TW200846356A - Preparation and use of variants of the kunitz domain 2 of the human placental bikunin gene - Google Patents

Abstract

The present invention relates to variants of the Kunitz domain 2 of the human placental bikunin gene (hBikD2) having good recombinant expression in suitable expression systems, favourable enzyme-inhibitory, immunological and anti-inflammatory properties and a decreased accumulation in the kidney, and to their preparation and use.

Description

200846356 IX. Description of invention: [Technical field to which the invention belongs]

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A variant of Kunitz's work energy site 2 with a good recombinant expression in a suitable expression system, which is good for enzyme inhibition, immune and anti-inflammatory properties, and reduced intrarenal accumulation, and the Moonbone a bikumn gene (hBik D2) , as well as its preparation and use. [Prior Art] The Kumtz functional site is a polypeptide of % to 6 amino acids (4) in length, and the same degree of inhibition inhibits many silk fibroin proteases. It usually contains =疋a white matter and three disulfide bonds that determine its three-dimensional structure. Its main Kumtz functional part of the N_terminal region of about 9 amino acid length of the ring #二, peach Μ * enzymes interact. This loop binds to the heart of the protease and thus prevents the breakage of the corresponding protease receptors just ki and ato '1980, Bode and Huber, 1992). 2〇, 卩 peptide (Aprotinin), also known as bovine trypsin inhibitor (BpTI), is considered to be a prototype of the genus Kunitz functional site (Fritz and Wunderer, 1983). It is a 58-month-old female acid length test protein that can be isolated from various organs of cattle, especially the pancreas, lung, liver and heart. Aprotinin is stabilized by three disulfide bonds 1~=55; Cys 14 ~Cys 38, ·Cys 3〇~CyS 51), specifically as trypsin, plasmin and blood A potent inhibitor of kallikrein. By the χ-photostructure analysis between the aprotinin and the bovine trypsin complex, it can show that the contact region of the aprotinin with the catalytic center of the protease 5 200846356 is a ring composed of 11 to 19 amino acid residues. It is formed (see Β〇 (^ and Huber, 1992 and references cited here). The key to the inhibition of aprotinin is the amino acid residue Lys/K 15, which is particularly intimately contacted with proteases. Reactive serine residue. Thus the amino acid Lys/K 15 is called the Pl residue (Schechter and Berger, 1967). The end of Lys/K 15 is the residue P2, P3, etc.; and the amine of Lys/K 15 The C-terminus of the acid is called hydrazine, P2, etc. It has been shown in the earlier stage that the inhibition of aprotinin can be modified by selective replacement of the amino acid in the range of residue 11 to residue 19 ( 〇tlewski et al., 2001; Apeler et al., 2004; Krowarsch et al., 2005). Amino acids at positions 36 to 39 are more important for aprotinin activity (Fritz and

