Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat hemophilia

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC’s anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC’s cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC’s anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection

Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability In vitro assays were established as part of a pre-clinical screening program used to characterize multiple discovery antibodies as part of a drug development project. These well-established assays were used to characterize the antibodies found in this publication. N=2-3 were used as part of the screening process with duplicate analysis of a sample as standard part of aPTT and TGA procedures for each N, including the data presented here. Since these assays were part of a screening cascade with multiple antibodies tested and corresponding data in multiple assays, the limited N was considered sufficient.
The N selected for the in vivo studies was based on pilot NHP studies with tool compounds and the number of animals needed to reach a statistically significant outcome. The pilot data with tool compounds and reproduction of those findings in the studies presented here supported the N selection for the in vivo studies.
No data were excluded for analysis.
All attempts at replication were successful.
Cynomolgus monkeys were randomized by evenly distributing the weights per group.
The dosing solutions were made by people outside the vivarium and then handed to the animal technicians inside, blinded. So the techs supposedly did not know what they were dosing.
The mouse anti-hAPC antibody HAPC1573 from Oklahoma Medical Research Foundation was humanized to generate the antiAPC type II mAb. The type I mAb was identified by panning the n-CoDeR® phage-display library of human antibody Fab fragment purchased from BioInvent International AB, Sweden.
Antibodies were validated by antigen-antibody binding assays (ELISA, SPR) and DNA sequencing, as well as by APC chromogenic assay/FVa inactivation assay/aPTT clotting assay/TGA.