Human antibodies against activated protein C and uses thereof

Abstract

The present invention provides antibodies against Activated Protein C (APC). Certain disclosed antibodies inhibit the anticoagulant activity of APC while 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 / 316,928, 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 "172085.00118_ST26.xml", 412,954 bytes in size, created on February 28, 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 isolated antibodies that specifically bind to activated protein C (APC) and minimally bind to inactivated protein C (PC). In some embodiments, the heavy chain variable region (V H ) comprises a CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 118. In some embodiments, these antibodies comprise a V selected from the group consisting of SEQ ID NO: 61-80 and SEQ ID NO: 473-496. H In some embodiments, the light chain variable region (V L ) comprises a CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 23 and SEQ ID NO: 138-140. In some embodiments, these antibodies comprise a V selected from the group consisting of SEQ ID NO: 24-35 and SEQ ID NO: 141-151. L In some embodiments, these antibodies comprise a V H and V L In some embodiments, these antibodies comprise a heavy chain and a light chain both from a single antibody described herein.

[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 1 TABLE 1 is a table presenting the results of a screening assay used to evaluate Panel 1 20 antibodies. These results include protein expression levels in transiently transfected HEK293 cells, amidolytic activity ( PCa), surface plasmon resonance (SPR)-based binding to PC (i.e., K D ), and the aggregation temperature based on UNcle (Tagg). The results were compared with those generated with the recombinant parent antibody TPP-24727 (highlighted in bold).

[0029] Figure 2 is a table presenting the binding kinetics and affinity data for Panel 1 antibodies. These data were generated by performing SPR binding assays using human APC and PC as analytes and then fitting the resulting sensorgrams to a 1:1 Langmuir binding model. The results are compared to those generated with the parental antibody TPP-24727 (highlighted in bold).

[0030] Figure 3 is a table presenting expression, purity and stability data for four antibodies in Panel 1. IgG concentrations were quantified via sandwich ELISA. Melting temperatures (Tm) were determined via differential scanning calorimetry using a MicroCal VP-capillary DSC apparatus according to the manufacturer's protocol.

[0031] Figure 4 is a table presenting data on the amidolytic activity of human APC in the presence of 8 nM and 3.2 nM of antibodies from Panel 1. The results are compared to those generated with the parental antibody TPP-24727 (highlighted in bold).

[0032] Figure 5 is a set of three graphs presenting the results of the FXa and thrombin generation assay, which is used to measure the ability of Panel 1 antibodies to inhibit the inactivation of FVa by APC. In this assay, FVa was incubated with each antibody at concentrations ranging from 0.5 nM to 1000 nM. After 30 minutes, a prothrombinase mixture containing FXa, prothrombin, and a chromogenic substrate for thrombin (S-2238) was added and substrate hydrolysis was monitored using a plate reader.

[0033] FIG. 6A to FIG. 6E The results for testing the effect of Group 1 antibodies on the clotting time of normal human plasma are shown. Results of the activated partial thromboplastin time (APTT) assay. This assay was performed in two independent screens (i.e., Screen 1 and Screen 2) on plasma samples containing different concentrations of antibody (i.e., 10 nM or 30 nM). The results are Fig. 6A and Figure 6B The clotting time at 30 nM versus the clotting time at 10 nM is presented in a graph and summarized in Figure 6C The results are still in Fig.6D and Fig. 6E A graphic showing the rank order in Screen 2 versus the rank order in Screen 1 is presented in FIG.

[0034] Figure 7 is a table showing secreted embryonic alkaline phosphatase (SEAP)-proteinase activated receptor 1 (PAR1) cleavage data by human APC in the presence of panel 1 antibodies. For this assay, SEAP-PAR1 was expressed on HEK293 cells with wild-type endoglin C receptor (wt-EPCR). APC were pre-incubated with antibodies for 30 minutes before adding them to confluent SEAP–PAR1 / wt-EPCR cells. After 60 minutes, release of SEAP was measured using one-step p-nitrophenyl phosphate.

[0035] FIG. 8A to FIG. 8BResults of an endothelial barrier integrity assay on human APCs in the presence of Panel 1 antibodies are shown. Results were compared to those generated with the parental antibody TPP-24727. TM The system measures endothelial cell permeability in real time. Confluent EA.hy926 endothelial cells were exposed to APC (50 nM) in the presence and absence of antibodies. After the addition of thrombin (2 nM), the permeability of the confluent monolayers was determined 3 hours after treatment with APC. The results are presented in Fig. 8A The percentage of barrier protection versus antibody concentration is presented graphically in Figure 8B in the table.

[0036] 9A to 9C Results of histone H3 cleavage assays on human APC in the presence of panel 1 antibodies are shown. A. APC (50 nM) and antibodies (500 nM) were pre-incubated together for 30 minutes. After incubation, the mixture was added to 100 μg / ml histone H3. The mixture was subsampled at different time points over 2 hours to monitor the digestion of histone H3 over time. Fig. 9A Results generated with the parental antibody TPP-24727 are shown. Fig. 9B Results generated with each antibody from Panel 1 are shown. Fig. 9C Shown are the results of a modified assay in which digestion of histone H3 was measured in the presence of different ratios of antibody to APC after 1 hour of incubation.

[0037] Fig.10 The table in shows the results of an in vitro plasma half-life assay of human APC in the presence of panel 1 antibodies. In this assay, APC and antibodies were pre-incubated for 30 minutes before the addition of normal pooled plasma. Samples were taken at different time points and the residual chromogenic activity of APC was determined by fitting a one-phase exponential decay curve.

[0038] Fig.11 is a table summarizing the mutations present in Group 2 antibodies.

[0039] Fig.12 TABLE 2 is a table showing the results of a screening assay for evaluating Panel 2 antibodies. These results include SPR-based measurements of antibody binding to APC and PC (i.e., K D ), the effect of antibodies on the amidolytic activity of APC ( PCa), and UNcle-based aggregation temperature (Tagg). Hyphens indicate that the specified data were not collected for the specified antibody.

