Potency of a human monoclonal antibody to diphtheria toxin relative to equine diphtheria anti-toxin in a guinea pig intoxication model

Prompt administration of anti-toxin reduces mortality following Corynebacterium diphtheriae infection. Current treatment relies upon equine diphtheria anti-toxin (DAT), with a 10% risk of serum sickness and rarely anaphylaxis. The global DAT supply is extremely limited; most manufacturers have ceased production. S315 is a neutralizing human IgG1 monoclonal antibody to diphtheria toxin that may provide a safe and effective alternative to equine DAT and address critical supply issues. To guide dose selection for IND-enabling pharmacology and toxicology studies, we dose-ranged S315 and DAT in a guinea pig model of diphtheria intoxication based on the NIH Minimum Requirements potency assay. Animals received a single injection of antibody premixed with toxin, were monitored for 30 days, and assigned a numeric score for clinical signs of disease. Animals receiving \u3e /= 27.5 microg of S315 or \u3e /= 1.75 IU of DAT survived whereas animals receiving \u3c /= 22.5 microg of S315 or \u3c /= 1.25 IU of DAT died, yielding a potency estimate of 17 microg S315/IU DAT (95% CI 16-21) for an endpoint of survival. Because some surviving animals exhibited transient limb weakness, likely a systemic sign of toxicity, DAT and S315 doses required to prevent hind limb paralysis were also determined, yielding a relative potency of 48 microg/IU (95% CI 38-59) for this alternate endpoint. To support advancement of S315 into clinical trials, potency estimates will be used to evaluate the efficacy of S315 versus DAT in an animal model with antibody administration after toxin exposure, more closely modeling anti-toxin therapy in humans

Identification of a Human Monoclonal Antibody to Replace Equine Diphtheria Anti-toxin for the Treatment of Diphtheria

Diphtheria anti-toxin (DAT) has been used to treat Corynebacterium diphtheriae infection for over one hundred years. While the global incidence of diphtheria has declined in the 20th century, the disease remains endemic in many parts of the world and significant outbreaks still occur. Diphtheria anti-toxin is an equine polyclonal antibody with considerable side effects that is in critically short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we cloned human monoclonal antibodies (HuMabs) directly from antibody secreting cells of human volunteers immunized with Td vaccine. We isolated a diverse panel of HuMabs that recognized diphtheria toxoid and recombinant protein fragments of diphtheria toxin. Forty-one unique HuMabs were expressed in 293T cells and tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays. The lead candidate HuMab, 315C4 potently neutralized diphtheria toxin with an EC50 of 0.65 ng/mL. Additionally, 25 μg of 315C4 completely protected guinea pigs in an in vivo lethality model. In comparison, 1.6 IU of DAT was necessary for full protection resulting in an estimated relative potency of 64 IU/mg for 315C4. We further established that our lead candidate HuMab binds to the receptor binding domain of diphtheria toxin and blocks the toxin from binding to the putative receptor, heparin binding-epidermal growth factor like growth factor. The discovery of a specific and potent neutralizing antibody against diphtheria toxin holds promise as a potential human therapeutic and is being developed for human use

Human Monoclonal Antibody HCV1 Effectively Prevents and Treats HCV Infection in Chimpanzees

Hepatitis C virus (HCV) infection is a leading cause of liver transplantation and there is an urgent need to develop therapies to reduce rates of HCV infection of transplanted livers. Approved therapeutics for HCV are poorly tolerated and are of limited efficacy in this patient population. Human monoclonal antibody HCV1 recognizes a highly-conserved linear epitope of the HCV E2 envelope glycoprotein (amino acids 412-423) and neutralizes a broad range of HCV genotypes. In a chimpanzee model, a single dose of 250 mg/kg HCV1 delivered 30 minutes prior to infusion with genotype 1a H77 HCV provided complete protection from HCV infection, whereas a dose of 50 mg/kg HCV1 did not protect. In addition, an acutely-infected chimpanzee given 250 mg/kg HCV1 42 days following exposure to virus had a rapid reduction in viral load to below the limit of detection before rebounding 14 days later. The emergent virus displayed an E2 mutation (N415K/D) conferring resistance to HCV1 neutralization. Finally, three chronically HCV-infected chimpanzees were treated with a single dose of 40 mg/kg HCV1 and viral load was reduced to below the limit of detection for 21 days in one chimpanzee with rebounding virus displaying a resistance mutation (N417S). The other two chimpanzees had 0.5-1.0 log(10) reductions in viral load without evidence of viral resistance to HCV1. In vitro testing using HCV pseudovirus (HCVpp) demonstrated that the sera from the poorly-responding chimpanzees inhibited the ability of HCV1 to neutralize HCVpp. Measurement of antibody responses in the chronically-infected chimpanzees implicated endogenous antibody to E2 and interference with HCV1 neutralization although other factors may also be responsible. These data suggest that human monoclonal antibody HCV1 may be an effective therapeutic for the prevention of graft infection in HCV-infected patients undergoing liver transplantation

