Camelid antibodies against activated protein C and uses thereof - Patents.com

Abstract

The present invention provides antibodies against activated protein C (APC). Certain disclosed antibodies inhibit the anticoagulant activity of APC while maintaining its beneficial cytoprotective functions. The present invention also provides nucleic acids, vectors, and host cells for producing the antibodies disclosed herein, as well as methods of using the antibodies to treat medical conditions such as hemorrhage, sepsis, and inflammation.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 316,927, filed March 4, 2022, the contents of which are incorporated by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing in XML format entitled "172085.00117_ST26.xml", with a size of 643,566 bytes and a creation date of February 21, 2023. The Sequence Listing has been submitted electronically via the Patent Center together with this application and is hereby incorporated by reference in its entirety.

[0003] This application relates to antibodies, antibody fragments, and other binding agents of activated protein C (APC), as well as related formulations, nucleic acids, methods of preparation, and methods of use.

[0004] Protein C (PC) is an enzyme precursor that is synthesized in the liver as a single-chain 461-amino acid precursor (SEQ ID NO: 633) and secreted into the blood. Prior to secretion, the single-chain precursor is converted to a heterodimer by removing the lysine-arginine dipeptide (amino acids 198-199 of SEQ ID NO: 633) and the 42 amino acid pre-proleader (amino acids 1-42 of SEQ ID NO: 633). The heterodimeric form (417 amino acids) consists of a light chain (155 amino acids, 21 kDa) and a heavy chain (262 amino acids, 41 kDa) linked by a disulfide bridge. The light chain contains one gamma-carboxyglutamic acid (Gla) domain (45 amino acids), two EGF-like domains (46 amino acids) and a linker sequence, whereas the heavy chain contains a highly polar activation peptide (amino acids 200-211 of SEQ ID NO: 633) and a catalytic domain with a typical serine protease catalytic triad. PC zymogen is cleaved at the thrombin cleavage site to remove the activation peptide and activate PC to yield activated PC (APC; SEQ ID NO: 634; 405 amino acids).

[0005] Human PC undergoes extensive post-translational modifications, including glycosylation, vitamin K-dependent gamma-carboxylation, and gamma-hydroxylation. It contains four potential N-linked glycosylation sites, one in the light chain (Asn97) and three in the heavy chain (Asn248, Asn313, and Asn329). Its Gla domain contains nine Gla residues and contributes to the calcium-dependent binding of PC to negatively charged phospholipid membranes. The Gla domain can also bind to the endothelial protein C receptor (EPCR), which aligns thrombin and thrombomodulin on the endothelial membrane during PC activation.

[0006] PC activation occurs in a two-step process at the surface of endothelial cells. It requires binding of PC (via the Gla domain) to EPCR on endothelial cells, followed by proteolytic cleavage of PC by the thrombin / thrombomodulin complex at Arg12 of the heavy chain. This single cleavage liberates a 12-amino acid activation peptide and converts the zymogen PC into the active serine protease APC.

[0007] Under physiological conditions, APC circulates in human blood at very low concentrations (1-2 ng / ml or 40 pM) and has a half-life of 20-30 minutes, whereas PC circulates at much higher concentrations (3-5 μg / ml or 65 nM) and has a half-life of 6-8 hours.

[0008] The PC pathway plays a key role in regulating coagulation and functions as a natural defense mechanism against thrombosis by amplifying the anticoagulant response as a coagulation response mount. Upon injury, thrombin is generated to initiate coagulation. Thrombin also triggers the anticoagulant response by binding to thrombomodulin in the inner layer of the vascular surface, thereby promoting PC activation. As a result, APC generation correlates with thrombin concentration and PC levels. APC functions as an anticoagulant by inactivating two coagulation cofactors (i.e., factors Va and VIIIa) via proteolytic cleavage, thereby inhibiting thrombin generation. APC also directly contributes to the enhancement of the fibrinolytic response via complex formation with plasminogen activator inhibitor.

[0009] Bleeding is a major problem in both medical conditions and medical treatments. For example, bleeding causes complications following surgery, organ transplants, intracranial hemorrhage, aortic aneurysm, postpartum hemorrhage, trauma, and overdosing of certain anticoagulants. In fact, the leading cause of death in humans aged 1-44 years is acute hemorrhage associated with traumatic injury. Currently, there are few options for treating life-threatening non-compressible hemorrhage. Bleeding disorders can be treated by inhibiting APC to promote thrombin generation and clotting. Thus, a first objective of the present invention is to provide a safe and effective treatment for hemorrhage.

[0010] In addition to its anticoagulant function, APC also induces cytoprotective effects through its antiapoptotic, anti-inflammatory, and endothelial barrier stabilizing activities, all of which contribute to the regenerative outcomes associated with APC (e.g., stimulation of neurogenesis, angiogenesis, and wound healing).

[0011] Ischemia is a life-threatening condition resulting from insufficient blood supply to an organ or tissue. Insufficient blood supply can lead to oxygen-starved tissues, resulting in heart attack or stroke. In metabolically active tissues such as the heart and brain, ischemia can cause irreversible damage in as little as 3-4 minutes. The cytoprotective effects of APC include reducing apoptosis and inhibiting the expression of inflammatory mediators after ischemia. Therefore, a second object of the present invention is to provide a safe and effective treatment for ischemia.

[0012] Studies of human diseases and animal models suggest that extracellular histones are involved in either the onset or development of inflammatory processes in various organs (Moiana et al., Clin Biochemistry 94(2021)12-19, p. 15). For this reason, histones are being investigated as potential therapeutic targets in diseases in which inflammation and thrombosis play important pathophysiological roles. APC reduces the cytotoxicity of extracellular histones by histone proteolysis. The function of APC depends on negatively charged exosites containing Glu330 and Glu333.

[0013] Extracellular histones are known to be the main mediator of death in sepsis. Sepsis is a life-threatening condition that occurs when the body's response to infection causes damage to its own tissues and organs. Sepsis is the leading cause of morbidity and mortality in the United States, accounting for nearly 270,000 deaths annually. The cytoprotective effects of APC include the inhibition of histone toxicity, which is associated with death in sepsis. Therefore, the third object of the present invention is to provide a safe and effective treatment for sepsis.

[0014] Histones form part of neutrophil extracellular traps (NETs), a network of extracellular fibers composed mainly of DNA from neutrophils. NETs have been shown to be involved in the development and progression of cancer. Cancer is a leading cause of death worldwide, accounting for approximately 10 million deaths in 2020, or one in six deaths. NETs affect cancer through several mechanisms, including the establishment of an inflammatory microenvironment and interactions with other tumor-promoting mechanisms such as inflammasomes and autophagy (Shao et al., Frontiers in Oncology vol.11 article 714357(2021)p.1). Evidence suggests that NETs play a role in various cancer types, including breast, lung, colon, pancreatic, hematological, neurological, and skin cancers (ibid. p.6). Thus, interventions that affect PC pathways may have an impact on cancer, and the fourth objective of the present invention is to provide a safe and effective treatment for cancer.

[0015] APC-based therapeutics have been in development for several years. Genetic engineering of APC has produced APC variants that selectively reduce either anticoagulant or cytoprotective activity. (Griffin et al., Blood 125:2898, 2015). For example, one such variant, 3K3A-APC (Lys191-193Ala), has less than 10% of wild-type APC's anticoagulant activity while retaining its full cytoprotective activity and has progressed into clinical trials for ischemic stroke. However, previous attempts to translate research findings into APC-based medicines have failed, including early attempts to use APC as a treatment for sepsis. Promising clinical trial results led to the approval of a recombinant APC protein-based drug (drotrecogin alfa (activated), recombinant human activated protein C (rhAPC), trade name Xigris™) in 2001 (N Engl J Med 344:699, 2001), but subsequent randomized clinical trials failed to replicate the positive initial findings and the drug was withdrawn from the market.

[0016] One possible problem with using exogenous proteins such as APC and APC variants as therapeutic agents is the possibility that the recipient's immune system will generate anti-drug antibodies (ADA) against them. The production of ADA can have dramatic consequences, such as loss of drug efficacy and neutralization, or other adverse events (J Pharm Sci 110(7):2575-2584,2021). For this reason, the fifth objective of the present invention is to provide an APC-based therapeutic agent with a lower immunogenicity risk.

[0017] Antibodies against APCs have been explored as a means to selectively enhance or inhibit different functions of APCs (see, e.g., U.S. Patent Application Publication No. 2015 / 0307625, published October 29, 2015, by Zhao et al., and U.S. Patent Application Publication No. 2018 / 326053, published November 15, 2018, by Egner et al.). For example, Magisetty et al. reported that a mouse antibody that selectively inhibited the anticoagulant activity of APC while preserving its cytoprotective activity significantly reduced the severity of joint bleeding in a mouse model of hemophilic arthropathy, whereas an antibody that inhibited both the anticoagulant and cytoprotective activities of APC did not (Blood(2022)blood.2021013119). In Zhao et al., two different antibodies against APC were evaluated as APC-based therapy for hemophilia (Nat Commun 11(1):2992,2020). One of the antibodies, called class I, targeted the active site of APC, and the other, called class II, targeted the exosite of APC. Administration of these antibodies to monkeys increased thrombin generation and promoted plasma clotting. However, while the class II antibodies were well tolerated, the class I antibodies resulted in fat in the animals. Nevertheless, the class II antibodies still partially inhibited the active site of APC, suggesting that it may partially inhibit the cytoprotective effect of APC. Thus, the sixth object of the present invention is to provide an anti-APC antibody that maintains or enhances the cytoprotective effect of APC. Summary of the Invention

[0018] In a first aspect, the invention provides an isolated antibody that specifically binds activated protein C (APC) and minimally binds non-activated protein C (PC). In some embodiments, the antibody comprises a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 317-474. In some embodiments, the antibody comprises a heavy chain variable domain (VD) selected from the group consisting of SEQ ID NOs: 475-632. H ).

[0019] In a second aspect, the invention provides a nucleic acid encoding an antibody as disclosed herein.

[0020] In a third aspect, the present invention provides a vector comprising a nucleic acid disclosed herein.

[0021] In a fourth aspect, the present invention provides a host cell comprising a nucleic acid or vector disclosed herein.

[0022] In a fifth aspect, the present invention provides a method of producing an antibody disclosed herein, the method comprising (a) culturing a host cell disclosed herein under conditions resulting in the production of the antibody, and (b) isolating the antibody from the host cell.

[0023] In a sixth aspect, the present invention provides a pharmaceutical composition comprising an antibody disclosed herein and a pharma- ceutically acceptable carrier.

[0024] In a seventh aspect, the present invention provides a method for treating or preventing a condition in a subject. The method includes administering to the subject a therapeutically effective amount of an antibody disclosed herein, an antibody disclosed herein specifically bound to an exogenous APC protein or variant thereof, or a pharmaceutical composition disclosed herein. In some embodiments, the condition is a condition that can be treated or prevented by tethering or inhibiting the anticoagulant function of APC. In other embodiments, the condition is a condition that can be treated or prevented by enhancing or inhibiting the cytoprotective function of APC.

[0025] In an eighth aspect, the present invention provides the use of an antibody disclosed herein as a medicament. [Brief description of the drawings]

