Production and Characterization of Protective Human Antibodies against Shiga Toxin 1

Mukherjee, Jean; Chios, Kerry; Fishwild, Dianne M.; Hudson, Deborah; O'Donnell, Susan L.; Rich, Stephen M.; Donohue‐Rolfe, Arthur; Tzipori, Saul

doi:10.1128/iai.70.10.5896-5899.2002

Cited by 55 publications

(54 citation statements)

References 29 publications

(23 reference statements)

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“…Several mouse strains harboring human Ig loci have been generated over the last years as an alternative strategy for the generation of human mAb of therapeutic value, and in a number of cases, the efforts have been quite successful [19][20][21][22][23]. However, in a previous study, immunization of mice transgenic for human l, c1, and j germ-line genes (HuMab-Mice) with Torpedo AChR or the fusion protein Trx-Ha1-210 did not result in the isolation of anti-AChR mAb [17].…”

Section: Discussionmentioning

confidence: 99%

Isolation and characterization of human anti-acetylcholine receptor monoclonal antibodies from transgenic mice expressing human immunoglobulin loci

Protopapadakis¹,

Kokla²,

Tzartos³

et al. 2005

Eur. J. Immunol.

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The isolation of human antibodies against muscle acetylcholine receptor (AChR), the autoantigen involved in myasthenia gravis (MG), is important for the development of therapeutically useful reagents. Monovalent antibody fragments from monoclonal antibodies against the main immunogenic region (MIR) of AChR protect the receptor from the destructive activity of MG autoantibodies. Human anti-AChR a-subunit antibody fragments with therapeutic potential have been isolated using phage display antibody libraries. An alternative approach for obtaining human mAb has been provided by the development of humanized mice. In this report, we show that immunization of transgenic mouse strains with the extracellular domain of the human AChR a-subunit results in antibody responses and isolation of hybridomas producing human mAb. Four specific IgM mAb were isolated and analyzed. mAb170 recognized the native receptor the best and was capable of inducing AChR antigenic modulation, suggesting its specificity for a pathogenic epitope. Moreover, the recombinant antigen-binding (Fab) fragment of this mAb competed with an anti-MIR mAb, revealing that its antigenic determinant lies in or near the MIR. Finally, Fab170 was able to compete with MG autoantibodies and protect the AChR against antigenic modulation induced by MG sera. This approach will be useful for isolating additional mAb with therapeutic potential against the other AChR subunits.

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Section: Discussionmentioning

confidence: 99%

Isolation and characterization of human anti-acetylcholine receptor monoclonal antibodies from transgenic mice expressing human immunoglobulin loci

Protopapadakis¹,

Kokla²,

Tzartos³

et al. 2005

Eur. J. Immunol.

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“…The production, characterization, and evaluation of a panel of human monoclonal antibodies (HuMAbs) against Stx1 and Stx2 in transgenic mice was shown to effectively inhibit cytotoxicity in HeLa cells and protect mice and piglets (22,23). Stx2 A-subunit-specific HuMAb, 5C12, currently undergoing phase I clinical trials, was selected on the basis of its superior efficacy in protecting mice against lethal challenge with Stx2 (30) and Stx2 (31) variants.…”

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confidence: 99%

Intracellular Neutralization of Shiga Toxin 2 by an A Subunit-Specific Human Monoclonal Antibody

et al. 2008

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Infection of children with Shiga toxin (Stx)-producing Escherichia coli (STEC) is the leading cause of hemolyticuremic syndrome (HUS). Stx2, one of two toxins liberated by the bacteria, is directly linked with HUS. We have previously shown that Stx2-specific human monoclonal antibodies (HuMAbs) protect mice and piglets from fatal systemic complications of Stx2. The present study investigates the mechanisms by which our most efficacious A-and B-subunit-specific HuMAbs neutralize the cytotoxic effects of Stx2 in vitro. Whereas the B-subunit-specific HuMAb 5H8 blocked binding of Stx2 to its receptor on the cell surface, the A-subunit-specific HuMAb 5C12 did not interfere with the toxin-receptor binding. Further investigations revealed that 5C12 did not block endocytosis of Stx2 by HeLa cells as both Stx2 and 5C12 colocalized with early endosomes. However, 5C12 blocked the retrograde transport of the toxin into the Golgi and the endoplasmic reticulum, preventing the toxin from entering the cytosol where the toxin exerts its cytotoxic effect. The endocytosed 5C12/Stx2 complexes appear to be rapidly transported to the plasma membrane and/or to the slow recycling perinuclear compartments, followed by their slow recycling to the plasma membrane, and release into the extracellular environment.Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) can become life threatening if it induces systemic complications, mainly hemolytic-uremic syndrome (HUS), the leading cause of acute renal failure in children (2,11,21,25). Of Stx1 and Stx2, the two immunologically distinct Stxs produced by STEC, strains producing only Stx2 are more frequently associated with HUS (10, 27). Stx1 and Stx2 are similar in basic structure, binding specificity, and mode of action (9). The Stx molecule consists of an A-subunit monomer and a B-subunit pentamer. The pentameric B subunit binds to its cell surface receptor CD77, also called globotriaosylceramide (Gb 3 ). This triggers endocytosis of the holotoxin, mainly through clathrin-coated pits (16). Internalized Stx is then delivered to the trans-Golgi network, where it is carried by retrograde transport to the endoplasmic reticulum (ER), and then to the cytosol (28). During this process, the A subunit is nicked by the membrane bound protease furin, generating a catalytically active N-terminal A1 fragment, while a C-terminal A2 fragment remains linked by a disulfide bond (28). This disulfide bond is subsequently reduced to release the active A1 component. The released A1 fragment has RNA N-glycosidase activity that removes a single adenine from the 28S rRNA, thereby inhibiting protein synthesis in the intoxicated cells (7).Several therapeutic approaches that attempted to neutralize Stx either in the gut or in the circulation include the use of synthetic Gb 3 analogues, genetically manipulated bacteria expressing Gb 3 , and Stx-specific neutralizing antibodies (32). The systemic administration of Stx-specific neutralizing antibodies, we believe, is currently the most promising approach for...

