Monoclonal antibodies against human activated protein C and their preparation and application

Abstract

The present invention belongs to the field of monoclonal antibody technology and relates to a monoclonal antibody targeting activated protein C (aPC). The amino acid sequences of the heavy and light chains of the monoclonal antibody are shown in SEQ ID NOs. 5 to 26, and the CDR sequences thereof are shown in SEQ ID NOs. 27 to 92. The present invention also provides biomaterials related to the monoclonal antibody, which selectively bind to aPC but not to inactivated protein C. The present invention also provides the use of the monoclonal antibody and related biomaterials in the preparation of drugs for treating coagulation deficiencies or defects, as well as related treatment methods, which have broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of monoclonal antibodies and relates to monoclonal antibodies targeting human activated protein C (aPC) and applications thereof. Background Art

[0002] The protein C (PC) pathway plays a key role in regulating coagulation and inflammation. Thrombin forms a complex with thrombin modulator (TM) on vascular endothelial cells, activating protein C. Activated protein C (aPC) cleaves activated factor V and factor VIII, negatively downregulating thrombin formation and maintaining the balance between thrombosis and hemostasis in vivo. aPC protects animals from endotoxin (LPS) or Escherichia coli. A recombinant human aPC product was approved by the FDA as the first drug to treat severe sepsis in humans. The molecular mechanisms of aPC's anti-inflammatory and cytoprotective effects are not fully understood. Mutational studies have shown that aPC's anticoagulant activity is not necessarily required for its anti-apoptotic effects on endothelial cells. The endothelial protein C receptor (EPCR) is primarily expressed on the endothelium of large vessels and enhances protein C activation via the thrombin-TM complex. Recently, aPC bound to EPCR was shown to cleave PAR-1 on endothelial cells, and this study suggests that all anti-inflammatory and anti-apoptotic effects of aPC signaling are mediated by PAR-1 in endothelial cells. However, upon lethal LPS challenge, PAR-1-deficient mice displayed a phenotype similar to their wild-type controls, suggesting that PAR-1 may not be the only player in aPC signaling regulating inflammation and apoptosis.

[0003] Given the central role of aPC in regulating pathophysiological functions in vivo, a monoclonal antibody, HAPC1573, has been produced that specifically recognizes the active form of human protein C (aPC) rather than protein C (Xu, US8153766B2, US9127072B2). This antibody alters the amidolytic activity of aPC towards chromogenic peptide substrates and blocks the anticoagulant activity of aPC in plasma. More importantly, this antibody does not affect aPC's cleavage of extracellular histones H3 and H4, nor does it affect aPC's cytoprotective activity against thrombin-induced permeability of endothelial cells. MAPC1591 is a monoclonal antibody against mouse aPC that has similar functions to HAPC1573, blocking the anticoagulant activity of mouse aPC and maintaining its cytoprotective activity, thereby protecting hemophilia A from traumatic bleeding, suggesting that anti-aPC antibodies may be applicable to clinical settings such as hemophilia, trauma, and sepsis.

[0004] Zhao et al. reported the CDR sequences of HAPC1573 and humanized HAPC1573 that target only the anticoagulant activity and not other activities of aPC, which showed in vivo protection against bleeding in an acute acquired hemophilia A monkey model (Zhao, US9657111B2, WO2017 / 087391A1, Nat. Communications 2020). The X-ray crystal structure of the aPC and humanized HAPC1573 Fab complex showed that HAPC 1573 may not bind to the active site of aPC, but rather bind to the aPC autolytic loop containing the known aPC-FVa binding site. Therefore, humanized HAPC1573 may inhibit the anticoagulant function of aPC by interfering with the interaction between aPC and FVa. The inhibitory effect of humanized HAPC1573 is limited to the anticoagulant activity of aPC, without affecting its cytoprotective, anti-inflammatory and cell signaling functions. The concentration of aPC in human plasma is reported to be approximately 40 μM. Surface plasmon resonance analysis indicates that humanized HAPC 1573 has an affinity for aPC of 10 nM. A Protac-based APTT coagulation assay demonstrated a concentration-dependent effect of HAPC 1573 in shortening the APTT prolongation in human FVIII-deficient and FIX-deficient plasma. Even at a concentration of 50 μg / ml, HAPC 1573 failed to achieve maximal inhibition of aPC's anticoagulant activity in this in vitro assay. This observation is consistent with in vivo observations of humanized HAPC 1573 in an acutely acquired monkey hemophilia model. In animal studies, higher doses of HAPC 1573, 10 mg / kg and 30 mg / kg, were found to provide significant protection. However, such high doses are not suitable for clinical use. Therefore, the development of humanized HAPC 1573 with higher affinity is a prerequisite and guarantee for the future potential therapeutic application of aPC. Summary of the Invention

[0005] One aspect of the present invention is to provide an anti-aPC monoclonal antibody, comprising an original antibody and a mutant antibody obtained by performing one or more mutations in the following six mutation sites on the original antibody:

[0006] Wherein, the original antibody comprises:

[0007] (a) a heavy chain CDR represented by any one or more of SEQ ID NOs: 27, 28, and 29; and / or,

[0008] (b) a light chain CDR represented by any one or more of SEQ ID NOs: 30, 31, and 32;

[0009] Among them, the 6 mutation sites are:

[0010] 1) N57R / W located in VL CDR2;

[0011] 2) E59G / P in VL CDR2;

[0012] 3) Q93V located in VL CDR3;

[0013] 4) N31F located in VH CDR1;

[0014] 5) S55K located in VH CDR2;

[0015] 6) Y104T in VH CDR3.

[0016] In the present invention, the constant region of the anti-aPC monoclonal antibody comprises one or more of the human IgG1, IgG2, IgG3, and IgG4 sequences.

[0017] Preferably, in the present invention, the mutation sites of the anti-aPC monoclonal antibody in the original antibody include at least: N31F in VH CDR1; S55K in VH CDR2; N57R in VL CDR2; and E59G in VL CDR2.

[0018] Preferably, in the present invention, the mutation sites of the anti-aPC monoclonal antibody in the original antibody include at least: N57R in VL CDR2; E59G in VL CDR2; Q93V in VL CDR3; N31F in VH CDR1; and S55K in VH CDR2.

[0019] Another aspect of the present invention is to provide the anti-aPC monoclonal antibody-related biological material, which is any one or more of the following 1) to 10):

[0020] 1) A nucleic acid molecule encoding an anti-aPC monoclonal antibody as described above;

[0021] 2) an expression cassette containing the nucleic acid molecule described in 1);

[0022] 3) a construct comprising the nucleic acid molecule described in 1);

[0023] 4) a construct containing the expression cassette described in 2);

[0024] 5) an expression system containing the nucleic acid molecule described in 1);

[0025] 6) an expression system comprising the expression cassette described in 2);

[0026] 7) an expression system containing the construct described in 3);

[0027] 8) an expression system containing the recombinant vector described in 4);

[0028] 9) a primer pair for amplifying a nucleic acid molecule encoding the amino acid sequence of the anti-aPC monoclonal antibody as described above;

[0029] 10) A fusion antibody comprising the anti-aPC monoclonal antibody as described above.

[0030] Another aspect of the present invention is to provide a pharmaceutical product, which is a pharmaceutical composition or a detection kit; the pharmaceutical product comprises any one or more of the anti-aPC monoclonal antibodies or related biological materials described above. When the pharmaceutical composition is a pharmaceutical composition, it further comprises a pharmaceutically acceptable carrier.

[0031] Another aspect of the present invention is to provide a method for treating a coagulation-related disorder, comprising administering an effective amount of the anti-aPC monoclonal antibody or a related biomaterial as described above to a subject in need of treatment, wherein the subject suffers from a coagulation deficiency or defect, or requires blood coagulation treatment, or suffers from sepsis or hemophilia, or requires regulation of hemostasis.

[0032] Another aspect of the present invention provides the use of the anti-aPC monoclonal antibody or related biomaterials in the preparation of a medicament for preventing and / or treating coagulation deficiencies or defects. These coagulation deficiencies or defects include coagulation disorders caused by coagulation factor deficiencies and resulting in bleeding, as well as coagulation disorders caused by trauma or surgery. Furthermore, these coagulation deficiencies or defects include hemophilia, sepsis, and traumatic bleeding.

[0033] Another aspect of the present invention is to provide the use of the anti-aPC monoclonal antibody or its related biomaterial in the preparation of an enhancer that enhances the ability of aPC to cleave histones H3 and H4, or in the preparation of a drug that inhibits the anticoagulant activity of activated protein C in an individual, or in the preparation of a drug for preventing and / or treating an individual requiring blood coagulation, or in the preparation of a drug for preventing and / or treating an individual suffering from sepsis, or in the preparation of a drug for preventing and / or treating an individual suffering from hemophilia, or in the preparation of a drug for regulating hemostasis in an individual, or in immunological examinations and analyses for non-disease diagnosis and treatment purposes, or in the preparation of aPC detection reagents or kits, or in combination with other reagents or drugs.

[0034] Another aspect of the present invention provides a method for preparing the anti-aPC monoclonal antibody as described above, comprising the steps of: culturing the anti-aPC monoclonal antibody expression system under conditions suitable for expressing the anti-aPC monoclonal antibody, thereby expressing the anti-aPC monoclonal antibody.

[0035] The beneficial effects of the present invention are that the anti-aPC monoclonal antibody provided by the present invention is a humanized antibody, which is obtained by humanizing HAPC1573, or by simultaneously performing mutation screening of single sites and combined sites. It selectively binds to activated protein C and inhibits activated protein C, but does not bind to inactivated protein C or inhibit inactivated protein C. It selectively binds to human activated protein C, but does not bind to activated protein C or inactivated protein C of other species. The affinity of the antibody is significantly improved, and the affinity of some antibodies is 100 times higher than that of HAPC 1573. In the Protac-based APTT coagulation analysis, the present invention uses a very small amount of antibody, such as 1 μg / ml, to produce the maximum inhibitory effect on the anticoagulant activity of aPC. The antibody can potentially block the anticoagulant activity of aPC and has broad application prospects in the treatment of coagulation deficiency or defect diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG1 shows the binding affinity of humanized anti-aPC monoclonal antibodies (Humab001-1 to Humab001-28 or HAPC-1 to HAPC-28) to human aPC protein.

[0037] FIG2 shows the binding affinity of humanized anti-aPC monoclonal antibodies (Humab001-1 to Humab001-28 or HAPC-1 to HAPC-28) to human PC protein.

[0038] FIG3 shows the binding force between HAPC-14 mutant antibodies (HAPC14-1 to HAPC14-10 or HAPC-14-1 to HAPC-14-10) and hAPC / hPC; Figures 3A to 3D The horizontal axes are 10 -6 , 10 -4 , 10 -2 , 10 0 , 10 2 , 10 4 nM.

[0039] FIG4 shows the affinity determination of mutant antibodies of HAPC-14 (HAPC14-1 to HAPC14-10 or HAPC-14-1 to HAPC-14-10).

[0040] Figure 5 The effect of the mutant antibody HAPC-14-6 of HAPC-14 on the ability of aPC to cleave histones.

[0041] Figure 6 shows an in vitro pharmacodynamics study. HAPC-14 mutant antibodies HAPC-14-3 and HAPC-14-6 inhibit the anticoagulant activity of aPC in factor-deficient plasma; A: FVIII-deficient plasma, B: FIX-deficient plasma, C: FXI-deficient plasma, D: FVII-deficient plasma, E: FXII-deficient plasma, F: FV-deficient plasma, G: FX-deficient plasma, H: VWF-deficient plasma.

[0042] Figure 7 shows in vivo pharmacodynamics experiments. A: Effects of injecting FVIII, HAPC1573, and HAPC-14-6 into FVIII-deficient mice on their coagulation function; B: Effects of injecting different doses of HAPC-14-6 into FVIII-deficient mice on their coagulation function; C: Effects of injecting HAPC1573 and different doses of HAPC-14-6 into FIX-deficient mice on their coagulation function. DETAILED DESCRIPTION

[0043] The present invention humanized HAPC 1573 and then optimized its affinity. The optimized mutation sites were screened using Fab, ultimately resulting in six optimal sites. Further combined mutations yielded an optimized mutant with an affinity 100-fold higher than that of HAPC 1573. Furthermore, in a Protac-based APTT coagulation assay, a mutant antibody dosage of 1 μg / ml was sufficient to achieve maximum inhibition of aPC anticoagulant activity, suggesting the possibility of further clinical application.

