Camelidae animal 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 retaining its beneficial cytoprotective function. The 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 bleeding, sepsis, and inflammation.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 316,927, filed on March 4, 2022, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application includes a sequence listing in XML format, named "172085.00117_ST26.xml", 643,566 bytes in size and created on February 21, 2023. The sequence listing is submitted electronically with the application via the Patent Center and is incorporated herein by reference in its entirety. Technical Field

[0005] The present invention relates to antibodies, antibody fragments and other conjugates of activated protein C (APC) and related preparations, nucleic acids, preparation methods and use methods. Background Art

[0006] Protein C (PC) is a zymogen, synthesized in the liver as a single-chain 461 amino acid precursor (SEQ ID NO:633) and secreted into the blood. Before secretion, the single-chain precursor is converted into a heterodimer by removing a lysine-arginine dipeptide (amino acid 198-199 in SEQ ID NO:633) and a 42 amino acid pre-leader sequence (amino acid 1-42 in SEQ ID NO:633). The heterodimer form (417 amino acids) is composed of a light chain (155 amino acids, 21 kDa) and a heavy chain (262 amino acids, 41 kDa) connected by a disulfide bridge. The light chain comprises a γ-carboxyglutamic acid (Gla) domain (45 amino acids), two EGF-like domains (46 amino acids) and a linker sequence, while the heavy chain comprises a high polarity activation peptide (amino acid 200-211 in SEQ ID NO:633) and a catalytic domain with a typical serine protease catalytic triad. Cleavage of the PC zymogen at the thrombin cleavage site results in the removal of the activation peptide and activation of PC to activated PC (APC; SEQ ID NO: 634; 405 amino acids).

[0007] Human PC undergoes a number of post-translational modifications, including glycosylation, vitamin K-dependent γ-carboxylation, and γ-hydroxylation. It contains four potential N-linked glycosylation sites: one on the light chain (Asn97) and three on the heavy chain (Asn248, Asn313, and Asn329). Its Gla domain contains nine Gla residues and is responsible for the calcium-dependent binding of PC to negatively charged phospholipid membranes. The Gla domain can also bind to the endothelial protein C receptor (EPCR), aligning thrombin and thrombomodulin on the endothelial membrane during PC activation.

[0008] PC activation occurs on the surface of endothelial cells in a two-step process. It requires PC to bind to EPCR on endothelial cells (via the Gla domain), followed by proteolytic cleavage of PC at Arg12 of the heavy chain by the thrombin / thrombomodulin complex. This single cleavage releases a 12-amino acid activation peptide that converts the zymogen PC into the active serine protease APC.

[0009] Under physiological conditions, APC circulates in human blood at very low concentrations (1-2 ng / ml or 40 pM) with a half-life of 20-30 minutes, while PC circulates at much higher concentrations (3-5 μg / ml or 65 nM) with a half-life of 6-8 hours.

[0010] The PC pathway plays a key role in regulating blood coagulation, and by amplifying the anticoagulant reaction when the coagulation reaction is enhanced, it is used as a natural defense mechanism for thrombosis. After injury, thrombin is generated to start coagulation. Thrombin also triggers the anticoagulant reaction by binding to the thrombomodulin on the inner wall of the vascular surface, thereby promoting PC activation. Therefore, APC generation is related to thrombin concentration and PC level. APC plays the role of an anticoagulant by inactivating two coagulation cofactors (i.e., factor Va and factor VIIIa) via proteolytic cleavage, thereby inhibiting thrombin generation. APC also directly contributes to the enhancement of fibrinolytic reaction by forming a complex with plasminogen activator inhibitor.

[0011] Bleeding is a major problem in both medical conditions and in connection with medical procedures. For example, bleeding leads to complications following surgery, organ transplantation, intracranial hemorrhage, aortic aneurysm, postpartum hemorrhage, trauma, and overdose of certain anticoagulants. In fact, the leading cause of death in people aged 1-44 years is acute bleeding associated with trauma. There are currently few options for treating fatal non-compressible bleeding. Bleeding disorders can be treated by inhibiting APC to promote thrombin generation and coagulation. Therefore, a first object of the present invention is to provide a safe and effective treatment for bleeding.

[0012] In addition to their anticoagulant function, APCs also induce cytoprotection through their antiapoptotic, anti-inflammatory, and endothelial barrier-stabilizing activities, all of which contribute to the regenerative outcomes associated with APCs (e.g., stimulation of neurogenesis, angiogenesis, and wound healing).

[0013] Ischemia is a fatal condition due to insufficient blood supply to an organ or tissue. Insufficient blood supply leads to tissue hypoxia and can potentially lead to heart attack or stroke. In metabolically highly active tissues (such as the heart and brain), ischemia may cause irreversible damage in just 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 ischemic treatment.

[0014] Studies of human diseases and animal models have shown that extracellular histones are involved in the development or progression of inflammatory processes in multiple organs (Moiana et al., Clin Biochemistry 94 (2021) 12-19 at 15). Therefore, histones are being studied as potential therapeutic targets for diseases in which inflammation and thrombosis play a key pathophysiological role. APC reduces the cytotoxicity of extracellular histones by histone proteolysis. This function of APC depends on negatively charged exosites, including Glu330 and Glu333.

[0015] It is known that extracellular histones are the main mediators of death in sepsis. Sepsis is a fatal condition that occurs when the body's response to an infection causes damage to its own tissues and organs. Sepsis is a leading cause of morbidity and mortality in the United States, resulting in nearly 270,000 deaths each year. The cytoprotective effects of APCs include inhibition of histone toxicity associated with death in sepsis. Therefore, a third object of the present invention is to provide a safe and effective treatment for sepsis.

[0016] Histones constitute part of neutrophil extracellular traps (NETs), an extracellular fibrous network, mainly composed of neutrophil DNA. NET has been shown to be involved in the pathogenesis and progression of cancer. Cancer is the leading cause of death worldwide, causing nearly 10 million deaths or one-sixth of deaths in 2020. NET affects cancer through several mechanisms, including the establishment of an inflammatory microenvironment and interaction with other tumor-promoting mechanisms such as inflammasomes and autophagy (Shao et al., Frontiers in Oncology Vol. 11 No. 714357 (2021) Page 1). Evidence shows that NET plays a role in a variety of cancer types, including breast cancer, lung cancer, colorectal cancer, pancreatic cancer, blood cancer, neural cancer and skin cancer (Ibid., Page 6). Therefore, interventions affecting PC pathways can potentially affect cancer, and a fourth object of the present invention is to provide a safe and effective cancer treatment.

[0017] APC-based therapeutics have been under development for several years. Genetic engineering of APC has produced APC variants that selectively reduce their 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 the anticoagulant activity of wild-type APC while retaining its full cytoprotective activity and has entered clinical trials for ischemic stroke. However, previous attempts to translate research observations into APC-based drugs have failed, including initial attempts to use APC to treat sepsis. Although in 2001, encouraging clinical trial results led to the development of drugs based on recombinant APC proteins (drotrecogin alfa (activated), recombinant human activated protein C (rhAPC), trade name Xigris TM ) was approved (NEngl J Med 344:699, 2001), but the initial positive results were not replicated in subsequent randomized clinical trials, and the drug was withdrawn from the market.

[0018] A potential 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 generation of ADA may have serious consequences, such as loss of drug efficacy and neutralization or other adverse events (J Pharm Sci 110 (7): 2575-2584, 2021). Therefore, a fifth object of the present invention is to provide an APC-based therapeutic agent with a lower risk of immunogenicity.

[0019] Antibodies to APC have been explored as a means of selectively enhancing or inhibiting different functions of APC (see, e.g., Zhao et al., U.S. Patent Application Publication 20150307625, published on October 29, 2015 and Egner et al., U.S. Patent Application Publication 2018326053, published on November 15, 2018). For example, Magisetty et al. reported that murine antibodies that selectively inhibited the anticoagulant activity of APC while retaining its cytoprotective activity significantly reduced the severity of joint bleeding in a mouse model of hemophilic arthropathy, while antibodies that inhibited both the anticoagulant and cytoprotective activities of APC did not (Blood (2022) blood. 2021013119). In Zhao et al., two different antibodies to APC were evaluated as APC-based hemophilia treatments (Nat Commun 11(1):2992, 2020). One antibody, referred to as class I, targets the active site of APC, while the other antibody, referred to as class II, targets an external site of APC. When these antibodies were administered to monkeys, thrombin generation increased and plasma coagulation was promoted. However, while class II antibodies were well tolerated, class I antibodies caused animal death. However, class II antibodies still partially inhibited the active site of APC, suggesting that it may partially inhibit the cytoprotective effect of APC. Therefore, 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

[0020] In a first aspect, the invention provides an isolated antibody that specifically binds to activated protein C (APC) and minimally binds to inactivated protein C (PC). In some embodiments, the antibody comprises a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 317-474. In some embodiments, the antibody comprises a heavy chain variable domain (VD) selected from the group consisting of SEQ ID NOs: 475-632. H ).

[0021] In a second aspect, the invention provides nucleic acids encoding the antibodies disclosed herein.

[0022] In a third aspect, the invention provides a vector comprising a nucleic acid disclosed herein.

[0023] In a fourth aspect, the invention provides a host cell comprising a nucleic acid or a vector disclosed herein.

[0024] In a fifth aspect, the present invention provides a method for producing an antibody disclosed herein. The method comprises: (a) culturing a host cell disclosed herein under conditions leading to production of the antibody; and (b) isolating the antibody from the host cell.

[0025] In a sixth aspect, the present invention provides a pharmaceutical composition comprising an antibody disclosed herein and a pharmaceutically acceptable carrier.

[0026] In a seventh aspect, the present invention provides a method for treating or preventing a condition in a subject. The method comprises administering to the subject a therapeutically effective amount of an antibody disclosed herein, an antibody disclosed herein that specifically binds to an exogenous APC protein or a 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 cell protection function of APC.

[0027] In an eighth aspect, the present invention provides the use of an antibody disclosed herein as a medicament. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1A-1F The results of epitope binning and affinity analysis of nano antibodies LP1-LP21 against human activated protein C (APC) by biolayer interferometry (BLI) are shown. The binding of APC to nano antibodies is detected based on the spectral shift of reflected light on the sensor surface loaded with nano antibodies. The biosensor top with nano antibody-APC complex is then immersed in TPP-4885 Fab solution. Figure 1A 8 clones were found not to block TPP-4885Fab binding to APC (upper panel) and 13 clones were found to block binding based on the spectral shift. Figure 1B The results of additional binning experiments with nanobodies that do not block TPP-4885 Fab binding to human APC are shown. LP7, LP9, LP12 and LP18 were found not to compete with LP2, LP3 and LP10 for binding to APC. The values ​​given in the table represent the binding response under background (red = competition between analyte and ligand for APC, orange = competition but with measurable analyte binding, green = no competition). Figure 1C Shown are the results of cluster analysis, which identified 2 Nanobody clusters with an AU p-value cutoff of 95% (red = AU approximate unbiased p-value, green = bp bootstrap probability, grey = number of edges). Figure 1D Included are a table summarizing the Nanobody clusters (top panel) and a graph of the median CDR3 length for each cluster (bottom panel). Figure 1E Figure 2 shows the K values ​​of LP1-LP21 generated using BLI. D Figure 2. Graph of the measurement results. Each Nanobody was tested against 5 different APC concentrations. The R 2 The range is 0.92 to 0.99. Figure 1FRepresentative sensorgrams generated by antibody LP6 in the presence of BSA.

