GSK3732394: a Multi-specific Inhibitor of HIV Entry.

Long-acting antiretrovirals could provide a useful alternative to daily oral therapy for HIV-1-infected individuals. Building on a bi-specific molecule with adnectins targeting CD4 and gp41, a potential long-acting biologic, GSK3732394, was developed with three independent and synergistic modes of HIV entry inhibition that potentially could be self-administered as a long-acting subcutaneous injection. Starting with the bi-specific inhibitor, an α-helical peptide inhibitor was optimized as a linked molecule to the anti-gp41 adnectin, with each separate inhibitor exhibiting at least single-digit nanomolar (or lower) potency and a broad spectrum. Combination of the two adnectins and peptide activities into a single molecule was shown to have synergistic advantages in potency, the resistance barrier, and the ability to inhibit HIV-1 infections at low levels of CD4 receptor occupancy, showing that GSK3732394 can work in trans on a CD4+ T cell. Addition of a human serum albumin molecule prolongs the half-life in a human CD4 transgenic mouse, suggesting that it may have potential as a long-acting agent. GSK3732394 was shown to be highly effective in a humanized mouse model of infection. GSK3732394 is currently in clinical trials.IMPORTANCE There continue to be significant unmet medical needs for patients with HIV-1 infection. One way to improve adherence and decrease the likelihood of drug-drug interactions in HIV-1-infected patients is through the development of long-acting biologic inhibitors. Building on a bi-specific inhibitor approach targeting CD4 and gp41, a tri-specific molecule was generated with three distinct antiviral activities. The linkage of these three biologic inhibitors creates synergy that offers a series of advantages to the molecule. The addition of human serum albumin to the tri-specific inhibitor could allow it to function as a long-acting self-administered treatment for patients with HIV infection. This molecule is currently in early clinical trials.

A Novel gp41-Binding Adnectin with Potent Anti-HIV Activity Is Highly Synergistic when Linked to a CD4-Binding Adnectin.

The N17 region of gp41 in HIV-1 is the most conserved region in gp160. mRNA selection technologies were used to identify an adnectin that binds to this region and inhibits gp41-induced membrane fusion. Additional selection conditions were used to optimize the adnectin to greater potency (5.4 ± 2.6 nM) against HIV-1 and improved binding affinity for an N17-containing helical trimer (0.8 ± 0.4 nM). Resistance to this adnectin mapped to a single Glu-to-Arg change within the N17 coding region. The optimized adnectin (6200_A08) exhibited high potency and broad-spectrum activity against 123 envelope proteins and multiple clinical virus isolates, although certain envelope proteins did exhibit reduced susceptibility to 6200_A08 alone. The reduced potency could not be correlated with sequence changes in the target region and was thought to be the result of faster kinetics of fusion mediated by these envelope proteins. Optimized linkage of 6200_A08 with a previously characterized adnectin targeting CD4 produced a highly synergistic molecule, with the potency of the tandem molecule measured at 37 ± 1 pM. In addition, these tandem molecules now exhibited few potency differences against the same panel of envelope proteins with reduced susceptibility to 6200_A08 alone, providing evidence that they did not have intrinsic resistance to 6200_A08 and that coupling 6200_A08 with the anti-CD4 adnectin may provide a higher effective on rate for gp41 target engagement.IMPORTANCE There continue to be significant unmet medical needs for patients with HIV-1 infection. One way to improve adherence and decrease the likelihood of drug-drug interactions in HIV-1-infected patients is through the development of long-acting biologic inhibitors. This study describes the development and properties of an adnectin molecule that targets the most conserved region of the gp41 protein and inhibits HIV-1 with good potency. Moreover, when fused to a similar adnectin targeted to the human CD4 protein, the receptor for HIV-1, significant synergies in potency and efficacy are observed. These inhibitors are part of an effort to develop a larger biologic molecule that functions as a long-acting self-administered regimen for patients with HIV-1 infection.

A Functional NaV1.7-NaVAb Chimera with a Reconstituted High-Affinity ProTx-II Binding Site.

