Human antibodies against activated protein C and uses thereof

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 / 316,928, 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 "172085.00118_ST26.xml", with a size of 412,954 bytes and a creation date of February 28, 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. [Background technology]

[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. This 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 treatments 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 caused the death of 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 present 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 heavy chain variable region (V) comprising a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 118. H In some embodiments, the antibody comprises a V selected from the group consisting of SEQ ID NOs: 61-80 and 473-496. H In some embodiments, the antibody comprises a light chain variable region (V) comprising a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 and 138-140. L In some embodiments, the antibody comprises a V selected from the group consisting of SEQ ID NOs: 24-35 and 141-151. L In some embodiments, the antibody comprises a V H and V L In some embodiments, the antibody comprises a heavy chain and a light chain that are both derived from a single antibody as described herein.

[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 that result 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 enhancing 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 1] 1 is a table presenting the results of screening assays used to evaluate 20 Set 1 antibodies. These results include protein expression levels in transiently transfected HEK293 cells, amidolytic activity (SPECTROZYME® PCa), binding to PC (i.e., KD)-based surface plasmon resonance (SPR), and UNcle-based aggregation temperature (Tagg). Results are compared to those generated with reformatted parent antibody TPP-24727 (highlighted in bold). [Diagram 2] 1 is a table presenting binding kinetics and affinity data for Set 1 antibodies. These data were generated by performing SPR binding assays using human APC and PC as analytes and fitting the resulting sensorgrams to a 1:1 Langmuir binding model. Results are compared to those generated with the parent antibody TPP-24727 (highlighted in bold). [Diagram 3]1 is a table presenting expression, purity, and stability data for four of the Set 1 antibodies. IgG concentrations were quantified via sandwich ELISA. Melting temperatures (Tm) were determined via differential scanning calorimetry using a MicroCal VP-Capillary DSC instrument according to the manufacturer's protocol. [Figure 4] 1 is a table presenting human APC amidolytic activity data in the presence of 8 nM and 3.2 nM of Set 1 antibodies. Results are compared to those generated with the parent antibody TPP-24727 (highlighted in bold). [Figure 5-1] A set of three plots and tables presenting the results of FXa and thrombin generation assays used to measure the ability of Set 1 antibodies to inhibit FVa inactivation of APC. In this assay, FVa was incubated with each antibody at concentrations ranging from 0.5 to 1000 nM. After 30 minutes, a prothrombinase mixture containing FXa, prothrombin, and a thrombin chromogenic substrate (S-2238) was added and substrate hydrolysis was monitored using a plate reader. [Figure 5-2] A set of three plots and tables presenting the results of FXa and thrombin generation assays used to measure the ability of Set 1 antibodies to inhibit FVa inactivation of APC. In this assay, FVa was incubated with each antibody at concentrations ranging from 0.5 to 1000 nM. After 30 minutes, a prothrombinase mixture containing FXa, prothrombin, and a thrombin chromogenic substrate (S-2238) was added and substrate hydrolysis was monitored using a plate reader. [Figure 5-3] A set of three plots and tables presenting the results of FXa and thrombin generation assays used to measure the ability of Set 1 antibodies to inhibit FVa inactivation of APC. In this assay, FVa was incubated with each antibody at concentrations ranging from 0.5 to 1000 nM. After 30 minutes, a prothrombinase mixture containing FXa, prothrombin, and a thrombin chromogenic substrate (S-2238) was added and substrate hydrolysis was monitored using a plate reader. [Figure 6A] 6A and 6B show the results of a Protac® Activated Partial Thromboplastin Time (APTT) assay used to test the effect of Set 1 antibodies on the clotting time of normal human plasma. The assay was performed on plasma samples containing different concentrations of antibody (i.e., 10 nM or 30 nM) in two independent screens (i.e., screen 1 and screen 2). The results are presented as plots of clotting time at 30 nM versus clotting time at 10 nM in Figures 6A and 6B and summarized in the table in Figure 6C. The results are also presented as plots of rank order of screen 2 versus rank order of screen 1 in Figures 6D and 6E. [Figure 6B] 6A and 6B show the results of a Protac® Activated Partial Thromboplastin Time (APTT) assay used to test the effect of Set 1 antibodies on the clotting time of normal human plasma. The assay was performed on plasma samples containing different concentrations of antibody (i.e., 10 nM or 30 nM) in two independent screens (i.e., screen 1 and screen 2). The results are presented as plots of clotting time at 30 nM versus clotting time at 10 nM in Figures 6A and 6B and summarized in the table in Figure 6C. The results are also presented as plots of rank order of screen 2 versus rank order of screen 1 in Figures 6D and 6E. [Figure 6C] 6A and 6B show the results of a Protac® Activated Partial Thromboplastin Time (APTT) assay used to test the effect of Set 1 antibodies on the clotting time of normal human plasma. The assay was performed on plasma samples containing different concentrations of antibody (i.e., 10 nM or 30 nM) in two independent screens (i.e., screen 1 and screen 2). The results are presented as plots of clotting time at 30 nM versus clotting time at 10 nM in Figures 6A and 6B and summarized in the table in Figure 6C. The results are also presented as plots of rank order of screen 2 versus rank order of screen 1 in Figures 6D and 6E. [Figure 6D]6A and 6B show the results of a Protac® Activated Partial Thromboplastin Time (APTT) assay used to test the effect of Set 1 antibodies on the clotting time of normal human plasma. The assay was performed on plasma samples containing different concentrations of antibody (i.e., 10 nM or 30 nM) in two independent screens (i.e., screen 1 and screen 2). The results are presented as plots of clotting time at 30 nM versus clotting time at 10 nM in Figures 6A and 6B and summarized in the table in Figure 6C. The results are also presented as plots of rank order of screen 2 versus rank order of screen 1 in Figures 6D and 6E. [Figure 6E] 6A and 6B show the results of a Protac® Activated Partial Thromboplastin Time (APTT) assay used to test the effect of Set 1 antibodies on the clotting time of normal human plasma. The assay was performed on plasma samples containing different concentrations of antibody (i.e., 10 nM or 30 nM) in two independent screens (i.e., screen 1 and screen 2). The results are presented as plots of clotting time at 30 nM versus clotting time at 10 nM in Figures 6A and 6B and summarized in the table in Figure 6C. The results are also presented as plots of rank order of screen 2 versus rank order of screen 1 in Figures 6D and 6E. [Figure 7] 1 is a table showing secreted embryonic alkaline phosphatase (SEAP)-protease-activated receptor 1 (PAR1) cleavage data for human APCs in the presence of Set 1 antibodies. For this assay, SEAP-PAR1 was expressed on HEK293 cells with wild-type endothelial protein C receptor (wt-EPCR). APCs were pre-incubated with the antibodies for 30 minutes before being added to confluent SEAP-PAR1 / wt-EPCR cells. After 60 minutes, SEAP release was measured using a one-step p-nitrophenyl phosphate. [Figure 8A]Figure 8 shows the results of an endothelial barrier integrity assay on human APC in the presence of Set 1 antibodies. Results are compared to those generated with the parent antibody TPP-24727. Endothelial cell permeability was measured in real time using the iCELLigence™ system. Confluent EA.hy926 endothelial cells were exposed to APC (50 nM) in the presence and absence of antibodies. Permeability of confluent monolayers after treatment with APC for 3 hours was determined upon addition of thrombin (2 nM). Results are presented as a plot of percent barrier protection versus antibody concentration in Figure 8A and summarized in the table in Figure 8B. [Figure 8B] Figure 8 shows the results of an endothelial barrier integrity assay on human APC in the presence of Set 1 antibodies. Results are compared to those generated with the parent antibody TPP-24727. Endothelial cell permeability was measured in real time using the iCELLigence™ system. Confluent EA.hy926 endothelial cells were exposed to APC (50 nM) in the presence and absence of antibodies. Permeability of confluent monolayers after treatment with APC for 3 hours was determined upon addition of thrombin (2 nM). Results are presented as a plot of percent barrier protection versus antibody concentration in Figure 8A and summarized in the table in Figure 8B. [Figure 9A] Figure 9 shows the results of a histone H3 cleavage assay on human APC in the presence of Set 1 antibodies. A. APC (50 nM) and nanobody (500 nM) were pre-incubated together for 30 min. After incubation, the mixture was added to 100 μg / ml histone H3. Over a 2 hour period, the mixture was subsampled at different time points to monitor the digestion of histone H3 over time. Figure 9A shows results generated with the parent antibody TPP-24727. Figure 9B shows results generated with each Set 1 antibody. Figure 9C shows the results of a modified assay measuring histone H3 digestion in the presence of different antibody to APC ratios after 1 hour of incubation. [Figure 9B]Figure 9 shows the results of a histone H3 cleavage assay on human APC in the presence of Set 1 antibodies. A. APC (50 nM) and nanobody (500 nM) were pre-incubated together for 30 min. After incubation, the mixture was added to 100 μg / ml histone H3. Over a 2 hour period, the mixture was subsampled at different time points to monitor the digestion of histone H3 over time. Figure 9A shows results generated with the parent antibody TPP-24727. Figure 9B shows results generated with each Set 1 antibody. Figure 9C shows the results of a modified assay measuring histone H3 digestion in the presence of different