[0029] Figure 2A-2C The effect of Nanobodies LP1-LP21 on the anticoagulant activity of APC is shown. Figure 2A is protein C (PC)-depleted plasma diluted in normal human plasma (o) (to achieve PC levels ranging from 0.78% to 100%) The standard curve of clotting time is also shown in the absence of Normal human plasma (·) and PC-consumed plasma APTT clotting time. Figure 2B The results of two independent experiments (Screen 1 and Screen 2) were compared in the presence of Nanobodies (300 nM). Figure 2 shows the clotting time. Consistent ordering was observed with respect to coagulation. Figure 2C The average plasma concentration of each Nanobody is shown. Figure 2 shows the effect of Nanobodies on the clotting time and the percentage of APC activity (repeated in 2 independent experiments). The clotting time has multiple effects. Among the nanobodies, LP11, LP8 and LP20 showed the highest potency in inhibiting anticoagulant activity.

[0030] Figure 3A-Figure 3F The effect of nanobody LP1-LP21 on APC-mediated histone H3 cleavage is shown. APC (50nM) and nanobody (500nM) were pre-incubated together for 30 minutes. After incubation, the APC-nanobody mixture was added to histone H3 (100μg / mL). During 2 hours, samples were collected at different time points and added to reducing sample buffer for SDS-PAGE analysis. A 20kDa molecular marker was used as a reference for normalization between gels. Figure 3A is a SDS-PAGE gel showing the time course of histone H3 cleavage by APC in the absence of Nanobodies (representative of 3 experiments). Figure 3B is a plot of histone H3 cleavage versus time for the 9 nanobodies that had the least effect on APC-mediated H3 cleavage. The percentage of H3 cleavage was calculated by normalizing with the H3 band intensity at time = 0 min: 100% - (intensity t＝x min / strength t＝0min ). Figure 3C is a SDS-PAGE gel showing that Nanobody LP14 enhances H3 cleavage. Figure 3Dis a graph of histone H3 cleavage versus time for 12 Nanobodies that inhibit APC-mediated H3 cleavage. Figure 3E is a SDS-PAGE gel showing that Nanobody LP21 effectively inhibits H3 cleavage. Figure 3F The table presents the data shown in (B) and (D) expressed as numerical values.

[0031] Figure 4A-4E Shown are the effects of Nanobodies LP1-LP21 on APC-mediated cleavage of SEAP protease-activated receptor 1 (PAR1). Figure 4A is a series of graphs showing normalized SEAP-PAR1 cleavage for each nanobody at a range of nanobody concentrations. APC and nanobodies were added to HEK293 cells expressing SEAP-PAR1 and EPCR. It was found that nanobodies modulated APC-mediated PAR1 cleavage to varying degrees. LP8, LP17, LP19 and LP21 showed the most potent inhibition, and LP3, LP9, LP11 and LP18 showed the lowest inhibition even at the highest concentration of 500 nM (n=3 independent experiments). PAR1 cleavage is expressed as a percentage of total SEAP activity present on cells relative to background. Figure 4B is a table describing the R41Q-SEAP-PAR1 and R46Q-SEAP-PAR1 cell lines used to evaluate the cleavage pattern of APC (50 nM) at R46 and R41, respectively. Figure 4C Is a series of graphs showing the R46 / R41 cleavage ratios of nanobodies LP8 and LP20 that inhibit PAR1 cleavage. These nanobodies inhibit APC cleavage at R46 and R41 in a dose-dependent manner. At high concentrations of LP8 and LP20, the cleavage ratio shifts to less than 1, i.e., the cell protective PAR1 cleavage characteristics are low (n=3 independent experiments). Figure 4D Is a series of graphs showing the R46 / R41 cleavage ratios of Nanobodies LP11 and LP18 that retain PAR1 cleavage, which induce curvilinear cleavage reactions at R46 and R41. At all tested concentrations, the R46 / R41 cleavage ratios were above 1, indicating that LP11 and LP18 can enhance the cytoprotective effect of APC (n=3 independent experiments). Figure 4E The table presents the data shown in (C) and (D) expressed as numerical values.

[0032] Figure 5A-5H Shown is the effect of LP11 on APC-mediated endothelial barrier function. Figure 5Ais a graph of the normalized cell index (NCI) versus thrombin addition time (n=4), which demonstrates the barrier protection effect of APC (40 nM) against barrier permeability induced by thrombin (0.25 nM) in the presence of different concentrations of LP11. Figure 5B is a graph quantifying the data shown in (A) based on the lowest NCI value for each time course, with the effect of APC in the absence of LP11 set to 100% and the thrombin alone group set to 0%. Figure 5C is a graph showing transient endothelial barrier disruption shortly after APC-priming of EA.hy926 cells (n=4). Figure 5D is a graph quantifying the data shown in (C). Figure 5E is a graph showing inhibition of transient barrier disruption initiated by APC (40 nM) using the PAR1 antagonist vorapaxar (1 μM) or SCH79797 (1 μM) (n=3). Fig. 5F is a graph showing transient endothelial cell barrier disruption following inhibition of APC priming (40 nM) in the presence of LP11 (n=4). Figure 5G is a graph quantifying the data shown in (F). Figure 5H is a graph showing the overall characteristics of NCI changes plotted against the time of addition of APCs to cells (APC priming).

[0033] Figure 6A-6B Shown are purified Nanobodies (LP1-LP21) run on reducing SDS-PAGE gel (12% Bis-Tris and MOPS running buffer). Fig. 6A Gels with LP1 to LP7 are shown. Figure 6B Gels with LP8 to LP21 are shown.

[0034] Figure 7 A schematic outlining the process of selecting Nanobodies LP1-LP21 from the Nanobody library for further studies is shown.

[0035] Figure 8 ELISA screening of 158 unique APC-PPACK specific clones against APC, FXa-PPACK and PBS coated wells (n=2) is shown. Selective binding of these clones to APC relative to FXa-PPACK and PC was observed. Twenty-one nanobodies (named LP1 to LP21) belonging to 18 sequence diversity families were selected for further characterization.

[0036] Fig. 9Nanobody LP1-LP21 is shown for the ELISA screening results of the wells coated with APC, FVIIa, FIXa and FXIa. All 21 nanobodies observed selective binding to APC (n=3 independent experiments). The ELISA plate was coated with 10 μg / mL APC, FVIIa, FIXa or FXIa in 100 μL of PBS for 1 hour, followed by 200 μL PBS1% casein (pH7.2) blocking for 1 hour. The plate was washed 4 times with PBS-T (0.05%, pH 7.2). Nanobody (100 μL, 2 μg / mL, in PBS) was added to the plate. After incubation for 1 hour, the plate was washed 4 times with PBS-T and incubated with 1:25,000 HRP-anti-V5 (100 μL). After incubation for 1 hour, the plate was washed 4 times with PBS-T, and the amount of nanobody bound to the plate coated with APC was detected using TMB substrate. The reaction was terminated with 100 μL 2N H2SO4.

[0037] Fig.10 The effect of nanobodies on S-2366 cleavage by APC is shown. The effect of nanobodies on the amidolytic activity of APC against the small chromogenic substrate S-2366 was measured by the change in absorbance at 405 nm over time of a reaction consisting of APC (20 nM), nanobodies (0-80 nM) and S-2366 (1 mM) in HBS / 0.1% BSA. APC-mediated S-2366 cleavage was enhanced by 8 nanobodies, partially inhibited by 5 nanobodies, and unaffected by 8 nanobodies (n=3).

[0038] Fig.11 Lineweaver-Burke plots of APC-mediated S-2366 cleavage in the presence of Nanobodies that enhance or inhibit cleavage are shown (n=3). Reactions consisted of APC (16.7 nM), Nanobodies (67 nM) and S-2366 (0.075-1.2 mM) in HBS / 0.1% BSA.

[0039] Fig.12 Indicated in LP11 in the presence of PC activators shortened the clotting time in plasma prolonged by FVIII inhibitors (n=3).

[0040] Figure 13A-13C Characterization of LP11 expressed in mammalian cells is shown. Fig.13A is a SDS-PAGE gel showing purified mammalian LP11. Fig. 13B yes A graph of clotting time versus LP11 concentration showing the effect of mammalian LP11 on of inhibition. Fig. 13C is a set of graphs showing the effect of mammalian LP11 on cleavage in R41Q-SEAP-PAR1 and R46Q-SEAP-PAR1 cell lines.

[0041] Figure 14A-14B Summarizes the library panning experiments. Fig.14A is a table summarizing the library panning process. On the plate coated overnight at 4°C, a reduced APC-PPACK concentration (100, 50, 10 and 1 nM) and an increased number of washes and durations were used for consecutive rounds of selection (R1, R2, R3 and R4). Negative selection was performed with factor Xa-PPACK, PC, and blank wells were closed with 2% bovine serum albumin (BSA) to remove non-specific binders. The enrichment of APC-PPACK binders with minimal binding to factor Xa-PPACK and PC was confirmed by phage pool ELISA between these rounds. Phages bound to APC-PPACK were eluted using 100 mM triethylamine and subsequently neutralized with 1 M Tris-HCl (pH 7.5). The eluted phages were used to infect TG1 cells. The output was titrated and plated on 2YTCG (2YT medium; 50 μg / mL carbenicillin, 2% glucose) plates and grown overnight at 30°C. A second set of panning (R2' and R3') was performed with a more stringent negative selection against PC. CFU, colony forming units. Fig. 14B is a table summarizing the phage pool output from panning rounds R2, R3, R4, and R3'.

[0042] Fig.15 Comparison of the structure of conventional antibodies with camelid antibodies and V H Schematic representation of the structure of the H antibody. DETAILED DESCRIPTION

[0043] The present disclosure relates to antibodies that specifically bind to activated protein C (APC). In preferred embodiments, the antibodies bind to the activated form of the enzyme (i.e., APC) and minimally bind to the zymogen form of the enzyme (i.e., protein C (PC)). In some embodiments, the disclosed antibodies inhibit the anticoagulant activity of APC while at least partially retaining or even enhancing the pleiotropic cytoprotective functions of APC. In some embodiments, the disclosed antibodies enhance the cytoprotective functions of APC while partially or completely retaining the anticoagulant activity of APC. The present invention also provides polynucleotides encoding these antibodies, pharmaceutical compositions comprising these antibodies, methods for preparing these antibodies, and methods for treating conditions by administering a therapeutically effective amount of these antibodies.

