First-in-Human Clinical Trial to Assess the Safety, Tolerability and Pharmacokinetics of Single Doses of NTM-1633, a Novel Mixture of Monoclonal Antibodies against Botulinum Toxin E.

Botulism is a rare, life-threatening paralytic disease caused by botulinum neurotoxin (BoNT). Available treatments including an equine antitoxin and human immune globulin are given postexposure and challenging to produce and administer. NTM-1633 is an equimolar mixture of 3 human IgG monoclonal antibodies, E1, E2, and E3, targeting BoNT serotype E (BoNT/E). This first-in-human study assessed the safety, tolerability, pharmacokinetics (PK), and immunogenicity of NTM-1633. This double-blind, single-center, placebo-controlled dose escalation study randomized 3 cohorts of healthy volunteers to receive a single intravenous dose of NTM-1633 (0.033, 0.165, or 0.330 mg/kg) or saline placebo. Safety monitoring included physical examinations, clinical laboratory studies, and vital signs. Blood sampling was performed at prespecified time points for PK and immunogenicity analyses. Twenty-four subjects received study product (18 NTM-1633; 6 placebo), and no deaths were reported. An unrelated serious adverse event was reported in a placebo subject. Adverse events in the NTM-1633 groups were generally mild and similar in frequency and severity to the placebo group, and no safety signal was identified. NTM-1633 has a favorable PK profile with a half-life >10 days for the 0.330 mg/kg dose and an approximately linear relationship with respect to maximum concentration and area under the concentration-time curve (AUC0→t). NTM-1633 also demonstrated low immunogenicity. NTM-1633 is well tolerated at the administered doses. The favorable safety, PK, and immunogenicity profile supports further development as a treatment for BoNT/E intoxication and postexposure prophylaxis.

Safety, Tolerability, and Pharmacokinetics of NTM-1632, a Novel Mixture of Three Monoclonal Antibodies against Botulinum Toxin B.

Botulism is a rare, life-threatening paralytic disease caused by Clostridium botulinum neurotoxin (BoNT). Available treatments, including an equine antitoxin and human immune globulin, are given postexposure and challenging to produce and administer. NTM-1632 is an equimolar mixture of 3 human IgG monoclonal antibodies, B1, B2, and B3, targeting BoNT serotype B (BoNT/B). This first-in-human study assessed the safety, tolerability, pharmacokinetics (PK), and immunogenicity of NTM-1632. This double-blind, single-center, placebo-controlled dose escalation study randomized 3 cohorts of healthy volunteers to receive a single intravenous dose of NTM-1632 (0.033, 0.165, or 0.330 mg/kg) or saline placebo. Safety monitoring included physical examinations, clinical laboratory studies, and vital signs. Blood sampling was performed at prespecified time points for PK and immunogenicity analyses. Twenty-four subjects received study product (18 NTM-1632; 6 placebo), and no deaths or serious adverse events were reported. Adverse events in the NTM-1632 groups were generally mild and similar in frequency and severity to the placebo group, and no safety signal was identified. NTM-1632 has a favorable PK profile with a half-life of >20 days for the 0.330-mg/kg dose and an approximately linear relationship with respect to maximum concentration and area under the concentration-time curve (AUC0→t). NTM-1632 demonstrated low immunogenicity with only a few treatment-emergent antidrug antibody responses in the low and middle dosing groups and none at the highest dose. NTM-1632 is well tolerated at the administered doses. The favorable safety, PK, and immunogenicity profile of NTM-1632 supports further clinical development as a treatment for BoNT/B intoxication and postexposure prophylaxis. (This study has been registered at ClinicalTrials.gov under identifier NCT02779140.).

The antithrombin P1 residue is important for target proteinase specificity but not for heparin activation of the serpin. Characterization of P1 antithrombin variants with altered proteinase specificity but normal heparin activation.