antibody to APC ratios after 1 hour of incubation. [Figure 9C] Figure 9 shows the results of a histone H3 cleavage assay on human APC in the presence of Set 1 antibodies. A. APC (50 nM) and nanobody (500 nM) were pre-incubated together for 30 min. After incubation, the mixture was added to 100 μg / ml histone H3. Over a 2 hour period, the mixture was subsampled at different time points to monitor the digestion of histone H3 over time. Figure 9A shows results generated with the parent antibody TPP-24727. Figure 9B shows results generated with each Set 1 antibody. Figure 9C shows the results of a modified assay measuring histone H3 digestion in the presence of different antibody to APC ratios after 1 hour of incubation. [Figure 10] 1 is a table showing the results of an in vitro plasma half-life assay of human APC in the presence of Set 1 antibodies. In this assay, APC and antibodies are pre-incubated for 30 minutes and normal pooled plasma is added. Samples are taken at different time points and the residual chromogenic activity of APC is determined by single-phase exponential decay curve fitting. [Figure 11] 1 is a table outlining the mutations present in Set 2 antibodies. [Figure 12]1 is a table showing the results of screening assays used to evaluate Set 2 antibodies. These results include a measure of antibody binding to APC and PC based on SPR (i.e., KD), the effect of the antibody on amidolytic activity of APC (SPECTROZYME® PCa), and UNcle-based aggregation temperature (Tagg). A hyphen indicates that the indicated data was not collected for the indicated antibody. [Figure 13] Figure 1 shows the results of a Protac®-APTT assay used to test the effect of Set 2 antibodies on the clotting time of normal human plasma. The assay was performed in two independent screens (i.e., Screen 1 and Screen 2) on plasma samples containing 30 nM of antibody. The results are presented as a plot of clotting time in Screen 1 vs. Screen 2 (left) and summarized in a table (right). [Figure 14A-1] Figure 14 shows the results of a histone H3 cleavage assay on human APC in the presence of Set 2 antibodies. APC (50 nM) and antibodies (500 nM) were pre-incubated for 30 min. After incubation, the mixture was added to 100 μg / mL histone H3. Over a 2-hour period, the mixture was subsampled at different time points to monitor the digestion of histone H3 over time. Digestion in the presence of each antibody was compared to digestion in the presence of TPP-26870. The results are presented as a plot of histone H3 cleavage versus time in Figure 14A and summarized in the table in Figure 14B. [Figure 14A-2] Figure 14 shows the results of a histone H3 cleavage assay on human APC in the presence of Set 2 antibodies. APC (50 nM) and antibodies (500 nM) were pre-incubated for 30 min. After incubation, the mixture was added to 100 μg / mL histone H3. Over a 2-hour period, the mixture was subsampled at different time points to monitor the digestion of histone H3 over time. Digestion in the presence of each antibody was compared to digestion in the presence of TPP-26870. The results are presented as a plot of histone H3 cleavage versus time in Figure 14A and summarized in the table in Figure 14B. [Figure 14A-3]Figure 14 shows the results of a histone H3 cleavage assay on human APC in the presence of Set 2 antibodies. APC (50 nM) and antibodies (500 nM) were pre-incubated for 30 min. After incubation, the mixture was added to 100 μg / mL histone H3. Over a 2-hour period, the mixture was subsampled at different time points to monitor the digestion of histone H3 over time. Digestion in the presence of each antibody was compared to digestion in the presence of TPP-26870. The results are presented as a plot of histone H3 cleavage versus time in Figure 14A and summarized in the table in Figure 14B. [Figure 14A-4] Figure 14 shows the results of a histone H3 cleavage assay on human APC in the presence of Set 2 antibodies. APC (50 nM) and antibodies (500 nM) were pre-incubated for 30 min. After incubation, the mixture was added to 100 μg / mL histone H3. Over a 2-hour period, the mixture was subsampled at different time points to monitor the digestion of histone H3 over time. Digestion in the presence of each antibody was compared to digestion in the presence of TPP-26870. The results are presented as a plot of histone H3 cleavage versus time in Figure 14A and summarized in the table in Figure 14B. [Figure 14A-5] Figure 14 shows the results of a histone H3 cleavage assay on human APC in the presence of Set 2 antibodies. APC (50 nM) and antibodies (500 nM) were pre-incubated for 30 min. After incubation, the mixture was added to 100 μg / mL histone H3. Over a 2-hour period, the mixture was subsampled at different time points to monitor the digestion of histone H3 over time. Digestion in the presence of each antibody was compared to digestion in the presence of TPP-26870. The results are presented as a plot of histone H3 cleavage versus time in Figure 14A and summarized in the table in Figure 14B. [Figure 14A-6]Figure 14 shows the results of a histone H3 cleavage assay on human APC in the presence of Set 2 antibodies. APC (50 nM) and antibodies (500 nM) were pre-incubated for 30 min. After incubation, the mixture was added to 100 μg / mL histone H3. Over a 2-hour period, the mixture was subsampled at different time points to monitor the digestion of histone H3 over time. Digestion in the presence of each antibody was compared to digestion in the presence of TPP-26870. The results are presented as a plot of histone H3 cleavage versus time in Figure 14A and summarized in the table in Figure 14B. [Figure 14B] Figure 14 shows the results of a histone H3 cleavage assay on human APC in the presence of Set 2 antibodies. APC (50 nM) and antibodies (500 nM) were pre-incubated for 30 min. After incubation, the mixture was added to 100 μg / mL histone H3. Over a 2-hour period, the mixture was subsampled at different time points to monitor the digestion of histone H3 over time. Digestion in the presence of each antibody was compared to digestion in the presence of TPP-26870. The results are presented as a plot of histone H3 cleavage versus time in Figure 14A and summarized in the table in Figure 14B. [Figure 15] 1 is a table showing the results of an in vitro plasma half-life assay of human APC in the presence of Set 2 antibodies. In this assay, APC and antibodies are pre-incubated for 30 minutes and normal pooled plasma is added. Samples are taken at different time points and the residual chromogenic activity of APC is determined by single-phase exponential decay curve fitting. [Figure 16]1 is a table in which the 20 Set 1 antibodies, as determined in Example 3, are divided into seven functional categories based on their pharmacological properties. In the anticoagulant activity column, + indicates that the antibody is more inhibitory than the parent antibody TPP-24727, while - indicates that the antibody is less inhibitory than TPP-24727. In the SEAP-PAR1 cleavage column, + indicates that the antibody causes more PAR1 cleavage than TPP-24727, while - indicates that the antibody causes less PAR1 cleavage than TPP-24727, and ND indicates that data was not collected. In the histone cleavage column, + indicates that the antibody causes more histone cleavage than TPP-24727, while - indicates that the antibody causes less histone cleavage than TPP-24727, and = indicates that the antibody causes similar histone cleavage to TPP-24727. In the Sensitivity to Physiological Inhibitors column, + indicates that the antibody renders APC more sensitive to physiological inhibitors than TPP-24727, while - indicates that the antibody renders APC less sensitive to physiological inhibitors than TPP-24727, and ND indicates that data was not collected. [Figure 17] 1 is a table in which the 24 Set 2 antibodies, as determined in Example 4, are divided into 11 functional categories based on their pharmacological properties. In the anticoagulant activity column, + indicates that the antibody is more inhibitory than the parent antibody TPP-26870, while - indicates that the antibody is less inhibitory than TPP-26870. In the histone cleavage column, + indicates that the antibody causes more histone cleavage than TPP-26870, while - indicates that the antibody causes less histone cleavage than TPP-26870. In the sensitivity to physiological inhibitors column, + indicates that the antibody renders the APC more sensitive to physiological inhibitors than TPP-26870, while - indicates that the antibody renders the APC less sensitive to physiological inhibitors than TPP-26870, and = indicates that the antibody renders the APC sensitive similar to TPP-26870. [Figure 18]FIG. 1 is a schematic diagram outlining how Set 1 and Set 2 antibodies were generated and screened. [Figure 19] 1 is a table showing the effect of humanizing the TPP-24727 heavy chain on immunogenicity score, expression levels, and APC binding kinetics. The table is divided into a first group of antibodies that showed an overall improvement in these parameters, and a second group of antibodies that were deemed suboptimal based on this analysis. A third group of antibodies disclosed in International Patent Application Publication No. WO 2023 / 284012 is included for comparison (referred to as "OMRF" in the figure). [Figure 20] 1 is a table showing the effect of humanizing the TPP-24727 light chain on immunogenicity score, expression level, and APC binding kinetics. The table is divided into a first group of antibodies that showed an overall improvement in these parameters, and a second group of antibodies that were deemed suboptimal based on this analysis. A third group of antibodies disclosed in International Patent Application Publication No. 2023 / 284012 is included for comparison (referred to as "OMRF" in the figure). The immunogenicity scores of the improved antibodies were substantially lower than those of the antibodies disclosed in WO2023 / 284012. 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 functions 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 functions 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 VH 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 (see, 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 certain preferred embodiments, the antibody is in the IgG4 format. The IgG4 antibody 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. Furthermore, IgG4 antibodies are easy to produce and are used in several marketed therapeutics, such as natalizumab (Tysabri®), gemtuzumab (Mylotarg®), dupilumab (Dupixant®), and nivolumab (Keytruda®).