Wunder, 1983; Krowarsch et al., 2005). Aprotinin is currently used primarily in the heart under the trade name of Trasylol. During surgery, clinical trials have shown that aprotinin treatment significantly reduces the need for the surgery and reduces postoperative bleeding (Royston, 1992). Its clinical role is attributed to inhibition of fibrinolysis and reduction of thrombin formation (Blauhut et al., 1991; Dietrich et al., 1995). Therefore, the inhibition of fibrinase and jk plasmin-releasing enzyme is extremely important for the action of aprotinin. Aprotinin results in the formation of antibodies in humans due to the presence of bovine proteins. 20 Allergic reactions (anaphylactic shock) occur when Trasylol is administered repeatedly. Its risk rate is 2.8%, so the repeated administration of aprotinin is strictly restricted (Dietrich et al., 2001; Beierlein et al., 2005). Therefore, there is a need in the medical field for an active substance which does not produce an allergic reaction and which has a clinical effect similar to or superior to aprotinin. 200846356 Despite extensive research, it has not been possible to confirm any of the human abdomin homologous proteins of bovine aprotinin. However, in the human genome, many existing genes contain one or more Kunitz functional sites (KD) such as amyloid A4 protein; isoforms a and b (APP4 or nexin 2, Ponte et al; Li et al, 5 1998) Hepatocyte growth factor activation inhibitors 1 and 2 (ΗΑΙβ1, 'Shimomura et al., 1997; ΗΑΙ-2, alias SPINT2 or human placenta bikunin, Delaria et al., 1997; Kawaguchi et al., 1997; Marlor _ et al., 1997 Or tissue factor pathway inhibitors 1 and 2 (TFPI-1, Wun et al., 1988; TRPI-2, Sprecher et al., 1994). 10 Human placenta bikunin is a protein encoding 252 amino acids having a molecular weight of 28.2 million Daltons (kD) and two Kunitz functional sites containing similar and structural features to the Kunitz-type serine protein S# sequence ( KD1 · aa38~88 'KD2 · aa 133~183) (Marlor et al., 1997) 〇• The protein has a signal peptide sequence (aa 1~27) at the N-terminus, at the C-terminus (aa 15 198~218) ) followed by a membrane-associated region that may be located in the cytoplasmic sequence unit (aa 219~252). Bikunin is expressed in the placenta, kidney, pancreas, prostate gland, testis, sacral glands, and trachea (Kawaguchi et al., 997). Bikunin has also been described as KOP (a κ-band-containing functional protein overexpressed in pancreatic cancer) (Muller-Pillasch et al., 1998), which is overexpressed in the pancreas as compared to normal pancreatic tissue 20. Mkunin not only strongly inhibits hepatocyte growth factor activator (HGFA) but also inhibits serine protease, which plays the role of fibrin protein > gluten and coagulation such as fibrin, plasma kallikrein and XIa (Delaria et al. 1997). The display of his functional sites i and 2 can be achieved by systemic preparation of peptides and recombinant preparation of bikunin 7 200846356 U; (1997) °Kunitz functional site 2 (hBlkD2) more specifically inhibits pancreatic egg 2 without its aprotinin The enzyme has a very similar inhibition curve (potent > this enzyme, plasma kallikrein and neoinhibitory enzyme. The required inhibition of fibrin is equivalent to a more obvious peptide-releasing enzyme and XIa factor. Site = two tLblkunm refers to the Kunltz functional indication of this protein has allergies already described in τ_,: the cause of recombination of the human platform plate bikunin based on this κ-tz functional site variant / target for injury combined The following properties of #good recombination can be prepared; 15 • Equivalent to the inhibition of fibrin by aprotinin; • The reduction of the heart factor of the sputum of the sputum is lower than that of the aprotinin (four degrees); Intrarenal accumulation compared to aprotinin. [Study of the invention] Placenta bikunin gene The purpose of the present invention is to recombinantly prepare human Kuntz functional part of human 8 20 200846356 (hBik D2) Yang 9 ^ 々 this. Variant of 丨 2 It is the inhibition of seric acid egg The effect, lowering the immunogen intensity compared to aprotinin, = [in the accumulation of the same or the same effect as aprotinin. By ^ D2 and aprotinin from the single _ (four) and kiss and money technology k slave nature The mutation can form an amino acid residue of the genus. The non-natural version of the functional site can form a hBik D2 variant. The second preparation can be recombined in an appropriate amount of 10 15 20 (Example 6, Sequence C, sequence (10)); Violation of the if: protein_inhibition is equivalent to aprotinin (Table); the inhibition of the month too cool release enzyme and ΧΙ & factor has an improved effect compared to aprotinin (Table) 1);, flutter =: 1 = (good; 6) and coagulation (Fig. 7) action phase :: :: aprotinin can shorten the bleeding time (Fig. 8); Antithrombotic effect of peptidase (Fig. 9, 10); ^;: peptidase can reduce the accumulation of kidneys (Table 2); (Table 3);, @cell 1& original decision (four) line towel display is lower The immunogenicity and the antiseptic activity of the ancient inhibitory enzyme did not show cross-reactivity; and the base S t, the inflammatory properties (Figures 11, 12 and 13) and the subsequent mutation t example 2 (preparation of the ship) M variant, "cool The heart protein "large part" f is suitable for recording different human Kunitz functions. A defined mid-section of the Kunitz functional part is combined with its 9 200846356. The different N-terminus and C-terminus of its fUnitZ functional part (Example 7). This can be formed - Different Kumtz functional sites in Dali can be analyzed for their biochemical and physiological properties and further altered by subsequent mutations. '5 The inventive Ku nitz functional site of the invention has 55 to 62 amino acid residues. A homolog of aprotinin, which typically contains six cysteine residues and three disulfide bonds formed between Cys 5 to Cys 55, Cys 14 to Cys 38 and Cys 30 to Cys 51, respectively (according to Calculation of aprotinin). 10 Kunitz inhibitors have been found in various vertebrates and invertebrates (Laskowski and Kato, 1980). These natural Kunitz functional sites - selected in this application as "natural Kunitz functional sites, (Shimomura, et al., 1997; Li et al., 1998; Ponte et al., 1988; Richardson et al. 2001; Wun et al. , 1988; Marlor et al., 1997; Petersen 15 et al., I"4; Vetr and Gebhard, 1990; Chun et al., 1990; • parente et al., 1991; Noiris et al., 1993; Claus et al., 2002;

Trexler et al., 2001, 2002). The Kunitz functional site, which is modified by genetic engineering, for example by substitution and/or deletion of an amino acid residue that does not occur in nature, is selected as the "non-natural Kunitz functional site," (US 6010880, US 5795865, WO 92/06111, US 5777568, US 6482798, P 0 238993, EP 0 307592, EP 0 821007, US 5863893, US 5914315, US 7019123) Peptidase is a fibrin, plasma kinin The 200846356 Strong Inhibitor of Release of Enzymes and XIa Factor's Clinical Role is Due to Inhibition of Fibrinolysis, Inhibition = Source Twins 'Spiral (Contact Activation) and Reduced Thrombin Formation (Blauhut et al' 1991; Dietrich Et al., 1995). The inhibitory enzymes are responsible for the formation of antibodies in humans due to the presence of bovine proteins. ^ Allergic reactions (allergic shock) occur when administered with Trasylol, thus strictly limiting the repeated administration of aprotinin (Dietrich) Etc., 2〇〇丨; such as Shen (10) et al., 2005).

