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Botulinum neurotoxins (BoNT) are extremely potent and can induce respiratory failure, requiring long-term intensive care to prevent death. Recombinant monoclonal antibodies (mAbs) hold considerable promise as BoNT therapeutics and prophylactics. In contrast, equine antitoxin cannot be used prophylactically and has a short half-life. Two three-mAb combinations are in development that specifically neutralize BoNT serotype A (BoNT/A) and B (BoNT/B). The three-mAb combinations addressing a single serotype provided pre-exposure prophylaxis in the guinea pig inhalation model. A lyophilized co-formulation of six mAbs, designated G03-52-01, that addresses both A and B serotypes is in development. Here, we investigated the efficacy of G03-52-01 to protect guinea pigs against an aerosol exposure challenge of BoNT/A1 or BoNT/B1. Previously, it was found that each antibody demonstrated a dose-dependent exposure and reached maximum circulating concentrations within 48 h after intramuscular (IM) or intravenous (IV) injection. Here we show that G03-52-01, in a single IM injection of G03-52-01 administered 48 h pre-exposure, protected guinea pigs against an aerosol challenge of up to 238 LD50s of BoNT/A1 and 191 LD50s of BoNT/B1. These data suggest that a single IM administration of G03-52-01 provides pre-exposure prophylaxis against botulism from an aerosol exposure of BoNT/A1 or BoNT/B1.
Doris Snow; Ronald Cobb; Juan Martinez; Isaac Finger-Baker; Laura Collins; Sara Terpening; Emily Syar; Nancy Niemuth; Dean Kobs; Roy Barnewall; Shauna Farr-Jones; James Marks; Milan Tomic. A Monoclonal Antibody Combination against both Serotypes A and B Botulinum Toxin Prevents Inhalational Botulism in a Guinea Pig Model. Toxins 2021, 13, 31 .
AMA StyleDoris Snow, Ronald Cobb, Juan Martinez, Isaac Finger-Baker, Laura Collins, Sara Terpening, Emily Syar, Nancy Niemuth, Dean Kobs, Roy Barnewall, Shauna Farr-Jones, James Marks, Milan Tomic. A Monoclonal Antibody Combination against both Serotypes A and B Botulinum Toxin Prevents Inhalational Botulism in a Guinea Pig Model. Toxins. 2021; 13 (1):31.
Chicago/Turabian StyleDoris Snow; Ronald Cobb; Juan Martinez; Isaac Finger-Baker; Laura Collins; Sara Terpening; Emily Syar; Nancy Niemuth; Dean Kobs; Roy Barnewall; Shauna Farr-Jones; James Marks; Milan Tomic. 2021. "A Monoclonal Antibody Combination against both Serotypes A and B Botulinum Toxin Prevents Inhalational Botulism in a Guinea Pig Model." Toxins 13, no. 1: 31.
Ephrin receptor A2 (EphA2) is a member of the Ephrin/Eph receptor cell-to-cell signaling family of molecules, and it plays a key role in cell proliferation, differentiation, and migration. EphA2 is overexpressed in a broad range of cancers, and its expression is in many cases associated with poor prognosis. We recently developed a novel EphA2-targeting antibody-directed nanotherapeutic encapsulating a labile prodrug of docetaxel (EphA2-ILs-DTXp) for the treatment of EphA2-expressing malignancies. Here, we characterized the expression of EphA2 in bladder cancer using immunohistochemistry in 177 human bladder cancer samples and determined the preclinical efficacy of EphA2-ILs-DTXp in four EphA2-positive patient-derived xenograft (PDX) models of the disease, either as a monotherapy, or in combination with gemcitabine. EphA2 expression was detected in 80–100% of bladder cancer samples and correlated with shorter patient survival. EphA2 was found to be expressed in tumor cells and/or tumor-associated blood vessels in both primary and metastatic lesions with a concordance rate of approximately 90%. The EphA2-targeted antibody-directed nanotherapeutic EphA2-ILs-DTXp controlled tumor growth, mediated greater regression, and was more active than free docetaxel at equitoxic dosing in all four EphA2-positive bladder cancer PDX models. Combination of EphA2-ILs-DTXp and gemcitabine in one PDX model led to improved tumor growth control compared to monotherapies or the combination of free docetaxel and gemcitabine. These data demonstrating the prevalence of EphA2 in bladder cancers and efficacy of EphA2-ILs-DTXp in PDX models support the clinical exploration of EphA2 targeting in bladder cancer.
