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Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117....."new modality for HIV-1 prevention, therapy and cure."
 
 
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Nature April 8 2015
 
"Given the difficulties in developing an HIV-1 vaccine and in eradicating established infection, passive transfer of monoclonal antibodies is being considered for HIV-1 prevention, therapy, and cure. Our data establish the principle that monoclonal antibodies can be both safe and effective against HIV-1 in humans. Antibody-mediated immunotherapy differs from currently available drugs in that it has the potential to affect the course of HIV-1 infection by engaging host immunity directly."
 
"Combinations of antiretroviral drugs are the standard of care for HIV-1 infection because resistance develops to single agents26. Similarly, monotherapy with 3BNC117 alone is insufficient to control infection, and we expect that antibody-drug or antibody-antibody combinations will be required for complete viraemic control. Although the current generation of drugs is less expensive than antibodies, the latter have very long half-lives and have the potential to kill infected cells and to enhance host immunity by engaging Fc receptors19, 27. Moreover, anti-HIV-1 antibodies can be made 100-fold more potent by molecular engineering28. Finally, the combination of antibodies with agents that activate latent viruses can interfere with the HIV-1 reservoir in hu-mice29 and may be critical to HIV-1 eradication strategies."
 
Marina Caskey1*, Florian Klein1*, Julio C. C. Lorenzi1, Michael S. Seaman2, AnthonyP.West Jr3,Noreen Buckley1, GiselaKremer4,5, Lilian Nogueira1, Malte Braunschweig1,6, Johannes F. Scheid1, Joshua A. Horwitz1, Irina Shimeliovich1, Sivan Ben-Avraham1, MaggiWitmer-Pack1, Martin Platten4,7, Clara Lehmann4,7, Leah A. Burke1,8, Thomas Hawthorne9, Robert J. Gorelick10, Bruce D. Walker11, Tibor Keler9, Roy M. Gulick8, Gerd Fa tkenheuer4,7, Sarah J. Schlesinger1 & Michel C. Nussenzweig1,12
 
1Laboratory of Molecular Immunology, The Rockefeller University, New York, New York 10065, USA. 2Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA. 3Division of Biology, California Institute of Technology, Pasadena, California 91125, USA. 4First Department of Internal Medicine, University Hospital of Cologne, D-50924 Cologne, Germany. 5Clinical Trials Center Cologne, ZKS Ko ln, BMBF 01KN1106, University of Cologne, Cologne, Germany. 6Albert Ludwigs University of Freiburg, 79085 Freiburg, Germany. 7German Center for Infection Research (DZIF), partner site Bonn-Cologne, Cologne, Germany. 8Division of Infectious Diseases, Weill Medical College of Cornell University, New York, New York 10065, USA. 9Celldex Therapeutics, Inc., Hampton, New Jersey 08827, USA. 10AIDS and Cancer Virus Program, Leidos Biomedical Research, Frederick, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, USA. 11Ragon Institute of MGH, MIT and Harvard, Howard Hughes Medical Institute, Massachusetts General Hospital and Harvard Medical School, Cambridge,Massachusetts 02139, USA. 12Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10065, USA.
 
HIV-1 immunotherapy with a combination of first generation monoclonal antibodies was largely ineffective in pre-clinical and clinical settings and was therefore abandoned1, 2, 3. However, recently developed single-cell-based antibody cloning methods have uncovered a new generation of far more potent broadly neutralizing antibodies to HIV-1 (refs 4, 5). These antibodies can prevent infection and suppress viraemia in humanized mice and nonhuman primates, but their potential for human HIV-1 immunotherapy has not been evaluated6, 7, 8, 9, 10. Here we report the results of a first-in-man dose escalation phase 1 clinical trial of 3BNC117, a potent human CD4 binding site antibody11, in uninfected and HIV-1-infected individuals. 3BNC117 infusion was well tolerated and demonstrated favourable pharmacokinetics. A single 30 mg kg-1 infusion of 3BNC117 reduced the viral load in HIV-1-infected individuals by 0.8-2.5 log10 and viraemia remained significantly reduced for 28 days. Emergence of resistant viral strains was variable, with some individuals remaining sensitive to 3BNC117 for a period of 28 days. We conclude that, as a single agent, 3BNC117 is safe and effective in reducing HIV-1 viraemia, and that immunotherapy should be explored as a new modality for HIV-1 prevention, therapy and cure.
 
