Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants.

Although plant biotechnology has been widely investigated for the production of clinical-grade monoclonal antibodies, no antibody products derived from transgenic plants have yet been approved by pharmaceutical regulators for clinical testing. In the Pharma-Planta project, the HIV-neutralizing human monoclonal antibody 2G12 was expressed in transgenic tobacco (Nicotiana tabacum). The scientific, technical and regulatory demands of good manufacturing practice (GMP) were addressed by comprehensive molecular characterization of the transgene locus, confirmation of genetic and phenotypic stability over several generations of transgenic plants, and by establishing standard operating procedures for the creation of a master seed bank, plant cultivation, harvest, initial processing, downstream processing and purification. The project developed specifications for the plant-derived antibody (P2G12) as an active pharmaceutical ingredient (API) based on (i) the guidelines for the manufacture of monoclonal antibodies in cell culture systems; (ii) the draft European Medicines Agency Points to Consider document on quality requirements for APIs produced in transgenic plants; and (iii) de novo guidelines developed with European national regulators. From the resulting process, a GMP manufacturing authorization was issued by the competent authority in Germany for transgenic plant-derived monoclonal antibodies for use in a phase I clinical evaluation. Following preclinical evaluation and ethical approval, a clinical trial application was accepted by the UK national pharmaceutical regulator. A first-in-human, double-blind, placebo-controlled, randomized, dose-escalation phase I safety study of a single vaginal administration of P2G12 was carried out in healthy female subjects. The successful completion of the clinical trial marks a significant milestone in the commercial development of plant-derived pharmaceutical proteins.

Delay of HIV-1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies.

To determine the protective potential of the humoral immune response against HIV-1 in vivo we evaluated the potency of three neutralizing antibodies (2G12, 2F5 and 4E10) in suppressing viral rebound in six acutely and eight chronically HIV-1-infected individuals undergoing interruption of antiretroviral treatment (ART). Only two of eight chronically infected individuals showed evidence of a delay in viral rebound during the passive immunization. Rebound in antibody-treated acutely infected individuals upon cessation of ART was substantially later than in a control group of 12 individuals with acute infection. Escape mutant analysis showed that the activity of 2G12 was crucial for the in vivo effect of the neutralizing antibody cocktail. By providing further direct evidence of the potency, breadth and titers of neutralizing antibodies that are required for in vivo activity, these data underline both the potential and the limits of humoral immunity in controlling HIV-1 infection.

Inhibition of in vivo HIV infection in humanized mice by gene therapy of human hematopoietic stem cells with a lentiviral vector encoding a broadly neutralizing anti-HIV antibody.

Due to the inherent immune evasion properties of the HIV envelope, broadly neutralizing HIV-specific antibodies capable of suppressing HIV infection are rarely produced by infected individuals. We examined the feasibility of utilizing genetic engineering to circumvent the restricted capacity of individuals to endogenously produce broadly neutralizing HIV-specific antibodies. We constructed a single lentiviral vector that encoded the heavy and light chains of 2G12, a broadly neutralizing anti-HIV human antibody, and that efficiently transduced and directed primary human B cells to secrete 2G12. To evaluate the capacity of this approach to provide protection from in vivo HIV infection, we used the humanized NOD/SCID/gamma(c)(null) mouse model, which becomes populated with human B cells, T cells, and macrophages after transplantation with human hematopoietic stem cells (hu-HSC) and develops in vivo infection after inoculation with HIV. The plasma of the irradiated NOD/SCID/gamma(c)(null) mice transplanted with hu-HSC transduced with the 2G12-encoding lentivirus contained 2G12 antibody, likely secreted by progeny human lymphoid and/or myeloid cells. After intraperitoneal inoculation with high-titer HIV-1(JR-CSF), mice engrafted with 2G12-transduced hu-HSC displayed marked inhibition of in vivo HIV infection as manifested by a profound 70-fold reduction in plasma HIV RNA levels and an almost 200-fold reduction in HIV-infected human cell numbers in mouse spleens, compared to control hu-HSC-transplanted NOD/SCID/gamma(c)(null) mice inoculated with equivalent high-titer HIV-1(JR-CSF). These results support the potential efficacy of this new gene therapy approach of using lentiviral vectors encoding a mixture of broadly neutralizing HIV antibodies for the treatment of HIV infection, particularly infection with multiple-drug-resistant isolates.

