Development of Humanized Monoclonal Antibody TMA-15 Which Neutralizes Shiga Toxin 2

Kimura, T.; Co, Man Sung; Vásquez, Maximiliano; Wei, Sharon; Xu, Hattie; Tani, Shinobu; Sakai, Yoshiko; Kawamura, Takashi; Matsumoto, Yoh-ichi; Nakao, Hiroshi; Takeda, Tae

doi:10.1089/153685902760173872

Cited by 29 publications

(27 citation statements)

References 35 publications

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“…With this in mind, we developed HuMAbs to neutralize Stx (17)(18)(19), which were shown to be highly effective in protecting STEC-infected piglets (17,20,21). Other investigators have also developed Stx-neutralizing chimeric (47) or humanized (48,49) MAbs. HuMAb 5C12, specific for Stx2, was found to be highly effective in protecting piglets 48 h after STEC infection; a dose of 0.4 mg/kg of body weight protected 80% of piglets (20).…”

Section: Discussionmentioning

confidence: 99%

Adenovirus Vector Expressing Stx1/Stx2-Neutralizing Agent Protects Piglets Infected with Escherichia coli O157:H7 against Fatal Systemic Intoxication

et al. 2015

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b Hemolytic-uremic syndrome (HUS), caused by Shiga toxin (Stx)-producing Escherichia coli (STEC), remains untreatable. Production of human monoclonal antibodies against Stx, which are highly effective in preventing Stx sequelae in animal models, is languishing due to cost and logistics. We reported previously that the production and evaluation of a camelid heavy-chain-only V H domain (VHH)-based neutralizing agent (VNA) targeting Stx1 and Stx2 (VNA-Stx) protected mice from Stx1 and Stx2 intoxication. Here we report that a single intramuscular (i.m.) injection of a nonreplicating adenovirus (Ad) vector carrying a secretory transgene of VNA-Stx (Ad/VNA-Stx) protected mice challenged with Stx2 and protected gnotobiotic piglets infected with STEC from fatal systemic intoxication. One i.m. dose of Ad/VNA-Stx prevented fatal central nervous system (CNS) symptoms in 9 of 10 animals when it was given to piglets 24 h after bacterial challenge and in 5 of 9 animals when it was given 48 h after bacterial challenge, just prior to the onset of CNS symptoms. All 6 placebo animals died or were euthanized with severe CNS symptoms. Ad/VNA-Stx treatment had no impact on diarrhea. In conclusion, Ad/VNA-Stx treatment is effective in protecting piglets from fatal Stx2-mediated CNS complications following STEC challenge. With a low production cost and further development, this could presumably be an effective treatment for patients with HUS and/or individuals at high risk of developing HUS due to exposure to STEC. I nfection with Shiga toxin (Stx)-producing Escherichia coli (STEC)is the most significant cause of hemolytic-uremic syndrome (HUS), the leading cause of acute renal failure in children (1-4) and in some adults. Of the two antigenically distinct toxins, Stx1 and Stx2, Stx2 is more firmly linked with the development of HUS, since STEC strains producing this toxin are more frequently associated with HUS than strains that produce both Stx1 and Stx2, while Stx1 alone has rarely been associated with HUS (5-7). Stx1 and Stx2 are similar in basic structure (8), binding specificity (8), and mode of action (9, 10). Both toxins consist of an A-subunit monomer and a B-subunit pentamer (8,11,12). The pentameric B subunit binds to its cell surface receptor, CD77, also called globotriaosyl ceramide (Gb3; Gal␣1-4 Gal␤1-4 glucosyl ceramide) (13,14). This binding triggers endocytosis of the holotoxin, mainly through clathrin-coated pits (15). Internalization of the catalytically active A subunit, delivered to the cytosol via retrograde transport, causes the shutdown of protein synthesis and leads to cell death (9, 10). In addition to blocking protein synthesis, a longterm effect of the toxin in several types of cells is the induction of apoptosis (16).We previously reported the production of human monoclonal antibodies (HuMAbs) against Stx1 and Stx2 and their evaluation in animal models for efficacy against systemic toxin challenge (17-19) or oral STEC infection (17,(19)(20)(21). Clinical evaluation of these monoclonal antibodies has been s...

