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Three-in-One Antibody Protects Monkeys from SHIV - HIV-Like Virus
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Download the PDF here
Download the PDF here
NIH Press Release: Three-in-One Antibody Protects Monkeys from HIV-Like Virus - NIH and Sanofi Scientists Prepare to Test Antibody in People
Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques
Science Sept 20, 2017
Abstract
The development of an effective AIDS vaccine has been challenging due to viral genetic diversity and the difficulty in generating broadly neutralizing antibodies (bnAbs). Here, we engineered trispecific antibodies (Abs) that allow a single molecule to interact with three independent HIV-1 envelope determinants: 1) the CD4 binding site, 2) the membrane proximal external region (MPER) and 3) the V1V2 glycan site. Trispecific Abs exhibited higher potency and breadth than any previously described single bnAb, showed pharmacokinetics similar to human bnAbs, and conferred complete immunity against a mixture of SHIVs in non-human primates (NHP) in contrast to single bnAbs. Trispecific Abs thus constitute a platform to engage multiple therapeutic targets through a single protein, and could be applicable for diverse diseases, including infections, cancer and autoimmunity.
Among the classes of bnAbs, we found that trispecific Abs derived from bnAbs with CD4bs, MPER, and V1V2 glycan specificities had broad specificity, were potent and could be produced in sufficient quantities to allow evaluation in NHP, and eventually in humans. When tested in NHPs with viruses resistant to individual parental bnAbs, the trispecific Ab demonstrated complete protection against both viruses whereas infection was established in most animals treated with individual parental antibodies VRC01 and PDGM1400. In addition, the ability of this trispecific Ab to target three independent epitopes may improve treatment efficacy in humans.
The availability of a single protein that targets multiple independent epitopes on virus also reduces the potential generation of escape mutations. This advantage in part could relate to the presence of three independent binding specificities at all times in contrast to mixtures of antibodies where selective pressure by individual mAbs with shorter half-lives may wane.
In HIV-1 infected patients, reductions in viral load have been observed after one infusion of a single bnAb, thus demonstrating biological activity of HIV bnAbs (31-34). A modest extension of viral rebound was also observed when individual bnAbs were infused after antiretroviral drugs were discontinued in previously suppressed HIV-infected subjects (32, 33). NHP and human passive transfer studies have also suggested that such bnAbs can enhance anti-viral immunity that may contribute to improved viral control (35, 36). In addition, NHP studies demonstrate the importance of mAb potency and prolonged antibody half-life in mediating protection against infection (26, 29). The generation of trispecific Abs with improved potency and breadth may further enhance the efficacy of either passive immunity or passive-active immunization strategies.
The administration of a bispecific antibody to the human cytokines IL-4 and IL 13, which uses a related format and linkers (44), may provide guidance in this regard. This bispecific antibody has been evaluated in humans where single subcutaneous doses of SAR156597, ranging from 10-300 mg/kg, were well tolerated in healthy subjects, with low titers of ADA in only 4 of 36 subjects (44). Importantly, it showed a mean half-life of about two weeks (44), similar to natural monoclonal antibodies.
FULL TEXT BELOW FOLLOWING NIH Press RELEASE
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NIH and Sanofi Scientists Prepare to Test Antibody in People: "Sanofi is manufacturing the trispecific antibody for use in a Phase 1 clinical trial that will be conducted by NIAID to test the antibody's safety and pharmacokinetics in healthy people beginning in late 2018. Discussions also are under way with the NIAID-funded AIDS Clinical Trials Group to conduct a separate Phase 1 clinical trial of the antibody in people living with HIV."
September 20, 2017
NIH Press Release
Three-in-One Antibody Protects Monkeys from HIV-Like Virus
The three-pronged antibody, created by investigators from the National Institutes of Health (NIH) and the Paris-based pharmaceutical company Sanofi, also stopped a greater number of HIV strains from infecting cells in the laboratory more potently than natural, single antibodies. This new broadly neutralizing antibody binds to three different critical sites on HIV.