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“…In contrast, neutralization of the toxin in the blood circulation, close to the organ target, is much more efficient and requires only small quantities of an antitoxin agent, which in the case of antibody, has a relatively long halflife. We have previously reported the production and characterization of human monoclonal antibodies (HuMAbs) against Stx1 and Stx2 (28,29) in transgenic mice which produce fully human antibodies in the absence of mouse antibodies (16,23). These HuMAbs effectively neutralize the cytotoxic effects of the toxins in HeLa cells and are highly protective in both mice and gnotobiotic piglets (14,28,29,46).…”

mentioning

confidence: 99%

“…Several therapeutic agents have been developed based on the concept that if the toxin(s) can be absorbed or neutralized in either the gastrointestinal tract or in the circulation, the development of HUS can be prevented. These agents include polymers of trisaccharide of Gb 3 (3,11,12,20,53,58), carbosilane dendrimers with trisaccharides of Gb 3 located at their termini (SUPER TWIG [30]), bacteria with Stx-specific glycolipid receptors (34,35), and Stx-specific antibodies (15,18,28,29,32,37,50). One limitation of most of these therapeutic agents, with the exception of antibodies, is that they are orally administered, which considerably reduces their efficacy.…”

mentioning

confidence: 99%

“…We have previously reported the production and characterization of human monoclonal antibodies (HuMAbs) against Stx1 and Stx2 (28,29) in transgenic mice which produce fully human antibodies in the absence of mouse antibodies (16,23). These HuMAbs effectively neutralize the cytotoxic effects of the toxins in HeLa cells and are highly protective in both mice and gnotobiotic piglets (14,28,29,46). The most efficacious of the Stx2-specific HuMAbs are those directed against the A subunit of Stx2, of which 5C12 is the most promising (28).…”

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confidence: 99%

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Characterization of a Human Monoclonal Antibody against Shiga Toxin 2 Expressed in Chinese Hamster Ovary Cells

et al. 2005

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Shiga toxin-producing Escherichia coli infections can often lead to the development of hemolytic-uremic syndrome (HUS) in a small percentage of infected humans. Patients with HUS receive only supportive treatment as the benefit of antibiotic therapy remains uncertain. We have previously reported the generation and preclinical evaluation of neutralizing human monoclonal antibodies (HuMAbs) against the Shiga toxins (Stx). In this paper, we describe the expression in Chinese hamster ovary (CHO) cells of 5C12 HuMAb, which is directed against the A subunit of Stx2. The cDNAs of the light and heavy chain immunoglobulin (Ig) variable regions of 5C12 HuMAb were isolated and cloned into an expression vector containing human IgG1 constant regions. The vector was transfected into CHO cells, and transfectants secreting Stx2-specific antibody were screened by an Stx2-specific enzyme-linked immunosorbent assay. The CHO-produced recombinant 5C12 (r5C12) showed similar specificity and binding affinity to Stx2 as the parent hybridoma-produced 5C12. More significantly, the r5C12 displayed the same neutralizing activity as the parent 5C12 in vitro and in vivo. In the mouse toxicity model, both antibodies significantly and equally prolonged survival at a dose of 0.312 g/mouse. The data showed that since r5C12, produced in CHO cells, was equally effective as the parent 5C12, it is our choice candidate as a potential prophylactic or therapeutic agent against hemolytic-uremic syndrome.

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Production and Characterization of Protective Human Antibodies against Shiga Toxin 1

Abstract: Hemolytic-uremic syndrome (HUS) is a serious complication which is predominantly associated in children with infection by Shiga toxin-producing

Cited by 55 publications

References 29 publications

Isolation and characterization of human anti-acetylcholine receptor monoclonal antibodies from transgenic mice expressing human immunoglobulin loci

Isolation and characterization of human anti-acetylcholine receptor monoclonal antibodies from transgenic mice expressing human immunoglobulin loci

Intracellular Neutralization of Shiga Toxin 2 by an A Subunit-Specific Human Monoclonal Antibody

Characterization of a Human Monoclonal Antibody against Shiga Toxin 2 Expressed in Chinese Hamster Ovary Cells

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