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

Identification of human monoclonal antibodies specific for human SOD1 recognizing distinct epitopes and forms of SOD1.

Mutations in the gene encoding human SOD1 (hSOD1) can cause amyotrophic lateral sclerosis (ALS) yet the mechanism by which mutant SOD1 can induce ALS is not fully understood. There is currently no cure for ALS or treatment that significantly reduces symptoms or progression. To develop tools to understand the protein conformations present in mutant SOD1-induced ALS and as possible immunotherapy, we isolated and characterized eleven unique human monoclonal antibodies specific for hSOD1. Among these, five recognized distinct linear epitopes on hSOD1 that were not available in the properly-folded protein but were available on forms of protein with some degree of misfolding. The other six antibodies recognized conformation-dependent epitopes that were present in the properly-folded protein with two different recognition profiles: three could bind hSOD1 dimer or monomer and the other three were specific for hSOD1 dimer only. Antibodies with the capacity to bind hSOD1 monomer were able to prevent increased hydrophobicity when mutant hSOD1 was exposed to increased temperature and EDTA, suggesting that the antibodies stabilized the native structure of hSOD1. Two antibodies were tested in a G93A mutant hSOD1 transgenic mouse model of ALS but did not yield a statistically significant increase in overall survival. It may be that the two antibodies selected for testing in the mouse model were not effective for therapy or that the model and/or route of administration were not optimal to produce a therapeutic effect. Therefore, additional testing will be required to determine therapeutic potential for SOD1 mutant ALS and potentially some subset of sporadic ALS

Identification and Characterization of Broadly Neutralizing Human Monoclonal Antibodies Directed against the E2 Envelope Glycoprotein of Hepatitis C Virus▿

Nearly all livers transplanted into hepatitis C virus (HCV)-positive patients become infected with HCV, and 10 to 25% of reinfected livers develop cirrhosis within 5 years. Neutralizing monoclonal antibody could be an effective therapy for the prevention of infection in a transplant setting. To pursue this treatment modality, we developed human monoclonal antibodies (HuMAbs) directed against the HCV E2 envelope glycoprotein and assessed the capacity of these HuMAbs to neutralize a broad panel of HCV genotypes. HuMAb antibodies were generated by immunizing transgenic mice containing human antibody genes (HuMAb mice; Medarex Inc.) with soluble E2 envelope glycoprotein derived from a genotype 1a virus (H77). Two HuMAbs, HCV1 and 95-2, were selected for further study based on initial cross-reactivity with soluble E2 glycoproteins derived from genotypes 1a and 1b, as well as neutralization of lentivirus pseudotyped with HCV 1a and 1b envelope glycoproteins. Additionally, HuMAbs HCV1 and 95-2 potently neutralized pseudoviruses from all genotypes tested (1a, 1b, 2b, 3a, and 4a). Epitope mapping with mammalian and bacterially expressed proteins, as well as synthetic peptides, revealed that HuMAbs HCV1 and 95-2 recognize a highly conserved linear epitope spanning amino acids 412 to 423 of the E2 glycoprotein. The capacity to recognize and neutralize a broad range of genotypes, the highly conserved E2 epitope, and the fully human nature of the antibodies make HuMAbs HCV1 and 95-2 excellent candidates for treatment of HCV-positive individuals undergoing liver transplantation

Accumulation of succinate controls activation of adipose tissue thermogenesis.

Thermogenesis by brown and beige adipose tissue, which requires activation by external stimuli, can counter metabolic disease1. Thermogenic respiration is initiated by adipocyte lipolysis through cyclic AMP-protein kinase A signalling; this pathway has been subject to longstanding clinical investigation2-4. Here we apply a comparative metabolomics approach and identify an independent metabolic pathway that controls acute activation of adipose tissue thermogenesis in vivo. We show that substantial and selective accumulation of the tricarboxylic acid cycle intermediate succinate is a metabolic signature of adipose tissue thermogenesis upon activation by exposure to cold. Succinate accumulation occurs independently of adrenergic signalling, and is sufficient to elevate thermogenic respiration in brown adipocytes. Selective accumulation of succinate may be driven by a capacity of brown adipocytes to sequester elevated circulating succinate. Furthermore, brown adipose tissue thermogenesis can be initiated by systemic administration of succinate in mice. Succinate from the extracellular milieu is rapidly metabolized by brown adipocytes, and its oxidation by succinate dehydrogenase is required for activation of thermogenesis. We identify a mechanism whereby succinate dehydrogenase-mediated oxidation of succinate initiates production of reactive oxygen species, and drives thermogenic respiration, whereas inhibition of succinate dehydrogenase supresses thermogenesis. Finally, we show that pharmacological elevation of circulating succinate drives UCP1-dependent thermogenesis by brown adipose tissue in vivo, which stimulates robust protection against diet-induced obesity and improves glucose tolerance. These findings reveal an unexpected mechanism for control of thermogenesis, using succinate as a systemically-derived thermogenic molecule

Antibody delivery to hSOD1-G93A transgenic mice.

<p>(A) HuMabs 37_L-63 (red) and 120_c (green) and an irrelevant isotype-matched HuMab (IR Mab, blue) were delivered to the lumbar intrathecal space of hSOD1-G93A transgenic mice via osmotic pump from mouse age 65 to 115 (days). Complete two limb paralysis was used as an endpoint of disease and the day of disease endpoint was used to calculate the percent survival each day. For each group, the mean days to reach the endpoint (mean survival) and P value from the Mantel-Cox test (log-rank) were calculated using JMP and are listed below the graph. (B) HuMab 37_L-63 (red) and an irrelevant isotype-matched HuMab (IR Mab, blue) were delivered by intraperitoneal injection to hSOD1-G93A transgenic mice. Antibody dosing was initiated at mouse age of 65 days and continued once per week for the duration of the mouse survival. Endpoint and statistics were calculated as above.</p

HuMabs recognize different regions of the hSOD1 protein.

