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Comparative Study
. 2020 Mar 29;12(4):215.
doi: 10.3390/toxins12040215.

A Humanized Monoclonal Antibody Cocktail to Prevent Pulmonary Ricin Intoxication

Yinghui Rong  1 , Michael Pauly  2 , Adrian Guthals  2 , Henry Pham  2 , Dylan Ehrbar  1 , Larry Zeitlin  2 , Nicholas J Mantis  1
Affiliations
Comparative Study

A Humanized Monoclonal Antibody Cocktail to Prevent Pulmonary Ricin Intoxication

Yinghui Rong et al. Toxins (Basel). .

Abstract

PB10 IgG1, a monoclonal antibody (MAb) directed against an immunodominant epitope on the enzymatic subunit (RTA) of ricin toxin (RT), has been shown to passively protect mice and non-human primates from an aerosolized lethal-dose RT challenge. However, it was recently demonstrated that the therapeutic efficacy of PB10 IgG1 is significantly improved when co-administered with a second MAb, SylH3, targeting RT's binding subunit (RTB). Here we report that the PB10/SylH3 cocktail is also superior to PB10 alone when used as a pre-exposure prophylactic (PrEP) in a mouse model of intranasal RT challenge. The benefit of the PB10/SylH3 cocktail prompted us to engineer a humanized IgG1 version of SylH3 (huSylH3). The huPB10/huSylH3 cocktail proved highly efficacious in the mouse model, thereby opening the door to future testing in non-human primates.

Keywords: antibody; biodefense; lung; prophylactic; toxin.

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Conflict of interest statement

M.P., (A.G.), and H.P. are (were) Mapp Biopharmaceutical employees. M.P. is also a shareholder. L.Z. is an employee, shareholder, and co-owner of MappBiopharmaceutical. The other authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Benefit of PB10/SylH3 in protecting mice against intranasal ricin toxin (RT) exposure. Groups of mice received an intranasal instillation of PB10/SylH3 monoclonal antibody (Mab) cocktail (2 mg/kg), PB10 MAb (2 mg/kg) or vehicle (saline) 1 h before an RT challenge by the IN route. The dose of 2 mg/kg (40 µg per mouse) was chosen because previous studies had indicated that this was the minimum amount of antibody required to passively protect against intranasal ricin challenge. Following RT challenge, the mice were monitored for (A) survival and (B) weight loss for a period of 14 days, as described in the materials and methods section. The control animals that received saline prior to 10 x LD50 RT challenge experienced a rapid decline in body weight and expired (or were euthanized) within 72 h. Mice that received PB10 prior to RT challenge survived for the duration of the experiment (14 days) but experienced weight loss. In contrast, mice that were pre-treated with the PB10/SylH3 MAb cocktail survived RT challenge without a demonstrable change in weight, demonstrating that the PB10/SylH3 MAb cocktail is superior to PB10 when employed as a PrEP.
Figure 2
Figure 2
Prophylactic potential of the PB10/SylH3 cocktail in a mouse model of intranasal ricin challenge. Groups of BALB/c mice (n = 3 per group) were administered the PB10/SylH3 cocktail (2 mg/kg) by the intranasal route at the time points indicated (−72, −48, −24, −8 and −4 h) prior to 10 x LD50 RT challenge by the same route. The mice were then monitored for (A) survival and (B–F) weight loss for a two-week period. The RT group received RT without antibody, while the control group received vehicle only (saline). For the treatment groups, each mouse received a total of 40 µg of antibody (20 µg PB10 plus 20 µg SylH3 for the cocktail; 40 µg of PB10 alone). (Panel A) Kaplan-Meier survival plot. Only animals in the RT only (red square) and −72 h treatment groups (red circle) succumbed to ricin intoxication. All other animals survived RT challenge (overlapping green circle) although mice in the −48 h treatment group displayed hunching and solitary nesting (clinical score of 2), and mice in the −24 and −8 groups had ruffled fur (clinical score 1). (Panels B–F) Weight per day per group (average with SEM). Statistical analysis of weight loss (* indicates significant loss compared to pre-challenge values) was performed using Friedman tests with Dunn’s multiple comparison tests. During the course of the study, mice were weighed daily and visually inspected twice daily.
Figure 3
Figure 3
PB10/SylH3 levels in BAL fluid and serum following intranasal instillation in mice. Groups of mice (n = 3) were administered the PB10/SylH3 cocktail (2 mg/kg) by the intranasal route. (A) BAL fluids and (B) serum samples were collected from groups of animals at the indicated time points (4, 24, 48, 72 h) and then assessed for PB10/SylH3 levels by RT ELISA [10]. In panel A, the numbers adjacent to each symbol correspond to number of mice that survived per group (survivors/group) from Figure 1, where mice received same dose regimens of PB10/SylH3 cocktail as in this figure except that they were subsequently challenged with RT.
Figure 4
Figure 4
PrEP with the huPB10/huSylH3 cocktail protects mice against lethal-dose intranasal ricin challenge. (Panels A,B) Groups of BALB/c mice (n = 5 mice/group) were administered RT in vehicle RT plus huPB10 (green triangle) or RT plus huSylH3 at time 0. (Panels C,D) Groups of mice (n = 5 mice/group) received huPB10/huSylH3 cocktail at the indicated time points prior to RT challenge. MAbs and the MAb cocktails were given at a final dose of 2 mg/kg by the intranasal route. Mice were challenged with 10 x LD50 RT and then monitored for 14 days. (Panels A,C) Kaplan-Meier survival plots. (Panel B,D) Weight per day per group (average with SEM). Statistical analysis of weight loss was performed using Friedman tests with Dunn’s multiple comparison tests. In panel B, mice that received RT plus huPB10 displayed ruffled fur, hunching, ataxia, weakness, and/or reduced movement (clinical score, 2). Mice that received RT plus huSylH3 displayed severe weakness, tremors, head tilt, seizures (clinical score, 3) and were euthanized. In panel D, the group of animals that received the cocktail at −48 h (pink circles) experienced significant weight loss on days 4, 6, and 7 post-challenge (as indicated by asterisk and horizontal bar) and a single mouse succumbed to intoxication. The mouse that succumbed to challenge on day 4 was excluded from that point forward from the weight loss analysis. During the course of the study, mice were weighed daily and visually inspected twice daily.
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