Plans are under way to conduct early-phase clinical trials of the "trispecific" antibody in healthy people and in people living with HIV in the hope that it could eventually be used for long-acting HIV prevention and treatment. By binding to three different sites on the virus, the new antibody should be harder for HIV to dodge than natural, single antibodies.
"Combinations of antibodies that each bind to a distinct site on HIV may best overcome the defenses of the virus in the effort to achieve effective antibody-based treatment and prevention," said Anthony S. Fauci, M.D., director of the National Institute of Allergy and Infectious Diseases, part of NIH. "The concept of having a single antibody that binds to three unique sites on HIV is certainly an intriguing approach for investigators to pursue."
The trispecific antibody reported today was created and tested through a collaborative research and development agreement between NIAID and Sanofi under the leadership of John R. Mascola, M.D., director of the NIAID Vaccine Research Center (VRC), and Sanofi Chief Scientific Officer and Senior Vice President Gary J. Nabel, M.D., Ph.D. The three HIV-binding segments of the antibody are based on three individual HIV antibodies, each of which powerfully neutralizes many strains of the virus. Scientists at NIAID and the IAVI Neutralizing Antibody Consortium of The Scripps Research Institute in La Jolla, California, previously isolated these individual antibodies from people living with HIV.
Researchers at the VRC and Sanofi tested dozens of bispecific and trispecific antibodies in the laboratory to find the best-performing combination. Individual antibodies were combined into trispecific antibodies using technology proprietary to Sanofi. The most successful formula combines the unique structures of the broadly neutralizing HIV antibodies called VRC01, PGDM1400, and 10E8v4.
VRC scientists tested this trispecific antibody in an experiment involving monkeys and two strains of SHIV. One SHIV strain is sensitive to neutralization by VRC01 and the trispecific antibody, but resistant to neutralization by PGDM1400. The other SHIV strain is sensitive to neutralization by PGDM1400 and the trispecific antibody, but resistant to neutralization by VRC01. Scientists from the Center for Virology and Vaccine Research at Beth Israel Deaconess Medical Center in Boston provided these SHIV strains.
The VRC researchers gave infusions of VRC01 to eight monkeys, PGDM1400 to another eight monkeys, and the trispecific antibody to a third group of eight monkeys. Five days later, the scientists exposed all 24 monkeys to both SHIV strains. Five of the eight monkeys that received PGDM1400 and six of the eight monkeys that received VRC01 became infected with SHIV, but none of the monkeys that received the trispecific antibody became infected.
Sanofi is manufacturing the trispecific antibody for use in a Phase 1 clinical trial that will be conducted by NIAID to test the antibody's safety and pharmacokinetics in healthy people beginning in late 2018. Discussions also are under way with the NIAID-funded AIDS Clinical Trials Group to conduct a separate Phase 1 clinical trial of the antibody in people living with HIV.
"The partnership between NIAID and Sanofi has been invaluable and allows us to move this trispecific antibody from the lab and preclinical testing into the clinic," said Dr. Mascola.
The ability of trispecific antibodies to bind to three independent targets at once could make them a useful prototype for treatments developed not only for HIV but also for other infectious diseases, autoimmune diseases and cancers, according to the study authors.
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Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques
Ling Xu,1* Amarendra Pegu,2* Ercole Rao,1 Nicole Doria-Rose,2 Jochen Beninga,1 Krisha McKee,2 Dana M. Lord,1 Ronnie R. Wei,1 Gejing Deng,1 Mark Louder,2 Stephen D. Schmidt,2 Zachary Mankoff,2 Lan Wu,1 Mangaiarkarasi Asokan,2 Christian Beil,1 Christian Lange,1 Wulf Dirk Leuschner,1 Jochen Kruip,1 Rebecca Sendak,1 Young Do Kwon,2 Tongqing Zhou,2 Xuejun Chen,2 Robert T. Bailer,2 Keyun Wang,2 Misook Choe,2 Lawrence J. Tartaglia,3,4 Dan H. Barouch,3,4 Sijy O'Dell,2 John-Paul Todd,2 Dennis R. Burton,4,5 Mario Roederer,2 Mark Connors,6 Richard A. Koup,2 Peter D. Kwong,2 Zhi-yong Yang,1 John R. Mascola,2† Gary J. Nabel1†
1Sanofi, 640 Memorial Drive, Cambridge, MA 02139, USA. 2Vaccine Research Center, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA. 3Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA. 4Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA. 5Department of Immunology and Microbiology, IAVI Neutralizing Antibody Center, Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA. 6National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.