[0026] [Figure 1A]Figure 1 shows the results of epitope binning and affinity analysis of nanobodies LP1-LP21 for human activation protein C (APC) by biolayer interferometry (BLI). Binding of APC to nanobodies was detected based on the spectral shift of reflected light at the nanobody-loaded sensor surface. The biosensor tip with the nanobody-APC complex was then immersed in a TPP-4885 Fab solution. Figure 1A shows a table of results. Based on the spectral shift, 8 clones were found not to block TPP-4885 Fab binding to APC (top) and 13 clones to block binding. Figure 1B shows the results of an additional binning experiment performed with nanobodies that did not block TPP-4885 Fab binding to human APC. LP7, LP9, LP12, and LP18 were found not to compete with LP2, LP3, and LP10 for APC binding. Values ​​presented in the table represent the binding response over background (red = competition between analyte and ligand for APC, orange = competition but with measurable analyte binding, green = no competition). Figure 1C shows the results of a clustering analysis that identified two clusters of nanobodies at a 95% AU p-value cutoff (red = approximately unbiased p-value for AU, green = bp bootstrap probability, grey = number of edges). Figure 1D includes a table summarizing the nanobody clusters (top) and a graph of the median CDR3 length for each cluster (bottom). Figure 1E is a graph showing KD measurements for LP1 to LP21 generated using BLI. Each nanobody was tested against five different concentrations of APC. The R2 for these measurements ranged from 0.92 to 0.99. Figure 1F is a representative sensor diagram generated for antibody LP6 in the presence of BSA. [Figure 1B]Figure 1 shows the results of epitope binning and affinity analysis of nanobodies LP1-LP21 for human activation protein C (APC) by biolayer interferometry (BLI). Binding of APC to nanobodies was detected based on the spectral shift of reflected light at the nanobody-loaded sensor surface. The biosensor tip with the nanobody-APC complex was then immersed in a TPP-4885 Fab solution. Figure 1A shows a table of results. Based on the spectral shift, 8 clones were found not to block TPP-4885 Fab binding to APC (top) and 13 clones to block binding. Figure 1B shows the results of an additional binning experiment performed with nanobodies that did not block TPP-4885 Fab binding to human APC. LP7, LP9, LP12, and LP18 were found not to compete with LP2, LP3, and LP10 for APC binding. Values ​​presented in the table represent the binding response over background (red = competition between analyte and ligand for APC, orange = competition but with measurable analyte binding, green = no competition). Figure 1C shows the results of a clustering analysis that identified two clusters of nanobodies at a 95% AU p-value cutoff (red = approximately unbiased p-value for AU, green = bp bootstrap probability, grey = number of edges). Figure 1D includes a table summarizing the nanobody clusters (top) and a graph of the median CDR3 length for each cluster (bottom). Figure 1E is a graph showing KD measurements for LP1 to LP21 generated using BLI. Each nanobody was tested against five different concentrations of APC. The R2 for these measurements ranged from 0.92 to 0.99. Figure 1F is a representative sensor diagram generated for antibody LP6 in the presence of BSA. [Figure 1C]Figure 1 shows the results of epitope binning and affinity analysis of nanobodies LP1-LP21 for human activation protein C (APC) by biolayer interferometry (BLI). Binding of APC to nanobodies was detected based on the spectral shift of reflected light at the nanobody-loaded sensor surface. The biosensor tip with the nanobody-APC complex was then immersed in a TPP-4885 Fab solution. Figure 1A shows a table of results. Based on the spectral shift, 8 clones were found not to block TPP-4885 Fab binding to APC (top) and 13 clones to block binding. Figure 1B shows the results of an additional binning experiment performed with nanobodies that did not block TPP-4885 Fab binding to human APC. LP7, LP9, LP12, and LP18 were found not to compete with LP2, LP3, and LP10 for APC binding. Values ​​presented in the table represent the binding response over background (red = competition between analyte and ligand for APC, orange = competition but with measurable analyte binding, green = no competition). Figure 1C shows the results of a clustering analysis that identified two clusters of nanobodies at a 95% AU p-value cutoff (red = approximately unbiased p-value for AU, green = bp bootstrap probability, grey = number of edges). Figure 1D includes a table summarizing the nanobody clusters (top) and a graph of the median CDR3 length for each cluster (bottom). Figure 1E is a graph showing KD measurements for LP1 to LP21 generated using BLI. Each nanobody was tested against five different concentrations of APC. The R2 for these measurements ranged from 0.92 to 0.99. Figure 1F is a representative sensor diagram generated for antibody LP6 in the presence of BSA. [Figure 1D]Figure 1 shows the results of epitope binning and affinity analysis of nanobodies LP1-LP21 for human activation protein C (APC) by biolayer interferometry (BLI). Binding of APC to nanobodies was detected based on the spectral shift of reflected light at the nanobody-loaded sensor surface. The biosensor tip with the nanobody-APC complex was then immersed in a TPP-4885 Fab solution. Figure 1A shows a table of results. Based on the spectral shift, 8 clones were found not to block TPP-4885 Fab binding to APC (top) and 13 clones to block binding. Figure 1B shows the results of an additional binning experiment performed with nanobodies that did not block TPP-4885 Fab binding to human APC. LP7, LP9, LP12, and LP18 were found not to compete with LP2, LP3, and LP10 for APC binding. Values ​​presented in the table represent the binding response over background (red = competition between analyte and ligand for APC, orange = competition but with measurable analyte binding, green = no competition). Figure 1C shows the results of a clustering analysis that identified two clusters of nanobodies at a 95% AU p-value cutoff (red = approximately unbiased p-value for AU, green = bp bootstrap probability, grey = number of edges). Figure 1D includes a table summarizing the nanobody clusters (top) and a graph of the median CDR3 length for each cluster (bottom). Figure 1E is a graph showing KD measurements for LP1 to LP21 generated using BLI. Each nanobody was tested against five different concentrations of APC. The R2 for these measurements ranged from 0.92 to 0.99. Figure 1F is a representative sensor diagram generated for antibody LP6 in the presence of BSA. [Figure 1E]Figure 1 shows the results of epitope binning and affinity analysis of nanobodies LP1-LP21 for human activation protein C (APC) by biolayer interferometry (BLI). Binding of APC to nanobodies was detected based on the spectral shift of reflected light at the nanobody-loaded sensor surface. The biosensor tip with the nanobody-APC complex was then immersed in a TPP-4885 Fab solution. Figure 1A shows a table of results. Based on the spectral shift, 8 clones were found not to block TPP-4885 Fab binding to APC (top) and 13 clones to block binding. Figure 1B shows the results of an additional binning experiment performed with nanobodies that did not block TPP-4885 Fab binding to human APC. LP7, LP9, LP12, and LP18 were found not to compete with LP2, LP3, and LP10 for APC binding. Values ​​presented in the table represent the binding response over background (red = competition between analyte and ligand for APC, orange = competition but with measurable analyte binding, green = no competition). Figure 1C shows the results of a clustering analysis that identified two clusters of nanobodies at a 95% AU p-value cutoff (red = approximately unbiased p-value for AU, green = bp bootstrap probability, grey = number of edges). Figure 1D includes a table summarizing the nanobody clusters (top) and a graph of the median CDR3 length for each cluster (bottom). Figure 1E is a graph showing KD measurements for LP1 to LP21 generated using BLI. Each nanobody was tested against five different concentrations of APC. The R2 for these measurements ranged from 0.92 to 0.99. Figure 1F is a representative sensor diagram generated for antibody LP6 in the presence of BSA. [Figure 1F]Figure 1 shows the results of epitope binning and affinity analysis of nanobodies LP1-LP21 for human activation protein C (APC) by biolayer interferometry (BLI). Binding of APC to nanobodies was detected based on the spectral shift of reflected light at the nanobody-loaded sensor surface. The biosensor tip with the nanobody-APC complex was then immersed in a TPP-4885 Fab solution. Figure 1A shows a table of results. Based on the spectral shift, 8 clones were found not to block TPP-4885 Fab binding to APC (top) and 13 clones to block binding. Figure 1B shows the results of an additional binning experiment performed with nanobodies that did not block TPP-4885 Fab binding to human APC. LP7, LP9, LP12, and LP18 were found not to compete with LP2, LP3, and LP10 for APC binding. Values ​​presented in the table represent the binding response over background (red = competition between analyte and ligand for APC, orange = competition but with measurable analyte binding, green = no competition). Figure 1C shows the results of a clustering analysis that identified two clusters of nanobodies at a 95% AU p-value cutoff (red = approximately unbiased p-value for AU, green = bp bootstrap probability, grey = number of edges). Figure 1D includes a table summarizing the nanobody clusters (top) and a graph of the median CDR3 length for each cluster (bottom). Figure 1E is a graph showing KD measurements for LP1 to LP21 generated using BLI. Each nanobody was tested against five different concentrations of APC. The R2 for these measurements ranged from 0.92 to 0.99. Figure 1F is a representative sensor diagram generated for antibody LP6 in the presence of BSA. [Figure 2A]Figure 2 shows the effect of nanobodies LP1 to LP21 on APC anticoagulant activity. Figure 2A shows a standard curve of Protac®-APTT clotting times in protein C (PC)-depleted plasma diluted with normal human plasma (o) to achieve PC levels ranging from 0.78% to 100%. APTT clotting times in normal human plasma (·) and PC-depleted plasma (∇) in the absence of Protac® are also shown. Figure 2B shows a graph comparing Protac®-APTT clotting times in the presence of nanobodies (300 nM) in two independent experiments (screen 1 and screen 2). A consistent rank order was observed in the shortening of Protac®-APTT clotting. Figure 2C shows the mean Protac®-APTT clotting times and percent APC activity in plasma for each nanobody (replicated in two independent experiments). The graph shows that the nanobodies have a wide range of effects on Protac®-APTT clotting times. Among the nanobodies, LP11, LP8 and LP20 showed the highest potency in inhibiting anticoagulant activity. [Figure 2B] Figure 2 shows the effect of nanobodies LP1 to LP21 on APC anticoagulant activity. Figure 2A shows a standard curve of Protac®-APTT clotting times in protein C (PC)-depleted plasma diluted with normal human plasma (o) to achieve PC levels ranging from 0.78% to 100%. APTT clotting times in normal human plasma (·) and PC-depleted plasma (∇) in the absence of Protac® are also shown. Figure 2B shows a graph comparing Protac®-APTT clotting times in the presence of nanobodies (300 nM) in two independent experiments (screen 1 and screen 2). A consistent rank order was observed in the shortening of Protac®-APTT clotting. Figure 2C shows the mean Protac®-APTT clotting times and percent APC activity in plasma for each nanobody (replicated in two independent experiments). The graph shows that the nanobodies have a wide range of effects on Protac®-APTT clotting times. Among the nanobodies, LP11, LP8 and LP20 showed the highest potency in inhibiting anticoagulant activity. [Figure 2C] Figure 2 shows the effect of nanobodies LP1 to LP21 on APC anticoagulant activity. Figure 2A shows a standard curve of Protac®-APTT clotting times in protein C (PC)-depleted plasma diluted with normal human plasma (o) to achieve PC levels ranging from 0.78% to 100%. APTT clotting times in normal human plasma (·) and PC-depleted plasma (∇) in the absence of Protac® are also shown. Figure 2B shows a graph comparing Protac®-APTT clotting times in the presence of nanobodies (300 nM) in two independent experiments (screen 1 and screen 2). A consistent rank order was observed in the shortening of Protac®-APTT clotting. Figure 2C shows the mean Protac®-APTT clotting times and percent APC activity in plasma for each nanobody (replicated in two independent experiments). The graph shows that the nanobodies have a wide range of effects on Protac®-APTT clotting times. Among the nanobodies, LP11, LP8 and LP20 showed the highest potency in inhibiting anticoagulant activity. [Figure 3A]Figure 3 shows the effect of nanobodies LP1-LP21 on APC-mediated histone H3 cleavage. APC (50 nM) and nanobodies (500 nM) were pre-incubated together for 30 min. After incubation, the APC-nanobody mixture was added to histone H3 (100 μg / mL). Over a period of 2 h, samples were taken at different time points and added to reducing sample buffer for SDS-PAGE analysis. A molecular marker of 20 kDa was used as a standard for normalization between gels. Figure 3A is an SDS-PAGE gel showing the time course of histone H3 cleavage by APC in the absence of nanobody (representative of three experiments). Figure 3B is a graph of histone H3 cleavage versus time for nine nanobodies that had minimal effects on APC-mediated H3 cleavage. The H3 cleavage percentage was calculated via normalization using the H3 band intensity at time = 0 min: 100% (intensity t = x min / intensity t = 0 min). Figure 3C is an SDS-PAGE gel showing that nanobody LP14 enhances H3 cleavage. Figure 3D is a graph of histone H3 cleavage versus time for 12 nanobodies that inhibit APC-mediated H3 cleavage. Figure 3E is an SDS-PAGE gel showing that nanobody LP21 potently inhibits H3 cleavage. Figure 3F is a table presenting the data shown in (B) and (D) as numerical values. [Figure 3B]Figure 3 shows the effect of nanobodies LP1-LP21 on APC-mediated histone H3 cleavage. APC (50 nM) and nanobodies (500 nM) were pre-incubated together for 30 min. After incubation, the APC-nanobody mixture was added to histone H3 (100 μg / mL). Over a period of 2 h, samples were taken at different time points and added to reducing sample buffer for SDS-PAGE analysis. A molecular marker of 20 kDa was used as a standard for normalization between gels. Figure 3A is an SDS-PAGE gel showing the time course of histone H3 cleavage by APC in the absence of nanobody (representative of three experiments). Figure 3B is a graph of histone H3 cleavage versus time for nine nanobodies that had minimal effects on APC-mediated H3 cleavage. The H3 cleavage percentage was calculated via normalization using the H3 band intensity at time = 0 min: 100% (intensity t = x min / intensity t = 0 min). Figure 3C is an SDS-PAGE gel showing that nanobody LP14 enhances H3 cleavage. Figure 3D is a graph of histone H3 cleavage versus time for 12 nanobodies that inhibit APC-mediated H3 cleavage. Figure 3E is an SDS-PAGE gel showing that nanobody LP21 potently inhibits H3 cleavage. Figure 3F is a table presenting the data shown in (B) and (D) as numerical values. [Figure 3C]Figure 3 shows the effect of nanobodies LP1-LP21 on APC-mediated histone H3 cleavage. APC (50 nM) and nanobodies (500 nM) were pre-incubated together for 30 min. After incubation, the APC-nanobody mixture was added to histone H3 (100 μg / mL). Over a period of 2 h, samples were taken at different time points and added to reducing sample buffer for SDS-PAGE analysis. A molecular marker of 20 kDa was used as a standard for normalization between gels. Figure 3A is an SDS-PAGE gel showing the time course of histone H3 cleavage by APC in the absence of nanobody (representative of three experiments). Figure 3B is a graph of histone H3 cleavage versus time for nine nanobodies that had minimal effects on APC-mediated H3 cleavage. The H3 cleavage percentage was calculated via normalization using the H3 band intensity at time = 0 min: 100% (intensity t = x min / intensity t = 0 min). Figure 3C is an SDS-PAGE gel showing that nanobody LP14 enhances H3 cleavage. Figure 3D is a graph of histone H3 cleavage versus time for 12 nanobodies that inhibit APC-mediated H3 cleavage. Figure 3E is an SDS-PAGE gel showing that nanobody LP21 potently inhibits H3 cleavage. Figure 3F is a table presenting the data shown in (B) and (D) as numerical values. [Figure 3D]Figure 3 shows the effect of nanobodies LP1-LP21 on APC-mediated histone H3 cleavage. APC (50 nM) and nanobodies (500 nM) were pre-incubated together for 30 min. After incubation, the APC-nanobody mixture was added to histone H3 (100 μg / mL). Over a period of 2 h, samples were taken at different time points and added to reducing sample buffer for SDS-PAGE analysis. A molecular marker of 20 kDa was used as a standard for normalization between gels. Figure 3A is an SDS-PAGE gel showing the time course of histone H3 cleavage by APC in the absence of nanobody (representative of three experiments). Figure 3B is a graph of histone H3 cleavage versus time for nine nanobodies that had minimal effects on APC-mediated H3 cleavage. The H3 cleavage percentage was calculated via normalization using the H3 band intensity at time = 0 min: 100% (intensity t = x min / intensity t = 0 min). Figure 3C is an SDS-PAGE gel showing that nanobody LP14 enhances H3 cleavage. Figure 3D is a graph of histone H3 cleavage versus time for 12 nanobodies that inhibit APC-mediated H3 cleavage. Figure 3E is an SDS-PAGE gel showing that nanobody LP21 potently inhibits H3 cleavage. Figure 3F is a table presenting the data shown in (B) and (D) as numerical values. [Figure 3E]Figure 3 shows the effect of nanobodies LP1-LP21 on APC-mediated histone H3 cleavage. APC (50 nM) and nanobodies (500 nM) were pre-incubated together for 30 min. After incubation, the APC-nanobody mixture was added to histone H3 (100 μg / mL). Over a period of 2 h, samples were taken at different time points and added to reducing sample buffer for SDS-PAGE analysis. A molecular marker of 20 kDa was used as a standard for normalization between gels. Figure 3A is an SDS-PAGE gel showing the time course of histone H3 cleavage by APC in the absence of nanobody (representative of three experiments). Figure 3B is a graph of histone H3 cleavage versus time for nine nanobodies that had minimal effects on APC-mediated H3 cleavage. The H3 cleavage percentage was calculated via normalization using the H3 band intensity at time = 0 min: 100% (intensity t = x min / intensity t = 0 min). Figure 3C is an SDS-PAGE gel showing that nanobody LP14 enhances H3 cleavage. Figure 3D is a graph of histone H3 cleavage versus time for 12 nanobodies that inhibit APC-mediated H3 cleavage. Figure 3E is an SDS-PAGE gel showing that nanobody LP21 potently inhibits H3 cleavage. Figure 3F is a table presenting the data shown in (B) and (D) as numerical values. [Figure 3F]Figure 3 shows the effect of nanobodies LP1-LP21 on APC-mediated histone H3 cleavage. APC (50 nM) and nanobodies (500 nM) were pre-incubated together for 30 min. After incubation, the APC-nanobody mixture was added to histone H3 (100 μg / mL). Over a period of 2 h, samples were taken at different time points and added to reducing sample buffer for SDS-PAGE analysis. A molecular marker of 20 kDa was used as a standard for normalization between gels. Figure 3A is an SDS-PAGE gel showing the time course of histone H3 cleavage by APC in the absence of nanobody (representative of three experiments). Figure 3B is a graph of histone H3 cleavage versus time for nine nanobodies that had minimal effects on APC-mediated H3 cleavage. The H3 cleavage percentage was calculated via normalization using the H3 band intensity at time = 0 min: 100% (intensity t = x min / intensity t = 0 min). Figure 3C is an SDS-PAGE gel showing that nanobody LP14 enhances H3 cleavage. Figure 3D is a graph of histone H3 cleavage versus time for 12 nanobodies that inhibit APC-mediated H3 cleavage. Figure 3E is an SDS-PAGE gel showing that nanobody LP21 potently inhibits H3 cleavage. Figure 3F is a table presenting the data shown in (B) and (D) as numerical values. [Figure 4A-1]Figure 4 shows the effect of nanobodies LP1-LP21 on APC-mediated cleavage of SEAP protease-activated receptor 1 (PAR1). Figure 4A is a series of graphs showing normalized SEAP-PAR1 cleavage for each nanobody over a range of nanobody concentrations. APC and nanobodies were added to HEK293 cells expressing SEAP-PAR1 and EPCR. The nanobodies were found to modulate APC-mediated PAR1 cleavage to different extents. LP8, LP17, LP19, and LP21 showed the most potent inhibition, whereas LP3, LP9, LP11, and LP18 showed only minimal inhibition even at the highest concentration of 500 nM (n=3 independent experiments). PAR1 cleavage is expressed as a percentage of the total SEAP activity present on the cells, relative to background. Figure 4B is a table illustrating the R41Q-SEAP-PAR1 and R46Q-SEAP-PAR1 cell lines used to evaluate the cleavage patterns of APC (50 nM) with R46 and R41, respectively. Figure 4C is a series of graphs showing the R46 / R41 cleavage ratios of the PAR1 cleavage inhibitory nanobodies LP8 and LP20. These nanobodies inhibited APC cleavage in a dose-dependent manner with both R46 and R41. At higher concentrations of LP8 and LP20, the cleavage ratio shifted to less than 1, i.e., a less cytoprotective PAR1 cleavage profile (n=3 independent experiments). Figure 4D is a series of graphs showing the R46 / R41 cleavage ratios for the less PAR1 cleaving nanobodies LP11 and LP18, which induced a curvilinear cleavage response with both R46 and R41. The R46 / R41 cleavage ratio was greater than 1 at all concentrations tested, suggesting that LP11 and LP18 can enhance cytoprotection by APC (n=3 independent experiments). Figure 4E is a table presenting the data shown in (C) and (D) as numerical values. [Figure 4A-2]Figure 4 shows the effect of nanobodies LP1-LP21 on APC-mediated cleavage of SEAP protease-activated receptor 1 (PAR1). Figure 4A is a series of graphs showing normalized SEAP-PAR1 cleavage for each nanobody over a range of nanobody concentrations. APC and nanobodies were added to HEK293 cells expressing SEAP-PAR1 and EPCR. The nanobodies were found to modulate APC-mediated PAR1 cleavage to different extents. LP8, LP17, LP19, and LP21 showed the most potent inhibition, whereas LP3, LP9, LP11, and LP18 showed only minimal inhibition even at the highest concentration of 500 nM (n=3 independent experiments). PAR1 cleavage is expressed as a percentage of the total SEAP activity present on the cells, relative to background. Figure 4B is a table illustrating the R41Q-SEAP-PAR1 and R46Q-SEAP-PAR1 cell lines used to evaluate the cleavage patterns of APC (50 nM) with R46 and R41, respectively. Figure 4C is a series of graphs showing the R46 / R41 cleavage ratios of the PAR1 cleavage inhibitory nanobodies LP8 and LP20. These nanobodies inhibited APC cleavage in a dose-dependent manner with both R46 and R41. At higher concentrations of LP8 and LP20, the cleavage ratio shifted to less than 1, i.e., a less cytoprotective PAR1 cleavage profile (n=3 independent experiments). Figure 4D is a series of graphs showing the R46 / R41 cleavage ratios for the less PAR1 cleaving nanobodies LP11 and LP18, which induced a curvilinear cleavage response with both R46 and R41. The R46 / R41 cleavage ratio was greater than 1 at all concentrations tested, suggesting that LP11 and LP18 can enhance cytoprotection by APC (n=3 independent experiments). Figure 4E is a table presenting the data shown in (C) and (D) as numerical values. [Figure 4B]Figure 4 shows the effect of nanobodies LP1-LP21 on APC-mediated cleavage of SEAP protease-activated receptor 1 (PAR1). Figure 4A is a series of graphs showing normalized SEAP-PAR1 cleavage for each nanobody over a range of nanobody concentrations. APC and nanobodies were added to HEK293 cells expressing SEAP-PAR1 and EPCR. The nanobodies were found to modulate APC-mediated PAR1 cleavage to different extents. LP8, LP17, LP19, and LP21 showed the most potent inhibition, whereas LP3, LP9, LP11, and LP18 showed only minimal inhibition even at the highest concentration of 500 nM (n=3 independent experiments). PAR1 cleavage is expressed as a percentage of the total SEAP activity present on the cells, relative to background. Figure 4B is a table illustrating the R41Q-SEAP-PAR1 and R46Q-SEAP-PAR1 cell lines used to evaluate the cleavage patterns of APC (50 nM) with R46 and R41, respectively. Figure 4C is a series of graphs showing the R46 / R41 cleavage ratios of the PAR1 cleavage inhibitory nanobodies LP8 and LP20. These nanobodies inhibited APC cleavage in a dose-dependent manner with both R46 and R41. At higher concentrations of LP8 and LP20, the cleavage ratio shifted to less than 1, i.e., a less cytoprotective PAR1 cleavage profile (n=3 independent experiments). Figure 4D is a series of graphs showing the R46 / R41 cleavage ratios for the less PAR1 cleaving nanobodies LP11 and LP18, which induced a curvilinear cleavage response with both R46 and R41. The R46 / R41 cleavage ratio was greater than 1 at all concentrations tested, suggesting that LP11 and LP18 can enhance cytoprotection by APC (n=3 independent experiments). Figure 4E is a table presenting the data shown in (C) and (D) as numerical values. [Figure 4C]Figure 4 shows the effect of nanobodies LP1-LP21 on APC-mediated cleavage of SEAP protease-activated receptor 1 (PAR1). Figure 4A is a series of graphs showing normalized SEAP-PAR1 cleavage for each nanobody over a range of nanobody concentrations. APC and nanobodies were added to HEK293 cells expressing SEAP-PAR1 and EPCR. The nanobodies were found to modulate APC-mediated PAR1 cleavage to different extents. LP8, LP17, LP19, and LP21 showed the most potent inhibition, whereas LP3, LP9, LP11, and LP18 showed only minimal inhibition even at the highest concentration of 500 nM (n=3 independent experiments). PAR1 cleavage is expressed as a percentage of the total SEAP activity present on the cells, relative to background. Figure 4B is a table illustrating the R41Q-SEAP-PAR1 and R46Q-SEAP-PAR1 cell lines used to evaluate the cleavage patterns of APC (50 nM) with R46 and R41, respectively. Figure 4C is a series of graphs showing the R46 / R41 cleavage ratios of the PAR1 cleavage inhibitory nanobodies LP8 and LP20. These nanobodies inhibited APC cleavage in a dose-dependent manner with both R46 and R41. At higher concentrations of LP8 and LP20, the cleavage ratio shifted to less than 1, i.e., a less cytoprotective PAR1 cleavage profile (n=3 independent experiments). Figure 