Cost-effective production of a vaginal protein microbicide to prevent HIV transmission.

A series of small-molecule microbicides has been developed for vaginal delivery to prevent heterosexual HIV transmission, but results from human clinical trials have been disappointing. Protein-based microbicides, such as HIV-specific monoclonal antibodies, have been considered as an alternative approach. Despite their promising safety profile and efficacy, the major drawback of such molecules is the economy of large-scale production in mammalian cells, the current system of choice. Here, we show that an alternative biomanufacturing platform is now available for one of the most promising anti-HIV antibodies (2G12). Our data show that the HIV-neutralization capability of the antibody is equal to or superior to that of the same antibody produced in CHO cells. We conclude that this protein production system may provide a means to achieve microbicide ingredient manufacture at costs that would allow product introduction and manufacture in the developing world.

Potent human immunodeficiency virus-neutralizing and complement lysis activities of antibodies are not obligatorily linked.

To evaluate the contribution of complement-mediated lysis to the in vivo activities of neutralizing antibodies, we analyzed the influence of complement activation on treatment success in a recent passive immunization trial with the neutralizing monoclonal antibodies 2G12, 2F5, and 4E10. Administration of monoclonal antibodies led to an immediate, high activation of the complement system even in the absence of viremia in the 14 participating human immunodeficiency virus-infected individuals. Lysis activity measured in patient plasma increased during passive immunization; however, the increases were modest and only partially attributable to the administration of antibodies. We found that unlike neutralization activity, lysis activity was not associated with treatment success in this trial. Compared to complement lysis mounted by the polyclonal antibody response in vivo, monoclonal antibodies were weak inducers of this activity, suggesting that polyclonal responses are more effective in reaching the required threshold of complement activation. Importantly, strong neutralization activity of the monoclonal antibodies did not predict complement lysis activity against patient and reference viruses, suggesting that these activities are not linked. In summary, our data support the notion that the in vivo activities of 2G12, 2F5, and 4E10 are likely due to direct neutralization or Fc receptor-mediated mechanisms such as phagocytosis and antibody-dependent cellular cytotoxicity.

In vivo efficacy of human immunodeficiency virus neutralizing antibodies: estimates for protective titers.

The definition of plasma neutralizing antibody titers capable of controlling human immunodeficiency virus (HIV) infection in vivo is considered a critical step in vaccine development. Here we provide estimates for effective neutralization titers by assessing samples from a recent passive immunization trial with the neutralizing monoclonal antibodies (MAbs) 2G12, 2F5, and 4E10 using an analytic strategy that dissects the contributions of these MAbs to the total neutralization activity in patient plasma. Assessment of neutralization activities for six responding patients with partial or complete control of viremia during the MAb treatment and for the eight nonresponding patients revealed a significant difference between these groups: Among responders, MAb-mediated activity exceeded the autologous neutralization response by 1 to 2 log units (median difference, 43.3-fold), while in the nonresponder group, the autologous activity prevailed (median difference, 0.63-fold). In order to reach a 50% proportion of the responders in our study cohort, MAb neutralizing titers higher than 1:200 were required based on this analysis. The disease stage appears to have a significant impact on the quantities needed, since titers above 1:1,000 were needed to reach the same effect in chronic infection. Although our analysis is based on very small sample numbers and thus cannot be conclusive, our data provide a first estimate on how in vitro-measured neutralizing antibody activity can relate to in vivo efficacy in controlling HIV infection and may therefore provide valuable information for vaccine development. Interestingly, lower neutralizing antibody levels showed an effect in acute compared to chronic infection, suggesting that in early disease stages, therapeutic vaccination may show promise. Equally, this raises hopes that a preventive vaccine could become effective at comparatively lower neutralizing antibody titers.