[0040] Fig.13 The results for testing the effect of Group 2 antibodies on the clotting time of normal human plasma are shown. Results of the assay. The assay was performed on plasma samples containing 30 nM antibody in two independent screens (i.e., Screen 1 and Screen 2). The results are presented as a clotting time graph for Screen 1 versus Screen 2 (left) and summarized in a table (right).

[0041] FIG. 14A to FIG. 14B The results of a histone H3 cleavage assay on human APC in the presence of panel 2 antibodies are shown. APC (50 nM) and antibodies (500 nM) were pre-incubated for 30 minutes. After incubation, the mixture was added to 100 μg / mL histone H3. The mixture was subsampled at different time points over 2 hours to monitor the digestion of histone H3 over time. Digestion in the presence of each antibody was compared to digestion in the presence of TPP-26870. The results are shown in Fig.14A The histone H3 cleavage versus time graph is presented in Fig. 14B in the table.

[0042] Fig.15 The table in shows the results of an in vitro plasma half-life assay of human APC in the presence of panel 2 antibodies. In this assay, APC and antibodies were pre-incubated for 30 minutes before the addition of normal pooled plasma. Samples were taken at different time points and the residual chromogenic activity of APC was determined by fitting a one-phase exponential decay curve.

[0043] exist Fig.16 In the table, the first group of 20 antibodies are divided into 7 functional categories based on their pharmacological properties, as determined in Example 3. In the anticoagulant activity column, a+ indicates that the antibody is more inhibitory than the parent antibody TPP-24727, while a- indicates that the antibody is less inhibitory than TPP-24727. In the SEAP-PAR1 cleavage column, a+ indicates that the antibody causes more PAR1 cleavage than TPP-24727, while a- indicates that the antibody causes less PAR1 cleavage than TPP-24727, and ND indicates that no data were collected. In the histone cleavage column, a+ indicates that the antibody causes more histone cleavage than TPP-24727, while a- indicates that the antibody causes less histone cleavage than TPP-24727, and an= indicates that the antibody causes histone cleavage similar to TPP-24727. In the Sensitivity to Physiological Inhibitors column, a+ indicates that the antibody renders APC more sensitive to the physiological inhibitor than TPP-24727, while a- indicates that the antibody renders APC less sensitive to the physiological inhibitor than TPP-24727, and ND indicates that data were not collected.

[0044] exist Fig.17In the table, the 24 antibodies in Group 2 are divided into 11 functional categories based on their pharmacological properties, as determined in Example 4. In the anticoagulant activity column, a+ indicates that the antibody is more inhibitory than the parent antibody TPP-26870, while a- indicates that the antibody is less inhibitory than TPP-26870. In the histone cleavage column, a+ indicates that the antibody causes more histone cleavage than TPP-26870, while a- indicates that the antibody causes less histone cleavage than TPP-26870. In the sensitivity to physiological inhibitors column, a+ indicates that the antibody makes APC more sensitive to physiological inhibitors than TPP-26870, while a- indicates that the antibody makes APC less sensitive to physiological inhibitors than TPP-26870, and an= indicates that the antibody causes similar APC sensitivity as TPP-26870.

[0045] Fig.18 is a schematic diagram outlining how Group 1 and Group 2 antibodies were generated and screened.

[0046] Fig.19 is a table showing the effect of humanizing the TPP-24727 heavy chain on immunogenicity scores, expression levels, and APC binding kinetics. The table is divided into a first group of antibodies that showed overall improvements in these parameters and a second group of antibodies that were considered suboptimal based on this analysis. A third group of antibodies disclosed in International Patent Application Publication WO2023284012 is included for comparison (referred to as "OMRF" in the figure).

[0047] Fig. 20 It is a table showing the effect of humanizing the light chain of TPP-24727 on immunogenicity scores, expression levels, and APC binding kinetics. The table is divided into a first group of antibodies and a second group of antibodies, the first group of antibodies showing overall improvements in these parameters, and the second group of antibodies being considered suboptimal based on this analysis. A third group of antibodies disclosed in International Patent Application Publication WO2023284012 is included for comparison (referred to as "OMRF" in the figure). The immunogenicity scores of the improved antibodies are much lower than those of the antibodies disclosed in WO2023284012. DETAILED DESCRIPTION

[0048] 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.

[0049] 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.

[0050] Antibody:

[0051] 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.

[0052] 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); (vii) minibodies, diabodies, triabodies, tetrabodies and κ 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.

[0053] 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 certain preferred embodiments, the antibody is in the form of an IgG4 antibody. The IgG4 antibody 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). In addition, IgG4 antibodies are easy to manufacture and have been used in several commercially available therapeutic agents (e.g., natalizumab). Gemtuzumab Dupilumab Nivolumab )middle.

[0054] 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: 242) 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 (K D ) 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.

[0055] 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.

[0056] 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: 241) or a variant thereof.

[0057] 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.

[0058] 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 SPR analysis.

[0059] 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 The ability of anti-APC antibodies to inhibit the anticoagulant activity of APC was tested by modified APTT coagulation assay (see Figures 6 and Fig.13 ).

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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, and / or (2) enhances, minimally affects or inhibits APC-mediated endothelial barrier protection. In the examples, the effect of anti-APC antibodies on APC-mediated histone cleavage was tested using a histone H3 cleavage assay (see Figures 9 and 14), and the effect of anti-APC antibodies on APC-mediated endothelial barrier protection was tested using an in vitro endothelial barrier function assay (see Figure 8).

[0065] 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, the effects of anti-APC antibodies on the half-life of an APC were tested using an in vitro plasma half-life assay (see Tables 10 and Fig.15 ).

[0066] The antibodies disclosed herein bind to the exosites (i.e., sites other than the active site) of APC. These antibodies are generated by the parent antibody TPP-4885 that binds to the exonuclease cleavage site. It has been shown that TPP-4885 inhibits the antithrombotic activity of APC while retaining its beneficial cytoprotective function (Nat Commun 11(1):2992,2020). The 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 acid residues that mediate the interaction of APC with cofactors and substrates are present in the exosite away from the active site.