4D is a series of graphs showing the R46 / R41 cleavage ratios for the less PAR1 cleaving nanobodies LP11 and LP18, which induced a curvilinear cleavage response with both R46 and R41. The R46 / R41 cleavage ratio was greater than 1 at all concentrations tested, suggesting that LP11 and LP18 can enhance cytoprotection by APC (n=3 independent experiments). Figure 4E is a table presenting the data shown in (C) and (D) as numerical values. [Figure 4D-1]Figure 4 shows the effect of nanobodies LP1-LP21 on APC-mediated cleavage of SEAP protease-activated receptor 1 (PAR1). Figure 4A is a series of graphs showing normalized SEAP-PAR1 cleavage for each nanobody over a range of nanobody concentrations. APC and nanobodies were added to HEK293 cells expressing SEAP-PAR1 and EPCR. The nanobodies were found to modulate APC-mediated PAR1 cleavage to different extents. LP8, LP17, LP19, and LP21 showed the most potent inhibition, whereas LP3, LP9, LP11, and LP18 showed only minimal inhibition even at the highest concentration of 500 nM (n=3 independent experiments). PAR1 cleavage is expressed as a percentage of the total SEAP activity present on the cells, relative to background. Figure 4B is a table illustrating the R41Q-SEAP-PAR1 and R46Q-SEAP-PAR1 cell lines used to evaluate the cleavage patterns of APC (50 nM) with R46 and R41, respectively. Figure 4C is a series of graphs showing the R46 / R41 cleavage ratios of the PAR1 cleavage inhibitory nanobodies LP8 and LP20. These nanobodies inhibited APC cleavage in a dose-dependent manner with both R46 and R41. At higher concentrations of LP8 and LP20, the cleavage ratio shifted to less than 1, i.e., a less cytoprotective PAR1 cleavage profile (n=3 independent experiments). Figure 4D is a series of graphs showing the R46 / R41 cleavage ratios for the less PAR1 cleaving nanobodies LP11 and LP18, which induced a curvilinear cleavage response with both R46 and R41. The R46 / R41 cleavage ratio was greater than 1 at all concentrations tested, suggesting that LP11 and LP18 can enhance cytoprotection by APC (n=3 independent experiments). Figure 4E is a table presenting the data shown in (C) and (D) as numerical values. [Figure 4D-2]Figure 4 shows the effect of nanobodies LP1-LP21 on APC-mediated cleavage of SEAP protease-activated receptor 1 (PAR1). Figure 4A is a series of graphs showing normalized SEAP-PAR1 cleavage for each nanobody over a range of nanobody concentrations. APC and nanobodies were added to HEK293 cells expressing SEAP-PAR1 and EPCR. The nanobodies were found to modulate APC-mediated PAR1 cleavage to different extents. LP8, LP17, LP19, and LP21 showed the most potent inhibition, whereas LP3, LP9, LP11, and LP18 showed only minimal inhibition even at the highest concentration of 500 nM (n=3 independent experiments). PAR1 cleavage is expressed as a percentage of the total SEAP activity present on the cells, relative to background. Figure 4B is a table illustrating the R41Q-SEAP-PAR1 and R46Q-SEAP-PAR1 cell lines used to evaluate the cleavage patterns of APC (50 nM) with R46 and R41, respectively. Figure 4C is a series of graphs showing the R46 / R41 cleavage ratios of the PAR1 cleavage inhibitory nanobodies LP8 and LP20. These nanobodies inhibited APC cleavage in a dose-dependent manner with both R46 and R41. At higher concentrations of LP8 and LP20, the cleavage ratio shifted to less than 1, i.e., a less cytoprotective PAR1 cleavage profile (n=3 independent experiments). Figure 4D is a series of graphs showing the R46 / R41 cleavage ratios for the less PAR1 cleaving nanobodies LP11 and LP18, which induced a curvilinear cleavage response with both R46 and R41. The R46 / R41 cleavage ratio was greater than 1 at all concentrations tested, suggesting that LP11 and LP18 can enhance cytoprotection by APC (n=3 independent experiments). Figure 4E is a table presenting the data shown in (C) and (D) as numerical values. [Figure 4E]Figure 4 shows the effect of nanobodies LP1-LP21 on APC-mediated cleavage of SEAP protease-activated receptor 1 (PAR1). Figure 4A is a series of graphs showing normalized SEAP-PAR1 cleavage for each nanobody over a range of nanobody concentrations. APC and nanobodies were added to HEK293 cells expressing SEAP-PAR1 and EPCR. The nanobodies were found to modulate APC-mediated PAR1 cleavage to different extents. LP8, LP17, LP19, and LP21 showed the most potent inhibition, whereas LP3, LP9, LP11, and LP18 showed only minimal inhibition even at the highest concentration of 500 nM (n=3 independent experiments). PAR1 cleavage is expressed as a percentage of the total SEAP activity present on the cells, relative to background. Figure 4B is a table illustrating the R41Q-SEAP-PAR1 and R46Q-SEAP-PAR1 cell lines used to evaluate the cleavage patterns of APC (50 nM) with R46 and R41, respectively. Figure 4C is a series of graphs showing the R46 / R41 cleavage ratios of the PAR1 cleavage inhibitory nanobodies LP8 and LP20. These nanobodies inhibited APC cleavage in a dose-dependent manner with both R46 and R41. At higher concentrations of LP8 and LP20, the cleavage ratio shifted to less than 1, i.e., a less cytoprotective PAR1 cleavage profile (n=3 independent experiments). Figure 4D is a series of graphs showing the R46 / R41 cleavage ratios for the less PAR1 cleaving nanobodies LP11 and LP18, which induced a curvilinear cleavage response with both R46 and R41. The R46 / R41 cleavage ratio was greater than 1 at all concentrations tested, suggesting that LP11 and LP18 can enhance cytoprotection by APC (n=3 independent experiments). Figure 4E is a table presenting the data shown in (C) and (D) as numerical values. [Figure 5A]Figure 5 shows the effect of LP11 on APC-mediated endothelial barrier function. Figure 5A is a graph of normalized cell index (NCI) versus time of thrombin addition (n=4), which shows the barrier-protecting effect of APC (40 nM) in the presence of various concentrations of LP11 on barrier permeability induced by thrombin (0.25 nM). Figure 5B is a graph quantifying the data shown in (A) based on the lowest NCI value for each time course, with the APC effect in the absence of LP11 set to 100% and the thrombin alone group set to 0%. Figure 5C is a graph showing transient endothelial barrier disruption immediately after APC priming of EA.hy926 cells (n=4). Figure 5D is a graph quantifying the data shown in (C). Figure 5E is a graph showing inhibition of transient barrier disruption by APC priming (40 nM) using PAR1 antagonists, vorapaxal (1 μM), or SCH79797 (1 μM) (n=3). Figure 5F is a graph showing inhibition of transient endothelial cell barrier disruption upon APC priming (40 nM) in the presence of LP11 (n=4). Figure 5G is a graph quantifying the data shown in (F). Figure 5H is a graph showing the overall profile of changes in NCI plotted against time of APC addition to cells (APC priming). [Figure 5B]Figure 5 shows the effect of LP11 on APC-mediated endothelial barrier function. Figure 5A is a graph of normalized cell index (NCI) versus time of thrombin addition (n=4), which shows the barrier-protecting effect of APC (40 nM) in the presence of various concentrations of LP11 on barrier permeability induced by thrombin (0.25 nM). Figure 5B is a graph quantifying the data shown in (A) based on the lowest NCI value for each time course, with the APC effect in the absence of LP11 set to 100% and the thrombin alone group set to 0%. Figure 5C is a graph showing transient endothelial barrier disruption immediately after APC priming of EA.hy926 cells (n=4). Figure 5D is a graph quantifying the data shown in (C). Figure 5E is a graph showing inhibition of transient barrier disruption by APC priming (40 nM) using PAR1 antagonists, vorapaxal (1 μM), or SCH79797 (1 μM) (n=3). Figure 5F is a graph showing inhibition of transient endothelial cell barrier disruption upon APC priming (40 nM) in the presence of LP11 (n=4). Figure 5G is a graph quantifying the data shown in (F). Figure 5H is a graph showing the overall profile of changes in NCI plotted against time of APC addition to cells (APC priming). [Figure 5C]Figure 5 shows the effect of LP11 on APC-mediated endothelial barrier function. Figure 5A is a graph of normalized cell index (NCI) versus time of thrombin addition (n=4), which shows the barrier-protecting effect of APC (40 nM) in the presence of various concentrations of LP11 on barrier permeability induced by thrombin (0.25 nM). Figure 5B is a graph quantifying the data shown in (A) based on the lowest NCI value for each time course, with the APC effect in the absence of LP11 set to 100% and the thrombin alone group set to 0%. Figure 5C is a graph showing transient endothelial barrier disruption immediately after APC priming of EA.hy926 cells (n=4). Figure 5D is a graph quantifying the data shown in (C). Figure 5E is a graph showing inhibition of transient barrier disruption by APC priming (40 nM) using PAR1 antagonists, vorapaxal (1 μM), or SCH79797 (1 μM) (n=3). Figure 5F is a graph showing inhibition of transient endothelial cell barrier disruption upon APC priming (40 nM) in the presence of LP11 (n=4). Figure 5G is a graph quantifying the data shown in (F). Figure 5H is a graph showing the overall profile of changes in NCI plotted against time of APC addition to cells (APC priming). [Figure 5D]Figure 5 shows the effect of LP11 on APC-mediated endothelial barrier function. Figure 5A is a graph of normalized cell index (NCI) versus time of thrombin addition (n=4), which shows the barrier-protecting effect of APC (40 nM) in the presence of various concentrations of LP11 on barrier permeability induced by thrombin (0.25 nM). Figure 5B is a graph quantifying the data shown in (A) based on the lowest NCI value for each time course, with the APC effect in the absence of LP11 set to 100% and the thrombin alone group set to 0%. Figure 5C is a graph showing transient endothelial barrier disruption immediately after APC priming of EA.hy926 cells (n=4). Figure 5D is a graph quantifying the data shown in (C). Figure 5E is a graph showing inhibition of transient barrier disruption by APC priming (40 nM) using PAR1 antagonists, vorapaxal (1 μM), or SCH79797 (1 μM) (n=3). Figure 5F is a graph showing inhibition of transient endothelial cell barrier disruption upon APC priming (40 nM) in the presence of LP11 (n=4). Figure 5G is a graph quantifying the data shown in (F). Figure 5H is a graph showing the overall profile of changes in NCI plotted against time of APC addition to cells (APC priming). [Figure 5E]Figure 5 shows the effect of LP11 on APC-mediated endothelial barrier function. Figure 5A is a graph of normalized cell index (NCI) versus time of thrombin addition (n=4), which shows the barrier-protecting effect of APC (40 nM) in the presence of various concentrations of LP11 on barrier permeability induced by thrombin (0.25 nM). Figure 5B is a graph quantifying the data shown in (A) based on the lowest NCI value for each time course, with the APC effect in the absence of LP11 set to 100% and the thrombin alone group set to 0%. Figure 5C is a graph showing transient endothelial barrier disruption immediately after APC priming of EA.hy926 cells (n=4). Figure 5D is a graph quantifying the data shown in (C). Figure 5E is a graph showing inhibition of transient barrier disruption by APC priming (40 nM) using PAR1 antagonists, vorapaxal (1 μM), or SCH79797 (1 μM) (n=3). Figure 5F is a graph showing inhibition of transient endothelial cell barrier disruption upon APC priming (40 nM) in the presence of LP11 (n=4). Figure 5G is a graph quantifying the data shown in (F). Figure 5H is a graph showing the overall profile of changes in NCI plotted against time of APC addition to cells (APC priming). [Figure 5F]Figure 5 shows the effect of LP11 on APC-mediated endothelial barrier function. Figure 5A is a graph of normalized cell index (NCI) versus time of thrombin addition (n=4), which shows the barrier-protecting effect of APC (40 nM) in the presence of various concentrations of LP11 on barrier permeability induced by thrombin (0.25 nM). Figure 5B is a graph quantifying the data shown in (A) based on the lowest NCI value for each time course, with the APC effect in the absence of LP11 set to 100% and the thrombin alone group set to 0%. Figure 5C is a graph showing transient endothelial barrier disruption immediately after APC priming of EA.hy926 cells (n=4). Figure 5D is a graph quantifying the data shown in (C). Figure 5E is a graph showing inhibition of transient barrier disruption by APC priming (40 nM) using PAR1 antagonists, vorapaxal (1 μM), or SCH79797 (1 μM) (n=3). Figure 5F is a graph showing inhibition of transient endothelial cell barrier disruption upon APC priming (40 nM) in the presence of LP11 (n=4). Figure 5G is a graph quantifying the data shown in (F). Figure 5H is a graph showing the overall profile of changes in NCI plotted against time of APC addition to cells (APC priming). [Figure 5G]Figure 5 shows the effect of LP11 on APC-mediated endothelial barrier function. Figure 5A is a graph of normalized cell index (NCI) versus time of thrombin addition (n=4), which shows the barrier-protecting effect of APC (40 nM) in the presence of various concentrations of LP11 on barrier permeability induced by thrombin (0.25 nM). Figure 5B is a graph quantifying the data shown in (A) based on the lowest NCI value for each time course, with the APC effect in the absence of LP11 set to 100% and the thrombin alone group set to 0%. Figure 5C is a graph showing transient endothelial barrier disruption immediately after APC priming of EA.hy926 cells (n=4). Figure 5D is a graph quantifying the data shown in (C). Figure 5E is a graph showing inhibition of transient barrier disruption by APC priming (40 nM) using PAR1 antagonists, vorapaxal (1 μM), or SCH79797 (1 μM) (n=3). Figure 5F is a graph showing inhibition of transient endothelial cell barrier disruption upon APC priming (40 nM) in the presence of LP11 (n=4). Figure 5G is a graph quantifying the data shown in (F). Figure 5H is a graph showing the overall profile of changes in NCI plotted against time of APC addition to cells (APC priming). [Figure 5H-1]Figure 5 shows the effect of LP11 on APC-mediated endothelial barrier function. Figure 5A is a graph of normalized cell index (NCI) versus time of thrombin addition (n=4), which shows the barrier-protecting effect of APC (40 nM) in the presence of various concentrations of LP11 on barrier permeability induced by thrombin (0.25 nM). Figure 5B is a graph quantifying the data shown in (A) based on the lowest NCI value for each time course, with the APC effect in the absence of LP11 set to 100% and the thrombin alone group set to 0%. Figure 5C is a graph showing transient endothelial barrier disruption immediately after APC priming of EA.hy926 cells (n=4). Figure 5D is a graph quantifying the data shown in (C). Figure 5E is a graph showing inhibition of transient barrier disruption by APC priming (40 nM) using PAR1 antagonists, vorapaxal (1 μM), or SCH79797 (1 μM) (n=3). Figure 5F is a graph showing inhibition of transient endothelial cell barrier disruption upon APC priming (40 nM) in the presence of LP11 (n=4). Figure 5G is a graph quantifying the data shown in (F). Figure 5H is a graph showing the overall profile of changes in NCI plotted against time of APC addition to cells (APC priming). [Figure 5H-2]Figure 5 shows the effect of LP11 on APC-mediated endothelial barrier function. Figure 5A is a graph of normalized cell index (NCI) versus time of thrombin addition (n=4), which shows the barrier-protecting effect of APC (40 nM) in the presence of various concentrations of LP11 on barrier permeability induced by thrombin (0.25 nM). Figure 5B is a graph quantifying the data shown in (A) based on the lowest NCI value for each time course, with the APC effect in the absence of LP11 set to 100% and the thrombin alone group set to 0%. Figure 5C is a graph showing transient endothelial barrier disruption immediately after APC priming of EA.hy926 cells (n=4). Figure 5D is a graph quantifying the data shown in (C). Figure 5E is a graph showing inhibition of transient barrier disruption by APC priming (40 nM) using PAR1 antagonists, vorapaxal (1 μM), or SCH79797 (1 μM) (n=3). Figure 5F is a graph showing inhibition of transient endothelial cell barrier disruption upon APC priming (40 nM) in the presence of LP11 (n=4). Figure 5G is a graph quantifying the data shown in (F). Figure 5H is a graph showing the overall profile of changes in NCI plotted against time of APC addition to cells (APC priming). [Figure 6A] Figure 6 shows purified nanobodies (LP1-LP21) run on a reducing SDS-PAGE gel (12% Bis-Tris with MOPS running buffer). Figure 6A shows a gel of LP1-LP7. Figure 6B shows a gel of LP8-LP21. [Figure 6B] Figure 6 shows purified nanobodies (LP1-LP21) run on a reducing SDS-PAGE gel (12% Bis-Tris with MOPS running buffer). Figure 6A shows a gel of LP1-LP7. Figure 6B shows a gel of LP8-LP21. [Figure 7] A schematic diagram summarizing the process by which nanobodies LP1–LP21 were selected from the nanobody library for further studies is shown. [Figure 8-1]ELISA screening of 158 unique APC-PPACK specific clones against APC, FXa-PPACK and PBS coated wells (n=2). For these clones, selective binding to APC over FXa-PPACK and PC was observed. Twenty-one nanobodies, designated LP1-LP21, belonging to 18 sequence diverse families were selected for further characterization. [Figure 8-2] ELISA screening of 158 unique APC-PPACK specific clones against APC, FXa-PPACK and PBS coated wells (n=2). For these clones, selective binding to APC over FXa-PPACK and PC was observed. Twenty-one nanobodies, designated LP1-LP21, belonging to 18 sequence diverse families were selected for further characterization. [Figure 8-3] ELISA screening of 158 unique APC-PPACK specific clones against APC, FXa-PPACK and PBS coated wells (n=2). For these clones, selective binding to APC over FXa-PPACK and PC was observed. Twenty-one nanobodies, designated LP1-LP21, belonging to 18 sequence diverse families were selected for further characterization. [Figure 9]Figure 1 shows the results of ELISA screening of nanobodies LP1-LP21 against wells coated with APC, FVIIa, FIXa, and FXIa. For all 21 of these nanobodies, selective binding to APC was observed (n=3 independent experiments). ELISA plates were coated with 100 μL of 10 μg / mL APC, FVIIa, FIXa, or FXIa in PBS for 1 h and then blocked with 200 μL of 1% casein pH 7.2 in PBS for 1 h. Plates were washed 4 times with PBS-T (0.05%, pH 7.2). Nanobodies (100 μL at 2 μg / mL in PBS) were added to the plates. After 1 h of incubation, plates were washed 4 times with PBS-T and incubated with 100 μL of HRP-anti-V5 at 1:25,000. After 1 h of incubation, plates were washed 4 times with PBS-T and the amount of nanobody binding to the APC-coated plates was detected using TMB substrate. The reaction was terminated with 100 μL of 2N H2SO4. [Figure 10-1] Figure 1 shows the effect of nanobodies on S-2366 cleavage by APC. The effect of nanobodies on the amidolytic activity of APC towards the small chromogenic substrate S-2366 was measured by the change in absorbance at 405 nm over time in reactions consisting of APC (20 nM), nanobodies (0-80 nM) and S-2366 (1 mM) in HBS / 0.1% BSA. APC-mediated S-2366 cleavage was enhanced by 8 nanobodies, partially inhibited by 5 nanobodies and unaffected by 8 nanobodies (n=3). [Figure 10-2] Figure 1 shows the effect of nanobodies on S-2366 cleavage by APC. The effect of nanobodies on the amidolytic activity of APC towards the small chromogenic substrate S-2366 was measured by the change in absorbance at 405 nm over time in reactions consisting of APC (20 nM), nanobodies (0-80 nM) and S-2366 (1 mM) in HBS / 0.1% BSA. APC-mediated S-2366 cleavage was enhanced by 8 nanobodies, partially inhibited by 5 nanobodies and unaffected by 8 nanobodies (n=3). [Figure 10-3]Figure 1 shows the effect of nanobodies on S-2366 cleavage by APC. The effect of nanobodies on the amidolytic activity of APC towards the small chromogenic substrate S-2366 was measured by the change in absorbance at 405 nm over time in reactions consisting of APC (20 nM), nanobodies (0-80 nM) and S-2366 (1 mM) in HBS / 0.1% BSA. APC-mediated S-2366 cleavage was enhanced by 8 nanobodies, partially inhibited by 5 nanobodies and unaffected by 8 nanobodies (n=3). [Figure 10-4] Figure 1 shows the effect of nanobodies on S-2366 cleavage by APC. The effect of nanobodies on the amidolytic activity of APC towards the small chromogenic substrate S-2366 was measured by the change in absorbance at 405 nm over time in reactions consisting of APC (20 nM), nanobodies (0-80 nM) and S-2366 (1 mM) in HBS / 0.1% BSA. APC-mediated S-2366 cleavage was enhanced by 8 nanobodies, partially inhibited by 5 nanobodies and unaffected by 8 nanobodies (n=3). [Figure 10-5] Figure 1 shows the effect of nanobodies on S-2366 cleavage by APC. The effect of nanobodies on the amidolytic activity of APC towards the small chromogenic substrate S-2366 was measured by the change in absorbance at 405 nm over time in reactions consisting of APC (20 nM), nanobodies (0-80 nM) and S-2366 (1 mM) in HBS / 0.1% BSA. APC-mediated S-2366 cleavage was enhanced by 8 nanobodies, partially inhibited by 5 nanobodies and unaffected by 8 nanobodies (n=3). [Figure 10-6]Figure 1 shows the effect of nanobodies on S-2366 cleavage by APC. The effect of nanobodies on the amidolytic activity of APC towards the small chromogenic substrate S-2366 was measured by the change in absorbance at 405 nm over time in reactions consisting of APC (20 nM), nanobodies (0-80 nM) and S-2366 (1 mM) in HBS / 0.1% BSA. APC-mediated S-2366 cleavage was enhanced by 8 nanobodies, partially inhibited by 5 nanobodies and unaffected by 8 nanobodies (n=3). [Figure 11-1] Lineweaver-Burk plots of APC-mediated S-2366 cleavage in the presence of nanobodies that enhanced or inhibited cleavage (n=3). Reactions consisted of APC (16.7 nM), nanobodies (67 nM), and S-2366 (0.075-1.2 mM) in HBS / 0.1% BSA. [Figure 11-2] Lineweaver-Burk plots of APC-mediated S-2366 cleavage in the presence of nanobodies that enhanced or inhibited cleavage (n=3). Reactions consisted of APC (16.7 nM), nanobodies (67 nM), and S-2366 (0.075-1.2 mM) in HBS / 0.1% BSA. [Figure 12] FIG. 1 shows that LP11 shortened the long clotting time in plasma with a FVIII inhibitor in the presence of the PC activator Protac® (n=3). [Figure 13A] Figure 13 shows characterization of LP11 expressed in mammalian cells. Figure 13A is an SDS-PAGE gel showing purified mammalian LP11. Figure 13B is a graph of Protac®-APTT clotting time versus LP11 concentration, showing inhibition of Protac®-APTT by mammalian LP11. Figure 13C is a set of graphs showing the effect of mammalian LP11 on cleavage in R41Q-SEAP-PAR1 and R46Q-SEAP-PAR1 cell lines. [Figure 13B]Figure 13 shows characterization of LP11 expressed in mammalian cells. Figure 13A is an SDS-PAGE gel showing purified mammalian LP11. Figure 13B is a graph of Protac®-APTT clotting time versus LP11 concentration, showing inhibition of Protac®-APTT by mammalian LP11. Figure 13C is a set of graphs showing the effect of mammalian LP11 on cleavage in R41Q-SEAP-PAR1 and R46Q-SEAP-PAR1 cell lines. [Figure 13C] Figure 13 shows characterization of LP11 expressed in mammalian cells. Figure 13A is an SDS-PAGE gel showing purified mammalian LP11. Figure 13B is a graph of Protac®-APTT clotting time versus LP11 concentration, showing inhibition of Protac®-APTT by mammalian LP11. Figure 13C is a set of graphs showing the effect of mammalian LP11 on cleavage in R41Q-SEAP-PAR1 and R46Q-SEAP-PAR1 cell lines. [Figure 14A]Library panning experiments are summarized. Figure 14A is a table summarizing the library panning process. Successive rounds of selection (R1, R2, R3, and R4) were performed on plates coated overnight at 4°C with decreasing APC-PPACK concentrations (100, 50, 10, and 1 nM) and increasing number and duration of washes. Negative selection was performed with factor Xa-PPACK, PC, and empty wells blocked with 2% bovine serum albumin (BSA) to remove non-specific binders. Enrichment of APC-PPACK binders, which had minimal binding to factor Xa-PPACK and PC, was confirmed via phage pool ELISA between rounds. Phage bound to APC-PPACK were eluted using 100 mM triethylamine and then neutralized with 1 M Tris-HCl pH 7.5. Eluted phage were used to infect TG1 cells. The output was titered and plated on 2YTCG (2YT medium, 50 μg / mL carbenicillin, 2% glucose) plates and grown overnight at 30° C. The second panning arm (R2′ and R3′) was performed using more stringent negative selection on PC. CFU, colony forming units. FIG. 14B is a table summarizing the output phage pools from panning rounds R2, R3, R4, and R3′. [Figure 14B]Library panning experiments are summarized. Figure 14A is a table summarizing the library panning process. Successive rounds of selection (R1, R2, R3, and R4) were performed on plates coated overnight at 4°C with decreasing APC-PPACK concentrations (100, 50, 10, and 1 nM) and increasing number and duration of washes. Negative selection was performed with factor Xa-PPACK, PC, and empty wells blocked with 2% bovine serum albumin (BSA) to remove non-specific binders. Enrichment of APC-PPACK binders, which had minimal binding to factor Xa-PPACK and PC, was confirmed via phage pool ELISA between rounds. Phage bound to APC-PPACK were eluted using 100 mM triethylamine and then neutralized with 1 M Tris-HCl pH 7.5. Eluted phage were used to infect TG1 cells. The output was titered and plated on 2YTCG (2YT medium, 50 μg / mL carbenicillin, 2% glucose) plates and grown overnight at 30° C. The second panning arm (R2′ and R3′) was performed using more stringent negative selection on PC. CFU, colony forming units. FIG. 14B is a table summarizing the output phage pools from panning rounds R2, R3, R4, and R3′. [Figure 15] FIG. 1 is a schematic diagram comparing the structure of conventional antibodies with that of camelid and VHH antibodies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present disclosure relates to antibodies that specifically bind to activated protein C (APC). In preferred embodiments, the antibodies bind to the active form of the enzyme (i.e., APC) and minimally bind to the proenzyme form of the enzyme (i.e., protein C (PC)). In some embodiments, the disclosed antibodies at least partially maintain or even enhance the multifaceted cytoprotective function of APC while inhibiting the anticoagulant activity of APC. In some embodiments, the disclosed antibodies partially or fully maintain the anticoagulant activity of APC while enhancing the cytoprotective function of APC. The present invention also provides polynucleotides encoding these antibodies, pharmaceutical compositions comprising these antibodies, methods of making these antibodies, and methods for treating conditions by administering a therapeutically effective amount of these antibodies.