Recombinant antibody 2G12 produced in maize endosperm efficiently neutralizes HIV-1 and contains predominantly single-GlcNAc N-glycans.

Antibody 2G12 is one of a small number of human immunoglobulin G (IgG) monoclonal antibodies exhibiting potent and broad human immunodeficiency virus-1 (HIV-1)-neutralizing activity in vitro, and the ability to prevent HIV-1 infection in animal models. It could be used to treat or prevent HIV-1 infection in humans, although to be effective it would need to be produced on a very large scale. We have therefore expressed this antibody in maize, which could facilitate inexpensive, large-scale production. The antibody was expressed in the endosperm, together with the fluorescent marker protein Discosoma red fluorescent protein (DsRed), which helps to identify antibody-expressing lines and trace transgenic offspring when bred into elite maize germplasm. To achieve accumulation in storage organelles derived from the endomembrane system, a KDEL signal was added to both antibody chains. Immunofluorescence and electron microscopy confirmed the accumulation of the antibody in zein bodies that bud from the endoplasmic reticulum. In agreement with this localization, N-glycans attached to the heavy chain were mostly devoid of Golgi-specific modifications, such as fucose and xylose. Surprisingly, most of the glycans were trimmed extensively, indicating that a significant endoglycanase activity was present in maize endosperm. The specific antigen-binding function of the purified antibody was verified by surface plasmon resonance analysis, and in vitro cell assays demonstrated that the HIV-neutralizing properties of the maize-produced antibody were equivalent to or better than those of its Chinese hamster ovary cell-derived counterpart.

Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure.

A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic human N-glycosylation (i.e. the presence of beta1,2-xylosylation and core alpha1,3-fucosylation). In this study, RNA interference (RNAi) technology was used to obtain a targeted down-regulation of the endogenous beta1,2-xylosyltransferase (XylT) and alpha1,3-fucosyltransferase (FucT) genes in Nicotiana benthamiana, a tobacco-related plant species widely used for recombinant protein expression. Three glyco-engineered lines with significantly reduced xylosylated and/or core alpha1,3-fucosylated glycan structures were generated. The human anti HIV monoclonal antibody 2G12 was transiently expressed in these glycosylation mutants as well as in wild-type plants. Four glycoforms of 2G12 differing in the presence/absence of xylose and core alpha1,3-fucose residues in their N-glycans were produced. Notably, 2G12 produced in XylT/FucT-RNAi plants was found to contain an almost homogeneous N-glycan species without detectable xylose and alpha1,3-fucose residues. Plant-derived glycoforms were indistinguishable from Chinese hamster ovary (CHO)-derived 2G12 with respect to electrophoretic properties, and exhibited functional properties (i.e. antigen binding and HIV neutralization activity) at least equivalent to those of the CHO counterpart. The generated RNAi lines were stable, viable and did not show any obvious phenotype, thus providing a robust tool for the production of therapeutically relevant glycoproteins in plants with a humanized N-glycan structure.

Functional analysis of the broadly neutralizing human anti-HIV-1 antibody 2F5 produced in transgenic BY-2 suspension cultures.

We report the production of an important human therapeutic antibody in plant cell suspension cultures and the functional analysis of that antibody, including a comparison with the same antibody produced in CHO cells. We established transgenic tobacco BY2 suspension cell cultures expressing the human monoclonal antibody 2F5, which shows broadly neutralizing activity against HIV-1. The antibody was directed to the endoplasmic reticulum of the plant cells and was isolated by cell disruption, followed by protein A chromatography. The plant-derived antibody was shown to be largely intact by SDS-PAGE and immunoblot. Antigen binding activity was investigated by electrophoretic mobility shift assay and quantitatively determined by ELISA and Biacore biosensor technology. Ligand binding properties were analyzed using the ectodomain of human Fc gammaRI for kinetic analysis. The plant-derived antibody showed similar kinetic properties and 89% of the binding capacity of its CHO-derived counterpart, but was only 33% as efficient in HIV-1 neutralization assays. Our results show that plant suspension cultures can be used to produce human antibodies efficiently and that the analysis methods used in this study, including biosensor technology, provide useful functional data about antibody performance. This highlights important issues raised by the use of plant systems to produce human biologics.

