Monoclonal antibodies directed against human activated protein C and their production and application

Abstract

The present invention belongs to the technical field of monoclonal antibodies and relates to a monoclonal antibody directed against activated protein C (aPC). The heavy and light chain amino acid sequences of the monoclonal antibody are represented by SEQ ID NOs. 5 to 26, and the CDR sequences are represented by SEQ ID NOs. 27 to 92. The present invention also provides biomaterials related to the monoclonal antibody. The monoclonal antibody can selectively bind to aPC but does not bind to unactivated protein C. The present invention also provides the use of the monoclonal antibody and related biomaterials in the manufacture of drugs for treating blood coagulation deficiencies or disorders, and related treatment methods. The present invention has broad applicability.

Description

[Technical Field]

[0001] The present invention belongs to the technical field of monoclonal antibodies, and relates to a monoclonal antibody directed against human activated protein C (aPC) and its applications. [Background technology]

[0002] The protein C (PC) pathway plays an important role in regulating blood coagulation and inflammation. Protein C is activated by the complex formation between thrombin and thrombomodulin (TM) on vascular endothelial cells. Activated protein C (aPC) negatively downregulates thrombin formation by cleaving activated factors V and VIII, thereby maintaining the balance between thrombosis and hemostasis in the body. aPC also protects animals, reducing the invasion of endotoxin (LPS) and Escherichia coli. Recombinant human aPC products have been approved by the FDA as the first treatment for severe sepsis in humans. However, the molecular mechanisms underlying the anti-inflammatory and cytoprotective effects of aPC are currently incompletely understood. Mutational studies have demonstrated that the anticoagulant activity of aPC is by no means essential for its anti-apoptotic effect on endothelial cells. The endothelial cell protein C receptor (EPCR), expressed primarily on the endothelium of large blood vessels, enhances the activation of protein C by the thrombin-TM complex. Recent evidence has shown that aPC bound to EPCR can degrade PAR-1 on endothelial cells. Furthermore, this study revealed that all of the anti-inflammatory and anti-apoptotic effects of aPC signaling are mediated by PAR-1 in endothelial cells. However, after lethal LPS challenge, the phenotype of PAR-1-deficient mice was similar to that of wild-type controls, suggesting that PAR-1 is not the only pathway involved in the regulation of inflammation and apoptosis by aPC signaling.

[0003] In light of the central role of aPC in regulating pathological and physiological functions in the body, the monoclonal antibody HAPC1573 has been produced. This antibody specifically recognizes human activated protein C (aPC) but not protein C (Xu, Patent Documents 1 and 2). This antibody alters the amidolytic activity of a chromogenic peptide substrate by aPC and blocks the anticoagulant activity of aPC in plasma. More importantly, this antibody does not affect the degradation of extracellular histones H3 and H4 by aPC, nor does it affect the cytoprotective activity of aPC against thrombin-induced permeability of endothelial cells. MAPC1591 is an anti-mouse aPC monoclonal antibody with similar functions to HAPC1573. MAPC1591 blocks the anticoagulant activity of mouse aPC while maintaining its cytoprotective activity, thereby eliminating the risk of traumatic bleeding even in patients with hemophilia A. This means that anti-aPC antibodies can be applied to clinical settings such as hemophilia, trauma, and sepsis.

[0004] Zhao et al. reported the CDR sequences of HAPC1573 and humanized HAPC1573, which target only the anticoagulant activity but not other activities of aPC. They demonstrated in vivo protection against bleeding in a monkey model of acute acquired hemophilia A (Zhao, Patent Documents 3 and 4, and Non-Patent Document 1). In these reports, the X-ray crystal structure of the aPC-humanized HAPC1573 Fab complex indicated that HAPC1573 does not bind to the active site of aPC, but rather to the aPC autolysis loop, including the known aPC-FVa binding site. Thus, humanized HAPC1573 may inhibit the anticoagulant function of aPC by interfering with the interaction between aPC and FVa. Furthermore, the inhibitory effect of humanized HAPC1573 was limited to the anticoagulant activity of aPC, without affecting its cytoprotective, anti-inflammatory, or cell signaling functions. The reported concentration of aPC in human plasma is approximately 40 pM, and surface plasmon resonance analysis demonstrated that the affinity of humanized HAPC1573 for aPC was 10 nM. Furthermore, a Protac-based APTT blood coagulation assay demonstrated a concentration-dependent effect of HAPC1573 in shortening the APTT prolongation in human FVIII-deficient and FIX-deficient plasma. However, even at a concentration of 50 μg / ml, HAPC1573 failed to achieve maximal inhibition of the anticoagulant activity of aPC in this in vitro assay. This observation was consistent with in vivo results obtained using humanized HAPC1573 in an acute acquired simian hemophilia model. Animal studies have shown that significant protective effects were only achieved at high doses of 10 mg / kg and 30 mg / kg. However, such high doses are not suitable for clinical use, and therefore, the development of a more affinity humanized HAPC1573 is a prerequisite and guarantee for the potential future application of aPC in therapy. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 8,153,766 [Patent Document 2] U.S. Patent No. 9,127,072 [Patent Document 3] U.S. Patent No. 9,657,111 [Patent Document 4] International Publication No. 2017 / 087391 [Non-patent literature]

[0006] [Non-Patent Document 1] Zhao, Nat. Communications 2020 Summary of the Invention

[0007] In one aspect, the present invention provides anti-aPC monoclonal antibodies, which include an original antibody and mutant antibodies obtained by mutating one or more of the following six mutation positions in the original antibody:

[0008] The original antibody comprises (a) heavy chain CDRs represented by any one or more of the sequences of SEQ ID NOs:27, 28, and 29, and / or (b) light chain CDRs represented by any one or more of the sequences of SEQ ID NOs:30, 31, and 32.

[0009] The six mutation positions are: 1) N57R / W located in VL CDR2, 2) E59G / P located in VL CDR2, 3) Q93V located in VL CDR3, 4) N31F located in VH CDR1, 5) S55K located in VH CDR2, and 6) Y104T located in VH CDR3.

[0010] In the present invention, the constant region of the anti-aPC monoclonal antibody comprises one or more of the sequences of human IgG1, IgG2, IgG3, and IgG4.

[0011] Preferably, in the present invention, the mutation positions to be performed on the original antibody of the anti-aPC monoclonal antibody include at least N31F located in VH CDR1, S55K located in VH CDR2, N57R located in VL CDR2, and E59G located in VL CDR2.

[0012] Preferably, in the present invention, the mutation positions to be performed on the original antibody of the anti-aPC monoclonal antibody include at least N57R located in VL CDR2, E59G located in VL CDR2, Q93V located in VL CDR3, N31F located in VH CDR1, and S55K located in VH CDR2.

[0013] In another aspect, the present invention provides one or more of the following 1) to 10) as biomaterials related to the above-mentioned anti-aPC monoclonal antibodies:

[0014] 1) A nucleic acid molecule encoding the above anti-aPC monoclonal antibody.

[0015] 2) An expression cassette containing the nucleic acid molecule described in 1).

[0016] 3) A construct containing the nucleic acid molecule described in 1).

[0017] 4) A construct containing the expression cassette described in 2).

[0018] 5) An expression system comprising the nucleic acid molecule described in 1).

[0019] 6) An expression system containing the expression cassette described in 2).

[0020] 7) An expression system comprising the construct described in 3).

[0021] 8) An expression system comprising the recombinant vector described in 4).

[0022] 9) A primer pair that amplifies a nucleic acid molecule encoding the amino acid sequence of the anti-aPC monoclonal antibody.

[0023] 10) A fusion antibody comprising the above anti-aPC monoclonal antibody.

[0024] In another aspect, the present invention provides a pharmaceutical product. The pharmaceutical product is a drug composition or a detection reagent kit. The pharmaceutical product contains one or more of the anti-aPC monoclonal antibodies or related biomaterials described above. When the pharmaceutical product is a drug composition, it further contains a pharmaceutically acceptable vector.

[0025] In another aspect, the present invention provides a method related to the treatment of blood coagulation. The method comprises administering an effective amount of the anti-aPC monoclonal antibody or a related biomaterial to an individual in need of treatment. The individual suffers from a blood coagulation deficiency or defect, or is in need of blood coagulation treatment, or is suffering from sepsis or hemophilia, or is in need of hemostasis regulation.

[0026] In another aspect, the present invention provides the use of the anti-aPC monoclonal antibody or its related biomaterial in the manufacture of a drug for preventing and / or treating blood coagulation deficiencies or disorders. Examples of blood coagulation deficiencies or disorders include diseases in which bleeding occurs due to a deficiency in blood coagulation factors, as well as disorders of blood coagulation caused by trauma or surgery. Further examples of blood coagulation deficiencies or disorders include hemophilia, sepsis, and traumatic bleeding.

[0027] In another aspect, the present invention provides the use of the anti-aPC monoclonal antibody or related biomaterial in the manufacture of an enhancer that enhances the ability of aPC to cleave histones H3 and H4, or in the manufacture of a drug that inhibits the anticoagulant activity of activated protein C in an individual, or in the manufacture of a drug for the prevention and / or treatment of an individual in need of blood coagulation, or in the manufacture of a drug for the prevention and / or treatment of an individual suffering from sepsis, or in the manufacture of a drug for the prevention and / or treatment of an individual suffering from hemophilia, or in the manufacture of a drug for regulating hemostasis in an individual, or in immunological tests and analyses for the diagnosis or treatment of non-diseases, or in the manufacture of reagents or reagent kits for the detection of aPC, or in the use in combination with other reagents or drugs.