[0044] The anti-APC antibodies of the present invention provide several advantages over recombinant APC protein therapeutics currently on the market or under development. For example, anti-APC antibodies have a lower risk of immunogenicity than therapeutics containing exogenous APC proteins and variants thereof (e.g., 3K3A-APC). Anti-drug antibodies (ADA) directed against exogenous APC proteins may cross-react with endogenous PC / APC proteins, leading to autoimmune reactions. For example, the APC variant 3K3A-APC from ZZ Biotech, LLC is in clinical trials, in which it is being administered to patients in large repeated doses to treat ischemic stroke (ClinicalTrials.gov Trial Id. NCT02222714). This can effectively immunize patients to produce anti-APC antibodies that can cross-react with endogenous APC. In contrast, ADA directed against anti-APC antibodies should not cross-react with endogenous PC / APC proteins, making the use of anti-APC antibodies a safer treatment strategy. Furthermore, depending on the antibody format selected, fewer doses of anti-APC antibodies may be required to achieve the desired therapeutic effect compared to the number of doses required for APC protein-based drugs.

[0045] Antibody:

[0046] In a first aspect, the present invention provides an antibody that specifically binds to an APC. As used herein, the term "antibody" refers to a protein that comprises at least one antigen binding domain from an immunoglobulin. The term encompasses full-length immunoglobulins and antigen binding fragments thereof.

[0047] As mentioned 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., heavy chain variable regions (V H ), light chain variable region (V L ), a monovalent fragment consisting of the constant domain of the kappa light chain (CL) and the first constant domain of the heavy chain (CH1); (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 monovalent fragment consisting of the constant domain of the V H and CH1; (iv) a fragment consisting of the V L and V H (v) dAb fragment (Nature 341: 544-546, 1989), which is composed of V H (vi) isolated complementarity determining regions (CDRs);

[0048] (vii) minibodies, diabodies, triabodies, tetrabodies and kappa bodies (see, e.g., Protein Eng 10:949-57, 1997); (viii) fragments of camelid antibodies, including V H H antibody, V H H dimer and V H H-Fc fusions; (ix) fragments of cartilaginous fish antibodies, including VNAR antibodies.

[0049] In some embodiments, the antibody is selected from the group consisting of IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, IgM antibodies, IgA1 antibodies, IgA2 antibodies, secretory IgA antibodies, IgD antibodies, IgE antibodies, and antigen-binding fragments thereof. In other embodiments, the antibody comprises an alternative scaffold, such as a non-immunoglobulin binding protein (e.g., Affilin TM , Alphabody Atrimer,Avimer,Centyrin, Fynomer, Kunitz domain, OBody, In some embodiments, the antibody is in the form of an IgG4. This form provides therapeutic advantages because IgG4 is the only IgG subclass that does not mediate common IgG effector functions (such as antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity).

[0050] In a preferred embodiment, the antibody is a nanobody. As used herein, the term "nanobody" refers to an antibody fragment consisting of a single monomeric variable antibody domain. Nanobodies may also be referred to as "single domain antibodies". In conventional antibodies, the antigen binding region consists of the heavy chain variable region (V H ) and light chain variable region (V L ). Therefore, nanobodies can be made from a single variable region (V H or V L ) are obtained. However, Nanobodies can also be obtained from heavy chain antibodies. "Heavy chain antibodies" or "heavy chain only antibodies" are antibodies composed of two heavy chains and lacking the two light chains found in conventional antibodies. Heavy chain antibodies use only V H The V domain of the heavy chain antibody binds to the antigen. H The V domain is an excellent candidate for use as a nanobody. Heavy chain antibodies are naturally produced by camelids (e.g., llamas, alpacas, and camels) and cartilaginous fish (e.g., sharks). Camelids produce heavy chain antibodies, from which the V domain can be obtained. HNanobodies are single domain antibodies of H antibodies, and cartilaginous fish produce heavy chain antibodies, known as immunoglobulin new antigen receptors (IgNARs), from which single domain antibodies known as VNAR antibodies can be obtained. Thus, in some embodiments, Nanobodies are V H H antibody or VNAR antibody.

[0051] The antibody library screened by the inventors in the examples is a V H Therefore, in a preferred embodiment, the antibodies of the present invention are camelid V H H antibodies. Camelid antibodies contain two heavy chains that lack the first constant Ig domain (CH1) found in typical heavy chains ( Fig.15 The variable part of these camelid heavy chains is called the single variable domain (V H H). Therefore, the V H The nanobody composed of the H part is called "V H H antibody". V H The H antibody has a V H Similar structural architecture and comprises four conserved framework regions (FR1 / 2 / 3 / 4) surrounding three hypervariable antigen binding loops (CDR1 / 2 / 3) called complementarity determining regions ( Fig.15 ). Therefore, V H The paratope of the H antibody consists of (1) three CDRs that directly contact the antigen and participate in antigen binding and (2) four framework regions that maintain the overall structure. H The H antibody can be prepared using methods known in the art (see, e.g., U.S. Pat. No. 6,765,087, U.S. Pat. No. 6,838,254, WO 06 / 079372).

[0052] Recently, V-based H For example, the first V-based H For a variety of reasons, V H H antibodies are very suitable for use as therapeutic agents. First, V H The H antibody is very robust, providing a long shelf life at 4°C and -20°C, resistance to proteolytic degradation, and tolerance to elevated temperatures (60°C-80°C, or several weeks at 37°C), exposure to non-physiological pH (pH range 3.0-9.0), high pressure (500-750 MPa), and chemical denaturants (2-3 M guanidine chloride, 6-8 M urea). Notably, V H The robustness of H antibodies is attributed to their efficient refolding after chemical or thermal denaturation.H The monomeric structure of H antibodies and their lack of post-translational modifications allow them to be expressed in microbial systems, including Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. H H antibodies can be produced at low cost by producing them in shake flasks at milligrams per liter of culture. H The H antibody has low immunogenicity and is therefore suitable for human administration. H H antibody and human immunoglobulin family III V H The sequence identity between the camelid germline IGHV family 3 and its human FR counterpart is greater than 80%. It was found that the camelid germline IGHV family 3 has 95% sequence identity with its human FR counterpart. However, if desired, the V H H antibodies can be "humanized". Fourth, compared with traditional antibodies, V H H antibodies provide increased flexibility for antigen recognition. Within the variable domain, CDR3 is the major contributor to antigen recognition and specificity, while CDR1 and CDR2 contribute to binding strength. H The average length of the CDR3 of H antibodies is 18 amino acids, which is larger than that of mouse or human antibodies, respectively. H The CDR3 domain is significantly longer, with an average of 12 or 14 amino acids. H The extended CDR3 loops of the H antibody can form finger-like structures or convex paratopes, which can penetrate into small cavities or interact with concave surfaces on surface antigens, respectively. This allows V H H antibodies target epitopes that are inaccessible to conventional antibodies. This increased flexibility allows for increased specificity, making some V H The H antibody can distinguish between different isoforms of the same protein. This increased specificity can be used to develop antibodies that specifically recognize APC (rather than PC) because these proteins differ by only 12 amino acids. H The small size of H antibodies allows high tissue penetration, and some nanobodies have been reported to even cross the blood-brain barrier.

[0053] 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 APC. Preferably, the antibody specifically binds to a human APC (which has an amino acid sequence of SEQ ID NO: 634) or a variant thereof. As used herein, the term "specific binding" refers to the ability to bind to a specific antigen (e.g., APC) in preference to other molecules. Typically, an antibody that exhibits specific binding has a binding affinity of at least about 10 -5 The equilibrium dissociation constant (KD ) binds to the antigen with a higher affinity (e.g., at least two times higher) than its binding affinity to an unrelated protein (e.g., BSA, casein). Generally, a higher affinity (i.e., a lower K D , for example, in the low nanomolar range) into a more effective and specific therapeutic agent because it increases the probability that the antibody will localize and bind to its target antigen.

[0054] 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 given the 1700-fold difference in plasma concentrations of APC (about 40 pM) and PC (70 nM). Therefore, it is preferred that the antibodies of the invention minimally bind to PC. An antibody that "minimally binds" to a particular antigen (a) does not bind to the antigen at a detectable level, or (b) binds to the antigen at a level less than about 10 -2 The equilibrium dissociation constant (K D ) binds to the antigen.

[0055] The term "Protein C" or "PC" may refer to any variant, isoform or homolog of the zymogen PC. Preferably, PC is human PC (which has the amino acid sequence of SEQ ID NO: 633) or a variant thereof.

[0056] The term "epitope" refers to a region of an antigen to which an antibody specifically binds. For examples of APC epitopes, see U.S. Patent Application Publication 2018 / 326053, which describes an epitope outside the catalytic triad of the active site of human APC. Conversely, the term "paratope" refers to a region of an antibody to which an antigen specifically binds.

[0057] Antibody binding activity can 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, to ensure that the antibodies disclosed herein have an association constant (i.e., k) suitable for use as human therapeutics, on ), and these antibodies were evaluated by BLI analysis.

[0058] In some embodiments, the antibodies of the present invention inhibit the anticoagulant activity of APC (i.e., relative to the anticoagulant activity of the no antibody control), thereby promoting blood clot formation. In some embodiments, the antibody inhibits 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 antibody can play this role by blocking the ability of APC to inactivate coagulation factors Factor Va and Factor VIIIa, thereby increasing the generation of thrombin. Suitable assays for assessing the ability of a specific 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 reduce clotting time can be measured using an activated partial thromboplastin time (APTT) coagulation assay. For a description of these assays, see Nat Commun 11(1):2992, 2020. In the examples, the S-2366 cleavage assay and The modified APTT assay tested the ability of 21 anti-APC antibodies to inhibit the anticoagulant activity of APC (see Figures 2, Fig.10 , Fig.11 and Table 6).

[0059] For some indications, it may be preferred to use an anti-APC antibody that maintains or enhances the anticoagulant activity of APC. For example, such an antibody may be used to treat patients with cancer-related thrombosis, which is a major cause of death in cancer patients. Thus, in other embodiments, the antibodies of the invention enhance or minimally affect the anticoagulant activity of APC.

[0060] As used herein, an antibody "minimall 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 "minimall affects" an APC activity is an antibody that has no detectable effect on APC activity (i.e., an antibody that has the same level of APC activity in the presence of the antibody 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.

[0061] For most human therapeutic agents, anti-APC antibodies are preferred to maintain or enhance the pleiotropic 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 leading to systemic inflammation. Therefore, the histone cleavage function of APC reduces apoptosis, endothelial barrier destruction and inflammation.