[0032] 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: 242 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.

[0033] 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.

[0034] 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: 241 or a variant thereof.

[0035] 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.

[0036] 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 ) was assessed via SPR analysis to ensure that the

[0037] 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 anti-APC antibodies to inhibit the anticoagulant activity of APC was tested using a Protac® adjusted APTT clotting assay (see Figures 6 and 13).

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] Thus, in some embodiments, the antibodies of the present invention enhance, minimally affect, or inhibit the cytoprotective function of APCs (i.e., compared to the cytoprotective function in a no-antibody control). Specifically, in some embodiments, the antibodies (1) enhance, minimally affect, or inhibit APC-mediated histone cleavage, and / or (2) enhance, minimally affect, or inhibit APC-mediated endothelial barrier protection. In the Examples, the effect of anti-APC antibodies on APC-mediated histone cleavage was tested using a histone H3 cleavage assay (see Figures 9 and 14), and the effect of anti-APC antibodies on APC-mediated endothelial barrier protection was tested using an in vitro endothelial barrier function assay (see Figure 8).

[0043] 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 anti-APC antibodies on APC half-life was tested using an in vitro plasma half-life assay (see Figures 10 and 15).

[0044] The antibodies disclosed herein bind to exosites (i.e., sites other than the active site) of APC. The antibodies were generated from the exosite-binding parent antibody TPP-4885. TPP-4885 has been shown to inhibit the antithrombotic activity of APC 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 acid residues that mediate APC's interactions with cofactors and substrates are found in the exosite, which is removed far from the active site.

[0045] Example 1 describes the methods used to optimize the parent antibody TPP-4885. First, TPP-4885 (an IgG2 antibody) was reformatted into an IgG4 antibody and renamed TPP-24727. The IgG4 format used contains a serine 228 to proline (S228P) mutation that serves to prevent Fab arm exchange. Next, a stretch of framework sequence from TPP-24727 was replaced with human germline sequence to produce a more germline-like antibody framework, generating 96 different antibody light chains and 96 different antibody heavy chains. The modified light and heavy chains were evaluated against the corresponding chains of the parent antibody (i.e., TPP-24727) using expression assays, enzyme activity assays, and APC binding assays. Based on these assays, 13 light chains and 6 heavy chains were selected and combined to generate 78 optimized antibodies. The 78 new antibodies were rescreened using the same assays, and 20 antibodies were selected for further study. These 20 antibodies are referred to in this application as "Set 1 antibodies." The generation and characterization of the Set 1 antibodies are described in more detail in Examples 1-3. The amino acid and DNA sequence sequence identifiers of the Set 1 antibodies are listed in Tables 1 and 2, respectively. A second set of 24 antibodies, referred to herein as "Set 2 antibodies," were generated and evaluated as described in Example 4. The Set 2 antibodies include 22 antibody variants of the promising Set 1 antibody TPP-26870 that were generated via random mutagenesis. The Set 2 antibodies also include two additional antibodies (i.e., TPP-26922 and TPP-26939) that are not related to any of the Set 1 antibodies. The amino acid and DNA sequence sequence identifiers of the Set 2 antibodies are listed in Tables 3 and 4, respectively. A schematic outlining how the Set 1 and Set 2 antibodies were generated and screened is provided in FIG. 18. [Table 1] [Table 2] [Table 3] [Table 4]

[0046] 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.

[0047] The classical representation of an antibody is as a Y-shaped molecule composed of four polypeptide subunits. Each antibody contains two identical copies of a longer heavy chain and two identical copies of a shorter light chain. The light chains of an antibody can be classified as either kappa (κ) or lambda (λ), based on minor differences in the polypeptide sequence. The composition of the heavy chain determines the overall class of each antibody. Each heavy chain contains a heavy chain constant region and a heavy chain variable region (V H Similarly, each light chain has two regions: a light chain constant region and a light chain variable region (V L The constant region is identical in all antibodies of the same isotype but differs between antibodies of different isotypes, whereas the variable region is antibody specific and depends on the B cell that produced it. Together, they form two V H and two V L These form the variable regions of the antibody which function as the antigen-binding sites.

[0048] Each V of the antibody H and V L The term "complementarity determining region" or "CDR" refers to hypervariable regions that together form an antigen-binding surface that is complementary to the three-dimensional structure of the antigen.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). In a conventional antibody (i.e., an antibody comprising two heavy chains and two light chains), the antigen-binding site is comprised of six CDRs, i.e., the V H The three CDRs and V L and the three CDRs:

[0049] 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.

[0050] Of the three CDRs, CDR3 is believed to be the primary 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 invention comprise a CDR3 of an antibody disclosed herein. Specifically, in some embodiments, the antibodies of the invention comprise a V CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 118. H Further, in some embodiments, the antibody comprises a VDR3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 and 138-140.L Includes.

[0051] In other embodiments, the antibodies of the invention further comprise the CDR1 and CDR2 of an antibody disclosed herein. H may comprise (a) a CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4 and 109 to 113, (b) a CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 8 and 114 to 117, or (c) both a CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4 and 109 to 113 and a CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 8 and 114 to 117. Furthermore, in some embodiments, the V L may comprise (a) a CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 to 19 and 130 to 131; (b) a CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 to 22 and 132 to 137; or (c) both a CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 to 19 and 130 to 131 and a CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 to 22 and 132 to 137.