[0044] The present invention provides an anti-aPC antibody, namely anti-aPC monoclonal antibody 14 (HAPC-14), comprising:

[0045] (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,

[0046] (b) a light chain comprising the light chain CDRs represented by any one or more of SEQ ID NOs: 30, 31 and 32.

[0047] The anti-aPC monoclonal antibody 14 (HAPC-14) provided by the present invention has a heavy chain sequence as shown in SEQ ID NO: 5, and a light chain sequence as shown in SEQ ID NO: 6.

[0048] The present invention also provides mutant antibodies of a monoclonal antibody against human activated protein C (aPC) (anti-aPC monoclonal antibody 14). Based on the sequence of the aforementioned anti-aPC monoclonal antibody 14, these mutant antibodies were designed, screened, and statistically analyzed. It was found that mutations at one or more of the following six sites significantly affect the binding ability of the monoclonal antibody Fab fragment to the capture antibody, resulting in high affinity, high functional activity, and low divergence from the germline sequence. These six sites are:

[0049] a) N57R / W in VL CDR2 (N57R is superior to N57W);

[0050] b) E59G / P in VL CDR2 (E59G is preferred over E59P);

[0051] c) Q93V in VL CDR3;

[0052] d) N31F located in VH CDR1;

[0053] e) S55K located in VH CDR2;

[0054] f) Y104T in VH CDR3.

[0055] Among them, the most critical mutation sites are N57R in the light chain and N31F in the heavy chain. In some embodiments, the anticoagulant activity of aPC is completely inhibited in a humanized protein C (haPC) hemophilia mouse model, and the concentration of anti-aPC monoclonal antibody required to restore coagulation function to normal is as low as 1 μg / ml.

[0056] For the convenience of description, the combined mutations are described below in the form of abbreviations, where N57R refers to N57R located in VLCDR2, N57W refers to N57W located in VL CDR2, E59G refers to E59G located in VL CDR2, E59P refers to E59P located in VLCDR2, Q93V refers to Q93V located in VL CDR3, N31F refers to N31F located in VH CDR1, S55K refers to S55K located in VHCDR2, and Y104T refers to Y104T located in VH CDR3.

[0057] In the present invention, the mutation combinations of the above six sites include but are not limited to: (1) mutations comprising only any one of the following sites: N57R, N57W, E59G, E59P, Q93V, N31F or S55K; or (2) mutations comprising two of the following sites at the same time: 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, N57R and E59G, N57R and E59P, N57R and Q93V, N57R and N31F, N57R and S55K, N57R and E59P, N57W and Q93V, N57R and N31F. 7W and N31F, N57W and S55K, E59G and Q93V, E59G and N31F, E59G, S55K, E59P and N31F, E59P and S55K, Q93V and S55K; or (3) mutations simultaneously comprising three of the following sites: 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, E 59G and N31F, N57R, E59G and S55K, N57R, E59P and Q93V, N57R, E59P and N31F, N57R, E59P and S55K, N57R, Q93V and N31F, N57R, Q93V and S55K, N57R, N31F and S55K, N57W, E59G, E59P, N57W, E59G, Q93V, N57W, E59G and N31F, N57W, E59G and S55K, N57W, E59P and Q93V, N57W, E59P and N31F, N57 W, E59P and S55K, N57W, Q93V and N31F, N57W, Q93V and S55K, N57W, N31F and S55K, E59G, E59P and Q93V, E59G, E59P and N31F, E59G, 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;or (4) mutations simultaneously comprising the following four sites: 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, E59G, E59P and S55K, N57R, E59G, Q93V and N31F, N57R, E59G, Q93V and S55K, N57R, E59P, N 31F and S55K, N57R, Q93V, 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 S55K, E59G, Q93V, N31F and S55K, E59P, Q93V, N31F and S55K;or (5) or mutations simultaneously comprising the following five sites: N57R, N57W, E59G, E59P and Q93V, N57R, N57W, E59G, E59P and N31F, N57R, N57W, E59G, Q93V and N31F, N57R, N57W, E59P, Q93V and N31F, N57R, E59G, E59P, Q93V and N31F, N57R, N57W, E59G, E59P, Q93V and N31F, N57R, N57W, E59G, E59P and S55K, N57R, N57W, E59G, E59P and N55K. 9G, Q93V and S55K, N57R, N57W, E59P, Q93V and S55K, N57R, E59G, E59P, Q93V and S55K, N57W, E59G, E59P, Q93V and S55K, N57R, N57W, E59G, N31F and S55K, N57R, N57W, E59P, N31F and S55K, N57R, E59G, E59P, N31F and S55K, N57W, E59G, E59P, N31F and S55K, N57R, N57W, Q93V, N31F and S55K, N57R, E59G, Q93V, N31F and S55K, N57W, E59G, Q93V, N31F and S55K, N57R, E59P, Q93V, N31F and S55K, N57W, E59P, Q93V, N31F and S55K, E59G, E59P, Q93V, N31F and S55K; or (6) or mutations simultaneously comprising the following six positions: N57R, N57W, E59G, E59P, Q93V and N31F, N57R, N57W, E59G, E59P, Q93V and S5 5K, N57R, N57W, E59G, E59P, N31F and S55K, N57R, N57W, E59G, 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) or mutations simultaneously comprising the following seven sites: N57R, N57W, E59G, E59P, Q93V, N31F, S55K. ;

[0058] In one embodiment, the present invention provides an anti-aPC monoclonal antibody comprising:

[0059] The heavy chain CDR1 represented by SEQ ID NO: 27 or 33; the heavy chain CDR2 represented by SEQ ID NO: 28 or 40; one or two or three of CDR1, CDR2, and CDR3 in the heavy chain CDR3 represented by SEQ ID NO: 29 or 77; and / or,

[0060] The light chain CDR1 represented by SEQ ID NO: 30; the light chain CDR2 represented by any one of SEQ ID NOs: 31, 37, 43, 49, and 73; and one, two, or three of CDR1, CDR2, and CDR3 among the light chain CDR3 represented by SEQ ID NO: 32 or 44.

[0061] In one embodiment, the antibody is selected from the group consisting of:

[0062] comprising (a) the heavy chain CDRs represented by SEQ ID NOs: 27, 28, and 29; and / or,

[0063] (b) antibodies having light chain CDRs represented by SEQ ID NOs: 30, 31, and 32;

[0064] (c) comprising the heavy chain CDRs represented by SEQ ID NOs: 33, 34, and 35; and / or,

[0065] (d) antibodies having light chain CDRs represented by SEQ ID NOs: 36, 37, and 38;

[0066] comprising (e) the heavy chain CDRs represented by SEQ ID NOs: 39, 40, and 41; and / or,

[0067] (f) antibodies having light chain CDRs represented by SEQ ID NOs: 42, 43, and 44;

[0068] comprising (g) the heavy chain CDRs represented by SEQ ID NOs: 45, 46, and 47; and / or,

[0069] (h) antibodies having light chain CDRs represented by SEQ ID NOs: 48, 49, and 50;

[0070] comprising (i) the heavy chain CDRs represented by SEQ ID NOs: 51, 52, and 53; and / or,

[0071] (j) antibodies having light chain CDRs represented by SEQ ID NOs: 54, 55, and 56;

[0072] comprising (k) the heavy chain CDRs represented by SEQ ID NOs: 57, 58, and 59; and / or,

[0073] (1) an antibody having a light chain CDR represented by SEQ ID NOs: 60, 61, and 62;

[0074] comprising (m) the heavy chain CDRs represented by SEQ ID NOs: 63, 64, and 65; and / or,

[0075] (n) an antibody having a light chain CDR represented by SEQ ID NOs: 66, 67, and 68;

[0076] comprising (o) the heavy chain CDRs represented by SEQ ID NOs: 69, 70, and 71; and / or,

[0077] (p) an antibody having a light chain CDR represented by SEQ ID NOs: 72, 73, and 74;

[0078] comprising (q) the heavy chain CDRs represented by SEQ ID NOs: 75, 76, and 77; and / or,

[0079] (r) antibodies having light chain CDRs represented by SEQ ID NOs: 78, 79, and 80;

[0080] comprising(s) the heavy chain CDRs represented by SEQ ID NOs: 81, 82, and 83; and / or,

[0081] (t) an antibody having a light chain CDR represented by SEQ ID NOs: 84, 85, and 86;

[0082] comprising (u) the heavy chain CDRs represented by SEQ ID NOs: 87, 88, and 89; and / or,

[0083] (v) Light chain CDRs represented by SEQ ID NOs: 90, 91 and 92.

[0084] In the present invention, the heavy chain framework region of the anti-aPC monoclonal antibody can be any existing framework region suitable for exerting the function of the anti-aPC monoclonal antibody of the present invention, and can be of human or mouse origin.

[0085] In the present invention, the light chain framework region of the anti-aPC monoclonal antibody can be any existing framework region suitable for exerting the function of the anti-aPC monoclonal antibody of the present invention, and can be of human or mouse origin.

[0086] In one embodiment, the anti-aPC monoclonal antibody comprises:

[0087] A heavy chain sequence as shown in any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25; and / or

[0088] A light chain sequence as shown in any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26.

[0089] In one embodiment, the anti-aPC monoclonal antibody is selected from the following:

[0090] The antibody HaPC14 comprises the heavy chain sequence shown in SEQ ID NO: 5 and the light chain sequence shown in SEQ ID NO: 6;

[0091] The antibody HaPC14-1 comprises the heavy chain sequence shown in SEQ ID NO:7 and the light chain sequence shown in SEQ ID NO:8;

[0092] The antibody HaPC14-2 comprises the heavy chain sequence shown in SEQ ID NO: 9 and the light chain sequence shown in SEQ ID NO: 10;

[0093] The antibody HaPC14-3 comprises the heavy chain sequence shown in SEQ ID NO: 11 and the light chain sequence shown in SEQ ID NO: 12;

[0094] The antibody HaPC14-4 comprises the heavy chain sequence shown in SEQ ID NO: 13 and the light chain sequence shown in SEQ ID NO: 14;

[0095] The antibody HaPC14-5 comprises the heavy chain sequence shown in SEQ ID NO: 15 and the light chain sequence shown in SEQ ID NO: 16;

[0096] The antibody HaPC14-6 comprises the heavy chain sequence shown in SEQ ID NO: 17 and the light chain sequence shown in SEQ ID NO: 18;

[0097] The antibody HaPC14-7 comprises the heavy chain sequence shown in SEQ ID NO: 19 and the light chain sequence shown in SEQ ID NO: 20.

[0098] The antibody HaPC14-8 comprises the heavy chain sequence shown in SEQ ID NO: 21 and the light chain sequence shown in SEQ ID NO: 22.

[0099] The antibody HaPC14-9 comprises the heavy chain sequence shown in SEQ ID NO: 23 and the light chain sequence shown in SEQ ID NO: 24.

[0100] The antibody HaPC14-10 comprises the heavy chain sequence shown in SEQ ID NO: 25 and the light chain sequence shown in SEQ ID NO: 26.

[0101] In the present invention, some sequences have the same composition but different numbering (for details, please refer to the specific amino acid sequences in the sequence table).

[0102] For example, the sequence of antibody heavy chain CDR1 is as follows: sequences 27 and 87 are identical; and sequences 33, 39, 45, 51, 57, 63, 69, 75, and 81 are identical.

[0103] The sequence of antibody heavy chain CDR2 is shown as follows: sequences 28, 34, 52, and 70 are identical; sequences 40, 46, 58, 64, 76, 82, and 88 are identical.

[0104] The sequence of antibody heavy chain CDR3 is shown as follows: sequences 29, 35, 41, 47, 53, 59, 65, and 71 are identical; sequences 77, 83, and 89 are identical.

[0105] The sequence of the antibody light chain CDR1 is shown: sequences 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, and 90 are identical.

[0106] The sequence of the antibody light chain CDR2 is shown: sequences 37 and 61 are identical; sequences 49, 55, 67, 79, 85, and 91 are identical.