[0062] However, for some indications, the use of anti-APC antibodies that block the cytoprotective function of APCs may be preferred. For example, short-term disruption of endothelial barrier function can be used to temporarily increase vascular permeability to induce inflammation for therapeutic purposes. For cancer treatment, blocking cytoprotective activity to promote an immune response against tumors may be advantageous.

[0063] Thus, in some embodiments, the antibodies of the present invention enhance, minimally affect or inhibit the cytoprotective function of APC (i.e., relative to the cytoprotective function of the no antibody control). Specifically, in some embodiments, the antibody (1) enhances, minimally affects or inhibits APC-mediated histone cleavage, (2) enhances, minimally affects or inhibits APC-mediated protease activated receptor 1 (PAR1) cleavage at residue R46, (3) inhibits, minimally affects or inhibits APC-mediated PAR1 cleavage at residue R41; (4) increases, minimally affects or decreases the ratio of APC-mediated PAR1 cleavage at residue R46 to APC-mediated PAR1 cleavage at residue R41; and / or (5) enhances, minimally affects or inhibits APC-mediated endothelial barrier protection. In the examples, the effects of 21 anti-APC antibodies on APC-mediated histone cleavage were tested using a histone H3 cleavage assay (see FIG. 3 ). In addition, the effects of these 21 antibodies on PAR1 cutting were tested using secreted embryonic alkaline phosphatase (SEAP)-PAR1 cutting assay (see Figure 4). Using this assay, two antibodies (i.e., LP11 and LP18) were identified that preferentially inhibit APC-mediated PAR1 cutting at residue R41 (compared to residue R46), which enhances cell protective PAR1 signaling. Finally, an anti-APC antibody (i.e., LP11) was tested for the effects of APC-mediated endothelial barrier protection using an in vitro endothelial barrier function assay (see Figure 5).

[0064] In some embodiments, the antibodies of the invention increase or decrease the half-life of an APC (i.e., relative to the half-life in the absence of the antibody). In other embodiments, the antibodies minimally affect the half-life of an APC. In the Examples, 21 anti-APC antibodies were tested for their effects on the half-life of an APC using an in vitro plasma half-life assay (see Table 8).

[0065] The antibodies disclosed herein bind to an exosite of APC (i.e., a site other than the active site). The antibodies are generated against an active site-blocked APC protein, referred to herein as "APC-PPACK," which comprises an APC bound to its active site by the tripeptide inhibitor Phe-Pro-Arg-chloromethylketone (PPACK). Antibodies targeting the exosite of APC have been shown to inhibit the antithrombotic activity of the enzyme while retaining its beneficial cytoprotective functions (Nat Commun 11(1):2992, 2020). Separation of the anticoagulant and cytoprotective functions of APC is possible because these functions involve different sites on the protein surface, i.e., the amino acids that mediate the interaction of APC with cofactors and substrates are present in exosites away from the active site.

[0066] The antibodies disclosed herein were customized by panning H H phage library, select antibodies that bind to human APC-PPACK and do not select antibodies that bind to human PC and XPPACK to identify. "XPPACK" is another serine kinase bound by PPACK. Therefore, selection against XPPACK reduces the possibility that the selected antibody has affinity for PPACK instead of APC. H H phage libraries were constructed from heavy chain antibodies generated by immunizing llamas with APC-PPACK. These libraries contained only the V H The H segments, although very small (usually 12-14 kDa), still provide full antigen binding potential. Each V H Sequences of the H antibody and its CDRs. The sequence identifiers of the protein sequences of the 158 unique anti-APC antibodies identified in the Examples are listed in Table 1. The sequence identifiers of the DNA sequences of the top 21 antibodies selected for further analysis by functional assays are listed in Table 2.

[0067] Table 1. CDRs and full length V sequences for each of the 158 unique anti-APC antibodies disclosed herein. H Sequence identifiers for the amino acid sequences of the H regions. The names of antibodies selected for further analysis in the Examples are highlighted in bold, and the new names given to these antibodies (ie, LP1-LP21) are shown in parentheses.

[0068]

[0069]

[0070]

[0071] Table 2. CDRs and full-length V sequences of 21 antibodies selected for further analysis in functional assays H Sequence identifier of the DNA sequence of the H region.

[0072]

[0073]

[0074] The antibodies of the present invention are "isolated", meaning that they are substantially free of other biomolecules, including antibodies with different antigenic specificities and other cellular material. In some embodiments, the isolated antibodies are 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, isolated antibodies that bind to human APCs may have cross-reactivity with other closely related antigens (e.g., APC homologs from other species).

[0075] The term "complementarity determining region" or "CDR" refers to the hypervariable regions that together form an antigen binding surface that is complementary to the three-dimensional structure of the antigen. H and V L Contains three complementary determining regions. CDR from V H or V L The numbers are "CDR1", "CDR2" and "CDR3" starting from the N-terminus of the heavy chain (see Proc Natl Acad Sci USA 72(12):5107, 1975; J Exp Med 132(2):211, 1970). Thus, in a conventional antibody (i.e., an antibody comprising two heavy chains and two light chains), the antigen binding site comprises six CDRs: V H The three CDRs and V L However, in heavy chain antibodies, the antigen binding site only contains V H The three CDRs ( Fig.15 ).

[0076] Because CDR sequences are responsible for most of the antibody-antigen interactions, recombinant antibodies that simulate the properties of specific naturally occurring antibodies can be created by grafting the CDR sequences of naturally occurring antibodies into the framework sequences of different antibodies with different properties (see, e.g., 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 including germline antibody gene sequences. Therefore, in some embodiments, the CDRs of the antibodies described herein are grafted into another antibody framework.

[0077] The CDRs of SEQ ID NOs: 1-474 disclosed herein are the camelid-derived VDRs selected in the screen described in the Examples. H The CDRs of the H antibody. Of the three CDRs, CDR3 is considered to be the main contributor to antigen recognition and specificity, and CDR1 and CDR2 are considered to contribute to binding strength (J Mol Biol 430:4369, 2018; Protein Eng Des Sel 31:267, 2018; Proteins 86:697, 2018). Therefore, in some embodiments, the antibodies of the present invention comprise the V disclosed herein. H CDR3 of H antibody. Specifically, in some embodiments, the antibody of the present invention comprises a CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 317-474.

[0078] In other embodiments, the antibodies of the invention further comprise a V disclosed herein. H CDR1 and CDR2 of H antibody. Specifically, these antibodies further comprise (a) CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-158, (b) CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 159-316, or (c) CDR1 and CDR2, the CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-158, the CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 159-316.

[0079] In certain embodiments, the antibodies of the invention comprise all of the individual V sequences disclosed herein. H In other words, these antibodies comprise the V HThe paratope of the H antibody (i.e., a set of three CDRs forming an antigen binding region). Specifically, these antibodies comprise a V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 475-632. H CDR1, CDR2 and CDR3 of H antibody.

[0080] In addition, the present invention provides a V disclosed herein H H or antibodies consisting thereof. Specifically, these antibodies comprise a V selected from the group consisting of SEQ ID NOs: 475-632. H H or consisting thereof.

[0081] 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 deletions, additions, and substitutions of one or more amino acid residues. The antibodies of the present invention may comprise any combination of amino acid modifications, as long as they retain the ability to bind to APCs while minimally or not binding to PCs. 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 selected from SEQ ID NOs: 475-632. H H variants comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications relative to the parent antibody. In other embodiments, the antibody comprises a CDR3 variant selected from SEQ ID NO: 317-474 comprising at least 1 amino acid modification.

[0082] In some embodiments, one or more amino acid modifications are conservative substitutions. As used herein, the term "conservative substitution" refers to an amino acid substitution that substantially retains the structure and function of a native polypeptide. Specifically, conservative substitutions generally maintain the structure of the polypeptide backbone around (a) the substitution (e.g., as a beta sheet or alpha spiral), the charge or hydrophobicity of the molecule at the substitution site, and / or the main body of the side chain (c). Examples of conservative substitutions are shown in Table 3.

[0083] Table 3. List of conservative amino acid substitutions

[0084]

[0085]

[0086] In some embodiments, an antibody of the invention comprises a V 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: 475-632. H H or consists of. In some embodiments, the antibody comprises a CDR3 having at least 99, 98, 97, 96, 95, 94, 93, 92, 91 or 90 percent sequence identity to one of the amino acid sequences of SEQ ID NOs: 317-474.

[0087] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window. Aligned sequences may include additions or deletions (i.e., gaps) relative to each other for optimal alignment. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences; dividing the number of matched 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 a search tool based on a local alignment algorithm ("BLAST") 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-partition 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-ranking segment pairs is evaluated using a statistical significance formula (Proc. Natl. Acad. Sci. USA (1990) 87: 2267-2268). The BLAST program can be used with default parameters or modified parameters provided by the user.

[0088] The present invention further provides antibodies that compete with the antibodies described herein for binding to APC. Like other antibodies described herein, these antibodies specifically bind to APC and bind to PC to a minimum extent. If the binding of one antibody causes the binding of other antibodies to decrease, the antibody is considered to "compete" for the binding of a specific epitope with another antibody. Competition can occur because antibodies bind to the same epitope, or because the binding of one antibody spatially interferes with the binding of other antibodies or causes conformational changes that interfere with the binding of other antibodies. In some cases, the first antibody can inhibit the binding of the second antibody to its epitope, while the second antibody does not inhibit the binding of the first antibody to its epitope. However, in the case where the first antibody and the second antibody detectably inhibit the binding of other antibodies (whether to the same, greater or lesser extent), the antibodies are considered to "cross-compete" with each other to bind to their epitopes. The present invention encompasses antibodies that compete or cross-compete with the antibodies described herein for binding to APC.

[0089] Nucleic acids, vectors and host cells:

[0090] The antibodies of the present invention can be produced by introducing nucleic acids encoding the antibodies into host cells and providing conditions suitable for protein expression. Therefore, in a second aspect, the present invention provides nucleic acids encoding the antibodies disclosed herein. Specifically, the present invention includes nucleic acids encoding the following: (1) comprising V H H antibody, the V H H comprises or consists of an amino acid sequence selected from SEQ ID NOs: 475-632, (2) an antibody comprising a CDR3 comprising or consists of an amino acid sequence selected from SEQ ID NOs: 317-474, or (3) any other antibody described herein. In some embodiments, the nucleic acid comprises (1) a nucleic acid encoding a CDR1 selected from SEQ ID NOs: 635-655, (2) a nucleic acid encoding a CDR2 selected from SEQ ID NOs: 656-676, and / or (3) a nucleic acid encoding a CDR3 selected from SEQ ID NOs: 677-697. In some embodiments, the nucleic acid encodes a V selected from SEQ ID NOs: 698-718. H H.