[0052] In certain embodiments, an antibody of the invention comprises CDR1, CDR2, and CDR3, all of which are derived from a single variable region of an antibody disclosed herein. H V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 15 and 119 to 129 H In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 of L V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 to 35 and 141 to 151 LIn some embodiments, the antibody comprises all six CDRs (i.e., V H CDR1, CDR2, and CDR3 of, and V L Such antibodies comprise the paratope (i.e., the set of six CDRs that form the antigen-binding region) of an antibody disclosed herein.

[0053] In addition, the present invention provides antibodies comprising or consisting of a full-length variable region disclosed herein. Specifically, in some embodiments, the antibody comprises a V sequence selected from the group consisting of SEQ ID NOs: 10-15 and 119-129. H Further, in some embodiments, the antibody comprises a V selected from the group consisting of SEQ ID NOs: 24-35 and 141-151. L Includes.

[0054] In some embodiments, the antibodies of the invention have the V H and V of the antibodies disclosed herein L For example, in some embodiments, the antibodies disclosed herein comprise at least a portion (e.g., CDR3) of both of SEQ ID NOs: 10-15 and 119-129. H and V selected from the group consisting of SEQ ID NOs: 24 to 35 and 141 to 151. L In some embodiments, V H and V L is from a single antibody disclosed herein (V for each antibody disclosed herein H Sequence and V L (See Tables 1 and 3 for sequences). In another embodiment, V H and V L are derived from two different antibodies disclosed herein.

[0055] 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 an APC with minimal or no binding to a 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: 10-15 and 119-129, which 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 V selected from SEQ ID NOs: 24-35 and 141-151 that contains 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. L In other embodiments, the antibody comprises a CDR3 selected from SEQ ID NOs: 9, 23, 118, and 138-140 comprising at least one amino acid modification.

[0056] 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 5. [Table 5]

[0057] In some embodiments, the antibody of the present invention comprises a V having an amino acid sequence having 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: 10-15 and 119-129, or 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: 9 and 118. H In some embodiments, the antibody of the present invention comprises a V having an amino acid sequence having 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: 24-35 and 141-151, or 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: 23 and 138-140. L In some embodiments, the antibody of the present invention comprises a heavy chain having an amino acid sequence having 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: 36-41 and 152-162. In some embodiments, the antibody of the present invention comprises a heavy chain consisting of one of the amino acid sequences of SEQ ID NOs: 36-41 and 152-162. In some embodiments, the antibody of the present invention comprises a light chain having an amino acid sequence having at least 99, 98, 97, 96, 95, 04, 93, 92, 91, 90, 85, 80, or 75 percent sequence identity to one of the amino acid sequences of SEQ ID NOs: 42-53 and 163-173. In some embodiments, the antibody comprises a light chain consisting of one of the amino acid sequences of SEQ ID NOs: 42-53 and 163-173.

[0058] "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.

[0059] 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.

[0060] 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 nucleic acid encoding an antibody comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 10 to 15 and 119 to 129; H (2) an antibody comprising an amino acid sequence selected from SEQ ID NOs: 24 to 35 and 141 to 151, or consisting of the same; L (3) an antibody comprising a CDR3 comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 9, 23, 118, and 138-140, or (4) a nucleic acid encoding any other antibody described herein. The present invention further includes nucleic acids comprising or consisting of the DNA sequences provided in Table 2 or Table 4 (i.e., DNA sequences selected from SEQ ID NOs: 54-102 and 174-240) and variants thereof.

[0061] 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.

[0062] 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.

[0063] 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 can be introduced into the host cell using standard techniques, including, for example, electroporation, heat shock, lipofection, microinjection, and particle bombardment.

[0064] 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).

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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 a variant thereof (e.g., 3K3A-APC) that specifically binds to one or more of the antibodies disclosed herein.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] In some embodiments, the methods of the invention are used to treat or prevent a condition that can be treated or prevented by inhibiting or enhancing the anticoagulant function of APC, 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 APC. 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.

[0074] 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 provides a method for treating inflammation in acute ischemic disease (e.g., by providing neuroprotection in ischemic stroke (Ann Neurol 85:125-136, 2019) or 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).

[0075] 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.

[0076] 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.

[0077] 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. Although no significant differences were observed between the ability of the two antibodies to inhibit the anticoagulant activity of APC, administration of MAPC1591, but not MPC1609, significantly reduced the severity of hemophilic arthropathy.

[0078] TPP-24727 is the parent antibody from which the antibodies of the present invention are derived. TPP-24727 inhibits APC anticoagulant activity, enhances APC histone H3 cleavage activity, and partially reduces APC-mediated PAR1 cleavage. Compared to this parent antibody, the promising Set 1 antibody TPP-26870 inhibits APC anticoagulant activity more strongly, resulting in enhanced pro-hemostatic effects. Furthermore, TPP-26870 inhibits PAR1 cleavage less than the parent antibody and does not interfere with APC-mediated histone cleavage. Anti-APC antibodies that provide improved cytoprotective effects compared to TPP-24727 or directly enhance APC cytoprotective activity may also be useful in the treatment of conditions such as sepsis and inflammation in acute ischemic diseases. As summarized in Figure 16, Set 1 antibodies can be divided into at least seven functional categories based on the pharmacological properties assayed in Example 3. Similarly, as summarized in FIG. 17, Set 2 antibodies can be divided into at least 11 functional categories based on the pharmacological properties assayed in Example 4. EXAMPLES

[0079] Example 1 - Optimization of parent antibodies A previous study (Nat Commun 11(1):2992,2020) reported two antibodies against APC that were shown to increase thrombin generation and promote plasma clotting in monkeys. One of these antibodies, designated TPP-4885, was well tolerated in monkeys and was determined to target an exosite on the APC protein. However, this antibody still partially inhibits the active site of APC and may therefore partially inhibit the cytoprotective effects of this enzyme.

[0080] The following examples describe the methods used to optimize TPP-4885. First, this IgG2 antibody was reformatted into an IgG4 antibody and renamed TPP-24727. The IgG kappa S228P format was used, which contains a serine 228 to proline (S228P) mutation that serves to prevent Fab arm exchange. Reformatting was performed using standard cloning techniques. Next, stretches of framework sequences from TPP-24727 were replaced with human germline sequences, i.e., KV2-28, KV3-20, KV3-11, V3-48-01.1, V3-15-01.1, and V1-69-04.1, to produce antibody frameworks that are more germline-like. Additionally, a deamidation site containing two asparagine residues was inserted into the heavy chain variable region (V) in a subset of constructs (i.e., TPP-24737, TPP-24738, TPP-24739, TPP-24743, TPP-24745, TPP-24750, TPP-24766, TPP-24769, TPP-24777, and TPP-24815). H ) was removed from the CDR2 of TPP-24727. This exchange process generated 96 different antibody light chains and 96 different antibody heavy chains. The modified antibody chains were evaluated by pairing each modified chain with the other unmodified chain from the parent antibody (i.e., each modified light chain was paired with the unmodified heavy chain of TPP-24727 and each modified heavy chain was paired with the unmodified light chain of TPP-24727).

[0081] As shown in Figures 19 and 20, the sequence exchange resulted in improved immunogenicity, expression levels, and / or K D The sequence identifiers associated with each of the heavy chains characterized in FIG. 19 are listed in Table 6, and the sequence identifiers associated with each of the light chains characterized in FIG. 20 are listed in Table 7. [Table 6] [Table 7]

[0082] Each modified antibody chain was tested for expression levels in transiently transfected HEK293 cells, amidolytic activity (SPECTROZYME® PCa), surface plasmon resonance (SPR) based binding (i.e., kon, koff, kD) to both human APC and PC, and aggregation temperature (Tagg) based on UNcle. Based on these measurements, 13 light chains and 6 heavy chains were selected and combined to generate 78 optimized antibodies. The 78 new antibodies were rescreened using the same assay, and 20 antibodies were selected for further study. These 20 optimized antibodies are referred to herein as Set 1 antibodies. Results of the initial screening assay for Set 1 antibodies are shown in FIG. 1.