[0067] Example 1 describes a method for optimizing the parent antibody TPP-4885. First, TPP-4885 (an IgG2 antibody) was recombined into an IgG4 antibody and renamed TPP-24727. The IgG4 form used contained a serine 228 to proline (S228P) mutation, which was used to prevent Fab arm exchange. Next, the framework sequence fragment from TPP-24727 was exchanged for a human germline sequence to produce an antibody framework more similar to the germline, thereby generating 96 different antibody light chains and 96 different antibody heavy chains. Using expression assays, enzyme activity assays, and APC binding assays, the modified light and heavy chains were evaluated against the corresponding chains of the parent antibody (i.e., TPP-24727). Based on these assays, 13 light chains and 6 heavy chains were selected to generate 78 optimized antibodies. These 78 new antibodies were rescreened using the same assay, and 20 antibodies were selected for further study. These 20 antibodies are referred to as "Group 1 antibodies" in this application. The generation and characterization of the first group of antibodies are described in more detail in Examples 1 to 3. The sequence identifiers of the amino acid sequences and DNA sequences of the first group of antibodies are listed in Tables 1 and 2, respectively. As described in Example 4, a second group of 24 antibodies, referred to herein as "the second group of antibodies," was generated and evaluated. The second group of antibodies includes 22 antibody variants generated via random mutagenesis of the promising first group of antibodies TPP-26870. The second group of antibodies also includes two additional antibodies (i.e., TPP-26922 and TPP-26939), which are not related to any of the first group of antibodies. The sequence identifiers of the amino acid sequences and DNA sequences of the second group of antibodies are listed in Tables 3 and 4, respectively. Fig.18 A schematic outlining how Group 1 and Group 2 antibodies were generated and screened is provided in .

[0068] Table 1. CDRs, V H 、V L , full-length heavy chain and full-length light chain amino acid sequence identifiers

[0069]

[0070] Table 2. CDRs, V H 、V L , full-length heavy chain and full-length light chain DNA sequence identifier

[0071]

[0072] Table 3. CDRs, V H 、V L , full-length heavy chain and full-length light chain amino acid sequence identifiers

[0073]

[0074] Table 4. CDRs, V H 、V L , full-length heavy chain and full-length light chain DNA sequence identifier

[0075]

[0076] 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).

[0077] The classic representation of an antibody is a Y-shaped molecule composed of four polypeptide subunits. Each antibody contains two identical copies of a longer heavy chain and two identical copies of a shorter light chain. Based on small differences in the polypeptide sequence, the light chains of an antibody can be classified as either kappa (κ) or lambda (λ). The heavy chain composition determines the overall class of each antibody. Each heavy chain has two regions, the heavy chain constant region and the heavy chain variable region (V H Similarly, each light chain comprises a light chain constant region and a light chain variable region (V L The constant region is identical in all antibodies of the same isotype, but differs between antibodies of different isotypes; the variable region is antibody-specific and differs depending on the B cell that produced it. H and two V L Together they form the variable region of the antibody, which serves as the antigen binding site.

[0078] Each V of the antibody H and VL 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 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). 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 Three CDRs.

[0079] 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.

[0080] 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 CDR3 of the antibodies disclosed herein. Specifically, in some embodiments, the V H comprising a CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 118 or consisting of the amino acid sequence. In addition, in some embodiments, the V L It comprises a CDR3 having or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 23 and SEQ ID NO: 138-140.

[0081] In some embodiments, the antibodies of the present invention further comprise CDR1 and CDR2 of the antibodies disclosed herein. Specifically, in some embodiments, the V H The antibody may comprise (a) a CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4 and SEQ ID NOs: 109-113 or consisting of the amino acid sequence, (b) a CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-8 and SEQ ID NOs: 114-117 or consisting of the amino acid sequence, or (c) both of the following: a CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4 and SEQ ID NOs: 109-113 or consisting of the amino acid sequence, and a CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-8 and SEQ ID NOs: 114-117 or consisting of the amino acid sequence. In addition, in some embodiments, the V L It may comprise (a) a CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-19 and SEQ ID NOs: 130-131 or consisting of the amino acid sequence, (b) a CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-22 and SEQ ID NOs: 132-137 or consisting of the amino acid sequence, or (c) both of the following: a CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-19 and SEQ ID NOs: 130-131 or consisting of the amino acid sequence, and a CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-22 and SEQ ID NOs: 132-137 or consisting of the amino acid sequence.

[0082] In certain embodiments, the antibodies of the invention comprise CDR1, CDR2, and CDR3 all derived from a single variable region of an antibody disclosed herein. Specifically, in some embodiments, the V H comprising a V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-15 and SEQ ID NOs: 119-129 H In addition, in some embodiments, the V L comprising a V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-35 and SEQ ID NOs: 141-151 L In some embodiments, these antibodies comprise all six CDRs (i.e., V HCDR1, CDR2 and CDR3, and V L Such antibodies comprise the paratope (i.e., a set of six CDRs that form an antigen binding region) of the antibodies disclosed herein.

[0083] In addition, the present invention provides antibodies comprising or consisting of the full-length variable regions disclosed herein. Specifically, in some embodiments, these antibodies comprise a V selected from the group consisting of SEQ ID NOs: 10-15 and SEQ ID NOs: 119-129. H In addition, in some embodiments, these antibodies comprise a V selected from the group consisting of SEQ ID NOs: 24-35 and SEQ ID NOs: 141-151. L .

[0084] In some embodiments, the antibodies of the invention comprise V or V of the antibodies disclosed herein. H and V of the antibodies disclosed herein L At least a portion (e.g., CDR3) of both. For example, in some embodiments, the antibodies disclosed herein comprise a V selected from the group consisting of SEQ ID NOs: 10-15 and SEQ ID NOs: 119-129. H and V selected from the group consisting of SEQ ID NOs: 24-35 and SEQ ID NOs: 141-151 L In some embodiments, V H and V L From the individual antibodies disclosed herein (see Table 1 and Table 3 for V values ​​of each antibody disclosed herein) H Sequence and V L In other embodiments, V H and V L From two different antibodies disclosed herein.