[0107] The sequence of antibody light chain CDR3 is shown as follows: sequences 32, 38, 68, 74, and 86 are identical; sequences 44, 50, 56, 62, 80, and 92 are identical.

[0108] In one embodiment, the present invention further provides a biological material related to the anti-aPC monoclonal antibody, which is any one or more of the following 1) to 10):

[0109] 1) a nucleic acid molecule encoding the anti-aPC monoclonal antibody as described above;

[0110] 2) an expression cassette containing the nucleic acid molecule described in 1);

[0111] 3) a construct comprising the nucleic acid molecule described in 1);

[0112] 4) a construct containing the expression cassette described in 2);

[0113] 5) an expression system containing the nucleic acid molecule described in 1);

[0114] 6) an expression system comprising the expression cassette described in 2);

[0115] 7) an expression system containing the construct described in 3);

[0116] 8) an expression system containing the recombinant vector described in 4);

[0117] 9) a primer pair for amplifying a nucleic acid molecule encoding the amino acid sequence of the anti-aPC monoclonal antibody described above;

[0118] 10) A fusion antibody comprising the anti-aPC monoclonal antibody as described above, wherein the expression system can be a cell or cell line encoding the anti-aPC monoclonal antibody.

[0119] In a specific embodiment, the present invention provides a preparation product, which is a pharmaceutical composition or a detection reagent or a detection kit.

[0120] In one embodiment, the present invention provides a pharmaceutical composition comprising an anti-aPC monoclonal antibody as described above, or any one or more of the biological materials as described above, and a pharmaceutically acceptable carrier. The carrier may include various excipients and diluents, which are not essential active ingredients and are not unduly toxic upon administration. Suitable carriers are well known to those skilled in the art; for example, pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ, 1991).

[0121] In one embodiment, the present invention provides a detection reagent or a detection kit comprising the anti-aPC monoclonal antibody described above, or any one or more of the biomaterials described above, wherein the antibody is labeled.

[0122] The labeling can be performed in any existing manner, such as using chemiluminescent labels, fluorophores, radioactive labels or chromophores.

[0123] The detection reagent or kit may further include a buffer, a diluent, instructions for use, and the like.

[0124] The antibody may be in any form suitable for its work or activity, such as in the form of an aqueous suspension.

[0125] In some embodiments, the anti-aPC monoclonal antibodies of the present invention enhance the ability of aPC to cleave histones H3 and H4, indicating that the anti-aPC monoclonal antibodies can provide cytoprotection against the cytotoxicity of histones H3 and H4.

[0126] In one embodiment, the present invention provides a method for treating a coagulation deficiency or defect, as defined above, specifically providing a method for treating inherited and acquired coagulation deficiencies or defects, such as hemophilia A and 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 requiring blood coagulation therapy). It also provides a method for inhibiting the anticoagulant activity of activated protein C in a subject. It also provides a method for regulating thrombosis in a subject. The methods of the present invention comprise administering an effective amount of the anti-aPC monoclonal antibody described above to a subject in need of treatment. It also provides a method for producing anti-human aPC monoclonal antibodies.

[0127] In one embodiment, the present invention provides a method for treating a coagulation deficiency or defect disorder, comprising administering an effective amount of the anti-aPC monoclonal antibody or its related biological material as described above to an individual in need of treatment.

[0128] In one embodiment, the present invention provides a method for treating coagulation-related diseases, comprising administering an effective amount of any one or more of the anti-aPC monoclonal antibodies described above or the biomaterials described above to an individual in need of treatment; wherein the individual suffers from a coagulation deficiency or defect, or the individual needs blood coagulation treatment, or the individual suffers from sepsis or hemophilia, or the individual needs regulation of hemostasis.

[0129] Among them, the coagulation deficiency or defect disease can be hereditary or acquired, including diseases caused by coagulation disorders and bleeding due to the lack of one or several coagulation factors in the plasma; specifically including: hemophilia A, hemophilia B, hemophilia C - caused by the lack of coagulation factors VIII, IX and XI; rare coagulation factor deficiency - bleeding diseases caused by the absence or abnormal function of one or more of other coagulation factors (i.e., coagulation factors I, II, V, V+VIII, VII, X, or XIII), etc., such as coagulation disorders caused by trauma, excessive bleeding caused by treatment of sepsis, transplantation, heart surgery, plastic surgery, etc.

[0130] In a specific embodiment, the present invention provides a method for enhancing the ability of aPC to cleave histones H3 and H4. After administering the anti-aPC monoclonal antibody or its related biomaterial or pharmaceutical composition, the ability of aPC to cleave histones H3 and H4 can be enhanced, thereby providing a cytoprotective effect against the cytotoxicity of histones H3 and H4.

[0131] In a specific embodiment, the present invention provides a method for inhibiting the anticoagulant activity of activated protein C in an individual. After administering the anti-aPC monoclonal antibody or its related biomaterial or pharmaceutical composition, the anticoagulant activity of activated protein C in the individual can be inhibited, thereby regulating the individual's coagulation function.

[0132] In one embodiment, the present invention provides the anti-aPC monoclonal antibody or its related biological materials

[0133] i) use in the preparation of a medicament for preventing and / or treating coagulation deficiency or defect disorders;

[0134] ii) application in the preparation of a promoter for enhancing the ability of aPC to cleave histones H3 and H4;

[0135] iii) use in the preparation of a medicament for inhibiting the anticoagulant activity of activated protein C in an individual;

[0136] iv) use in the preparation of a medicament for treating an individual requiring blood coagulation;

[0137] vi) use in the preparation of a medicament for regulating hemostasis in an individual;

[0138] vii) Use in immunological examination and analysis for the purpose of disease or non-disease diagnosis and treatment, or in the preparation of aPC detection reagents or kits;

[0139] viii) Use in combination with other agents or drugs.

[0140] In a specific embodiment, the present invention also provides a method for preparing the anti-aPC monoclonal antibody as described above, comprising the following steps: culturing the anti-aPC monoclonal antibody expression system under conditions suitable for expressing the anti-aPC monoclonal antibody, thereby expressing the anti-aPC monoclonal antibody.

[0141] In the present invention, polypeptides having a certain degree of amino acid sequence identity with the anti-aPC monoclonal antibody are also within the scope of protection of the present invention, such as antibodies or antibody fragments having 70%, 75%, 80%, 85%, 90%, 95%, or 99% or greater sequence identity with the anti-aPC monoclonal antibody and possessing the functions of the anti-aPC monoclonal antibody (having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity). Specifically, the anti-aPC monoclonal antibody is obtained by substituting, deleting, or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or by adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids to the N-terminus and / or C-terminus, and a polypeptide fragment having the function of the anti-aPC monoclonal antibody.

[0142] The term "protein C" or "PC" refers to any variant, isoform and / or species homolog of protein C in its zymogen form that is naturally expressed by cells and present in plasma, and is distinct from the activated form of protein C.

[0143] The term "activated protein C" or "aPC" refers to the activated form of protein C.

[0144] The term "anti-aPC monoclonal antibody" refers to an antibody that specifically binds to an epitope of aPC, and is an anti-human aPC monoclonal antibody. The anti-aPC monoclonal antibody can be in various antibody forms, including but not limited to whole antibodies, antibody fragments, human antibodies, humanized antibodies, and genetically engineered antibodies, such as monoclonal antibodies, chimeric antibodies, or recombinant antibodies, as well as fragments of these antibodies, provided that they retain the properties of the present invention.

[0145] The antibody fragments are further defined as Fab', Fab, F(ab')2, single-domain antibodies, Fv, or scFv. The anti-aPC monoclonal antibodies described herein refer to antibody molecule preparations consisting 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 germline immunoglobulin sequences. For example, a Fab fragment is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; a F(ab')2 fragment is a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; and an Fv fragment is an Fv fragment consisting of the VL and VH domains of a single antibody arm.

[0146] The term "identity" can be evaluated with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0147] The term "subject" generally includes humans, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cows, etc., who can benefit from treatment with the above-mentioned antibodies, drugs, compositions, related preparations, kits or combined preparations.

[0148] The term "therapeutically effective amount" generally refers to an amount that can achieve the effect of treating the diseases listed above after an appropriate administration period.

[0149] The term "monoclonal antibody" refers to a preparation of antibody molecules of a single amino acid composition. It may have variable and constant regions derived from human germline immunoglobulin sequences. In one embodiment, the monoclonal antibody is produced by a hybridoma comprising a B cell obtained from a transgenic non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a human light chain transgene fused to immortalized cells.

[0150] The term "fusion antibody" refers to a product obtained by fusing an antibody fragment with another biologically active protein using genetic engineering techniques; a target gene is linked to an Ig fragment gene at the genetic level, and a recombinant protein with these two structural domains is expressed in eukaryotic or prokaryotic cells. Depending on the different Ig fragments to which the target protein is linked, it can be divided into two major categories: Fab (Fv) fusion proteins and Fc fusion proteins. Two different proteins are linked into one large molecule. This can be achieved by chemical connection or gene fusion. Methods for preparing fusion antibodies involve conventional recombinant DNA and gene transfection techniques well known in the art. Due to the different fusion proteins, such antibody fusion proteins have multiple biological functions, and the expressed recombinant protein neither affects the antigen-binding ability of the single-chain antibody nor the biological properties of the protein to which it is fused.

[0151] The term "humanized antibody" refers to antibodies in which the framework or "complementarity determining regions" (CDRs) have been modified to encompass an immunoglobulin with a different specificity than that of the parent immunoglobulin.

[0152] When referring to inhibiting and / or blocking the binding of an aPC substrate to aPC, the terms inhibiting and blocking also include a measurable decrease in the binding affinity of aPC for a physiological substrate when contacted with an anti-aPC antibody as compared to when aPC is not contacted with the anti-aPC antibody, for example, 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% blocking the interaction of aPC with its substrates, including Factor Va, or with Factor VIIIa.

[0153] "Complementarity determining region" or "CDR" refers to one of the three hypervariable regions within the heavy or light chain variable region of an antibody molecule that forms the N-terminal antigen-binding surface that is complementary to the three-dimensional structure of the bound antigen. Starting from the N-terminus of the heavy or light chain, these complementarity determining regions are designated "CDR1," "CDR2," and "CDR3," respectively. CDRs are involved in antigen-antibody binding, and CDR3 contains a unique region that is specific for antigen-antibody binding. Thus, an antigen-binding site may include six CDRs, comprising CDR regions from each of the heavy and light chain V regions.

[0154] The term "epitope" means the area or region of an antigen to which an antibody specifically binds or interacts, which in some embodiments indicates where the antigen is in physical contact with the antibody.

[0155] The term "conservative substitution" refers to a polypeptide modification that involves replacing one or more amino acids with amino acids that have similar biochemical properties but do not cause a loss of biological or biochemical function of the polypeptide. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. Common conservative substitutions are, for example, the mutual substitution between the aromatic amino acids Phe, Trp, and Tyr; the mutual substitution between the hydrophobic amino acids Leu, Ile, and Val; the mutual substitution between the polar amino acids Gln and Asn; the mutual substitution between the basic amino acids Lys, Arg, and His; the mutual substitution between the acidic amino acids Asp and Glu; and the mutual substitution between the hydroxyl amino acids Ser and Thr.

[0156] The term "identity" refers to the proportion of nucleotides that are identical when two nucleic acids or two polypeptides are compared, such as at least about 80% of the nucleotides or amino acids, usually at least about 85%, 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 identical.

[0157] Nucleic acids can be present in whole cells, in cell lysates, or in partially purified or substantially pure form. A nucleic acid is "isolated" or "made substantially pure" when it is purified from other cellular components with which it is normally associated in its natural environment. Nucleic acids can be isolated using techniques well known in the art, such as alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and the like.

[0158] The relevant terms used in this invention are explained as follows:

[0159]

[0160]

[0161] The present invention will be further described below with reference to the examples. These examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The processes, conditions, experimental methods, etc. for carrying out the present invention, except for those specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited in the present invention.

