Evaluation of strategies to modify Anti-SARS-CoV-2 monoclonal antibodies for optimal functionality as therapeutics.

The current global COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a public health crisis with more than 168 million cases reported globally and more than 4.5 million deaths at the time of writing. In addition to the direct impact of the disease, the economic impact has been significant as public health measures to contain or reduce the spread have led to country wide lockdowns resulting in near closure of many sectors of the economy. Antibodies are a principal determinant of the humoral immune response to COVID-19 infections and may have the potential to reduce disease and spread of the virus. The development of monoclonal antibodies (mAbs) represents a therapeutic option that can be produced at large quantity and high quality. In the present study, a mAb combination mixture therapy was investigated for its capability to specifically neutralize SARS-CoV-2. We demonstrate that each of the antibodies bind the spike protein and neutralize the virus, preventing it from infecting cells in an in vitro cell-based assay, including multiple viral variants that are currently circulating in the human population. In addition, we investigated the effects of two different mutations in the Fc portion (YTE and LALA) of the antibody on Fc effector function and the ability to alleviate potential antibody-dependent enhancement of disease. These data demonstrate the potential of a combination of two mAbs that target two different epitopes on the SARS-CoV2 spike protein to provide protection against SARS-CoV-2 infection in humans while extending serum half-life and preventing antibody-dependent enhancement of disease.

A combination of two human neutralizing antibodies prevents SARS-CoV-2 infection in cynomolgus macaques.

BACKGROUND: Human monoclonal antibody (mAb) treatments are promising for COVID-19 prevention or therapy. The pre-exposure prophylactic efficacy of neutralizing antibodies that are engineered with mutations to extend their persistence in human serum and the neutralizing antibody titer in serum required for protection against SARS-CoV-2 infection remain poorly characterized. METHODS: The Fc region of two neutralizing mAbs (COV2-2130 and COV2-2381) targeting non-overlapping epitopes on the receptor binding domain of SARS-CoV-2 spike protein was engineered to extend their persistence in humans and reduce interactions with Fc gamma receptors. We assessed protection by individual antibodies or a combination of the two antibodies (designated ADM03820) given prophylactically by an intravenous or intramuscular route in a non-human primate (NHP) model of SARS-CoV-2 infection. FINDINGS: Passive transfer of individual mAbs or ADM03820 conferred virological protection in the NHP respiratory tract in a dose-dependent manner, and ADM03820 potently neutralized SARS-CoV-2 variants of concern in vitro. We defined a protective serum-neutralizing antibody titer and concentration in NHPs for passively transferred human antibodies that acted by direct viral neutralization. CONCLUSIONS: In summary, we demonstrate that neutralizing antibodies with extended half-life and lacking Fc-mediated effector functions are efficient for pre-exposure prophylaxis of SARS-CoV-2 infection in NHPs. These results support clinical development of ADM03820 for COVID-19 prevention. FUNDING: This research was supported by a contract from the JPEO-CBRND (W911QY-20-9-003, 20-05); the Joint Sciences and Technology Office and Joint Program Executive Office (MCDC-16-01-002 JSTO, JPEO); a DARPA grant (HR0011-18-2-0001); an NIH grant (R01 AI157155); and the 2019 Future Insight Prize from Merck KGaA.

Neutralizing Concentrations of Anti-Botulinum Toxin Antibodies Positively Correlate with Mouse Neutralization Assay Results in a Guinea Pig Model.

Botulinum neurotoxins (BoNT) are some of the most toxic proteins known and can induce respiratory failure requiring long-term intensive care. Treatment of botulism includes the administration of antitoxins. Monoclonal antibodies (mAbs) hold considerable promise as BoNT therapeutics and prophylactics, due to their potency and safety. A three-mAb combination has been developed that specifically neutralizes BoNT serotype A (BoNT/A), and a separate three mAb combination has been developed that specifically neutralizes BoNT serotype B (BoNT/B). A six mAb cocktail, designated G03-52-01, has been developed that combines the anti-BoNT/A and anti-BoNT/B mAbs. The pharmacokinetics and neutralizing antibody concentration (NAC) of G03-52-01 has been determined in guinea pigs, and these parameters were correlated with protection against an inhalation challenge of BoNT/A1 or BoNT/B1. Previously, it was shown that each antibody demonstrated a dose-dependent mAb serum concentration and reached maximum circulating concentrations within 48 h after intramuscular (IM) or intraperitoneal (IP) injection and that a single IM injection of G03-52-01 administered 48 h pre-exposure protected guinea pigs against an inhalation challenge of up to 93 LD50s of BoNT/A1 and 116 LD50s of BoNT/B1. The data presented here advance our understanding of the relationship of the neutralizing NAC to the measured circulating antibody concentration and provide additional support that a single IM or intravenous (IV) administration of G03-52-01 will provide pre-exposure prophylaxis against botulism from an aerosol exposure of BoNT/A and BoNT/B.