[0028] In another aspect, the present invention provides a method for producing the above-mentioned anti-aPC monoclonal antibody, which comprises the step of culturing an expression system for the anti-aPC monoclonal antibody under conditions suitable for the expression of the anti-aPC monoclonal antibody, thereby expressing the anti-aPC monoclonal antibody. [Effects of the Invention]

[0029] The beneficial effects of the present invention are as follows: The anti-aPC monoclonal antibody provided by the present invention is a humanized antibody obtained by humanizing HAPC1573 or by simultaneously screening for mutations at both single and multiple positions. The anti-aPC monoclonal antibody selectively binds to and inhibits activated protein C, but does not bind to or inhibit inactivated protein C. The anti-aPC monoclonal antibody selectively binds to human activated protein C, but does not bind to activated protein C or inactivated protein C from other species. The affinity of the antibody is significantly improved, with some antibodies exhibiting an affinity 100-fold improvement compared to HAPC1573. Protac-based APTT blood coagulation assays demonstrated that the present invention can achieve maximum inhibitory effects on the anticoagulant activity of aPC with only a small amount of antibody (e.g., 1 μg / ml). Because the antibody can potentially block the anticoagulant activity of aPC, it has broad applicability in the treatment of blood coagulation deficiencies or disorders. [Brief explanation of the drawings]

[0030] [Figure 1A] FIG. 1 shows the binding affinity of humanized anti-aPC monoclonal antibodies (Humab001-1 to Humab001-28, or HAPC-1 to HAPC-28) to human aPC. [Figure 1B] FIG. 1 shows the binding affinity of humanized anti-aPC monoclonal antibodies (Humab001-1 to Humab001-28, or HAPC-1 to HAPC-28) to human aPC. [Figure 1C] FIG. 1 shows the binding affinity of humanized anti-aPC monoclonal antibodies (Humab001-1 to Humab001-28, or HAPC-1 to HAPC-28) to human aPC. [Figure 1D] FIG. 1 shows the binding affinity of humanized anti-aPC monoclonal antibodies (Humab001-1 to Humab001-28, or HAPC-1 to HAPC-28) to human aPC. [Figure 2A]FIG. 1 shows the binding affinity of humanized anti-aPC monoclonal antibodies (Humab001-1 to Humab001-28, or HAPC-1 to HAPC-28) to human PC. [Figure 2B] FIG. 1 shows the binding affinity of humanized anti-aPC monoclonal antibodies (Humab001-1 to Humab001-28, or HAPC-1 to HAPC-28) to human PC. [Figure 2C] FIG. 1 shows the binding affinity of humanized anti-aPC monoclonal antibodies (Humab001-1 to Humab001-28, or HAPC-1 to HAPC-28) to human PC. [Figure 3A] 3A shows the binding avidity of HAPC-14 mutant antibodies (HAPC14-1 to HAPC14-10, or HAPC-14-1 to HAPC-14-10) to hAPC / hPC. The abscissa in FIG. 3A is 10-6 nM, 10-4 nM, 10-2 nM, 100 nM, 102 nM, and 104 nM, respectively. [Figure 3B] 3B shows the binding avidity of HAPC-14 mutant antibodies (HAPC14-1 to HAPC14-10, or HAPC-14-1 to HAPC-14-10) to hAPC / hPC. The abscissa in FIG. 3B is 10-6 nM, 10-4 nM, 10-2 nM, 100 nM, 102 nM, and 104 nM, respectively. [Figure 3C] This figure shows the binding avidity of HAPC-14 mutant antibodies (HAPC14-1 to HAPC14-10, or HAPC-14-1 to HAPC-14-10) to hAPC / hPC. The abscissa in Figure 3C is 10-6 nM, 10-4 nM, 10-2 nM, 100 nM, 102 nM, and 104 nM, respectively. [Figure 3D] This figure shows the binding avidity of HAPC-14 mutant antibodies (HAPC14-1 to HAPC14-10, or HAPC-14-1 to HAPC-14-10) to hAPC / hPC. The abscissa in Figure 3D is 10-6 nM, 10-4 nM, 10-2 nM, 100 nM, 102 nM, and 104 nM, respectively. [Figure 4A]FIG. 1 shows the affinity measurements of mutant HAPC-14 antibodies (HAPC14-1 to HAPC14-10, or HAPC-14-1 to HAPC-14-10). [Figure 4B] FIG. 1 shows the affinity measurements of mutant HAPC-14 antibodies (HAPC14-1 to HAPC14-10, or HAPC-14-1 to HAPC-14-10). [Figure 4C] FIG. 1 shows the affinity measurements of mutant HAPC-14 antibodies (HAPC14-1 to HAPC14-10, or HAPC-14-1 to HAPC-14-10). [Figure 4D] FIG. 1 shows the affinity measurements of mutant HAPC-14 antibodies (HAPC14-1 to HAPC14-10, or HAPC-14-1 to HAPC-14-10). [Figure 4E] FIG. 1 shows the affinity measurements of mutant HAPC-14 antibodies (HAPC14-1 to HAPC14-10, or HAPC-14-1 to HAPC-14-10). [Figure 4F] FIG. 1 shows the affinity measurements of mutant HAPC-14 antibodies (HAPC14-1 to HAPC14-10, or HAPC-14-1 to HAPC-14-10). [Figure 4G] FIG. 1 shows the affinity measurements of mutant HAPC-14 antibodies (HAPC14-1 to HAPC14-10, or HAPC-14-1 to HAPC-14-10). [Figure 4H] FIG. 1 shows the affinity measurements of mutant HAPC-14 antibodies (HAPC14-1 to HAPC14-10, or HAPC-14-1 to HAPC-14-10). [Figure 4I] FIG. 1 shows the affinity measurements of mutant HAPC-14 antibodies (HAPC14-1 to HAPC14-10, or HAPC-14-1 to HAPC-14-10). [Figure 4J] FIG. 1 shows the affinity measurements of mutant HAPC-14 antibodies (HAPC14-1 to HAPC14-10, or HAPC-14-1 to HAPC-14-10). [Figure 4K]FIG. 1 shows the affinity measurements of mutant HAPC-14 antibodies (HAPC14-1 to HAPC14-10, or HAPC-14-1 to HAPC-14-10). [Figure 5] FIG. 1 shows the effect of the mutant HAPC-14 antibody HAPC-14-6 on the histone cleavage ability of aPC. [Figure 6A] This figure shows an in vitro efficacy study, demonstrating the ability of HAPC-14 mutant antibodies HAPC-14-3 and HAPC-14-6 to inhibit the anticoagulant activity of aPC in plasma deficient in blood coagulation factors. A in the figure corresponds to plasma deficient in FVIII blood coagulation factor. [Figure 6B] This figure shows an in vitro efficacy study, demonstrating the ability of HAPC-14 mutant antibodies HAPC-14-3 and HAPC-14-6 to inhibit the anticoagulant activity of aPC in plasma deficient in blood coagulation factors. B in the figure corresponds to plasma deficient in FIX blood coagulation factors. [Figure 6C] This figure shows an in vitro efficacy study, demonstrating the ability of HAPC-14 mutant antibodies HAPC-14-3 and HAPC-14-6 to inhibit the anticoagulant activity of aPC in plasma deficient in blood coagulation factors. C in the figure corresponds to plasma deficient in FXI blood coagulation factor. [Figure 6D] This figure shows an in vitro efficacy study, demonstrating the ability of HAPC-14 mutant antibodies HAPC-14-3 and HAPC-14-6 to inhibit the anticoagulant activity of aPC in plasma deficient in blood coagulation factors. D in the figure corresponds to plasma deficient in FVII blood coagulation factor. [Figure 6E] This figure shows an in vitro efficacy study, demonstrating the ability of HAPC-14 mutant antibodies HAPC-14-3 and HAPC-14-6 to inhibit the anticoagulant activity of aPC in plasma deficient in blood coagulation factors. E in the figure corresponds to plasma deficient in blood coagulation factor FXII. [Figure 6F]This figure shows an in vitro efficacy study, demonstrating the ability of HAPC-14 mutant antibodies HAPC-14-3 and HAPC-14-6 to inhibit the anticoagulant activity of aPC in plasma deficient in blood coagulation factors. F in the figure corresponds to FV blood coagulation factor-deficient plasma. [Figure 6G] This figure shows an in vitro efficacy study, demonstrating the ability of HAPC-14 mutant antibodies HAPC-14-3 and HAPC-14-6 to inhibit the anticoagulant activity of aPC in plasma deficient in blood coagulation factors. G in the figure corresponds to plasma deficient in FX blood coagulation factors. [Figure 6H] This figure shows an in vitro efficacy study, demonstrating the ability of HAPC-14 mutant antibodies HAPC-14-3 and HAPC-14-6 to inhibit the anticoagulant activity of aPC in plasma deficient in blood coagulation factors. H in the figure corresponds to VWF-deficient plasma. [Figure 7A] 1 shows an in vivo efficacy test, in which A shows the effect on the blood coagulation function of FVIII blood coagulation factor-deficient mice when injected with FVIII blood coagulation factor, HAPC1573, or HAPC-14-6. [Figure 7B] 1 shows an in vivo efficacy test, in which B shows the effect on the blood coagulation function of FVIII blood coagulation factor-deficient mice when different doses of HAPC-14-6 are injected into the deficient mice. [Figure 7C] 1 shows an in vivo efficacy test, in which C shows the effect on the blood coagulation function of FIX blood coagulation factor-deficient mice when HAPC1573 and different doses of HAPC-14-6 are injected into the deficient mice. DETAILED DESCRIPTION OF THE INVENTION

[0031] In the present invention, HAPC1573 was humanized and then affinity-optimized. Optimized mutation sites were then screened using a Fab fragment, ultimately selecting six favorable sites. Further mutation combinations resulted in a 100-fold increase in affinity of the resulting optimized mutants compared to HAPC1573. Furthermore, Protac-based APTT blood coagulation assays showed that a maximum inhibitory effect on the anticoagulant activity of aPC could be achieved at a concentration of 1 μg / ml of the mutant antibody, indicating potential for further clinical application.

[0032] The present invention provides an anti-aPC antibody, i.e., anti-aPC monoclonal antibody 14 (HAPC-14), which comprises (a) a heavy chain comprising the heavy chain CDRs represented by any one or more of SEQ ID NOs:27, 28, and 29, and / or (b) a light chain comprising the light chain CDRs represented by any one or more of SEQ ID NOs:30, 31, and 32.

[0033] The anti-aPC monoclonal antibody 14 (HAPC-14) provided by the present invention has a heavy chain sequence represented by SEQ ID NO:5 and a light chain sequence represented by SEQ ID NO:6.

[0034] The present invention also provides mutant antibodies of a monoclonal antibody (anti-aPC monoclonal antibody 14) directed against human activated protein C (aPC). Based on the sequence of the above-described anti-aPC monoclonal antibody 14, the mutant antibodies were designed, screened, and statistically analyzed. It was found that the inclusion of one or more mutations at the following six positions significantly affected the binding ability of the monoclonal antibody's Fab fragment to the capture antibody, resulting in high affinity, excellent functional activity, and minimal phylogenetic sequence variation. The six positions are: a) N57R / W (N57R is superior to N57W) at the VL CDR2; b) E59G / P (E59G is superior to E59P) at the VL CDR2; c) Q93V at the VL CDR3; d) N31F at the VH CDR1; e) S55K at the VH CDR2; and f) Y104T at the VH CDR3.

[0035] Among these, the most significant mutations are N57R in the light chain and N31F in the heavy chain. In some examples, humanized protein C (haPC) completely inhibited the anticoagulant activity of aPC in a hemophilia mouse model and reduced the concentration of anti-aPC monoclonal antibody required to restore normal blood clotting function to less than 1 μg / ml.

[0036] For ease of description, each mutation combination will be described below using abbreviations: N57R is N57R located at VL CDR2, N57W is N57W located at VL CDR2, E59G is E59G located at VL CDR2, E59P is E59P located at VL CDR2, Q93V is Q93V located at VL CDR3, N31F is N31F located at VH CDR1, S55K is S55K located at VH CDR2, and Y104T is Y104T located at VH CDR3.