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20 The main inhibitory enzyme repeats have a positive effect on the rodents and dogs when administered at high doses due to the positive charge of the protein (Glasser et al., 1972). Therefore, the goal is to identify a source a white or protein variant having a functional activity comparable to aprotinin. This protein or protein variant must have the same or better protease inhibitory properties as aprotinin, which has potent inhibitory effects on fibrin, plasma kallikrein and XIa and reduces immunogenicity. And reduce the accumulation in the kidneys. l The Kunitz functional part of the placenta bikunin gene (hBik D2) inhibits human different serine proteases: the equivalent of aprotinin II, a white enzyme inhibition 'hBik D2 on plasma kallikrein and nucleus The inhibition of the subunit is even stronger than that of aprotinin. Therefore, the sub- (10) is a protein having the above-mentioned |± shell. To date, it has not been possible to recombinantly prepare an appropriate amount of a brush. One method is to replace hBik D2, N- and C with a corresponding amino acid of aprotinin to form a protein having the above-mentioned desired properties. By two PCR reactions (Example 2 and 笙, π, 曰 (and diagram), human placenta bikunin gene 1 Λ U U KUmtz functional site 2 of 5, and 3, the region is corresponding to the sequence of bovine suppression 11 200846356 peptidase Partially substituted. Therefore, the following mixed protein called sandwich protein (Example 6, the sequence has 58 amino acid lengths and the end of the aprotinase sequence (amine & acid 1) ~15) and C-terminus (amino acid 39~58), including position 15 (''ρι locus = lysine/K linonic acid, which is made up of the catalytically active serine residue of the protease) Contact. The middle region of the sandwich protein (amino acid 16-38) is derived from hBikD2 and contains a specific amino group which specifically contributes to this protease inhibitor. This clip, read white (Example 6, sequence Q) It was sub-selected in the mother secreted protein vector (P1U10.10.W* plUl〇1〇M, Example 4; Figures 4 and 5), and successfully expressed in baker's yeast (Examples 8, 9; Table) 1) In an in vitro transcription and translation system (Example 3), the sandwich egg that was successfully sub-selected in the vector plVEX213d was also expressed. White, a protein of about 8 Torrton. By subsequent selective mutation (Example 5), the lysine residue at position 15 of sequence c is replaced by the arginine residue (K15R)m to form the sequence number. 1 (KD-A_BD2_A-mutl, see Example 0). The sequence number i was selected to be secreted into the yeast and successfully produced in the face (four) mother (four). Similar to the sequence number 1 'N- and C-end of the sequence C The amino acid is replaced by the corresponding amino acid (hBik D2) in sequence B (Example 6, SEQ ID NO: 2 to 76), which can form a natural or non-natural Kunitz functional site via mutation. Other deletion-containing variants It has been described, for example, in SEQ ID NO: 32, 33, 36 and 65 to 75 (delD3). Other sandwich proteins similar to the examples}, 2, 4, 5 and 6 (Example 7) can also be prepared. Expression of the variants produced (Example 8, 200846356 9) and purification (Case 1G) and their potency to inhibit human-derived different serine proteases (Example 12) are illustrated in Sequence C, Sequence Number}, 7, 9, 18 Examples of Tables 1 of 1, ^, 53, 56, and 57. Also listed in the table is the theoretical isoelectric point of a variant that provides the original Kunitz Information on the charge of functional heterozygous (sequence A, B), sandwich protein (sequence C) and various variants (sequence number 丨, 7, 9, i8 35, 53, 56 and 57). Sequence c and All variants were expressed in baker's yeast g (> 1 G microgram/ml). Sequence c, SEQ ID NO: 1 and 7 (iv) mesitidase (about 5 nal trypsin, fibrin and plasma kallikrein, Factor XI is only inhibited by sequence C at a more southern concentration. SEQ ID NO: 9, 18, 35 n 56 and 57 y (4) mesitidase (four) degree (about 5 ns) inhibitory protease 'the plasma kallikrein and melon factor have a more inhibitory effect (plasma excimer release) Enzyme < 丨Nike molecule; Nike molecule). 50 15 20 In vitro determination of serial number % for human bark acid 2 white dissolution and coagulation _, one heart .... The antifibrinolytic action of the sequence 5 Tiger 35 was excellent or the same (Fig. 6). The simultaneous addition of the second A of the sequence number 35 was comparatively increased (Fig. 7). Investigating the blood stasis;;^ 35 A ^ = (different 8 figure). In the large-venous (AV) shunt mode (examples of SEQ ID NO: 35, "Tie-like comparison with Sequence A = #U 35 shows that comparison with Sequence A shows improved anti-thrombotic effects (Figure 9, Figure 1). 13 200846356 To date, it has not been possible to pre-clinically determine the immunogenic potency of therapeutic proteins. One possible way to determine its efficacy is by analyzing the determinants of the computer model (Example 18). The methods in this computer can be listed. A, SEQ ID NO: 35 and SEQ ID NO: 53 and Confirmed SEQ ID NO: 35 and SEQ ID NO: 53 (hBikD2 variant) clearly have a lower number of τ_cell' epitopes than sequence A (Aprotinin/ΒΡΤΙ, see Table 3) The sequence Α(ΒΡΤΙ) and sequence number 35 (hBikD2, mut34) have a similarity of 55.3% amino acid sequence. Check the patient's serum (example n), which has formed an anti-Trasylol treatment The antibody to bovine aprotinin, and the sequence 1 of the sequence 1 showed a negative result, ie the patient serum containing the anti-apoptinase antibody did not show cross-reactivity with SEQ ID NO: 35. The accumulation of the hBik D2 variant in the kidney tissue Word, with sequence a and Column No. 35 treated the rats and then allowed the kidneys to work (Example 19). Compared to Sequence-Column A, there was a significantly lower content of 15 in the kidney homogenate of SEQ ID NO: 35. _ Trauma, reperfusion and Cardiopulmonary bypass is responsible for the complex inflammatory process that will activate the activation of humoral and cellular cascade systems. These will lead to the activation of, inter alia, white blood cells and gray platelets, which will produce clinically observable side effects such as the formation of edema and organs. Injury (Hess, 2005). Various studies have shown the possible effect of aprotinin on the inflammatory state of patients undergoing bypass surgery (Asimakopoulos et al., 2000). Inflammation sites release CAP37 by activation of neutrophils (also It is called heparin-binding protein/HBP or blue bite such as 111*〇(^(^11);?6 generations, such as 1995, used for 'food test') to affect the permeability of blood vessels (Gautam et al., 2001). 14 200846356 CAP37 is similar to elastase, which has protease activity. However, the human Kunitz-type peptone inhibitors, for example, inhibit three different assays (chemical tropism, smooth muscle cells/SMC in sputum, and permeability). Determination of sequence number 35 ( Dayton said the two