[0028] The anti-APC antibodies of the present invention provide several advantages over recombinant APC protein therapeutics currently on the market or in development. For example, anti-APC antibodies have a lower risk of immunogenicity than therapeutics that include exogenous APC proteins and their variants (e.g., 3K3A-APC). Anti-drug antibodies (ADA) against exogenous APC proteins can cross-react with endogenous PC / APC proteins and result in autoimmune reactions. For example, ZZ Biotech, LLC's APC variant 3K3A-APC is in clinical trials, where it is administered to patients in multiple repeated doses to treat ischemic stroke (ClinicalTrials.gov Trial Id. NCT02222714). This can effectively immunize patients and cause them to develop anti-APC antibodies that can cross-react with endogenous APC. In contrast, ADA against anti-APC antibodies should not cross-react with endogenous PC / APC proteins, making the use of anti-APC antibodies a safer therapeutic strategy. Furthermore, depending on the antibody format selected, fewer doses of the anti-APC antibody may be required to achieve the desired therapeutic effect compared to the number of doses required for an APC protein-based drug.

[0029] antibody: In the first aspect, the present invention provides an antibody that specifically binds to APC. As used herein, the term "antibody" refers to a protein that comprises at least one antigen-binding domain from an immunoglobulin. This term encompasses both full-length immunoglobulins and their antigen-binding fragments.

[0030] As noted above, the term "antibody" includes fragments of full-length immunoglobulins that contain an antigen-binding domain. Examples of antigen-binding fragments include, but are not limited to, (i) Fab fragments, i.e., fragments that contain the heavy chain variable region (V H ), light chain variable region (V L (ii) a F(ab')2 fragment, i.e. a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and CH1, (iv) a V of a single arm of the antibody; L and V H (v) an Fv fragment consisting of V H (Nature 341:544-546, 1989), (vi) isolated complementarity determining regions (CDRs), (vii) minibodies, diabodies, triabodies, tetrabodies, and kappabodies (e.g., Protein Eng 10:949-57, 1997), (viii) V H H antibody, V H H dimer and V H (ix) fragments of camelid antibodies, including H-Fc fusions, and (ix) fragments of cartilaginous antibodies, including VNAR antibodies.

[0031] In some embodiments, the antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secretory IgA, IgD, IgE, and antigen-binding fragments thereof. In other embodiments, the antibody comprises an alternative scaffold, such as a scaffold comprising a non-immunoglobulin binding protein (e.g., Affibody®, Affilin™, Affimer®, Alphabody, Anticalin®, Atrimer, Avimer, Centyrin, DARPin®, Fynomer, Kunitz domain, OBody, Pronectin®, or repebody). In some embodiments, the antibody is in IgG4 format. This format offers therapeutic advantages since IgG4 is the only subclass of IgG that does not mediate common IgG effector functions such as antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity.

[0032] In a preferred embodiment, the antibody is a nanobody. As used herein, the term "nanobody" refers to an antibody fragment consisting of a single monomeric variable antibody domain. Nanobodies may also be referred to as "single domain antibodies". In conventional antibodies, the antigen-binding region is composed of the heavy chain variable region (V H ) and the light chain variable region (V L Nanobodies therefore consist of a single variable region (V) of a conventional antibody. H Or V L ). However, nanobodies may also be derived from heavy chain antibodies. A "heavy chain antibody" or "heavy chain only antibody" is an antibody that consists of two heavy chains and lacks the two light chains found in conventional antibodies. Heavy chain antibodies are H The V domain of a heavy chain antibody is the only one that binds to the antigen. H Heavy chain antibodies are naturally produced by camelids (e.g., llamas, alpacas, and camels) and cartilaginous fish (e.g., sharks). Camelids produce heavy chain antibodies from which V HSingle domain antibodies, termed H antibodies, can be derived from cartilaginous fish, and cartilaginous fish produce heavy chain antibodies, termed immunoglobulin new antigen receptors (IgNARs), from which single domain antibodies, termed VNAR antibodies, can be derived. Thus, in some embodiments, nanobodies are V H It is an H antibody or a VNAR antibody.