[0091] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to polymers of DNA or RNA that may be single-stranded or double-stranded, synthetic, or obtained (e.g., isolated and / or purified) from natural sources. Nucleic acids may contain natural, non-natural, or altered nucleotides, and may contain natural, non-natural, or altered internucleotide bonds (e.g., phosphoramidate bonds or phosphorothioate bonds). In some embodiments, nucleic acids of the invention are "isolated," meaning that they are separated from other cellular material.

[0092] 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 transport a specific DNA segment (i.e., a DNA segment contained in a vector) to a host cell. Some vectors are capable of autonomous replication in a host cell (e.g., bacterial vectors and additional mammalian vectors containing a replication origin). Other vectors can be integrated into the genome of a host cell so that they replicate with the host genome (e.g., viral vectors and transposons). The vector may contain heterologous genetic elements necessary for vector propagation or expression of an encoded gene product (e.g., a promoter). The 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. Vectors suitable for use in the present invention include DNA segments encoding antibodies described herein and heterologous sequences that allow the encoded antibodies to be expressed.

[0093] In a fourth aspect, the invention provides host cells comprising nucleic acids and vectors disclosed herein. The term "host cell" means a transgenic cell in which heterologous DNA can be expressed. Nucleic acids or vectors disclosed herein can be introduced into host cells using standard techniques, including, for example, electroporation, heat shock, lipofection, microinjection, and particle bombardment. It is generally advantageous to express antibodies in eukaryotic host cells to ensure that they are properly modified, folded, and secreted. However, because the V H H antibodies have a monomeric structure and lack post-translational modifications, so they can be easily produced by prokaryotic cells such as Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris.

[0094] In a fifth aspect, the present invention also provides a method for producing antibodies using the host cells disclosed herein. The method comprises: (a) culturing the host cells disclosed herein under conditions that result in antibody production; and (b) isolating the antibodies from the host cells. In these methods, antibodies are produced by culturing the host cells for a sufficient time to allow the antibodies to be expressed in the host cells. Standard protein purification methods can then be used to recover the antibodies from the cell culture, 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, e.g., Nat Methods 5(2):135–146, 2008).

[0095] Pharmaceutical composition:

[0096] In a sixth aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of an antibody disclosed herein and a pharmaceutically 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 can be aqueous or non-aqueous solutions, suspensions, and 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, alcohol / water solutions, emulsions, and suspensions, including saline and buffered media.

[0097] The compositions of the invention may further comprise diluents of various pH, ionic strength and buffering components (e.g., Tris-HCl, acetate, phosphate); additives to prevent absorption to surfaces; detergents (e.g., Tween 20, Tween 80, Pluronic F68); solubilizers (e.g., glycerol, polyethylene glycol); antioxidants (e.g., ascorbic acid, sodium metabisulfite, L-methionine); fillers; or tonicity modifiers (e.g., sucrose, mannitol). In the compositions, the antibodies may be covalently linked to polymers (e.g., polyethylene glycol), complexed with metal ions, or incorporated into or on particulate preparations of polymeric compounds (e.g., polylactic acid, polyglycolic acid, hydrogels), or incorporated into liposomes, microemulsions, micelles, multilamellar vesicles, erythrocyte ghosts, or spheroplasts. The inclusion of such compounds in the compositions will affect the physical state, solubility, stability, in vivo release rate, and in vivo clearance rate. The compositions can also be formulated in lipophilic depots (eg, fatty acids, waxes, oils) for controlled or sustained release.

[0098] method:

[0099] In a seventh aspect, the present invention provides a method for treating or preventing a disease, comprising administering to a subject a therapeutically effective amount of an antibody or pharmaceutical composition disclosed herein.

[0100] The antibodies of the present invention can be optionally administered in combination with exogenous APC proteins to confer additional properties that cannot be achieved by binding of the antibodies to endogenous APC proteins. Therefore, in some embodiments, the method comprises administering to the subject an exogenous APC protein or variant thereof (e.g., 3K3A-APC) that specifically binds to one or more antibodies disclosed herein.

[0101] As used herein, the term "treatment" describes the management and care of a patient for the purpose of combating a condition. Treatment includes administering 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 having certain characteristics and / or symptoms. The term condition is meant to encompass diseases, illnesses, syndromes, and the like.

[0102] As used herein, the term "prevention" describes the management and care of a patient with the purpose of preventing the onset of symptoms or complications of a disorder.

[0103] As used herein, the term "administering" refers to a method of providing a drug 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, inhalation administration, nasal administration, topical administration, intravaginal administration, ocular administration, intraauricular 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 may be continuous or intermittent. In some embodiments, administration is systemic rather than local.

[0104] The term "therapeutically effective amount" refers to an amount sufficient to achieve a beneficial or desired biological or clinical result. The result can be a reduction, alleviation, inhibition or prevention of 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 blood clot 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 skilled in the art. The therapeutically effective amount will vary with several factors, including, for example, the formulation of the composition for therapy, the purpose of the therapy, and the subject to be treated. A therapeutically effective amount of the composition can be delivered by single or multiple administrations. For example, a suitable daily dose may be in 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.

[0105] In some embodiments, the method of the present invention is used to treat or prevent a condition that can be treated or prevented by enhancing or inhibiting the anticoagulant function of APC. For example, in some embodiments, the method is used to treat a condition in which blood coagulation is desired by inhibiting the anticoagulant function of APC. Suitable conditions where blood coagulation is desired include, but are not limited to, bleeding (e.g., intracranial hemorrhage, diffuse alveolar hemorrhage, intracerebral hemorrhage), contusions (e.g., cerebral contusions), burns, gastrointestinal bleeding, uncontrolled bleeding, bleeding caused by transplantation (e.g., stem cell transplantation, liver transplantation) or resection, bleeding caused by surgery (e.g., cardiac surgery, spinal surgery, orthopedic surgery, neurological surgery, tumor surgery, or postpartum surgery), varicose bleeding, thrombocytopenia, idiopathic thrombocytopenic purpura, hemophilia, aortic aneurysm, reversal of anticoagulants or antithrombotics (e.g., warfarin, heparin), bleeding caused by trauma (e.g., penetrating trauma or blunt trauma), menorrhagia, bleeding in cirrhosis (e.g., active varicose or non-varicose), coagulation factor deficiencies (e.g., factor VII), Glanzmann's thrombasthenia (e.g., platelet transfusion refractoriness), and Bernard-Soulier syndrome. syndrome). In some embodiments, the disorder is an acute bleeding disorder. In some embodiments, the disorder is an inherited bleeding disorder.

[0106] 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 cytoprotective functions of an APC. For example, in some embodiments, the method can be used to treat or prevent sepsis (Biochem Soc Trans 43:691-5, 2015), inflammation in acute ischemic disease (e.g., by providing neuroprotection in ischemic stroke (Ann Neurol 85:125-136, 2019) or 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 SOD1 mutation (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 method can be used to treat or prevent central nervous system injury (e.g., spinal cord ischemia), ischemic stroke, Alzheimer's disease, acute kidney injury, lung disease (e.g., acute lung injury, acute respiratory distress syndrome) or acute pancreatitis (Zhao et al. Int. J. Mol. Sci. 2019, 20, 903, page 12 in 20).In some embodiments, the method is used to treat or prevent a disorder associated with histones or NETs, ​​such as cancer (e.g., breast cancer, lung cancer, colorectal cancer, pancreatic cancer, blood cancer, neural cancer, skin cancer) or an inflammatory disease or autoimmune disease (e.g., psoriasis, rheumatoid arthritis, systemic lupus erythematosus).

[0107] The "subject" to which the method is applied can be a mammal or a non-mammal, such as a bird. Suitable mammals include, but are not limited to, humans, cattle, horses, sheep, pigs, goats, rabbits, dogs, cats, bats, mice, and rats. In certain embodiments, the method can be performed on laboratory animals (e.g., mice, rats) for research purposes. In other embodiments, the method is used to treat commercially important farm animals (e.g., cattle, horses, pigs, rabbits, goats, sheep, chickens) or companion animals (e.g., cats, dogs). In a preferred embodiment, the subject is a human.

[0108] In an eighth aspect, the present invention provides the use of an antibody disclosed herein as a medicament. The medicament can be used to treat or prevent (1) a condition that can be treated or prevented by inhibiting the anticoagulant function of an APC, and / or (2) a condition that can be treated or prevented by enhancing one or more pleiotropic cytoprotective functions of an APC. Examples of such conditions are provided above.

[0109] The results of the functional assays provided in the embodiments can be used to determine whether a specific anti-APC antibody described herein can be used for a specific indication. For example, antibodies that inhibit the anticoagulant activity of APC while maintaining its cytoprotective activity can be used to treat diseases that require blood coagulation (e.g., acute bleeding). For such indications, the importance of maintaining the cytoprotective activity of APC was demonstrated 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 the anticoagulant and cytoprotective activity of APC. The results of this study indicate that retaining the cytoprotective activity of APC can be used to treat hemophilic arthropathy. Although no significant differences were observed between the ability of the two antibodies to inhibit the anticoagulant activity of APC, the administration of MAPC1591 (rather than MPC1609) significantly reduced the severity of hemophilic arthropathy. Antibodies that enhance the cytoprotective activity of APCs can be used to treat diseases such as inflammation in sepsis and acute ischemic disease. Table 7 provides some examples of potential uses of specific antibodies based on functional assay results.

[0110] Example

[0111] Activated protein C (APC) is a multi-effect coagulation protease with anticoagulant, anti-inflammatory and cytoprotective activities. The selective regulation of these APC activities helps us understand the regulation of these physiological mechanisms and allows the development of therapeutic agents for pathologies associated with these pathways. As described in the following examples, a llama-derived nano antibody library targeting the inactive site of APC was generated. 21 nano antibodies that selectively recognize APC compared to protein C (PC) zymogen were identified from the library. In general, three nano antibody clusters were identified based on competition to APC in the study of biolayer interference technology. The diversity of antibodies was understood using APC functional assays (i.e., anticoagulant activity, histone H3 cleavage and protease activated receptor 1 (PAR1) cleavage). These functional assays revealed the APC activity characteristics induced by 13 new nano antibodies by selectively regulating the APC multi-effect activity, with the potential for regulating specific mechanisms for therapeutic purposes. Among these antibodies, three nano antibodies (LP2, LP8, LP17) inhibited all three APC functions. Four nano antibodies (LP1, LP5, LP16, LP20) only inhibit two of the three APC functions. Only two nano antibodies (LP9, LP11) observed a single functional inhibition specifically for APC anticoagulant activity. It was also found that LP11 transformed the ratio of PAR1 cutting at R46 relative to R41 of APC, which resulted in APC-mediated biased PAR1 signaling and APC cell protection. Therefore, LP11 has an active feature that is believed to promote hemostasis and cell protection of bleeding associated with hemophilia or coagulopathy by selectively regulating APC anticoagulation and PAR1 cutting features.