<p>(A) Full-length hSOD1 (amino acids 1–153) and various portions of the protein were expressed in bacteria fused to the carboxy-terminus (C-terminus) of thioredoxin (Trx) and containing a C-terminal 6-histidine tag used for purification (Trx-hSOD1-WT-his). Each protein is represented in the figure as a hashed line with the beginning and ending amino acid number listed below the line. The proteins were coated on ELISA plates and binding of HuMabs (listed at the top right) detected with goat-anti-human antibody conjugated to alkaline phosphatase followed by PNPP substrate addition. ELISA results are listed to the right of the schematic; positive recognition is indicated by a plus sign while signals equivalent to background are indicated by a minus sign. HuMabs recognizing only full-length hSOD1 were designated conformation dependent and are noted below with a C. HuMabs with an epitope that mapped to a linear sequence of amino acids are noted below with an L. (B) Minimal linear epitopes were determined with amino-terminal biotin-labeled overlapping peptides coated on streptavidin ELISA plates. Binding of HuMabs was assessed as described in A. The epitopes are noted as a grey box for each linear-epitope HuMab with the amino acids (aa) bound indicated. To distinguish these epitopes throughout the rest of the manuscript each linear-epitope HuMab has a subscripted L for linear followed by the initial amino acid of the epitope.</p

Increased hydrophobicity of hSOD1 can be inhibited by a subset of hSOD1 specific HuMabs.

<p>(A) hSOD1-G85R, hSOD1-G93A, various hSOD1-specific HuMabs, and an irrelevant isotype-matched antibody (IR Mab) were separately incubated with 5 mM EDTA at room temperature (RT) or 45°C for 4 hrs. Hydrophobicity was measured with ANS dye fluorescence using a Victor 3 plate reader at 390 nm excitation and 460 nm emission and is reported as the fold change in relative fluorescence units from RT to 45°C. hSOD1-G85R (B) and hSOD1-G93A (C) were mixed with irrelevant antibody or various HuMabs with 5 mM EDTA and incubated at RT or 45°C for 4 hrs. Hydrophobicity was measured as indicated above. The mean of replicates is noted with a thick black bar with the standard deviation indicated. Samples with insufficient material for replicates are indicated with the single data point as a thick grey bar. Statistical significant differences from irrelevant antibody were determined using a Student’s t-test with P<0.01 (***), <0.05 (**), and <0.10 (*) indicated above the black bar for each sample.</p

HuMab epitope location in predicted hSOD1 structure.

<p>(A) The crystal structure of one monomer of a hSOD1 dimer (2C9V) is indicated in ribbon model from Rasmol. The bound zinc is indicated as a yellow sphere. The two cysteines involved in the intramolecular disulfide bond (C57 and C146) are displayed as wireframe in light blue. The phenylalanine at position 50 and glycine at position 51, two residues mutated at the dimer interface to generate apo-hSOD1-monomer, are displayed as wireframe in dark grey. HuMab epitopes are indicated as follows: 16_L-40 amino acids 40–47 in yellow, 3_L-42 amino acids 42–49 in red, the overlap of these two epitopes is orange, 37_L-63 amino acids 63–71 in dark blue, 11_L-80 amino acids 80–88 in purple, and 33_L-112 amino acids 112–121 in green. (B) The same orientation molecule in (A) is displayed as a space-filling model. (C) The ribbon model from (A) is rotated 90 degrees on the x-axis to view from the bottom of the molecule in (A). (D) The same orientation molecule in (C) is displayed as a space-filling model.</p

HuMab immunoprecipitation of WT and mutant hSOD1 from mammalian cells.

<p>A human derived cell line (293T) was transiently transfected with vectors engineered to express myc-tagged wild type (WT) or mutant hSOD1 (A4V, G85R, or G93A), or with empty vector as a control (neg). (A) Lysate from transfected cells was subjected to SDS-PAGE and immunoblot. Myc tagged proteins were detected with a mouse monoclonal antibody specific for the myc tag followed by goat anti-mouse HRP conjugate and chemiluminescence. An arrow to the right of the blots indicates a band present at the expected size for SOD1 (16 kDa). Lysate from transfected cells was mixed with an irrelevant isotype-matched antibody (B), HuMab 120_c (C), and HuMab 11_L-80 (D) and incubated at ambient temperature for 2 hrs followed by immunoprecipitation (IP) with protein A sepharose beads. Precipitated material was subjected to SDS-PAGE and immunoblotted with the anti-myc antibody. (E) Additional immunoprecipitations where performed with the remaining HuMabs, and the presence or absence of a band at the appropriate size for SOD1 in anti-myc immunoblots is indicated with a plus or minus.</p

Competition of various HuMabs for binding to Trx-hSOD1-WT-his.

<p>(A) HuMabs designated as conformation-dependent where assayed for simultaneous binding of hSOD1. The HuMab listed to the left of the graph was bound to an anti-human biosensor and then allowed to bind Trx-hSOD1-WT-his. The second antibody (listed at the top of the graph) was then assayed for binding to the biosensor through interaction with Trx-hSOD1-WT-his. Simultaneous binding of both antibodies is indicated as a white box. Competing antibodies are indicated as a dark grey box. (B) The epitopes of the conformation-dependent HuMabs are represented graphically by overlapping circles. Circles that do not overlap represent antibodies that can bind the protein simultaneously. Circles that overlap are competing epitopes. Based on this profile, the conformation-dependent epitopes were grouped into five different binding profiles numbered from one to five.</p