[0033] The antibody library that the inventors screened in the Examples was V H H antibody library. Thus, in a preferred embodiment, the antibodies of the present invention are H Camelid antibodies are H antibodies. Camelid antibodies contain two heavy chains that lack the first constant Ig domain (CH1) found in typical heavy chains (Figure 15). The variable portion of these camelid heavy chains is the heavy chain (V H The V of a camelid antibody is called the single variable domain on the H Nanobodies consisting of the H portion are called "V H These are called "H antibodies". H H antibodies are typical of human immunoglobulin V H It has a similar structural organization to V and contains four conserved framework regions (FR1 / 2 / 3 / 4) surrounding three hypervariable antigen-binding loops called complementarity determining regions (CDR1 / 2 / 3) (Figure 15). H The paratope of an H antibody consists of (1) three CDRs that directly contact the antigen and are involved in antigen binding, and (2) four framework regions that maintain the overall structure. H H antibodies can be prepared using methods known in the art (see, for example, US Pat. No. 6,765,087, US Pat. No. 6,838,254, WO06 / 079372).

[0034] V H H antibody-based therapeutics have been developed recently. For example, the first V H Caplacizumab, an H antibody-based drug, was approved in 2018. H H antibodies are well suited for use as therapeutic agents for several reasons. First, V HH antibodies are extremely robust, offering extended shelf life at both 4°C and -20°C, resistance to proteolytic degradation, and resistance to elevated temperatures (60-80°C or several weeks at 37°C), non-physiological pH (pH range 3.0-9.0), high pressure (500-750 MPa), and exposure to chemical denaturants (2-3M guanidine chloride, 6-8M urea). H The robustness of VH antibodies is due to their efficient refolding after chemical or thermal denaturation. H The monomeric structure of H antibodies and their lack of post-translational modifications allows for their expression in microbial systems, including Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. H H antibodies can be produced at low cost by producing them in milligram quantities per liter of culture in shake flasks. H H antibodies are suitable for human administration due to their low immunogenicity profile. H H antibodies and V of human immunoglobulins of family III H The sequence identity between the camelid germline IGHV family 3 and its human FR counterpart is greater than 80%. The camelid germline IGHV family 3 was found to have 95% sequence identity with its human FR counterpart. Nevertheless, the V H The VH antibody can be "humanized" if desired. H H antibodies offer greater flexibility for antigen recognition compared to conventional antibodies. Within the variable domain, CDR3 is the main contributor to antigen recognition and specificity, while CDR1 and CDR2 contribute to binding strength. H The H antibody CDR3 is on average 18 amino acids long, which corresponds to the V H significantly longer than the average 12 or 14 amino acid CDR3 of the domain. H The extended CDR3 loop of the H antibody can form a finger-like structure or a convex paratope that can penetrate into a small cavity or interact with a concave surface on the surface antigen, respectively. HH antibodies can target epitopes that are inaccessible to conventional antibodies. This increased flexibility increases specificity and, as a result, some V H This allows the VH antibody to distinguish between different isoforms of the same protein. This increased specificity is useful for developing antibodies that specifically recognize APCs as opposed to PCs, because these proteins differ by only 12 amino acids. H The small size of H antibodies allows for high tissue penetration, and some nanobodies have even been reported to cross the blood-brain barrier.

[0035] The antibody of the present invention is an anti-APC antibody. As used herein, the term "anti-APC antibody" refers to an antibody that specifically binds to an epitope of an APC. Preferably, the antibody specifically binds to a human APC having the amino acid sequence of SEQ ID NO: 634 or a variant thereof. As used herein, the term "specific binding" refers to the ability to bind to a particular antigen (e.g., APC) in preference to other molecules. Typically, an antibody that exhibits specific binding has a specific binding activity of at least about 10 -5 The equilibrium dissociation constant of M (K D ) and binds to the antigen with an affinity that is higher (e.g., at least 2-fold higher) than its binding affinity to an unrelated protein (e.g., BSA, casein). In many cases, an antibody will bind to an antigen with a higher affinity (i.e., a lower K, e.g., in the low nanomolar range) because it increases the likelihood that the antibody will locate and bind to its target antigen. D ) represents a more potent and specific therapeutic agent.

[0036] The antibodies disclosed herein have high specificity for the activated form of protein C (APC) as opposed to the inactive zymogen form (PC). This is useful considering the 1700-fold difference in plasma concentrations of APC (about 40 pM) and PC (70 nM). For this reason, it is preferred that the antibodies of the invention only minimally bind to PC. They either (a) do not bind to the antigen at detectable levels, or (b) bind to the antigen at about 10 -2 A lower equilibrium dissociation constant (K D) or binds to the antigen at a specific antigen.

[0037] The term "protein C" or "PC" may refer to any variant, isoform, or homologue of zymogen PC. Preferably, the PC is human PC having the amino acid sequence of SEQ ID NO: 633 or a variant thereof.

[0038] The term "epitope" refers to the area of ​​an antigen to which an antibody specifically binds. For examples of APC epitopes, see US Patent Application Publication No. 2018 / 326053, which describes epitopes outside the catalytic triad of the active site of human APC. Conversely, the term "paratope" refers to the area of ​​an antibody to which an antigen specifically binds.

[0039] Antibody binding activity may be assessed using methods known in the art, including enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), radioimmunoassay, biolayer interferometry (BLI), etc. For example, the antibodies disclosed herein may have association constants (i.e., k on ) were assessed via BLI analysis to ensure that the

[0040] In some embodiments, the antibodies of the invention inhibit the anticoagulant activity of APC (i.e., compared to the anticoagulant activity in a control without the antibody), thereby promoting thrombus formation. In some embodiments, the antibodies inhibit the anticoagulant activity of APC by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%. The antibodies may exert this effect by increasing thrombin generation by blocking the ability of APC to inactivate the clotting factors Factor Va and Factor VIIIa. Suitable assays for assessing the ability of a particular antibody to inhibit the anticoagulant activity of APC include, but are not limited to, amidolytic activity assays, substrate cleavage assays (e.g., S-2366), and thrombin generation assays. Alternatively, the ability of an antibody to decrease clotting time may be measured using an activated partial thromboplastin time (APTT) clotting assay. For a description of these assays, see Nat Commun 11(1):2992, 2020. In the Examples, the ability of 21 anti-APC antibodies to inhibit the anticoagulant activity of APC was tested using both the S-2366 cleavage assay and the Protac® adjusted APTT assay (see Figures 2, 10, 11, and Table 6).

[0041] For some indications, it may be preferable to use anti-APC antibodies that maintain or enhance the anticoagulant activity of APC. For example, such antibodies may be useful in treating patients with cancer-related thrombosis, which is a major cause of death in cancer patients. Thus, in other embodiments, the antibodies of the present invention enhance or only minimally affect the anticoagulant activity of APC.

[0042] As used herein, an antibody "minimally affects" a particular APC activity if the level of APC activity in the presence of the antibody is within + / - 20% of the level of APC activity in a no-antibody control. In some cases, an antibody that "minimally affects" APC activity is an antibody that has no detectable effect on APC activity (i.e., an antibody in the presence of the antibody has the same level of APC activity as the level of APC activity in a no-antibody control). As used herein, a "no-antibody control" is a comparable sample to which no anti-APC antibody is added.

[0043] For most human therapeutics, anti-APC antibodies preferably maintain or enhance the multifaceted cytoprotective functions of APC. The terms "cytoprotective function" and "cytoprotective activity" are used interchangeably herein to describe the anti-apoptotic, anti-inflammatory, and endothelial barrier stabilizing functions of APC, all of which contribute to the regenerative outcomes associated with APC. These functions are often interrelated. For example, cell death (e.g., due to injury or infection) results in the release of histones into the extracellular space where they interact with endothelial cells, triggering endothelial cell apoptosis and contributing to systemic inflammation. Thus, the histone cleavage function of APC reduces apoptosis, endothelial barrier disruption, and inflammation.

[0044] However, in some indications, it may be preferable to use anti-APC antibodies that block the cytoprotective function of APC. For example, short-term disruption of vascular endothelial barrier function can be used to temporarily increase vascular permeability and induce inflammation for therapeutic purposes. For cancer therapy, it may be advantageous to block cytoprotective activity to promote immune responses against tumors.

[0045] Thus, in some embodiments, the antibodies of the invention enhance, minimally affect, or inhibit APC cytoprotective function (i.e., compared to cytoprotective function in a no-antibody control). Specifically, in some embodiments, the antibodies (1) enhance, minimally affect, or inhibit APC-mediated histone cleavage, (2) enhance, minimally affect, or inhibit APC-mediated protease-activated receptor 1 (PAR1) cleavage at residue R46, (3) inhibit, minimally affect, or inhibit APC-mediated PAR1 cleavage at residue R41, (4) increase, minimally affect, or decrease the ratio of APC-mediated PAR1 cleavage at residue R46 to APC-mediated PAR1 cleavage at residue R41, and / or (5) enhance, minimally affect, or inhibit APC-mediated endothelial barrier protection. In the examples, the effect of 21 anti-APC antibodies on APC-mediated histone cleavage was tested using a histone H3 cleavage assay (see FIG. 3). Furthermore, the effect of these 21 antibodies on PAR1 cleavage was tested using a secreted embryonic alkaline phosphatase (SEAP)-PAR1 cleavage assay (see FIG. 4). Using this assay, two antibodies (i.e., LP11 and LP18) were identified that preferentially inhibited APC-mediated cleavage of PAR1 at residue R41 compared to residue R46, which enhances cytoprotective PAR1 signaling. Finally, the effect of one anti-APC antibody (i.e., LP11) on APC-mediated endothelial barrier protection was tested using an in vitro endothelial barrier function assay (see FIG. 5).

[0046] In some embodiments, the antibodies of the invention increase or decrease the half-life of APCs (i.e., compared to the half-life in the absence of the antibody). In other embodiments, the antibodies only minimally affect the half-life of APCs. In the Examples, the effect of 21 anti-APC antibodies on APC half-life was tested using an in vitro plasma half-life assay (see Table 8).

[0047] The antibodies disclosed herein bind to exosites (i.e., sites other than the active site) of APC. The antibodies were generated against an active site-blocked APC protein, referred to herein as "APC-PPACK," which contains APC bound at its active site by the tripeptide inhibitor Phe-Pro-Arg-chloromethylketone (PPACK). Antibodies targeting the exosites of APC have been shown to inhibit the antithrombotic activity of this enzyme while maintaining its beneficial cytoprotective functions (Nat Commun 11(1):2992, 2020). Separation of the anticoagulant and cytoprotective functions of APC is possible because these functions require distinct sites on the protein surface, i.e., amino acids that mediate APC's interactions with cofactors and substrates are found in the exosite, far removed from the active site.

[0048] The antibodies disclosed herein are custom V H H phage library was panned to select for antibodies that bind to human APC-PPACK and deselect for antibodies that bind to human PC and XPPACK. "XPPACK" is another serine kinase that PPACK binds to. Therefore, selecting against XPPACK reduces the chance that the selected antibodies have affinity for PPACK rather than APC. H The H phage library was constructed from heavy chain antibodies generated by immunizing llamas with APC-PPACK. The library consisted of the V H Each V contains only the H portion, which, despite being significantly smaller (usually 12-14 kDa), provides full antigen-binding capacity. H The sequences of H antibodies and their CDRs were identified via sequencing. The sequence identifiers for the protein sequences of the 158 unique anti-APC antibodies identified in the Examples are listed in Table 1. The sequence identifiers for the DNA sequences of the top 21 antibodies selected for further analysis via functional assays are listed in Table 2.

[0049] [Table 1] TIFF2025509184000003.tif220170TIFF2025509184000004.tif219170TIFF2025509184000005.tif187170 [Table 2]

[0050] The antibodies of the present invention are "isolated", meaning that they are substantially free of other biological molecules, including antibodies with different antigen specificities and other cellular materials. In some embodiments, an isolated antibody is at least about 75%, about 80%, about 90%, about 95%, about 97%, about 99%, about 99.9%, or about 100% pure by dry weight. Purity can be measured using standard methods such as column chromatography, polyacrylamide gel electrophoresis, or high performance liquid chromatography (HPLC). However, an isolated antibody that binds to human APC may have cross-reactivity to other closely related antigens, such as APC homologs from other species.

[0051] The term "complementarity determining region" or "CDR" refers to the hypervariable regions that together form an antigen-binding surface that is complementary to the three-dimensional structure of the antigen. H and V L The CDRs are V H Or V L Starting from the N-terminus of each CDR, the CDRs are numbered as "CDR1," "CDR2," and "CDR3" (see Proc Natl Acad Sci USA 72(12):5107, 1975; J Exp Med 132(2):211, 1970). Thus, in a conventional antibody (i.e., an antibody comprising two heavy chains and two light chains), the antigen-binding site is composed of six CDRs, i.e., V H The three CDRs and V L However, in heavy chain antibodies, the antigen-binding site comprises three CDRs: V H It contains only the three CDRs (Figure 15).

[0052] Since CDR sequences are involved in most antibody-antigen interactions, it is possible to create recombinant antibodies that mimic the properties of a particular naturally occurring antibody by grafting the CDR sequences of a naturally occurring antibody onto the framework sequences of a different antibody with different properties (see, for example, Nature 332:323-327,1998; Nature 321:522-525,1986; Proc Natl Acad Sci USA 86:10029-10033,1989). Such framework sequences can be obtained from public databases that contain germline antibody gene sequences. Thus, in some embodiments, the CDRs of the antibodies described herein are grafted onto another antibody framework.

[0053] The CDRs disclosed herein as SEQ ID NOs: 1-474 are derived from Camelidae V-seq. selected in the screen described in the Examples. H These are the CDRs of the VH antibody. Of the three CDRs, CDR3 is believed to be the main contributor to antigen recognition and specificity, while CDR1 and CDR2 are believed to contribute to binding strength (J Mol Biol 430:4369,2018; Protein Eng Des Sel 31:267,2018; Proteins 86:697,2018). Thus, in some embodiments, the antibodies of the present invention have the VH antibody CDRs disclosed herein. H The antibody of the present invention comprises a CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 317-474.

[0054] In other embodiments, the antibodies of the invention comprise the V HThese antibodies further comprise CDR1 and CDR2 of an H antibody. Specifically, these antibodies further comprise (a) a CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 158, (b) a CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 159 to 316, or (c) both a CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 158 and a CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 159 to 316.

[0055] In certain embodiments, an antibody of the invention comprises CDR1, CDR2, and CDR3, all of which are contained within a single V domain as disclosed herein. H In other words, these antibodies are derived from the VH antibodies disclosed herein. H The H antibody paratope (i.e., a set of three CDRs that form the antigen-binding region) is included. Specifically, these antibodies include a VH antibody having an amino acid sequence selected from the group consisting of SEQ ID NOs: 475 to 632. H It includes CDR1, CDR2, and CDR3 of the H antibody.

[0056] In addition, the present invention relates to the V H Specifically, these antibodies comprise or consist of a VH selected from the group consisting of SEQ ID NOs: 475 to 632. H Contains or consists of H.

[0057] In some embodiments, the antibody comprises one or more amino acid modifications. As used herein, the term "amino acid modification" refers to a change in a polypeptide sequence. Amino acid modifications include deletion, addition, and substitution of one or more amino acid residues. The antibody of the present invention may comprise any combination of amino acid modifications, so long as it retains the ability to bind to APC with minimal or no binding to PC. In some embodiments, the antibody comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more amino acid modifications. For example, in some embodiments, the antibody comprises a V-type antibody selected from SEQ ID NOs: 475-632 that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications compared to the parent antibody. H In other embodiments, the antibody comprises a variant of CDR3 selected from SEQ ID NOs: 317-474 that contains at least one amino acid modification.

[0058] In some embodiments, one or more of the amino acid modifications are conservative substitutions. As used herein, the term "conservative substitution" refers to an amino acid substitution that substantially preserves the structure and function of the native polypeptide. Specifically, a conservative substitution generally maintains (a) the structure of the polypeptide backbone (e.g., as a beta sheet or alpha helix) around the substitution, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and / or (c) the bulk of the side chain. Examples of conservative substitutions are shown in Table 3. [Table 3]

[0059] In some embodiments, the antibodies of the invention comprise a V that has at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, or 75 percent sequence identity to one of the amino acid sequences of SEQ ID NOs: 475-632. HH. In some embodiments, the antibody comprises a CDR3 having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, or 90 percent sequence identity to one of the amino acid sequences of SEQ ID NOs: 317-474.

[0060] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window. Aligned sequences may contain additions or deletions (i.e., gaps) relative to each other for optimal alignment. The percentage is calculated by determining the number of matching positions where the same nucleic acid base or amino acid residue occurs in both sequences, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100. Protein and nucleic acid sequence identity can be determined using the Basic Local Alignment Search Tool (BLAST), which is well known in the art (Proc. Natl. Acad. Sci. USA (1990) 87:2267-2268, Nucl. Acids Res. (1997) 25:3389-3402). The BLAST program identifies homologous sequences by identifying similar segments, referred to herein as "high-scoring segment pairs," between a query amino acid or nucleic acid sequence and a test sequence, preferably obtained from a protein or nucleic acid sequence database. Preferably, the statistical significance of high-scoring segment pairs is evaluated using the statistical significance formula (Proc. Natl. Acad. Sci. USA (1990) 87:2267-2268). The BLAST programs can be used with default parameters or with modified parameters provided by the user.