Walid Kamoun; Elden Swindell; Christine Pien; Lia Luus; Jason Cain; Minh Pham; Irawati Kandela; Zhaohua Richard Huang; Suresh K. Tipparaju; Alexander Koshkaryev; Vasileios Askoxylakis; Dmitri B. Kirpotin; Troy Bloom; Mari Mino-Kenudson; James D. Marks; Alena Zalutskaya; Wiam Bshara; Carl Morrison; Daryl C. Drummond. Targeting EphA2 in Bladder Cancer Using a Novel Antibody-Directed Nanotherapeutic. Pharmaceutics 2020, 12, 996 .
AMA StyleWalid Kamoun, Elden Swindell, Christine Pien, Lia Luus, Jason Cain, Minh Pham, Irawati Kandela, Zhaohua Richard Huang, Suresh K. Tipparaju, Alexander Koshkaryev, Vasileios Askoxylakis, Dmitri B. Kirpotin, Troy Bloom, Mari Mino-Kenudson, James D. Marks, Alena Zalutskaya, Wiam Bshara, Carl Morrison, Daryl C. Drummond. Targeting EphA2 in Bladder Cancer Using a Novel Antibody-Directed Nanotherapeutic. Pharmaceutics. 2020; 12 (10):996.
Chicago/Turabian StyleWalid Kamoun; Elden Swindell; Christine Pien; Lia Luus; Jason Cain; Minh Pham; Irawati Kandela; Zhaohua Richard Huang; Suresh K. Tipparaju; Alexander Koshkaryev; Vasileios Askoxylakis; Dmitri B. Kirpotin; Troy Bloom; Mari Mino-Kenudson; James D. Marks; Alena Zalutskaya; Wiam Bshara; Carl Morrison; Daryl C. Drummond. 2020. "Targeting EphA2 in Bladder Cancer Using a Novel Antibody-Directed Nanotherapeutic." Pharmaceutics 12, no. 10: 996.
Background: Botulinum neurotoxins (BoNTs) comprise seven agreed-on serotypes, A through G. In 2014, a novel chimeric neurotoxin produced by clostridial strain IBCA10-7060 was reported as BoNT/H, with subsequent names of BoNT/FA or BoNT/HA based on sequence homology of the N-terminus to BoNT/F, the C-terminus to BoNT/A and neutralization studies. The purpose of this study was to define the immunologic identity of the novel BoNT. Methods: monoclonal antibodies (mAbs) to the novel BoNT/H N-terminus were generated by antibody repertoire cloning and yeast display after immunization with BoNT/H LC-HN or BoNT/F LC-HN. Results: 21 unique BoNT/H LC-HN mAbs were obtained; 15 from the BoNT/H LC-HN immunized library (KD 0.78 nM to 182 nM) and six from the BoNT/F-immunized libraries (KD 20.5 nM to 1490 nM). A total of 15 of 21 mAbs also bound catalytically inactive BoNT/H holotoxin. The mAbs bound nine non-overlapping epitopes on the BoNT/H LC-HN. None of the mAbs showed binding to BoNT serotypes A-G, nor any of the seven subtypes of BoNT/F, except for one mAb that weakly bound BoNT/F5. Conclusions: The results, combined with the chimeric structure and neutralization by anti-A, but not anti-F antitoxin indicate that immunologically the novel BoNT is BoNT/HA. This determination has significant implications for existing countermeasures and potential vulnerabilities.
Yongfeng Fan; Jason R. Barash; Fraser Conrad; Jianlong Lou; Christina Tam; Luisa W. Cheng; Stephen S. Arnon; James D. Marks. The Novel Clostridial Neurotoxin Produced by Strain IBCA10-7060 Is Immunologically Equivalent to BoNT/HA. Toxins 2019, 12, 9 .
AMA StyleYongfeng Fan, Jason R. Barash, Fraser Conrad, Jianlong Lou, Christina Tam, Luisa W. Cheng, Stephen S. Arnon, James D. Marks. The Novel Clostridial Neurotoxin Produced by Strain IBCA10-7060 Is Immunologically Equivalent to BoNT/HA. Toxins. 2019; 12 (1):9.