Comparison of a novel human rabies monoclonal antibody to human rabies immunoglobulin for post-exposure prophylaxis: A phase 2/3 randomized, single blind, non-inferiority, controlled study

Background: Lack of access to rabies immunoglobulin (RIG) contributes to the high rabies mortality. A recombinant human monoclonal antibody (SII RMAb) was tested in a post-exposure prophylaxis (PEP) regimen in comparison to a human RIG (HRIG)-containing PEP regimen. Methods: This was a Phase 2/3 randomized, single blind, non-inferiority study conducted in 200 participants with WHO category III suspected rabies exposures. On Day 0, participants received either SII RMAb or HRIG (1:1 ratio) into wounds and if required, intramuscularly (IM) along with five doses of rabies vaccine IM on Days 0, 3, 7, 14 and 28. The primary endpoint was the ratio of Day 14 geometric mean concentration (GMC) of RVNA activity as measured by RFFIT for SII RMAb recipients relative to HRIG recipients. Results: 199 participants received SII RMAb (101) or HRIG (98) and at least one dose of vaccine. The Day 14 GMC ratio of SII RMAb group to HRIG group was 4.23 (96.9018% CI 2.59 - 6.94) with the GMC for SII RMAb recipients of 24.90 IU/mL (95% CI 18.94 - 32.74) and 5.88 IU/mL (95% CI 4.11 - 8.41) for HRIG recipients. Majority of local injection site and systemic adverse reactions reported from bothgroups were mild to moderate in severity. Conclusions: A PEP regimen containing SII RMAb was safe and demonstrated non-inferiority to HRIG PEP in RVNA production. The novel monoclonal potentially offers a safe and potent alternative for the passive component of PEP and could significantly improve the management of bites from suspected rabid animals. Trial registration number: CTRI/2012/05/002709

Circulating Levels of sFlt1 Splice Variants as Predictive Markers for the Development of Preeclampsia

Angiogenic biomarkers, including soluble fms-like tyrosine kinase 1 (sFlt1), are thought to be predictors of preeclampsia onset; however, improvement is needed before a widespread diagnostic test can be utilized. Here we describe the development and use of diagnostic monoclonal antibodies specific to the two main splice variants of sFlt1, sFlt1-1 and sFlt1-14. These antibodies were selected for their sensitivity and specificity to their respective sFlt1 isoform in a capture ELISA format. Data from this pilot study suggest that sFlt1-1 may be more predictive of preeclampsia than total sFlt1. It may be possible to improve current diagnostic platforms if more specific antibodies are utilized

Amino acid alterations identified in H77 HCV E1/E2 envelope glycoproteins after initial viral infection in untreated (#1) and treated (#2) chimpanzees.

<p>Amino acid alterations identified in H77 HCV E1/E2 envelope glycoproteins after initial viral infection in untreated (#1) and treated (#2) chimpanzees.</p

HCV1 does not bind mutant E2 envelope glycoprotein produced in serum-containing medium.

<p>A vector encoding wild-type E2₆₆₀ was transfected into CHO cells grown in defined media (<b>A</b>) or serum-containing medium (<b>B</b>). In addition, a vector encoding E2₆₆₀–N415K was also transfected into CHO cells grown in defined media (<b>C</b>) or serum-containing media (<b>D</b>). All proteins were purified from culture supernatant using nickel-affinity chromatography, quantified, assessed for purity and coated on ELISA plates. Varying dilutions of HCV1 (red circles) was applied to each protein and binding assessed. In addition, varying dilutions of 96-2 (epitope II specific, blue squares), irrelevant antibody (blue triangles) and a mouse antibody specific to the (His)₆ tag (blue crosses) at the C-terminus of E2₆₆₀ were assayed to control for protein coating and non-specific binding. For HCV1, 96-2 and irrelevant mAb, bound antibody was detected employing goat anti–human secondary antibody conjugated to alkaline phosphatase, followed by development with PNPP substrate, and the absorbance at 405 nm for each dilution was plotted. For the mouse anti-(His)₆ antibody, goat anti-mouse secondary antibody was used.</p

Amino acid alterations in E1/E2 envelope glycoproteins following viral rebound in treated chimpanzee B.

<p>Amino acid alterations in E1/E2 envelope glycoproteins following viral rebound in treated chimpanzee B.</p