[0085] 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: 10-15 and SEQ ID NOs: 119-129. HVariants 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 a parent antibody. In other embodiments, the antibody comprises a V selected from SEQ ID NOs: 24-35 and SEQ ID NOs: 141-151. L , which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications relative to the parent antibody. In other embodiments, the antibody comprises a CDR3 selected from SEQ ID NO: 9, 23, 118 and 138-140 with at least 1 amino acid modification.

[0086] 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 typically 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 5.

[0087] Table 5. List of conservative amino acid substitutions

[0088] Original residue Conservative substitution Ala Gly,Ser Arg His,Lys Asn Asp,Gln,His Asp Asn,Glu Cys Ala,Ser Gln Asn,Glu,His Glu Asp,Gln,His Gly Ala His Asn,Arg,Gln,Glu Ile Leu,Val Leu Ile, Val Lys Arg,Gln,Glu Met Leu,Ile Phe His,Met,Leu,Trp,Tyr Ser Cys,Thr Thr Ser, Val Trp Phe, Tyr Tyr His,Phe,Trp Val Ile,Leu,Thr

[0089] In some embodiments, the V of an antibody of the invention is H The invention also provides an antibody having an amino acid sequence that has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, or 75% sequence identity to one of the amino acid sequences of SEQ ID NOs: 10-15 and 119-129, or a CDR3 that has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to one of the amino acid sequences of SEQ ID NOs: 9 and 118. In some embodiments, the V Lhaving an amino acid sequence that has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% or 75% sequence identity to one of the amino acid sequences of SEQ ID NOs: 24-35 and 141-151, or comprising a CDR3 that has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% sequence identity to one of the amino acid sequences of SEQ ID NOs: 23 and 138-140. In some embodiments, the heavy chain of the antibody of the invention has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% or 75% identical to one of the amino acid sequences of SEQ ID NOs: 36-41 and 152-162. In some embodiments, these antibodies comprise a heavy chain consisting of one of the amino acid sequences of SEQ ID NOs: 36-41 and 152-162. In some embodiments, the light chain of the antibody of the invention has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% or 75% identical to one of the amino acid sequences of SEQ ID NOs: 42-53 and 163-173. In some embodiments, these antibodies comprise a light chain consisting of one of the amino acid sequences of SEQ ID NOs: 42-53 and 163-173.

[0090] "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.

[0091] 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.

[0092] Nucleic acids, vectors and host cells:

[0093] The antibodies of the present invention can be produced by introducing a nucleic acid encoding the antibody into a host cell and providing conditions suitable for protein expression. Therefore, in a second aspect, the present invention provides a nucleic acid encoding the antibody disclosed herein. Specifically, the present invention includes nucleic acids encoding the following: (1) a V sequence having an amino acid sequence selected from SEQ ID NOs: 10-15 and 119-129 or consisting of the amino acid sequence; H The antibody (2) comprises a V having an amino acid sequence selected from SEQ ID NOs: 24-35 and 141-151 or consisting of the amino acid sequence L (3) an antibody comprising a CDR3 having an amino acid sequence selected from SEQ ID NOs: 9, 23, 118 and 138-140 or consisting of the amino acid sequence, or (4) any other antibody described herein. The present invention also includes nucleic acids having or consisting of the DNA sequences provided in Table 2 or Table 4 (i.e., a DNA sequence selected from SEQ ID NOs: 54-102 and 174-240) and variants thereof.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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).

[0098] Pharmaceutical composition:

[0099] 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.

[0100] 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.

[0101] method:

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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).

[0110] 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 preferred embodiments, the subject is a human.

[0111] 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.

[0112] 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 requiring blood coagulation (e.g., acute bleeding). For such indications, the importance of maintaining the cytoprotective activity of APC is demonstrated by Magisetty et al. (Blood (2022) blood.2021013119) discussed above, where 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 severity of hemophilic arthropathy was significantly reduced by administering MAPC1591 (rather than MPC1609).

[0113] TPP-24727 is the parent antibody from which the antibodies of the present invention are derived. TPP-24727 inhibits the anticoagulant activity of APC, enhances the histone H3 cleavage activity of APC, and partially reduces APC-mediated PAR1 cleavage. Compared to the parent antibody, the promising Group 1 antibody TPP-26870 more strongly inhibits the anticoagulant activity of APC, resulting in an enhanced pro-hemostatic effect. In addition, TPP-26870 exhibits less PAR1 cleavage inhibition than the parent antibody and does not interfere with APC-mediated histone cleavage. Anti-APC antibodies that provide improved cytoprotection compared to TPP-24727, or that directly enhance the cytoprotective activity of APC, can also be used to treat conditions such as sepsis and inflammation in acute ischemic diseases. Fig.16 As summarized in , based on the pharmacological properties determined in Example 3, Group 1 antibodies can be divided into at least 7 functional categories. Fig.17 As summarized in , based on the pharmacological properties determined in Example 4, Group 2 antibodies can be divided into at least 11 functional categories.

[0114] Example

[0115] Example 1 - Optimization of parent antibodies

[0116] A previous study (Nat Commun 11(1):2992, 2020) reported two antibodies against APC that were shown to increase thrombin generation and promote plasma coagulation in monkeys. One of these antibodies, called TPP-4885, was well tolerated in monkeys and was determined to target an exonuclease cleavage site on the APC protein. However, the antibody still partially inhibited the active site of APC and therefore may partially inhibit the cytoprotective effect of this enzyme.

[0117] The following example describes a method for optimizing TPP-4885. First, the IgG2 antibody was recombined into an IgG4 antibody and renamed TPP-24727. The IgGκS228P form was used, which contained a serine 228 to proline (S228P) mutation to prevent Fab arm exchange. Standard cloning techniques were used for recombination. Next, the framework sequence fragments from TPP-24727 were exchanged for human germline sequences, namely KV2-28, KV3-20, KV3-11, V3-48-01.1, V3-15-01.1, and V1-69-04.1, to produce an antibody framework that is more similar to the germline. In addition, a deamidation site containing two asparagine residues was removed from the heavy chain variable region (V H ) was removed from the CDR2 of TPP-24727. This exchange process produced 96 different antibody light chains and 96 different antibody heavy chains. The modified antibody chains were evaluated by pairing each modified chain with another unmodified chain from the parent antibody (i.e., each modified light chain was paired with an unmodified heavy chain of TPP-24727, and each modified heavy chain was paired with an unmodified light chain of TPP-24727).