[0037] In the present invention, the mutation combinations at the above six positions include (but are not limited to):

[0038] (1) Contains only one mutation at any one of positions N57R, N57W, E59G, E59P, Q93V, N31F, or S55K. Or (2) mutations at two positions simultaneously: N57R and N57W, E59G and E59P, Q93V and N31F, N31F and S55K, N57R and E59G, N57R and E59P, N57R and Q93V, N57R and N31F, N57R and S55K, N57W and E59P, N57W and Q93V, N57W and N31F, N57W and S55K, E59G and Q93V, E59G and N31F, E59G and S55K, E59P and N31F, E59P and S55K, Q93V and S55K. Or, (3) N57R, N57W and E59G, N57R, N57W and E59P, N57R, N57W and Q93V, N57R, N57W and N31F, N57R, N57W and S55K, N57R, E59G and E59P, N57R, E59G and Q93V, N57R, E59G and N31F, N57R, E59G and S 55K, N57R and E59P and Q93V, N57R and E59P and N31F, N57R and E59P and S55K, N57R and Q93V and N31F, N57R and Q93V and S55K, N57R and N31F and S55K, N57W and E59G and E59P, N57W and E59G and Q93V, N57W and E59G and N31F , N57W and E59G and S55K, N57W and E59P and Q93V, N57W and E59P and N31F, N57W and E59P and S55K, N57W and Q93V and N31F, N57W and Q93V and S55K, N57W and N31F and S55K, E59G and E59P and Q93V, E59G and E59P and N31F, E5 It simultaneously contains mutations at three positions: 9G, E59P, and S55K, E59G, Q93V, and N31F, E59G, Q93V, and S55K, E59G, N31F, and S55K, E59P, Q93V, and N31F, E59P, Q93V, and S55K, E59P, N31F, and S55K, and Q93V, N31F, and S55K.Or, (4) N57R, N57W, E59G, and E59P, N57R, N57W, E59G, and Q93V, N57R, N57W, E59G, and N31F, N57R, N57W, E59G, and S55K, N57R, E59G, E59P, and Q93V, N57R, E59G, E59P, and N31F , N57R and E59G and E59P and S55K, N57R and E59G and Q93V and N31F, N57R and E59G and Q93V and S55K, N57R and E59P and Q93V and N31F, N57R and E59P and Q93V and S55K, N57R and E59P and N31F and S55K, N57R and Q It contains mutations at four positions simultaneously: 93V, N31F, and S55K, N57W, E59G, E59P, and Q93V, N57W, E59G, E59P, and N31F, N57W, E59G, E59P, and S55K, N57W, E59P, Q93V, and N31F, N57W, E59P, Q93V, and S55K, N57W, E59P, N31F, and S55K, N57W, Q93V, N31F, and S55K, E59G, E59P, Q93V, and N31F, E59G, E59P, Q93V, and N31F, E59G, E59P, Q93V, and S55K, E59G, Q93V, N31F, and S55K, and E59P, Q93V, N31F, and S55K.Or, (5) N57R and N57W and E59G and E59P and Q93V, N57R and N57W and E59G and E59P and N31F, N57R and N57W and E59G and Q93V and N31F, N57R and N57W and E59P and Q93V and N31F, N57R and E59G and E59P and Q93V and N31F, N57W and E 59G and E59P and Q93V and N31F, N57R and N57W and E59G and E59P and S55K, N57R and N57W and E59G and Q93V and S55K, N57R and N57W and E59P and Q93V and S55K, N57R and E59G and E59P and Q93V and S55K, N57W and E59G and E59P and Q93V and S55K, N57R and N57W and E59G and N31F and S55K, N57R and N57W and E59P and N31F and S55K, N57R and E59G and E59P and N31F and S55K, N57W and E59G and E59P and N31F and S55K, N57R and N57W and Q93V and N31F and S55K, N57R and E5 It simultaneously contains mutations at five positions: 9G, Q93V, N31F, and S55K; N57W, E59G, Q93V, N31F, and S55K; N57R, E59P, Q93V, N31F, and S55K; N57W, E59P, Q93V, N31F, and S55K; and E59G, E59P, Q93V, N31F, and S55K. Or (6) containing mutations at six positions simultaneously: N57R, N57W, E59G, E59P, Q93V, and N31F; N57R, N57W, E59G, E59P, Q93V, and S55K; N57R, N57W, E59G, E59P, N31F, and S55K; N57R, N57W, E59G, E59P, Q93V, N31F, and S55K; N57R, N57W, E59P, Q93V, N31F, and S55K; N57R, E59G, E59P, Q93V, N31F, and S55K; N57W, E59G, E59P, Q93V, N31F, and S55K. or (7) containing mutations at seven positions simultaneously: N57R, N57W, E59G, E59P, Q93V, N31F, and S55K.

[0039] In a specific embodiment, the present invention provides an anti-aPC monoclonal antibody comprising one, two, or three of the CDR1, CDR2, and CDR3 of a heavy chain CDR1 represented by SEQ ID NO:27 or 33, a heavy chain CDR2 represented by SEQ ID NO:28 or 40, and a heavy chain CDR3 represented by SEQ ID NO:29 or 77, and / or one, two, or three of the CDR1, CDR2, and CDR3 of a light chain CDR1 represented by SEQ ID NO:30, a light chain CDR2 represented by any of the sequences of SEQ ID NO:31, 37, 43, 49, and 73, and a light chain CDR3 represented by SEQ ID NO:32 or 44.

[0040] In a specific embodiment, the antibody is an antibody comprising (a) a heavy chain CDR represented by SEQ ID NOs: 27, 28, and 29, and / or (b) a light chain CDR represented by SEQ ID NOs: 30, 31, and 32; (c) a heavy chain CDR represented by SEQ ID NOs: 33, 34, and 35, and / or (d) a light chain CDR represented by SEQ ID NOs: 36, 37, and 38; (e) a heavy chain CDR represented by SEQ ID NOs: 39, 40, and 41, and / or (f) a light chain CDR represented by SEQ ID NOs: 42, 43, and 44; (g) a heavy chain CDR represented by SEQ ID NOs: 45, 46, and 47, and / or (h) a light chain CDR represented by SEQ ID NOs: 48, 49, and 50; and (i) a heavy chain CDR represented by SEQ ID NOs: 51, 52, and 53; and / or (j) a heavy chain CDR represented by SEQ ID NOs: 54, 55, 56, and 57. (k) an antibody comprising a heavy chain CDR represented by SEQ ID NOs: 57, 58 and 59 and / or (l) an antibody comprising a light chain CDR represented by SEQ ID NOs: 60, 61 and 62; (m) an antibody comprising a heavy chain CDR represented by SEQ ID NOs: 63, 64 and 65 and / or (n) an antibody comprising a light chain CDR represented by SEQ ID NOs: 66, 67 and 68; (o) an antibody comprising a heavy chain CDR represented by SEQ ID NOs: 69, 70 and 71 and / or (p) an antibody comprising a light chain CDR represented by SEQ ID NOs: 72, 73 and 74; (q) an antibody comprising a heavy chain CDR represented by SEQ ID NOs: 75, 76 and 77 and / or (r) an antibody comprising a light chain CDR represented by SEQ ID NOs: 78, 79 and 80; (t) an antibody comprising heavy chain CDRs represented by SEQ ID NOs: 81, 82 and 83, and / or light chain CDRs represented by SEQ ID NOs: 84, 85 and 86; (u) an antibody comprising heavy chain CDRs represented by SEQ ID NOs: 87, 88 and 89, and / or (v) an antibody comprising light chain CDRs represented by SEQ ID NOs: 90, 91 and 92.

[0041] In the present invention, the heavy chain framework region of the anti-aPC monoclonal antibody may be any conventional framework region suitable for exerting the function of the anti-aPC monoclonal antibody of the present invention, and may be derived from either human or mouse.

[0042] In the present invention, the light chain framework region of the anti-aPC monoclonal antibody may be any conventional framework region suitable for exerting the function of the anti-aPC monoclonal antibody of the present invention, and may be derived from either human or mouse.

[0043] In a specific embodiment, the anti-aPC monoclonal antibody comprises a heavy chain sequence represented by any one of SEQ ID NOs:5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25, and / or a light chain sequence represented by any one of SEQ ID NOs:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26.

[0044] In a specific embodiment, the anti-aPC monoclonal antibody is selected from the group consisting of antibody HaPC14 comprising a heavy chain sequence represented by SEQ ID NO:5 and a light chain sequence represented by SEQ ID NO:6, antibody HaPC14-1 comprising a heavy chain sequence represented by SEQ ID NO:7 and a light chain sequence represented by SEQ ID NO:8, antibody HaPC14-2 comprising a heavy chain sequence represented by SEQ ID NO:9 and a light chain sequence represented by SEQ ID NO:10, antibody HaPC14-3 comprising a heavy chain sequence represented by SEQ ID NO:11 and a light chain sequence represented by SEQ ID NO:12, antibody HaPC14-4 comprising a heavy chain sequence represented by SEQ ID NO:13 and a light chain sequence represented by SEQ ID NO:14, antibody HaPC14-5 comprising a heavy chain sequence represented by SEQ ID NO:15 and a light chain sequence represented by SEQ ID NO:16, antibody HaPC14-6 comprising a heavy chain sequence represented by SEQ ID NO:17 and a light chain sequence represented by SEQ ID NO:18, and antibody HaPC14-7 comprising a heavy chain sequence represented by SEQ ID NO:19 and a light chain sequence represented by SEQ ID NO:20. The antibody HaPC14-7 comprises a heavy chain sequence represented by SEQ ID NO:19 and a light chain sequence represented by SEQ ID NO:20; the antibody HaPC14-8 comprises a heavy chain sequence represented by SEQ ID NO:21 and a light chain sequence represented by SEQ ID NO:22; the antibody HaPC14-9 comprises a heavy chain sequence represented by SEQ ID NO:23 and a light chain sequence represented by SEQ ID NO:24; and the antibody HaPC14-10 comprises a heavy chain sequence represented by SEQ ID NO:25 and a light chain sequence represented by SEQ ID NO:26.

[0045] In the present invention, some sequences have the same structure but different numbers (for specific examples, see the specific amino acid sequences in the sequence listing).

[0046] For example, among sequences representing the heavy chain CDR1 of an antibody, sequences 27 and 87 are the same, and sequences 33, 39, 45, 51, 57, 63, 69, 75, and 81 are the same.

[0047] Furthermore, in the sequences representing the heavy chain CDR2 of the antibody, sequences 28, 34, 52, and 70 are the same, and sequences 40, 46, 58, 64, 76, 82, and 88 are the same.

[0048] Furthermore, in the sequences representing the antibody heavy chain CDR3, the sequences 29, 35, 41, 47, 53, 59, 65, and 71 are the same, and the sequences 77, 83, and 89 are the same.