Heparin has been proposed to conformationally activate the serpin, antithrombin, by making the reactive center loop P1 arginine residue accessible to proteinases. To evaluate this proposal, we determined the effect of mutating the P1 arginine on antithrombin's specificity for target and nontarget proteinases in both native and heparin-activated states of the serpin. As expected, mutation of the P1 arginine to tryptophan, histidine, leucine, and methionine converted the specificity of antithrombin from a trypsin inhibitor (k(assoc) = 2 x 10(5) M(-1) s(-1)) to a chymotrypsin inhibitor (k(assoc) = 10(3)-10(5) M(-1) s(-1)). However, heparin pentasaccharide activation increased the reactivity of the P1 variants with chymotrypsin or of the wild-type inhibitor with trypsin only 2-6-fold, implying that the P1 residue had similar accessibilities to these proteinases in native and activated states. Mutation of the P1 arginine greatly reduced k(assoc) for antithrombin inhibition of thrombin and factor Xa from 40- to 5000-fold, but heparin normally accelerated the reactions of the variant antithrombins with these enzymes to make them reasonably efficient inhibitors (k(assoc) = 10(3)-10(4) M(-1) s(-1)). Fluorescence difference spectra of wild-type and P1 tryptophan variant antithrombins showed that the P1 tryptophan exhibited fluorescence properties characteristic of a solvent-exposed residue which were insignificantly affected by heparin activation. Moreover, all P1 variant antithrombins bound heparin with approximately 2-3-fold higher affinities than the wild type. These findings are consistent with the P1 mutations disrupting a P1 arginine-serpin body interaction which stabilizes the native low-heparin affinity conformation, but suggest that this interaction is of low energy and unlikely to limit the accessibility of the P1 residue. Together, these findings suggest that the P1 arginine residue is similarly accessible to proteinases in both native and heparin-activated states of the serpin and contributes similarly to the specificity of antithrombin for thrombin and factor Xa in the two serpin conformational states. Consequently, determinants other than the P1 residue are responsible for enhancing the specificity of antithrombin for the two proteinases when activated by heparin.

Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin.

Heparin activates the primary serpin inhibitor of blood clotting proteinases, antithrombin, both by an allosteric conformational change mechanism that specifically enhances factor Xa inactivation and by a ternary complex bridging mechanism that promotes the inactivation of thrombin and other target proteinases. To determine whether the factor Xa specificity of allosterically activated antithrombin is encoded in the reactive center loop sequence, we attempted to switch this specificity by mutating the P6-P3' proteinase binding sequence excluding P1-P1' to a more optimal thrombin recognition sequence. Evaluation of 12 such antithrombin variants showed that the thrombin specificity of the serpin allosterically activated by a heparin pentasaccharide could be enhanced as much as 55-fold by changing P3, P2, and P2' residues to a consensus thrombin recognition sequence. However, at most 9-fold of the enhanced thrombin specificity was due to allosteric activation, the remainder being realized without activation. Moreover, thrombin specificity enhancements were attenuated to at most 5-fold with a bridging heparin activator. Surprisingly, none of the reactive center loop mutations greatly affected the factor Xa specificity of the unactivated serpin or the several hundred-fold enhancement in factor Xa specificity due to activation by pentasaccharide or bridging heparins. Together, these results suggest that the specificity of both native and heparin-activated antithrombin for thrombin and factor Xa is only weakly dependent on the P6-P3' residues flanking the primary P1-P1' recognition site in the serpin-reactive center loop and that heparin enhances serpin specificity for both enzymes through secondary interaction sites outside the P6-P3' region, which involve a bridging site on heparin in the case of thrombin and a previously unrecognized exosite on antithrombin in the case of factor Xa.

Localization and environment of tryptophans in soluble and membrane-bound states of a pore-forming toxin from Staphylococcus aureus.