[0162] Example 1 Humanization of mouse anti-aPC monoclonal antibody

[0163] The variable region sequence of the mouse hybridoma antibody was humanized. The VH / VL amino acid sequence was annotated using the Kabat system. Secondly, the VH / VL sequence was analyzed for hot spot sites, including unpaired cysteine residues, N-glycosylation sites, and deamination sites within the CDR. N-glycosylation sites close to the CDR will also be considered high-risk sites. If such sites are identified, they need to be removed. Based on the alignment of the VH / VL sequences, the framework sequence of the best human germline gene is selected, and the J region sequence is selected for splicing based on the best homology. Homology modeling of the mouse VH / VL sequence is performed to form a 3D structure. FR residues within a certain distance and residues on the VH / VL binding surface that may affect antigen binding after modification are considered potential sites for back mutation. Computer analysis is then used to determine the importance of back mutations, and different combinations of single or multiple back mutations are designed.

[0164] The target DNA segment was amplified by PCR to construct a plasmid encoding the humanized antibody sequence. Transient transfection and expression were performed, and the antibody was purified using a protein A affinity chromatography column. The humanized antibodies were analyzed and identified by SEC-HPLC and SDS-PAGE, resulting in 28 humanized anti-aPC monoclonal antibody clones.

[0165] 1.1 ELISA determination of the affinity of anti-aPC monoclonal antibodies to human aPC protein (haPC)

[0166] The affinity of the chimeric antibody and the above 28 humanized antibodies to haPC was determined and compared by ELISA and FACS.

[0167] As shown in Figure 1, humanized antibodies 1-28 (Humab001-1 to 1-28 or HAPC-1 to HAPC28), the 3657 positive control antibody (3657PC mAb), and the chimeric antibody (30120-mAb001×hIgG) all showed strong binding to haPC. Antibodies 13-20 had lower EC50 values, indicating stronger binding to haPC.

[0168] 1.2 ELISA determination of the binding affinity of anti-aPC monoclonal antibodies to human PC protein (hPC)

[0169] Humanized anti-aPC monoclonal antibodies No. 13-20 (Humab001-13 to Humab001-20 or HAPC-13 to HAPC-20) were selected to determine their binding ability to hPC.

[0170] The results are shown in FIG2 , which indicate that humanized anti-aPC monoclonal antibodies No. 13-20 did not bind to hPC at all.

[0171] Among them, Antibody 13 (HAPC-13), Antibody 14 (HAPC-14), and Antibody 16 (HAPC-16) are the three parent antibodies selected for affinity optimization. The framework regions of the heavy chains of these three antibodies are the same, but the framework regions of the light chains are different. HAPC-13 is the original, HAPC-14 has one back mutation based on HAPC-13, and HAPC-16 has five back mutations based on HAPC-13. In the following examples, HAPC-14 antibody was selected for subsequent affinity optimization.

[0172] Example 2 Affinity Optimization of Anti-aPC Monoclonal Antibody HAPC-14

[0173] Based on the aforementioned antibody No. 14 (Humab001-14 or HAPC14 or HAPC-14), antibody affinity optimization design and mutant construction were performed.

[0174]

[0175]

[0176] 2.1 Site-directed mutagenesis

[0177] Using site-directed mutagenesis, each amino acid in the six complementarity-determining regions (CDRs) of the parental HAPC-14 Fab clone was mutated to the other 19 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 used for site-directed mutagenesis reactions.

[0178] 2.2 PCR reaction

[0179] 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.

[0180] The PCR product was purified and then electroporated into E. coli TG1 for colony formation and production of Fab fragments.

[0181] 2.3 Primary Fab mutant library screening

[0182] Initial screening involved single-point ELISA (SPE) analysis, which was performed by capture ELISA.

[0183] The process involves coating each well of a 96-well plate with an anti-Fab antibody in a coating buffer PBS at pH 7.4 at 4°C. The next day, the plate was blocked with casein in PBS at 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. This was followed by incubation with SA horseradish peroxidase (HRP) conjugate at 25°C for 1 hour. HRP activity was detected with tetramethylbenzidine (TMB) substrate, and the reaction was quenched with 2M HCl. The plate was read at 450nM. Clones exhibiting an optical density (OD) signal 3 times greater than that of the parent clone at 450nm were selected and sequenced. The binding improved mutants of HAPC-14 with a unique sequence were confirmed by capture ELISA at pH 7.4. The binding improved mutants with a unique sequence of HAPC-14 were sorted by direct antigen coating ELISA. As shown in Table 1 below.

[0184] Table 1

[0185]

[0186] As shown in Table 1 , nine individual affinity-improving mutations (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) identified in the primary screening of HAPC-14 were further used to design and construct a combinatorial mutant library.

[0187] 2.4 Validation of the primary Fab mutant library by direct KD ranking ELISA

[0188] The KD ranking of the improved binding Fab for each mutant was determined by KD ELISA using direct antigen coating ELISA.

[0189] Each well of a 96-well plate was coated with hPro1.biotin in 1xPBS buffer at pH 7.4 and incubated overnight at 4°C. The next day, the plate was blocked with casein in 1xPBS buffer at pH 7.4 at 25°C for 1 hour. Fab PE diluted at a single concentration for each mutant was added to the plate and incubated at 25°C for 1 hour. This was followed by incubation with goat anti-c-myc HRP conjugated at 25°C for 1 hour. HRP activity was detected using tetramethylbenzidine (TMB) substrate, and the reaction was quenched with 2M HCl. The OD value of each plate was read at 450 nM.

[0190] 2.5 Combinatorial Screening of HAPC-14 Fab Library

[0191] Point mutations in VH and VL that were determined to be beneficial for antigen binding were further combined to obtain additional binding synergy. Briefly, each degenerate primer was phosphorylated and then used with uracil-terminated ssDNA at a 10:1 ratio. The mixture was heated to 85°C for 5 minutes and then cooled to 55°C for more than 1 hour. Afterwards, T4 ligase and T4 DNA polymerase were added, and the mixture was incubated at 37°C for 1.5 hours. 100 ng of the combined library DNA was electroporated into TG1 cells to form colonies and produce Fab fragments.

[0192] Combination mutants were expressed as Fabs and screened using capture ELISA. Clones that exhibited a 3-fold optical density (OD) signal at 450 nm greater than the first-round lead clone were sequenced. Binding improved mutants with unique sequences were further confirmed by capture ELISA and ranked by direct antigen coating ELISA.

[0193] Multiple combinatorial mutants of HAPC-14 were screened, and the top 16 combinatorial mutants with significantly improved affinity were confirmed by KD ELISA (see No. 1-16 in Table 2 ).

[0194] Table 2

[0195]

[0196] After screening and statistics, it was found that mutations at the following six sites significantly affected the binding ability of the Fab fragment to the capture antibody. These six sites are:

[0197] a) N57R / W in VL CDR2 (N57R is superior to N57W)

[0198] b) E59G / P in VL CDR2 (E59G is preferred over E59P);

[0199] c) Q93V in VL CDR3;

[0200] d) N31F located in VH CDR1;

[0201] e) S55K located in VH CDR2;

[0202] f) Y104T in VH CDR3;

[0203] The most critical mutation sites are N57R in the light chain and N31F in the heavy chain.

[0204] Example 3 Anti-aPC Monoclonal Antibodies Against HAPC-14 Combined Mutation Sites (Construction of Whole IgG1)

[0205] 3.1 Construction of anti-aPC monoclonal antibodies (HAPC14-1 to HAPC14-10) by combining HAPC14 mutation sites

[0206] Based on the six mutation sites in Example 2, a combinatorial construction was performed to obtain 10 mutants (14-1 to 14-10 in Table 3 below) (HAPC14-1 to HAPC14-10). The sequence was then constructed into a complete IgG form for expression and purification to examine its antibody expression level, affinity, and other biological activities.

[0207] The amino acid sequences of HAPC-14 and its 10 mutants are shown in SEQ ID NOs. 5 to 26.

[0208] Humanized antibody 13 (HAPC-13)

[0209] Heavy chain (SEQ ID NO. 1)

[0210] EVQLVESGGGLVQPGGSLRLSCAASGFTFS NYYLN WVRQAPGKGLEWVG DIRLKSNNYEKHYAESVKG RFTISRDDSKSITYLQMNSLRAEDTAVYYCAR EGDYFDY WGQGTTVTVSS

[0211] Light chain (SEQ ID NO.2)

[0212] DIQMTQSPSSLSASVGDRVTITC RASESVDSFGATFMH WYQQKPGKAPKLLIY LASNLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQNNEDPYT FGQGTKLEIK

[0213] Humanized antibody 16 (HAPC-16)

[0214] Heavy chain (SEQ ID NO.3)

[0215] EVQLVESGGGLVQPGGSLRLSCAASGFTFS NYYLN WVRQAPGKGLEWVG DIRLKSNNYEKHYAESVKG RFTISRDDSKSITYLQMNSLRAEDTAVYYCAR EGDYFDY WGQGTTVTVSS

[0216] Light chain (SEQ ID NO.4)

[0217] N I VL TQSPSSLSASVGDRVTITC RASESVDSFGATFMH WYQQKPGK P PKLLIY LASNLES GVPSRFSGSGS R TDFTLTISSLQPEDFATYYC QQNNEDPYT FGQGTKLEIK

[0218] Humanized antibody 14 (HAPC-14)

[0219] Heavy chain (SEQ ID NO.5)

[0220] EVQLVESGGGLVQPGGSLRLSCAASGFTFS NYYLN WVRQAPGKGLEWVG DIRLKSNNYEKHYAESVKG RFTISRDDSKSITYLQMNSLRAEDTAVYYCAR EGDYFDY WGQGTTVTVSS

[0221] Light chain (SEQ ID NO.6)

[0222] DIQ L TQSPSSLSASVGDRVTITC RASESVDSFGATFMH WYQQKPGKAPKLLIY LASNLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC<� QQNNEDPYT FGQGTKLEIK

[0223] HAPC-14-1 [[ID=2^]]

[0224] Heavy chain (SEQ ID NO.7)

[0225] EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYYLNWVRQAPGKGLEWVGDIRLKSNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDYFDYWGQGTTVTVSS

[0226] Light chain (SEQ ID NO.8)

[0227] DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASWLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIK

[0228] HAPC-14-2

[0229] Heavy chain (SEQ ID NO.9)

[0230] EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYYLNWVRQAPGKGLEWVGDIRLKKNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDYFDYWGQGTTVTVSS

[0231] Light chain (SEQ ID NO.10)

[0232] DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASWLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQNNEDPYTFGQGTKLEIK

[0233] HAPC-14-3

[0234] Heavy chain (SEQ ID NO.11)

[0235] EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYYLNWVRQAPGKGLEWVGDIRLKKNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDYFDYWGQGTTVTVSS

[0236] Light chain (SEQ ID NO.12)

[0237] DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASRLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQNNEDPYTFGQGTKLEIK

[0238] HAPC-14-4

[0239] Heavy chain (SEQ ID NO.13)

[0240] EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYYLNWVRQAPGKGLEWVGDIRLKSNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDYFDYWGQGTTVTVSS

[0241] Light chain (SEQ ID NO.14)

[0242] DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASRLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQNNEDPYTFGQGTKLEIK

[0243] HAPC-14-5

[0244] Heavy chain (SEQ ID NO.15)

[0245] EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYYLNWVRQAPGKGLEWVGDIRLKKNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDYFDYWGQGTTVTVSS

[0246] Light chain (SEQ ID NO.16)

[0247] DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASWLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQNNEDPYTFGQGTKLEIK

[0248] HAPC-14-6

[0249] Heavy chain (SEQ ID NO.17)

[0250] EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYYLNWVRQAPGKGLEWVGDIRLKKNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDYFDYWGQGTTVTVSS

[0251] Light chain (SEQ ID NO.18)

[0252] DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASRLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIK

[0253] HAPC-14-7

[0254] Heavy chain (SEQ ID NO.19)

[0255] EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYYLNWVRQAPGKGLEWVGDIRLKSNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDYFDYWGQGTTVTVSS

[0256] Light chain (SEQ ID NO.20)

[0257] DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASRLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIK

[0258] HAPC-14-8

[0259] Heavy chain (SEQ ID NO.21)

[0260] EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYYLNWVRQAPGKGLEWVGDIRLKKNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDTFDYWGQGTTVTVSS

[0261] Light chain (SEQ ID NO.22)

[0262] DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASRLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQNNEDPYTFGQGTKLEIK

[0263] HAPC-14-9

[0264] Heavy chain (SEQ ID NO.23)

[0265] EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYYLNWVRQAPGKGLEWVGDIRLKKNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDTFDYWGQGTTVTVSS

[0266] Light chain (SEQ ID NO.24)

[0267] DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASRLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIK

[0268] HAPC-14-10

[0269] Heavy chain (SEQ ID NO.25)

[0270] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYLNWVRQAPGKGLEWVGDIRLKKNNYEKHYAESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCAREGDTFDYWGQGTTVTVSS

[0271] Light chain (SEQ ID NO.26)

[0272] DIQLTQSPSSLSASVGDRVTITCRASESVDSFGATFMHWYQQKPGKAPKLLIYLASRLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQNNEDPYTFGQGTKLEIK

[0273] HAPC-14

[0274] Heavy chain CDR1 (SEQ ID NO.27)

[0275] NYYLN

[0276] HAPC-14

[0277] Heavy chain CDR2 (SEQ ID NO.28)

[0278] DIRLKSNNYEKHYAESVKG

[0279] HAPC-14

[0280] Heavy chain CDR3 (SEQ ID NO.29)

[0281] EGDYFDY

[0282] HAPC-14

[0283] Light chain CDR1 (SEQ ID NO.30)

[0284] RASESVDSFGATFMH

[0285] HAPC-14

[0286] Light chain CDR2 (SEQ ID NO.31)

[0287] LASNLES<​​​​​​​QQNNEDPYT

[0291] HAPC-14-1

[0292] Heavy chain CDR1 (SEQ ID NO. 33)

[0293] FYYLN

[0294] HAPC-14-1

[0295] Heavy chain CDR2 (SEQ ID NO. 34)

[0296] DIRLKSNNYEKHYAESVKG

[0297] HAPC-14-1

[0298] Heavy chain CDR3 (SEQ ID NO. 35)

[0299] EGDYFDY

[0300] HAPC-14-1

[0301] Light chain CDR1 (SEQ ID NO. 36)

[0302] RASESVDSFGATFMH

[0303] HAPC-14-1

[0304] Light chain CDR2 (SEQ ID NO. 37)

[0305] LASWLGS

[0306] HAPC-14-1

[0307] Light chain CDR3 (SEQ ID NO. 38)

[0308] QQNNEDPYT

[0309] HAPC-14-2

[0310] Heavy chain CDR1 (SEQ ID NO. 39)

[0311] FYYLN

[0312] HAPC-14-2 (SEQ ID NO. 40)

[0313] Heavy chain CDR2

[0314] DIRLKKNNYEKHYAESVKG

[0315] HAPC-14-2

[0316] Heavy chain CDR3 (SEQ ID NO. 41)

[0317] EGDYFDY

[0318] HAPC-14-2

[0319] Light chain CDR1 (SEQ ID NO. 42)

[0320] RASESVDSFGATFMH

[0321] HAPC-14-2

[0322] Light chain CDR2 (SEQ ID NO. 43)

[0323] LASWLES

[0324] HAPC-14-2

[0325] Light chain CDR3 (SEQ ID NO. 44)

[0326] VQNNEDPYT

[0327] HAPC-14-3

[0328] Heavy chain CDR1 (SEQ ID NO. 45)

[0329] FYYLN

[0330] HAPC-14-3

[0331] Heavy chain CDR2 (SEQ ID NO. 46)

[0332] DIRLKKNNYEKHYAESVKG

[0333] HAPC-14-3

[0334] Heavy chain CDR3 (SEQ ID NO. 47)

[0335] EGDYFDY

[0336] HAPC-14-3

[0337] Light chain CDR1 (SEQ ID NO. 48)

[0338] RASESVDSFGATFMH

[0339] HAPC-14-3

[0340] Light chain CDR2 (SEQ ID NO. 49)

[0341] LASRLGS

[0342] HAPC-14-3

[0343] Light chain CDR3 (SEQ ID NO. 50)

[0344] VQNNEDPYT

[0345] HAPC-14-4

[0346] Heavy chain CDR1 (SEQ ID NO. 51)

[0347] FYYLN

[0348] HAPC-14-4

[0349] Heavy chain CDR2 (SEQ ID NO. 52)

[0350] DIRLKSNNYEKHYAESVKG

[0351] HAPC-14-4

[0352] Heavy chain CDR3 (SEQ ID NO. 53)

[0353] EGDYFDY

[0354] HAPC-14-4

[0355] Light chain CDR1 (SEQ ID NO. 54)

[0356] RASESVDSFGATFMH

[0357] HAPC-14-4

[0358] Light chain CDR2 (SEQ ID NO. 55)

[0359] LASRLGS

[0360] HAPC-14-4

[0361] Light chain CDR3 (SEQ ID NO. 56)

[0362] VQNNEDPYT

[0363] HAPC-14-5

[0364] Heavy chain CDR1 (SEQ ID NO. 57)

[0365] FYYLN

[0366] HAPC-14-5

[0367] Heavy chain CDR2 (SEQ ID NO. 58)

[0368] DIRLKKNNYEKHYAESVKG

[0369] HAPC-14-5

[0370] Heavy chain CDR3 (SEQ ID NO. 59)

[0371] EGDYFDY

[0372] HAPC-14-5

[0373] Light chain CDR1 (SEQ ID NO. 60)

[0374] RASESVDSFGATFMH

[0375] HAPC-14-5

[0376] Light chain CDR2 (SEQ ID NO. 61)

[0377] LASWLGS

[0378] HAPC-14-5

[0379] Light chain CDR3 (SEQ ID NO. 62)

[0380] VQNNEDPYT

[0381] HAPC-14-6

[0382] Heavy chain CDR1 (SEQ ID NO. 63)

[0383] FYYLN

[0384] HAPC-14-6

[0385] Heavy chain CDR2 (SEQ ID NO. 64)

[0386] DIRLKKNNYEKHYAESVKG

[0387] HAPC-14-6

[0388] Heavy chain CDR3 (SEQ ID NO. 65)

[0389] EGDYFDY

[0390] HAPC-14-6

[0391] Light chain CDR1 (SEQ ID NO. 66)

[0392] RASESVDSFGATFMH

[0393] HAPC-14-6

[0394] Light chain CDR2 (SEQ ID NO. 67)

[0395] LASRLGS

[0396] HAPC-14-6

[0397] Light chain CDR3 (SEQ ID NO. 68)

[0398] QQNNEDPYT

[0399] HAPC-14-7

[0400] Heavy chain CDR1 (SEQ ID NO. 69)

[0401] FYYLN

[0402] HAPC-14-7

[0403] Heavy chain CDR2 (SEQ ID NO. 70)

[0404] DIRLKSNNYEKHYAESVKG

[0405] HAPC-14-7

[0406] Heavy chain CDR3 (SEQ ID NO. 71)

[0407] EGDYFDY

[0408] HAPC-14-7

[0409] Light chain CDR1 (SEQ ID NO. 72)

[0410] RASESVDSFGATFMH

[0411] HAPC-14-7

[0412] Light chain CDR2 (SEQ ID NO. 73)

[0413] LASRLES

[0414] HAPC-14-7

[0415] Light chain CDR3 (SEQ ID NO. 74)

[0416] QQNNEDPYT

[0417] HAPC-14-8

[0418] Heavy chain CDR1 (SEQ ID NO. 75)

[0419] FYYLN

[0420] HAPC-14-8

[0421] Heavy chain CDR2 (SEQ ID NO. 76)

[0422] DIRLKKNNYEKHYAESVKG

[0423] HAPC-14-8

[0424] Heavy chain CDR3 (SEQ ID NO. 77)

[0425] EGDTFDY

[0426] HAPC-14-8

[0427] Light chain CDR1 (SEQ ID NO. 78)

[0428] RASESVDSFGATFMH

[0429] HAPC-14-8

[0430] Light chain CDR2 (SEQ ID NO. 79)

[0431] LASRLGS

[0432] HAPC-14-8

[0433] Light chain CDR3 (SEQ ID NO. 80)

[0434] VQNNEDPYT

[0435] HAPC-14-9

[0436] Heavy chain CDR1 (SEQ ID NO. 81)

[0437] FYYLN

[0438] HAPC-14-9

[0439] Heavy chain CDR2 (SEQ ID NO. 82)

[0440] DIRLKKNNYEKHYAESVKG

[0441] HAPC-14-9

[0442] Heavy chain CDR3 (SEQ ID NO. 83)

[0443] EGDTFDY

[0444] HAPC-14-9

[0445] Light chain CDR1 (SEQ ID NO. 84)

[0446] RASESVDSFGATFMH

[0447] HAPC-14-9

[0448] Light chain CDR2 (SEQ ID NO. 85)

[0449] LASRLGS

[0450] HAPC-14-9

[0451] Light chain CDR3 (SEQ ID NO. 86)

[0452] QQNNEDPYT

[0453] HAPC-14-10

[0454] Heavy chain CDR1 (SEQ ID NO. 87)

[0455] NYYLN

[0456] HAPC-14-10

[0457] Heavy chain CDR2 (SEQ ID NO. 88)

[0458] DIRLKKNNYEKHYAESVKG

[0459] HAPC-14-10

[0460] Heavy chain CDR3 (SEQ ID NO. 89)

[0461] EGDTFDY

[0462] HAPC-14-10

[0463] Light chain CDR1 (SEQ ID NO.90)

[0464] RASESVDSFGATFMH

[0465] HAPC-14-10

[0466] Light chain CDR2 (SEQ ID NO.91)

[0467] LASRLGS

[0468] HAPC-14-10

[0469] Light chain CDR3 (SEQ ID NO.92)

[0470] VQNNEDPYT

[0471] See Table 3 for detailed information.

[0472] Table 3

[0473] Backbone hIgG1

[0474]

[0475]

[0476] Among them, the underlined regions are CDRs regions, and the others are framework regions.

[0477] Humanized antibody 14 (anti-aPC monoclonal antibody 14)

[0478] Heavy chain

[0479]

[0480] light chain

[0481]

[0482] Among them, the underlined area is the CDRs region, the others are framework regions, and the white fonts indicate the humanized sites.

[0483] Protein number mutation sites HPAC14-1 L57W,L59G,H31F HPAC 14-2 L57W,L93V,H31F,H55K HPAC 14-3 L57R,L59G,L93V,H31F,H55K HPAC 14-4 L57R,L59G,L93V,H31F HPAC 14-5 L57W,L59G,L93V,H31F,H55K HPAC 14-6 L57R,L59G,H31F,H55K HPAC 14-7 L57R,H31F HPAC 14-8 L57R,L59G,L93V,H31F,H55K,H104T HPAC 14-9 L57R,L59G,H31F,H55K,H104T HPAC 14-10 L57R,L59G,L93V,H55K,H104T HAPC14

[0484] 3.2 Binding Ability Determination of Anti-aPC Monoclonal Antibodies Constructed with Combined Mutation Sites in HAPC14 to HaPC / hPC (HAPC14-1 to HAPC14-10)

[0485] Determination method:

[0486] The binding affinity of anti-aPC monoclonal antibody to haPC / hPC was determined using ELISA.

[0487] 1. Coat ELISA plates with haPC / hPC (1 μg / mL in PBS), 100 μL / well, at 4°C overnight.

[0488] 2. Wash the plate three times with PBST (0.1% Tween 20), 200 μL / well.

[0489] 3. Block the plate with 2% BSA (PBS solution), 200 μL / well, at room temperature for 1 hour.

[0490] 4. Wash the plate three times with 200 μL / well of PBST (0.1% Tween 20).

[0491] 5. Incubate the plate with 100 μL / well of anti-aPC monoclonal antibody as the primary antibody (diluted from 10 μg / mL in 2% BSA) for 1 hour at room temperature.

[0492] 6. Wash the plate three times with PBST (0.1% Tween 20), 200 μL / well.

[0493] 7. Incubate the plate with anti-hIgG-Fc antibody-HRP (diluted 5000-fold in 2% BSA buffer) at room temperature for 1 hour.

[0494] 8. Wash the plate 6 times with PBST (0.1% Tween 20), 200 μL / well.