Reassessment of autoreactivity of the broadly neutralizing HIV antibodies 4E10 and 2F5 and retrospective analysis of clinical safety data.

BACKGROUND: The broadly neutralizing recombinant human HIV-1 antibodies 4E10, 2F5 and Igh1b12 are reported to have autoreactive potential, which is significant for HIV-1 vaccine development and passive immunotherapy using these antibodies. OBJECTIVE: To investigate the clinical relevance of these findings in subjects receiving passive immunotherapy with these antibodies. METHODS: Four types of investigations were performed: (1) Investigation of clotting parameters in an ongoing clinical study with 4E10, 2F5 and 2G12. (2) Mixing experiments of pooled plasma with the same antibodies. (3) Retrospective analysis of serum from patients who received passive immunotherapy with 4E10, 2F5 and 2G12 either alone or in combination. (4) Assessment of clinical safety data obtained after 418 infusions with these antibodies. RESULTS: Standard clinical assays confirmed that 4E10 showed low-level cross-reactivity with cardiolipin, while previously reported cardiolipin cross-reactivity for 2F5 could not be confirmed. High serum titers of 4E10 induced mild prolongation of the activated partial thromboplastin time, which resolved with the wash out of 4E10. Neither 2F5 nor 2G12 affected coagulation. Repeated high-dose infusions of the monoclonal antibody combination were well tolerated with no incidence for thrombotic complications after 418 infusions in 39 subjects. CONCLUSIONS: Monoclonal antibody 4E10 but not 2F5 or 2G12 showed autoreactive binding specificities. Infusion of 4E10 resulted in transient low anticardiolipin titers. Although an increased thromboembolic risk cannot definitely be excluded, this risk appears to be low and likely depend on underlying disorders.

Selection of a novel gp41-specific HIV-1 neutralizing human antibody by competitive antigen panning.

The HIV envelope glycoprotein (Env) is composed of two non-covalently associated subunits: gp120 and gp41. Panning of phage-displayed antibody libraries against Env-based antigens has resulted mostly in selection of anti-gp120 antibodies. Native gp41 in the absence of gp120 is unstable. The use of gp41 fragments as antigens has resulted in selection of antibodies with only relatively modest neutralizing activity. To enhance selection of antibodies specific for gp41 in the context of the whole Env we developed a methodology termed competitive antigen panning (CAP). Using CAP, we identified a novel gp41-specific human monoclonal antibody (hmAb), m48, from an immune library derived from long-term nonprogressors with high titers of broadly cross-reactive neutralizing antibodies (bcnAbs). Selection of m48 was only successful using CAP and not through the conventional pre-incubation methodology. In assays based on spreading infection in peripheral blood mononuclear cells (PBMCs) m48 neutralized a panel of HIV-1 primary isolates from different clades more potently than the well-characterized broadly cross-reactive HIV-1-neutralizing antibodies IgG1 4E10 and Fab Z13. These results may have implications for the selection of novel gp41-specific bcnAbs and other antibodies, and for the development of HIV-1 inhibitors and vaccine immunogens.

HIV-1 mutants escaping neutralization by the human antibodies 2F5, 2G12, and 4E10: in vitro experiments versus clinical studies.