The location and environment of tryptophans in the soluble and membrane-bound forms of Staphylococcus aureus alpha-toxin were monitored using intrinsic tryptophan fluorescence. Fluorescence quenching of the toxin monomer in solution indicated varying degrees of tryptophan burial within the protein interior. N-Bromosuccinimide readily abolished 80% of the fluorescence in solution. The residual fluorescence of the modified toxin showed a blue-shifted emission maximum, a longer fluorescence lifetime as compared to the unmodified and membrane-bound alpha-toxin, and a 5- to 6-nm red edge excitation shift, all indicating a restricted tryptophan environment and deeply buried tryptophans. In the membrane-bound form, the fluorescence of alpha-toxin was quenched by iodide, indicating a conformational change leading to exposure of some tryptophans. A shorter average lifetime of tryptophans in the membrane-bound alpha-toxin as compared to the native toxin supported the conclusions based on iodide quenching of the membrane-bound toxin. Fluorescence quenching of membrane-bound alpha-toxin using brominated and spin-labeled fatty acids showed no quenching of fluorescence using brominated lipids. However, significant quenching was observed using 5- and 12-doxyl stearic acids. An average depth calculation using the parallax method indicated that the doxyl-quenchable tryptophans are located at an average depth of 10 A from the center of the bilayer close to the membrane interface. This was found to be in striking agreement with the recently described structure of the membrane-bound form of alpha-toxin.

Movement of a loop in domain 3 of aerolysin is required for channel formation.

Aerolysin is a channel-forming toxin that must oligomerize in order to become insertion-competent. Modeling based on the crystal structure of the proaerolysin dimer and electron microscopic images of the oligomer indicated that a loop in domain 3 must move away from the beta-sheet that forms the main body of the protein before oligomerization can proceed. In order to determine if movement actually occurs, strategically located amino acids in the loop and in the sheet were replaced with cysteines by site-directed mutagenesis. A double mutant was produced in which the new cysteines, at position 253 on the loop and position 300 in the sheet, were close enough together to allow formation of a disulfide bridge. The double mutant was unable to oligomerize, and it was completely inactive, showing not only that the bridge had formed but also that movement of the loop was essential for formation of the oligomer. The existence of the bridge was confirmed by X-ray crystallography. The reduced form of the protein and the single mutants T253C and A300C were as active as wild type, indicating that the amino acid replacements themselves had no functional consequences. Labeling studies using an environment-sensitive fluorescent sulfhydryl-reactive probe confirmed that the structure of the protein changes in the loop region as a consequence of proteolytic activation of proaerolysin, a step which also must precede oligomerization.

Glycosylphosphatidylinositol anchors of membrane glycoproteins are binding determinants for the channel-forming toxin aerolysin.

Cells that are sensitive to the channel-forming toxin aerolysin contain surface glycoproteins that bind the toxin with high affinity. Here we show that a common feature of aerolysin receptors is the presence of a glycosylphosphatidylinositol anchor, and we present evidence that the anchor itself is an essential part of the toxin binding determinant. The glycosylphosphatidylinositol (GPI)-anchored T-lymphocyte protein Thy-1 is an example of a protein that acts as an aerolysin receptor. This protein retained its ability to bind aerolysin when it was expressed in Chinese hamster ovary cells, but could not bind the toxin when expressed in Escherichia coli, where the GPI anchor is absent. An unrelated GPI-anchored protein, the variant surface glycoprotein of trypanosomes, was shown to bind aerolysin with similar affinity to Thy-1, and this binding ability was significantly reduced when the anchor was removed chemically. Cathepsin D, a protein with no affinity for aerolysin, was converted to an aerolysin binding form when it was expressed as a GPI-anchored hybrid in COS cells. Not all GPI-anchored proteins bind aerolysin. In some cases this may be due to differences in the structure of the anchor itself. Thus the GPI-anchored proteins procyclin of Trypanosoma congolense and gp63 of Leishmania major did not bind aerolysin, but when gp63 was expressed with a mammalian GPI anchor in Chinese hamster ovary cells, it bound the toxin.

The glycosylphosphatidylinositol-anchored surface glycoprotein Thy-1 is a receptor for the channel-forming toxin aerolysin.

Aerolysin is a channel-forming protein secreted by virulent Aeromonas spp. Some eucaryotic cells, including T-lymphocytes, are sensitive to very low concentrations of the toxin (<10(-9) M). Here we show that aerolysin binds selectively and with high affinity to the glycosylphosphatidylinositol (GPI)-anchored surface protein Thy-1, which is found on T-lymphocyte populations as well as in brain. Less than 1 ng of purified Thy-1 could be detected by probing Western blots with the toxin. Mutant T-cell lines that lack the ability to add GPI anchors to Thy-1 and other surface proteins were much less sensitive to aerolysin, as were wild-type cells that were pretreated with phosphatidylinositol-specific phospholipase C to remove GPI-anchored proteins. Phosphatidylcholine/cholesterol liposomes containing purified Thy-1 in their membranes were much more sensitive to aerolysin than protein-free liposomes.