Main
 
A fraction of HIV-1-infected individuals develop potent neutralizing serologic activity against diverse viral isolates4, 5. Single-cell cloning methods to isolate antibodies from these individuals12 revealed that broad and potent neutralization can be achieved by antibodies targeting many sites on the viral envelope5, 13, 14. Many of these antibodies can prevent infection, and some can suppress active infection in humanized mice (hu-mice) or macaques6, 7, 8, 9, 10. Therefore, it is generally accepted that a vaccine eliciting such antibodies is likely to be protective against HIV-1. However, potent anti-HIV-1 broadly neutralizing antibodies (bNAbs) are highly somatically mutated and many carry other uncommon features such as insertions, deletions, or long complementary determining regions4, 5, 11, 12, 15, which may account for the difficulty in eliciting such antibodies by immunization. In view of the efficacy of passive bNAb administration in hu-mice and macaques6, 7, 8, 9, 16, it has been suggested that bNAbs should be administered passively, or by viral vectors for prevention and immunotherapy4, 9, 16. However, their safety and efficacy has not been tested in humans.
 
To determine whether the new generation of more potent bNAbs are safe and active against HIV-1 in humans, we initiated an open label phase 1 study (Fig. 1a) with 3BNC117, an anti-CD4 binding site antibody cloned from a viraemic controller11. 3BNC117 neutralizes 195 out of 237 HIV-1 strains comprising 6 different clades with an average half-maximal inhibitory concentration (IC50) of 0.08 μg ml-1 (Extended Data Fig. 1)11. 12 uninfected and 17 HIV-1-infected individuals (Table 1) were administered a single intravenous dose of 1, 3, 10 or 30 mg kg-1 of 3BNC117 (Extended Data Table 1a). 3BNC117 serum concentrations, plasma HIV-1 viral loads (VL), CD4+ and CD8+ T-cell counts, and safety were monitored closely (Fig. 1a, Extended Data Figs 2, 3, and Extended Data Tables 1b, 2). The two groups were comparable for gender, race and age (Table 1).
 
Figure 1: Pharmacokinetics of 3BNC117 in uninfected and HIV-1-infected individuals.

Figure1.gif

a, Diagrammatic representation of the study. Time of 3BNC117 infusion indicated by the red arrow, and sampling for 3BNC117 serum levels, HIV-1 viral load, CD4+/CD8+ T cell counts and env sequencing as indicated below. b, Antibody decay measured in TZM.bl assays (solid lines) and ELISA (dotted lines). Mean values and s.e.m. for uninfected individuals (3 per group) are shown in blue and for HIV-1-infected individuals (2-5 per group) in red. Light grey indicates lower level of accuracy by the ELISA assay and dark grey by the TZM.bl assay. Open circles indicate levels lower than the accuracy threshold.
 
3BNC117 was generally safe and well tolerated at all doses tested in both uninfected and HIV-1-infected individuals. No grade 3, 4 or serious adverse events and no treatment-related laboratory changes were observed during 56 days of follow up (Extended Data Table 1b). CD4+ or CD8+ T-cell counts did not change after 3BNC117 infusion in the HIV-1-infected group, possibly because initial CD4+ T-cell counts were near normal in most participants (mean absolute CD4+ T-cell count was 655 cells per μl, Extended Data Fig. 2).
 
Two different assays were used to measure 3BNC117 levels in serum: TZM.bl neutralization assay to measure activity, and anti-idiotype-specific ELISA to measure antibody protein levels (Fig. 1b, Extended Data Fig. 3 and Extended Data Tables 4, 5). With few exceptions the two assays were generally in agreement in both groups (Fig. 1b and Extended Data Fig. 3). However, elimination of 3BNC117 activity was more rapid in the HIV-1-infected group, resulting in an estimated average t1/2 of around 9 days as opposed to around 17 days in uninfected individuals (Fig. 1b and Extended Data Tables 4, 5). We conclude that 3BNC117 has pharmacokinetic properties consistent with a typical human IgG1 in uninfected individuals and a somewhat faster decay rate in HIV-1-viraemic individuals. Similar antigen-dependent enhanced clearance has been reported with anti-cancer antibodies17. Although there may be other explanations, we speculate that the increased rate of antibody elimination in the presence of HIV-1 is due to accelerated clearance of antigen-antibody complexes.
 