[0049] Furthermore, in the sequences representing the antibody light chain CDR1, the sequences 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, and 90 are the same.

[0050] Furthermore, in the sequences representing the antibody light chain CDR2, sequences 37 and 61 are the same, and sequences 49, 55, 67, 79, 85, and 91 are the same.

[0051] Furthermore, in the sequences representing the antibody light chain CDR3, the sequences 32, 38, 68, 74, and 86 are the same, and the sequences 44, 50, 56, 62, 80, and 92 are the same.

[0052] In a specific embodiment, the present invention further provides any one or more of the following 1) to 10) as biomaterials related to the above-mentioned anti-aPC monoclonal antibodies:

[0053] 1) A nucleic acid molecule encoding the above anti-aPC monoclonal antibody.

[0054] 2) An expression cassette containing the nucleic acid molecule described in 1).

[0055] 3) A construct containing the nucleic acid molecule described in 1).

[0056] 4) A construct containing the expression cassette described in 2).

[0057] 5) An expression system comprising the nucleic acid molecule described in 1).

[0058] 6) An expression system containing the expression cassette described in 2).

[0059] 7) An expression system comprising the construct described in 3).

[0060] 8) An expression system comprising the recombinant vector described in 4).

[0061] 9) A primer pair that amplifies a nucleic acid molecule encoding the amino acid sequence of the anti-aPC monoclonal antibody.

[0062] 10) A fusion antibody comprising the above anti-aPC monoclonal antibody.

[0063] The expression system may be a cell or a cell line, and the cell or cell line encodes the anti-aPC monoclonal antibody.

[0064] In a specific embodiment, the present invention provides a pharmaceutical product, which is a drug composition or a detection reagent or a detection reagent kit.

[0065] In a specific embodiment, the pharmaceutical composition provided by the present invention comprises one or more of the anti-aPC monoclonal antibodies or biomaterials described above and a pharmaceutically acceptable vector. The vector may contain various excipients and diluents, but these are not essential active ingredients and do not pose excessive toxicity after administration. Suitable vectors are well known to those skilled in the art; for example, Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ, 1991) provides information on pharmaceutically acceptable vectors.

[0066] In a specific embodiment, the detection reagent or detection reagent kit provided herein comprises any one or more of the anti-aPC monoclonal antibodies or biomaterials described above, and the antibodies are labeled.

[0067] The labeling can be done in any conventional manner, such as with a chemiluminescent label, a fluorophore, a radioactive label or a chromophore.

[0068] The detection reagent or reagent kit may further include a buffer, a diluent, an instruction manual, and the like.

[0069] The antibody may be in any form suitable for its function or activity, for example, in the form of an aqueous suspension.

[0070] In some embodiments, the anti-aPC monoclonal antibodies of the present invention enhance the ability of aPC to degrade histones H3 and H4, suggesting that the anti-aPC monoclonal antibodies may provide cytoprotection against the cytotoxicity of histones H3 and H4.

[0071] In a specific embodiment, the present invention provides a method for treating a blood coagulation deficiency or defect. The definition of the blood coagulation deficiency or defect is as described above. Specifically, the present invention provides a method for treating inherited and acquired blood coagulation deficiencies or defects (e.g., hemophilia A, hemophilia B, or sepsis). It also provides a method for shortening bleeding time by administering an anti-aPC monoclonal antibody to a patient in need thereof (e.g., a trauma patient or a patient in need of blood clotting). It also provides a method for inhibiting the anticoagulant activity of activated protein C in a subject. It also provides a method for modulating thrombus formation in a subject. The methods of the present invention involve administering an effective amount of the anti-aPC monoclonal antibody of the present invention to an individual in need of treatment. It also provides a method for producing an anti-human aPC monoclonal antibody.

[0072] In a specific embodiment, the present invention provides a method for treating a blood coagulation deficiency or defect comprising administering to an individual in need thereof an effective amount of the above-described anti-aPC monoclonal antibody or related biomaterial.

[0073] In a specific embodiment, the present invention provides a method relating to the treatment of blood clotting, the method comprising administering an effective amount of any one or more of the anti-aPC monoclonal antibodies or biomaterials described above to an individual in need of treatment. The individual is suffering from a blood clotting deficiency or defect, or is in need of blood clotting treatment, or is suffering from sepsis or hemophilia, or is in need of hemostatic regulation.

[0074] The blood coagulation deficiency or defect may be hereditary or acquired, and includes diseases in which a deficiency of one or more blood coagulation factors in plasma causes blood coagulation disorders and bleeding, specifically, rare blood coagulation factor deficiencies such as hemophilia A, hemophilia B, and hemophilia C, which are caused by a deficiency of blood coagulation factors VIII, IX, and XI, and bleeding disorders associated with a deficiency or abnormal function of one or more other blood coagulation factors (i.e., blood coagulation factors I, II, V, V+VIII, VII, X, or XIII) (e.g., excessive bleeding caused by disrupted blood coagulation following trauma, treatment of sepsis, transplantation, cardiac surgery, or orthopedic surgery).

[0075] In a specific embodiment, the method of the present invention for enhancing the ability of aPC to degrade histones H3 and H4 can enhance the ability of aPC to degrade histones H3 and H4 after administration of the anti-aPC monoclonal antibody or its related biomaterial or pharmaceutical composition, thereby providing cytoprotection against the cytotoxicity of histones H3 and H4.

[0076] In a specific embodiment, the method of the present invention for inhibiting the anticoagulant activity of activated protein C in an individual can regulate the blood coagulation function of an individual by administering the anti-aPC monoclonal antibody or its related biomaterial or pharmaceutical composition, thereby inhibiting the anticoagulant activity of activated protein C in the individual.

[0077] In specific embodiments, the present invention provides for the use of the anti-aPC monoclonal antibody or related biomaterials in: i) the manufacture of a medicament for preventing and / or treating a blood coagulation deficiency or defect; ii) the manufacture of a promoter that enhances the ability of aPC to degrade histones H3 and H4; iii) the manufacture of a medicament that inhibits the anticoagulant activity of activated protein C in an individual; iv) the manufacture of a medicament for treating an individual in need of blood coagulation; vi) the manufacture of a medicament for regulating hemostasis in an individual; vii) the manufacture of a reagent or reagent kit for immunological tests and assays for the diagnosis or treatment of disease or non-disease, or for the detection of aPC; and viii) the use in combination with other reagents or drugs.

[0078] In a specific embodiment, the present invention further provides a method for producing the above-described anti-aPC monoclonal antibody, the method comprising the step of culturing an expression system for the anti-aPC monoclonal antibody under conditions suitable for expression of the anti-aPC monoclonal antibody to express the anti-aPC monoclonal antibody.

[0079] The present invention also covers polypeptides having a certain degree of identity with the amino acid sequence of the anti-aPC monoclonal antibody, such as antibodies or antibody fragments having 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity with the anti-aPC monoclonal antibody and the functions of the anti-aPC monoclonal antibody (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity). Specifically, examples of such polypeptide fragments include those obtained by substituting, deleting, or adding one or more amino acids (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2, or 3) to the anti-aPC monoclonal antibody, or those obtained by adding one or more amino acids (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2, or 3) to the N-terminus and / or C-terminus, and which have the functions of the anti-aPC monoclonal antibody.

[0080] The term "protein C" or "PC" refers to any variant, isotype, and / or homolog of the proenzyme form of protein C that is naturally expressed by cells and present in plasma, and that is distinct from the activated form of protein C.

[0081] The term "activated protein C" or "aPC" refers to the activated form of protein C.

[0082] The term "anti-aPC monoclonal antibody" refers to an antibody that specifically binds to an epitope on aPC, and is an anti-human aPC monoclonal antibody. The anti-aPC monoclonal antibody can be in a variety of antibody formats, including, but not limited to, a complete antibody, an antibody fragment, a human antibody, a humanized antibody, and a recombinant antibody (e.g., a monoclonal antibody, a chimeric antibody, or a recombinant antibody, and fragments of these antibodies), provided that the properties of the present invention are maintained.

[0083] The antibody fragments are further defined as Fab', Fab, F(ab')2, single-domain antibody, Fv, scFv, etc. The anti-aPC monoclonal antibody described herein refers to an antibody molecule preparation of a single molecular component. The monoclonal antibody component exhibits a single binding specificity and affinity for a specific epitope and may have variable and constant regions derived from human immunoglobulin sequences. For example, a Fab fragment refers to a monovalent fragment consisting of the VL, VH, CL, and CH1 domains, and a F(ab')2 fragment refers to a bivalent fragment containing two Fab fragments connected by a disulfide bond at the hinge region. Furthermore, an Fv fragment refers to an Fv fragment consisting of the VL and VH domains in one arm of an antibody.

[0084] The term "identity" can be evaluated by eye or by computer software. When using computer software, the identity between two or more sequences can be expressed as a percentage (%). This percentage can be used to evaluate the identity between related sequences.

[0085] The term "individual" generally includes humans and non-human primates (e.g., mammals, dogs, cats, horses, sheep, pigs, cows, etc.) that can benefit from treatment with the antibodies, drugs, compositions, related formulations, reagent kits, or combinations described above.

[0086] The term "therapeutically effective amount" generally refers to a dose that can achieve the effect of treating the above-listed diseases after an appropriate administration period.

[0087] The term "monoclonal antibody" refers to an antibody molecule preparation of a single amino acid composition. A "monoclonal antibody" may have variable and constant regions derived from human immunoglobulin sequences. In one embodiment, the monoclonal antibody is produced from a hybridoma. The hybridoma contains B cells obtained from a transgenic non-human animal (e.g., a transgenic mouse) whose genome contains a human heavy chain transgene and a human light chain transgene that are fused to an immortalized cell.

[0088] The term "fusion antibody" refers to a product obtained by fusing an antibody fragment with an activated protein from another organism using genetic engineering techniques. Specifically, it refers to a recombinant protein containing the domains of two Ig fragments, which is produced by linking a target gene to a target Ig fragment gene at the genetic level and expressing it in eukaryotic or prokaryotic cells. Depending on which Ig fragment the target protein is linked to, "fusion antibodies" can be broadly classified into two types: Fab (Fv) fusion proteins and Fc fusion proteins. Furthermore, two different proteins are linked to form a single polymer. Linkage can be achieved by chemical methods or gene fusion. Methods for producing fusion antibodies involve common DNA recombination and gene transfer techniques known in the art. Depending on the fusion protein, such antibody fusion proteins can have multiple biological functions. Furthermore, the expressed recombinant protein does not affect the antigen-binding ability of the single-chain antibody or the biological properties of the fused protein.

[0089] The term "humanized antibody" means that the framework regions or "complementarity determining regions" (CDRs) have been modified to contain CDRs of an immunoglobulin with different specificity compared to the parent immunoglobulin.