[0118] like Fig.19 and Fig. 20 As demonstrated in the literature, sequence swapping resulted in antibodies with improved immunogenicity, expression levels and / or K compared to the parent antibody TPP-24727. D Several heavy and light chains. Fig.19 The sequence identifiers associated with each heavy chain characterized in are listed in Table 6, Fig. 20 The sequence identifiers associated with each light chain characterized in are listed in Table 7.

[0119] Table 6. Assignment to Fig.19 The CDRs and V H The amino acid sequence identifier

[0120]

[0121] Table 7. Assignment to Fig. 20 The CDRs and V L The sequence identifier of the amino acid sequence

[0122]

[0123] Each modified antibody chain was tested for expression level, amidolytic activity ( PCa), surface plasmon resonance (SPR)-based binding to both human APCs and PCs (i.e., kon, koff, kD), and UNcle-based aggregation temperature (Tagg). Based on these measurements, 13 light chains and 6 heavy chain combinations were selected to generate 78 optimized antibodies. These 78 new antibodies were rescreened using the same assay, and 20 antibodies were selected for further study. These 20 optimized antibodies are referred to herein as Group 1 antibodies. The initial screening assay results for Group 1 antibodies are shown in Figure 1 middle.

[0124] The first group of antibodies includes antibodies with a variety of improved property combinations compared to the parent antibody TPP-24727. They also include a series of various heavy and light chains, providing a wide range of optimization space for further affinity maturation. The sequences of the variable domains of the first group of antibodies are summarized in Table 1. In preparation for extended functional evaluation and chemical, manufacturing and control (CMC) evaluations (including those described in Example 2), scaled-up production of the first group of 20 antibodies was carried out.

[0125] Material

[0126] Plasma-derived human APC and human PC were purchased from Enzyme Research Laboratories (South Bend, IN, USA). Human APC substrate PCa was purchased from American Diagnostica (Pfungstadt, Germany).

[0127] Expression and purification

[0128] The genes encoding the heavy and light chains of the antibodies were individually subcloned into the expression vector pTT5 (National Research Council, Canada) and then co-transfected into HEK293 cells (National Research Council, Canada). The cells were maintained in a medium supplemented with 2 mM GlutaMAX TM Supplement (Invitrogen), 0.1% Pluronic TM FreeStyle F-68 nonionic surfactant (Sigma-Aldrich) and 0.5% PenStrep (penicillin-streptomycin solution) (Invivogen) TMAfter 5 days of transient expression, the cleared supernatant was affinity purified using protein A beads (GE Healthcare) and then purified using Zeba TM Buffer exchange was performed using a spin desalting column (Thermo Fisher) into PBS. IgG concentrations were determined using anti-human Fc (Sigma) for capture and Amplex TM Quantification was performed via sandwich ELISA using anti-human IgG (Fc specific)-peroxidase antibody (Sigma) detected with the Sigma Red Hydrogen Peroxide / Peroxidase Assay Kit (Invitrogen). Human reference serum (Bethyl) was used as IgG concentration standard.

[0129] Aggregation temperature

[0130] The aggregation onset temperature (Tagg) was determined using the UNcle apparatus (integrated biologics stabilization platform; UNCHAINED Labs) according to the manufacturer's protocol. The machine was used to measure static scattered light at 266 nm from protein solutions, and the UNcle software was then used to calculate the aggregation onset. Supernatants were collected from HEK293 cells induced to express the antibody of interest (see above) and the supernatants were collected using 500 centrifugal concentrator device (10000MWCO PES, Sartorius) concentrated and buffer exchanged (exchanged into PBS). Before analysis, the final protein solution (in PBS buffer, <0.5mg / mL) was centrifuged at 8000g for 2 minutes. The following temperature gradient was used for analysis: starting temperature: 30°C, rate: 0.33°C / min, final temperature: 95°C.

[0131] Affinity and binding kinetics

[0132] Surface plasmon resonance (SPR) assays were performed to evaluate the binding kinetics and affinity of anti-APC antibodies. TM Binding assays were performed on a T200 instrument (GE Healthcare) at 25°C using assay buffer (HBS-P+, 1 mg / ml BSA, 0.01% Na-azide, 300 mM NaCl, 2.75 mM MgCl2, 0.75 mM CaCl2). Antibodies were captured using anti-human Fc IgG covalently coupled (via amine) to a CM5 sensor chip. Human APC and PC were used as analytes at concentrations of 100 nM and 1 μM, respectively. In order to obtain reliable dissociation rate constants (k d), after 180 seconds of association, the dissociation rate was set to 600 seconds. The resulting sensorgrams were fitted to a 1:1 Langmuir binding model to obtain kinetic and affinity data. The resulting sensorgrams were fitted to a 1:1 Langmuir binding model to obtain kinetic and affinity data.

[0133] Effects on the amidolytic activity of APC

[0134] The amidolytic activity assay was performed to evaluate the effect of antibody binding on the amidolytic activity of human APC (hAPC). hAPC (20 nM) was mixed with various concentrations (20 nM, 4 nM and 0.5 nM) of each monoclonal antibody (mAb) in equal volumes in a 96-well dilution plate and then pre-incubated for 20 minutes at room temperature. Thereafter, the APC / antibody mixture was transferred to a 384-well plate in quadruplicate at 10 μL / well and 10 μL of substrate was taken. PCa (0.1 mM) was added to each well. Substrate hydrolysis was monitored using a chromogenic readout at 405 nm using an M1000Pro plate reader.