OBJECTIVE: The human monoclonal antibodies (mAb) 2F5, 2G12, and 4E10 are three of the most broadly neutralizing antibodies against HIV-1. Although they have been shown to prevent de novo infection in vivo, their potential for treatment of chronic infection is less clear. One major obstacle may be the emergence of resistant viruses during mAb treatment. DESIGN: To assess whether escape mutants can be generated in vitro which are resistant to all three mAbs, two neutralization-sensitive T-cell line-adapted viruses and two primary isolates were passaged in the presence of increasing concentrations of 2F5, 2G12, 4E10, and a 1: 1: 1 mixture. To get insight into viral escape in vivo, viruses were isolated from eight patients treated with repeated infusions of 2F5/2G12/4E10. RESULTS: In vitro, viruses resistant to a single mAb emerged after 3-22 weeks. Generation of viruses resistant to the triple-combination was a slower process characterized by recurrent loss of virus replication. Some generated triple-resistant viruses seemed to be impaired in their replicative fitness. Neutralization resistance to 2F5 and partly 4E10 could be attributed to amino acid mutations in the mAb epitopes, but not for 2G12. In vivo, none of the patients developed detectable viruses that escaped neutralization by all three mAbs within the 77-day observation period. Virus escape occurred only to 2G12 in three patients. CONCLUSIONS: In summary, the findings of the in vivo study and the difficulty in generating multi-resistance in vitro together with the fact that some generated viruses seemed to have impaired replication fitness indicate that 2F5, 2G12, and 4E10 may be useful for therapy in HIV-1 infection.

Anti-idiotypic antibody Ab2/3H6 mimics the epitope of the neutralizing anti-HIV-1 monoclonal antibody 2F5.

Anti-idiotypic antibodies directed against potentially neutralizing anti-HIV-1 antibodies may mimic epitopes on gp41 otherwise cryptic to the immune system. This study reports the generation of murine monoclonal antibody Ab2/3H6 blocking the binding of human Ab1 2F5 to the synthetic epitope and to gp160 in an enzyme-linked immunosorbent competition assay. Ab2/2H6 diminished the neutralizing potency of 2F5 in an in-vitro neutralization assay. Ab2/3H6 Fab fragments were capable of inducing neutralizing immune and 2F5-specific responses in B6D2F1 mice applying a simple prime-boost regimen of immunization.

Antiviral activity of the neutralizing antibodies 2F5 and 2G12 in asymptomatic HIV-1-infected humans: a phase I evaluation.

BACKGROUND: The human monoclonal antibodies (MAbs) 2F5 and 2G12 were identified to be two of the most potent neutralizing antibodies against HIV-1. In a first human study they have been shown to be safe after repeated intravenous infusions to asymptomatic HIV-1-infected individuals. However, the antiviral effects of antibody treatment have not been fully analyzed in this first clinical trial. METHODS: The aim of the present study was to gain a preliminary insight into the antiviral effects of 2F5 and 2G12 in humans. For this purpose, plasma samples obtained from the previous phase I study were studied for RNA copy numbers by reverse transcriptase-polymerase chain reaction. As a measure for activation of complement levels of the major complement factor C3 were measured by enzyme-linked immunosorbent assay. Flow cytometry was used to study T-lymphocyte counts and the amount of infected peripheral blood mononuclear cells (PBMC) was determined by co-culture with uninfected donor PBMC. Virus escape from antibody neutralization was determined in vitro in a PBMC neutralization assay. RESULTS: Transient reduction in viral loads was observed in five of seven patients. Vigorous complement activation was observed directly after HIV-specific antibody infusions. The number of infective peripheral blood mononuclear cells was reduced in some patients whereas CD4+ T-lymphocyte counts and CD4+/CD8+ ratios were transiently increased in all patients. Virus escape occurred only against 2G12. CONCLUSIONS: Analysis of disease progression markers indicate that antibody therapy may have antiviral effects. These findings suggest that neutralizing antibodies should be further evaluated as an alternative therapeutic approach in HIV-1 disease.

A phase I trial with two human monoclonal antibodies (hMAb 2F5, 2G12) against HIV-1.