[0090] When referring to inhibiting and / or blocking the binding of an aPC substrate to aPC, the terms inhibit and block also include a measurable decrease in the binding affinity of aPC for a physiological substrate upon contact with an anti-aPC antibody compared to when the aPC is not contacted with the anti-aPC antibody, e.g., blocking the interaction between aPC and its substrate (including factor Va or factor VIIIa) by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0091] "Complementarity-determining region" or "CDR" refers to one of the three hypervariable regions in the heavy or light chain variable region of an antibody molecule, which forms the N-terminal antigen-binding surface complementary to the three-dimensional structure of the antigen to be bound. From the N-terminus of the heavy or light chain, these complementarity-determining regions are designated as "CDR1," "CDR2," and "CDR3," respectively. CDRs are involved in antigen-antibody binding, and CDR3 contains the unique region having specificity for antigen-antibody binding. Thus, an antigen-binding site may contain six CDRs, including the CDR regions from each of the heavy and light chain V regions.

[0092] The term "epitope" refers to the area or region of an antigen to which an antibody specifically binds or interacts, and in some embodiments refers to the point at which the antigen makes physical contact with the antibody.

[0093] The term "conservative substitution" refers to a polypeptide modification in which one or more amino acids are replaced with amino acids having similar biochemical properties but without abolishing the biological or biochemical function of the polypeptide. A "conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Common conservative substitutions include, for example, the intersubstitution of aromatic amino acids Phe, Trp, and Tyr; the intersubstitution of hydrophobic amino acids Leu, Ile, and Val; the intersubstitution of polar amino acids Gln and Asn; the intersubstitution of alkaline amino acids Lys, Arg, and His; the intersubstitution of acidic amino acids Asp and Glu; and the intersubstitution of hydroxyl amino acids Ser and Thr.

[0094] The term "identity" refers to the percentage of nucleotides (e.g., at least about 80% (usually at least about 85%) of nucleotides or amino acids) that are the same when comparing two nucleic acids or two polypeptides. In some embodiments, about 90%, 91%, 92%, 93%, 94%, or 95%, and in at least one embodiment, at least about 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% of the nucleotides or amino acids are the same.

[0095] Nucleic acids may be present in whole cells, cell lysates, or in a partially purified or substantially purified form. Nucleic acids are "isolated" or "substantially purified" when they are purified from other cellular components with which they are normally associated in their natural environment. Nucleic acids can be separated by techniques well known in the art, such as, for example, alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, etc.

[0096] The relevant terms used in this invention are explained below.

[0097] [Table 1]

[0098] The present invention will be described in more detail below with reference to examples. These examples are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. The processes, conditions, experimental methods, etc. for carrying out the present invention are all common knowledge and common general knowledge in the relevant field, except as specifically mentioned below, and therefore the present invention does not particularly limit the contents thereof. [Example]

[0099] Humanization of mouse anti-aPC monoclonal antibody Humanization was designed for the variable region sequences of mouse hybridoma antibodies. First, the VH / VL amino acid sequences were numbered using the Kabat system. Next, the VH / VL sequences were analyzed for the presence of hot spots, including unpaired cysteine ​​residues, N-glycosylation sites, and deamination sites within the CDRs. N-glycosylation sites close to the CDRs were identified as high-risk sites. If such sites were identified, they needed to be removed. Based on comparison of the VH / VL sequences, optimal human gene frame sequences were selected. J region sequences were then selected and linked based on optimal homology. Subsequently, homology modeling was performed on the mouse VH / VL sequences to generate 3D structures. After modifying FR residues within 5 Å of the CDRs and the VH / VL binding interface, residues potentially affecting antigen binding were identified as sites requiring backmutation. Additionally, the importance of the back mutations was determined by computer analysis, and different combinations containing one or more back mutations were designed.

[0100] The target segment DNA was amplified by PCR to construct a plasmid encoding the humanized antibody sequence. Subsequently, after transient transfection and expression, the antibody was purified using a protein A affinity chromatography column. The humanized antibody was then analyzed and characterized by SEC-HPLC and SDS-PAGE, and 28 humanized anti-aPC monoclonal antibody clones were isolated.

[0101] 1.1 Measurement of the affinity of anti-aPC monoclonal antibodies to human aPC (haPC) by ELISA The affinities of the chimeric antibody and the 28 humanized antibodies against haPC were measured and compared by ELISA and FACS.

[0102] As shown in Figure 1, the humanized antibodies 1 to 28 (Humab001-1 to 1-28 or HAPC-1 to HAPC28), the 3657 positive control antibody (3657 PC mAb), and the chimeric antibody (30120-mAb001 x hIgG) all bound well to haPC. Furthermore, the antibodies 13 to 20 had low EC50 values, indicating that these antibodies had stronger binding avidity to haPC.

[0103] 1.2 Measurement of binding affinity of anti-aPC monoclonal antibodies to human PC (hPC) by ELISA Humanized anti-aPC monoclonal antibodies Nos. 13 to 20 (Humab001-13 to Humab001-20, or HAPC-13 to HAPC-20) were selected and their binding affinity to hPC was measured.

[0104] The results are shown in Figure 2. The results demonstrated that the humanized anti-aPC monoclonal antibodies Nos. 13 to 20 did not bind to hPC at all.

[0105] Antibody No. 13 (HAPC-13), antibody No. 14 (HAPC-14), and antibody No. 16 (HAPC-16) were the three parent antibodies selected for affinity optimization. These three antibodies had the same heavy chain framework regions but different light chain framework regions. HAPC-13 was the original antibody, HAPC-14 had one back mutation based on HAPC-13, and HAPC-16 had five back mutations based on HAPC-13. In the following examples, antibody HAPC-14 was selected for subsequent affinity optimization. [Example]

[0106] Affinity optimization of anti-aPC monoclonal antibody HAPC-14 Based on the above antibody No. 14 (Humab001-14, HAPC14, or HAPC-14), antibody affinity optimization design and mutants were created.

[0107] [Table 2]

[0108] 2.1 Site-directed mutagenesis Using site-directed mutagenesis, each amino acid in the six complementarity-determining regions (CDRs) of the parental HAPC-14 Fab clone was mutated to 19 different amino acids. DNA primers containing NNS codons encoding 20 amino acids were used to introduce mutations at each target CDR position. Phosphorylated degenerate primers were also used in the site-directed mutagenesis reactions.

[0109] 2.2 PCR reaction PCR conditions were 94°C for 2 min (94°C for 30 s, 55°C for 30 s, 72°C for 5 min), 16 cycles, and 72°C for 10 min.

[0110] After purification of the PCR product, it was electroporated into E. coli TG1 for colony formation and production of Fab fragments.

[0111] 2.3 Primary screening of the Fab mutant library The primary screen included a one-point ELISA (SPE) assay, which was also performed by capture ELISA.

[0112] Specifically, each well of a 96-well microplate was coated with an anti-Fab antibody in coating buffer PBS, pH 7.4, at 4°C. On the second day, the plate was blocked with casein in PBS, pH 7.4, for 1 hour at 25°C. Fab PE was then added to the plate and incubated at 25°C for 1 hour. After washing, biotinylated antigen was added to the wells and incubated at 25°C for 1 hour. Subsequently, the plate was incubated with SA-horseradish peroxidase (HRP) conjugate for 1 hour at 25°C. HRP activity was detected using tetramethylbenzidine (TMB) substrate and quenched with 2M HCl. The plate was read at 450 nM. Clones that showed an optical density (OD) signal at 450 nm threefold greater than that of the parent clone were selected and sequenced. Thus, mutants with improved binding to HAPC-14 bearing unique sequences were demonstrated by capture ELISA at pH 7.4. We also sorted mutants with improved binding to the unique sequence of HAPC-14 by direct antigen-coating ELISA. The results are shown in Table 3.

[0113] [Table 3]

[0114] As shown in Table 3, affinity-improving mutations in nine individuals identified in the primary HAPC-14 screening (HAPC-14-R1-1H1-F2, HAPC-14-R1-2H6-C2, HAPC-14-R1-3H4-C6, HAPC-14-R1-2L4-B2, HAPC-14-R1-2L4-D4, HAPC-14-R1-2L6-G1, HAPC-14-R1-2L6-G11, HAPC-14-R1-3L1-D11, and HAPC-14-R1-3L1-G8) were further used to design and construct combinatorial mutant libraries.

[0115] 2.4 Validation of the primary Fab mutant library by direct KD sorting ELISA The KD level of the Fab with improved binding for each mutant was determined by KD ELISA in a direct antigen coating ELISA.

[0116] Each well of a 96-well microplate was coated with hPro1.biotin in 1x PBS buffer, pH 7.4, and incubated overnight at 4°C. On the second day, the plate was blocked with casein in 1x PBS buffer, pH 7.4, at 25°C for 1 hour. Then, a single dilution of Fab PE from each mutant was added to the plate and incubated at 25°C for 1 hour. The plate was then incubated with coupled goat anti-c-myc HRP for 1 hour at 25°C. HRP activity was detected using tetramethylbenzidine (TMB) substrate and quenched with 2M HCl. The OD of each plate was then read at 450 nM.

[0117] 2.5 Combinatorial screening of the HAPC-14 Fab library Point mutations in VH and VL favoring antigen binding were identified and further combined to obtain synergistic binding effects. Briefly, each degenerate primer was phosphorylated and then used with uracillated ssDNA at a 10:1 ratio. The mixture was then heated to 85°C for 5 minutes, cooled to 55°C, and incubated for at least 1 hour. T4 ligase and T4 DNA polymerase were then added, and the mixture was incubated at 37°C for 1.5 hours. 100 ng of combinatorial library DNA was then electroporated into TG1 to allow colony formation and production of Fab fragments.

[0118] The combined mutants were then screened by capture ELISA. Clones that showed an optical density (OD) signal at 450 nm three times greater than that of the primary lead clone were sequenced. Thus, mutants with improved binding and unique sequences were identified by capture ELISA and sorted by direct antigen-coating ELISA.

[0119] Several combination mutants of HAPC-14 were screened, and the top 16 combination mutants with significantly improved affinity were identified by KD ELISA (see Nos. 1 to 16 in Table 4).

[0120] [Table 4]

[0121] Screening and statistical results revealed that mutations at the following six positions significantly affected the binding ability of the Fab fragment to the capture antibody: a) N57R / W located in VL CDR2 (N57R is better than N57W), b) E59G / P located in VL CDR2 (E59G is better than E59P), c) Q93V located in VL CDR3, d) N31F located in VH CDR1, e) S55K located in VH CDR2, and f) Y104T located in VH CDR3.

[0122] Among these, the most significant mutation positions are N57R located in the light chain and N31F located in the heavy chain. [Example]

[0123] Anti-aPC monoclonal antibodies (generation of full IgG1) for combination mutation positions in HAPC-14 3.1 Generation of anti-aPC monoclonal antibodies (HAPC14-1 to HAPC14-10) for combined mutation sites of HAPC-14 Ten mutants were obtained by combining and creating the six mutation positions in Example 2 (see 14-1 to 14-10 in Table 5 below) (HAPC14-1 to HAPC14-10). The sequences were then constructed into a complete IgG format, which was then expressed and purified, and the expression level, affinity, and other biological activities of the antibody were examined.