[0495] 9. Incubate the plate with TMB substrate (prepared with A and B solutions in a 1:1 (v / v) ratio) at 100 μL / well in the dark for 10 minutes.

[0496] 10. Quench the reaction with HCl (1 M), 100 μL / well, and read the OD450.

[0497] The measurement results are shown in FIG3 , Table 4 and Table 5 , which show that the anti-aPC monoclonal antibody binds to hAPCs but does not bind to hPCs.

[0498] Table 4

[0499]

[0500] Table 5

[0501]

[0502] 3.3 Affinity Determination of Anti-aPC Monoclonal Antibodies Constructed from HAPC-14 Combination Mutation Sites (HAPC-14-1 to HAPC-14-10) Using the Octet Biomolecular Interaction System

[0503] Assay method: Octet analysis was performed at 25°C using a Protein A Octet biosensor in 1x Kinetics buffer. For the kinetic assay, the first baseline step was run for 120 seconds in 1x Kinetics buffer, followed by 240 seconds after loading the antibody on the biosensor, followed by a second baseline step of 120 seconds in 1x Kinetics buffer. The biosensor with captured antibody was then immersed in wells containing antigen (huAPC) at 5 dilutions (3.13, 6.25, 12.5, 25, and 50 nM) for 180 seconds and then dissociated in 1x Kinetics buffer for 400 seconds. 1xKinetics buffer served as a blank control to show the natural dissociation of the antibody in the buffer. 10 mM glycine pH 1.5 was used for the regeneration step. Binding kinetics were calculated using Octet data analysis software, and curve fitting was performed using a 1:1 binding model.

[0504] The affinity of the mutant antibody was measured at pH 7.4. The results are shown in Figure 4 and Table 6:

[0505] Table 6

[0506] Sample KD(M) kon(1 / Ms) kdis(1 / s) Full R^2 HAPC14-1 3.83E-09 3.06E+05 1.17E-03 0.9932 HAPC14-2 3.78E-09 3.07E+05 1.16E-03 0.9877 HAPC14-3 2.67E-09 3.20E+05 8.54E-04 0.9933 HAPC14-4 4.06E-09 3.10E+05 1.26E-03 0.9911 HAPC14-5 2.83E-09 3.22E+05 9.11E-04 0.9924 HAPC14-6 3.64E-09 2.97E+05 1.08E-03 0.9917 HAPC14-7 7.72E-09 2.56E+05 1.97E-03 0.9929 HAPC14-8 1.05E-08 2.50E+05 2.62E-03 0.9901 HAPC14-9 9.69E-09 2.45E+05 2.38E-03 0.9917 HAPC14-10 1.90E-08 2.25E+05 4.28E-03 0.9906 HAPC14 2.54E-07 6.40E+04 1.62E-02 0.9866

[0507] As can be seen from the results in Figure 4 and Table 6, the mutated antibodies can still bind to haPC but not to hPC, and the affinity is improved compared with the unmutated antibody No. 14 HAPC-14, especially No. 6 (HAPC14-6) and No. 3 (HAPC14-3), which are nearly 100-fold improved.

[0508] Example 4 Histone Cleavage by Anti-aPC Monoclonal Antibody HAPC14-6 and aPC

[0509] Determination method:

[0510]

[0511]

[0512] The promotion or inhibition of aPC cleavage of histones by aPC was observed by SDS-PAGE gel electrophoresis followed by Coomassie Brilliant Blue staining.

[0513] The results of the test are as follows Figure 5 As shown ( Figure 5From left to right: histones only; histones and APC; histones, APC, and HAPC1573; histones, APC, and HAPC14-6). As can be seen from the figure, HAPC1573 and HAPC14-6 enhance the ability of aPC to cleave histones, indicating that the HAPC14-6 monoclonal antibody of the present invention can provide cytoprotection against the cytotoxicity of histones H3 and H4.

[0514] Consistent with HAPC1573, HAPC-14-6 did not inhibit but actually enhanced aPC cleavage of histones H3 and H4. Unlike HAPC1573, HAPC-14-6 slightly inhibited aPC cleavage of histone H1, suggesting that HAPC-14-6 may provide cytoprotection against the cytotoxic effects of histones H3 and H4.

[0515] Example 5 In vitro pharmacodynamics study of anti-aPC monoclonal antibodies HAPC-14-3 and HAPC-14-6

[0516] This example measures the effects of different antibodies at different concentrations on coagulation factor-deficient plasma.

[0517] The protc-induced APTT experiment was used to detect the minimum dose of anti-monoclonal antibodies to inhibit the anticoagulant activity of aPC in human plasma deficient in multiple coagulation factors. A concentration of only 1ug / ml was required to inhibit more than 95% of the aPC anticoagulant activity, while HAPC1573 required a concentration of more than 100ug / ml.

[0518] 5.1 Experimental methods and principles

[0519] Activated partial thromboplastin time (APTT) assay principle: Under 37℃, factors XII and XI are activated by kaolin, and platelet factor III is replaced by phospholipids (partial thromboplastin). 2+ The activated partial thromboplastin time (APTT) is the most sensitive and commonly used screening test for the intrinsic coagulation system.

[0520] The newly developed anti-aPC monoclonal antibody HAPC-14-6 can inhibit the anticoagulant activity of aPC at a concentration of up to 1 μg / ml in human plasma with various coagulation factor deficiencies, including factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, and VWF.

[0521] 5.2 Determination of the Effect of Antibodies on Coagulation Factor-Deficient Plasma

[0522] (1) Experimental results of different antibodies at different concentrations on plasma deficient in coagulation factors VIII and VIX

[0523] Experimental method: Antibodies were diluted to the corresponding concentration with OWREN-KOLLER buffer;

[0524] Coagulation factor FVIII-deficient plasma+ Table 7 Antibody dilutions (final concentration) + 0.05U / ml Protac (final concentration) +OWREN-KOLLER Buffer , room temperature 15min;

[0525] 150μl total 150ul

[0526] Control: 150 μl of plasma deficient in coagulation factor FVIII + 150 μl of OWREN-KOLLER buffer;

[0527] Measurement method: STAGO Compact Max APTT-procedure.

[0528] Coagulation factor FIX-deficient plasma+ Table 7 Antibody dilutions (final concentration) + 0.01 U / ml Protac (final concentration) + OWREN-KOLLER buffer , room temperature 15min;

[0529] 150μl total 150ul

[0530] Control: 150 μl of coagulation factor FIX-deficient plasma + 150 μl of OWREN-KOLLER buffer;

[0531] Measurement method: STAGO Compact Max APTT-procedure.

[0532] Assay Results: As shown in Table 7, anti-aPC monoclonal antibodies HAPC14-1 to HAPC14-10 inhibited the anticoagulant activity of aPC to varying degrees in plasma deficient in coagulation factors VIII and VIX. Furthermore, at the same antibody concentration, anti-aPC monoclonal antibodies HAPC14-1 to HAPC14-10 were more effective than mouse antibodies 1573, HAPC13, and HAPC14 in inhibiting the anticoagulant activity of aPC.

[0533] Table 7

[0534]

[0535]

[0536] (2) Experimental results of different antibodies at different concentrations on plasma deficient in coagulation factor FVIII

[0537] Experimental method: Antibodies were diluted to the corresponding concentration with OWREN-KOLLER buffer;

[0538] Coagulation factor FVIII-deficient plasma+ Figure 6A Each dilution antibody (final concentration) + 0.05U / ml Protac (final concentration degrees) + OWREN-KOLLER buffer, room temperature 15min;

[0539] 150μl total 150ul

[0540] Control: 150 μl of plasma deficient in coagulation factor FVIII + 150 μl of OWREN-KOLLER buffer;

[0541] Measurement method: STAGO Compact Max APTT-procedure.

[0542] Experimental results: Figure 6A As shown, anti-aPC monoclonal antibodies completely inhibited the anticoagulant activity of aPC in plasma deficient in coagulation factor FVIII.

[0543] (3) Experimental results of different antibodies and concentrations on plasma deficient in coagulation factor FIX

[0544] Experimental method: Antibodies were diluted to the corresponding concentration with OWREN-KOLLER buffer;

[0545] Coagulation factor FIX-deficient plasma+ Figure 6B Each dilution antibody (final concentration) + 0.01U / ml Protac (final concentration) + OWREN-KOLLER buffer , room temperature 15min;

[0546] 150μl total 150ul

[0547] Control: 150 μl of coagulation factor FIX-deficient plasma + 150 μl of OWREN-KOLLER buffer;

[0548] Measurement method: STAGO Compact Max APTT-procedure.

[0549] Test results: Figure 6B As shown, anti-aPC monoclonal antibodies completely inhibited the anticoagulant activity of aPC in plasma deficient in coagulation factor FIX.

[0550] (4) Experimental results of different antibodies at different concentrations on plasma deficient in coagulation factor FXI

[0551] Experimental method: Antibodies were diluted to the corresponding concentration with OWREN-KOLLER buffer;

[0552] Coagulation factor FXI-deficient plasma+ Figure 6C Each dilution antibody (final concentration) + 0.06U / ml Protac (final concentration) + OWREN-KOLLER buffer , room temperature 15min;

[0553] 150μl total 150ul

[0554] Control: 150 μl of coagulation factor FXI-deficient plasma + 150 μl of OWREN-KOLLER buffer;

[0555] Measurement method: STAGO Compact Max APTT-procedure.

[0556] Test results: Figure 6C As shown, anti-aPC monoclonal antibodies completely inhibited the anticoagulant activity of aPC in plasma deficient in coagulation factor FXI.

[0557] (5) Experimental results of different antibodies and different concentrations on coagulation factor FVII plasma

[0558] Experimental method: Antibodies were diluted to the corresponding concentration with OWREN-KOLLER buffer;

[0559] Coagulation factor FVII-deficient plasma+ Figure 6D Each dilution antibody (final concentration) + 0.1U / ml Protac (final concentration) + OWREN-KOLLER buffer , room temperature 15min;

[0560] 150μl total 150ul

[0561] Control: 150 μl of coagulation factor FVII-deficient plasma + 150 μl of OWREN-KOLLER buffer;

[0562] Measurement method: STAGO Compact Max APTT-procedure.

[0563] Test results: Figure 6D As shown, anti-aPC monoclonal antibodies completely inhibited the anticoagulant activity of aPC in FVII-deficient plasma.

[0564] (6) Experimental results of different antibodies at different concentrations on plasma deficient in coagulation factor FXII

[0565] Experimental method: Antibodies were diluted to the corresponding concentration with OWREN-KOLLER buffer;

[0566] Coagulation factor FXII-deficient plasma+ Figure 6E Each dilution antibody (final concentration) + 0.025U / ml Protac (final concentration degrees) + OWREN-KOLLER buffer , room temperature 15min;

[0567] 150μl total 150ul

[0568] Control: 150 μl of plasma deficient in coagulation factor FXII + 150 μl of OWREN-KOLLER buffer;

[0569] Assay method: STAGO Compact Max APTT-Procedure;

[0570] Test results: Figure 6EAs shown, anti-aPC monoclonal antibodies completely inhibited the anticoagulant activity of aPC in plasma deficient in coagulation factor FXII.

[0571] (7) Experimental results of different antibodies and concentrations on coagulation factor FV plasma

[0572] Experimental method: Antibodies were diluted to the corresponding concentration with OWREN-KOLLER buffer;

[0573] Coagulation factor FV deficient plasma (containing 5% standard plasma) + Figure 6F Each dilution antibody (final concentration) + 0.05U / ml Protac (final concentration) + OWREN-KOLLER buffer , room temperature 15min;

[0574] 150μl total 150ul

[0575] Control: coagulation factor FV-deficient plasma (containing 5% standard plasma) 150 μl + OWREN-KOLLER buffer 150 μl;

[0576] Measurement method: STAGO Compact Max APTT-procedure.

[0577] Test results: Figure 6F As shown, anti-aPC monoclonal antibodies completely inhibited the anticoagulant activity of aPC in plasma deficient in coagulation factor FV.