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Section: Discussionmentioning

confidence: 99%

Adenovirus Vector Expressing Stx1/Stx2-Neutralizing Agent Protects Piglets Infected with Escherichia coli O157:H7 against Fatal Systemic Intoxication

et al. 2015

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“…However, our research group is one of several that investigate passive immunization strategies to neutralize the Stxs associated with STEC infections (3,4,10,13,19,20). Our passive immunization strategy is based on murine monoclonal antibodies (MAbs) developed in this laboratory that specifi-cally bind to and neutralize Stx/Stx1 or Stx2 (21,28).…”

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confidence: 99%

Monoclonal Antibody 11E10, Which Neutralizes Shiga Toxin Type 2 (Stx2), Recognizes Three Regions on the Stx2 A Subunit, Blocks the Enzymatic Action of the Toxin In Vitro, and Alters the Overall Cellular Distribution of the Toxin

et al. 2009

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Monoclonal antibody (MAb) 11E10 recognizes the Shiga toxin type 2 (Stx2) A 1 subunit. The binding of 11E10 to Stx2 neutralizes both the cytotoxic and lethal activities of Stx2, but the MAb does not bind to or neutralize Stx1 despite the 61% identity and 75% similarity in the amino acids of the A 1 fragments. In this study, we sought to identify the segment or segments on Stx2 that constitute the 11E10 epitope and to determine how recognition of that region by 11E10 leads to inactivation of the toxin. Toward those objectives, we generated a set of chimeric Stx1/Stx2 molecules and then evaluated the capacity of 11E10 to recognize those hybrid toxins by Western blot analyses and to neutralize them in Vero cell cytotoxicity assays. We also compared the amino acid sequences and crystal structures of Stx1 and Stx2 for stretches of dissimilarity that might predict a binding epitope on Stx2 for 11E10. Through these assessments, we concluded that the 11E10 epitope is comprised of three noncontiguous regions surrounding the Stx2 active site. To determine how 11E10 neutralizes Stx2, we examined the capacity of 11E10/Stx2 complexes to target ribosomes. We found that the binding of 11E10 to Stx2 prevented the toxin from inhibiting protein synthesis in an in vitro assay but also altered the overall cellular distribution of Stx2 in Vero cells. We propose that the binding of MAb 11E10 to Stx2 neutralizes the effects of the toxin by preventing the toxin from reaching and/or inactivating the ribosomes.

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“…Additionally, a third MAb (2H3) that recognizes StxB1, but not StxB1d, has been described, and residue 55 may play a role in that difference as well (3). Finally, another MAb, VTm1.1 (later humanized and called TMA-15), recognizes StxB2 and neutralizes Stx2 (12,22) but fails to recognize StxB2 when the 56th amino acid is mutated (E56H). The 55th residue of StxB1 and the 56th residue of StxB2 are both located on the outside of the B monomers in approximately the same location.…”

Section: Discussionmentioning

confidence: 99%

“…Several recent vaccine candidates not described in the above-mentioned reviews include Stx1 and Stx2 genetic toxoids, a plantbased Stx2 toxoid, and a chimeric StxA2/StxB1 toxoid that elicits a neutralizing antibody response and provides protection against a lethal challenge of both Stx1 and Stx2 (25,33,34). Several groups are pursuing passive immunization strategies that utilize humanized or human monoclonal antibodies (MAb) that neutralize one or more Stxs (6,7,12,18,19,35). STEC may produce one or more types of Stxs.…”