[0083] Set 1 antibodies include antibodies with a diverse combination of improved properties compared to the parent antibody TPP-24727. They also contain a diverse array of heavy and light chains, providing a wide optimization space for further affinity maturation. The variable domain sequences of Set 1 antibodies are outlined in Table 1. Scale-up production of 20 Set 1 antibodies was performed in preparation for expanded functional and chemical, manufacturing, and control (CMC) evaluations, including those described in Example 2.

[0084] material Plasma-derived human APCs and human PCs were obtained from Enzyme Research Laboratories (South Bend, IN, USA), and the human APC substrate SPECTROZYME® PCa was obtained from American Diagnostica (Pfungstadt, Germany).

[0085] Expression and purification Genes encoding antibody heavy and light chains were subcloned separately into expression vector pTT5 (National Research Council, Canada) and co-transfected into HEK293 cells (National Research Council, Canada). Cells were maintained in FreeStyle™ F17 expression medium (Invitrogen) supplemented with 2 mM GlutaMAX™ supplement (Invitrogen), 0.1% Pluronic™ F-68 non-ionic surfactant (Sigma-Aldrich), and 0.5% PenStrep (penicillin-streptomycin solution) (Invivogen). After 5 days of transient expression, cleared supernatants were affinity purified using Protein A beads (GE Healthcare) and subsequently buffer exchanged into PBS using Zeba™ Spin desalting columns (Thermo Fisher). IgG concentrations were quantified by sandwich ELISA using the Amplex™ Red hydrogen peroxide / peroxidase assay kit (Invitrogen) with anti-human Fc (Sigma) for capture and anti-human IgG (Fc specific)-peroxidase antibody (Sigma) for detection. Human reference serum (Bethyl) was used as the IgG concentration standard.

[0086] Coagulation temperature The aggregation onset temperature (Tagg) was determined using an UNcle instrument (All-in-one biologics stability platform, UNCHAINED Labs) according to the manufacturer's protocol. The instrument was used to measure the static light scattering of the protein solution at 266 nm, and the onset of aggregation was calculated using UNcle software. Supernatants were collected from HEK293 cells induced to express the antibody of interest (see above), concentrated, and buffer exchanged (into PBS) using VIVASpin® 500 centrifugal concentrators (10000 MWCO PES, Sartorius). The final protein solution (<0.5 mg / mL in PBS buffer) was centrifuged at 8000 g for 2 min before analysis. The following temperature gradient was used for the analysis: start temperature: 30° C., speed: 0.33° C. / min, end temperature: 95° C.

[0087] Affinity and binding kinetics Surface plasmon resonance (SPR) assays were performed to evaluate the binding kinetics and affinity of anti-APC antibodies. Binding assays were performed on a Biacore™ T200 instrument (GE Healthcare) at 25° C. using assay buffer (HBS-P+, 1 mg / ml BSA, 0.01% Na-azide, 300 mM NaCl, 2.75 mM MgCl2, 0.75 mM CaCl2). Antibodies were captured using anti-human Fc IgG covalently coupled (via amines) to a CM5 sensor chip. Human APC and PC were used as analytes at concentrations of 100 nM and 1 μM, respectively. Reliable dissociation rate constants (k d The dissociation rate was set to 600 seconds after 180 seconds of association to obtain the kinetic and affinity data. The resulting sensorgrams were fitted to a 1:1 Langmuir binding model to derive kinetic and affinity data. The resulting sensorgrams were fitted to a 1:1 Langmuir binding model to derive kinetic and affinity data.

[0088] Effect of APC on amidolytic activity An amidolytic activity assay was performed to evaluate the effect of antibody conjugation on amidolytic activity of human APCs (hAPCs). hAPCs (20 nM) were combined in equal volumes with each monoclonal antibody (mAb) at various concentrations (20 nM, 4 nM, and 0.5 nM) in a 96-well dilution plate and pre-incubated for 20 min at room temperature. The APC / antibody mixtures were then transferred to a 384-well plate at 10 μL / well in quadruplicate, and 10 μL of the substrate SPECTROZYME® PCa (0.1 mM) was added to each well. Substrate hydrolysis was monitored at 37° C. using a Tecan Infinite® M1000Pro reader at 405 nm for chromogenic reading.

[0089] Example 2 - Screening of Set 1 Antibodies The following example describes several assays that were used to evaluate the top 20 anti-APC antibodies (i.e., Set 1 antibodies) identified in Example 1. The purpose of these evaluations was to evaluate the effect of the antibodies on the anticoagulant activity and cytoprotective function of APC.

[0090] Affinity and binding kinetics To evaluate the binding kinetics and affinity of Set 1 antibodies to APC, binding assays were performed using surface plasmon resonance (SPR). Binding assays were performed on a Biacore™ T200 instrument (GE Healthcare) at 25° C. using assay buffer (HBS-P+, 1 mg / ml BSA, 0.01% Na-azide, 300 mM NaCl, 2.75 mM MgCl2, 0.75 mM CaCl2). Antibodies were captured by anti-human Fc IgG covalently coupled to a CM5 sensor chip. Human APC and PC were used as analytes at concentrations of 100 nM and 1 μM, respectively. Reliable dissociation rate constants (k d The dissociation rate was set to 600 seconds after 180 seconds of association to obtain a k σ ... d 11 antibodies showed improved K D showed.

[0091] Protein concentration IgG concentrations were quantified by sandwich ELISA using the Amplex™ Red hydrogen peroxide / peroxidase assay kit (Invitrogen) with anti-human Fc (Sigma) for capture and anti-human IgG (Fc specific)-peroxidase antibody (Sigma) for detection. Human reference serum (Bethyl) was used as an IgG concentration standard.

[0092] Agglomeration and Melting Temperatures The aggregation onset temperature (Tagg) was determined using an UNcle instrument (UNCHAINED Labs) according to the manufacturer's protocol. This instrument was used to measure the static light scattering of protein solutions at 266 nm, and the onset of aggregation was calculated using UNcle software. Additionally, the melting temperature (Tm) was determined via differential scanning calorimetry using a MicroCal VP-Capillary DSC instrument according to the manufacturer's protocol. A reference protein solution (0.5 mg / mL in PBS, pH 7.4) was centrifuged at 8000 g for 2 min before analysis. The following temperature gradient was used for the analysis: start temperature: 25° C., heating scan rate: 2° C. / min, end temperature: 95° C. The heat capacity signals were deconvoluted to obtain the basic peaks for different melting temperatures (for CH2 / CH3 and Fab). HEK293 supernatants containing antibodies from transient expression (see above) were concentrated and buffer exchanged (into PBS) using VIVASpin® 500 centrifugal concentrators (10000 MWCO PES, Satorius). The final protein solution (<0.5 mg / mL in PBS buffer) was centrifuged at 8000 g for 2 min before analysis. The following temperature gradient was used for the analysis: start temperature: 30° C., speed: 0.33° C. / min, end temperature: 95° C. The results of this analysis are depicted in FIG. 3. Of the 20 Set 1 antibodies, all were shown to have high purity and percent monomer, and solubility above 188 mg / mL. Among them, TPP-26882 has the highest expression titer (332 mg / mL) and thermal stability (Tm=82.0° C.).

[0093] Effect of APC on amidolytic activity To evaluate the effect of Set 1 antibodies on the amidolytic activity of APC, 20 nM human APC was combined in equal volumes with 20 antibodies at different concentrations in a 96-well plate and pre-incubated for 20 min at room temperature. The APC / antibody mixture was then transferred to a 384-well assay plate at 10 μL / well in quadruplicate and 10 μL of substrate SPECTROZYME® PCa (0.1 mM) was added. Substrate hydrolysis was monitored using a Tecan Infinite® M1000Pro reader at 37° C. and 405 nm for chromogenic reading. The results of this assay are presented in FIG. 4. Anti-APC antibodies were tested at 8 nM and 3.2 nM. At 8 nM, 12 antibodies produced higher amidolytic activity compared to the parent antibody TPP-24727. At 3.2 nM, 7 antibodies produced higher activity than TPP-24727.