[0578] (8) Experimental results of different antibodies and concentrations on plasma deficient in coagulation factor FX

[0579] Experimental method: Antibodies were diluted to the corresponding concentration with OWREN-KOLLER buffer;

[0580] Coagulation factor FX deficient plasma (containing 1% standard plasma) + Figure 6G Each dilution antibody (final concentration) + 0.05U / ml Protac (final concentration) + OWREN-KOLLER buffer , room temperature 15min;

[0581] 150μl total 150ul

[0582] Control: coagulation factor FX-deficient plasma (containing 1% standard plasma) 150 μl + OWREN-KOLLER buffer 150 μl;

[0583] Measurement method: STAGO Compact Max APTT-procedure.

[0584] Test results: Figure 6G As shown, anti-aPC monoclonal antibodies completely inhibited the anticoagulant activity of aPC in plasma deficient in coagulation factor FX.

[0585] (9) Experimental results of different antibodies and concentrations on plasma lacking coagulation factor VWF

[0586] Experimental method: Antibodies were diluted to the corresponding concentration with OWREN-KOLLER buffer;

[0587] Coagulation factor VWF-deficient plasma+ Figure 6H Each dilution antibody (final concentration) + 0.01U / ml Protac (final concentration) + OWREN-KOLLER buffer , room temperature 15min;

[0588] 150μl total 150ul

[0589] Control: 150 μl of plasma lacking coagulation factor VWF + 150 μl of OWREN-KOLLER buffer.

[0590] Measurement method: STAGO Compact Max APTT-procedure.

[0591] Test results: Figure 6H As shown, anti-aPC monoclonal antibodies completely inhibited the anticoagulant activity of aPC in plasma deficient in coagulation factor VWF.

[0592] Example 6 In vivo pharmacodynamics experiment

[0593] In a humanized protein C hemophilia mouse model (see paragraphs

[0230] to

[0238] of Chinese patent application CN201911027061.6), after injection of a certain dose of anti-aPC monoclonal antibody, the amount of bleeding after tail amputation of the mouse is examined to check whether the anti-aPC monoclonal antibody achieves the coagulation effect by inhibiting the anticoagulant function of aPC.

[0594] 6.1 Experimental methods

[0595] The drug was administered intravenously at the canthus of the mouse eye. One hour after administration, the mouse was anesthetized by intraperitoneal injection of sodium pentobarbital. After anesthesia, the mouse tail was cut off 2 mm from the end of the tail tip, and the tail wound was placed in 15 ml of 37°C normal saline to collect the blood flowing out of the mouse. Collection was stopped after 20 minutes. The collected fluid was lysed with red blood cells and centrifuged to collect the supernatant. The supernatant was detected using an ultraviolet spectrophotometer at a wavelength of 600 nm. The measured OD value was used to represent the amount of bleeding in the mouse, and the amount of bleeding in the mouse was used to measure the coagulation function of the mouse. WT refers to normal mice, and untreated refers to untreated humanized protein C hemophilia A mice or untreated humanized protein C hemophilia B mice.

[0596] 6.2 Experimental Results

[0597] like Figure 7AAs shown, humanized protein C hemophilia A mice were injected with 1 U / mouse of coagulation factor FVIII, 20 μg / mouse of mouse anti-APC 1573, and 1 μg / mouse of HAPC-14-6. The coagulation function of mice injected with the new anti-APC monoclonal antibody HAPC-14-6 was restored to the level of normal mice, consistent with the effect of mice treated with coagulation factor FVIII.

[0598] like Figure 7B As shown, humanized protein C hemophilia A mice were injected with different doses of HAPC-14-6, and up to 1 μg of HAPC-14-6 could restore the coagulation function of the defective mice to normal levels.

[0599] like Figure 7C As shown, humanized protein C hemophilia B mice were injected with different doses of HAPC-14-6, and up to 1 μg of HAPC-14-6 could restore the coagulation function of the defective mice to normal levels.

[0600] The results show that in the present invention, the APTT test induced by protein C activating enzyme (protac) was used to detect the minimum dose of anti-aPC monoclonal antibody to inhibit the anticoagulant activity of aPC in human plasma deficient in multiple coagulation factors. A concentration of only 1 μg / ml was required for complete inhibition, while mouse anti-HAPC1573 required a concentration of more than 100 μg / ml.

[0601] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the same. Although the present invention has been described in detail with reference to preferred embodiments, persons skilled in the art should understand that the specific implementation methods of the present invention may still be modified or some technical features may be replaced by equivalents. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be conceived by those skilled in the art should be included in the scope of the technical solutions claimed for protection by the present invention. Sequence Listing <110> Shanghai RAAS Blood Products Co., Ltd. <120> Monoclonal antibodies against human activated protein C and their preparation and application <160> 92 <170> SIPOSequenceListing 1.0 <210> 1 <211> 118 <212> PRT <213> Artificial Sequence <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Asp Ile Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Thr Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 2 <211> 111 <212> PRT <213> Artificial Sequence <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Ser Phe 20 25 30 Gly Ala Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 3 <211> 118 <212> PRT <213> Artificial Sequence <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Asp Ile Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Thr Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 4 <211> 111 <212> PRT <213> Artificial Sequence <400> 4 Asn Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Ser Phe [[ID=三十七]]20 25 30 Gly Ala Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 5 <211> 118 <212> PRT <213> Artificial Sequence <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Asp Ile Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr Ala Glu [[ID=�6]]50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Thr Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 6 <211> 111 <212> PRT <213> Artificial Sequence <400> 6 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Ser Phe 20 25 30 Gly Ala Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 7 <211> 118 <212> PRT <213> Artificial Sequence <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr 20 25 30 Tyr Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Asp Ile Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Thr Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 8 <211> 111 <212> PRT <213> Artificial Sequence <400> 8 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Ser Phe 20 25 30 Gly Ala Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Trp Leu Gly Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 9 <211> 118 <212> PRT <213> Artificial Sequence <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr 20 25 30 Tyr Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Asp Ile Arg Leu Lys Lys Asn Asn Tyr Glu Lys His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Thr Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 10 <211> 111 <212> PRT <213> Artificial Sequence <400> 10 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Ser Phe 20 25 30 Gly Ala Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Trp Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Asn Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 11 <211> 118 <212> PRT <213> Artificial Sequence <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr 20 25 30 Tyr Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Asp Ile Arg Leu Lys Lys Asn Asn Tyr Glu Lys His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Thr Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 12 <211> 111 <212> PRT <213> Artificial Sequence <400> 12 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Ser Phe 20 25 30 Gly Ala Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Arg Leu Gly Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Asn Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 13 <211> 118 <212> PRT <213> Artificial Sequence <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr 20 25 30 Tyr Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Asp Ile Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Thr Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 14 <211> 111 <212> PRT <213> Artificial Sequence <400> 14 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Ser Phe 20 25 30 Gly Ala Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Arg Leu Gly Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Asn Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 15 <211> 118 <212> PRT <213> Artificial Sequence <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr 20 25 30 Tyr Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Asp Ile Arg Leu Lys Lys Asn Asn Tyr Glu Lys His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Thr Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser [[ID=z7]]115 <210> 16 <211> 111 <212> PRT <213> Artificial Sequence <400> 16 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Ser Phe 20 25 30 Gly Ala Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Trp Leu Gly Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Asn Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 17 <211> 118 <212> PRT[[ID=2⑤]] <213> Artificial Sequence <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr 20 25 30 Tyr Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Asp Ile Arg Leu Lys Lys Asn Asn Tyr Glu Lys His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Thr Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 18 <211> 111 <212> PRT <213> Artificial Sequence <400> 18 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Ser Phe 20 25 30 Gly Ala Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Arg Leu Gly Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 19 <211> 118< <212> PRT <213> Artificial Sequence <400> 19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr 20 25 30 Tyr Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Asp Ile Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Thr Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 20 <211> 111 <212> PRT <213> Artificial Sequence <400> 20 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Ser Phe 20 25 30 Gly Ala Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Arg Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 21 <211> 118 <212> PRT <213> Artificial Sequence <400> 21 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr 20 25 30 Tyr Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Asp Ile Arg Leu Lys Lys Asn Asn Tyr Glu Lys His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Thr Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Asp Thr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 22 <211> 111 <212> PRT <213> Artificial Sequence <400> 22 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Ser Phe 20 25 30 Gly Ala Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Arg Leu Gly Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Asn Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 23 <211> 118 <212> PRT <213> Artificial Sequence <400> 23 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr 20 25 30 Tyr Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Asp Ile Arg Leu Lys Lys Asn Asn Tyr Glu Lys His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Thr Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Asp Thr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 24 <211> 111 <212> PRT <213> Artificial Sequence <400> 24 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Ser Phe 20 25 30 Gly Ala Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Arg Leu Gly Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 25 <211> 118 <212> PRT <213> Artificial Sequence <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr[[ID=o27]] 20 25 30 Tyr Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Asp Ile Arg Leu Lys Lys Asn Asn Tyr Glu Lys His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Thr Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Asp Thr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 26 <211> 111 <212> PRT <213> Artificial Sequence <400> 26 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Ser Phe 20 25 30 Gly Ala Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Arg Leu Gly Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Asn Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 27 <211> 5 <212> PRT <213> Artificial Sequence <400> 27 Asn Tyr Tyr Leu Asn 1 5 <210> 28 <211> 19 <212> PRT <213> Artificial Sequence <400> 28 Asp Ile Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <400> 29 Glu Gly Asp Tyr Phe Asp Tyr 1 5 <210> 30 <211> 15 <212> PRT <213> Artificial Sequence <400> 30 Arg Ala Ser Glu Ser Val Asp Ser Phe Gly Ala Thr Phe Met His 1 5 10 15 <210> 31 <211> 7 <212> PRT <213> Artificial Sequence <400> 31 Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <400> 32 Gln Gln Asn Asn Glu Asp Pro Tyr Thr 1 5 <210> 33 <211> 5 <212> PRT <213> Artificial Sequence <400> 33 Phe Tyr Tyr Leu Asn 1 5 <210> 34 <211> 19 <212> PRT <213> Artificial Sequence <400> 34 Asp Ile Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly <210> 35 <211> 7 <212> PRT <213> Artificial Sequence <400> 35 Glu Gly Asp Tyr Phe Asp Tyr 1 5 <210> 36 <211> 15 <212> PRT <213> Artificial Sequence <400> 36 Arg Ala Ser Glu Ser Val Asp Ser Phe Gly Ala Thr Phe Met His 1 5 10 15 <210> 37 <211> 7 <212> PRT <213> Artificial Sequence <400> 37 Leu Ala Ser Trp Leu Gly Ser 1 5 <210> 38 <211> 9 <212> PRT <213> Artificial Sequence <400> 38 Gln Gln Asn Asn Glu Asp Pro Tyr Thr 1 5 <210> 39 <211> 5 <212> PRT <213> Artificial Sequence <400> 39 Phe Tyr Tyr Leu Asn 1 5 <210> 40 <211> 19 <212> PRT <213> Artificial Sequence <400> 40 Asp Ile Arg Leu Lys Lys Asn Asn Tyr Glu Lys His Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly <210> 41 <211> 7 <212> PRT <213> Artificial Sequence <400> 41 Glu Gly Asp Tyr Phe Asp Tyr 1 5 <210> 42 <211> 15 <212> PRT <213> Artificial Sequence <400> 42 Arg Ala Ser Glu Ser Val Asp Ser Phe Gly Ala Thr Phe Met His 1 5 10 15 <210> 43 <211> 7 <212> PRT <213> Artificial Sequence <400> 43 Leu Ala Ser Trp Leu Glu Ser 1 5 <210> 44 <211> 9 <212> PRT <213> Artificial Sequence <400> 44 Val Gln Asn Asn Glu Asp Pro Tyr Thr 1 5 <210> 45 <211> 5 <212> PRT <213> Artificial Sequence <400> 45 Phe Tyr Tyr Leu Asn 1 5 <210> 46 <211> 19 <212> PRT <213> Artificial Sequence <400> 46 Asp Ile Arg Leu Lys Lys Asn Asn Tyr Glu Lys His Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly <210> 47 <211> 7 <212> PRT <213> Artificial Sequence <400> 47 Glu Gly Asp Tyr Phe Asp Tyr 1 5 <210> 48 <211> 15 <212> PRT <213> Artificial Sequence <400> 48 Arg Ala Ser Glu Ser Val Asp Ser Phe Gly Ala Thr Phe Met His 1 5 10 15 <210> 49 <211> 7 <212> PRT <213> Artificial Sequence <400> 49 Leu Ala Ser Arg Leu Gly Ser 1 5 <210> 50 <211> 9 <212> PRT <213> Artificial Sequence <400> 50 Val Gln Asn Asn Glu Asp Pro Tyr Thr 1 5 <210> 51 <211> 5 <212> PRT <213> Artificial Sequence <400> 51 Phe Tyr Tyr Leu Asn 1 5 <210> 52 <211> 19 <212> PRT <213> Artificial Sequence <400> 52 Asp Ile Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly <210> 53 <211> 7 <212> PRT <213> Artificial Sequence <400> 53 Glu Gly Asp Tyr Phe Asp Tyr 1 5 <210> 54 <211> 15 <212> PRT <213> Artificial Sequence <400> 54 Arg Ala Ser Glu Ser Val Asp Ser Phe Gly Ala Thr Phe Met His 1 5 10 15 <210> 55 <211> 7 <212> PRT <213> Artificial Sequence <400> 55 Leu Ala Ser Arg Leu Gly Ser 1 5 <210> 56 <211> 9 <212> PRT <213> Artificial Sequence <400> 56 Val Gln Asn Asn Glu Asp Pro Tyr Thr 1 5 <210> 57 <211> 5 <212> PRT <213> Artificial Sequence <400> 57 Phe Tyr Tyr Leu Asn 1 5 <210> 58 <211> 19 <212> PRT <213> Artificial Sequence <400> 58 Asp Ile Arg Leu Lys Lys Asn Asn Tyr Glu Lys His Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly <210> 59 <211> 7 <212> PRT <213> Artificial Sequence <400> 59 Glu Gly Asp Tyr Phe Asp Tyr 1 5 <210> 60 <211> 15 <212> PRT <213> Artificial Sequence <400> 60 Arg Ala Ser Glu Ser Val Asp Ser Phe Gly Ala Thr Phe Met His 1 5 10 15 <210> 61 <211> 7 <212> PRT <213> Artificial Sequence <400> 61 Leu Ala Ser Trp Leu Gly Ser 1 5 <210> 62 <211> 9 <212> PRT <213> Artificial Sequence <400> 62 Val Gln Asn Asn Glu Asp Pro Tyr Thr 1 5 <210> 63 <211> 5 <212> PRT <213> Artificial Sequence <400> 63 Phe Tyr Tyr Leu Asn 1 5 <210> 64 <211> 19 <212> PRT <213> Artificial Sequence <400> 64 Asp Ile Arg Leu Lys Lys Asn Asn Tyr Glu Lys His Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly <210> 65 <211> 7 <212> PRT <213> Artificial Sequence <400> 65 Glu Gly Asp Tyr Phe Asp Tyr 1 5 <210> 66 <211> 15 <212> PRT <213> Artificial Sequence <400> 66 Arg Ala Ser Glu Ser Val Asp Ser Phe Gly Ala Thr Phe Met His 1 5 10 15 <210> 67 <211> 7 <212> PRT <213> Artificial Sequence <400> 67 Leu Ala Ser Arg Leu Gly Ser 1 5 <210> 68 <211> 9 <212> PRT <213> Artificial Sequence <400> 68 Gln Gln Asn Asn Glu Asp Pro Tyr Thr 1 5 <210> 69 <211> 5 <212> PRT <213> Artificial Sequence <400> 69 Phe Tyr Tyr Leu Asn 1 5 <210> 70 <211> 19 <212> PRT <213> Artificial Sequence <400> 70 Asp Ile Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly <210> 71 <211> 7 <212> PRT <213> Artificial Sequence <400> 71 Glu Gly Asp Tyr Phe Asp Tyr 1 5 <210> 72 <211> 15 <212> PRT <213> Artificial Sequence <400> 72 Arg Ala Ser Glu Ser Val Asp Ser Phe Gly Ala Thr Phe Met His 1 5 10 15 <210> 73 <211> 7 <212> PRT <213> Artificial Sequence <400> 73 Leu Ala Ser Arg Leu Glu Ser 1 5 <210> 74 <211> 9 <212> PRT <213> Artificial Sequence <400> 74 Gln Gln Asn Asn Glu Asp Pro Tyr Thr 1 5 <210> 75 <211> 5 <212> PRT <213> Artificial Sequence <400> 75 Phe Tyr Tyr Leu Asn 1 5 <210> 76 <211> 19 <212> PRT <213> Artificial Sequence <400> 76 Asp Ile Arg Leu Lys Lys Asn Asn Tyr Glu Lys His Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly <210> 77 <211> 7 <212> PRT <213> Artificial Sequence <400> 77 Glu Gly Asp Thr Phe Asp Tyr 1 5 <210> 78 <211> 15 <212> PRT <213> Artificial Sequence <400> 78 Arg Ala Ser Glu Ser Val Asp Ser Phe Gly Ala Thr Phe Met His 1 5 10 15 <210> 79 <211> 7 <212> PRT <213> Artificial Sequence <400> 79 Leu Ala Ser Arg Leu Gly Ser 1 5 <210> 80 <211> 9 <212> PRT <213> Artificial Sequence <400> 80 Val Gln Asn Asn Glu Asp Pro Tyr Thr 1 5 <210> 81 <211> 5 <212> PRT <213> Artificial Sequence <400> 81 Phe Tyr Tyr Leu Asn 1 5 <210> 82 <211> 19 <212> PRT <213> Artificial Sequence <400> 82 Asp Ile Arg Leu Lys Lys Asn Asn Tyr Glu Lys His Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly <210> 83 <211> 7 <212> PRT <213> Artificial Sequence <400> 83 Glu Gly Asp Thr Phe Asp Tyr 1 5 <210> 84 <211> 15 <212> PRT <213> Artificial Sequence <400> 84 Arg Ala Ser Glu Ser Val Asp Ser Phe Gly Ala Thr Phe Met His 1 5 10 15 <210> 85 <211> 7 <212> PRT <213> Artificial Sequence <400> 85 Leu Ala Ser Arg Leu Gly Ser 1 5 <210> 86 <211> 9 <212> PRT <213> Artificial Sequence <400> 86 Gln Gln Asn Asn Glu Asp Pro Tyr Thr 1 5 <210> 87 <211> 5 <212> PRT <213> Artificial Sequence <400> 87 Asn Tyr Tyr Leu Asn 1 5 <210> 88 <211> 19 <212> PRT <213> Artificial Sequence <400> 88 Asp Ile Arg Leu Lys Lys Asn Asn Tyr Glu Lys His Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly <210> 89 <211> 7 <212> PRT <213> Artificial Sequence <400> 89 Glu Gly Asp Thr Phe Asp Tyr 1 5 <210> 90 <211> 15 <212> PRT <213> Artificial Sequence <400> 90 Arg Ala Ser Glu Ser Val Asp Ser Phe Gly Ala Thr Phe Met His 1 5 10 15 <210> 91 <211> 15 <212> PRT <213> Artificial Sequence <400> 91 Arg Ala Ser Glu Ser Val Asp Ser Phe Gly Ala Thr Phe Met His 1 5 10 15 <210> 92 <211> 9 <212> PRT <213> Artificial Sequence <400> 92 Val Gln Asn Asn Glu Asp Pro Tyr Thr 1 5