[0061] The present invention further provides antibodies that compete with the antibodies described herein for binding to APC. As with other antibodies described herein, these antibodies specifically bind to APC and minimally bind to PC. An antibody is said to "compete" with the binding of another antibody for a particular epitope if the binding of one antibody reduces the binding of the other antibody. Competition can occur because the antibodies bind to the same epitope, or because the binding of one antibody sterically interferes with the binding of the other antibody or causes a confirmatory change that interferes with the binding of the other antibody. In some cases, a first antibody can inhibit the binding of a second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its epitope. However, if both the first antibody and the second antibody detectably inhibit the binding of the other antibody (to the same extent, to a greater extent, or to a lesser extent), the antibodies are said to "cross-compete" with each other for binding their epitopes. Antibodies that compete or cross-compete with the antibodies described herein for binding to APC are encompassed by the present invention.

[0062] Nucleic acids, vectors, and host cells: The antibodies of the present invention can be produced by introducing a nucleic acid encoding the antibody into a host cell and providing suitable conditions for protein expression. Thus, in a second aspect, the present invention provides a nucleic acid encoding the antibody disclosed herein. Specifically, the present invention provides: (1) a VRNA comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 475 to 632; H In some embodiments, the nucleic acid comprises a nucleic acid encoding an antibody comprising a CDR1 selected from SEQ ID NOs: 635-655, (2) a nucleic acid encoding a CDR2 selected from SEQ ID NOs: 656-676, and / or (3) a nucleic acid encoding a CDR3 selected from SEQ ID NOs: 677-697. In some embodiments, the nucleic acid comprises a nucleic acid encoding a VH selected from SEQ ID NOs: 698-718. H Code for H.

[0063] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably to refer to polymers of DNA or RNA, which may be single-stranded or double-stranded, and may be synthetic or obtained (e.g., isolated and / or purified) from natural sources. Nucleic acids may contain natural, non-natural, or modified nucleotides and may contain natural, non-natural, or modified internucleotide linkages (e.g., phosphoramidate or phosphorothioate linkages). In some embodiments, the nucleic acids of the invention are "isolated," meaning that they are separated from other cellular material.

[0064] In a third aspect, the present invention provides a vector comprising a nucleic acid disclosed herein. The term "vector" refers to a DNA molecule used to carry a particular DNA segment (i.e., a DNA segment contained in a vector) into a host cell. Some vectors can replicate autonomously in a host cell (e.g., bacterial vectors containing an origin of replication and episomal mammalian vectors). Other vectors can integrate into the genome of a host cell so that they are replicated along with the host genome (e.g., viral vectors and transposons). A vector may contain heterologous genetic elements necessary for the propagation of the vector or for the expression of an encoded gene product (e.g., a promoter). A vector may also contain a reporter gene and / or a selectable marker gene. Suitable vectors include plasmids (i.e., circular double-stranded DNA molecules) and minichromosomes. A vector suitable for use in the present invention comprises a DNA segment encoding an antibody described herein and a heterologous sequence that allows for the expression of the encoded antibody.

[0065] In a fourth aspect, the present invention provides a host cell comprising the nucleic acid and vector disclosed herein. The term "host cell" is meant to refer to a transgenic cell in which heterologous DNA can be expressed. The nucleic acid or vector disclosed herein may be introduced into a host cell using standard techniques, including, for example, electroporation, heat shock, lipofection, microinjection, and particle bombardment. It is generally advantageous to express antibodies in eukaryotic host cells to ensure that they are properly modified, folded, and secreted. However, the V of the present invention may be used in combination with other methods. H Because the H antibody has a monomeric structure and lacks post-translational modifications, it can be easily produced by prokaryotic cells such as Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris.

[0066] In a fifth aspect, the present invention also provides a method for producing an antibody using a host cell disclosed herein. The method includes (a) culturing a host cell disclosed herein under conditions that result in the production of the antibody, and (b) isolating the antibody from the host cell. In these methods, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell. The antibody can then be recovered from the cell culture using standard protein purification methods such as ultrafiltration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, and centrifugation. Methods for expressing and purifying proteins are well known in the art (see, for example, Nat Methods 5(2):135-146,2008).

[0067] Pharmaceutical Compositions: In a sixth aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an antibody disclosed herein and a pharma- ceutically acceptable carrier. "Pharmaceutically acceptable" carriers are known in the art and include, but are not limited to, diluents, preservatives, solubilizers, emulsifiers, liposomes, nanoparticles, and adjuvants. Pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include isotonic solutions, alcoholic / aqueous solutions, emulsions, and suspensions, including saline and buffered media.

[0068] The compositions of the invention may further include diluents of various pH, ionic strength, and buffer components (e.g., Tris-HCl, acetate, phosphate), additives to prevent absorption to surfaces, surfactants (e.g., Tween 20, Tween 80, Pluronic F68), solubilizers (e.g., glycerol, polyethyleneglycerol), antioxidants (e.g., ascorbic acid, sodium metabisulfite, L-methionine), bulking substances, or tonicity adjusting agents (e.g., sucrose, mannitol). Within the compositions, the antibodies may be covalently attached to polymers (e.g., polyethylene glycol), complexed with metal ions, incorporated within or on particulate preparations of polymeric compounds (e.g., polylactic acid, polyglycolic acid, hydrogels), or on liposomes, microemulsions, micelles, multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Inclusion of such compounds in the composition affects the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. The compositions may also be formulated in lipophilic depots (e.g., fatty acids, waxes, oils) for controlled or sustained release.

[0069] method: In a seventh aspect, the present invention provides a method for treating or preventing a condition, the method comprising administering to a subject a therapeutically effective amount of an antibody or pharmaceutical composition disclosed herein.

[0070] The antibodies of the invention can optionally be administered in combination with an exogenous APC protein to confer additional properties not achievable via antibody binding to an endogenous APC protein. Thus, in some embodiments, the methods include administering to a subject an exogenous APC protein or variant thereof (e.g., 3K3A-APC) that specifically binds to one or more of the antibodies disclosed herein.

[0071] As used herein, the term "treating" describes the management and care of a patient for the purpose of addressing a condition. Treating includes the administration of an antibody or pharmaceutical composition of the invention to alleviate the symptoms or complications of a condition or to eliminate the condition. As used herein, the term "condition" is used to refer to a health problem with certain characteristics and / or symptoms. The term condition is meant to encompass diseases, disorders, syndromes, and the like.

[0072] As used herein, the term "preventing" describes the management and care of a patient for the purpose of preventing the onset of the symptoms or complications of a condition.

[0073] As used herein, the term "administering" refers to a method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, intranasal administration, topical administration, intravaginal administration, ocular administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, intravenous administration, intraarterial administration, intramuscular administration, intradermal administration, intrathecal administration, and subcutaneous administration. Administration can be continuous or intermittent. In some embodiments, administration is systemic rather than local.

[0074] The term "therapeutically effective amount" refers to an amount sufficient to produce a beneficial or desired biological or clinical result. The result may be to reduce, alleviate, inhibit, or prevent one or more symptoms of a condition, or any other desired change in a biological system. For example, in some embodiments, a therapeutically effective amount is an amount suitable for promoting thrombus formation. In other embodiments, a therapeutically effective amount is an amount suitable for treating sepsis. Methods for determining a therapeutically effective amount are well known to those of skill in the art. A therapeutically effective amount will vary depending on several factors, including, for example, the formulation of the composition used in the therapy, the purpose of the therapy, and the subject being treated. A therapeutically effective amount of the composition may be delivered via single or multiple administrations. For example, a suitable daily dose may be within the range of 3-20 mg / patient per day, 1-3 mg / patient per day, 20-100 mg / patient per day, or 20-50 mg / patient per day.

[0075] In some embodiments, the methods of the invention are used to treat or prevent a condition that can be treated or prevented by enhancing or inhibiting the anticoagulant function of APCs, for example, in some embodiments, the methods are used to treat a condition in which blood clotting is desirable by inhibiting the anticoagulant function of APCs. Suitable conditions in which blood clotting is desirable include, but are not limited to, bleeding (e.g., intracranial hemorrhage, diffuse alveolar hemorrhage, intracerebral hemorrhage), contusion (e.g., cerebral contusion), burns, gastrointestinal bleeding, uncontrolled bleeding, bleeding due to transplantation (e.g., stem cell transplantation, liver transplantation) or resection procedures, bleeding due to surgery (e.g., cardiac, spinal, orthopedic, neurological, oncology, or postpartum surgery), variceal bleeding, thrombocytopenia, idiopathic thrombocytopenic purpura, hemophilia, aortic aneurysm, reversal of anticoagulant or antithrombotic agents (e.g., warfarin, heparin), bleeding due to traumatic injury (e.g., penetrating or blunt traumatic injury), menorrhagia, bleeding in cirrhosis of the liver (e.g., active variceal or non-variceal), clotting factor deficiency (e.g., Factor VII), Glanzmann thrombasthenia (e.g., refractory to platelet transfusions), and Bernard-Soulier syndrome. In some embodiments, the condition is an acute bleeding disorder. In some embodiments, the condition is an inherited bleeding disorder.

[0076] In other embodiments, the methods of the invention are used to treat or prevent a condition that can be treated or prevented by enhancing or inhibiting one or more of the cytoprotective functions of APCs. For example, in some embodiments, the method is directed to treating sepsis (Biochem Soc Trans 43:691-5,2015), inflammation in acute ischemic disease (e.g., by providing neuroprotection in ischemic stroke (Ann Neurol 85:125-136,2019) or by providing cardioprotection in ischemic heart disease or heart failure (Int J Mol Sci 20:1762-1774,2019)), coronavirus disease 2019 (COVID-19), diabetes (e.g., type 1 diabetes (J Biol Chem 287:16356-16364,2012), diabetic nephropathy (Proc Natl Acad Sci USA 110:648-653,2013; Nature Med 13:1349-1358,2007; J Thromb Haemost 10:337-346,2012, Blood 119:874-883,2012), diabetic ulcers, wounds (Am J Pathol 179:2233-2242,2011, Wound Repair Regen 13:284-294,2005, Circ Res 95:34-41,2004, J Invest Dermatol 125:1279-1285,2005, J Biol Chem 286:6742-6750,2011, Clin Haemorheol Microcirc 34:153-161,2006, Arch Dermatol 144:1479-1483,2008, Intern J Low Extrem Wounds 10:146-151, 2011), amyotrophic lateral sclerosis (ALS) caused by mutations in SOD1 (J Clin Invest 119:3437-3449, 2009), and multiple sclerosis (Nature 451:1076-1081, 2008, J Immunol 191:3764-3777, 2013).In some embodiments, the methods may be used to treat or prevent central nervous system injury (e.g., spinal cord ischemia), ischemic stroke, Alzheimer's disease, acute kidney injury, pulmonary disorders (e.g., acute lung injury, acute respiratory distress syndrome), or acute pancreatitis (Zhao et al., Int. J. Mol. Sci. 2019, 20, 903, p. 12 of 20). In some embodiments, the methods are used to treat or prevent conditions associated with histones or NETs, ​​such as cancer (e.g., breast cancer, lung cancer, colon cancer, pancreatic cancer, hematological cancer, neurological cancer, skin cancer), or inflammatory or autoimmune diseases (e.g., psoriasis, rheumatoid arthritis, systemic lupus erythematosus).

[0077] The "subject" to which the method is applied may be a mammal or a non-mammalian animal such as a bird. Suitable mammals include, but are not limited to, humans, cows, horses, sheep, pigs, goats, rabbits, dogs, cats, bats, mice, and rats. In certain embodiments, the method may be performed on laboratory animals (e.g., mice, rats) for research purposes. In other embodiments, the method is used to treat commercially important livestock (e.g., cows, horses, pigs, rabbits, goats, sheep, chickens) or companion animals (e.g., cats, dogs). In a preferred embodiment, the subject is a human.

[0078] In an eighth aspect, the present invention provides the use of an antibody disclosed herein as a medicament. The medicament may be for the treatment or prevention of (1) a condition that can be treated or prevented by inhibiting the anticoagulant function of APCs and / or (2) a condition that can be treated or prevented by enhancing one or more of the multifaceted cytoprotective functions of APCs. Examples of such conditions are provided above.

[0079] The results of the functional assays provided in the Examples can be used to determine whether a particular anti-APC antibody described herein is useful for a particular indication. For example, an antibody that inhibits the anticoagulant activity of APC is useful for treating disorders requiring blood clotting (e.g., acute hemorrhage) while maintaining its cytoprotective activity. The importance of maintaining the cytoprotective activity of APC for such indications is supported by Magisetty et al. (Blood (2022) blood.2021013119), discussed above, in which the ability of two anti-APC antibodies to treat hemophilic arthropathy was evaluated in a mouse model. One of these antibodies, MAPC1591, inhibits the anticoagulant activity of APC without inhibiting its cytoprotective activity. The second antibody, MPC1609, inhibits both the anticoagulant and cytoprotective activities of APC. The results of this study suggest that preserving the cytoprotective activity of APC is useful for treating hemophilic arthritis. No significant difference was observed between the ability of the two antibodies to inhibit the anticoagulant activity of APC, but administration of MAPC1591, but not MPC1609, significantly reduced the severity of hemophilic arthropathy. Antibodies that enhance the cytoprotective activity of APC may be useful in treating disorders such as sepsis and inflammation in acute ischemic diseases. Table 7 provides examples of some possible uses of specific antibodies based on the results of functional assays. EXAMPLES

[0080] Activated protein C (APC) is a multifaceted coagulation protease with anticoagulant, anti-inflammatory, and cytoprotective activities. Selective modulation of these APC activities contributes to the understanding of the regulation of these physiological mechanisms and allows the development of therapeutics against pathologies associated with these pathways. As described in the Examples below, a llama-derived nanobody library was generated that targets non-active sites of APC. From the library, 21 nanobodies were identified that selectively recognize APC compared to protein C (PC) zymogens. Overall, three clusters of nanobodies were identified based on competition for APC in biolayer interferometry studies. To understand the diversity of the antibodies, APC functional assays (i.e., anticoagulant activity, histone H3 cleavage, and protease-activated receptor 1 (PAR1) cleavage) were used. These functional assays revealed 13 novel nanobody-induced APC activity profiles via selective modulation of APC multifaceted activities with the potential to modulate specific mechanisms for therapeutic purposes. Of these, three nanobodies (LP2, LP8, LP17) inhibited all three APC functions. Four nanobodies (LP1, LP5, LP16, LP20) inhibited only two of the three APC functions. Specific monofunctional inhibition of APC anticoagulant activity was observed with only two nanobodies (LP9, LP11). LP11 was also found to shift the ratio of APC cleavage of PAR1 at R46 to R41, resulting in APC-mediated biased PAR1 signaling and APC cytoprotective effects. Thus, LP11 has an activity profile that is believed to promote hemostasis and cytoprotection in bleeding associated with hemophilia or coagulation disorders by selectively modulating APC anticoagulation and PAR1 cleavage profiles.

[0081] introduction: APC is a serine protease with physiological functions in anticoagulation and cytoprotection (1, 2). APC circulates at very low concentrations (40 pM) in human blood, whereas its zymogen, protein C (PC), circulates at much higher concentrations (65 nM) (3). Thrombin / thrombomodulin activates PC via proteolytic cleavage at Arg169 and release of a 12-amino acid peptide. APC downregulates coagulation by inactivating factor Va (FVa) and factor VIIIa (FVIIIa), terminating thrombin generation once hemostasis is achieved. This role in anticoagulation makes APC a possible therapeutic target for bleeding. Hemophilia patients with APC-resistant FV Leiden variants were observed to have reduced bleeding (4-6). Mice with acute traumatic coagulopathy have been shown to have APC-resistant super Treatment with the FVa variant resulted in significantly less bleeding (7). In addition, inhibitory antibodies specific to the anticoagulant activity of APC have been effective in hemophilic monkeys (8) and mice (9, 10). Furthermore, an engineered KRK-α1-antitrypsin specific for inhibition of APC has also shown efficacy in reducing annualized bleeding rates in hemophilia studies (11, 12). These findings indicate that APC inhibition promotes a procoagulant state.

[0082] APC also induces regenerative effects, including neurogenesis (13, 14) and wound healing (15). In addition, APC has a protective effect against extracellular histones released from immune cells or necrotic cells (16), which, in pathological amounts, can cause systemic inflammation, organ failure, and even death (17). Protease-activated receptor 1 (PAR1) is the major mediator of APC cytoprotective activity. PAR1 has two cleavage sites for thrombin and APC. Cleavage at R41 by thrombin induces a proinflammatory response. In contrast, cleavage at R46 by APC excites biased PAR1 signaling and activation of cytoprotective pathways (18, 19). Oligonucleotide-based aptamers, such as APC-167 (20), HS02 (21, 22), and G-NB3 (23), have been developed to target APC non-active sites and inhibit anticoagulant activity without interfering with cytoprotective functions. A recombinant APC protein (drotrecogin alfa (activated)) was developed as a treatment for severe sepsis (24). However, its initial efficacy was not replicated in subsequent trials (25). As the mechanism of action was further understood, it was proposed that APC may provide benefit in septic settings with an optimized dosing regimen to maximize cytoprotective signaling (2). More recently, 3K3A-APC (K191A / K192A / K193A), which has less than 10% APC anticoagulant activity while retaining cytoprotective activity, has advanced into clinical trials for ischemic stroke (26, 27). These cytoprotective roles of APC are important, as complete blockade of the APC active site by antibodies results in many adverse effects, including death (8, 28, 29).