Viral loads were measured by standard assays or by single-copy assays. Baseline VLs in HIV-1-infected individuals not on anti-retroviral therapy (ART) varied from 640 to 53,470 copies ml-1 (mean 9,420 copies ml-1). Two participants were on ART at the time of antibody infusion but had detectable baseline VLs (30 and 100 copies ml-1). (Table 1, Extended Data Table 2a).
 
Virologic responses correlated with antibody dose. Infected individuals receiving 1 or 3 mg kg-1 3BNC117 doses showed only small and transient changes in viraemia consisting of increases of up to threefold 1 day after infusion, followed by a short temporary decrease, and rapid return to baseline (Fig. 2, Extended Data Fig. 4, and Extended Data Table 2a). The magnitude and kinetics of the initial increase in viraemia were consistent with those seen with viral entry inhibitors18.
 
Figure 2: HIV-1 viral load measurements.

Figure2.gif

3BNC117 dose indicated in red. Plots (left column) show absolute VLs in HIV-1 RNA copies ml-1 (y axis) versus time in days after infusion (x axis). Right column shows log10 changes in VL from day 0. Red line illustrates the average (least-squares means, by mixed-effect linear model). Individual subjects are indicated on the right. Subjects 2E1 to 2E5 were pre-screened for 3BNC117 sensitivity. At 30 mg kg-1 dose level, the change in viraemia was significant (P = 0.004, P < 0.001, P < 0.001, P < 0.001, P < 0.011 at days 4, 7, 14, 21, and 28, respectively) when compared to all available pretreatment values (Extended Data Table 2b).
 
In contrast, 10 out of 11 individuals receiving 10 or 30 mg kg-1 infusions responded by dropping their VLs by up to 2.5 log10 (Fig. 2, Extended Data Fig. 4 and Extended Data Table 2a). Two individuals off ART received the 10 mg kg-1 dose, of whom 1 responded with 1.36 log10 decline in viraemia and the other did not (Fig. 2 and Extended Data Table 2a). The individual that did not respond was infected with a virus that was completely resistant to 3BNC117 (2C4; IC50 > 20 μg ml-1; Fig. 3 and Extended Data Table 3). All 8 individuals that received the 30 mg kg-1 dose of 3BNC117 showed highly significant and rapid decreases in their viral loads that varied between -0.8 and -2.5 log10 (Figs 2 and 3 and Extended Data Table 2a, b). The magnitude of the decline was related to the starting VL and the sensitivity of the subjects' autologous virus to 3BNC117 (Figs 2 and 3, Extended Data Fig. 5). The median time to reach the lowest level in viraemia was 7 days, and the mean drop in VL was 1.48 log10 at lowest level. When compared to all available pre-treatment measurements, the drop in viraemia was highly significant from days 4 through 28 (Fig. 2 and Extended Data Table 2b). Although the limited data set does not allow us to determine viral set point, 4 of the 8 individuals receiving a single 30 mg kg-1 infusion did not entirely return to day 0 pre-infusion levels during the observation period of 56 days (Figs 2 and 3, Extended Data Table 2a).

Figure3.gif

3BNC117 dose is indicated at the top of the graphs. The left y axis shows log10 change in viraemia from day 0, and right y axis shows antibody level measured by ELISA. Blue line reflects change in VL and dotted grey line antibody level. Numbers indicate IC50 values for 3BNC117 of autologous viral isolates measured by TZM.bl assay, colour-coded as indicated in the key. Dotted line indicates lower level of accuracy.
 
To further examine the virologic effects of 3BNC117 immunotherapy, autologous viral isolates were obtained from cultured PBMCs before (day 0, day -7) and after (day 28) antibody infusion. Paired samples from 12 of the 17 HIV-1-infected individuals were tested for 3BNC117 sensitivity (Fig. 3 and Extended Data Table 3). Samples obtained from individuals infused with 1 mg kg-1 showed 35- and 13.5-fold decreases in 3BNC117 sensitivity, indicating that the antibody exerts selective pressure on HIV-1 even at the lowest dose (2A3, 2A4; Fig. 3). Similar changes in sensitivity were seen for some (2B1, 2C5) individuals treated with 3 and 10 mg kg-1, but others remained 3BNC117-sensitive throughout (2B3) (Fig. 3, Extended Data Table 3). Similarly, at 30 mg kg-1, only 2 out of 5 individuals tested showed greater than fivefold reduction in 3BNC117 sensitivity on day 28 (Extended Data Table 3). In contrast, 2C1, 2D1, 2D3 showed only 3.2-, 1.3- and 2.7-fold changes in sensitivity and these individuals did not rebound to baseline viraemia levels at day 28 (Figs 2 and 3). We conclude that, in some individuals, HIV-1 develops high-level resistance to 3BNC117 by 28 days after a single dose, while in others it does not.
 