[0094] FVa inactivation The ability of Set 1 antibodies to inhibit APC inactivation of FVa was measured using FXa and thrombin generation assays. In this assay, 20 pM FVa was incubated with 50 pM APC in assay buffer (25 mM Tris-HCl, 130 mM NaCl, 2.7 mM KCl, 25 μM phospholipids (PC:PE:PS 40:40:20), 5 mM CaCl2, and 1 mg / mL BSA) with each antibody at concentrations ranging from 0.5 to 1000 nM. After 30 min of incubation at room temperature, 100 μL of prothrombinase mixture containing 400 nM FXa, 1 μM prothrombin, and 0.6 mM thrombin chromogenic substrate (S-2238) was added to the 100 μL incubation mixture. The kinetics of thrombin-mediated substrate hydrolysis was monitored at 405 nm using a plate reader for 3 hours at 25° C. The results of this assay are presented in Figure 5. Of the antibodies tested, TPP-26783, TPP-26784, and TPP-26920 showed improved EC50s in inhibiting FVa compared to the parent antibody TPP-24727.

[0095] Example 3: Functional evaluation of Set 1 antibodies In the following examples, the top 20 anti-APC antibodies identified in Example 1 (i.e., Set 1 antibodies) are functionally characterized using several assays. Specifically, the effect of the antibodies on coagulation-related activities of APCs is assessed using a plasma clotting assay, the effect of the antibodies on cytoprotective activities of APCs is assessed using a PARl cleavage assay, an endothelial barrier integrity assay, and a histone H3 cleavage assay, and the effect of the antibodies on the sensitivity of APCs to physiological inhibitors is assessed using an in vitro plasma half-life assay.

[0096] The results of these functional assays will help determine whether a particular Set 1 antibody is useful for a particular indication. For example, an antibody that inhibits the anticoagulant activity of APC while maintaining its cytoprotective activity may be useful in treating disorders requiring blood clotting (e.g., acute hemorrhage), whereas an antibody that enhances the cytoprotective activity of APC may be useful in treating disorders such as sepsis and inflammation in acute ischemic disease.

[0097] result: Clotting time The ability of Set 1 antibodies to reduce clotting time was measured using the Protac®-APTT clotting assay. The results of this assay revealed that the antibodies reduced in vitro clotting time to various degrees (Figure 6). Antibodies TPP-26870, TPP-26935, TPP-26874, TPP-26873, TPP-26926, TPP-26864, TPP-26918, and TPP-26920 were shown to have improved potency in inhibiting the anticoagulant effect of APC and reducing plasma clotting time compared to the parent antibody TPP-24727. Of these antibodies, TPP-26870 showed the greatest reduction in clotting time.

[0098] PAR1 cleavage Cleavage and activation of protease-activated receptor 1 (PAR1) by APC alone induces cytoprotective effects, including protective gene expression, enhanced endothelial barrier function, reduced cytokine secretion, and protection against apoptosis. Therefore, the ability of Set 1 antibodies to inhibit APC-mediated PAR1 cleavage was tested using a secreted embryonic alkaline phosphatase (SEAP) protease-activated receptor 1 (PAR1) cleavage assay. The results of this assay are depicted in FIG. 7. Based on these results, antibodies with increased (e.g., TPP-26918) or decreased (e.g., TPP-26920) ability to inhibit APC-mediated PAR1 cleavage, and one antibody (i.e., TPP-26924) that did not show any inhibition of APC-mediated PAR1 cleavage at concentrations up to 500 nM were identified.

[0099] Endothelial Barrier Integrity APC has a protective effect on endothelial barrier function. Therefore, the effect of Set 1 antibodies on this cytoprotective activity was tested using an endothelial barrier integrity assay. The results of this assay are presented in Figure 8. Based on these results, several antibodies (e.g., TPP26926 and TPP26935) that enhance APC-mediated endothelial barrier protection were identified.

[0100] Histone cleavage Histones, when released into the extracellular space, act as damage-associated molecular patterns (DAMPs), resulting in systemic inflammatory and toxic responses. APC inhibits histone-induced cytotoxicity through proteolytic cleavage of histones. Therefore, the ability of Set 1 antibodies to inhibit APC-mediated histone cleavage was tested using a histone H3 cleavage assay. Based on the results of this assay, antibodies were identified that could either inhibit or enhance APC-mediated H3 cleavage (Figure 9). For example, TPP-26921 inhibited cleavage, while TPP-26862 enhanced both the cleavage rate and the total amount of cleavage.

[0101] Plasma half-life An in vitro plasma half-life assay was used to determine the effect of Set 1 antibodies on the sensitivity of APCs to physiological inhibitors. This assay identified antibodies that increased the serum half-life of APCs (FIG. 10). This increase in half-life indicates that binding of these antibodies reduces the sensitivity of APCs to inhibition by physiological inhibitors present in plasma.

[0102] Materials and Methods: Protac®-APTT Clotting Assay. Plasma samples (50 μL) containing different concentrations of Set 1 antibodies were mixed with Protac® (25 μL; 1 U / mL) and STA-PTT reagent (75 μL) at 37° C. After 4 min of incubation, clotting was initiated by adding 75 μL of 25 mM CaCl2 solution. In this assay, Protac® (a single-chain glycoprotein derived from snake venom that activates protein C) is added to plasma to activate protein C, reduce clotting activity, and prolong clotting time.

[0103] SEAP-PAR1 cleavage assay. A PAR1 cleavage reporter construct was made with the N-terminal SEAP domain. HEK293 cells expressing SEAP-PAR1 and wild-type (wt)-EPCR were grown to confluence in 96-well plates. On the day of the experiment, cells were washed with Hank's balanced salt solution supplemented with 1.3 mM CaCl2, 0.6 mM MgCl2, and 0.1% BSA (HMM2). Plasma-derived APC (50 or 100 nM) and antibodies were added to confluent SEAP-PAR1 / wt-EPCR cells in 96-well plates after preincubation for 30 min in HMM2. After 60 min, SEAP release was measured using one-step p-nitrophenyl phosphate (Pierce #37621). After correction for background activity in the absence of APC, values ​​were expressed as a percentage of the total SEAP activity present on the cells. Values ​​are normalized to the value obtained without antibody (=100%).

[0104] Endothelial Barrier Integrity Assay. Endothelial cell permeability was measured in real time using the iCELLigence™ System (ACEA, San Diego, CA), which measures changes in transepithelial electrical resistance (TEER) by electrical cell substrate impedance sensing (ECIS). Briefly, EA.hy926 endothelial cells were grown to confluence for 2-3 days in 8-well culture dishes containing gold film electrodes. DMEM (Dulbecco's Modified Eagle Medium, Invitrogen) containing 10% fetal bovine serum was replaced with serum-free medium containing 0.1% BSA 2 hours prior to the addition of APC (50 nM) in the presence and absence of antibodies. Permeability of confluent monolayers after treatment with APC for 3 hours was determined upon the addition of thrombin (2 nM). All comparisons of cell index were performed using normalized resistance. Results were expressed as a percentage of maximal barrier protection by APC in the absence of antibodies.