Chicago/Turabian StyleYongfeng Fan; Jason R. Barash; Fraser Conrad; Jianlong Lou; Christina Tam; Luisa W. Cheng; Stephen S. Arnon; James D. Marks. 2019. "The Novel Clostridial Neurotoxin Produced by Strain IBCA10-7060 Is Immunologically Equivalent to BoNT/HA." Toxins 12, no. 1: 9.
Botulinum neurotoxins (BoNT) are potential biothreat agents due to their high lethality, potency, and ease of distribution, thus the development of antitoxins is a high priority to the US government. This study examined pre-clinical pharmacokinetic studies in rats of four oligoclonal anti-BoNT mAb-based therapeutics (NTM-1631, NTM-1632, NTM-1633, NTM-1634) for five BoNT serotypes (A, B, E, C, and D). NTM-1631, NTM-1632, and NTM-1633 each consist of three IgG1 mAbs, each with a distinct human or humanized variable region which bind to distinct epitopes on BoNT serotype A, B, or E respectively. NTM-1634 consists of four human immunoglobulin G1 (IgG1) mAbs binding BoNT C/D mosaic toxins. The mechanism of these antitoxins requires that three antibodies simultaneously bind toxin to achieve rapid clearance. Rats (total 378) displayed no adverse clinical signs attributed to antibody treatment from any of the antitoxins. Pharmacokinetic evaluation demonstrated that the individual mAbs are slowly eliminated, exhibiting dose-dependent exposure and long elimination half-lives ranging from 6.5 days to 10 days. There were no consistent differences observed between males and females or among the individual antibodies in each formulation in half-life. Anti-drug antibodies (ADA) were observed, as expected for human antibodies administered to rats. The results presented were used to support the clinical investigation of antibody-based botulism antitoxins.
Yero Espinoza; David Wong; Ago Ahene; Kenneth Der; Zachary Martinez; John Pham; Ronald R. Cobb; Shauna Farr-Jones; James. D. Marks; Milan T. Tomic. Pharmacokinetics of Human Recombinant Anti-Botulinum Toxin Antibodies in Rats. Toxins 2019, 11, 345 .
AMA StyleYero Espinoza, David Wong, Ago Ahene, Kenneth Der, Zachary Martinez, John Pham, Ronald R. Cobb, Shauna Farr-Jones, James. D. Marks, Milan T. Tomic. Pharmacokinetics of Human Recombinant Anti-Botulinum Toxin Antibodies in Rats. Toxins. 2019; 11 (6):345.
Chicago/Turabian StyleYero Espinoza; David Wong; Ago Ahene; Kenneth Der; Zachary Martinez; John Pham; Ronald R. Cobb; Shauna Farr-Jones; James. D. Marks; Milan T. Tomic. 2019. "Pharmacokinetics of Human Recombinant Anti-Botulinum Toxin Antibodies in Rats." Toxins 11, no. 6: 345.
Botulinum neurotoxins (BoNT) are some of the most toxic proteins known, with a human LD50 of ~1 ng/kg. Equine antitoxin has a half-life in circulation of less than 1 day and is limited to a treatment rather than a prevention indication. The development of monoclonal antibodies (mAbs) may represent an alternative therapeutic option that can be produced at high quantities and of high quality and with half-lives of >10 days. Two different three mAb combinations are being developed that specifically neutralize BoNT serotypes A (BoNT/A) and B (BoNT/B). We investigated the pharmacokinetics of the anti-BoNT/A and anti-BoNT/B antibodies in guinea pigs (Cavia porcellus) and their ability to protect guinea pigs against an aerosol challenge of BoNT/A1 or BoNT/B1. Each antibody exhibited dose-dependent exposure and reached maximum circulating concentrations within 48 h post intraperitoneal or intramuscular injection. A single intramuscular dose of the three mAb combination protected guinea pigs against an aerosol challenge dose of 93 LD50 of BoNT/A1 and 116 LD50 of BoNT/B1 at 48 h post antibody administration. These mAbs are effective in preventing botulism after an aerosol challenge of BoNT/A1 and BoNT/B1 and may represent an alternative to vaccination to prevent type A or B botulism in those at risk of BoNT exposure.