The NaV1.7 voltage-gated sodium channel is implicated in human pain perception by genetics. Rare gain of function mutations in NaV1.7 lead to spontaneous pain in humans whereas loss of function mutations results in congenital insensitivity to pain. Hence, agents that specifically modulate the function of NaV1.7 have the potential to yield novel therapeutics to treat pain. The complexity of the channel and the challenges to generate recombinant cell lines with high NaV1.7 expression have led to a surrogate target strategy approach employing chimeras with the bacterial channel NaVAb. In this report we describe the design, synthesis, purification, and characterization of a chimera containing part of the voltage sensor domain 2 (VSD2) of NaV1.7. Importantly, this chimera, DII S1-S4, forms functional sodium channels and is potently inhibited by the NaV1.7 VSD2 targeted peptide toxin ProTx-II. Further, we show by [125I]ProTx-II binding and surface plasmon resonance that the purified DII S1-S4 protein retains high affinity ProTx-II binding in detergent. We employed the purified DII S1-S4 protein to create a scintillation proximity assay suitable for high-throughput screening. The creation of a NaV1.7-NaVAb chimera with the VSD2 toxin binding site provides an important tool for the identification of novel NaV1.7 inhibitors and for structural studies to understand the toxin-channel interaction.

Discovery and Characterization of a Novel CD4-Binding Adnectin with Potent Anti-HIV Activity.

A novel fibronectin-based protein (Adnectin) HIV-1 inhibitor was generated using in vitro selection. This inhibitor binds to human CD4 with a high affinity (3.9 nM) and inhibits viral entry at a step after CD4 engagement and preceding membrane fusion. The progenitor sequence of this novel inhibitor was selected from a library of trillions of Adnectin variants using mRNA display and then further optimized for improved antiviral and physical properties. The final optimized inhibitor exhibited full potency against a panel of 124 envelope (gp160) proteins spanning 11 subtypes, indicating broad-spectrum activity. Resistance profiling studies showed that this inhibitor required 30 passages (151 days) in culture to acquire sufficient resistance to result in viral titer breakthrough. Resistance mapped to the loss of multiple potential N-linked glycosylation sites in gp120, suggesting that inhibition is due to steric hindrance of CD4-binding-induced conformational changes.

The Sensitivity of HIV-1 gp120 Polymorphs to Inhibition by Temsavir Correlates to Temsavir Binding On-rate.

Temsavir binds directly to the HIV-1 envelope glycoprotein gp120 and selectively inhibits interactions between HIV-1 and CD4 receptors. Previous studies identified gp120 amino acid positions where substitutions are associated with reduced susceptibility to temsavir. The mechanism by which temsavir susceptibility is altered in these envelope glycoproteins was evaluated. Pseudoviruses encoding gp120 substitutions alone (S375H/I/M/N, M426L, M434I, M475I) or in combination (S375H + M475I) were engineered on a wild-type JRFL background. Temsavir-gp120 and CD4-gp120 binding kinetics and ability of temsavir to block CD4-gp120 binding were evaluated using the purified polymorphic gp120 proteins and a Creoptix® WAVE Delta grating-coupled interferometry system. The fold-change in half-maximal inhibitory concentration (IC50) in JRFL-based pseudoviruses containing the aforementioned polymorphisms relative to that of wild-type ranged from 4-fold to 29,726-fold, while temsavir binding affinity for the polymorphic gp120 proteins varied from 0.7-fold to 73.7-fold relative to wild-type gp120. Strong correlations between temsavir IC50 and temsavir binding affinity (r=0.7332; P=0.0246) as well as temsavir binding on-rate (r=-0.8940; P=0.0011) were observed. Binding affinity of gp120 proteins for CD4 varied between 0.4-fold and 3.1-fold compared with wild-type gp120; no correlations between temsavir IC50 and CD4 binding kinetic parameters were observed. For all polymorphic gp120 proteins, temsavir was able to fully block CD4 binding; 3 polymorphs required higher temsavir concentrations. Loss of susceptibility to temsavir observed for gp120 polymorphisms strongly correlated with reductions in temsavir binding on-rate. Nonetheless, temsavir retained the ability to fully block CD4-gp120 engagement given sufficiently high concentrations.

A Phase 1 randomized study of GSK3732394, an investigational long-acting biologic treatment regimen for HIV-1 infection.