[0105] Histone H3 cleavage assay. APC (50 nM) and antibodies (500 nM) were pre-incubated for 30 min in HBS buffer ((20 mM Hepes, 147 mM NaCl, 3 mM KCl) + 100 ug / ml BSA + 2 mM CaCl2, pH 7.4). After incubation, the mixture was added to 100 μg / ml histone H3 (H3, Roche). Over a period of 2 h, the mixture was subsampled at different time points and added to reducing sample buffer. Subsamples were then heated at 95°C for 15 min, centrifuged at 4000 rpm for 5 min, vortexed briefly, and loaded onto a 12% Bis-Tris gel containing MES running buffer (30 μl / well, 750 ng H3). Odyssey® markers (10 μL) were used as molecular weight standards with 20 kDa as a reference for normalization between gels. Gels were stained overnight with Biotium One-Step Blue® Protein Stain (50 ml / gel). Protein bands were scanned on a LI-COR imaging system (700 channel, intensity 6, 1 mm offset, resolution 169 um, best quality, manual background). Images were analyzed using Image Studio V5.2.

[0106] In vitro plasma half-life assay. APC (70 nM) and antibodies (700 nM) were pre-incubated for 30 min and normal pooled plasma was added to 90% (v / v). Samples were collected at various time points (0-90 min) and quenched with ice-cold TBS. Chromogenic activity of APC was determined using Pefachrome® PCa. Background chromogenic activity of plasma (no APC) was subtracted and APC activity was normalized to the t=0 time point. Half-life (T 1 / 2 ) was determined by monophasic exponential decay curve fitting.

[0107] Example 4 - Screening and Functional Evaluation of Set 2 Antibodies The following examples describe several assays that were used to evaluate a second set of 24 anti-APC antibodies, referred to herein as Set 2 antibodies. The Set 2 antibodies include 22 variants of the promising Set 1 antibody TPP-26870 that have higher affinity for APC compared to TPP-26870. These variants were generated by creating a single NNK site saturation mutagenesis library based on TPP-26870. The mutations found in each of these 22 antibody variants are outlined in FIG. 11. Set 2 also includes two additional antibodies (i.e., TPP-26922 and TPP-26939) that are not related to any of the Set 1 antibodies. The sequences of the variable domains of the Set 2 antibodies are outlined in Table 3.

[0108] Several assays were used to screen and evaluate the functional properties of Set 2 antibodies. Again, the goal of these evaluations was to assess the effect of the antibodies on the anticoagulant activity and cytoprotective function of APCs.

[0109] Screening assays Set 2 antibodies were screened using several assays described in Example 2. Specifically, (1) surface plasmon resonance (SPR) was used to assess the binding kinetics and affinity of the antibodies to APC and PC, (2) an amidolytic activity assay was used to assess the effect of the antibodies on the substrate cleavage activity of APC, and (3) the aggregation onset temperature (Tagg) of the antibodies was determined using an UNcle instrument. The results of these assays are presented in Figure 12. Each of these assays was performed as described in Example 2.

[0110] Clotting time The ability of Set 2 antibodies to reduce clotting time was measured using a Protac®-APTT clotting assay. The results of this assay revealed that some of the TPP-26870 variant antibodies (i.e., TPP-29843, TPP-29847, TPP-29850, TPP-29852, TPP-29853, TPP-29854, TPP-29855, TPP-29856, TPP-29857, TPP-29858, TPP-29864, and TPP-29865) showed enhanced inhibition of APC anticoagulant activity compared to the parent antibodies, resulting in reduced in vitro clotting times, while others (e.g., TPP-29851, TPP-29844) showed reduced inhibition of APC anticoagulant activity (Figure 13). The Protac®-APTT clotting assay was performed as described in Example 3.

[0111] Histone cleavage The ability of Set 2 antibodies to inhibit APC-mediated histone cleavage was assayed using a histone H3 cleavage assay. This assay identified antibodies that could inhibit or enhance histone cleavage to various degrees (Figure 14). For example, TPP-29864 inhibited APC-mediated H3 cleavage, while TPP-29848 enhanced the total amount of cleavage. The histone H3 cleavage assay was performed as described in Example 3.

[0112] Plasma half-life An in vitro plasma half-life assay was used to determine the effect of Set 2 antibodies on the sensitivity of APCs to physiological inhibitors. The results of this assay were used to identify antibodies that increase the serum half-life of APCs (Figure 15). This increase in half-life indicates that binding of these antibodies reduces the sensitivity of APCs to inhibition by physiological inhibitors present in plasma. The in vitro plasma half-life assay was performed as described in Example 3.

[0113] References: Each patent and non-patent publication cited herein is incorporated herein by reference in its entirety, including, but not limited to, the references listed below. 1. Peyvandi, F., Garagiola, I. & Young, G. The past and future of haemophilia:diagnosis, treatments, and its complications. Lancet 388,187-197(2016). 2. Lusher, JMInhibitor antibodies to factor VIII and factor IX:management. Semin. Thromb. Hemost. 26,179-188(2000). 3. Hartmann, J. & Croteau, SE2017 clinical trials update: innovations in hemophilia therapy. Am. J. Hematol. 91,1252-1260(2016). 4. Esmon,CTProtein C anticoagulant system-anti-inflammatory effects. Semin. Immunopathol. 34,127-132(2012). 5. Griffin, JH, Zlokovic, BV & Mosnier, LOActivated protein C: biased for translation. Blood 125, 2898-2907 (2015). 6. Polderdijk,S.G.et al.Design and characterization of an APC-specific serpin for the treatment of hemophilia. Blood 129,105-113(2017). 7. Escuriola Ettingshausen,C.et al.Symptomatic onset of severe hemophilia A in childhood is dependent on the presence of prothrombotic risk factors. Thromb. Haemost. 85,218-220(2001). 8. Vianello,F.et al.Mild bleeding diathesis in a boy with combined severe haemophilia B(C(10400)->T)and heterozygous factor V Leiden. Haemophilia 7,511-514(2001). 9. Nichols,W.C.et al.Moderation of hemophilia A phenotype by the factor V R506Q mutation. Blood 88,1183-1187(1996). 10. Xu,J.et al.Extracellular histones are major mediators of death in sepsis. Nat. Med. 15,1318-1321(2009). 11. Bae,J.S.,Yang,L.,Manithody,C.&Rezaie,A.R.Engineering a disulfide bond to stabilize the calcium-binding loop of activated protein C eliminates its anticoagulant but not its protective signaling properties. J.Biol. Chem. 282,9251-9259(2007). 12. Mosnier,L.O.,Gale,A.J.,Yegneswaran,S.&Griffin,J.H.Activated protein C variants with normal cytoprotective but reduced anticoagulant activity. Blood 104,1740-1744(2004). 13. Mosnier,L.O.et al.Hyperantithrombotic,noncytoprotective Glu149Ala activated protein C mutant. Blood 113,5970-5978(2009). 14. Lyden et al.,Ann Neural 85: 125(2019). 15. Mann,H.J.,Short,M.A.&Schlichting,D.E.Protein C in critical illness. Am. J.Health Syst. Pharm. 66,1089-1096(2009). 16. Ranieri,VM et al,N Engl J Med 366:2055(2012). 17. Angus,DC,Crit Care 16:107(2012). 18. Expert Opin Biol Ther. 13:1149(2013). 19. Appl Microbial Biotechnol. 77:13(2007). 20. Klarenbeek A,et al.Mabs-Austin.;7:693(2015). 21. Trends Biochem Sci. 26:230(2001). 22. Van Audenhove I,Gettemans J.EBioMedicine 8:40(2016). 23. Van de Water JA,et al.Proc Natl Acad Sci USA. 109:16642(2012).

Claims

1. A heavy chain variable region (V) comprising a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 118. Ｈ 1. An isolated antibody comprising:

2. The V Ｈ The antibody of claim 1, further comprising: (a) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4 and 109-113; (b) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-8 and 114-117; or (c) both a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4 and 109-113 and a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-8 and 114-117.

3. The V Ｈ having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-15 and 119-129 Ｈ The antibody of claim 2, comprising CDR1, CDR2, and CDR3 of:

4. V comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-15 and 119-129 Ｈ 1. An isolated antibody comprising:

5. A light chain variable region (V) comprising a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 and 138-140. Ｌ 13. An isolated antibody comprising:

6. The V Ｌ The antibody of claim 5, further comprising: (a) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-19 and 130-131; (b) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-22 and 132-137; or (c) both a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-19 and 130-131 and a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-22 and 132-137.