[0135] Example 2 - Screening of Group 1 Antibodies

[0136] The following examples describe several assays used to evaluate the first 20 anti-APC antibodies (ie, Panel 1 antibodies) identified in Example 1. The goal of these evaluations was to assess the effects of the antibodies on the anticoagulant activity and cytoprotective function of APC.

[0137] Affinity and binding kinetics

[0138] To evaluate the binding kinetics and affinity of Panel 1 antibodies to APC, binding assays were performed using surface plasmon resonance (SPR). TM Binding assays were performed on a T200 instrument (GE Healthcare) at 25°C using assay buffer (HBS-P+, 1 mg / ml BSA, 0.01% Na-azide, 300 mM NaCl, 2.75 mM MgCl2, 0.75 mM CaCl2). Antibodies were captured by anti-human Fc IgG covalently coupled to a CM5 sensor chip. Human APC and PC were used as analytes at concentrations of 100 nM and 1 μM, respectively. To obtain reliable dissociation rate constants (k d ), after 180 seconds of association, the dissociation rate was set to 600 seconds. The resulting sensorgram was fitted to a 1:1 Langmuir binding model to obtain Figure 2All 20 antibodies in group 1 showed improved ka and 1 antibody showed improved k compared to the parent antibody TPP-24727. d , 11 antibodies showed improved K D .

[0139] Protein concentration

[0140] IgG concentration was determined using anti-human Fc (Sigma) for capture and Amplex TM Quantification was performed via sandwich ELISA using anti-human IgG (Fc specific)-peroxidase antibody (Sigma) detected with the Sigma Red Hydrogen Peroxide / Peroxidase Assay Kit (Invitrogen). Human reference serum (Bethyl) was used as IgG concentration standard.

[0141] Aggregation temperature and melting temperature

[0142] The aggregation onset temperature (Tagg) was determined using a UNcle device (UNCHAINED Labs) according to the manufacturer's protocol. The machine was used to measure the static scattered light of the protein solution at 266nm, and the UNcle software was then used to calculate the aggregation onset. In addition, the melting temperature (Tm) was determined via differential scanning calorimetry using a MicroCal VP-capillary DSC device according to the manufacturer's protocol. Prior to analysis, a reference protein solution (0.5 mg / mL in PBS, pH 7.4) was centrifuged at 8000g for 2 minutes. The following temperature gradient was used for analysis: starting temperature: 25°C, heating scan rate: 2°C / min, final temperature: 95°C. The heat capacity signal was deconvoluted to produce base peaks at different melting temperatures (for CH2 / CH3 and Fab). The HEK293 supernatant containing antibodies from transient expression was concentrated (see above) and used. The protein was concentrated and buffer exchanged (into PBS) using a 500 centrifugal concentrator unit (10000 MWCO PES, Satorius). The final protein solution (<0.5 mg / mL in PBS buffer) was centrifuged at 8000 g for 2 min prior to analysis. For the analysis, the following temperature gradient was used: start temperature: 30 °C, rate: 0.33 °C / min, final temperature: 95 °C. The results of this analysis are presented in Figure 3 Among the 20 group 1 antibodies, all showed high purity and high monomer percentage, and had a solubility greater than 188 mg / mL. Among them, TPP-26882 had the highest expression titer (332 mg / mL) and thermal stability (Tm = 82.0°C).

[0143] Effects on the amidolytic activity of APC

[0144] To evaluate the effect of panel 1 antibodies on the amidolytic activity of APC, 20 nM human APC was mixed with different concentrations of the 20 antibodies in equal volumes in a 96-well plate and then pre-incubated at room temperature for 20 minutes. Thereafter, the APC / antibody mixture was transferred to a 384-well assay plate in quadruplicate at 10 μL / well, and 10 μL of substrate was added. PCa (0.1 mM). At 37°C, a Tecan The M1000Pro plate reader provides a chromogenic reading at 405 nm, thereby monitoring substrate hydrolysis. The results of this assay are presented in Figure 4 Anti-APC antibodies were tested at 8 nM and 3.2 nM concentrations. At 8 nM, 12 antibodies elicited higher amidolytic activity compared to the parent antibody TPP-24727. At 3.2 nM, 7 antibodies elicited higher activity than TPP-24727.

[0145] FVa inactivation

[0146] The ability of the first group of antibodies to inhibit the inactivation of FVa by APC was measured using a FXa and thrombin generation assay. In this assay, 20 pM FVa was incubated with 50 pM APC in an assay buffer (25 mM Tris-HCl, 130 mM NaCl, 2.7 mM KCl, 25 μM phospholipids (PC: PE: PS 40: 40: 20), 5 mM CaCl2, and 1 mg / mL BSA), with the concentration of each antibody ranging from 0.5 nM to 1000 nM. After incubation for 30 minutes at room temperature, 100 μL of a prothrombinase mixture containing 400 nM FXa, 1 μM prothrombin, and 0.6 mM thrombin chromogenic substrate (S-2238) was added to the 100 μL incubation mixture. The kinetics of thrombin-mediated substrate hydrolysis were monitored at 405 nm for 3 hours at 25 ° C using a plate reader. The results of this assay are presented in Figure 5 Among the antibodies tested, TPP-26783, TPP-26784, and TPP-26920 showed improved EC50 in inhibiting FVa compared to the parent antibody TPP-24727.

[0147] Example 3: Functional evaluation of group 1 antibodies

[0148] In the following examples, the top 20 anti-APC antibodies (i.e., Panel 1 antibodies) identified in Example 1 were functionally characterized using several assays. Specifically, the effects of these antibodies on the coagulation-related activities of APC were evaluated using a plasma coagulation assay; the effects of these antibodies on the cytoprotective activity of APC were evaluated using a PAR1 cleavage assay, an endothelial barrier integrity assay, and a histone H3 cleavage assay; and the effects of these antibodies on the sensitivity of APC to physiological inhibitors were evaluated using an in vitro plasma half-life assay.

