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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.
TGF-β is a promising immunotherapeutic target. It is expressed ubiquitously in a latent form that must be activated to function. Determination of where and how latent TGF-β (L-TGF-β) is activated in the tumor microenvironment could facilitate cell- and mechanism-specific approaches to immunotherapeutically target TGF-β. Binding of L-TGF-β to integrin αvβ8 results in activation of TGF-β. We engineered and used αvβ8 antibodies optimized for blocking or detection, which - respectively - inhibit tumor growth in syngeneic tumor models or sensitively and specifically detect β8 in human tumors. Inhibition of αvβ8 potentiates cytotoxic T cell responses and recruitment of immune cells to tumor centers - effects that are independent of PD-1/PD-L1. β8 is expressed on the cell surface at high levels by tumor cells, not immune cells, while the reverse is true of L-TGF-β, suggesting that tumor cell αvβ8 serves as a platform for activating cell-surface L-TGF-β presented by immune cells. Transcriptome analysis of tumor-associated lymphoid cells reveals macrophages as a key cell type responsive to β8 inhibition with major increases in chemokine and tumor-eliminating genes. High β8 expression in tumor cells is seen in 20%-80% of various cancers, which rarely coincides with high PD-L1 expression. These data suggest tumor cell αvβ8 is a PD-1/PD-L1-independent immunotherapeutic target.
Naoki Takasaka; Robert I. Seed; Anthony Cormier; Andrew J. Bondesson; Jianlong Lou; Ahmed Elattma; Saburo Ito; Haruhiko Yanagisawa; Mitsuo Hashimoto; Royce Ma; Michelle D. Levine; Jean Publicover; Rashaun Potts; Jillian M. Jespersen; Melody G. Campbell; Fraser Conrad; James D. Marks; Yifan Cheng; Jody L. Baron; Stephen L. Nishimura. Integrin αvβ8–expressing tumor cells evade host immunity by regulating TGF-β activation in immune cells. JCI Insight 2018, 3, 1 .
AMA StyleNaoki Takasaka, Robert I. Seed, Anthony Cormier, Andrew J. Bondesson, Jianlong Lou, Ahmed Elattma, Saburo Ito, Haruhiko Yanagisawa, Mitsuo Hashimoto, Royce Ma, Michelle D. Levine, Jean Publicover, Rashaun Potts, Jillian M. Jespersen, Melody G. Campbell, Fraser Conrad, James D. Marks, Yifan Cheng, Jody L. Baron, Stephen L. Nishimura. Integrin αvβ8–expressing tumor cells evade host immunity by regulating TGF-β activation in immune cells. JCI Insight. 2018; 3 (20):1.
Chicago/Turabian StyleNaoki Takasaka; Robert I. Seed; Anthony Cormier; Andrew J. Bondesson; Jianlong Lou; Ahmed Elattma; Saburo Ito; Haruhiko Yanagisawa; Mitsuo Hashimoto; Royce Ma; Michelle D. Levine; Jean Publicover; Rashaun Potts; Jillian M. Jespersen; Melody G. Campbell; Fraser Conrad; James D. Marks; Yifan Cheng; Jody L. Baron; Stephen L. Nishimura. 2018. "Integrin αvβ8–expressing tumor cells evade host immunity by regulating TGF-β activation in immune cells." JCI Insight 3, no. 20: 1.
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.