Milan T. Tomic; Yero Espinoza; Zachary Martinez; Khanh Pham; Ronald R. Cobb; Doris M. Snow; Christopher G. Earnhart; Traci Pals; Emily S. Syar; Nancy Niemuth; Dean J. Kobs; Shauna Farr-Jones; James D. Marks. Monoclonal Antibody Combinations Prevent Serotype A and Serotype B Inhalational Botulism in a Guinea Pig Model. Toxins 2019, 11, 208 .
AMA StyleMilan T. Tomic, Yero Espinoza, Zachary Martinez, Khanh Pham, Ronald R. Cobb, Doris M. Snow, Christopher G. Earnhart, Traci Pals, Emily S. Syar, Nancy Niemuth, Dean J. Kobs, Shauna Farr-Jones, James D. Marks. Monoclonal Antibody Combinations Prevent Serotype A and Serotype B Inhalational Botulism in a Guinea Pig Model. Toxins. 2019; 11 (4):208.
Chicago/Turabian StyleMilan T. Tomic; Yero Espinoza; Zachary Martinez; Khanh Pham; Ronald R. Cobb; Doris M. Snow; Christopher G. Earnhart; Traci Pals; Emily S. Syar; Nancy Niemuth; Dean J. Kobs; Shauna Farr-Jones; James D. Marks. 2019. "Monoclonal Antibody Combinations Prevent Serotype A and Serotype B Inhalational Botulism in a Guinea Pig Model." Toxins 11, no. 4: 208.
Botulism, caused by exposure to one or more of the eight serotypes of botulinum neurotoxins (BoNTs) (BoNT/A through H), is often fatal without rapid treatment.
Jianlong Lou; James D. Marks. Botulinum Neurotoxins (BoNTs)—Antibody and Vaccine. Toxins 2018, 10, 495 .
AMA StyleJianlong Lou, James D. Marks. Botulinum Neurotoxins (BoNTs)—Antibody and Vaccine. Toxins. 2018; 10 (12):495.
Chicago/Turabian StyleJianlong Lou; James D. Marks. 2018. "Botulinum Neurotoxins (BoNTs)—Antibody and Vaccine." Toxins 10, no. 12: 495.
Human botulism is most commonly caused by botulinum neurotoxin (BoNT) serotypes A, B, and E. For this work, we sought to develop a human monoclonal antibody (mAb)-based antitoxin capable of binding and neutralizing multiple subtypes of BoNT/E. Libraries of yeast-displayed single chain Fv (scFv) antibodies were created from the heavy and light chain variable region genes of humans immunized with pentavalent-toxoid- and BoNT/E-binding scFv isolated by Fluorescence-Activated Cell Sorting (FACS). A total of 10 scFv were isolated that bound one or more BoNT/E subtypes with nanomolar-level equilibrium dissociation constants (KD). By diversifying the V-regions of the lead mAbs and selecting for cross-reactivity, we generated three scFv that bound all four BoNT/E subtypes tested at three non-overlapping epitopes. The scFvs were converted to IgG that had KD values for the different BoNT/E subtypes ranging from 9.7 nM to 2.28 pM. An equimolar combination of the three mAbs was able to potently neutralize BoNT/E1, BoNT/E3, and BoNT/E4 in a mouse neutralization assay. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing multiple BoNT/E subtypes. A derivative of the three-antibody combination (NTM-1633) is in pre-clinical development with an investigational new drug (IND) application filing expected in 2018.
Consuelo Garcia-Rodriguez; Ali Razai; Isin Geren; Jianlong Lou; Fraser Conrad; Wei-Hua Wen; Shauna Farr-Jones; Theresa J. Smith; Jennifer L. Brown; Janet C. Skerry; Leonard A. Smith; James D. Marks. A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes. Toxins 2018, 10, 105 .
AMA StyleConsuelo Garcia-Rodriguez, Ali Razai, Isin Geren, Jianlong Lou, Fraser Conrad, Wei-Hua Wen, Shauna Farr-Jones, Theresa J. Smith, Jennifer L. Brown, Janet C. Skerry, Leonard A. Smith, James D. Marks. A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes. Toxins. 2018; 10 (3):105.
Chicago/Turabian StyleConsuelo Garcia-Rodriguez; Ali Razai; Isin Geren; Jianlong Lou; Fraser Conrad; Wei-Hua Wen; Shauna Farr-Jones; Theresa J. Smith; Jennifer L. Brown; Janet C. Skerry; Leonard A. Smith; James D. Marks. 2018. "A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes." Toxins 10, no. 3: 105.
