, P < 0.05; **, P < 0.01). Results are representative of those from 2 to 3 experiments.

FIG 6

Comparison of the effects of ITs on infected (H9/NL4-3) and transfected (293T/92UG or 293T/C97) cell lines. (A and B) Direct IT assay. The indicated cells were incubated with serial dilutions of 7B2-RAC in the presence or absence of sCD4₁₈₃ for 72 h, at which time MTS was added to measure cell viability. Nontransfected 293T cells served as specificity controls. The results are reported as percent cytotoxicity (means and SEMs for triplicate samples). (C) Indirect IT assay. Cell killing by MAbs to gp120 and gp41 was compared. Cells were incubated with the indicated MAb. One hour later, anti-human IgG conjugated to RAC (500 ng/ml) was added. Viability was measured 72 h later. (D) Binding of cell surface Env by MAbs, measured by indirect immunofluorescence and flow cytometry. Live cells (80,000) were incubated with the indicated MAb (3 μg/ml) for 1 h, washed, and incubated with fluorescent anti-human IgG (H+L chain). Cells were again washed and then fixed in 2% paraformaldehyde–PBS and analyzed by flow cytometry using an LSR II flow cytometer, capturing 10,000 events gated by forward and side scatter. (E) Direct immunofluorescence. Cells were incubated with Alexa Fluor 488-conjugated MAb 7B2 (18 μg/ml) overnight at 4°C, washed, and fixed in paraformaldehyde.

FIG 7

Relationship of IT toxicity and MAb binding to 293T/92UG cells. (A) Indirect IT assay. A panel of gp120 and gp41 MAbs (100 ng/ml) was tested for cytotoxicity on 293T/92UG cells in the presence or absence of sCD4₁₈₃. Results are means and SEMs from triplicate determinations. (B) Correlation of MAb binding and cytotoxicity. Percent cytotoxicity (x axis) is plotted against the median fluorescent intensity (MFI) (y axis), as determined by indirect immunofluorescence (2 μg/ml MAb) and flow cytometry. Correlations were performed by the method of Pearson. Neither correlation was statistically significant. Ag, antigen.