Abstract
The development of an effective AIDS vaccine has been challenging due to viral genetic diversity and the difficulty in generating broadly neutralizing antibodies (bnAbs). Here, we engineered trispecific antibodies (Abs) that allow a single molecule to interact with three independent HIV-1 envelope determinants: 1) the CD4 binding site, 2) the membrane proximal external region (MPER) and 3) the V1V2 glycan site. Trispecific Abs exhibited higher potency and breadth than any previously described single bnAb, showed pharmacokinetics similar to human bnAbs, and conferred complete immunity against a mixture of SHIVs in non-human primates (NHP) in contrast to single bnAbs. Trispecific Abs thus constitute a platform to engage multiple therapeutic targets through a single protein, and could be applicable for diverse diseases, including infections, cancer and autoimmunity.
A variety of broadly neutralizing antibodies (bnAbs) have been isolated from HIV-1 infected individuals (1-3), but their potential to treat or prevent infection in humans may be limited by the potency or breadth of viruses neutralized (4, 5). The targets of these antibodies have been defined based on an understanding of the HIV-1 envelope structure (6-9). While bnAbs occur in selected HIV-1 infected individuals, usually after several years of infection, it remains a challenge to elicit them by vaccination because broad and potent HIV-1 neutralization often requires unusual antibody characteristics, such as long hypervariable loops, interaction with glycans, as well as a substantial level of somatic mutation. Strategies have thus shifted from active to passive immunization to both protect against infection and to target latent virus (10-14). We and others have begun to explore combinations of bnAbs that optimize potency and breadth of protection, thus reducing the likelihood of resistance and viral escape (15-17). Antibodies directed to the CD4bs, MPER, and variable region glycans are among the combinations that so far provide optimal neutralization (18). In addition, alternative combinations have also been investigated for the immunotherapy of AIDS, by directing T lymphocytes to activate latent viral gene expression and enhance lysis of virally-infected cells (19, 20). Given that multiple antibodies may help to reduce the viral replication that sustains chronic HIV-1 infection, we report here the generation of multi-specific antibodies designed to increasing the efficacy of HIV therapy.
Design of bispecific antibodies and evaluation of neutralization breadth
Although individual anti-HIV-1 bnAbs can neutralize naturally occurring viral isolates with high potency, the percentage of strains inhibited by these mAbs varies (21, 22). In addition, resistant viruses can be found in the same patients from whom bnAbs were isolated, suggesting that immune pressure against a single epitope may not optimally protect or treat HIV-1 infection. We hypothesized that the breadth and potency of HIV-1 neutralization by a single antibody could be increased by combining the specificities against different epitopes into a single molecule. This strategy would be expected to not only improve efficacy, but also simplify both treatment regimens and the regulatory issues required for clinical development. To test this concept, we initially incorporated prototype bnAbs to the CD4bs and MPER sites into a modified bispecific Ab. When two variable regions are linked in tandem, the distal site typically retains its ability to bind antigen while the proximal binding is markedly diminished. We therefore utilized an alternative configuration, termed CODV-Ig, which introduced linkers and inverted the order of the antibody binding site in light and heavy chains to alter the orientation of the variable regions, allowing each region to interact with their target (23). Several known bnAbs were evaluated, including VRC01, 10E8, PGT121, and PGT128 [reviewed in (1)] for their ability to neutralize a select panel of viruses with known resistance or sensitivity to these antibodies (fig. S1). Initially, we determined whether the position of the variable regions from VRC01 and 10E8 in the proximal or distal positions (Fig. 1A) could affect neutralization breadth and potency. Inclusion of both variable regions in either orientation in the bispecific antibody reduced the number of resistant strains compared to the parental antibodies alone (Fig. 1B). Better potency was observed when VRC01 was proximal and 10E8 distal, though neither bispecific antibody was as potent as a mixture of the two antibodies alone.