OBJECTIVE: To study the safety, immunogenicity and pharmacokinetics of two intravenously administered human monoclonal antibodies (hMAb 2F5, 2G12) against HIV-1 in humans. DESIGN: Open label clinical phase I trial. SETTING: Primary institutional care. PATIENTS: Seven HIV-1-infected healthy volunteers with > or = 500 x 10(6)CD4 cells/l and < or = 10,000 HIV-1 RNA copies/ml, not treated with highly active antiretroviral therapy (HAART), entered and finished the study. INTERVENTIONS: and main outcome measures: Eight separate infusions of the hMAb were administered over a 4-week period (total dose 14 g). The safety was assessed by physical examination, blood chemistry, complete blood cell count and recording adverse events. 2F5 and 2G12 plasma levels were determined prior to and at the end of each infusion and during the follow-up period of 22 weeks. RESULTS: No clinical or laboratory abnormalities were observed throughout the study. The median distribution half-life (t(1/2 alpha)) of 2F5 and 2G12 was 1.02 (range, 0.77-1.47) days and 2.49 (range, 0.92-4.59) days, respectively. The elimination half-life (t(1/2 beta)) was calculated to be 7.94 (range, 3.46-8.31) days for 2F5 and 16.48 (range, 12.84-24.85) days for 2G12. The median plasma concentration immediately after the first infusion was 216 microg/ml (range, 158-409 microg/ml) for 2F5 and 238 microg/ml (range, 197-402 microg/ml) for 2G12. Multiple infusions resulted in maximum plasma concentrations of 374 microg/ml (range, 304-700 microg/ml) and 605 microg/ml (range, 479-897 microg/ml) for 2F5 and 2G12, respectively. CONCLUSIONS: This study showed that the hMAb 2F5 and 2G12 are safe and well tolerated by HIV-1-infected subjects.

Post-exposure prophylaxis with human monoclonal antibodies prevented SHIV89.6P infection or disease in neonatal macaques.

BACKGROUND: The majority of infants infected through maternal transmission acquire the virus during birth or postpartum through breastfeeding: mucosal exposure is considered to be a major route of infection. OBJECTIVES: To develop passive immunization with human neutralizing monoclonal antibodies (mAbs) against mother-to-child transmission of HIV during delivery and through breastfeeding. DESIGN: An oral challenge model in newborn rhesus macaques mimicked peri- and postpartum virus transmission. METHODS: Neonatal rhesus macaques were challenged orally with the highly pathogenic, chimeric simian-human immunodeficiency virus SHIV89.6P and given post-exposure prophylaxis with a quadruple combination of neutralizing human mAbs, IgG1b12, 2G12, 2F5, and 4E10, directed against conserved epitopes of HIV envelope glycoproteins. Control animals were virus challenged but left untreated. All infants were followed prospectively for signs of viremia and immunodeficiency. RESULTS: Two out of four macaque infants treated with neutralizing mAbs showed no evidence of infection; the other two maintained normal CD4 T cell counts. In contrast, all control animals became highly viremic and had profound CD4 T cell losses; three out of four died from AIDS within 1.5-6 weeks of the challenge. CONCLUSIONS: Passive immunization with this quadruple neutralizing mAbs combination may represent a promising approach to prevent peri- and postnatal HIV transmission. Furthermore, the epitopes recognized by the four neutralizing mAbs are key determinants to achieve complete protection and represent important targets against which to develop active, antibody-response-based AIDS vaccines.

Characterization of molecular features, antigen-binding, and in vitro properties of IgG and IgM variants of 4E10, an anti-HIV type 1 neutralizing monoclonal antibody.

The human monoclonal antibody 4E10 has been generated previously by immortalization of peripheral blood cells from an HIV-1-infected individual. This antibody binds to the linear epitope NWFDIT on gp41 and exhibits exceptional neutralizing activity against a broad spectrum of primary HIV-1 isolates. In the present study, molecular features, immunoreactivity, and functional activity of 4E10 were studied. The original hybridoma-derived 4E10 was of subtype IgG(3). Analysis of the variable segment of the heavy chain (VH) demonstrated extensive somatic mutations compared to the closest homologous germline gene VH1-69. Most amino acid substitutions occurred in the complementarity-determining region (CDR) 2, characteristic for an antigen-driven somatic maturation. The heavy chain of the CDR3 (H3) is of unusual length and cannot be attributed with certainty to any specific D(H) locus. To enable mass production and to prolong the in vivo half-life, 4E10 was subsequently cloned as IgG(1) in Chinese hamster ovary (CHO) cells. In additional studies, 4E10 was class switched to the IgM isotype. Binding to the linear epitope NWFDIT was not significantly changed after the cloning procedures. However, in vitro studies revealed dramatic differences in the neutralizing potential. The antiviral activity could be greatly enhanced by change of IgG(3) to IgG(1). In contrast, the IgM isotype almost completely lost its neutralizing potential.