(BPTI / aprotinin) including fire production 4 a l & - 吁 | | J A less into the fire resistance. Compare sequence A (BPTI/apopeptidase), sequence numbering in the fingertips, and effect (faces U, u, and 13). Change the fragrance

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20 The clip-protein is a non-ferrite = ltZ type protease inhibitor having a length of about 58 amino acids, and has the general formula 2 (amino end from 仏_(« end). Segments A, B and The amino acid of c may be derived from any desired protease inhibitor of the different species Kumtz(R), such as an invertebrate such as nemona, fruit scale, spirochete; or a vertebrate such as a fish, a bee, an eye = a snake: 雉•, However, in the case of, for example, the mammalian Kimitz35 protease inhibits K soil such as Mus musculus, house mouse, cattle, modern human. If the segment A, b C lines are derived from, for example, modern human varieties, they may be derived from different genes. The functional site of H Kuimz, such as the placenta secretory 匕 functional site 2, the phosphonase protein - is a connexin peptidase π or a tissue factor pathway ρ (tfpi) functional site 丨. The soft egg is prepared by, for example, pcR, and Other amino acid sequences of segments 8, B and C of any of the sandwich proteins can be altered by selective mutagenesis. Novel variants of the functional site 2 of the human placenta bikunin gene are used to treat the lower touch disorders. : Treatment of surgery/blood loss and thrombotic embolism with a high risk of i ( For example, after surgery and accidents, shock, multiple trauma, sepsis, diffuse intravascular blood clot (DIC), 15 200846356 organ failure (MOF), unstable angina, myocardial infarction, stroke, suppository, deep vein thrombosis , inflammatory diseases (such as rheumatism, asthma), invasive tumor growth and metastasis, pain and edema treatment (cerebral edema, spinal edema), prevention of dialysis treatment to start hemostasis, treatment of skin aging symptomatic tissue degeneration, atrophy , wrinkle formation, hemodynamic changes, pigmentation, solar keratosis, blackheads, cystices), wound healing, skin cancer, treatment of skin cancer (solar keratosis, basal cell carcinoma, squamous cells) Cancer, malignant melanoma), multiple sclerosis, fibrosis, cerebral hemorrhage, inflammation of the brain and spinal cord, brain infection, tendinopathy. [Embodiment] Molecular biology techniques Routine colonization operations were carried out as described by Sambrook et al. (Cold Spring Harbor Molecular Colonization, 1989). Plasmid DNA was isolated from E. coli using Qiagentips (Qiagen) ("small and medium preparation"). The host cell used for transformation was Escherichia coli strain DH5a (Stratagene). The DNA fragments were extracted from the agar gel by a Qiagen gel extraction kit according to the manufacturer's instructions (Qiagen). Oligonucleotides for "site-directed mutagenesis" experiments and primers for PCR and sequencing reactions were obtained from the operon (small ship), synthetic gene (suitable for Saccharomyces cerevisiae codon usage) from Geneart体外 In vitro mutations were performed by the Quick-change II XL site-directed mutagenesis kit according to the manufacturer's instructions (Stratagene). For the pCR experiment, 16 200846356 is not used according to the manufacturing instructions from Qiagen (Hot Star Mastermix),