[0112] Import:

[0113] APC is a serine protease with physiological functions in anticoagulation and cytoprotection (1,2). APC circulates in human blood at very low concentrations (40 pM), while its zymogen protein C (PC) circulates at much higher concentrations (65 nM) (3). Thrombin / thrombomodulin activates PC by proteolytic cleavage at Arg169 and release of a 12 amino acid peptide. Once hemostasis is achieved, APC downregulates coagulation by inactivating factor Va (FVa) and factor VIIIa (FVIIIa) to terminate thrombin generation. This role in anticoagulation makes APC a potential therapeutic target for bleeding. Reduced bleeding was observed in hemophiliacs with the APC-resistant FVLeiden variant (4–6). When treated with APC-resistant superMice with acute traumatic coagulopathy had significantly less bleeding when treated with FVa variants (7). In addition, inhibitory antibodies specifically targeting the anticoagulant activity of APC were effective in hemophilic monkeys (8) and mice (9,10). Furthermore, engineered KRK-α1-antitrypsin specifically targeted to inhibit APC also showed efficacy in reducing annual bleeding rates in hemophilia trials (11,12). These findings suggest that APC inhibition promotes a procoagulant state.

[0114] APC also induces regenerative effects, including neurogenesis (13, 14) and wound healing (15). In addition, APC has a protective effect against extracellular histones released by immune cells or necrotic cells (16), which can lead to systemic inflammation, organ failure, and even pathological amounts of death (17). Protease-activated receptor 1 (PAR1) is the main mediator of APC's cytoprotective activity. PAR1 has two cleavage sites for thrombin and APC. Cleavage at R41 by thrombin induces a proinflammatory response. In contrast, cleavage at R46 by APC triggers biased PAR1 signaling and activation of cytoprotective pathways (18, 19). Oligonucleotide-based aptamers, such as APC-167 (20), HS02 (21, 22), and G-NB3 (23), have been developed to target APC inactive sites to inhibit anticoagulant activity without interfering with cytoprotective function. Recombinant APC protein (drotrecogin alfa (activated)) is being developed as a treatment for severe sepsis (24). However, its initial efficacy was not replicated in subsequent trials (25). As the mechanism of action is further understood, it has been proposed that APC could provide benefit in septic settings with an optimized dosing regimen to maximize cytoprotective signaling (2). Recently, 3K3A-APC (K191A / K192A / K193A), which has less than 10% of the anticoagulant activity of APC while retaining cytoprotective activity, has entered clinical trials in ischemic stroke (26, 27). These APC cytoprotective effects are important because complete blockade of the APC active site by antibodies results in a number of adverse effects, including death (8, 28, 29).

[0115] Heavy chain IgG (hcIgG) is a camelid antibody composed of two heavy chains, each with a single variable domain (V H H) and two constant domains CH2 and CH3 (30, 31). H H-region antibodies (also called nanobodies) represent the smallest antibody format.

[0116] In order to selectively target different functions of APC, a nanobody phage library was constructed using peripheral blood mononuclear cells (PBMC) of llamas immunized with active site blocked APC (APC-PPACK). From this library, a set of 21 llama nanobodies specific for inactive sites of APC were identified. These 21 nanobodies have the potential to selectively modulate the pleiotropic activities of APC for therapeutic use.

[0117] Materials and methods:

[0118] Material

[0119] Human APC, human APC-PPACK (Phe-Pro-Arg-chloromethylketone), human protein C, and factor Xa-PPACK were obtained from Haematologic Technologies (Essex Junction, VT); oligo(dT) primers were obtained from Invitrogen (Waltham, MA); V5 tag monoclonal antibody and HRP-anti-M13 were obtained from ThermoFisher (Waltham, MA); Maxisorp ELISA plates were obtained from Nunc (Rochester, NY); MAB230P monoclonal antibody against His-tag was obtained from Maine Biotechnology (Portland, ME); llama pups were obtained from Capralogics (Hardwick, MA); obtained from Pentapharm (Parsippany, NJ); STA-PTT Automate 5, STA NeoplastinCIPlus 5 from Stago (Parsipanny, NJ); normal human plasma from George King Bio-Medical (Overland Park, KS); PC-depleted plasma from Affinity Biologicals (Anacaster, ON, Canada); TPP-4885 Fab anti-APC in IgG1 / κ framework (in-house); plasma-derived human APC and human PC from Enzyme Research Laboratories (South Bend, IN, USA); Spectrozyme PCa from American Diagnostica (Pfungstadt, Germany); calf thymus histone H3 from Sigma-Aldrich (St. Louis MO); Odyssey marker from LI-COR (Lincoln, NE); and one-step blue protein stain from Biotium (Fremont, CA).

[0120] Generation of llama nanobodies targeting inactive sites of human APC

[0121] Two male llamas were immunized with APC-PPACK on the 1st day and the 77th day. Peripheral blood mononuclear cells (PBMC) were isolated 5 days after the last immunization. Total RNA was extracted using RNeasy test kit (Qiagen). SuperScript III first-strand synthesis (ThermoFisher) of oligo (dT) primer and RT-PCR was used to synthesize cDNA from RNA. Nanobody phage display library was constructed by directly amplifying nanobody using framework region 1 (FR1) and hinge region specific primers and cloning the amplicon into a vector with V5 and His-tags. APC-PPACK conjugates were enriched by panning for APC-PPACK, and negatively not selecting factor Xa-PPACK and PC (Figure 14).

[0122] Phage and bacterial periplasmic extract (PPE) ELISA screening

[0123] The plates were coated with 2 μg / mL APC-PPACK, PC or FXa-PPACK at 4°C overnight, blocked with 2% BSA at room temperature (RT) for 1 hour, and used to screen clones from panning rounds R2, R3 and R3' (Figure 14). The phages were blocked in 4% BSA at RT for 1 hour and detected with 1:5000 HRP-anti-M13 mAb. The plates were developed with tetramethylbenzidine (TMB) and quenched with 2N H2SO4. The OD values ​​that were at least three times higher in the antigen-coated wells than in the blocked wells were selected. 450 The PPE generated from individual clones in panning round R4 were similarly screened by enzyme-linked immunosorbent assay (ELISA) using mouse anti-V5 monoclonal antibody (1:5000) as primary antibody and goat HRP-anti-mouse IgG (1:5000) as secondary antibody.

[0124] Nanobody expression and purification

[0125] E. coli grown in 2YTGC supplemented with 0.1% glucose were transformed with a plasmid encoding the Nanobody fused to a 6xHis-tag. At an OD of approximately 0.5 600 Nanobody expression was induced using 1 mM IPTG at 25 °C. Nanobodies in the periplasm were purified with 1 mL of immobilized metal chelate affinity chromatography (IMAC) resin at 4 °C. The resin was washed 3 times with 1 mL of 10 mM imidazole / PBS, and the bound antibodies were eluted with 3 mL of 100 mM imidazole / PBS. The buffer was replaced with PBS containing 1 mM CaCl2. The purity of the nanobody was characterized by SDS-PAGE (Fig. 6). LP11 was also expressed in mammalian CHO cells and purified by IMAC as described above.

[0126] Epitope binning on nanobodies

[0127] exist Binning experiments were performed on HTX at 25°C. Anti-V5 mouse antibody (5 μg / mL) was loaded onto the anti-mouse IgG Fc capture On HTX sensor. The loaded sensor was immersed in nano antibody solution (5 μg / mL) to capture V5-tagged nano antibodies as ligands. The sensor was immersed in 500nM APC for 80 seconds, then dissociated for 30 seconds, and in 200nM APC inactive site-specific TPP-4885Fab for 80 seconds. The combination of APC and nano antibodies was detected by measuring the reflectance spectrum shift on the sensor surface when nano antibodies-APC interact at 25 ° C. The combination of TPP-4885Fab and the nano antibody-APC complex on the sensor was similarly monitored.

[0128] To further understand the epitope diversity of nanobodies that do not compete with TPP-4885Fab, a classic sandwich BLI epitope assay was performed on nanobodies containing a V5 tag at 25°C. The anti-mouse IgG Fc capture sensor loaded with anti-V5 antibody was immersed in a nanobody solution (10 μg / mL), followed by 80 seconds in 500nM APC, 20 seconds of dissociation, and 80 seconds in 200nM nanobody analyte. The normalized binned data (49) were analyzed using the pvclust package. The y-axis of the cluster plot (called height) is a measure of the difference between nanobodies. Multiscale guided resampling was used to calculate the approximately unbiased (AU) p value to confirm the bin assignment.

[0129] Analysis of binding kinetics by bio-layer interferometry

[0130] With anti-mouse IgG The sensor surface of the HTX instrument (Sartorius, Fremont, CA) was loaded with anti-V5 mouse antibodies (10 μg / mL). The loaded sensor was immersed in a nanobody solution (5 μg / mL in the first analysis and 10 μg / mL in the second analysis) to immobilize the V5-tagged nanobody. The sensor with immobilized nanobody was immersed in an APC diluent (78.12-5000nM in PBS, 1 mM CaCl2, 0.1% w / v Tween20, with or without 0.5 mg / mL BSA) for 60 seconds. After APC binding, the surface was immersed in the assay buffer for 120 seconds for dissociation. Biolayer interferometry (BLI) measurements were collected during the binding and dissociation phases (25°C). The surface between samples was regenerated with 10 mM glycine buffer (pH 1.7). The kinetic constant (k) was calculated using a monovalent (1:1) binding model on , k off and K D ). Each nanobody was tested against 5 different APC concentrations.

[0131] Effect of Nano-antibodies on the Anticoagulant Activity of APC

[0132] Snake venom-derived reagents using activated PC APC was generated in normal plasma, and activated partial thromboplastin time (APTT) clotting time was measured to test the effect of nanobodies on the anticoagulant activity of APC. Normal plasma samples (50 μL) with anti-APC nanobodies were mixed with (25 μL; 1 U / mL) and Stago reagent STA-PTT (75 μL) were mixed at 37°C. After incubation for 4 minutes, 75 μL of 25 mM CaCl2 solution was added to initiate coagulation. Normal human plasma serially diluted with PC-depleted plasma was used. The clotting time was used as the standard curve. For FVIII inhibitor plasma, 0.5 μg / ml (16 BU / ml) of FVIII inhibitory antibody GMA-8015 (Green Mountain) was added to normal plasma and Incubate for 2 hours before assay. Effect of Nanobodies on APC-mediated Histone H3 Cleavage

[0133] APC (50nM) and anti-APC nano antibodies (500nM) were pre-incubated in HBS buffer (20mM Hepes, 147mM NaCl, 3mM KCl, pH 7.4) with 100μg / mL BSA and 2mM CaCl2. After 30 minutes, the mixture was added to 100μg / mL histone H3. During 2 hours, the mixture was subsampled at different time points and quenched with reducing sample buffer. The sample was heated at 95°C for 15min, centrifuged at 4000rpm for 5min, vortexed briefly, and loaded into a 12% Bis-Tris gel (Bio-Rad) with MES electrophoresis buffer (30μl / well; 750ng H3). The 20kDa marker in the Odyssey standard (10μL) was used as a reference for normalization between gels. The gel was stained overnight with Biotum one-step blue protein stain (50mL / gel). Protein bands were scanned on Licor Odyssey (700 channels, intensity 6, 1 mm offset, resolution 169 μm) and analyzed using Image Studio V5.2.