BACKGROUND: The GSK3732394 multivalent protein was developed as a novel, long-acting, antiretroviral biologic treatment regimen with three independent, non-cross-resistant mechanisms for inhibiting HIV-1 entry. METHODS: A single-centre, Phase 1, double-blind, randomized, placebo-controlled study was conducted in healthy volunteers, using a 2-part adaptive study design: in Part 1, participants were randomized to receive subcutaneous injection of GSK3732394 or placebo (3:1) as single ascending doses (10-mg starting dose); in Part 2, participants were intended to receive multiple ascending doses. Primary and secondary objectives included safety, pharmacokinetics (PK) and pharmacodynamics (PD; cluster of differentiation four receptor occupancy [CD4 RO]) of GSK3732394 in healthy adults; PK/PD results in healthy volunteers were used to project HIV-1 treatment success. RESULTS: The most frequently reported adverse event was injection site reactions (ISRs; 8/18 [44%]). Most ISRs were mild (Grade 1-2; n = 7); one participant experienced a Grade 3 ISR (erythema ≥10 cm). All ISRs were delayed in onset (after Day 10). GSK3732394 demonstrated linear PK across all cohorts. Clearance was faster than expected, and PK/PD results were lower than expected, with the maximum dose investigated (80 mg) achieving mean trough CD4 RO of ∼25% on Day 7. The study was terminated as the PK/PD model linking PK and CD4 RO indicated that the maximum planned doses would not achieve the desired therapeutic profile. CONCLUSIONS: This study demonstrated successful deployment of PK/PD dose relationships in the design and conduct of clinical trials by leveraging the findings toward predicting probability of success, resulting in appropriate early termination (ClinicalTrials.gov, NCT03984812).

Novel Bent Conformation of CD4 Induced by HIV-1 Inhibitor Indirectly Prevents Productive Viral Attachment.

GSK3732394 is a multi-specific biologic inhibitor of HIV entry currently under clinical evaluation. A key component of this molecule is an adnectin (6940_B01) that binds to CD4 and inhibits downstream actions of gp160. Studies were performed to determine the binding site of the adnectin on CD4 and to understand the mechanism of inhibition. Using hydrogen-deuterium exchange with mass spectrometry (HDX), CD4 peptides showed differential rates of deuteration (either enhanced or slowed) in the presence of the adnectin that mapped predominantly to the interface of domains 2 and 3 (D2-D3). In addition, an X-ray crystal structure of an ibalizumab Fab/CD4(D1-D4)/adnectin complex revealed an extensive interface between the adnectin and residues on CD4 domains D2-D4 that stabilize a novel T-shaped CD4 conformation. A cryo-EM map of the gp140/CD4/GSK3732394 complex clearly shows the bent conformation for CD4 while bound to gp140. Mutagenic analyses on CD4 confirmed that amino acid F202 forms a key interaction with the adnectin. In addition, amino acid L151 was shown to be a critical indirect determinant of the specificity for binding to the human CD4 protein over related primate CD4 molecules, as it appears to modulate CD4's flexibility to adopt the adnectin-bound conformation. The significant conformational change of CD4 upon adnectin binding brings the D1 domain of CD4 in proximity to the host cell membrane surface, thereby re-orienting the gp120 binding site in a direction that is inaccessible to incoming virus due to a steric clash between gp160 trimers on the virus surface and the target cell membrane.

Distinct in vitro binding properties of the anti-CD20 small modular immunopharmaceutical 2LM20-4 result in profound and sustained in vivo potency in cynomolgus monkeys.