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confidence: 99%

The 13C4 Monoclonal Antibody That Neutralizes Shiga Toxin Type 1 (Stx1) Recognizes Three Regions on the Stx1 B Subunit and Prevents Stx1 from Binding to Its Eukaryotic Receptor Globotriaosylceramide

et al. 2006

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The 13C4 monoclonal antibody (MAb) recognizes the B subunit of Stx1 (StxB1) and neutralizes the cytotoxic and lethal activities of Stx1. However, this MAb does not bind to the B polypeptide of Stx2, despite the 73% amino acid sequence similarity between StxB1 and StxB2. When we compared the amino acid sequences of StxB1 and StxB2, we noted three regions of dissimilarity (amino acids 1 to 6, 25 to 32, and 54 to 61) located near each other on the crystal structure of StxB1. To identify the 13C4 epitope, we generated seven Stx1/Stx2 B chimeric polypeptides that contained one, two, or three of the dissimilar StxB1 regions. The 13C4 MAb reacted strongly with StxB1 and the triple-chimeric B subunit but not with the other chimeras. Mice immunized with the triple-chimeric B subunit survived a lethal challenge with Stx1 but not Stx2, substantiating the identified regions as the 13C4 MAb epitope and suggesting that the incorporation of this epitope into StxB2 altered sites necessary for anti-Stx2-neutralizing Ab production. Next, single amino acid substitutions were made in StxB1 to mimic Stx1d, a variant not recognized by the 13C4 MAb. The 13C4 MAb reacted strongly to StxB1 with the T1A or G25A mutations but not with the N55T change. Finally, we found that the 13C4 MAb blocked the binding of Stx1 to its receptor, globotriaosyl ceramide. Taken together, these results indicate that the 13C4 MAb prevents the interaction of Stx1 with its receptor by binding three nonlinear regions of the molecule that span receptor recognition sites on StxB1, one of which includes the essential residue 55N.In the United States, Shiga toxin-producing Escherichia coli (STEC) strains account for about 110,000 infections per year (16). Enterohemorrhagic E. coli (EHEC) strains are a subset of STEC that contain a large pathogenicity island called the locus of enterocyte effacement and carry a 90-kb plasmid (13,30,32). Enterohemorrhagic E. coli serotype O157:H7 in particular is noted for it association with food-, water-, or petting zoo-linked outbreaks of Stx-mediated disease (4,23,28). A possible complication from an infection with an Stx-producing organism is the hemolytic uremic syndrome (HUS), a syndrome that is characterized by hemolytic anemia, thrombic thrombocytopenia, and renal failure. The majority of patients with HUS are children (5, 23), and there is a 5 to 10% fatality rate for those individuals. Furthermore, HUS survivors may have lasting kidney damage. Currently, there are no FDA-approved therapies or vaccines in the United States to combat or prevent illness from STEC, but several promising options for the future are under investigation. These options include receptor mimics and active and passive immunization strategies (reviewed in references 17 and 31). Several recent vaccine candidates not described in the above-mentioned reviews include Stx1 and Stx2 genetic toxoids, a plantbased Stx2 toxoid, and a chimeric StxA2/StxB1 toxoid that elicits a neutralizing antibody response and provides protection against a lethal challenge of b...

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Development of Humanized Monoclonal Antibody TMA-15 Which Neutralizes Shiga Toxin 2

Cited by 29 publications

References 35 publications

Adenovirus Vector Expressing Stx1/Stx2-Neutralizing Agent Protects Piglets Infected with Escherichia coli O157:H7 against Fatal Systemic Intoxication

Adenovirus Vector Expressing Stx1/Stx2-Neutralizing Agent Protects Piglets Infected with Escherichia coli O157:H7 against Fatal Systemic Intoxication

Monoclonal Antibody 11E10, Which Neutralizes Shiga Toxin Type 2 (Stx2), Recognizes Three Regions on the Stx2 A Subunit, Blocks the Enzymatic Action of the Toxin In Vitro, and Alters the Overall Cellular Distribution of the Toxin

The 13C4 Monoclonal Antibody That Neutralizes Shiga Toxin Type 1 (Stx1) Recognizes Three Regions on the Stx1 B Subunit and Prevents Stx1 from Binding to Its Eukaryotic Receptor Globotriaosylceramide

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