[0124] The amino acid sequences of HAPC-14 and the 10 mutants are shown in SEQ ID NOs. 5 to 26.

[0125] Humanized antibody 13 (HAPC-13) Heavy chain (SEQ ID NO. 1) EVQLVESGGGLVQPGGSLRLSCAASGFTFS NYYLN WVRQAPGKGLEWVG DIRLKSNNYEKHYAESVKG RFTISRDDSKSITYLQMNSLRAEDTAVYYCAR EGDYFDY WGQGTTVTVSS Light chain (SEQ ID NO. 2) DIQMTQSPSSLSASVGDRVTITC RASESVDSFGATFMH WYQQKPGKAPKLLIY LASNLES GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQNNEDPYT FGQGTKLEIK

[0126] Humanized antibody 16 (HAPC-16) Heavy chain (SEQ ID NO. 3) EVQLVESGGGLVQPGGSLRLSCAASGFTFS NYYLN WVRQAPGKGLEWVG DIRLKSNNYEKHYAESVKG RFTISRDDSKSITYLQMNSLRAEDTAVYYCAR EGDYFDY WGQGTTVTVSS Light chain (SEQ ID NO. 4) N I VL TQSPSSLSASVGDRVTITC RASESVDSFGATFMH WYQQKPGK P PKLLIY LASNLES GVPSRFSGSGS R TDFTLTISSLQPEDFATYYC QQNNEDPYT FGQGTKLEIK

[0127] Humanized antibody 14 (HAPC-14) Heavy chain (SEQ ID NO. 5) EVQLVESGGGLVQPGGSLRLSCAASGFTFS NYYLN WVRQAPGKGLEWVG DIRLKSNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAR EGDYFDY WGQGTTVTVSS Light chain (SEQ ID NO.6) DIQ L TQSPSSLSASVGDRVTITC RASESVDSFGATFMH WYQQKPGKAPKLLIY LASNLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQNNEDPYT FGQGTKLEIK

[0128] HAPC-14-1 Heavy chain (SEQ ID NO.7) EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYYLNWVRQAPGKGLEWVGDIRLKSNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDYFDYWGQGTTVTVSS Light chain (SEQ ID NO.8) DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASWLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIK

[0129] HAPC-14-2 Heavy chain (SEQ ID NO.9) EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYYLNWVRQAPGKGLEWVGDIRLKKNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDYFDYWGQGTTVTVSS Light chain (SEQ ID NO.10) DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASWLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQNNEDPYTFGQGTKLEIK

[0130] HAPC-14-3 Heavy chain (SEQ ID NO.11) EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYYLNWVRQAPGKGLEWVGDIRLKKNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDYFDYWGQGTTVTVSS Light chain (SEQ ID NO.12) DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASRLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQNNEDPYTFGQGTKLEIK

[0131] HAPC-14-4 Heavy chain (SEQ ID NO.13) EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYYLNWVRQAPGKGLEWVGDIRLKSNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDYFDYWGQGTTVTVSS Light chain (SEQ ID NO.14) DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASRLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQNNEDPYTFGQGTKLEIK

[0132] HAPC-14-5 Heavy chain (SEQ ID NO.15) EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYYLNWVRQAPGKGLEWVGDIRLKKNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDYFDYWGQGTTVTVSS Light chain (SEQ ID NO.16) DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASWLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQNNEDPYTFGQGTKLEIK

[0133] HAPC-14-6 Heavy chain (SEQ ID NO.17) EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYYLNWVRQAPGKGLEWVGDIRLKKNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDYFDYWGQGTTVTVSS Light chain (SEQ ID NO.18) DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASRLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIK

[0134] HAPC-14-7 Heavy chain (SEQ ID NO.19) EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYYLNWVRQAPGKGLEWVGDIRLKSNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDYFDYWGQGTTVTVSS Light chain (SEQ ID NO.20) DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASRLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIK

[0135] HAPC-14-8 Heavy chain (SEQ ID NO.21) EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYYLNWVRQAPGKGLEWVGDIRLKKNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDTFDYWGQGTTVTVSS Light chain (SEQ ID NO.22) DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASRLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQNNEDPYTFGQGTKLEIK [[ID=X]]

[0136] 这里原文中是

[0136] ,翻译后保留[[ID=X]] 这里原文中是 ,翻译后保留HAPC-14-9 Heavy chain (SEQ ID NO.23)<000A606>这里原文中是 ,翻译后保留 EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYYLNWVRQAPGKGLEWVGDIRLKKNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDTFDYWGQGTTVTVSS Light chain (SEQ ID NO.24) DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASRLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIK [[ID=X]]

[0137] 这里原文中是

[0137] ,翻译后保留 [[ID=X]] 这里原文中是 ,翻译后保留 HAPC-14-10 Heavy chain (SEQ ID NO.25) EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYLNWVRQAPGKGLEWVGDIRLKKNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDTFDYWGQGTTVTVSS [[ID=2X]]軽鎖(SEQ ID NO.26) 这里原文中是軽鎖(SEQ ID NO.26),推测是日语,保留原文 DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASRLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQNNEDPYTFGQGTKLEIK 说明:对于原文中一些不太明确用途的连续数字标签(如