7. The V Ｌ having an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-35 and 141-151 Ｌ The antibody of claim 6, comprising CDR1, CDR2, and CDR3 of:

8. V comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-35 and 141-151 Ｌ 1. An isolated antibody comprising:

9. V comprising a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 118 Ｈ The antibody of any one of claims 5 to 8, further comprising:

10. The V Ｈ The antibody of claim 9, further comprising: (a) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4 and 109-113; (b) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-8 and 114-117; or (c) both a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4 and 109-113 and a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-8 and 114-117.

11. The V Ｈ having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-15 and 119-129 Ｈ The antibody of claim 10, comprising CDR1, CDR2, and CDR3 of:

12. The V Ｈ The antibody of claim 10, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-15 and 119-129.

13. 1. An isolated antibody, comprising: 1) V of SEQ ID NO: 10 Ｈ and V of SEQ ID NO:24 Ｌ , 2) V of SEQ ID NO:11 Ｈ and V of SEQ ID NO:25 Ｌ , 3) V of SEQ ID NO: 11 Ｈ and V of SEQ ID NO:26 Ｌ , 4) V of SEQ ID NO:11 Ｈ and V of SEQ ID NO:27 Ｌ , 5) V of SEQ ID NO: 11 Ｈ and V of SEQ ID NO:28 Ｌ , 6) V of SEQ ID NO: 11 Ｈ and V of SEQ ID NO:29 Ｌ , 7) V of SEQ ID NO: 11 Ｈ and V of SEQ ID NO:30 Ｌ , 8) V of SEQ ID NO:11 Ｈ and V of SEQ ID NO:31 Ｌ , 9) V of SEQ ID NO: 11 Ｈ and V of SEQ ID NO:32 Ｌ , 10) V of SEQ ID NO: 11 Ｈ and V of SEQ ID NO:33 Ｌ , 11) V of SEQ ID NO: 12 Ｈ and V of SEQ ID NO:28 Ｌ , 12) V of SEQ ID NO: 13 Ｈ and V of SEQ ID NO:34 Ｌ , 13) V of SEQ ID NO: 14 Ｈ and V of SEQ ID NO:35 Ｌ , 14) V of SEQ ID NO: 14 Ｈ and V of SEQ ID NO:34 Ｌ , 15) V of SEQ ID NO: 14 Ｈ and V of SEQ ID NO:28 Ｌ , 16) V of SEQ ID NO: 14 Ｈ and V of SEQ ID NO:31 Ｌ , 17) V of SEQ ID NO: 14 Ｈ and V of SEQ ID NO:32 Ｌ , 18) V of SEQ ID NO: 14 Ｈ and V of SEQ ID NO:33 Ｌ , 19) V of SEQ ID NO: 15 Ｈ and V of SEQ ID NO:28 Ｌ , 20) V of SEQ ID NO: 15 Ｈ and V of SEQ ID NO:29 Ｌ , 21) V of SEQ ID NO: 14 Ｈ and V of SEQ ID NO:29 Ｌ , 22) V of SEQ ID NO: 15 Ｈ and V of SEQ ID NO:33 Ｌ , 23) V of SEQ ID NO: 119 Ｈ and V of SEQ ID NO:29 Ｌ , 24) V of SEQ ID NO: 120 Ｈ and V of SEQ ID NO:29 Ｌ , 25) V of SEQ ID NO: 121 Ｈ and V of SEQ ID NO:29 Ｌ , 26) V of SEQ ID NO: 122 Ｈ and V of SEQ ID NO:29 Ｌ , 27) V of SEQ ID NO: 11 Ｈ and V of SEQ ID NO: 141 Ｌ , 28) V of SEQ ID NO: 11 Ｈ and V of SEQ ID NO: 142 Ｌ , 29) V of SEQ ID NO: 11 Ｈ and V of SEQ ID NO: 143 Ｌ , 30) V of SEQ ID NO: 11 Ｈ and V of SEQ ID NO: 144 Ｌ , 31) V of SEQ ID NO: 123 Ｈ and V of SEQ ID NO:29 Ｌ , 32) V of SEQ ID NO: 124 Ｈ and V of SEQ ID NO:29 Ｌ , 33) V of SEQ ID NO: 125 Ｈ and V of SEQ ID NO:29 Ｌ , 34) V of SEQ ID NO: 11 Ｈ and V of SEQ ID NO: 145 Ｌ , 35) V of SEQ ID NO: 126 Ｈ and V of SEQ ID NO:29 Ｌ , 36) V of SEQ ID NO: 11 Ｈ and V of SEQ ID NO: 146 Ｌ , 37) V of SEQ ID NO: 11 Ｈ and V of SEQ ID NO: 147 Ｌ , 38) V of SEQ ID NO: 11 Ｈ and V of SEQ ID NO: 148 Ｌ , 39) V of SEQ ID NO: 11 Ｈ and V of SEQ ID NO: 149 Ｌ , 40) V of SEQ ID NO: 127 Ｈ and V of SEQ ID NO:29 Ｌ , 41) V of SEQ ID NO:11 Ｈ and V of SEQ ID NO: 150 Ｌ , 42) V of SEQ ID NO:11 Ｈ and V of SEQ ID NO: 151 Ｌ , 43) V of SEQ ID NO: 128 Ｈ and V of SEQ ID NO:29 Ｌ or 44) V of SEQ ID NO: 129 Ｈ and V of SEQ ID NO:29 Ｌ 1. An isolated antibody comprising:

14. The antibody of any one of claims 1 to 13, wherein the antibody binds to an exosite of an APC.

15. The antibody according to any one of claims 1 to 14, 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.

16. The antibody of claim 15, wherein the antibody is an IgG4 antibody.

17. The antibody, a) enhance, minimally affect, or inhibit the anticoagulant activity of APC; b) enhances, minimally affects, or inhibits APC-mediated histone cleavage; c) enhance, minimally affect, or inhibit the endothelial barrier protective activity of APCs; d) increase, have minimal effect on, or decrease the plasma half-life of APC; or e) An antibody according to any one of claims 1 to 9, which is any combination thereof.

18. 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.

19. A nucleic acid encoding an antibody according to any one of claims 1 to 18.

20. A vector comprising the isolated nucleic acid of claim 19.

21. 21. A host cell comprising the nucleic acid of claim 19 or the vector of claim 20.

22. 1. A method for producing an antibody, comprising: a) culturing the host cell of claim 21 under conditions conducive to the production of said antibody; b) isolating said antibody from said host cell.

23. A pharmaceutical composition comprising the antibody of any one of claims 1 to 18 and a pharma- ceutically acceptable carrier.

24. 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 18, an antibody according to any one of claims 1 to 18 specifically binding to an exogenous APC protein or a variant thereof, or a pharmaceutical composition according to claim 23.

25. 25. The method of claim 24, wherein the condition is a condition that can be treated or prevented by enhancing or inhibiting the anticoagulant function of APC.

26. 26. The method of claim 25, 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.

27. 25. The method of claim 24, wherein the condition is a condition that can be treated or prevented by enhancing or inhibiting one or more cytoprotective functions of APCs.

28. The state is 28. The method of claim 27, 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.

29. An antibody according to any one of claims 1 to 18 for use as a medicament for treating or preventing a condition.

30. 30. The antibody of claim 29, wherein the condition is a condition that can be treated or prevented by enhancing or inhibiting the anticoagulant function of APC.

31. 31. The antibody of claim 30, 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 deficiencies, Glanzmann thrombasthenia, and Bernard-Soulier syndrome.

32. The antibody of claim 29, wherein the condition is a condition that can be treated or prevented by enhancing or inhibiting one or more cytoprotective functions of APCs.

33. The state is 33. The antibody of claim 32, 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.

Images