[0149] The results of these functional assays will help determine whether a particular Group 1 antibody is useful for a particular indication. For example, antibodies that inhibit the anticoagulant activity of APCs while maintaining their cytoprotective activity may be used to treat conditions requiring blood coagulation (e.g., acute bleeding), while antibodies that enhance the cytoprotective activity of APCs may be used to treat conditions such as sepsis and inflammation in acute ischemic disease.

[0150] result:

[0151] Coagulation time

[0152] The ability of group 1 antibodies to shorten clotting time is used Coagulation assay was used to measure. The results of the assay revealed that these antibodies shortened the in vitro coagulation time to varying degrees (Fig. 6). It was confirmed that antibodies TPP-26870, TPP-26935, TPP-26874, TPP-26873, TPP-26926, TPP-26864, TPP-26918 and TPP-26920, compared with the parent antibody TPP-24727, inhibited the anticoagulant effect of APC and shortened the plasma coagulation time. Among these antibodies, TPP-26870 showed that the coagulation time was shortened to the greatest extent.

[0153] PAR1 cleavage

[0154] Cleavage and activation of PAR1 by APC alone induces cytoprotective effects, including protective gene expression, enhanced endothelial barrier function, reduced cytokine secretion, and protection against apoptosis. Therefore, the ability of panel 1 antibodies to inhibit APC-mediated PAR1 cleavage was tested using a secreted embryonic alkaline phosphatase (SEAP)-PAR1 cleavage assay. The results of this assay are presented in Figure 7 Based on these results, antibodies with increased (e.g., TPP-26918) or decreased (e.g., TPP-26920) ability to inhibit APC-mediated cleavage of PAR1 were identified, as well as one antibody (i.e., TPP-26924) that did not show any inhibitory effect on APC-mediated cleavage of PAR1 at concentrations up to 500 nM.

[0155] Endothelial barrier integrity

[0156] APC has a protective effect on endothelial barrier function. Therefore, the effect of Group 1 antibodies on this cell protective activity was tested using an endothelial barrier integrity assay. The results of this assay are presented in Figure 8. Based on these results, several antibodies (e.g., TPP26926 and TPP26935) that enhance APC-mediated endothelial barrier protection were identified.

[0157] Histone cleavage

[0158] Histones act as damage-associated molecular patterns (DAMPs) when released into the extracellular space, causing systemic inflammation and toxic reactions. APC inhibits the cytotoxicity of histone induction via the proteolytic cleavage of histones. Therefore, the ability of the first group of antibodies to inhibit APC-mediated histone cleavage was tested using histone H3 cleavage assay. Based on the results of the assay, antibodies (Fig. 9) that can inhibit or enhance APC-mediated H3 cleavage were identified. For example, TPP-26921 inhibits cleavage, while TPP-26862 both increases cleavage rate and increases cleavage total amount.

[0159] Plasma half-life

[0160] An in vitro plasma half-life assay was used to determine the effect of Panel 1 antibodies on the sensitivity of APC to physiological inhibitors. This assay identified antibodies that extended the serum half-life of APC ( Fig.10 ). This extension of half-life suggests that binding of these antibodies reduces the sensitivity of APCs to inhibition by physiological inhibitors present in plasma.

[0161] Materials and methods:

[0162] Coagulation assay. Plasma samples (50 μL) containing different concentrations of group 1 antibodies were mixed with (25 μL; 1 U / mL) and STA-PTT reagent (75 μL) were mixed at 37°C. After incubation for 4 minutes, 75 μL of 25 mM CaCl2 solution was added to initiate coagulation. (a single-chain glycoprotein derived from snake venom that activates protein C) is added to plasma to activate protein C, reduce coagulation activity, and prolong clotting time.

[0163] SEAP–PAR1 cleavage assay. A PAR1 cleavage reporter construct was prepared with an N-terminal SEAP domain. HEK293 cells expressing SEAP–PAR1 and wild-type (wt)-EPCR were grown in 96-well plates until confluent. On the day of the experiment, cells were washed with Hank's balanced salt solution supplemented with 1.3 mM CaCl2, 0.6 mM MgCl2, and 0.1% BSA (HMM2). Plasma-derived APC (50 nM or 100 nM) and antibodies were preincubated in HMM2 for 30 minutes before being added to confluent SEAP–PAR1 / wt-EPCR cells in 96-well plates. After 60 minutes, the release of SEAP was measured using one-step p-nitrophenyl phosphate (Pierce No. 37621). After correcting for background activity in the absence of APC, values ​​are expressed as a percentage of total SEAP activity present on the cells. Values ​​were normalized to the values ​​obtained in the absence of antibody (=100%).

[0164] Endothelial Barrier Integrity Assay Using iCELLigence TM Endothelial cell permeability was measured in real time by the ACEA system (ACEA, San Diego, CA), which measures changes in transendothelial resistance (TEER) by cell-substrate electrical impedance sensing (ECIS). In brief, EA.hy926 endothelial cells were grown to confluence for 2 to 3 days in an 8-well culture dish containing a gold film electrode. Two hours before the addition of APC (50nM), DMEM (Double's modified Eagle's medium; Invitrogen) containing 10% fetal bovine serum was replaced with serum-free medium containing 0.1% BSA in the presence and absence of antibodies. After the addition of thrombin (2nM), the permeability of the confluent monolayer after 3 hours of treatment with APC was determined. All comparisons of cell indices were performed using standardized resistance. The results are expressed as the percentage of the maximum barrier protection produced by APC in the absence of antibodies.

[0165] Histone H3 cleavage assay. APC (50nM) and antibody (500nM) were pre-incubated in HBS buffer ((20mM Hepes, 147mM NaCl, 3mM KCl) + 100μg / ml BSA + 2mM CaCl2, pH 7.4) for 30 minutes. After incubation, the mixture was added to 100μg / ml histone H3 (H3; Roche). Over 2 hours, the mixture was subsampled at different time points and added to reducing sample buffer. The subsampled samples were then heated at 95°C for 15 minutes, rotated at 4000rpm for 5 minutes, and loaded into a 12% Bis-Tris gel with MES running buffer (30μl / well; 750ng H3) after a brief vortex. Markers (10 μL) were used as molecular weight standards, and the 20 kDa marker was used as a reference for normalization between gels. Protein gel stain (50 ml / gel) was used for staining overnight. Protein bands were scanned on a LI-COR imaging system (700 channels, intensity 6, 1 mm offset, resolution 169 μm, highest quality, manual background). Images were analyzed using Image Studio V5.2.