[0136] 等),按照规则保留原样未进行翻译调整。对于“軽鎖”推测可能是日语,保留原文未翻译。你可根据实际情况进一步确认和调整。

[0138] HAPC-14 Heavy chain CDR1 (SEQ ID NO.27) NYYLN

[0139] HAPC-14 Heavy chain CDR2 (SEQ ID NO.28) DIRLKSNNYEKHYAESVKG

[0140] HAPC-14 Heavy chain CDR3 (SEQ ID NO.29) EGDYFDY

[0141] HAPC-14 Light chain CDR1 (SEQ ID NO.30) RASESVDSFGATFMH

[0142] HAPC-14 Light chain CDR2 (SEQ ID NO.31) LASNLES

[0143] <000064=3>HAPC-14 Light chain CDR3 (SEQ ID NO.32) QQNNEDPYT

[0144] HAPC-14-1 Heavy chain CDR1 (SEQ ID NO.33) FYYLN

[0145] HAPC-14-1 Heavy chain CDR2 (SEQ ID NO.34) DIRLKSNNYEKHYAESVKG

[0146] HAPC-14-1 Heavy chain CDR3 (SEQ ID NO.35) [[ID==1]]EGDYFDY

[0147] HAPC-14-1 Light chain CDR1 (SEQ ID NO.36) RASESVDSFGATFMH

[0148] HAPC-14-1 Light chain CDR2 (SEQ ID NO.37) LASWLGS

[0149] HAPC-14-1 Light chain CDR3 (SEQ ID NO.38) QQNNEDPYT

[0150] HAPC-14-2 Heavy chain CDR1 (SEQ ID NO.39) FYYLN

[0151] <00(00683>HAPC-14-2 (SEQ ID NO.40) Heavy chain CDR2 DIRLKKNNYEKHYAESVKG

[0152] HAPC-14-2 Heavy chain CDR3 (SEQ ID NO.41) EGDYFDY

[0153] HAPC-14-2 Light chain CDR1 (SEQ ID NO.42) RASESVDSFGATFMH

[0154] HAPC-14-2 Light chain CDR2 (SEQ ID NO.43) LASWLES

[0155] HAPC-14-2 Light chain CDR3 (SEQ ID NO.44) VQNNEDPYT

[0156] [[ID=7(0]]HAPC-14-3 Heavy chain CDR1 (SEQ ID NO.45) FYYLN

[0157] HAPC-14-3 Heavy chain CDR2 (SEQ ID NO.46) DIRLKKNNYEKHYAESVKG

[0158] HAPC-14-3 Heavy chain CDR3 (SEQ ID NO.47) EGDYFDY

[0159] HAPC-14-3 Light chain CDR1 (SEQ ID NO.48) RASESVDSFGATFMH

[0160] HAPC-14-3 Light chain CDR2 (SEQ ID NO.49) LASRLGS

[0161] HAPC-14-3 Light chain CDR3 (SEQ ID NO.50) VQNNEDPYT

[0162] HAPC-14-4 Heavy chain CDR1 (SEQ ID NO.51) FYYLN

[0163] HAPC-14-4 Heavy chain CDR2 (SEQ ID NO.52) DIRLKSNNYEKHYAESVKG

[0164] HAPC-14-4 Heavy chain CDR3 (SEQ ID NO.53) EGDYFDY

[0165] HAPC-14-4 Light chain CDR1 (SEQ ID NO.54) RASESVDSFGATFMH

[0166] HAPC-14-4 Light chain CDR2 (SEQ ID NO.55) LASRLGS

[0167] HAPC-14-4 Light chain CDR3 (SEQ ID NO.56) VQNNEDPYT

[0168] HAPC-14-5 Heavy chain CDR1 (SEQ ID NO.57) FYYLN

[0169] HAPC-14-5 Heavy chain CDR2 (SEQ ID NO.58) DIRLKKNNYEKHYAESVKG

[0170] HAPC-14-5 Heavy chain CDR3 (SEQ ID NO.59) EGDYFDY

[0171] HAPC-14-5 Light chain CDR1 (SEQ ID NO.60) RASESVDSFGATFMH

[0172] HAPC-14-5 Light chain CDR2 (SEQ ID NO.61) LASWLGS

[0173] HAPC-14-5 Light chain CDR3 (SEQ ID NO.62) VQNNEDPYT

[0174] HAPC-14-6 Heavy chain CDR1 (SEQ ID NO.63) FYYLN

[0175] HAPC-14-6 Heavy chain CDR2 (SEQ ID NO.64) DIRLKKNNYEKHYAESVKG

[0176] HAPC-14-6 SPECIFICATION CDR3(SEQ ID NO.65) EGDYFDY

[0177] HAPC-14-6 Page CDR1(SEQ ID NO.66) RASESVDSFGATFMH

[0178] HAPC-14-6 Page CDR2(SEQ ID NO.67) LASRLGS

[0179] HAPC-14-6 Page CDR3(SEQ ID NO.68) QQNNEDPYT

[0180] HAPC-14-7 SPECIFICATION CDR1(SEQ ID NO.69) FYYLN

[0181] HAPC-14-7 SPECIFICATION CDR2(SEQ ID NO.70) DIRLKSNNYEKHYAESVKG

[0182] HAPC-14-7 SPECIFICATION CDR3(SEQ ID NO.71) EGDYFDY

[0183] HAPC-14-7 Page CDR1(SEQ ID NO.72) RASESVDSFGATFMH

[0184] HAPC-14-7 Page CDR2(SEQ ID NO.73) LASRLES

[0185] HAPC-14-7 Page CDR3(SEQ ID NO.74) QQNNEDPYT

[0186] HAPC-14-8 SPECIFICATION CDR1(SEQ ID NO.75) FYYLN

[0187] HAPC-14-8 SPECIFICATION CDR2(SEQ ID NO.76) DIRLKKNNYEKHYAESVKG

[0188] HAPC-14-8 SPECIFICATION CDR3(SEQ ID NO.77) EGDTFDY

[0189] HAPC-14-8 Page CDR1(SEQ ID NO.78) RASESVDSFGATFMH

[0190] HAPC-14-8 Page CDR2(SEQ ID NO.79) LASRLGS

[0191] HAPC-14-8 Page CDR3(SEQ ID NO.80) VQNNEDPYT

[0192] HAPC-14-9 SPECIFICATION CDR1(SEQ ID NO.81) FYYLN

[0193] HAPC-14-9 SPECIFICATION CDR2(SEQ ID NO.82) DIRLKKNNYEKHYAESVKG

[0194] HAPC-14-9 SPECIFICATION CDR3(SEQ ID NO.83) EGDTFDY

[0195] HAPC-14-9 Light chain CDR1 (SEQ ID NO. 84) RASESVDSFGATFMH

[0196] HAPC-14-9 Light chain CDR2 (SEQ ID NO. 85) LASRLGS

[0197] HAPC-14-9 Light chain CDR3 (SEQ ID NO. 86) QQNNEDPYT

[0198] HAPC-14-10 Heavy chain CDR1 (SEQ ID NO. 87) NYYLN

[0199] HAPC-14-10 Heavy chain CDR2 (SEQ ID NO. 88) DIRLKKNNYEKHYAESVKG

[0200] HAPC-14-10 Heavy chain CDR3 (SEQ ID NO. 89) EGDTFDY

[0201] HAPC-14-10 Light chain CDR1 (SEQ ID NO. 90) RASESVDSFGATFMH

[0202] HAPC-14-10 Light chain CDR2 (SEQ ID NO. 91) LASRLGS

[0203] HAPC-14-10 Light chain CDR3 (SEQ ID NO. 92) VQNNEDPYT

[0204] Detailed information is shown in Table 5.

[0205] [Table 5] The underlined regions in the table are CDR regions, and the rest are framework regions.

[0206] [Table 6]

[0207] The underlined regions are CDRs, and the rest are framework regions. The white letters indicate the humanized positions.

[0208] [Table 7]

[0209] 3.2 Measurement of binding affinity of anti-aPC monoclonal antibodies generated for the combined mutation positions of HAPC14 to haPC / hPC (HAPC14-1 to HAPC14-10) Measurement method: The binding ability of the anti-aPC monoclonal antibody to haPC / hPC was measured by ELISA.

[0210] 1. ELISA plates were coated overnight with 100 μL / well of haPC / hPC (1 μg / ml in PBS) at 4° C.

[0211] 2. The plate was washed three times with 200 μL / well of PBST (0.1% Tween 20).

[0212] 3. The plate was blocked with 2% BSA (in PBS) at room temperature, 200 μL / well, for 1 hour.

[0213] 4. The plate was washed three times with 200 μL / well of PBST (0.1% Tween 20).

[0214] 5. Anti-aPC monoclonal antibody was used as the primary antibody (diluted from 10 μg / ml in 2% BSA), 100 μL / well, and the plates were incubated at room temperature for 1 hour.

[0215] 6. The palette was washed three times with 200 μL / well of PBST (0.1% Tween 20).

[0216] 7. The plate and anti-hIgG-Fc antibody-HRP (diluted 5000-fold in 2% BSA buffer) were incubated at room temperature for 1 hour.

[0217] 8. The palette was washed six times with 200 μL / well of PBST (0.1% Tween 20).

[0218] 9. Incubate the plate with TMB substrate (prepared using solution A and solution B in a 1:1 (v / v) ratio) at 100 μL / well and protect from light for 10 minutes.

[0219] 10. The reaction was quenched with HCl (1M) at 100 μL / well and the OD450 was read.

[0220] The measurement results are shown in Figure 3 and Tables 8 and 9. This demonstrates that the anti-aPC monoclonal antibody binds to hAPC but not to hPC.

[0221] [Table 8]

[0222] [Table 9]

[0223] 3.3 Affinity measurement of anti-aPC monoclonal antibodies (HAPC-14-1 to HAPC-14-10) generated by combinatorial mutation positions of HAPC-14 using the Octet biomolecular interaction system Measurement Method: Octet analysis was performed at 25°C using a Protein A Octet biosensor in 1x Kinetics buffer. For kinetic measurements, a first baseline step was performed in 1x Kinetics buffer for 120 seconds, followed by 240 seconds of antibody loading on the biosensor. A second baseline step was then performed in 1x Kinetics buffer for 120 seconds. The capture antibody biosensor was then immersed in wells containing five dilutions of antigen (huAPC) (3.13 nM, 6.25 nM, 12.5 nM, 25 nM, and 50 nM) for 180 seconds, followed by dissociation in 1x Kinetics buffer for 400 seconds. 1x Kinetics buffer was also used as a blank control to demonstrate the natural dissociation of the antibody in buffer. A regeneration step was performed using 10 mM glycine at pH 1.5. In addition, binding kinetics was calculated using Octet data analysis software, and curve fitting was performed using a 1:1 binding model.

[0224] The affinity of the mutant antibodies was measured at pH 7.4, and the results are shown in Figure 4 and Table 10.

[0225] [Table 10]

[0226] As is clear from the results in Figure 4 and Table 10, the mutated antibodies were still able to bind to haPC but were unable to bind to hPC. Furthermore, their affinity was improved compared to that of the unmutated antibody HAPC-14 (No. 14). In particular, antibodies No. 6 (HAPC14-6) and No. 3 (HAPC14-3) showed nearly 100-fold improved affinity. [Example]

[0227] Histone degradation by anti-aPC monoclonal antibody HAPC14-6 and aPC Measurement method: [Table 11] After SDS-PAGE gel electrophoresis, staining with Coomassie brilliant blue was performed to observe whether the HAPC monoclonal antibody promoted or inhibited histone degradation by aPC.

[0228] The measurement results are shown in Figure 5 (Figure 5 shows, from left to right, the results when the cells contained only histone, when they contained histone and APC, when they contained histone, APC and HAPC1573, and when they contained histone, APC and HAPC14-6). As is clear from the figure, HAPC1573 and HAPC14-6 enhanced the ability of aPC to degrade histone. This suggests that the HAPC14-6 monoclonal antibody of the present invention can provide cytoprotection against the cytotoxicity of histones H3 and H4.

[0229] Like HAPC1573, HAPC-14-6 substantially enhanced, but did not inhibit, the degradation of histones H3 and H4 by aPC. However, unlike HAPC1573, HAPC-14-6 only slightly inhibited the degradation of histone H1 by aPC, suggesting that HAPC-14-6 may provide cytoprotection against the cytotoxicity of histones H3 and H4. [Example]

[0230] In vitro efficacy study of anti-aPC monoclonal antibodies HAPC-14-3 and HAPC-14-6 In this example, the effects of different antibodies and different concentrations on factor-deficient plasma were measured.

[0231] Using a protac-induced APTT test, we determined the minimum dose of monoclonal antibodies required to inhibit the anticoagulant activity of aPC in human plasma deficient in multiple blood coagulation factors. While HAPC1573 required a concentration of 100 μg / ml or more, this study demonstrated that a maximum concentration of just 1 μg / ml was sufficient to inhibit the anticoagulant activity of aPC by 95% or more.

[0232] 5.1 Experimental method and principles Principle of measurement of activated partial thromboplastin time (APTT): At 37°C, factors XII and XI are activated with kaolin, and cephalin (partial thromboplastin) is used as a substitute for platelet factor 3. 2+ The time required for plasma clotting (i.e., activated partial thromboplastin time) was observed using the intrinsic coagulation system, which is a highly sensitive and most commonly used screening test.

[0233] The newly developed anti-aPC monoclonal antibody, HAPC-14-6, was able to inhibit the anticoagulant activity of aPC at a concentration of up to 1 μg / ml in human plasma deficient in multiple blood coagulation factors, including deficiencies of blood coagulation factors V, VII, VIII, IX, X, XI, XII, and VWF.

[0234] 5.2 Measurement of the effect of antibodies on blood coagulation factor-deficient plasma (1) Experimental results for different antibodies and different concentrations of VIII and VIX blood coagulation factor deficient plasma Experimental method: The antibodies were diluted to the corresponding concentrations using Owren-Koller buffer.

[0235] FVIII blood clotting factor deficient plasma+ Antibody (final concentration) at each dilution in Table 12 + 0.05U / ml Protac (final concentration) + OWREN-KOLLER buffer , room temperature 15 min 150μl Total 150μl Control: FVIII blood clotting factor deficient plasma 150 μl + OWREN-KOLLER buffer 150 μl Measurement method: STAGO Compact Max APTT program FIX blood coagulation factor deficient plasma+ Antibody (final concentration) at each dilution in Table 12 + 0.01 U / ml Protac (final concentration) + Owren-Koller buffer , room temperature 15 min 150μl Total 150μl Control: FIX blood coagulation factor-deficient plasma 150 μl + Owren-Koller buffer 150 μl Measurement method: STAGO Compact Max APTT program

[0236] Measurement results: As shown in Table 12, in plasma deficient in blood coagulation factors VIII and VIX, the anti-aPC monoclonal antibodies HAPC14-1 to HAPC14-10 were all able to inhibit the anticoagulant activity of aPC to different degrees. Furthermore, at the same antibody concentration, the inhibitory effect of anti-aPC monoclonal antibodies HAPC14-1 to HAPC14-10 on the anticoagulant activity of aPC was superior to that of rat antibodies 1573, HAPC13, and HAPC14.

[0237] [Table 12]

[0238] (2) Experimental results for different antibodies and different concentrations of FVIII blood coagulation factor deficient plasma Experimental method: The antibodies were diluted to the corresponding concentrations using Owren-Koller buffer.

[0239] FVIII blood clotting factor deficient plasma+ Antibody (final concentration) at each dilution in Figure 6A + 0.05 U / ml Protac (final concentration) + Owren-Koller buffer , room temperature 15 min 150μl Total 150μl Control: FVIII blood clotting factor deficient plasma 150 μl + OWREN-KOLLER buffer 150 μl Measurement method: STAGO Compact Max APTT program Experimental results: As shown in Figure 6A, the anti-aPC monoclonal antibody completely inhibited the anticoagulant activity of aPC in FVIII-deficient plasma.

[0240] (3) Experimental results for different antibodies and different concentrations of FIX blood coagulation factor deficient plasma Experimental method: The antibodies were diluted to the corresponding concentrations using Owren-Koller buffer.