A set of Stratagene (PfuUltra Hotstart DNA polymerase) or Novagen (KOD HiFi, Hot Start and XL DNA polymerase), and a PCR pure 5 kit from Qiagen for the purification of PCR fragments. For in vitro transcription and translation experiments, the rapid translation system (RTS) pi VEX HIS-tag, the second generation _ body group, was used according to the manufacturer's instructions (Roche). i Confirmation of all vectors by circular DNA sequencing using a fluorescent marker termination gene (Big Dye Termination Gene, I", Applied Biosystem) on a sequencer (3100 Αν ant gene analyzer, Applied Biosystem ι〇) Construction and mutation. Example 1 15 Selection of functional part 2 of human placenta bikunin gene (hBikD2) Functional part 2 of natural human placenta bikunin (hBik D2, locus)

Chain NM-021102, 043291) contains 58 amino acids (Tyr/Yl29~Gln/Q

186) and the synthetic gene ordered from Geneart - suitable for Saccharomyces cerevisiae codon usage. Further sequenced hBikD2 5, and 3, end coding sequence 2 被 column was sub-selected into the yeast secretion vector plUlO.lO.W and plU3.12.M (Apeler, 2005) 〇 was selected into the vector pPCR - The script's hBikD2 (Figure 1) is also ordered from Geneart. The DNA sequence for the synthetic gene of hBik D2 has the following sequence: 17 200846356

GGGCGAATTGGGTACCGATTCCCATCTATTTTTACTGCTGTTTTGTTTGCTGCTTCTTCT gctttggcttatgaagagtattgtactgctaatgctgttactggtccatgtagagcttct

TTTCCAAGATGGTATTTTGATGTTGAGAGAAATTCTTGTAACAACTTCATCTATGGTGGT TGTAGAGGTAACAAAAATTCTTATAGATCTGAAGAGGCTTGCATGTTGAGATGTTTTAGA 5 CAATAATAACTCGAGGAGCTCrAnrTTTTPxTTnnn The bottom line is the restriction enzyme recognition sequence (5, Kpnl: GGTACC, BsaBl: GATnnnnATC; 3, Xhol: CTCGAG, Sacl: GAGCTC). Example 2 ίο hBikD2 variant, preparation of "sandwich protein" The N- and C-terminal amino acids (aa) of hBik D2 were substituted with bovine aprotinin (BPTI) by two successive PCR reactions. Corresponding to the amino acid (see the schematic of Figure 2). The sandwich protein formed by this method was then cloned into the vector plVEX2.3d (Fig. 1) and assayed in an in vitro transcription and translation experiment 15 . The first PCR reaction (PCR#1) The nucleotide sequence used for the primers in this PCR was derived from BpTI and the N-terminus of aa 1 to 15 with BPTI and the C-terminus of aa 39 to 58. At its 5, 20-end, primer A (BPTI-kdl) has a flanking sequence containing a restriction cleavage site for Ncol, and the primer is called 赃阳2) correspondingly has a restriction cleavage site for χΜΐ χΜΐ The side sequence. Primers VIII and β have a length of 7 ι and 87 nucleotides, respectively, including flanking sequences, and similarity to 尬 μ μ

It is 50% (introduction) and 62% (introduction). This similarity data does not include primers A 18 200846356 and the flanking sequences within B. Primers A and B have the following sequences: Primer A (BPTI-kdl, 71-mer)

5 55-atagaaggagatataccMgggtAGACCAGATTTCTGCCTGGAACCACCATATAC TGGACCATGTAAGGCT-3, primer B (BPTI-kd2,87-mer)

