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

In Vivo Protection with Human Monoclonal Antibody S315 following Challenge with Diphtheria Toxin

Background: Morbidity and mortality from Corynebacterium diphtheriae is reduced by prompt administration of equine-derived diphtheria anti-toxin (DAT), which is in short supply worldwide. MassBiologics has developed a human monoclonal antibody (S315) to diphtheria toxin to provide a safer alternative to DAT and address critical supply issues. S315 prevents toxin binding to its putative host receptor and S315 pre-mixed with toxin increased survival in a guinea pig model of intoxication. To further evaluate the ability of S315 to provide in vivo protection, we established a post-exposure treatment model. Methods: Female Hartley guinea pigs (300-350g) were challenged subcutaneously with diphtheria toxin (0.03 to 0.09 Lf, limit of flocculation) to identify the minimum lethal dose. To evaluate anti-toxin efficacy, DAT or S315 was administered five hours post-toxin challenge and animals monitored for 30 days for signs of illness (lethargy, dehydration, weak limbs). Serum anti-diphtheria toxin antibodies were measured by ELISA and Vero cell toxin neutralization assays. Results: The minimum lethal toxin dose was 0.09 Lf. To determine the protective dose of DAT, 0.2 IU, 1.0 IU or 5.0 IU was administered intravenously post-toxin challenge (n=4/cohort). All 0.2 IU or 1.0 IU DAT-treated animals died, while one animal treated with 5.0 IU survived. DAT was subsequently evaluated at 5.0 IU, 10 IU, and 20 IU and compared to a cohort receiving 3.5 mg of S315. All untreated animals died within 72 hours and all antibody-treated animals survived. Dehydration was observed more frequently in the 5 IU and 10 IU DAT cohorts compared to the 20 IU and S315 cohorts. Conclusions: Treatment with S315 after diphtheria toxin exposure is protective; further studies will define a minimum effective dose of S315. This model mimics the route and timing of anti-toxin treatment in humans and provides a rigorous preclinical evaluation of a human antibody replacement for equine DAT

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

The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement

Lysosomotropic amines, such as ammonium chloride, are known to protect cells from the cytotoxic effects of diphtheria toxin. These drugs are believed to inhibit the transport of the toxin from a receptor at the cell exterior into the cytoplasm where a fragment of the toxin arrests protein synthesis. We studied the effects of lysosomotropic agents on the cytotoxic process to better understand how the toxin enters the cytoplasm. The cytotoxic effects of diphtheria toxin were not inhibited by antitoxin when cells were preincubated at 37 degrees C with toxin and ammonium chloride, exposed to antitoxin at 4 degrees C, washed to relieve the ammonium chloride inhibition, and finally warmed to 37 degrees C. The antigenic determinants of the toxin were, therefore, either altered or sheltered. It is likely that the combination of ammonium chloride and a low temperature trapped the toxin in an intracellular vesicle from which the toxin could proceed to the cytoplasm. Because lysosomotropic amines raise the pH within acidic intracellular vesicles, such as lysosomes, they could trap the toxin within such a vesicle if an acidic environment were necessary for the toxin to penetrate into the cytoplasm. We simulated acidic conditions which the toxin might encounter by exposing cells with toxin bound to their surface to acidic medium. We then measured the effects of lysosomotropic amines on the activity of the toxin to see if the acidic environment substituted for the function normally inhibited by the drugs. The drugs no longer protected the cells. This suggests that exposing the toxin to an acidic environment, such as that found within lysosomes, is an important step in the penetration of diphtheria toxin into the cytoplasm

Diphtheria anti-toxoid antibody levels among pre-clinical students and staff in an institute of higher learning in Malaysia: are they protected?

Introduction: Little is known about the sero-prevalence of diphtheria anti-toxoid antibody levels among medical students in Malaysia. They too, just like other health care workers (HCWs) are at risk of contracting and transmitting diphtheria. Fortunately, this can be prevented by giving a specific vaccine: the diphtheria, tetanus and pertussis (DTP) vaccine. Nonetheless, data from local or regional surveys are needed before any decision is made by the respective authorities. General objective: We studied the epidemiology of diphtheria anti-toxoid antibody levels and vaccination history amongst medical students and staff in Faculty of Medicine and Health Sciences, Universiti Putra Malaysia. Specific objectives: We determined the level of diphtheria anti-toxoid antibodies amongst pre-clinical students and staff. Methodology: A total of 152 sera were collected from subjects aged 19 to 63, and diphtheria anti-toxoid levels were measured by an enzyme-linked immunosorbent assay. Results: One hundred and fifty-two (94.4%) blood samples out of 161 participants were successfully withdrawn, which comprised 105 (69.1%) and 47 (30.9%) medical students and staff, respectively. A total of 77.6% and the other 22.4% of the subjects had full and basic protection, respectively. Higher levels were predominant amongst males and they were 1.3 times more protected than females in 20-29 year-old group (85.1% vs 66.2%; odd ratios 1.25 [95% CI 1.03-1.50]; P=0.03). No significant difference in the levels of immunity among subjects for ethnicity and academic position (P>0.05). Recommendations: Level of full protection against diphtheria toxin should be clearly defined by broad population based studies using several comparable detection methods. Medical students and staff with basic protection should be closely monitored or should be given a booster dose for those who are at high risk of acquiring the disease. Thus, a standard degree of coverage should be clearly determined for health workers to prevent a potential outbreak. Conclusion: Students and staff possess immunity towards diptheria toxin however the level of full protective antibody is yet to be determined in future

Studies on the denaturation of antibody. IV. The influence of pH and certain other factors on the rate of inactivation of Staphylococcus antitoxin in urea solutions

In previous work on the denaturation of antibody, studies have been made of some of the factors influencing the inactivation of diphtheria antitoxin in urea solutions (1, 2). A quantitative formulation of a simple kinetic theory was found to fit satisfactorily the experimental data and to offer a reasonable explanation of the deviation of the inactivation from simple first order behavior (2). In the present work we have studied the inactivation of Staphylococcus antitoxin, investigating the influence of certain new factors on the rate and course of the over-all reaction, with a view toward gaining further insight into the mechanism of the reactions, and reinvestigating the influence of pH, a factor studied previously with diphtheria antitoxin, in order to test the applicability of the proposed kinetic mechanism to different antibodies

Delivery of drugs, proteins and genes into cells using transferrin as a ligand for receptor-mediated endocytosis

Transferrin, an iron-transporting serum glycoprotein, is efficiently taken up into cells by the process of receptor-mediated endocytosis. Transferrin receptors are found on the surface of most proliferating cells, in elevated numbers on erythroblasts and on many kinds of tumors. The efficient cellular mechanism for uptake of transferrin has been subverted for the delivery of low-molecular-weight drugs, protein toxins, and liposomes by linkage of these agents to transferrin or to anti-transferrin receptor antibodies. Linkage may be via chemical conjugation procedures or by the generation of chimeric fusion proteins. Transferrin conjugated to DNA-binding compounds (e.g. polycations or intercalating agents) has been successfully used for the import of DNA molecules into cells. High-level gene expression is obtained only if endosome-disruptive agents such as influenza hemagglutinin peptides or adenovirus particles are included which release the DNA complex from intracellular vesicles into the cytoplasm