The standard of treatment for botulism, equine antitoxin, is a foreign protein with associated safety issues and a short serum half-life which excludes its use as a prophylactic antitoxin and makes it a less-than-optimal therapeutic. Due to these limitations, a recombinant monoclonal antibody (mAb) product is preferable. It has been shown that combining three mAbs that bind non-overlapping epitopes leads to highly potent botulinum neurotoxin (BoNT) neutralization. Recently, a triple human antibody combination for BoNT/A has demonstrated potent toxin neutralization in mouse models with no serious adverse events when tested in a Phase I clinical trial. However, a triple antibody therapeutic poses unique development and manufacturing challenges. Thus, potentially to streamline development of BoNT antitoxins, we sought to achieve the potency of multiple mAb combinations in a single IgG-based molecule that has a long serum half-life. The design, production, and testing of a single tri-epitopic IgG1-based mAb (TeAb) containing the binding sites of each of the three parental BoNT/A mAbs yielded an antibody of nearly equal potency to the combination. The approach taken here could be applied to the design and creation of other multivalent antibodies that could be used for a variety of applications, including toxin elimination.
Jianlong Lou; Weihua Wen; Fraser Conrad; Qi Meng; Jianbo Dong; Zhengda Sun; Consuelo Garcia-Rodriguez; Shauna Farr-Jones; Luisa W. Cheng; Thomas D. Henderson; Jennifer L. Brown; Theresa J. Smith; Leonard A. Smith; Anthony Cormier; James D. Marks. A Single Tri-Epitopic Antibody Virtually Recapitulates the Potency of a Combination of Three Monoclonal Antibodies in Neutralization of Botulinum Neurotoxin Serotype A. Toxins 2018, 10, 84 .
AMA StyleJianlong Lou, Weihua Wen, Fraser Conrad, Qi Meng, Jianbo Dong, Zhengda Sun, Consuelo Garcia-Rodriguez, Shauna Farr-Jones, Luisa W. Cheng, Thomas D. Henderson, Jennifer L. Brown, Theresa J. Smith, Leonard A. Smith, Anthony Cormier, James D. Marks. A Single Tri-Epitopic Antibody Virtually Recapitulates the Potency of a Combination of Three Monoclonal Antibodies in Neutralization of Botulinum Neurotoxin Serotype A. Toxins. 2018; 10 (2):84.
Chicago/Turabian StyleJianlong Lou; Weihua Wen; Fraser Conrad; Qi Meng; Jianbo Dong; Zhengda Sun; Consuelo Garcia-Rodriguez; Shauna Farr-Jones; Luisa W. Cheng; Thomas D. Henderson; Jennifer L. Brown; Theresa J. Smith; Leonard A. Smith; Anthony Cormier; James D. Marks. 2018. "A Single Tri-Epitopic Antibody Virtually Recapitulates the Potency of a Combination of Three Monoclonal Antibodies in Neutralization of Botulinum Neurotoxin Serotype A." Toxins 10, no. 2: 84.
We present a strategy to discover recombinant monoclonal antibodies (mAbs) to specific cancers and demonstrate this approach using basal subtype breast cancers. A phage antibody library was depleted of antibodies to common cell surface molecules by incubation with luminal breast cancer cell lines, and then selected on a single basal-like breast cancer cell line (MDA-MB-231) for binding associated receptor-mediated endocytosis. Additional profiling against two luminal and four basal-like cell lines revealed 61 unique basal-specific mAbs from a pool of 1440 phage antibodies. The unique mAbs were further screened on nine basal and seven luminal cell lines to identify those with the greatest affinity, specificity, and internalizing capability for basal-like breast cancer cells. Among the internalizing basal-specific mAbs were those recognizing four transmembrane receptors (EphA2, CD44, CD73 and EGFR), identified by immunoprecipitation-mass spectrometry and yeast-displayed antigen screening. Basal-like breast cancer expression of these four receptors was confirmed using a bioinformatic approach, and expression microarray data on 683 intrinsically subtyped primary breast tumors. This overall approach, which sequentially employs phage display antibody library selection, antigen identification and bioinformatic confirmation of antigen expression by cancer subtypes, offers efficient production of high-affinity mAbs with diagnostic and therapeutic utility against specific cancer subtypes.