[0134] Effects of Nanobodies on APC-mediated SEAP-PAR1 Cleavage

[0135] HEK293 cells expressing wild-type EPCR and a PAR1 cleavage reporter construct with N-terminal secreted embryonic alkaline phosphatase (SEAP-PAR1) (18, 38) were cultured in 96-well plates until confluence. The cells were washed with Hanks balanced salt solution supplemented with 1.3 mM CaCl2, 0.6 mM MgCl2 and 0.1% BSA (endotoxin-free HMM2). APC (50 or 100 nM) and nanobodies were preincubated in HMM2 for 30 minutes and then added to the cells. After 60 minutes, SEAP release was determined using p-nitrophenyl phosphate. PAR1 cleavage is expressed as a percentage of total SEAP activity present on the cells relative to background. The effect of nanobodies on the cleavage selectivity of APC at R46 relative to R41 was evaluated using R41Q-SEAP-PAR1 and R46Q-SEAP-PAR1 HEK293 cell lines (18).

[0136] Endothelial barrier integrity assay

[0137] The effect of nanobodies on APC-mediated endothelial barrier integrity was measured using the transendothelial electrical resistance (TEER) assay (iCelligence system, ACEA, San Diego, CA) as previously described (18, 39). EA.hy926 endothelial cells were plated in 8-well E-plates L8 (Agilent Technologies, Santa Clara, CA) at 2.5 x 10 per well. 4 Cells / 500 μL were cultured until confluence. Two hours before the addition of APC (40 nM) and LP11, DMEM (Invitrogen) containing 10% fetal bovine serum was replaced with serum-free medium containing 0.1% BSA. Thrombin (0.25 nM) was added 30 minutes later to induce endothelial permeability. The resistance change of the confluent monolayer was measured in real time as the normalized cell index.

[0138] In vitro plasma half-life determination.

[0139] The effect of Nanobodies on the sensitivity of APC to physiological inhibitors was evaluated using an in vitro plasma half-life assay. In this assay, APC (70 nM) and antibody (700 nM) were pre-incubated for 30 minutes and normal pooled plasma was added to 90% (v / v). Samples were collected at different time points (0-90 minutes) and quenched in ice-cold TBS. PCa determined the chromogenic activity of APC. The background chromogenic activity of plasma (without APC addition) was subtracted from the measured signal, and the APC activity was normalized to the t = 0 time point to illustrate the effect of the antibody on the chromogenic activity of APC. The half-life (T) was determined by fitting a single-phase exponential decay curve. 1 / 2 ).

[0140] result:

[0141] Generation of nanobodies specific for inactive sites on APC

[0142] APC-PPACK is used as an immunogen to generate nano antibodies against the inactive sites of APC in llama. After immunization with two doses of APC-PPACK, PBMCs are collected, and the nano antibody binding region is cloned to create a nano antibody library. The library is panned for APC-PPACK and PC to select clones that are specific to APC but not PC. After panning round R1, panning (Figure 14) is continued in two groups with different negative selection stringencies for PC. The first group (R1, R2, R3, R4) and the second group (R1, R2', R3') all result in the enrichment of APC-specific clones. Among a total of 1128 phage clones selected from R2, R3, R4 and R3' ( Figure 7), 725 clones were identified as selective APC-PPACK binders lacking nonspecific binding to PC or FXa-PPACK ( Figure 8 ). After sequencing, 158 clones were found to be unique. Based on their CDR1, CDR2 and CDR3 sequences, these 158 clones were divided into 18 families, from which the 21 strongest binding nanobodies in each family (named LP1 to LP21) were selected. Additional nonspecific binding studies against FVIIa, FIXa and FXIa were performed and further demonstrated the selectivity of LP1-LP21 nanobodies for APC ( Fig. 9 ).

[0143] Binding kinetics and epitope binning

[0144] To determine whether any of the Nanobodies bind to the same epitope as the previously reported non-active site-specific anti-APC Fab antibody TPP-4885 (8), a BLI-based epitope binning study was performed. Nanobodies were tested in pairs with TPP-4885 Fab. Thirteen clones blocked TPP-4885 Fab binding and were grouped into cluster 1 ( Figure 1A ). Nanobodies that did not block TPP-4885Fab binding to APC were selected for additional binning experiments by BLI, in addition to LP11, because it has greater than 90% CDR sequence identity with LP9 (Table 4). Therefore, LP9 was used as a surrogate for LP11. Based on the reaction measurements by BLI, LP7, LP9, LP12, and LP18 did not compete with LP2, LP3, and LP10 ( Figure 1B LP7 showed a weaker response as a ligand, but appeared to bind well as an analyte. This may be due to the faster k of LP7. off Cluster analysis showed that the clone could be divided into cluster 2 and cluster 3 ( Figure 1C ). Most Nanobodies (62%) were in Cluster 1. The lower number of Nanobodies in Clusters 2 (24%) and 3 (14%) means that these epitope boxes are less accessible and / or more complex for immune recognition and antibody development. These epitope boxes also appear to be targeted only by Nanobodies with longer CDR3s. Unlike Cluster 1 Nanobodies, which have a median CDR3 length of 9.5 amino acids, the median CDR3 lengths of Cluster 2 and Cluster 3 Nanobodies are 13.5 and 17 amino acids, respectively ( Figure 1D , Table 4). The k of these nanobodies on The value range is 1.26x10 5 Up to 6.22x10 5 1 / M. seconds, k off The range of values ​​is 2.59x10 -4 Up to 1.37x10 -31 / s. k of nanobody LP9 off The value is extremely low, estimated at about 6.33x10 -7 1 / s. Both nanobodies have subnanomolar K D values, while 15 nanobodies had single-digit nanomolar K D The K values ​​of the four nanobodies were D The values ​​ranged from 16.5 to 27.5 nM ( Figure 1E , Table 5).

[0145] Table 4. Amino acid sequences of CDR1, CDR2 and CDR3 of LP1-LP21

[0146]

[0147]

[0148] Table 5. k of 21 nanobodies on , k off and K D Each nanobody was tested against 5 different APC concentrations. The R 2 The range is 0.92 to 0.99.

[0149]

[0150]

[0151] Effects on APC-mediated S-2366 cleavage

[0152] The eight nanobodies had no effect on APC-mediated cleavage of the chromogenic substrate S-2366 ( Fig.10 ). Five nanobodies induced partial inhibition and eight nanobodies showed enhancement of S-2366 cleavage. Among the inhibitory nanobodies, two were non-competitive inhibitors and three were competitive inhibitors of S-2366 cleavage ( Fig.11 , Table 6). Among the 8 Nanobodies that enhanced S-2366 cleavage, 4 increased V max Enhanced cutting, the other 4 by reducing K M Enhanced cleavage. The lack of complete inhibition of S-2366 cleavage suggests that these 21 anti-APC nanobodies target inactive sites.

[0153] Table 6. Nanobodies against APC-mediated S-2366 cleavage V max and K m Summary of the impact

[0154]

[0155]

[0156] Effects on coagulation time in plasma

[0157] use The modified APTT assay was used to screen the effect of nanoantibodies on the anticoagulant activity of APC. (a single-chain glycoprotein derived from snake venom that activates protein C) is added to plasma to generate APCs. The APTT clotting time was prolonged from an average of 40.7 s to 262.0 s, depending on the concentration of PC in plasma ( Figure 2A ). The Nanobodies were screened twice at 300 nM and showed consistent potency ranking between the screens ( Figure 2B ). All nanobodies at least partially reversed the prolonged clotting time. LP2, LP8, LP9, LP11, LP17, and LP20 inhibited APC-induced coagulation by ≥90%, reversing the prolonged clotting time to less than 80 s. LP20 was the most potent inhibitor and almost completely corrected the clotting time ( Figure 2C These data suggest that there are at least three distinct epitopes on APC (Cluster 1, Cluster 2, and Cluster 3) that can be bound by Nanobodies to inhibit the anticoagulant activity of this protein.

[0158] Effects on APC-mediated histone H3 cleavage

[0159] APC inhibits histone-induced cytotoxicity by proteolytic cleavage (16). In this assay, APC is incubated with histone H3 in the presence of nanobodies, and histone H3 cleavage is monitored by SDS-PAGE ( Figure 3A The nine nanobodies had minimal effects on APC-mediated histone H3 cleavage ( Figure 3B ), one (LP14) enhances H3 cleavage ( Figure 3C ), twelve nanobodies inhibited APC-mediated H3 cleavage, resulting in residual activity <75% ( Figure 3D ), LP21 is the most effective ( Figure 3E ). Effects of APC-mediated SEAP-PAR1 cleavage

[0160] Nanobodies that inhibited PAR1 cleavage by APC were found in all three clusters, with LP1, LP8, LP17, LP19, and LP21 showing the strongest inhibition. In contrast, nanobodies LP3, LP9, LP11, and LP18 showed minimal inhibition even at their highest concentration of 500 nM ( Figure 4A). At a lower concentration range (0.7-6nM), these four nanobodies reduced PAR1 cleavage to 73%-82% of the control. These nanobodies belong to cluster 2 (LP9, LP11, LP18) and cluster 3 (LP3), respectively. All nanobodies in cluster 1 inhibit PAR1 cleavage. APC can cleave PAR1 at R41 and R46, and the latter cleavage leads to cell protective signaling. Therefore, the effects of two PAR1 cleavage inhibitory nanobodies and two nanobodies that retain PAR-1 cleavage on the APC cleavage pattern were evaluated using the R41Q-SEAP-PAR1 and R46Q-SEAP-PAR1 cell lines ( Figure 4B In the presence of inhibitory nanobodies LP8 and LP20, cleavage at both sites by APC (at 50 nM) was inhibited in a dose-dependent manner. At high concentrations of LP8 and LP20, the cleavage ratio shifted to below 1, with a lower cytoprotective PAR1 cleavage signature ( Figure 4C ). In contrast, LP11 and LP18 appear to induce a curvilinear cleavage response to R46 and R41. At 0.7 to 6 nM, R41 cleavage was inhibited to a greater extent than R46. At 6 nM and higher, the inhibition of cleavage was reversed. Interestingly, for the entire concentration range assayed, the R46 / R41 cleavage ratio was above 1, indicating enhanced cytoprotection by LP11 and LP18 ( Figure 4D ).