OBJECTIVES: To characterize the in vitro binding and effector function properties of CD20-directed small modular immunopharmaceutical (SMIP) 2LM20-4, and to compare its in vivo B-cell depletion activity with the mutated 2LM20-4 P331S [no in vitro complement-dependent cytotoxicity (CDC)] and rituximab in cynomolgus monkeys. METHODS: Direct binding is examined in flow cytometry, confocal microscopy, scatchard and lipid raft assays. Effector function assays include CDC and Fc-mediated cellular toxicity. In the 6-month-long in vivo B-cell depletion study, single i.v. dosages of 1 or 10 mg/kg of anti-CD20 proteins were administered to monkeys and B-cell counts were monitored in peripheral blood, bone marrow and lymph nodes. RESULTS: 2LM20-4 has lower saturation binding to human primary B cells and recruits fewer CD20 molecules into lipid rafts compared with rituximab; however, it induces higher in vitro CDC. In competitive binding, 2LM20-4 only partially displaces rituximab, suggesting that it binds to a fraction of CD20 molecules within certain locations of the plasma membrane as compared with rituximab. In monkeys, 2LM20-4 had more sustained B-cell depletion activity than rituximab in peripheral blood and had significantly more profound and sustained activity than 2LM20-4 P331S and rituximab in the lymph nodes. CONCLUSIONS: SMIP 2LM20-4, which binds to a fraction of CD20 molecules as compared with rituximab, has more potent in vitro CDC, and more potent and sustained B-cell depletion activity in cynomolgus monkeys. Our work has considerable clinical relevance since it provides novel insights related to the emerging B-cell depletion therapies in autoimmune diseases.

Complex pharmacokinetics of a humanized antibody against human amyloid beta peptide, anti-abeta Ab2, in nonclinical species.

PURPOSE: Anti-Aß Ab2 (Ab2) is a humanized monoclonal antibody against amino acids 3-6 of primate (but not rodent) amyloid ß (Aß) and is being evaluated for the treatment of Alzheimer's disease (AD). This study was conducted to predict the human pharmacokinetics of Ab2. METHODS: In vivo PK profile of Ab2 in preclinical species and in vitro mechanistic studies in preclinical and human systems were used for pharmacokinetic predictions. RESULTS: In Tg2576 and PSAPP mice that have ~100-fold higher circulating levels of human Aß compared to humans, elimination of Ab2 was target-mediated, such that exposure was 5-10 fold lower compared to wild-type rodents or to PDAPP mice that have human Aß concentrations in plasma similar to humans. In cynomolgus monkeys, the t(1/2) of Ab2 was faster (<2.5 days) compared to that of the control antibody (~13 days). The fast elimination of Ab2 in cynomolgus monkeys was linked to off-target binding to cynomolgus monkey fibrinogen that was also causing incomplete recovery of Ab2 in cynomolgus monkey serum in blood partitioning experiments. Ab2 had significantly weaker to undetectable binding to human (and mouse) fibrinogen and had good recovery in human serum in blood partitioning experiments. CONCLUSIONS: These data predict that elimination of Ab2 in healthy or AD humans is expected to be slow, with t(1/2) similar to that observed for other humanized antibodies.

High-throughput screening of chromatographic separations: III. Monoclonal antibodies on ceramic hydroxyapatite.

High-throughput screening (HTS) of chromatography resins for identifying optimal protein purification conditions is becoming an integral part of industrial process development. In this work, ceramic hydroxyapatite (cHA) chromatography of 15 humanized monoclonal antibodies (mAbs) was examined by HTS. MAb binding, as quantified by partition coefficient (K(p)), was measured under 92 combinations of sodium chloride, phosphate, and pH. Binding varied inversely with these variables for all mAbs tested. However, the magnitudes of binding among mAbs under identical conditions varied significantly, showing a >1.5 log range in K(p). Analysis of variance (ANOVA) techniques were used to describe the binding of each mAb as a function of the three screen variables. Linear models relating log K(p) to the pH, log[sodium chloride], and log[phosphate] fit the data for each antibody with 93-96% accuracy. From these models, characteristic charge values for the cation exchange and metal coordination components of the multi-modal mAb/cHA interaction varied twofold across the mAbs, reflecting inherent variability in the number of contacts between a particular mAb and the cHA surface. Furthermore, we reduced the number of test conditions required from 92 to 8 while maintaining an accurate representation of the full binding response surface. This eight-point modeling method accurately predicted the binding behavior of mAbs as well as mAb aggregates, a common impurity in crude mAb preparations. Using this eight-point modeling method, binding and selectivity information for mAb and aggregate can be obtained from less than two milligrams of protein, making the method attractive for early manufacturability assessments.

Developing Adnectins that target SRC co-activator binding to PXR: a structural approach toward understanding promiscuity of PXR.