Yu Zhou; Hao Zou; Christina Yau; Lequn Zhao; Steven C Hall; Daryl C Drummond; Shauna Farr-Jones; John W Park; Christopher C Benz; James D Marks. Discovery of internalizing antibodies to basal breast cancer cells. Protein Engineering, Design, and Selection 2017, 31, 17 -28.
AMA StyleYu Zhou, Hao Zou, Christina Yau, Lequn Zhao, Steven C Hall, Daryl C Drummond, Shauna Farr-Jones, John W Park, Christopher C Benz, James D Marks. Discovery of internalizing antibodies to basal breast cancer cells. Protein Engineering, Design, and Selection. 2017; 31 (1):17-28.
Chicago/Turabian StyleYu Zhou; Hao Zou; Christina Yau; Lequn Zhao; Steven C Hall; Daryl C Drummond; Shauna Farr-Jones; John W Park; Christopher C Benz; James D Marks. 2017. "Discovery of internalizing antibodies to basal breast cancer cells." Protein Engineering, Design, and Selection 31, no. 1: 17-28.
Human botulism is primarily caused by botulinum neurotoxin (BoNT) serotypes A, B and E, with around 1% caused by serotype F (BoNT/F). BoNT/F comprises at least seven different subtypes with the amino acid sequence difference between subtypes as high as 36%. The sequence differences present a significant challenge for generating monoclonal antibodies (mAbs) that can bind, detect and neutralize all BoNT/F subtypes. We used repertoire cloning of immune mouse antibody variable (V) regions and yeast display to generate a panel of 33 lead single chain Fv (scFv) mAbs that bound one or more BoNT/F subtypes with a median equilibrium dissociation constant (KD) of 4.06 × 10−9 M. By diversifying the V-regions of the lead mAbs and selecting for cross reactivity we generated five mAbs that bound each of the seven subtypes. Three scFv binding non-overlapping epitopes were converted to IgG that had KD for the different BoNT/F subtypes ranging from 2.2×10−8 M to 1.47×10−12 pM. An equimolar combination of the mAbs was able to potently neutralize BoNT/F1, F2, F4 and F7 in the mouse neutralization assay. The mAbs have potential utility as diagnostics capable of recognizing the known BoNT/F subtypes and could be developed as antitoxins to prevent and treat type F botulism.
Yongfeng Fan; Consuelo Garcia-Rodriguez; Jianlong Lou; Weihua Wen; Fraser Conrad; Wenwu Zhai; Theresa J. Smith; Leonard A. Smith; James D. Marks. A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes. PLOS ONE 2017, 12, e0174187 .
AMA StyleYongfeng Fan, Consuelo Garcia-Rodriguez, Jianlong Lou, Weihua Wen, Fraser Conrad, Wenwu Zhai, Theresa J. Smith, Leonard A. Smith, James D. Marks. A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes. PLOS ONE. 2017; 12 (3):e0174187.
Chicago/Turabian StyleYongfeng Fan; Consuelo Garcia-Rodriguez; Jianlong Lou; Weihua Wen; Fraser Conrad; Wenwu Zhai; Theresa J. Smith; Leonard A. Smith; James D. Marks. 2017. "A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes." PLOS ONE 12, no. 3: e0174187.
Background. Only Clostridium botulinum strain IBCA10-7060 produces the recently described novel botulinum neurotoxin type H (BoNT/H). BoNT/H (N-terminal two-thirds most homologous to BoNT/F and C-terminal one-third most homologous to BoNT/A) requires antitoxin to toxin ratios ≥1190:1 for neutralization by existing antitoxins. Hence, more potent and safer antitoxins against BoNT/H are needed.
Yongfeng Fan; Jason R. Barash; Jianlong Lou; Fraser Conrad; James D. Marks; Stephen S. Arnon. Immunological Characterization and Neutralizing Ability of Monoclonal Antibodies Directed Against Botulinum Neurotoxin Type H. Journal of Infectious Diseases 2016, 213, 1606 -1614.
AMA StyleYongfeng Fan, Jason R. Barash, Jianlong Lou, Fraser Conrad, James D. Marks, Stephen S. Arnon. Immunological Characterization and Neutralizing Ability of Monoclonal Antibodies Directed Against Botulinum Neurotoxin Type H. Journal of Infectious Diseases. 2016; 213 (10):1606-1614.