Claims

1. An anti-aPC monoclonal antibody, characterized in that The anti-aPC monoclonal antibody is selected from the following: comprising heavy chain CDR1 to CDR3 represented by SEQ ID NO: 33, 34 and 35; and an antibody having light chain CDR1 to 3 represented by SEQ ID NOs: 36, 37, and 38; or, comprising heavy chain CDR1 to CDR3 represented by SEQ ID NOs: 39, 40, and 41; and, an antibody having light chain CDR1 to 3 represented by SEQ ID NOs: 42, 43, and 44; or, comprising heavy chain CDR1 to CDR3 represented by SEQ ID NOs: 45, 46, and 47; and, an antibody having light chain CDR1 to 3 represented by SEQ ID NOs: 48, 49, and 50; or, comprising heavy chain CDR1 to CDR3 represented by SEQ ID NOs: 51, 52, and 53; and, an antibody having light chain CDR1 to 3 represented by SEQ ID NOs: 54, 55, and 56; or, comprising heavy chain CDR1 to CDR3 represented by SEQ ID NOs: 57, 58, and 59; and, an antibody having light chain CDRs represented by SEQ ID NOs: 60, 61, and 62; or, comprising heavy chain CDR1 to CDR3 represented by SEQ ID NOs: 63, 64, and 65; and, an antibody having light chain CDR1 to 3 represented by SEQ ID NOs: 66, 67, and 68; or, comprising heavy chain CDR1 to CDR3 represented by SEQ ID NOs: 69, 70, and 71; and, an antibody having light chain CDR1 to 3 represented by SEQ ID NOs: 72, 73, and 74; or, comprising heavy chain CDR1 to CDR3 represented by SEQ ID NOs: 75, 76, and 77; and, an antibody having light chain CDR1 to 3 represented by SEQ ID NOs: 78, 79, and 80; or, comprising heavy chain CDR1 to CDR3 represented by SEQ ID NOs: 81, 82, and 83; and, an antibody having light chain CDR1 to 3 represented by SEQ ID NOs: 84, 85, and 86; or, comprising heavy chain CDR1 to CDR3 represented by SEQ ID NOs: 87, 88, and 89; and, Light chain CDR1 to 3 represented by SEQ ID NOs: 90, 91, and 92.

2. The anti-aPC monoclonal antibody according to claim 1, characterized in that Include: The heavy chain shown in SEQ ID NO: 7; and The light chain shown in SEQ ID NO: 8; or, The heavy chain shown in SEQ ID NO: 9; and the light chain shown in SEQ ID NO: 10; or, The heavy chain shown in SEQ ID NO: 11; and the light chain shown in SEQ ID NO: 12; or, The heavy chain shown in SEQ ID NO: 13; and the light chain shown in SEQ ID NO: 14; or, The heavy chain shown in SEQ ID NO: 15; and the light chain shown in SEQ ID NO: 16; or, The heavy chain shown in SEQ ID NO: 17; and the light chain shown in SEQ ID NO: 18; or, The heavy chain shown in SEQ ID NO: 19; and the light chain shown in SEQ ID NO: 20; or, The heavy chain shown in SEQ ID NO: 21; and the light chain shown in SEQ ID NO: 22; or, The heavy chain shown in SEQ ID NO: 23; and the light chain shown in SEQ ID NO: 24; or, The heavy chain is shown in SEQ ID NO: 25; and the light chain is shown in SEQ ID NO:

26.

3. The anti-aPC monoclonal antibody according to claim 1, characterized in that The anti-aPC monoclonal antibody is a complete antibody or an antibody fragment thereof; wherein the antibody fragment is Fab', Fab, F(ab')2, Fv or scFv.

4. The anti-aPC monoclonal antibody-related biomaterial according to any one of claims 1 to 3, characterized in that: Any one of the following 1) to 3): 1) a nucleic acid molecule encoding the anti-aPC monoclonal antibody according to claim 1; 2) a construct comprising the nucleic acid molecule described in 1); 3) An expression system containing the nucleic acid molecule described in 1).

5. A preparation product, characterized in that: The preparation product is a pharmaceutical composition or a detection kit; The preparation product comprises any one or more of the anti-aPC monoclonal antibody according to claim 1 or the biomaterial according to claim 4; When the preparation product is a pharmaceutical composition, it further comprises a pharmaceutically acceptable carrier.

6. Any one or more of the following uses of the anti-aPC monoclonal antibody according to any one of claims 1 to 3, the biomaterial according to claim 4, or the preparation product according to claim 5: i) use in the preparation of a medicament for preventing and / or treating a coagulation deficiency or deficiency disorder, wherein the coagulation deficiency or deficiency disorder is a disorder in which a deficiency of coagulation factors VIII, VIX, FVIII, FIX, FXI, FVII, FXII, FV, FX and / or VWF causes a coagulation disorder and leads to bleeding; ii) use in the preparation of a medicament for treating sepsis; iii) use in the preparation of a medicament for treating traumatic bleeding; iv) Use in immunological examination and analysis of aPC for purposes other than disease diagnosis and treatment, or in the preparation of aPC detection reagents or kits.

Images