A survey of dermatophytosis in animals in Madras, India.

Two hundred and eleven dogs (including strictly house and stray dogs) and 170 cattle in and around the city of Madras, India were screened for the presence of dermatophytosis. 106 strains of dermatophytes (89 strains from dogs and 17 strains from bovines) were isolated. 57/106 strains were Trichophyton mentagrophytes var. mentagrophytes and 42/106 strains were of the Microsporum gypseum complex. 5 strains of T. rubrum and 2 strains of T. simii were also obtained in culture. A predominance of M. gypseum complex isolates was recorded in stray dogs and cattle and T. mentagrophytes var. mentagrophytes and T. rubrum in strictly house dogs. The family history of the owners of the most of the dogs had clear records of dermatophytosis. Further, the owners of the 11 dogs that yielded T. mentagrophytes var. mentagrophytes had either tinea corporis or tinea pedis. The etiological agent of all the 11 human cases was T. mentagrophytes var. interdigitale. Similarly the owners of 4 of the 5 dogs that yielded T. rubrum were known T. rubrum patients. All these patients responded to oral griseofulvin or ketaconozole, but the recurrence of lesions was noted with the cessation of treatment. None of the patients had onychomycosis and the family history of all the patients revealed no reports of T. rubrum infections. The pet dogs were presumed to be the source of re-infection. Reversed transmission of dermatophytes from humans to animals may be the reason for the selective predominance of these organisms in strictly house dogs. They also may act as sources of reinfection. Most of the animals had small, occult, scattered lesions. These lesions may either go unnoticed or are ignored by the owners of the animals. The taxonomic status of T. mentagrophytes and T. mentagrophytes var. interdigitale was aligned to their teleomorph Arthroderma vanbreuseghemii. Our study suggests that the periodic screening and medication of all live-stock are essential for the prevention and management of the public health problem caused by dermatophytes.

Photolabeling of a pore-forming toxin with the hydrophobic probe 2-[3H]diazofluorene. Identification of membrane-inserted segments of Staphylococcus aureus alpha-toxin.

The identification of membrane-inserted segments of pore-forming soluble proteins is crucial to understanding the action of these proteins at the molecular level. A distinct member of this class of proteins is alpha-toxin, a 293-amino acid-long 33-kDa hemolytic toxin secreted by Staphylococcus aureus that can form pores in both artificial and natural membranes. We have studied the interaction of alpha-toxin with single bilayer vesicles prepared from asolectin using a hydrophobic photoactivable reagent, 2-[3H]diazofluorene ([3H]DAF) (Pradhan, D., and Lala, A. K. (1987) J. Biol. Chem. 262, 8242-8251). This reagent readily partitions into the membrane hydrophobic core and on photolysis labels the lipid and protein segments that penetrate the membrane. Current models on the mode of action of alpha-toxin indicate that, on interaction with membranes, alpha-toxin forms an oligomer, which represents the active pore. In keeping with these models, we observe that [3H]DAF photolabels the membrane-bound alpha-toxin oligomer. Cyanogen bromide fragmentation of [3H]DAF-labeled alpha-toxin gave several fragments, which were subjected to Edman degradation. We could thus sequence residues 1-19, 35-60, 114-139, 198-231, and 235-258. Radioactive analysis and phenylthiohydantoin-derivative analysis during sequencing permitted analysis of DAF insertion sites. The results obtained indicated that the N and C termini (residues 235-258) have been extensively labeled. The putative pore-forming glycine-rich central hinge region was poorly labeled, indicating that the apposing side of the lumen of the pore does not form the lipid-protein interface. The DAF labeling pattern indicated that the major structural motif in membrane-bound alpha-toxin was largely beta-sheet.