[0083] Heavy chain IgG (hcIgG) is a type of camelid antibody that consists of two heavy chains, each of which is a heavy chain (V H H) has one single variable domain and two constant domains, CH2 and CH3 (30, 31). H Antibodies with only H regions (also known as nanobodies) represent the smallest antibody format.

[0084] To selectively target distinct functions of APCs, we constructed a nanobody phage library using peripheral blood mononuclear cells (PBMCs) from llamas immunized with active site-blocked APCs (APC-PPACK). From this library, we identified a set of 21 llama nanobodies specific for APC non-active sites. These 21 nanobodies may selectively modulate the pleiotropic activities of APCs for therapeutic applications.

[0085] Materials and Methods: material Human APC, human APC-PPACK (Phe-Pro-Arg-chloromethylketone), human protein C, and factor Xa-PPACK were from Biosciences Technologies (Essex Junction, VT), oligo(dT) primers from Invitrogen (Waltham, MA), V5 tag monoclonal antibody and HRP-anti-M13 from ThermoFisher (Waltham, MA), Maxisorp ELISA plates from Nunc (Rochester, NY), MAB230P monoclonal antibody against the His tag from Maine Biotechnology (Portland, ME), naive llama from Capralogics (Hardwick, MA), Protac® from Pentapharm (Parsippany, NJ), STA-PTT Automate 5, STA Neoplastin CI Plus 5 from Stago (Parsippany, NJ), normal human plasma from George King Bio-Medical (Overland PC-depleted plasma was from Affinity Biologicals (Anacaster, ON, Canada), TPP-4885 Fab anti-APC in an IgG1 / κ framework (self-made), plasma-depleted human APC and human PC were from Enzyme Research Laboratories (South Bend, IN, USA), Spectrozyme PCa was from American Diagnostica (Pfungstadt, Germany), calf thymus histone H3 was from Sigma-Aldrich (St. Louis MO), Odyssey marker was from LI-COR (Lincoln, NE), and One-Step Blue Protein Stain was from Biotium (Fremont, CA).

[0086] Generation of llama nanobodies against the non-active site of human APC Two male llamas were immunized with APC-PPACK on days 1 and 77. Peripheral blood mononuclear cells (PBMCs) were isolated 5 days after the last immunization. Total RNA was extracted using the RNeasy kit (Qiagen). cDNA was synthesized from RNA using oligo(dT) primers and SuperScript III First-Strand Synthesis for RT-PCR (ThermoFisher). A nanobody phage display library was constructed by directly amplifying nanobodies using framework region 1 (FR1) and hinge region specific primers and cloning the amplicons into a vector with V5 and His tags. APC-PPACK binders were enriched by panning against APC-PPACK and negatively deselected with factor Xa-PPACK and PC (Figure 14).

[0087] Phage and bacterial periplasmic extract (PPE) ELISA screening Plates were coated with 2 μg / mL APC-PPACK, PC, or FXa-PPACK overnight at 4° C., blocked with 2% BSA for 1 h at room temperature (RT), and used to screen clones from panning rounds R2, R3, and R3' ( FIG. 14 ). Phages were blocked in 4% BSA for 1 h at room temperature and detected with HRP-anti-M13 mAb at 1:5000. Plates were developed with tetramethylbenzidine (TMB) and quenched with 2N H2SO4. At least 3-fold higher OD in antigen-coated wells compared to blocked wells. 450 Clones that exhibited the above results were selected for sequencing. PPEs generated from individual clones in panning round R4 were similarly screened by enzyme-linked immunosorbent assay (ELISA) using mouse anti-V5 monoclonal antibody (1:5000) as the primary antibody and goat HRP-anti-mouse IgG (1:5000) as the secondary antibody.

[0088] Expression and purification of nanobodies E. coli grown in 2YTGC supplemented with 0.1% glucose was transformed with a plasmid encoding the nanobody fused to a 6xHis tag. Nanobody expression was detected at an OD of approximately 0.5. 600 The cells were induced with 1 mM IPTG at 4 °C (25 °C). Nanobodies in the periplasm were purified with 1 mL of immobilized metal chelate affinity chromatography (IMAC) resin at 4 °C. The resin was washed three times with 1 mL of 10 mM imidazole / PBS and bound antibodies were eluted with 3 mL of 100 mM imidazole / PBS. The buffer was exchanged into PBS containing 1 mM CaCl2. Nanobody purity was characterized by SDS-PAGE (Figure 6). LP11 was also expressed in mammalian CHO cells and purified by IMAC as described above.

[0089] Epitope binning on nanobodies Binning experiments were performed on Octet® HTX at 25° C. Anti-V5 mouse antibody (5 μg / mL) was loaded onto an anti-mouse IgG Fc-captured Octet® HTX sensor. The loaded sensor was immersed in a nanobody solution (5 μg / mL) to capture the V5-tagged nanobody as a ligand. The sensor was immersed in 500 nM APC for 80 s, then dissociated for 30 s and immersed in 200 nM APC non-active site specific TPP-4885 Fab for 80 s. Binding of APC to the nanobody was detected by measuring the reflected light spectral shift on the sensor surface upon nanobody-APC interaction at 25° C. Binding of TPP-4885 Fab to this nanobody-APC complex on the sensor was monitored similarly.

[0090] To further understand the epitope diversity of nanobodies that do not compete with TPP-4885 Fab, a classical sandwich BLI epitope assay was performed with nanobodies that contained a V5 tag at 25 °C. Anti-mouse IgG Fc capture sensors loaded with anti-V5 antibodies were immersed in nanobody solution (10 μg / mL), followed by immersion in 500 nM APC for 80 s, dissociated for 20 s, and immersed in 200 nM nanobody analyte for 80 s. Normalized binned data were analyzed using the pvclust package (49). The y-axis of the clustergram, referred to as height, is a measure of dissimilarity between nanobodies. Approximately unbiased (AU) p-values ​​were calculated using multiscale bootstrap resampling to confirm the bin assignments.

[0091] Binding dynamics analysis by biolayer interferometry Anti-V5 mouse antibody (10 μg / mL) was loaded onto the Octet® HTX instrument (Sartorius, Fremont, CA) sensor surface with anti-mouse IgG. The loaded sensor was immersed in nanobody solution (5 μg / mL for the first analysis, 10 μg / mL for the second analysis) to immobilize the V5-tagged nanobody. The nanobody-immobilized sensor was immersed in APC dilutions (78.12-5000 nM in PBS, 1 mM CaCl2, 0.1% w / v Tween20, with or without 0.5 mg / mL BSA) for 60 s. After APC binding, the surface was immersed in assay buffer for 120 s and allowed to dissociate. Biolayer Interferometry (BLI) measurements were collected during the association and dissociation phases (25 °C). The surface was regenerated between samples using 10 mM glycine buffer, pH 1.7. The kinetic constant (k on , k off , and K D ) was calculated using a monovalent (1:1) binding model. Each nanobody was tested against five different concentrations of APC.

[0092] Effect of nanobodies on APC anticoagulant activity APC was generated in normal plasma using the PC-activated snake venom-derived reagent Protac® and the effect of nanobodies on the anticoagulant activity of APC was tested by measuring the activated partial thromboplastin time (APTT) clotting time. Normal plasma samples (50 μL) with anti-APC nanobodies were mixed with Protac® (25 μL; 1 U / mL) and Stago reagent STA-PTT (75 μL) at 37° C. After 4 min of incubation, clotting was initiated by adding 75 μL of 25 mM CaCl2 solution. Protac®-APTT clotting times of normal human plasma serially diluted with PC-depleted plasma were used as a standard curve. For FVIII inhibitor plasma, 0.5 μg / ml (16 BU / ml) of the FVIII inhibitor antibody GMA-8015 (Green Mountain) was added to normal plasma and incubated for 2 h before the Protac®-APTT assay.

[0093] Effect of nanobodies on APC-mediated histone H3 cleavage APC (50 nM) and anti-APC nanobody (500 nM) were pre-incubated in HBS buffer (20 mM Hepes, 147 mM NaCl, 3 mM KCl, pH 7.4) with 100 μg / mL BSA and 2 mM CaCl2. After 30 min, the mixture was added to 100 μg / mL histone H3. Over a period of 2 h, the mixture was subsampled at different time points and quenched with reducing sample buffer. Samples were heated for 15 min at 95°C, centrifuged for 5 min at 4000 rpm, vortexed briefly, and loaded onto a 12% Bis-Tris gel (Bio-Rad) containing MES running buffer (30 μl / well, 750 ng H3). The 20 kDa marker in Odyssey standard (10 μL) was used as a reference for normalization between gels. Gels were stained overnight with Biotum One-Step Blue Protein Stain (50 mL / gel). Protein bands were scanned with a Licor Odyssey (700 channel, intensity 6, 1 mm offset, resolution 169 μm) and analyzed with Image Studio V5.2.

[0094] Effect of nanobodies on APC-mediated SEAP-PAR1 cleavage HEK293 cells expressing wild-type EPCR and a PAR1 cleavage reporter construct with N-terminal secreted embryonic alkaline phosphatase (SEAP-PAR1) were grown to confluence in 96-well plates (18, 38). Cells were washed with Hanks' balanced salt solution supplemented with 1.3 mM CaCl2, 0.6 mM MgCl2, and 0.1% BSA (endotoxin-free HMM2). APC (50 or 100 nM) and nanobodies were preincubated in HMM2 for 30 min before being added to the cells. After 60 min, SEAP release was determined using p-nitrophenyl phosphate. PAR1 cleavage was expressed as a percentage of the total SEAP activity present on the cells relative to background. The R41Q-SEAP-PAR1 and R46Q-SEAP-PAR1 HEK293 cell lines were used to evaluate the effect of nanobodies on the selectivity of APC for cleavage at R46 versus R41 (18).

[0095] Endothelial barrier integrity assay The effect of nanobodies on APC-mediated endothelial barrier integrity was measured using a transendothelial electrical resistance (TEER) assay (iCelligence system, ACEA, San Diego, CA) as previously described ( 18 , 39 ). EA.hy926 endothelial cells were cultured at 2.5 × 10 cells / well. 4 Cells / 500 μL were grown to confluence in 8-well E-plates L8 (Agilent Technologies, Santa Clara, CA). DMEM (Invitrogen) containing 10% fetal bovine serum was replaced with serum-free medium containing 0.1% BSA 2 h before addition of APC (40 nM) and LP11. Thrombin (0.25 nM) was added 30 min later to induce endothelial permeability. Changes in electrical resistance of confluent monolayers were measured in real time as a normalized cell index.

[0096] In vitro plasma half-life assay The effect of nanobodies on the sensitivity of APC to physiological inhibitors was assessed using an in vitro plasma half-life assay. In this assay, APC (70 nM) and antibodies (700 nM) are pre-incubated for 30 min and normal pooled plasma is added to 90% (v / v). Samples are collected at various time points (0-90 min) and quenched with ice-cold TBS. The chromogenic activity of APC is determined using Pefachrome® PCa. The background chromogenic activity of plasma (without APC added) is subtracted from the measured signal and APC activity is normalized to the time point t=0 to account for the effect of antibodies on the chromogenic activity of APC. The half-life (T 1 / 2 ) is determined by monophasic exponential decay curve fitting.

[0097] result: Generation of nanobodies specific for non-active sites of APC APC-PPACK was used as immunogen to generate nanobodies against non-active sites of llama APC. After immunization with two doses of APC-PPACK, PBMCs were collected and nanobody binding regions were cloned to create a nanobody library. The library was panned against APC-PPACK and PC to select clones specific for APC but not PC. After panning round R1, panning was continued with two arms with different stringency of negative selection for PC (Figure 14). Both the first arm (R1, R2, R3, R4) and the second arm (R1, R2', R3') led to enrichment of APC-specific clones. Of a total of 1128 phage clones selected from R2, R3, R4 and R3' (Figure 7), 725 clones were identified as selective APC-PPACK binders lacking non-specific binding to PC or FXa-PPACK (Figure 8). After sequencing, 158 clones were found to be unique. Based on their CDR1, CDR2, and CDR3 sequences, these 158 clones were grouped into 18 families from which 21 nanobodies with the highest binding within each family (designated LP1–LP21) were selected. Additional nonspecific binding studies against FVIIa, FIXa, and FXIa were performed to further demonstrate the selectivity of the LP1–LP21 nanobodies towards APCs (Figure 9).

[0098] Binding kinetics and epitope binning To determine whether any of the nanobodies bind to the same epitope as the previously reported non-active site-specific anti-APC Fab antibody TPP-4885 (8), a BLI-based epitope binning study was performed. The nanobodies were tested pairwise with the TPP-4885 Fab. Thirteen clones blocked TPP-4885 Fab binding and were binned into cluster 1 (Figure 1A). Nanobodies that did not block TPP-4885 Fab binding to APC were selected for further binning experiments by BLI, except for LP11, which has >90% CDR sequence identity with LP9 (Table 4). For this reason, LP9 was used as a surrogate for LP11. Based on response measurements by BLI, LP7, LP9, LP12, and LP18 did not compete with LP2, LP3, and LP10 (Figure 1B). LP7 had a weak response measurement as a ligand, but appeared to bind well as an analyte. This is due to the k off This may be due to the faster cloning. Cluster analysis showed that the clones could be binned into clusters 2 and 3 (Figure 1C). Most of the nanobodies (62%) are in cluster 1. The low number of nanobodies in clusters 2 (24%) and 3 (14%) means that these epitope bins were less accessible and / or more complex for immune recognition and antibody development. These epitope bins also seem to be targetable only by nanobodies with longer CDR3s. Unlike cluster 1 nanobodies, which had a median CDR3 length of 9.5 amino acids, the CDR3s of clusters 2 and 3 nanobodies had a median length of 13.5 and 17 amino acids, respectively (Figure 1D, Table 4). These nanobodies showed a k on The value is 1.26 x 10 5 ~6.22×10 5 1 / millisecond range, k off The value is 2.59 x 10 -4 ~1.37×10 -3 The k range was 1 / sec. off The value is particularly low, about 6.33 × 10 -7 The K Dvalues ​​are subnanomolar, and 15 nanobodies have K D values ​​are in the single digit nanomolar range, and four nanobodies have K D values ​​ranged from 16.5 to 27.5 nM (Figure 1E, Table 5). [Table 4] [Table 5]

[0099] Effect of S-2366 on APC-mediated cleavage Eight nanobodies had no effect on APC-mediated cleavage of the chromogenic substrate S-2366 (Figure 10). Five nanobodies induced partial inhibition and eight nanobodies showed enhancement of S-2366 cleavage. Of the inhibitory nanobodies, two were non-competitive inhibitors and three were competitive inhibitors of S-2366 cleavage (Figure 11, Table 6). Of the eight S-2366 cleavage enhancing nanobodies, four were V max The other four enhance cleavage by increasing K M The lack of complete inhibition of S-2366 cleavage indicated that these 21 anti-APC nanobodies target non-active sites. [Table 6]

[0100] Effect on clotting time in plasma The effect of nanobodies on APC anticoagulant activity was screened using a Protac® modified APTT assay. APC was generated by adding Protac®, a single-chain glycoprotein derived from snake venom that activates protein C, to plasma. Protac® extended the APTT clotting time from an average of 40.7 to 262.0 seconds depending on the concentration of PC in plasma (Figure 2A). Nanobodies were screened twice at 300 nM and showed consistent potency rankings between screens (Figure 2B). All nanobodies at least partially reversed the prolonged clotting time. LP2, LP8, LP9, LP11, LP17, and LP20 inhibited APC-induced clotting by ≧90% and reversed the prolonged clotting time to less than 80 seconds. LP20 was the most potent inhibitor with an almost completely corrected clotting time (Figure 2C). These data indicate that there are at least three distinct epitopes on APC (clusters 1, 2, and 3) to which nanobodies can bind in order to inhibit the anticoagulant activity of this protein.

[0101] Effect on APC-mediated histone H3 cleavage APC inhibits histone-induced cytotoxicity via proteolytic cleavage (16). In this assay, APC was incubated with histone H3 in the presence of nanobodies and cleavage of histone H3 was monitored by SDS-PAGE (Figure 3A). Nine nanobodies minimally affected APC-mediated histone H3 cleavage (Figure 3B), one (LP14) enhanced H3 cleavage (Figure 3C), and 12 nanobodies inhibited APC-mediated H3 cleavage with less than 75% residual activity (Figure 3D), with LP21 being the most potent (Figure 3E).