To explore whether other bnAbs could perform better in the bispecific format, we evaluated two different combinations, namely VRC01 plus PGT121, or VRC01 plus PGT128. For PGT121, expression was observed only with VRC01 in the distal position. When this antibody was compared to the parental antibodies alone, it provided marginally better neutralization (Fig. 2A). In contrast, VRC01 could be expressed with PGT128 in both positions, with greater breadth observed when VRC01 was distal (Fig. 2B). Together, these data indicated that improvements in breadth could be achieved with a bispecific format; however, the potency was not consistently improved compared to each Ab alone. We therefore sought an alternative format to improve the potency and breadth of neutralization.
Generation and comparison of broad and potent trispecific antibodies
To achieve our goal, we used a previously undescribed trispecific Ab format. Three specificities were combined by using knob-in-hole heterodimerization (24) to pair a single arm derived from a normal immunoglobulin (IgG) with a double-arm generated in the CODV-Ig. A panel of bnAbs was evaluated, including those directed against the CD4bs that included VRC01 and N6, as well as PGT121, PGDM1400 and 10E8 (fig. S1). A modified version of the latter, termed 10E8v4, was used because of its greater solubility (25). We first determined which bispecific arms showed the best potency, breadth and yield. This screening analysis revealed that combinations which contained PGDM1400, CD4bs, and 10E8v4 showed the highest level of production and greatest potency of neutralization (fig. S2).
We then evaluated different combinations of single arm and double arm specificities from PGDM1400, CD4bs, and 10E8v4 Abs for their expression levels and activity against a small panel of viruses (fig. S3), leading ultimately to the identification of trispecific antibodies VRC01/PGDM1400-10E8v4 and N6/PGDM1400-10E8v4 as lead candidates. When analyzed against a panel of 208 viruses (18) and compared to the parental antibodies alone, the highest neutralization potency and breadth was observed with N6/PGDM1400-10E8v4, with only 1 of the 208 viruses showing neutralization resistance and a median IC50 of less than 0.02 μg/ml (Fig. 3A). VRC01/PGDM1400-10E8v4 also displayed high potency and breadth, and only 4 resistant viruses were found. While some parental mAbs displayed either high breadth (e.g., 10E8, N6) or high potency (PGDM1400), none displayed a combination of breadth and potency as optimal as the trispecific Abs (Fig. 3B). For example, the most potent and broad parental mAb, N6, was around 5-fold less potent than the N6/PGDM1400-10E8v4 trispecific Ab and targeted only a single epitope, which could increase the chance of viral escape mutations. Importantly, as a single recombinant protein, the trispecific Abs demonstrated potency and breath superior to any single antibody yet defined (Fig. 3 and fig. S4). We also determined the binding affinity of each component of the trispecific Ab and compared each to its parental Fab. The equilibrium binding constant, Kd, of each binding site in the trispecific Ab, determined by surface plasmon resonance (SPR), was comparable to the affinity of the parental Fab, with PGDM1400 showing a slight decrease (~3-fold), and VRC01 and 10E8v4 exhibiting approximately a log increase in affinity (fig. S5). In addition, the trispecific Ab was able to bind sequentially to each of the three antigens (Fig. 3C), indicating that there is independent binding of each epitope.
The N6 trispecific Ab also showed greater potency and breadth compared to three related bispecific Abs when tested against a panel of 20 viruses that were selected for resistance to bnAbs (table S1). This finding is consistent with previous studies comparing the efficacy of mixtures of two vs. three bnAbs (18) and provides additional support for the multi-targeting concept. In addition to their greater efficacy, the trispecific Abs also yielded higher protein levels and greater solubility than the bispecific model (see fig. S2A vs. fig. S3), facilitating large scale production and clinical translation.
