Peer review of: Cyclic peptide FXII inhibitor provides safe anticoagulation in a thrombosis model and in (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

Using phage display the authors have developed a peptide inhibitor to FXIIa that excels, existing 
protein-based inhibitors, in its specificity, selectivity and half-life. Much of the manuscript focuses 
on improving another inhibitor from the same group (FXII618) to the new inhibitor FXII900. The 
advantage of FXIIa inhibition in inhibiting thrombosis, without bleeding complications, was further 
consolidated in mouse models of thrombosis and haemostasis. 

1. In Fig. 2, it would have been better to compare FXII900 to FXII901, the latter being the inactive 
control peptide. 
2. In Fig. 2, whereas the concentration-dependent inhibition, in aPTT is very convincing in the 
rabbit plasma (Fig 2a, bottom left panel), it is less so in human and mouse plasma. Does the 
inhibitory effect reach saturation, at all, in these two species? 
3. Why is it that much higher concentrations of FXII900 are needed to inhibit aPTT in plasma 
compared to the inhibition against pure FXIIa. This point needs to be addressed, atleast, in the 
discussion. 
4. In Fig. 3, the maximal concentration reached in mouse plasma is around 1 microM (Panel 3a) 
and aPTT is doubled at this time point. However, this concentration of inhibitor has no affect on 
aPTT in mouse plasma in Fig. 2a. This discrepency needs to be addressed. 
5. In Fig. 4; what is the effect of the negative control, FXII901, compared to PBS. This is an 
important control to exclude any spurious effects of the control peptide. 
6. Considering that the authors have focussed on improving the specificty of a previously existing 
peptide it may be useful to do modelling studies to explain why the new peptide is better. 

Reviewer #2 (Remarks to the Author): 

Inhibition of intrinsic coagulation factor XII (FXII) has the potential to become a safe novel 
antithrombotic therapy that, unlike the current anti-thrombotic therapies, may not coincide with 
bleeding as a side-effect. As such, also inhibition of the FXII-downstream coagulation factor XI is 
of interest and currently under investifgation. Antibody and antisense strategies have been 
developed that successfully inhibit/lower FXII for preclinical testing, however, synthetic small-
molecule approaches that are successful (in the preclinic) are not available yet. A small molecule-
based therapy may in some respects have advantages over antibody or antisense approaches 
(regarding synthesis, immunogenicity, and possibly oral availability). 
In the current work, Wilbs and colleagues started from their previously generated and reported 
bicyclic peptide, to generate a novel compound (FXII900) with strong FXIIa inhibition in the sub-
nanomolar range. This proved potent for inhibition of human FXIIa, and also for FXIIa from 
animals allowing preclinical studies (mouse, rabbit, pig). Apart from strong FXIIa inhibition, also 
high stability of FXII900 was demonstrated using plasmas of the total of four species. The 
experiments (including compound modifications) that were required to produce the strong 
inhibitory and high stability compound were well-executed and are clearly and well-described. 
Convincingly is also demonstrated that FXII900, for the multiple species evaluated, potently 
impacted intrinsic pathway (prolonged the aPTT), not extrinsic pathway (PT), as anticipated based 
on specific FXIIa inhibition. Also lack of impact of FXII900 on other related/relevant proteases was 
convincingly demonstrated. 
Pharmacokinetic studies were performed, and for the species analyzed, following 
subcutaneous/intravenous injection, FXII900 appeared to have a short half-life in the blood 
circulation. Consequently, a limited time frame is available where FXII900 can impact the 
functionality of the intrinsic pathway (as analyzed ex vivo using aPTT). The rapid clearance, 
despite stability of FXII900 in plasma, is attributed to a fast renal clearance. 
Subsequently, FXII900 was tested in a preclinical mouse thrombosis model i.e. the ferric chloride 
model on mesenteric arteries to demonstrate in vivo efficacy. Clearly, FXII900 inhibited thrombosis 

in this specific experimental model. Furthermore and importantly, the conditions that proved 
antithrombotic did not coincided with prolonged bleeding times in the commonly used mouse tail 
transection bleeding model. 

Comments 

Abstract: 

One important typical characteristic of the FXII900 compound is the short plasma half-life in vivo. 
This should be addressed in the abstract. 

In the abstract is claimed that FXII900 is a promising candidate for safe thromboprotection. In the 
discussion section, the application of FXII900 is presented with more restriction i.e. applicability in 
acute procedures such as extracorporeal membrane oxygenation (ECMO) or cardiopulmonary 
bypass (CPB) surgeries. This should be denoted in the abstract, as the abstract now suggests that 
FXII900 may also be used in treatment of/prophylaxis in venous and/or arterial thrombosis. 
However, this would requiring longer half-lives and functional availability of the compound (and 
optionally also oral bioavailability). 