Primary African HIV clade A and D isolates: effective cross-clade neutralization with a quadruple combination of human monoclonal antibodies raised against clade B.

We investigated the ability of several human neutralizing monoclonal antibodies (nmAbs), originally raised against human immunodeficiency virus (HIV) clade B isolates, to neutralize primary clade A and D isolates as single agents and in combinations. All four primary HIV clade A isolates and five primary HIV clade D isolates tested were neutralized >99% by the quadruple combination of nmAbs IgG1b12, 2G12, 2F5, and 4E10. These mAbs recognize conserved epitopes on HIV-1 envelope (Env), resulting in strong cross-clade neutralization. Previously, we showed synergistic neutralization of primary HIV-1 clade C isolates in vitro by the same nMAb combination. We and others also showed neutralization of primary HIV clade B strains. Together, our data show that the quadruple combination of mAbs effectively neutralized primary HIV clade A, B, C, and D isolates.

Development and antitumor activity of a BCL-2 targeted single-stranded DNA oligonucleotide.

PNT100 is a 24-base, chemically unmodified DNA oligonucleotide sequence that is complementary to a region upstream of the BCL-2 gene. Exposure of tumor cells to PNT100 results in suppression of proliferation and cell death by a process called DNA interference. PNT2258 is PNT100 that is encapsulated in protective amphoteric liposomes developed to efficiently encapsulate the PNT100 oligonucleotide, provide enhanced serum stability, optimized pharmacokinetic properties and antitumor activity of the nanoparticle both in vivo and in vitro. PNT2258 demonstrates broad antitumor activity against BCL-2-driven WSU-DLCL2 lymphoma, highly resistant A375 melanoma, PC-3 prostate, and Daudi-Burkitt's lymphoma xenografts. The sequence specificity of PNT100 was demonstrated against three control sequences (scrambled, mismatched, and reverse complement) all encapsulated in a lipid formulation with identical particle characteristics, and control sequences did not demonstrate antiproliferative activity in vivo or in vitro. PNT2258 is currently undergoing clinical testing to evaluate safety and antitumor activity in patients with recurrent or refractory non-Hodgkin's lymphoma and additional studies are planned.

Exposure of the membrane-proximal external region of HIV-1 gp41 in the course of HIV-1 envelope glycoprotein-mediated fusion.

The membrane-proximal external region (MPER) of HIV-1 gp41 is highly conserved and critical for the fusogenic ability of the virus. However, little is known about the activity of this region in the context of viral fusion. In this study we investigate the temporal exposure of MPER during the course of HIV-1 Env-mediated fusion. We employed the broadly neutralizing monoclonal antibodies 2F5 and 4E10, whose epitopes localize to this region as indicators for accessibility to this region. Time of addition experiments indicated that escape of HIV-1 infection inhibition by 2F5 and 4E10 occurred concomitantly with that of C34, a peptide that blocks the six-helix bundle formation and fusion, which was about 20 min later than escape of inhibition by the mAb b12 that blocks CD4-gp120 attachment. We also probed accessibility of the MPER region on fusion intermediates by measuring the binding of the monoclonal antibodies at different time points during the fusion reaction. Immunofluorescence and in-cell Western assays showed that binding of 2F5 and 4E10 decreased upon triggering HIV-1 Env-expressing cells with appropriate target cells. Addition of C34 did not counteract the loss of antibody binding, suggesting that changes in exposure of MPER occur independently of six-helix bundle formation.