[0241] FIX blood coagulation factor deficient plasma+ Antibody (final concentration) at each dilution in Figure 6B + 0.01 U / ml Protac (final concentration) + Owren-Koller buffer , room temperature 15 min 150μl Total 150μl Control: FIX blood coagulation factor deficient plasma 150 μl + Owren-Koller buffer 150 μl Measurement method: STAGO Compact Max APTT program Measurement results: As shown in FIG. 6B, the anti-aPC monoclonal antibody completely inhibited the anticoagulant activity of aPC in FIX blood coagulation factor-deficient plasma.

[0242] (4) Experimental results for different antibodies and different concentrations of FXI blood coagulation factor deficient plasma Experimental method: The antibodies were diluted to the corresponding concentrations using Owren-Koller buffer.

[0243] FXI Blood Coagulation Factor Deficiency Plasma+ Antibody (final concentration) at each dilution in Figure 6C + 0.06 U / ml Protac (final concentration) + Owren-Koller buffer , room temperature 15 min 150μl Total 150μl Control: FXI blood coagulation factor deficient plasma 150μl + OWREN-KOLLER buffer 150μl Measurement method: STAGO Compact Max APTT program Measurement results: As shown in FIG. 6C, the anti-aPC monoclonal antibody completely inhibited the anticoagulant activity of aPC in FXI blood coagulation factor-deficient plasma.

[0244] (5) Experimental results for different antibodies and different concentrations of FVII blood coagulation factor deficient plasma Experimental method: The antibodies were diluted to the corresponding concentrations using Owren-Koller buffer.

[0245] FVII blood coagulation factor deficient plasma+ Antibody (final concentration) at each dilution in Figure 6D + 0.1 U / ml Protac (final concentration) + Owren-Koller buffer , room temperature 15 min 150μl Total 150μl Control: FVII blood coagulation factor-deficient plasma 150 μl + Owren-Koller buffer 150 μl Measurement method: STAGO Compact Max APTT program Measurement results: As shown in FIG. 6D, the anti-aPC monoclonal antibody completely inhibited the anticoagulant activity of aPC in FVII blood coagulation factor-deficient plasma.

[0246] (6) Experimental results of different antibodies and different concentrations against FXII blood coagulation factor deficient plasma Experimental method: The antibodies were diluted to the corresponding concentrations using Owren-Koller buffer.

[0247] FXII blood coagulation factor deficient plasma+ Antibody (final concentration) at each dilution in Figure 6E + 0.025 U / ml Protac (final concentration) + Owren-Koller buffer , room temperature 15 min 150μl Total 150μl Control: FXII blood coagulation factor-deficient plasma 150 μl + Owren-Koller buffer 150 μl Measurement method: STAGO Compact Max APTT program Measurement results: As shown in Figure 6E, the anti-aPC monoclonal antibody completely inhibited the anticoagulant activity of aPC in FXII-deficient plasma.

[0248] (7) Experimental results for different antibodies and different concentrations against FV blood coagulation factor-deficient plasma Experimental method: The antibodies were diluted to the corresponding concentrations using Owren-Koller buffer.

[0249] FV blood coagulation factor deficient plasma (contains 5% normal plasma) + Antibody (final concentration) at each dilution in Figure 6F + 0.05 U / ml Protac (final concentration) + Owren-Koller buffer , room temperature 15 min 150μl Total 150μl Control: 150 μl of FV blood coagulation factor-deficient plasma (containing 5% normal plasma) + 150 μl of Owren-Koller buffer solution Measurement method: STAGO Compact Max APTT program Measurement results: As shown in FIG. 6F, the anti-aPC monoclonal antibody completely inhibited the anticoagulant activity of aPC in FV blood coagulation factor-deficient plasma.

[0250] (8) Experimental results for different antibodies and different concentrations of FX blood coagulation factor deficient plasma Experimental method: The antibodies were diluted to the corresponding concentrations using Owren-Koller buffer.

[0251] FX blood coagulation factor deficient plasma (contains 1% normal plasma) + Antibody (final concentration) at each dilution in Figure 6G + 0.05 U / ml Protac (final concentration) + Owren-Koller buffer , room temperature 15 min 150μl Total 150μl Control: FX blood coagulation factor-deficient plasma (containing 1% normal plasma) 150 μl + Owren-Koller buffer 150 μl Measurement method: STAGO Compact Max APTT program Measurement results: As shown in FIG. 6G, the anti-aPC monoclonal antibody completely inhibited the anticoagulant activity of aPC in FX blood coagulation factor-deficient plasma.

[0252] (9) Experimental results for different antibodies and different concentrations against VWF blood coagulation factor-deficient plasma Experimental method: The antibodies were diluted to the corresponding concentrations using Owren-Koller buffer.

[0253] VWF blood coagulation factor deficient plasma+ Antibody (final concentration) at each dilution in Figure 6H + 0.01 U / ml Protac (final concentration) + Owren-Koller buffer, room temperature for 15 min 150μl Total 150μl Control: VWF-deficient plasma 150 μl + Owren-Koller buffer 150 μl Measurement method: STAGO Compact Max APTT program Measurement results: As shown in FIG. 6H, the anti-aPC monoclonal antibody completely inhibited the anticoagulant activity of aPC in VWF blood coagulation factor-deficient plasma. [Example]

[0254] In vivo drug efficacy test In a humanized protein C hemophilia mouse model (see paragraphs

[0230] to

[0238] of Chinese Patent Application No. 201911027061.6), a certain dose of anti-aPC monoclonal antibody was injected, and the amount of bleeding after docking the mouse's tail was observed to examine the blood coagulation effect achieved by the anti-aPC monoclonal antibody inhibiting the anticoagulant function of aPC.

[0255] 6.1 Experimental Method Mice were administered the drug via intracanthal vein injection. One hour after administration, the mice were anesthetized with an intraperitoneal injection of pentobarbital. After anesthesia, the mice's tails were docked 2 mm from the end, and the wound was placed in 15 ml of 37°C saline to collect the blood flowing from the mice. Collection was stopped after 20 minutes. The collected fluid was centrifuged to lyse red blood cells and the supernatant was collected. The supernatant was then measured at a wavelength of 600 nm using a UV spectrophotometer, and the OD value measured represented the amount of bleeding. The amount of bleeding was used to evaluate the blood coagulation function of the mice. WT mice represent normal mice, and untreated mice represent untreated humanized protein C hemophilia A mice or untreated humanized protein C hemophilia B mice.

[0256] 6.2 Experimental results As shown in Figure 7A, humanized protein C hemophilia A mice were injected with 1 U / mouse of FVIII blood coagulation factor, 20 μg / mouse of mouse antibody 1573, and 1 μg / mouse of HAPC-14-6. The blood coagulation function of mice injected with the new anti-APC monoclonal antibody HAPC-14-6 was restored to the level of normal mice, and the effect was consistent with that of mice treated with FVIII blood coagulation factor.

[0257] As shown in Figure 7B, when humanized protein C hemophilia A mice were injected with different doses of HAPC-14-6, only 1 μg of HAPC-14-6 was able to restore the blood coagulation function of the deficient mice to normal levels.

[0258] As shown in Figure 7C, when humanized protein C hemophilia B mice were injected with different doses of HAPC-14-6, only 1 μg of HAPC-14-6 was able to restore the blood coagulation function of the deficient mice to normal levels.

[0259] In this study, we used a protein C kinase (APTT) assay to determine the minimum dose of anti-aPC monoclonal antibodies required to inhibit the anticoagulant activity of aPC in human plasma lacking multiple blood coagulation factors. While the mouse antibody HAPC1573 required a concentration of 100 μg / ml or more, the present invention demonstrated complete inhibition at a maximum concentration of only 1 μg / ml.

[0260] The above examples are for illustrative purposes only and do not limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications to the specific embodiments of the present invention and equivalent substitutions for some technical features are still possible. Furthermore, any modifications and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive idea are included in the scope of the technical solutions claimed by the present invention.

Claims

1. An antibody comprising a heavy chain represented by SEQ ID NO: 7 and a light chain represented by SEQ ID NO: 8; An antibody comprising a heavy chain represented by SEQ ID NO: 9 and a light chain represented by SEQ ID NO: 10; An antibody comprising a heavy chain represented by SEQ ID NO: 11 and a light chain represented by SEQ ID NO: 12; An antibody comprising a heavy chain represented by SEQ ID NO: 13 and a light chain represented by SEQ ID NO: 14; An antibody comprising a heavy chain represented by SEQ ID NO: 15 and a light chain represented by SEQ ID NO: 16; An antibody comprising a heavy chain represented by SEQ ID NO: 17 and a light chain represented by SEQ ID NO: 18; An antibody comprising a heavy chain represented by SEQ ID NO: 19 and a light chain represented by SEQ ID NO: 20; An antibody comprising a heavy chain represented by SEQ ID NO: 21 and a light chain represented by SEQ ID NO: 22; An antibody comprising a heavy chain represented by SEQ ID NO: 23 and a light chain represented by SEQ ID NO: 24; an antibody comprising a heavy chain represented by SEQ ID NO:25 and a light chain represented by SEQ ID NO:26;

2. A biomaterial related to the anti-aPC monoclonal antibody of claim 1, 1) a nucleic acid molecule encoding the anti-aPC monoclonal antibody of claim 1; 2) A structure comprising the nucleic acid molecule according to 1) above. 3) an expression system comprising the nucleic acid molecule according to 1) above.

3. A pharmaceutical product comprising: The pharmaceutical product is a drug composition or a detection reagent kit; The pharmaceutical product comprises any one or more of the anti-aPC monoclonal antibody of claim 1 or the biomaterial of claim 2; When the pharmaceutical product is a pharmaceutical composition, it further comprises a pharma- ceutically acceptable vector.

4. The anti-aPC monoclonal antibody according to claim 1 or the biomaterial according to claim 2, i) application in the manufacture of a medicament for preventing and / or treating blood coagulation deficiencies or defects; ii) Application in the manufacture of promoters that enhance the ability of aPC to degrade histones H3 and H4; iii) in the manufacture of a medicament for inhibiting the anticoagulant activity of activated protein C in an individual; iv) application in the manufacture of drugs to treat individuals in need of blood clotting; vi) application in the manufacture of a medicament for regulating hemostasis in an individual; vii) Applications in immunological testing and analysis for the diagnosis or treatment of disease or non-disease, or in the manufacture of reagents or reagent kits for detecting aPC; Use of any one or more of the following:

5. The use according to claim 4, characterized in that the blood clotting deficiency or defect includes diseases in which blood clotting disorders occur due to a deficiency of blood clotting factors, causing bleeding, and blood clotting disorders caused by trauma or surgery.

6. 5. The use according to claim 4, characterized in that the blood coagulation deficiency or defect includes hemophilia, sepsis, and traumatic hemorrhage.

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