[0102] Effect on APC-mediated SEAP-PAR1 cleavage Nanobodies inhibiting PAR1 cleavage by APC were found in all three clusters, with LP1, LP8, LP17, LP19, and LP21 showing the greatest inhibition. In contrast, nanobodies LP3, LP9, LP11, and LP18 showed minimal inhibition even at the highest concentration of 500 nM (Figure 4A). In the lower concentration range, 0.7-6 nM, these four nanobodies reduced PAR1 cleavage to 73-82% of the control. These nanobodies belonged to cluster 2 (LP9, LP11, LP18) and cluster 3 (LP3), respectively. All nanobodies in cluster 1 inhibited PAR1 cleavage. APC can cleave PAR1 at both R41 and R46, the latter cleavage resulting in cytoprotective signaling. Therefore, two PAR1 cleavage inhibitory nanobodies and two PAR-1 cleavage avoidance nanobodies were evaluated for their effect on APC cleavage patterns using the R41Q-SEAP-PAR1 and R46Q-SEAP-PAR1 cell lines (Figure 4B). In the presence of inhibitory nanobodies LP8 and LP20, cleavage at both sites by APC (at 50 nM) was inhibited in a dose-dependent manner. At higher concentrations of LP8 and LP20, the cleavage ratio shifted to less than 1 and the cytoprotective PAR1 cleavage profile was less (Figure 4C). In comparison, LP11 and LP18 appeared to induce a curvilinear cleavage response for both R46 and R41. At 0.7-6 nM, cleavage of R41 was inhibited to a greater extent than R46. At 6 nM and above, inhibition of cleavage was reversed. Interestingly, the R46 / R41 cleavage ratio was greater than 1 across the entire concentration range of the assay, suggesting enhanced cytoprotection by LP11 and LP18 (FIG. 4D).

[0103] Effect on plasma half-life of APC An in vitro plasma half-life assay was used to determine the effect of 21 nanobodies on the sensitivity of APCs to physiological inhibitors. This assay identified V that increases, decreases, and has no effect on the plasma half-life of APCs. HH antibodies were identified (Table 7). The increased half-life indicates that binding of these antibodies reduces the susceptibility of APC to inhibition by physiological inhibitors present in plasma.

[0104] [Table 7]

[0105] Activity Profile The 21 nanobodies were classified into 15 different activity profiles based on their clusters and effects in the three APC functional assays (Table 8). Three nanobodies (LP2, LP8, LP17) potently inhibited all three APC functional activities. Some nanobodies were found to inhibit only two of the three functions. LP16 and LP20 inhibit anticoagulation and PAR1 cleavage, while LP1 and LP5 inhibit H3 and PAR1 cleavage. These nanobodies exploit only one of the three APC functions and may be useful to dissect the pleiotropic effects of APC, especially in the in vivo setting. No nanobodies were identified that strongly inhibited only PAR1 cleavage or histone H3 cleavage. Potent monofunctional inhibition of APC anticoagulant activity was observed only for LP9 and LP11. [Table 8] TIFF2025509184000013.tif233111

[0106] Effect of LP11 on endothelial cell barrier function Nanobody LP11 is the best candidate for treating bleeding. LP11 inhibited APC in a dose-dependent manner, prolonged clotting times in the Protac®-APTT clotting assay of FVIII inhibitor plasma (Figure 12), inhibited anticoagulation and enhanced the PAR1 R46 / R41 cleavage ratio. Thus, LP11 supports prohemostatic activity and at the same time enhances APC-mediated cytoprotective effects.

[0107] To test the effect of LP11 on endothelial barrier function in vitro, LP11 was expressed in mammalian cells and purified. Characterization of CHO cell-derived LP11 showed that its properties were indistinguishable from E. coli-derived LP11 (i.e., inhibiting the anticoagulant activity of APC and significantly inhibiting R41 cleavage while only minimally affecting APC-mediated PAR1 cleavage at R46 (Figure 13)). A classical endothelial barrier function assay was performed using EA.hy926 cells to analyze APC-induced barrier protection against thrombin-induced permeability (18, 39). This assay showed that LP11 only minimally affected the barrier protection function of APC (Figure 5A-B), which is consistent with its minimal effect on APC-mediated PAR1 cleavage at R46. To evaluate the effect of LP11 on priming of endothelial cells by APC through PAR1 cleavage, the integrity of the endothelial cell layer was evaluated before barrier disruption was induced by thrombin. APC priming was observed to induce a dose-dependent transient barrier disruption (Fig. 5C-D), which could be inhibited by the PAR1 antagonists vorapaxal and SCH-79797 (Fig. 5E), consistent with the notion that APC priming encompasses the effects of APC-mediated cleavage of PAR1 at R41. LP11 inhibited the transient barrier disruption induced by APC priming in a dose-dependent manner, consistent with the inhibition of APC-mediated PAR1 cleavage at R41 (Fig. 5F-G). Most importantly, analysis of the overall barrier function profile showed that the normalized cell index of APC in the presence of LP11 reached higher values ​​compared to APC in the absence of LP11 (Fig. 5H), suggesting that LP11 results in a net enhancement of the barrier protective effect of APC. Notably, LP11 alone did not alter the electrical resistance of the endothelial layer. Thus, the data indicate that preferential inhibition of APC-mediated R41 cleavage relative to R46 cleavage during APC priming exerts a cytoprotective effect on endothelial cells.

[0108] Consideration: The APC pathway, which includes thrombin / thrombomodulin, PC, protein S, EPCR, and PAR, mediates multiple physiological functions including anticoagulant, anti-inflammatory, and cytoprotective functions. Targeting these proteins may provide clinical benefit for the myriad pathologies involved in this pathway. Currently, two drugs that target different components of this pathway, KRK-α1-antitrypsin and 3K3A-APC, are in clinical development for hemophilia and ischemic stroke, respectively (40). However, many of the possibilities of using APC and its substrates as therapeutic targets await further exploration. Studies based on single-molecule FRET and low-angle X-ray scattering have demonstrated that there are limited changes in the overall composition of PC upon activation, and that these changes are primarily conformational changes within the active site region (41, 42). Thus, identifying antibodies specific for the non-active site of APC and that do not bind to PC presents a challenge. This study addresses this challenge through the construction of a llama antibody library in which 21 nanobodies belonging to 18 families were selected and further characterized for their binding diversity. Epitope binding studies found only three clusters of nanobodies specific for three epitope bins. This low number of epitope bins indicates that only limited conformational differences exist between the non-active site region of APC and its zymogen PC, which is in agreement with previous reports (41, 42).

[0109] Based on APC functional studies, all three epitope bins appear to play multiple roles simultaneously in anticoagulation, H3 cleavage, and PAR1 cleavage, as none of the bins showed exclusive loss of only one role upon blocking. Nanobodies in cluster 1 competed with the previously reported antibody TPP-4885, which targets the APC autolysis loop and potentially also amino acids L429-N431 (8). Thus, nanobodies in cluster 1 are expected to bind to a similar region. The autolysis loop is a positively charged loop that plays a role in FVa cleavage (43), FVIIIa cleavage (44), and APC sensitivity to serpin inhibition (45, 46). Within the autolysis loop, residues R306, K308, K311, R312, and R314 are important for FVa cleavage, while R306, K311, and R314 are important for FVIIIa cleavage. Since all cluster 1 nanobodies reverse the prolonged Protac®-APTT clotting time to some extent, they all likely bind to these critical arginine and lysine residues on the autolysis loop. The observation that some cluster 1 nanobodies inhibit cleavage of H3 and / or PAR1 could be the result of a previously unreported interaction between the autolysis loop with H3 and PAR1. Alternatively, these nanobodies may bind to a region that is proximal, but not within the autolysis loop, that is important for the interaction of H3 and PAR1.

[0110] Clusters 2 and 3 contain nanobodies that did not compete with TPP-4885 Fab binding. Among them, LP9 and LP11 inhibited the anticoagulant activity of APC without interfering with the cleavage of H3 and PAR1. The activity profile of APC bound to LP9 or LP11 is similar to that of APC mutants designed to have minimal anticoagulant activity, such as 3K3A-APC and 5A-APC. 3K3A-APC (K191A / K192A / K193A) (47) has amino acid substitutions in loop 37, and 5A-APC (K191A / K192A / K193A / R229A / R230A) has substitutions in both loop 37 and the calcium-binding loop (48). The cytoprotective function of APC is based on its biased cleavage of PAR1 at R46. R46 / R41 cleavage ratio studies on the SEAP-PAR1 cell line indicate that LP11 increases the R46 / R41 cleavage ratio by up to 2.5-fold, resulting in an overall improvement in endothelial cell barrier function. Preclinical hemophilia and blood coagulation disorder studies have shown that the prohemostatic effects of APC inhibition without interfering with the cytoprotective function of APC should provide improved outcomes in hemophilic arthropathy (9) and survival outcomes in traumatic blood coagulation disorders (28). Thus, the novel properties of LP11 may further improve outcomes in these conditions.

[0111] In summary, the llama nanobody library yielded 21 nanobodies capable of selectively modulating APC anticoagulant and cytoprotective pathways, which are useful as novel therapeutic agents for the treatment of a number of APC-related indications, including acute hemorrhage, hemophilia, ischemia, and sepsis.

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Claims

1. 1. An isolated antibody comprising a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 317-474, wherein the antibody specifically binds to activated protein C (APC) and minimally binds to non-activated protein C (PC).

2. The antibody of claim 1, further comprising: (a) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-158; (b) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 159-316; or (c) both a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-158 and a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 159-316.

3. The antibody has a heavy chain variable region (V Ｈ 3. The antibody of claim 2, comprising CDR1, CDR2, and CDR3 of:

4. The antibody, 1) CDR1 of SEQ ID NO:45, CDR2 of SEQ ID NO:203, and CDR3 of SEQ ID NO:361; 2) CDR1 of SEQ ID NO:54, CDR2 of SEQ ID NO:212, and CDR3 of SEQ ID NO:370; 3) CDR1 of SEQ ID NO:61, CDR2 of SEQ ID NO:219, and CDR3 of SEQ ID NO:377; 4) CDR1 of SEQ ID NO:74, CDR2 of SEQ ID NO:232, and CDR3 of SEQ ID NO:390; 5) CDR1 of SEQ ID NO:82, CDR2 of SEQ ID NO:240, and CDR3 of SEQ ID NO:398; 6) CDR1 of SEQ ID NO:84, CDR2 of SEQ ID NO:242, and CDR3 of SEQ ID NO:400; 7) CDR1 of SEQ ID NO:88, CDR2 of SEQ ID NO:246, and CDR3 of SEQ ID NO:404; 8) CDR1 of SEQ ID NO:90, CDR2 of SEQ ID NO:248, and CDR3 of SEQ ID NO:406; 9) CDR1 of SEQ ID NO:95, CDR2 of SEQ ID NO:253, and CDR3 of SEQ ID NO:411; 10) CDR1 of SEQ ID NO: 101, CDR2 of SEQ ID NO: 259, and CDR3 of SEQ ID NO: 417; 11) CDR1 of SEQ ID NO: 103, CDR2 of SEQ ID NO: 261, and CDR3 of SEQ ID NO: 419; 12) CDR1 of SEQ ID NO: 105, CDR2 of SEQ ID NO: 263, and CDR3 of SEQ ID NO: 421; 13) CDR1 of SEQ ID NO: 109, CDR2 of SEQ ID NO: 267, and CDR3 of SEQ ID NO: 425; 14) CDR1 of SEQ ID NO: 116, CDR2 of SEQ ID NO: 274, and CDR3 of SEQ ID NO: 432; 15) CDR1 of SEQ ID NO: 142, CDR2 of SEQ ID NO: 300, and CDR3 of SEQ ID NO: 458; 16) CDR1 of SEQ ID NO: 144, CDR2 of SEQ ID NO: 302, and CDR3 of SEQ ID NO: 460; 17) CDR1 of SEQ ID NO: 146, CDR2 of SEQ ID NO: 304, and CDR3 of SEQ ID NO: 462; 18) CDR1 of SEQ ID NO: 147, CDR2 of SEQ ID NO: 305, and CDR3 of SEQ ID NO: 463; 19) CDR1 of SEQ ID NO: 149, CDR2 of SEQ ID NO: 307, and CDR3 of SEQ ID NO: 465; 20) CDR1 of SEQ ID NO: 150, CDR2 of SEQ ID NO: 308, and CDR3 of SEQ ID NO: 466; or 21) The antibody of claim 3, comprising CDR1 of SEQ ID NO: 157, CDR2 of SEQ ID NO: 315, and CDR3 of SEQ ID NO:

473.

5. V comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 475-632 Ｈ 1. An isolated antibody comprising:

6. The antibody of any one of claims 1 to 5, wherein the antibody binds to an exosite of an APC.

7. The antibody according to any one of claims 1 to 6, wherein the antibody is an antibody selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, an IgM antibody, an IgA1 antibody, an IgA2 antibody, a secretory IgA antibody, an IgD antibody, an IgE antibody, and an antigen-binding fragment thereof.

8. The antibody of any one of claims 1 to 6, wherein the antibody is a nanobody.

9. The antibody is V Ｈ The antibody of claim 8 which is an H antibody.

10. The antibody, a) enhance, minimally affect, or inhibit the anticoagulant activity of APC; b) enhances, minimally affects, or inhibits APC-mediated histone cleavage; c) enhances, minimally affects, or inhibits APC-mediated cleavage of protease-activated receptor 1 (PAR1) at residue R46; d) enhances, minimally affects, or inhibits APC-mediated PAR1 cleavage at residue R41; e) increasing, minimally affecting, or decreasing the ratio of APC-mediated PAR1 cleavage at residue R46 to APC-mediated PAR1 cleavage at residue R41; f) enhance, minimally affect, or inhibit the endothelial barrier protective activity of APCs; g) increasing, having minimal effect on, or decreasing the plasma half-life of APC; or h) The antibody according to any one of claims 1 to 9, in any combination thereof.

11. 13. An isolated antibody that specifically binds to APC, binds minimally to PC, and competes with the antibody of claim 1 for binding to APC.

12. A nucleic acid encoding the antibody of any one of claims 1 to 11.

13. A vector comprising the nucleic acid of claim 12.

14. A host cell comprising the nucleic acid of claim 12 or the vector of claim 13.

15. 1. A method for producing an antibody, comprising: a) culturing the host cell of claim 14 under conditions that result in the production of said antibody; b) isolating said antibody from said host cell.

16. A pharmaceutical composition comprising the antibody of any one of claims 1 to 11 and a pharma- ceutically acceptable carrier.

17. A method for treating or preventing a condition in a subject, comprising administering to the subject a therapeutically effective amount of an antibody according to any one of claims 1 to 11, an antibody according to any one of claims 1 to 11 specifically binding to an exogenous APC protein or a variant thereof, or a pharmaceutical composition according to claim 16.

18. 18. The method of claim 17, wherein the condition is a condition that can be treated or prevented by enhancing or inhibiting the anticoagulant function of APC.

19. 18. The method of claim 17, wherein the condition is selected from the group consisting of hemorrhage, bruising, burns, gastrointestinal bleeding, uncontrollable bleeding, bleeding due to transplantation or resection procedures, bleeding due to surgery, bleeding due to traumatic injury, variceal bleeding, bleeding in cirrhosis of the liver, thrombocytopenia, idiopathic thrombocytopenic purpura, hemophilia, aortic aneurysm, anticoagulant or antithrombotic drug overdose, menorrhagia, clotting factor deficiency, Glanzmann thrombasthenia, and Bernard-Soulier syndrome.

20. 18. The method of claim 17, wherein the condition is a condition that can be treated or prevented by enhancing or inhibiting one or more cytoprotective functions of APCs.

21. The state is 21. The method of claim 20, wherein the disease is selected from the group consisting of sepsis, inflammation in acute ischemic disease, coronavirus disease 2019 (COVID-19), diabetes, diabetic nephropathy, diabetic ulcers, wounds, amyotrophic lateral sclerosis (ALS), multiple sclerosis, central nervous system injury, ischemic stroke, Alzheimer's disease, acute kidney injury, lung injury, acute pancreatitis, cancer, inflammatory disease, and autoimmune disease.

22. An antibody according to any one of claims 1 to 11 for use as a medicament for treating or preventing a condition.

23. The antibody of claim 22, wherein the condition is a condition that can be treated or prevented by enhancing or inhibiting the anticoagulant function of APC.

24. 24. The antibody of claim 23, wherein the condition is selected from the group consisting of hemorrhage, bruising, burns, gastrointestinal bleeding, uncontrollable bleeding, bleeding due to transplantation or ablation procedures, bleeding due to surgery, bleeding due to traumatic injury, variceal bleeding, bleeding in cirrhosis of the liver, thrombocytopenia, idiopathic thrombocytopenic purpura, hemophilia, aortic aneurysm, anticoagulant or antithrombotic drug overdose, menorrhagia, clotting factor deficiency, Glanzmann thrombasthenia, and Bernard-Soulier syndrome.

25. The antibody of claim 22, wherein the condition is a condition that can be treated or prevented by enhancing or inhibiting one or more cytoprotective functions of APCs.

26. The state is 26. The antibody of claim 25, wherein the antibody is selected from the group consisting of sepsis, inflammation in acute ischemic disease, COVID-19, diabetes, diabetic nephropathy, diabetic ulcers, wounds, ALS, multiple sclerosis, central nervous system injury, ischemic stroke, Alzheimer's disease, acute kidney injury, lung injury, acute pancreatitis, cancer, inflammatory diseases, and autoimmune diseases.

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