[0161] Effect on the plasma half-life of APC

[0162] An in vitro plasma half-life assay was used to determine the effects of 21 Nanobodies on the sensitivity of APCs to physiological inhibitors. This assay identified V that increased, decreased, and had no effect on APC plasma half-life. H H antibodies (Table 7). The increase in half-life suggests that binding of these antibodies reduces the sensitivity of APCs to inhibition by physiological inhibitors present in plasma.

[0163] Table 7. The results of in vitro plasma half-life determination show that the average in vitro half-life (T 1 / 2 ) were used to determine the effect of the antibody-free condition. Standard deviation (SD) and P values ​​were generated by comparison with the no-antibody condition.

[0164]

[0165]

[0166] Active features

[0167] Based on their clusters and the effects in 3 APC functional assays, these 21 nanobodies are divided into 15 different activity profiles (Table 8). Three nanobodies (LP2, LP8, LP17) strongly inhibit all 3 APC functional activities. It was found that several nanobodies only inhibited 2 of the 3 functions: LP16 and LP20 inhibited anticoagulation and PAR1 cutting, and LP1 and LP5 inhibited H3 and PAR1 cutting. Since these nanobodies only retain 1 of the 3 APC functions, they can be used to analyze the pleiotropic effects of APC, particularly in an in vivo environment. Nanobodies that only strongly inhibit PAR1 cutting or histone H3 cutting have not been identified. Strong monofunctional inhibition of APC anticoagulant activity was observed only with LP9 and LP11.

[0168] Table 8. Classification of 21 Nanobodies into 15 antibody characteristic classes based on their ability to affect various activities of APCs.

[0169]

[0170]

[0171] Effects of LP11 on endothelial cell barrier function

[0172] Nanobody LP11 is the best candidate for the treatment of bleeding. LP11 inhibits APC in a dose-dependent manner and shortens the duration of FVIII inhibitor plasma. Prolonged clotting time in coagulation assays ( Fig.12 ), inhibits anticoagulation, and enhances the PAR1 R46 / R41 cleavage ratio. Thus, LP11 supports pro-hemostatic activity and simultaneously enhances APC-mediated cytoprotection.

[0173] To test the effect of LP11 on endothelial barrier function in vitro, LP11 was expressed in mammalian cells and purified. Characterization of CHO cell-derived LP11 showed that its properties were indistinguishable from those of E. coli-derived LP11 (i.e., it inhibited the anticoagulant activity of APC and had minimal effect on APC-mediated cleavage of PAR1 at R46, while significantly inhibiting R41 cleavage (Figure 13)). A classic endothelial barrier function assay was performed using EA.hy926 cells to analyze APC-induced barrier protection against thrombin-induced permeability (18,39). This assay showed that LP11 had minimal effect on the barrier protection function of APC ( Figure 5A-5B ), which is consistent with its minimal effect on APC-mediated PAR1 cleavage at R46. To evaluate the effect of LP11 on APC priming of endothelial cells via PAR1 cleavage, we assessed the integrity of the endothelial cell layer prior to thrombin-induced barrier disruption. APC priming was observed to induce a dose-dependent transient barrier disruption ( Figure 5C-5D ), which can be inhibited by the PAR1 antagonists vorapaxar and SCH-79797 ( Figure 5E ), which is consistent with the concept that APC priming encompasses the role of APC-mediated PAR1 cleavage at R41. LP11 inhibited transient barrier disruption induced by APC priming in a dose-dependent manner. This is consistent with its inhibition of APC-mediated PAR1 cleavage at R41 ( Figure 5F-5G Most importantly, analysis of the overall barrier function characteristics showed that the normalized cell index of APCs reached higher values ​​in the presence of LP11 compared to APCs in the absence of LP11 ( Figure 5H ), indicating that LP11 provides a net enhancement of the barrier-protective effects of APC. Notably, LP11 alone did not alter the electrical resistance of the endothelium. Thus, this data suggests that preferential inhibition of APC-mediated R41 cleavage over R46 cleavage during APC priming exerts a cytoprotective effect on endothelial cells.

[0174] discuss:

[0175] The APC pathway includes thrombin / thrombomodulin, PC, protein S, EPCR, and PAR, which mediate a variety of physiological functions, including anticoagulant, anti-inflammatory, and cytoprotective functions. Targeting of these proteins could provide clinical benefits for a myriad of pathologies involving this pathway. Currently, two drugs targeting different components of this pathway, KRK-α1-antitrypsin and 3K3A-APC, are in clinical development for hemophilia and ischemic stroke, respectively (40). However, much of the potential of using APC and its substrates as therapeutic targets remains to be explored. Studies based on single-molecule FRET and small-angle X-ray scattering have shown that the overall architecture of PC undergoes limited changes upon activation, and these changes are primarily conformational changes within the active site region (41,42). Therefore, it is challenging to identify antibodies that are specific for the inactive site of APC and do not bind PC. This work addressed this challenge by constructing a llama antibody library from which 21 nanobodies belonging to 18 families were selected and further characterized for their binding diversity. In the epitope binding studies, only three nanobody clusters with specificity for three epitope bins were found. This low number of epitope bins suggests limited conformational differences between the inactive site regions of APC and its proenzyme PC and is consistent with previous reports (41,42).

[0176] Based on APC functional studies, all 3 epitope boxes appear to play multiple roles simultaneously in anticoagulation, H3 cleavage, and PAR1 cleavage, as none of the epitope boxes showed exclusive loss of only one role upon blockade. Nanobodies within cluster 1 compete with the previously reported antibody TPP-4885, which targets the APC autolysis loop and potentially also targets amino acids L429-N431 (8). Therefore, nanobodies within cluster 1 are expected to bind to similar regions. The autolysis loop is a positively charged loop that plays a role in FVa cleavage (43), FVIIIa cleavage (44), and APC sensitivity to serine protease inhibition (45,46). Within the autolysis loop, residues R306, K308, K311, R312, and R314 are important for FVa cleavage, while R306, K311, and R314 are important for FVIIIa cleavage. Since all cluster 1 nanobodies reversed the prolonged The clotting time, so they are likely to bind to these important arginine and lysine residues on the autolytic loop. The observation that some cluster 1 nanobodies inhibit H3 and / or PAR1 cleavage may be the result of previously unreported interactions between the autolytic loop and H3 and PAR1. Alternatively, these nanobodies may bind to regions that are important for the interaction of H3 and PAR1, which are adjacent to but not within the autolytic loop.

[0177] Clusters 2 and 3 contain nanobodies that do not compete with TPP-4885Fab binding. Among them, LP9 and LP11 inhibit the anticoagulant activity of APC without interfering with H3 and PAR1 cleavage. The activity characteristics of APC bound by LP9 or LP11 are similar to those of APC mutants designed to have minimal anticoagulant activity (such as 3K3A-APC and 5A-APC). 3K3A-APC (K191A / K192A / K193A) (47) has amino acid substitutions in loop 37, and 5A-APC (K191A / K192A / K193A / R229A / R230A) (48) has substitutions in both loop 37 and the calcium binding loop. The cytoprotective function of APC is based on its biased PAR1 cleavage at R46. Studies of the R46 / R41 cleavage ratio in the SEAP-PAR1 cell line showed that LP11 increased the R46 / R41 cleavage ratio by up to 2.5-fold, which resulted in an overall improvement in endothelial cell barrier function. Preclinical hemophilia and coagulopathy studies have shown that the pro-hemostatic effects of APC inhibition should be elicited without interfering with the cytoprotective function of APCs to provide improved outcomes in hemophilic arthropathy (9) and survival outcomes in traumatic coagulopathy (28). Therefore, the novel properties of LP11 may further improve outcomes in these conditions.

[0178] In summary, the llama nanobody library generated 21 nanobodies that selectively modulate APC anticoagulant and cytoprotective pathways. These nanobodies can be used as new therapeutic agents for treating a range of APC-related indications, including acute bleeding, hemophilia, ischemia, and sepsis.

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Claims

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

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

3. The antibody of claim 2, wherein the antibody comprises a heavy chain variable region (V H) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 475-632. H )'s CDR1, CDR2 and CDR3.

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

473.

5. An isolated antibody comprising V H , the V H Comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 475-632, wherein the antibody specifically binds to APCs and minimally binds to PCs.

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

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

8. The antibody of any one of claims 1-6, wherein the antibody is a Nanobody.

9. The antibody of claim 8, wherein the antibody is V H H antibody.

10. The antibody of any one of claims 1 to 9, wherein the antibody: a) enhance, minimally affect, or inhibit the anticoagulant activity of APCs; b) enhance, minimally affect, or inhibit APC-mediated histone cleavage; c) enhance, minimally affect, or inhibit APC-mediated cleavage of protease-activated receptor 1 (PAR1) at residue R46; d) enhance, minimally affect, or inhibit APC-mediated cleavage of PAR1 at residue R41; e) increasing, minimally affecting, or decreasing the ratio of APC-mediated cleavage of PAR1 at residue R46 to APC-mediated cleavage of PAR1 at residue R41; f) enhance, minimally affect, or inhibit the endothelial barrier protection activity of APCs; g) increase, minimally affect, or decrease the plasma half-life of APC; or h) any combination thereof.

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

12. A nucleic acid encoding the antibody of any one of claims 1-11. A vector comprising the nucleic acid according to claim 12 .

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

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

16. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

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

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

19. The method of claim 17, wherein the condition is selected from the group consisting of hemorrhage, contusion, burn, gastrointestinal bleeding, uncontrolled bleeding, bleeding caused by transplantation or resection, bleeding caused by surgery, bleeding caused by trauma, varicose bleeding, cirrhosis bleeding, thrombocytopenia, idiopathic thrombocytopenic purpura, hemophilia, aortic aneurysm, overdose of anticoagulants or antithrombotics, menorrhagia, coagulation factor deficiency, Glanzmann thrombocytopenia, and Bernard-Soulier syndrome.

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

21. The method of claim 20, wherein the condition is selected from the group consisting of: Sepsis, inflammation in acute ischemic disease, 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 disease, acute pancreatitis, cancer, inflammatory diseases and autoimmune diseases.

22. The antibody of any one of claims 1-11 for use as a medicament for the treatment or prevention of a disorder.

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

24. The antibody of claim 23, wherein the condition is selected from the group consisting of hemorrhage, contusion, burn, gastrointestinal bleeding, uncontrolled bleeding, bleeding caused by transplantation or resection, bleeding caused by surgery, bleeding caused by trauma, varicose bleeding, cirrhosis bleeding, thrombocytopenia, idiopathic thrombocytopenic purpura, hemophilia, aortic aneurysm, overdose of anticoagulants or antithrombotics, menorrhagia, coagulation factor deficiency, Glanzmann thrombocytopenia, and Bernard-Soulier syndrome.

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

26. The antibody of claim 25, wherein the disorder 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 disease, acute pancreatitis, cancer, inflammatory diseases and autoimmune diseases.

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