A Monoclonal Antibody Combination against both Serotypes A and B Botulinum Toxin Prevents Inhalational Botulism in a Guinea Pig Model.

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.

Safety and Pharmacokinetics of a Four Monoclonal Antibody Combination Against Botulinum C and D Neurotoxins.

Botulism is caused by botulinum neurotoxin (BoNT), the most poisonous substance known. BoNTs are also classified as Tier 1 biothreat agents due to their high potency and lethality. The existence of seven BoNT serotypes (A-G), which differ between 35% to 68% in amino acid sequence, necessitates the development of serotype specific countermeasures. We present results of a Phase 1 clinical study of an anti-toxin to BoNT serotypes C and D, NTM-1634, which consists of an equimolar mixture of four fully human IgG1 monoclonal antibodies (mAbs), each binding to non-overlapping epitopes on BoNT serotypes C and D resulting in potent toxin neutralization in rodents. This first-in-human study evaluated the safety and pharmacokinetics of escalating doses of NTM-1634 administered intravenously to healthy adults (NCT03046550). Three cohorts of eight healthy subjects received a single intravenous dose of NTM-1634 or placebo at 0.33 mg/kg, 0.66 mg/kg or 1 mg/kg. Follow-up examinations and pharmacokinetic evaluations were continued up to 121 days post-infusion. Subjects were monitored using physical examinations, hematology and chemistry blood tests, and electrocardiograms. Pharmacokinetic parameters were estimated using noncompartmental methods. The results demonstrated that the materials were safe and well-tolerated with the expected half-lives for human mAbs and with minimal anti-drug antibodies detected over the dose ranges and duration of the study.

Monoclonal Antibody Combinations Prevent Serotype A and Serotype B Inhalational Botulism in a Guinea Pig Model.

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.

Vero cell technology for rapid development of inactivated whole virus vaccines for emerging viral diseases.

INTRODUCTION: Rapid development and production of vaccines against emerging diseases requires well established, validated, robust technologies to allow industrial scale production and accelerated licensure of products. Areas covered: A versatile Vero cell platform has been developed and utilized to deliver a wide range of candidate and licensed vaccines against emerging viral diseases. This platform builds on the 35 years' experience and safety record with inactivated whole virus vaccines such as polio vaccine. The current platform has been optimized to include a novel double inactivation procedure in order to ensure a highly robust inactivation procedure for novel emerging viruses. The utility of this platform in rapidly developing inactivated whole virus vaccines against pandemic (-like) influenza viruses and other emerging viruses such as West Nile, Chikungunya, Ross River and SARS is reviewed. The potential of the platform for development of vaccines against other emerging viruses such as Zika virus is described. Expert commentary: Use of this platform can substantially accelerate process development and facilitate licensure because of the substantial existing data set available for the cell matrix. However, programs to provide vaccines against emerging diseases must allow alternative clinical development paths to licensure, without the requirement to carry out large scale field efficacy studies.

Protection in mice passively immunized with serum from cynomolgus macaques and humans vaccinated with recombinant plague vaccine (rF1V).

Passive transfer models were developed to evaluate the ability of antibodies generated in cynomolgus macaques and humans vaccinated with a recombinant plague vaccine (rF1V) to protect naïve Swiss Webster mice against pneumonic plague. Development of the passive transfer model is intended to support clinical and nonclinical development of the rF1V vaccine. To evaluate protection, unfractionated serum collected from rF1V vaccinated cynomolgus macaques and human volunteers with known antibody titers to rF1, rV and rF1V was transferred into naïve Swiss Webster mice via the intraperitoneal route. Results of these studies demonstrated that passive immunization protected mice from challenge or extended mean survival time and that the passive transfer assay can be used to evaluate the functional role of antibodies induced by rF1V vaccination in protection against aerosol exposure.