5'-atgatRagaaccccccccggAGAGCACCACCACAAGTTCTCATACAATCTTCAGC AGACTTGAAATTGTTTCTCTTAGCTCTACAACC-3' Capital letters are specific nucleotides that recognize BPTI, lower case letters are flanking sequences that limit the cleavage site incl (Ncol ·· ccatgg, Xmal : cccggg) ο 50 of the first PCR reaction The total volume of microliters contains about 10 ng of hBikD2 plasmid DNA, 10 pm of primer A, 10 pimol of primers, dNTPs'lxPCR reaction buffer (Qiagen), 4.75 gram of MgCl2, 2.5 Unit of Taq DNA polymerase (Qiagen). The 7% '' condition is a one-person cycle of 15 minutes at 94 ° C, 1 minute at 94 ° C, 1 minute at 42 ° C, 1 minute at 72 ° C, and thereafter Incubate at 72 ° C for 1 minute. 19 200846356 Second PCR reaction (PCR#2) The primers C (23-mer) and D (21-mer) used for this PCR have 100% similarity to the primers A and B in the flanking region. The primers C and D have the following sequences: 5 primer C (23-mer) ·· 5'-atagaaggagatataccatgggt-3' primer D (21-mer): 5 '-atgatgagaaccccccccggg-3 ' | bottom line is restricted cut Site (Ncol : ccatgg, Xmal : cccggg) 〇ίο 1 μ·125-fold dilution of 1 μl of the first PCR reaction (template) in the 50 μl total volume of the second PCR reaction, 1 μpi The primer c, 10 Pimol primer D, 1 milligram of dNTPs, lxPCR reaction buffer (Novagen), 1 milligram of MgCl2, l unit of KODHotStart - DNA polymerase (Novagen). The "loop" condition is at 94. (: 30 minutes for 1 minute, 15 for 1 minute at 94 ° C, 1 minute for 5 〇, 1 minute for 72 _ ,, and 10 minutes for culturing at 72 ° C thereafter. Example 3 The colony protein was cloned in the vector plVEX2.3d. 20 The second PCR reaction was purified using a purification kit (QiaSen), and the restriction enzymes Ncol and Xmal were used for cleavage and ligation into a vector ρ1νΕχ2%, which also utilized Ncol and 11^1. Shearing was performed using a batch-type transformation of Escherichia coli DH5a. From the results of the selection, 20 200846356

It was determined by DNA sequence analysis to determine the sandwich structure that was successfully produced. The coding region of the sandwich protein contains 173 base pairs (bp) encoding a total of 71 amino acids, and has the following sequence··

MGRPDFCLFPPVTnPrvASFPPwvir^^p^^^^jy^^^RAKRNNFKSAEDCjVIRTCGGAFGGGSHHHHHH 5 • The amino acid (aa) at the N-terminus is derived from the carrier structure, and the amino acids 3 to 17 and the amino acid 41 to 60 are derived from BPTI/ Peptidase (bold and sputum line), amine _ acid 18~40 derived from hBikD2 (bold), amino acid 61~71 derived from carrier structure (aa 61~65 partition, followed by 6 groups) Amine acid residue). 10 The sandwich protein was named kd-A-BD2-A (kd=Kunitz functional site 'A= aprotinin, BD2=bikunin functional site 2). 3 inflammatory heart protein kd-A-BD2-A is applied to the in vitro transcription and translation system (RTS, Roche), and can produce a protein of the desired size (about 8 仟 耳 - 顿). _ Example 4 Sandwich protein kd_A_BD2-A in the yeast secretion vector ρΐυΐ〇·ι〇·\γ and ρ1ϋ3·12·ΜSelection modification in the market available Escherichia coli / Saccharomyces cerevisiae "shuttle 20 (shuttle) ''Vector pYES2 (Invitrogen) and the starting material used to construct the yeast secretion vectors plUlO.lO.W and plU3.12M (Apeler, 2005). By PCR and primers with appropriate restriction of the cleavage site (primer e~ H), the sandwich protein kd-A-BD2-A is once again selected from the vector plVEX2.3d into the yeast secretion vectors plUHUO.w and plU3.12.JVi. 200846356 is selected into the yeast secretion vector ρΐυΐ0·10· Λν is defined by limiting the cleavage site BsaBI (GAT tccc ATC in the primer 和) and Xliol (CTCGAG in the primer F), and using the restriction cleavage site Hindlll (AAGCT in the primer G) and BamHI (introduction) GGATCC) is selected into ριυ 5 3·12·Μ. These primers have the following sequences: Primer E (pRJlO-l)

• ^^TGAGATTCCCATCTATTTTCACTGCTGTCTTGTTCGCTGCTTCTTCTGCTTTGGCTA G ACC AG ATTTCTGCCTGG AAC-3 ' 10 Primer F(plU10-2) 5,-ATGCTCGAGCTATTAAGCACCACCACAAGTTCTCAT-3, Primer G(plU312-l) 15 55-GGTAAGCTTGGATAAAAGAAGAGATTTCTGCCTGGAACCACCATATA-3? ® Primer H(plU312-2) 5 '-TAGTGGATCCCGAGCTTGCCTATTAAGCACCACCACAAGTTCTCAT-3, 2〇 Purification of PCR using a purification kit (Qiagen) using restriction enzymes

BsaBI was cut and Xhol was selected into plUlO.lO.W and Hindlll and BamHI were used to select plU3.12.M and ligated into corresponding vectors. The DNA sequence of the clone obtained by batch-transformation of Escherichia coli DH5a and by the sequence of 22 200846356 was determined. The derivatized amino acid sequence of the sandwich expression structure was selected into the yeast secretion vector plUlO.lO.W (the leader of the MFa pairing factor α in the bottom line, the amino acid sequence of the sandwich protein in bold):