The human pregnane X receptor (PXR) is a promiscuous nuclear receptor that functions as a sensor to a wide variety of xenobiotics and regulates expression of several drug metabolizing enzymes and transporters. We have generated "Adnectins", derived from 10th fibronectin type III domain ((10)Fn3), that target the PXR ligand binding domain (LBD) interactions with the steroid receptor co-activator-1 (SRC-1) peptide, displacing SRC-1 binding. Adnectins are structurally homologous to the immunoglobulin superfamily. Three different co-crystal structures of PXR LBD with Adnectin-1 and CCR1 (CC chemokine receptor-1) antagonist Compound-1 were determined. This structural information was used to modulate PXR affinity for a related CCR1 antagonist compound that entered into clinical trials for rheumatoid arthritis. The structures of PXR with Adnectin-1 reveal specificity of Adnectin-1 in not only targeting the interface of the SRC-1 interactions but also engaging the same set of residues that are involved in binding of SRC-1 to PXR. Substituting SRC-1 with Adnectin-1 does not alter the binding conformation of Compound-1 in the ligand binding pocket. The structure also reveals the possibility of using Adnectins as crystallization chaperones to generate structures of PXR with compounds of interest.

The safety of methadone hydrochloride.

Methadone is an interesting analgesic for multiple reasons. The unique properties of the agent, low cost and widespread availability have led to increases in methadone prescribing. Despite advantages, methadone is challenging to work with, particularly in patients with high opioid requirements. Recent concerns regarding cardiac arrhythmias and respiratory depression have led to changes in the labeling of methadone. This editorial highlights some of these concerns and provides some recommendations for the appropriate use of methadone in the setting of pain.

A virus-virus interaction circumvents the virus receptor requirement for infection by pathogenic retroviruses.

During ongoing C-type retrovirus infection, the probability of leukemia caused by insertional gene activation is markedly increased by the emergence of recombinant retroviruses that repeatedly infect host cells. The murine mink cell focus-inducing (MCF) viruses with this property have acquired characteristic changes in the N-terminal domain of their envelope glycoprotein that specify binding to a different receptor than the parental ecotropic virus. In this report, we show that MCF virus infection occurs through binding to this receptor (termed Syg1) and, remarkably, by a second mechanism that does not utilize the Syg1 receptor. By the latter route, the N-terminal domain of the ecotropic virus glycoprotein expressed on the cell surface in a complex with its receptor activates the fusion mechanism of the MCF virus in trans. The rate of MCF virus spread through a population of permissive human cells was increased by establishment of trans activation, indicating that Syg1 receptor-dependent and -independent pathways function in parallel. Also, trans activation shortened the interval between initial infection and onset of cell-cell fusion associated with repeated infection of the same cell. Our findings indicate that pathogenic retrovirus infection may be initiated by virus binding to cell receptors or to the virus envelope glycoprotein of other viruses expressed on the cell surface. Also, they support a broader principle: that cooperative virus-virus interactions, as well as virus-host interactions, shape the composition and properties of the retrovirus quasispecies.

Structure and mechanism of a coreceptor for infection by a pathogenic feline retrovirus.

Infection of T lymphocytes by the cytopathic retrovirus feline leukemia virus subgroup T (FeLV-T) requires FeLIX, a cellular coreceptor that is encoded by an endogenous provirus and closely resembles the receptor-binding domain (RBD) of feline leukemia virus subgroup B (FeLV-B). We determined the structure of FeLV-B RBD, which has FeLIX activity, to a 2.5-A resolution by X-ray crystallography. The structure of the receptor-specific subdomain of this glycoprotein differs dramatically from that of Friend murine leukemia virus (Fr-MLV), which binds a different cell surface receptor. Remarkably, we find that Fr-MLV RBD also activates FeLV-T infection of cells expressing the Fr-MLV receptor and that FeLV-B RBD is a competitive inhibitor of infection under these conditions. These studies suggest that FeLV-T infection relies on the following property of mammalian leukemia virus RBDs: the ability to couple interaction with one of a variety of receptors to the activation of a conserved membrane fusion mechanism. A comparison of the FeLV-B and Fr-MLV RBD structures illustrates how receptor-specific regions are linked to conserved elements critical for postbinding events in virus entry.