Peer review of: Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat (2020)



Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
The authors have adequately addressed all of my prior concerns. The paper is much improved and will 
be a valuable contribution to the field. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
The manuscript is very much improved in the current form, especially due to the inclusion of several 
of key data not in the original submission. There are some outstanding issues that should be 
addressed. 
1) Reduction of PC level with type 1. In order to evaluate the difference between an active site 
inhibitor (type 1) and an exosite (near active site, type 2) binder in vivo, it is critical that evidence of 
binding to other proteins is absent. In this version of the manuscript the authors mention that the PC 
levels are reduced by 50% with type 1 and astoundingly that fVIII levels are massively reduced as 
well. This indicates that the type 1 antibody is not specific for APC, and one cannot rule out that other 
activities are contributing to the adverse events when given at high doses to monkeys. Why was this 
data left out of the original manuscript? Importantly, now that it is included one must acknowledge 
the possibility that the difference in tolerability of type 1 and 2 antibodies has nothing to do with APC 
signalling. 
2) The fact that type 1 reduces PC activation by thrombin-TM is also troubling and suggests an extra 
activity. This is given only as a ‘reviewer figure’, but should be included in the manuscript. Again, this 
should temper the contention that the type 1 demonstrates that active site inhibition the only cause of 
the adverse in vivo effects observed. 
3) At the end of the abstract there is a statement that ‘only type II …normalized hemostasis in 
monkeys’. Since only type 2 was tested, this is misleading. 
4) The sentence at the end of page 9 is nonsensical: ‘Whether the prolonged t1/2…’ 
5) The affinity of the antibodies to human APC and PC were tested by biocore and ELISA, but for NHP, 
it was only evaluated by ELISA and only for APC. One would think that the 50% reduction in PC levels 
in NHP would have prompted a thorough investigation into the binding of type 1 to cyno PC. These 
data need to be presented as they massively affect the interpretation of the cause of the adverse 
effects. 
6) The reduction in PC levels by the same extent (50%) for 10, 30 and 100mg/kg type 1 is not 
commented on. Could this be the result of a saturation of the receptors used to take up the PC-Mab 
complex? 
7) The most disturbing revelation in this new version is the catastrophic reduction in fVIII levels when 
dosed with type 1. Disturbing because it wasn’t mentioned before, and because it demonstrates at 
least one important cross-reactant. Was binding to fVIII or vWF not investigated? Why is this not 
discussed in more detail? How is it possible to suggest that the adverse events like leaky vessels and 
hematomas are solely caused by the inhibition of APC signalling?! 
8) The type 2 antibody clearly works in both hemostasis models, however, I am unclear as to why one 
is called acute and the other prophylactic. Indeed, I have no idea why the antibody was given 7 days 

before the induction of haemophilia and challenge with needle sticks. This alone doesn’t make it 
prophylactic. What would the difference have been if the antibody was given just after challenge? Was 
this tested? 
 
 
 
Reviewer #3: 
Remarks to the Author: 
The authors have satisfactorily addressed this reviewer's comments. The inclusion of a more thorough 
evaluation of the impact of each antibody on APC cytoprotective activity in vitro and in the 
haemophilia A monkey model significantly strengthens the conclusions of the study. 
 
Notably, the type II antibody does significantly attenuate EPCR-dependent APC proteolysis of PAR1 in 
transfected cells (Fig. 4b) and seems also to limit APC protection of the endothelial barrier at higher 
Ab concentrations (Fig. 4e), albeit not to the same extent as the type I antibody. Therefore, it seems 
possible that the type II antibody may impact APC cytoprotective activity when it is required in vivo. 
Experiments to assess this, are, however, beyond the scope of the existing study.