Discussion
Next generation HIV bnAbs
A hallmark of HIV infection is the remarkable genetic diversity of the virus. Since 2010, significant progress has been made in the identification of bnAbs that show exceptional breadth and potency (reviewed in ref. 1). Several of these antibodies have progressed into clinical trials for prevention or treatment, and there is renewed interest in exploring their potential in the clinical management of HIV infection (5, 12, 14). Here, we explored the potential of different bnAbs to combine into a single protein that confers protection against diverse HIV strains. Among the classes of bnAbs, we found that trispecific Abs derived from bnAbs with CD4bs, MPER, and V1V2 glycan specificities had broad specificity, were potent and could be produced in sufficient quantities to allow evaluation in NHP, and eventually in humans. When tested in NHPs with viruses resistant to individual parental bnAbs, the trispecific Ab demonstrated complete protection against both viruses whereas infection was established in most animals treated with individual parental antibodies VRC01 and PDGM1400. In addition, the ability of this trispecific Ab to target three independent epitopes may improve treatment efficacy in humans.
In HIV-1 infected patients, reductions in viral load have been observed after one infusion of a single bnAb, thus demonstrating biological activity of HIV bnAbs (31-34). A modest extension of viral rebound was also observed when individual bnAbs were infused after antiretroviral drugs were discontinued in previously suppressed HIV-infected subjects (32, 33). NHP and human passive transfer studies have also suggested that such bnAbs can enhance anti-viral immunity that may contribute to improved viral control (35, 36). In addition, NHP studies demonstrate the importance of mAb potency and prolonged antibody half-life in mediating protection against infection (26, 29). The generation of trispecific Abs with improved potency and breadth may further enhance the efficacy of either passive immunity or passive-active immunization strategies.
Although bnAbs show exceptional breadth and potency, resistant viral strains have been detected in patients who make these Abs (6, 37) and among natural viral isolates (38-40), raising the concern that resistance and escape mutations may arise. Such escape mutations are produced frequently with antiviral drug therapy (41), and countermeasures to reduce the likelihood of escape would increase the likelihood of developing a globally relevant therapy. Such breadth of coverage might alternatively be generated by administering multiple bnAbs, and protective efficacy in a NHP model has recently been demonstrated against a mixture of SHIV viruses using an antibody cocktail (42), providing further support for the multi-targeting concept. Combination mAb therapy increases the complexity, development pathway, cost and regulatory burdens of their use for treatment or prevention, in contrast to a single biologic therapy. The potency of the trispecific Abs described here also exceeds that of a broad and potent recombinant form of CD4 (43), termed eCD4-Ig (fig. S4), and this latter molecule is also directed to a single, albeit highly conserved, HIV Env epitope. The availability of a single protein that targets multiple independent epitopes on virus also reduces the potential generation of escape mutations. This advantage in part could relate to the presence of three independent binding specificities at all times in contrast to mixtures of antibodies where selective pressure by individual mAbs with shorter half-lives may wane.
Clinical translation
The trispecific Abs have not yet been evaluated for safety and efficacy in humans. While initial characterization of their half-life in NHPs suggests that they behave similarly to conventional antibodies, the question remains as to whether they could be immunogenic in vivo. The administration of a bispecific antibody to the human cytokines IL-4 and IL 13, which uses a related format and linkers (44), may provide guidance in this regard. This bispecific antibody has been evaluated in humans where single subcutaneous doses of SAR156597, ranging from 10-300 mg/kg, were well tolerated in healthy subjects, with low titers of ADA in only 4 of 36 subjects (44). Importantly, it showed a mean half-life of about two weeks (44), similar to natural monoclonal antibodies. While further human trials are needed to assess the full potential of the trispecific Ab platform, the data from the NHP challenge study described here, as well as the previous experience in humans with bispecific Abs (44), suggests that the approach merits further clinical investigation. Studies in HIV-infected subjects, alone or in combination with other immune interventions, will address the potential of trispecific Abs to provide durable protective immunity against infection or sustained viral control in HIV infected subjects during drug holidays or in the absence of antiretroviral therapy. The recognition of independent target sites with multi-specific antibodies can also be applied to other infectious diseases, cancer, and autoimmunity. These antibodies can promote recognition and binding to critical antigenic determinants on target cells and simultaneously allow engagement of immune cells that can stimulate relevant effector function without the complications and expense of delivering multiple recombinant proteins.
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