MRfPSIFTAYLFAASSALARPDFCLEPPYTGPCKASFPRWYFDVERNSCNNFIYGGCRA

KRNNFKSAEDCMRTCGGGA | The derivatized amino acid sequence of the sandwich expression structure was selected into the yeast secretion vector ρ11Ι3·12·Μ (the bottom line is the leader sequence of MFa pairing factor α, ίο italic is the original sequence of the MFa, bold type The amino acid sequence of the sandwich protein; the kd_A-BD2-A in the yeast secretion vector plU3.12.M is correctly processed, and is deleted after confirming the "1^_8!^, _5〇^ of the 1^11 protease ^ The proline in position 2 of the sandwich protein, p): - Guanchao / G Cong flaps

NNFKSAEDCMRTCGGA Two inflammatory protein expression structures (yeast secretory vectors plulG1〇w and kd-A-BD2-A in plU3.12.M) were successfully expressed in Baker's yeast 20 Example 5 Preparation of sandwich protein kd-A - mutant of BD2_A

Mutations were performed according to the manufacturer (stmtagene) instructions using the help of the n XL 23 200846356 site-directed mutation kit. The sequence of each mutant of the sandwich protein kd-A-BD2-A was determined by DNA sequence analysis. Preparation of Mutant 1 in Sandwich Protein kd-A-BD2-A 5 Insertion of arginine (R) at position 15 (K15R) of lysine (K) into the sandwich protein kd-A_BD2-A, which was selected Into the yeast secretion vector plUlO · 10.W. The mutation primers I and j used for this have the following sequences··

Initiator I i 5'-TATACTGGACCATGTAGAGCTTCTTTTCCAAGA-3,

10 primer J 5'-TCTTGGAAAAGAAGCTCTACATGGTCCAGTATA-3, • The obtained mutant has the following sequence and is named kd-A- - BD2-A-mutl(K15R) · 15 mrfpsiftavlfaasmlarpdfcleppytgpcrasfprwyfdvernscnnfiyg

GCRAKRNNFKSAEDCMRTCGGA The bottom line is the leader sequence of MFA (matching factor α), the bold word is sandwich protein, and the bottom line is the position of amino acid substitution of sandwich protein 20 . kd-A-BD2-A-mutl was successfully expressed in baker's yeast. By the primers plU312-1 and plU312-2 described in Example 3, kdj BD2-A-leg tl was also selected by PCR into the yeast secretion vector plU3.12.M and its sequence was determined by DNA sequence analysis. Its amino acid 24 200846356 sequence is ·

MKF?SIFTAVLFAASSA1AAPVNTTTEDETAQIPAEA VIGYSDLEGDFD VA VLPFSNSTNNGLLF /iVTr/^iSZM^E'GF^ZDiCRRDFCLEPPYTGPC^SFPRWYFDVERNSCNNFIYGGClL^KR NNFKSAEDCMRTCGGA * 5 The bottom line is the leading sequence of MFa (matching factor a), the italicized word is MFa-pre-sequence, italicized For the original sequence of the MFa, the bold word is the sandwich | protein, and the bottom line is the position of the amino acid substitution of the sandwich protein 15

(K15R). In order to correctly treat kd-A-ίο BD2-A_mutl in the yeast secretion vector plU3.12.M (after confirming the "Lys-Arg"-KR at the site of the KexII protease), the amino acid in position 2 of the sandwich protein was deleted. (Proline, P). ^ Example 6 Preparation of hBikD2 variant 15 Similar to Example 4, further mutations (kd-A-BD2_A-mut2 to kd-A_BD2-A-mut76) were introduced into the sandwich protein kd-A-BD2-A and tested for expression. If the mutant sandwich protein is selected into the yeast secretion vector plU3.12.M, the amino acid of position 2 (proline, P) in each case is deleted.

