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Isolation and chimerization of a highly neutralizing antibody conferring passive protection against lethal Bacillus anthracis infection.

Rosenfeld R, Marcus H, Ben-Arie E, Lachmi BE, Mechaly A, Reuveny S, Gat O, Mazor O, Ordentlich A - PLoS ONE (2009)

Bottom Line: Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD(50) of B. anthracis spores.The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol.The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Israel Institute for Biological Research, Ness-Ziona, Israel.

ABSTRACT
Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29). Guinea pigs were fully protected against infection by 40LD(50)B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD(50) of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax.

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Kinetic analysis of mAb 29 and cAb 29 binding to PA.SPR sensograms obtained during injection of (A) 100–2000 nM of PA on anti-human captured cAb 29 (∼300 RU), refers to the direct format; or (B) 5–100 nM of cAb 29 on immobilized PA (∼900 RU), refers to the indirect format. Similar sensograms were obtained for the mAb 29 (results not shown).
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pone-0006351-g004: Kinetic analysis of mAb 29 and cAb 29 binding to PA.SPR sensograms obtained during injection of (A) 100–2000 nM of PA on anti-human captured cAb 29 (∼300 RU), refers to the direct format; or (B) 5–100 nM of cAb 29 on immobilized PA (∼900 RU), refers to the indirect format. Similar sensograms were obtained for the mAb 29 (results not shown).

Mentions: To evaluate the PA-binding capabilities of the chimeric and murine #29 antibodies, Surface Plasmon Resonance (SPR) analysis was applied. Based on the binding characteristics, demonstrated earlier by ELISA, an indirect SPR format was applied for the kinetic measurements (Fig. 4; Table 2). Accordingly, the antibodies were attached to anti-Fc antibody pre-coated CM5 sensor chips (anti-human or anti-mouse for the cAb 29 and the mAb 29, respectively), followed by the injection of PA at different concentrations. Sensograms were fitted to a 1∶1 Langmuir model, and association (kon) and dissociation (koff) rate constants were obtained for both antibodies (Table 2). The dissociation constants (KD) deduced from these rate constants were similar for both antibodies, with calculated values of 6.6±2.8 nM and 8.5±4.8 nM for the chimeric and murine antibodies, respectively. When SPR analysis was performed in the direct format, in which PA was immobilized on the sensor chip, significantly lower affinities were obtained (Fig. 4; Table 2). In this assay format, a divalent analysis model was used, and KD values of 37±9 nM and 44±7 nM were calculated for the chimeric and the murine antibodies, respectively (Table 2). These results are in good agreement with the ELISA binding studies, in which antibody binding to surface-immobilized PA was impaired compared to that of soluble PA (indirect format), indicating that the epitope recognized by this antibody is probably disrupted when the PA is used as the capture moiety. Taken together, the binding studies, along with the previously described in vitro and in vivo protection analyses, indicate that the chimeric form of the #29 antibody retains the binding properties of its parental murine antibody.


Isolation and chimerization of a highly neutralizing antibody conferring passive protection against lethal Bacillus anthracis infection.

Rosenfeld R, Marcus H, Ben-Arie E, Lachmi BE, Mechaly A, Reuveny S, Gat O, Mazor O, Ordentlich A - PLoS ONE (2009)

Kinetic analysis of mAb 29 and cAb 29 binding to PA.SPR sensograms obtained during injection of (A) 100–2000 nM of PA on anti-human captured cAb 29 (∼300 RU), refers to the direct format; or (B) 5–100 nM of cAb 29 on immobilized PA (∼900 RU), refers to the indirect format. Similar sensograms were obtained for the mAb 29 (results not shown).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2710523&req=5

pone-0006351-g004: Kinetic analysis of mAb 29 and cAb 29 binding to PA.SPR sensograms obtained during injection of (A) 100–2000 nM of PA on anti-human captured cAb 29 (∼300 RU), refers to the direct format; or (B) 5–100 nM of cAb 29 on immobilized PA (∼900 RU), refers to the indirect format. Similar sensograms were obtained for the mAb 29 (results not shown).
Mentions: To evaluate the PA-binding capabilities of the chimeric and murine #29 antibodies, Surface Plasmon Resonance (SPR) analysis was applied. Based on the binding characteristics, demonstrated earlier by ELISA, an indirect SPR format was applied for the kinetic measurements (Fig. 4; Table 2). Accordingly, the antibodies were attached to anti-Fc antibody pre-coated CM5 sensor chips (anti-human or anti-mouse for the cAb 29 and the mAb 29, respectively), followed by the injection of PA at different concentrations. Sensograms were fitted to a 1∶1 Langmuir model, and association (kon) and dissociation (koff) rate constants were obtained for both antibodies (Table 2). The dissociation constants (KD) deduced from these rate constants were similar for both antibodies, with calculated values of 6.6±2.8 nM and 8.5±4.8 nM for the chimeric and murine antibodies, respectively. When SPR analysis was performed in the direct format, in which PA was immobilized on the sensor chip, significantly lower affinities were obtained (Fig. 4; Table 2). In this assay format, a divalent analysis model was used, and KD values of 37±9 nM and 44±7 nM were calculated for the chimeric and the murine antibodies, respectively (Table 2). These results are in good agreement with the ELISA binding studies, in which antibody binding to surface-immobilized PA was impaired compared to that of soluble PA (indirect format), indicating that the epitope recognized by this antibody is probably disrupted when the PA is used as the capture moiety. Taken together, the binding studies, along with the previously described in vitro and in vivo protection analyses, indicate that the chimeric form of the #29 antibody retains the binding properties of its parental murine antibody.

Bottom Line: Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD(50) of B. anthracis spores.The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol.The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Israel Institute for Biological Research, Ness-Ziona, Israel.

ABSTRACT
Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29). Guinea pigs were fully protected against infection by 40LD(50)B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD(50) of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax.

Show MeSH
Related in: MedlinePlus