A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes

Consuelo Garcia-Rodriguez; Ali Razai; Isin N 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

doi:10.3390/toxins10030105

A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes

Toxins (Basel). 2018 Mar 1;10(3):105. doi: 10.3390/toxins10030105.

Authors

Consuelo Garcia-Rodriguez¹, Ali Razai^{2

3}, Isin N Geren^{4

5}, Jianlong Lou⁶, Fraser Conrad⁷, Wei-Hua Wen⁸, Shauna Farr-Jones⁹, Theresa J Smith¹⁰, Jennifer L Brown¹¹, Janet C Skerry¹², Leonard A Smith¹³, James D Marks¹⁴

Affiliations

¹ Zuckerberg San Francisco General Hospital and Trauma Center, Room 3C-38, Department of Anesthesia and Perioperative Care, University of California, 1001 Potrero Avenue, San Francisco, CA 94110, USA. MariaConsuelo.Garcia@ucsf.edu.
² Zuckerberg San Francisco General Hospital and Trauma Center, Room 3C-38, Department of Anesthesia and Perioperative Care, University of California, 1001 Potrero Avenue, San Francisco, CA 94110, USA. ali.razai@knobbe.com.
³ Knobbe Martin, 2040 Main Street, 14th Floor, Irvine, CA 92614, USA. ali.razai@knobbe.com.
⁴ Zuckerberg San Francisco General Hospital and Trauma Center, Room 3C-38, Department of Anesthesia and Perioperative Care, University of California, 1001 Potrero Avenue, San Francisco, CA 94110, USA. isin.geren@medeniyet.edu.tr.
⁵ Department of Molecular Biology and Genetics, Faculty of Science, Istanbul Medeniyet University, Unalan Mahallesi, Unalan Sokak, D100 Karayolu, Uskudar-Istanbul 34700, Turkey. isin.geren@medeniyet.edu.tr.
⁶ Zuckerberg San Francisco General Hospital and Trauma Center, Room 3C-38, Department of Anesthesia and Perioperative Care, University of California, 1001 Potrero Avenue, San Francisco, CA 94110, USA. jianlong.lou@ucsf.edu.
⁷ Zuckerberg San Francisco General Hospital and Trauma Center, Room 3C-38, Department of Anesthesia and Perioperative Care, University of California, 1001 Potrero Avenue, San Francisco, CA 94110, USA. fraser.conrad@ucsf.edu.
⁸ Zuckerberg San Francisco General Hospital and Trauma Center, Room 3C-38, Department of Anesthesia and Perioperative Care, University of California, 1001 Potrero Avenue, San Francisco, CA 94110, USA. wei.wen@ucsf.edu.
⁹ Zuckerberg San Francisco General Hospital and Trauma Center, Room 3C-38, Department of Anesthesia and Perioperative Care, University of California, 1001 Potrero Avenue, San Francisco, CA 94110, USA. shauna.farr-jones@ucsf.edu.
¹⁰ Molecular and Translational Sciences Division, United States Army Medical Institute of Infectious Diseases (USAMRIID), Fort Detrick, MD 21702, USA. terrys2much@comcast.net.
¹¹ Ke'aki Technologies LLC, United States Army Medical Institute of Infectious Diseases, (USAMRIID) Fort Detrick, MD 21702, USA. jennifer.l.brown436.ctr@mail.mil.
¹² Ke'aki Technologies LLC, United States Army Medical Institute of Infectious Diseases, (USAMRIID) Fort Detrick, MD 21702, USA. janet.c.skerry.ctr@mail.mil.
¹³ Medical Countermeasures Technology, USAMRIID, Fort Detrick, MD 21702-5011, USA. leonard.a.smith@comcast.net.
¹⁴ Zuckerberg San Francisco General Hospital and Trauma Center, Room 3C-38, Department of Anesthesia and Perioperative Care, University of California, 1001 Potrero Avenue, San Francisco, CA 94110, USA. jim.marks@ucsf.edu.

Abstract

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 (K_D). 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 K_D 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.

Keywords: antibody engineering; botulinum antitoxin; botulinum neurotoxin; mouse neutralization assay; oligoclonal antibodies; recombinant antibodies; serotype E botulism.

Publication types

Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Antibodies, Monoclonal / immunology*
Antibodies, Neutralizing / immunology*
Botulinum Toxins / immunology*
Drug Combinations
Epitopes
Humans

Substances

Antibodies, Monoclonal
Antibodies, Neutralizing
Drug Combinations
Epitopes
Botulinum Toxins
botulinum toxin type E

Grants and funding

U01 AI056493/AI/NIAID NIH HHS/United States