Introduction: 

P2.“Mice lacking FXII have reduced risk…” Term “ risk” may be not fully appropriate for the 
experimental mouse condition and could be replaced by wording like: “ reduced thrombosis in 
mouse models of arterial and venous thrombosis” 

P3. When summarizing studies on antisense oligonucleotides and antibodies targeting FXII in 
thrombosis, the type of models used for which the response was recorded could be indicated. 

Results: 

The results describing the engineering of the compound and testing it’s inhibitory potential and 
stability are well-performed and well-described. 

Regarding in vivo administration of FXII900 in mice, rabbits and pigs; what is the effect of FXII900 
injection on parameters of acute toxicity? Such as hematology, circulating liver enzymes, plasma 
inflammation markers, acute phase proteins? 

What is known on these class of compounds regarding oral availability? Is FXII900 effective in the 
circulation of the animals when administered orally? When not tested, or when negative, one can 
comment on this in the discussion section, since one important reason to go for small synthetic 
molecule approach, and not for an antibody and antisense approach, is the potential that the 
compound can orally administered. This would be a major advantage, or even prerequisite, for 
application in thrombosis prophylaxis. 

Given the rather short plasma half-life of FXII900, and consequent restricted duration of impact of 
FXII900 on the ex vivo analyzed aPTT, FXII900 would be more suitable for applicability in acute 
procedures such as extracorporeal membrane oxygenation (ECMO) or cardiopulmonary bypass 
(CPB) surgeries. The authors outlined these applications for FXIIa inhibition already in the 
introduction and also in the discussion section. Although the antithrombotic effect of FXII900 in the 
‘ ferric chloride model’ is strong, this model is rather distant from (any form of) clinical thrombosis 
and also the suggested/anticipated application. Addition of studies demonstrating the performance 
of FXII900 in models for which use is anticipated such ECMO and CPB (or stroke) models would 
definitely strengthen this work. 

Figure 4c: the authors could comment on the bi-distribution in the heparin response regarding 

blood loss. Moreover, for heparin a mean bleeding time of 30 minutes is observed for all animals 
included though coinciding for 3 animals with only minimal blood loss (in microliters). Possibly for 
using the readout ‘blood loss’ the group size is too low for comparisons. 
The lines in figure 4c that likely indicate mean values should be clarified in the figure legends. In 
addition, when having a bi-distribution, one can question whether it is appropriate to use a ‘mean’ 
to describe these data and using of parametric testing for comparison. 

Discussion: 

It is outlined that small synthetic compounds for FXIIa inhibition are attractive. One of the number 
of advantages of small synthetic compounds may be the limited immunogenicity. On the other 
hand, the short half-life of FXII900 restricts its anticipated application to use solely in acute 
situations, with likely single administration, and thus limited chance of resulting in 
immunoreactivity. Hence, this advantage of the small molecule approach seems to have become 
irrelevant because of its short plasma half-life and restricted use. 

It is mentioned that FXII900 is rapidly cleared by renal filtration. The authors could outline and/or 
mention the data that support this conclusion. 

The half-life of FXII900 could also be discussed in the context of the half-life of other approaches 
to inhibit FXII and sofar developed and published (antisense and antibody 3F7 approach). 

Reviewer #3 (Remarks to the Author): 

The manuscript by Wilbs et al. describes the optimization of a macrocyclic peptide inhibitor of the 
coagulation pathway protease factor XII. The authors do a nice job of highlighting the potential for 
factor XII inhibitors for treatment of a number of conditions in which blood clotting is an issue. The 
current data from knockout mice and humans deficient for factor XII suggest that this protease will 
be a possible viable target to block clotting without inducing bleeding. 

This work builds on the prior work of the Heinis Lab in which they use a phage display method to 
identify high affinity binding bicyclic peptides for factor XII. Their prior efforts resulted in the 
development of FXII618 which was not viable as a lead molecule due to low potency and poor in 
vivo stability. In the current work, the authors use a combination of rational design and 
randomization methods to identify a lead molecule with greatly increased potency and stability. 
The authors show that this new lead molecule, FXII900, appears to be a significant improvement 
and thus suitable for validation studies in animal models of clotting disorders. The results 
presented are compelling and show that the molecule has an in vivo half life of 30-90 min in mice, 
rabbits and pigs. Most importantly, the authors show that the molecule can reduce clot formation 
in a mouse model without inducing the prolonged bleeding that occurs with heprin sulfate 
treatment. 

Overall, I think this manuscript presents some exciting results for a compound that could be a 
valuable new clinical lead molecule. The work is all performed with a high degree of rigor and I 
have no issue with any of the results and how they are presented. The only minor issue is that the 
molecules still have relatively short half-life in blood, which will limit their utility for some 
indications. However, this rapid clearance could be a benefit for other situations where only a 
short-term block of clotting is needed. The study is valuable, although not particularly novel, and 
is mainly a medicinal chemistry study so I am not sure about the fit of this work for Nature 
Communications. However, I think the potential impact as a clinical lead molecule may be 
sufficient to overcome any limitation in novelty of the approach. 

I have no specific suggestions and would suggest publication as is.