[0166] In vitro plasma half-life determination. APC (70 nM) and antibody (700 nM) were pre-incubated for 30 min, and then normal pooled plasma was added to 90% (v / v). Samples were collected at different time points (0 to 90 min) and quenched in ice-cold TBS. The chromogenic activity of APC was determined by PCa. The background chromogenic activity of plasma (without APC) was subtracted and the APC activity was normalized to the t=0 time point. The half-life (T) was determined by fitting a one-phase exponential decay curve. 1 / 2 ).

[0167] Example 4 - Screening and functional evaluation of panel 2 antibodies

[0168] The following examples describe several assays used to evaluate a second set of 24 anti-APC antibodies (referred to herein as Group 2 antibodies). Group 2 antibodies include 22 variants of the promising Group 1 antibody TPP-26870 that have greater affinity for APC than TPP-26870. These variants were generated by establishing a single NNK site saturation mutagenesis library based on TPP-26870. Fig.11 The mutations found in each of these 22 antibody variants are summarized in Table 3. Group 2 also includes two additional antibodies (i.e., TPP-26922 and TPP-26939) that are not related to any of the Group 1 antibodies. The sequences of the variable domains of the Group 2 antibodies are summarized in Table 3.

[0169] Several assays were used to screen and evaluate the functional characteristics of the antibodies in panel 2. Again, the goal of these evaluations was to assess the effects of the antibodies on the anticoagulant activity and cytoprotective function of APCs.

[0170] Screening assay

[0171] Panel 2 antibodies were screened using several assays described in Example 2. Specifically, (1) surface plasmon resonance (SPR) was used to assess the binding kinetics and affinity of these antibodies to APC and PC, (2) an amidolytic activity assay was used to assess the effect of these antibodies on the substrate cleavage activity of APC, and (3) the UNcle apparatus was used to determine the aggregation onset temperature (Tagg) of these antibodies. The results of these assays are presented in Fig.12 Each of these assays was performed as described in Example 2.

[0172] Coagulation time

[0173] The ability of group 2 antibodies to shorten clotting time was used The results of this assay revealed that several TPP-26870 variant antibodies (i.e., TPP-29843, TPP-29847, TPP-29850, TPP-29852, TPP-29853, TPP-29854, TPP-29855, TPP-29856, TPP-29857, TPP-29858, TPP-29864, and TPP-29865) exhibited enhanced inhibition of the anticoagulant activity of APC relative to the parent antibody and caused a shortened in vitro clotting time, while other variant antibodies (e.g., TPP-29851, TPP-29844) exhibited reduced inhibition of the anticoagulant activity of APC ( Fig.13 ). Carry out as described in Example 3 Coagulation assays.

[0174] Histone cleavage

[0175] The ability of the second group of antibodies to inhibit APC-mediated histone cleavage was determined using a histone H3 cleavage assay. The assay identified antibodies that can inhibit or enhance histone cleavage to varying degrees (Figure 14). For example, TPP-29864 inhibits APC-mediated H3 cleavage, while TPP-29848 enhances the total amount of cleavage. The histone H3 cleavage assay was performed as described in Example 3.

[0176] Plasma half-life

[0177] An in vitro plasma half-life assay was used to determine the effect of Panel 2 antibodies on the sensitivity of APC to physiological inhibitors. The results of this assay were used to identify antibodies that extend the serum half-life of APC ( Fig.15 ). This extension of half-life indicates that binding of these antibodies reduces the sensitivity of APC to inhibition by physiological inhibitors present in plasma. In vitro plasma half-life determinations were performed as described in Example 3.

[0178] References:

[0179] Each of the patent and non-patent publications cited herein is hereby incorporated by reference in its entirety, including but not limited to the references listed below.

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Claims

1. An isolated antibody comprising a heavy chain variable region (V H ), the heavy chain variable region comprising a CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:118, 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 V H It also comprises (a) a CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4 and SEQ ID NOs: 109-113, (b) a CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-8 and SEQ ID NOs: 114-117, or (c) both of the following: a CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4 and SEQ ID NOs: 109-113, and a CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-8 and SEQ ID NOs: 114-117.

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

4. An isolated antibody comprising a V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-15 and SEQ ID NOs: 119-129. H , wherein the antibody specifically binds to APCs and minimally binds to PCs.

5. An isolated antibody comprising a light chain variable region (V L ), wherein the light chain variable region comprises a CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:23 and SEQ ID NO:138-140, wherein the antibody specifically binds to APCs and minimally binds to PCs.

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

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

8. An isolated antibody comprising a V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-35 and SEQ ID NOs: 141-151. L , wherein the antibody specifically binds to APCs and minimally binds to PCs.

9. The antibody according to any one of claims 5 to 8, further comprising V H , which comprises a CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:

118.

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

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

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

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

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

15. The antibody of any one of claims 1-14, 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.

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

17. The antibody of any one of claims 1-9, 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 the endothelial barrier protection activity of APCs; d) increase, minimally affect, or decrease the plasma half-life of APC; or e) any combination thereof.

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

19. A nucleic acid encoding the antibody of any one of claims 1-18.

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

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

22. A method for producing an antibody, the method comprising: a) culturing the host cell of claim 21 under conditions that result in the production of the antibody; as well as b) isolating the antibody from the host cell.

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

24. A method for treating or preventing a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody as described in any one of claims 1-18, an antibody that specifically binds to an exogenous APC protein or a variant thereof as described in any one of claims 1-18, or a pharmaceutical composition as described in claim 23.

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

26. The method of claim 25, 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.

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

28. The method of claim 27, 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.

29. The antibody of any one of claims 1-18 for use as a medicament for the treatment or prevention of a disorder.

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

31. The antibody of claim 30, 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.

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

33. The antibody of claim 32, 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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