Chicago/Turabian StyleYongfeng Fan; Jason R. Barash; Jianlong Lou; Fraser Conrad; James D. Marks; Stephen S. Arnon. 2016. "Immunological Characterization and Neutralizing Ability of Monoclonal Antibodies Directed Against Botulinum Neurotoxin Type H." Journal of Infectious Diseases 213, no. 10: 1606-1614.
Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.
Yongfeng Fan; Jianbo Dong; Jianlong Lou; Weihua Wen; Fraser Conrad; Isin N. Geren; Consuelo Garcia-Rodriguez; Theresa J. Smith; Leonard A. Smith; Mengfei Ho; Melissa Pires-Alves; Brenda A. Wilson; James D. Marks. Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B. Toxins 2015, 7, 3405 -3423.
AMA StyleYongfeng Fan, Jianbo Dong, Jianlong Lou, Weihua Wen, Fraser Conrad, Isin N. Geren, Consuelo Garcia-Rodriguez, Theresa J. Smith, Leonard A. Smith, Mengfei Ho, Melissa Pires-Alves, Brenda A. Wilson, James D. Marks. Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B. Toxins. 2015; 7 (9):3405-3423.
Chicago/Turabian StyleYongfeng Fan; Jianbo Dong; Jianlong Lou; Weihua Wen; Fraser Conrad; Isin N. Geren; Consuelo Garcia-Rodriguez; Theresa J. Smith; Leonard A. Smith; Mengfei Ho; Melissa Pires-Alves; Brenda A. Wilson; James D. Marks. 2015. "Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B." Toxins 7, no. 9: 3405-3423.
The paralytic disease botulism is caused by botulinum neurotoxins (BoNT), multi-domain proteins containing a zinc endopeptidase that cleaves the cognate SNARE protein, thereby blocking acetylcholine neurotransmitter release. Antitoxins currently used to treat botulism neutralize circulating BoNT but cannot enter, bind to or neutralize BoNT that has already entered the neuron. The light chain endopeptidase domain (LC) of BoNT serotype A (BoNT/A) was targeted for generation of monoclonal antibodies (mAbs) that could reverse paralysis resulting from intoxication by BoNT/A. Single-chain variable fragment (scFv) libraries from immunized humans and mice were displayed on the surface of yeast, and 19 BoNT/A LC-specific mAbs were isolated by using fluorescence-activated cell sorting (FACS). Affinities of the mAbs for BoNT/A LC ranged from a KD value of 9.0×10−11 M to 3.53×10−8 M (mean KD 5.38×10−9 M and median KD 1.53×10−9 M), as determined by flow cytometry analysis. Eleven mAbs inhibited BoNT/A LC catalytic activity with IC50 values ranging from 8.3 ~73×10−9 M. The fine epitopes of selected mAbs were also mapped by alanine-scanning mutagenesis, revealing that the inhibitory mAbs bound the α-exosite region remote from the BoNT/A LC catalytic center. The results provide mAbs that could prove useful for intracellular reversal of paralysis post-intoxication and further define epitopes that could be targeted by small molecule inhibitors.
Yongfeng Fan; Isin Geren; Jianbo Dong; Jianlong Lou; Weihua Wen; Fraser Conrad; Theresa J. Smith; Leonard A. Smith; Mengfei Ho; Melissa Pires-Alves; Brenda A. Wilson; James D. Marks. Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity. PLOS ONE 2015, 10, e0135306 .
AMA StyleYongfeng Fan, Isin Geren, Jianbo Dong, Jianlong Lou, Weihua Wen, Fraser Conrad, Theresa J. Smith, Leonard A. Smith, Mengfei Ho, Melissa Pires-Alves, Brenda A. Wilson, James D. Marks. Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity. PLOS ONE. 2015; 10 (8):e0135306.
Chicago/Turabian StyleYongfeng Fan; Isin Geren; Jianbo Dong; Jianlong Lou; Weihua Wen; Fraser Conrad; Theresa J. Smith; Leonard A. Smith; Mengfei Ho; Melissa Pires-Alves; Brenda A. Wilson; James D. Marks. 2015. "Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity." PLOS ONE 10, no. 8: e0135306.