To examine the molecular nature of the changes in HIV-1 in response to 3BNC117, we cloned and sequenced HIV-1 envelopes from paired plasma samples from 10 individuals before and 28 days after infusion. Evidence for antibody-induced selection was seen in some but not all samples analysed (Fig. 4, Supplementary Fig. 1). For example, 2C5, who received a 10 mg kg-1 infusion, selected for a G459D mutation in 15 out of 23 env sequences, while the remainder showed a longer V5 loop. The G459D mutation alters the CD4 binding site and can result in resistance to 3BNC117 (ref. 9). Changes in the V5 loop can alter sensitivity to anti-CD4 binding site antibodies by steric clashing with the heavy or light chains of 3BNC117-type antibodies. Similarly, 10 or 30 mg kg-1 infusions selected single mutations at Q363H (2E1), S461D (2E2), and S274Y (2E2) (Fig. 4, Supplementary Fig. 1). These changes may alter sensitivity to 3BNC117 by interfering with binding5. Selection in these 3 individuals is also indicated by the emergence of a distinct group of closely related sequences in phylogenetic trees (Fig. 4, Supplementary Fig. 1). Consistent with the molecular analysis, and the viral culture data (Fig. 3), pseudoviruses produced from serum of 2C5 from days 0 and 28 showed high level 3BNC117 resistance, whereas the changes in pseudoviruses produced from 2C1, 2D1, 2E1 and 2E2 were modest (Fig. 4 and Extended Data Table 3). In contrast, autologous viral isolates from individuals who did not become resistant, or had only small changes in sensitivity, such as 2B3, showed little if any evidence of selection. We conclude that a single infusion of 3BNC117 leads to selection for high-level resistance in some but not all individuals.
 
Although immunotherapy was initially used to treat infectious diseases, the great majority of therapeutic monoclonal antibodies are currently used to treat cancer and autoimmune diseases. This form of therapy has been shown to be highly effective, well tolerated, and to function in large part by engaging the host immune system through Fc receptors19.
 
In contrast, a role for antibodies in controlling HIV-1 infection has been difficult to establish. For example, the overall course of infection is not thought to be altered in individuals that develop bNAbs20. Moreover, first generation anti-HIV-1 bNAbs with limited breadth and activity produced little if any measurable effects in hu-mice or viraemic individuals3, 4, 21. However, antibodies can put strong selective pressure on the virus in individuals that develop anti-HIV-1 antibody responses22, 23, 24. In addition, recent studies in hu-mice showed that, when administered as monotherapy, new generation bNAbs can transiently reduce VLs, and in combination they control viraemia for as long as concentrations remain in the therapeutic range6, 9. In contrast, single antibodies led to control of viraemia in SHIV-infected macaques for as long as antibody levels remained therapeutic, and immune escape was rarely observed7, 8. The surprising difference between hu-mice and macaques might be attributed in part to an intact host immune system in the macaque, including endogenous antibodies25, or differences between SHIV and HIV-1 infection. Our data establish that passive infusion of single bNAbs can have profound effects on HIV-1 viraemia in humans.
 
Combinations of antiretroviral drugs are the standard of care for HIV-1 infection because resistance develops to single agents26. Similarly, monotherapy with 3BNC117 alone is insufficient to control infection, and we expect that antibody-drug or antibody-antibody combinations will be required for complete viraemic control. Although the current generation of drugs is less expensive than antibodies, the latter have very long half-lives and have the potential to kill infected cells and to enhance host immunity by engaging Fc receptors19, 27. Moreover, anti-HIV-1 antibodies can be made 100-fold more potent by molecular engineering28. Finally, the combination of antibodies with agents that activate latent viruses can interfere with the HIV-1 reservoir in hu-mice29 and may be critical to HIV-1 eradication strategies.
 
Given the difficulties in developing an HIV-1 vaccine and in eradicating established infection, passive transfer of monoclonal antibodies is being considered for HIV-1 prevention, therapy, and cure. Our data establish the principle that monoclonal antibodies can be both safe and effective against HIV-1 in humans. Antibody-mediated immunotherapy differs from currently available drugs in that it has the potential to affect the course of HIV-1 infection by engaging host immunity directly.
 
 
 
 
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