20 Sequence A RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGA BPTI/Aprotinin

Sequence B YEEYCTANAVTGPCRAS FPRWYFDVERNS CNNFIYGGCRGNKNSYRSEEACMLRCFRQ BikD2 25 200846356 5

10 15

Sequence C RPDFCLEPPYTGPCKASFPRWYFDVERNSCNNFIYGGCRAKJCNOTKSAEDCMRTCGGA KD-A-BD2-A SEQ ID NO 1 RPDFCLEPPYTGPCEASFPRWYFDVERNSCNNFIYGGCRAKRNNFKSAEDCMRTCGGA KD-A-BD2-A-mut 1 SEQ ID NO 2 RPDFCLEPPVTGPCKASFPRWYFDVERNSCNNFIYGGCRAKRNNFKSAEDCMRTCGGA KB-A-BD2-A-nmt 2 SEQ ID NO 3 RPDFCLENAVTGPCKASFPRWYFDVERNSCNNFIYGGCRAKRNNFKSAEDCMRTCGGA KD-A-BD2-A- mut 3 SEQ ID NO 4 RPDFCLETAVTGPCKASFPRWYFDVERNSCNNFIYGGCRAKRNNFKSAEDCMRTCGGA KD-A-BD2-A-mut 3a SEQ ID NO 5 RPDFCLEPPYTGPCKASFPRWYFDVERNSCNNFIYGGCRGKRNNFKSAEDCMRTCGGA KD-A-BD2-A-mut 4 SEQ ID NO ό RPDFCLEFPYTGPCKASFPRWYFDVERNSCNNFIYGGCRGNRNNFKSAEDCMRTCGGA KD-A-BD2-A-mut 5 SEQ ID NO 7 RPDFCLEPPYTGPCKASFPRWYFDVERNSCNNFIYGGCRGNKNNFKSAEDCMRTCGGA KD- A-BD2-A-mut 6 SEQ ID NO 8 RPDFCLEPPYTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFKSAEDCMRTCGGA KD-A-BD2-A-mut 7 SEQ ID 9 RPDFCLEPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFKSAEDCMRTCGGA KD-A-BD2-A-mut 8 SEQ ID NO 10 RPDFCLEPPSTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFKSAEDCMRTCGGA KD-A-BD2-A_mut 9 sequence number 11 RPDFCLEPPSTGPCRASFPRWYFDV ERNSCNNFIYGGCRGNKNSFKSAEDCMRTCGGA KD-A-BD2-A-mut 10 26 20 200846356 Serial No. 12 RPDFCLEPPSTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYKSAEDCMRTCGGA KD-A-BD2-A-mut 11 SEQ ID NO 13 RPDFCLEPPSTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSAEDCMRTCGGA KD-A-BD2-A-mut 12 SEQ ID 14 RPDFCLEPPSTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEEDCMRTCGGA KD-A- BD2-A-mut 13 SEQ ID NO: 15 RPDFCLEPPSTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEEACMRTCGGA KD-A-BD2-A-mut 14 10 15

SEQ ID NO 1 ό RPDFCLEPPSTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNYRSEEACMRTCGGA KD-A-BD2-A-mut 15 SEQ ID NO Π RPDFCLEPPSTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSFRSEEACMRTCGGA KD-A-BD2-A-mut 16 SEQ ID NO 18 RPDFCLEPPSTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMRTCGGA KD-A-BD2-A-mut 17 SEQ ID NO 19 RPDFCLEPPSTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCGGA KD-A -BD2-A-mut 18 SEQ ID NO: 20 RPDFCLEPPSTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLRCGGA KD-A-BD2-A-mut 19 SEQ ID NO: 21 RPDFCLEPPSTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLRCFGA KD-A-BD2-A-mut 20 SEQ ID NO: 22 RPDFCLEPPSTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMIJRCFEA KD-A-BD2-A-mut 21 Sequence No. 23 RPDFCLEPPSTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLRCFRO KD-A-BD2-A-mut 22 27 20 200846356 SEQ ID NO 24 RPDFCLEPASTGPCRASFPRAVYFDVERNSCNNFIYGGCRGNKNNFRSEEACMRTCGGA KD-A-BD2-A-mut 23 SEQ ID NO 25 RPDFCLENASTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMRTCGGA KD-A-BD2-A-mut 24 SEQ ID NO 26 RPDFCLMASTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMRTCGGA KD- A-BD2- A-mut 25

10 15

SEQ ID NO 27 RPDFCTANASTGPCRASFPRWWDVERNSQsINFIYGGCRGNKNNFRSEEACMRTCGGA KD-A-BD2-A-mut 26 SEQ ID NO 28 RPDYCTANASTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMRTCGGA KD-A-BD2-A-mut 27 SEQ ID NO 29 RPEYCTANASTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMRTCGGA KD-A-BD2-A-mut 28 SEQ ID 30 YPEYCTANASTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMRTCGGA KD-A- BD2-A.-niut 29 SEQ ID NO: 31 RPDFCLEPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMRTCGGA KD-A-BD2-A-mut 30 SEQ ID NO: 32 AE-FCLEPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMRTCGGA KD-A-BD2-A-mut 31 SEQ ID NO: 33 AE-FCLEPPSTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMRTCGGA KD-A-BD2-A -mut 32 SEQ ID NO: 34 RPDFCLEPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCGGA KD-A-BD2-A-mut 33 SEQ ID NO: 35 AE-FCLEPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCGGA KD-A-BD2-A-mut 34 28 20 200846356 Serial Number 36 M-FCLEPPSTGPCEASFPRWYFDVERNSCNNFr^GGCRGMN^RSEEACMLTCGGA KD-A- BD2-A-iimt 35 _ Serial Number 37 RPDFCLEPMTGPCMSFPRWYFDVERNSCNNFIYGGCRGMNNFRSEEACMI^CFEQ KD-A-BD2-A-mu t 36 SERIES 38 RPDFCLEi^TGPCKASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACML^CFRQ KD-A-BD2-A-mut 37 SERIES 39 RPDFCLMMTGPCRASFPRWYFDVERNSCl^IYGGCRGMOiNFRSEEACMISCFRQ KD-A-BD2-A-mut 38

10 15

SEQ ID NO 40 RPDFCI ^ NMTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMIJRCFRQ KD-A-BD2-A-mut 39 SEQ ID NO 41 RPDYCT ^ NMTGPCEASFPRWYFDVERNSCNNFIYGGCRGMNNFRSEEACMLRCFRQ KD-A-BD2-A-mut 40 SEQ ID NO 42 RPEYCTANASTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLRCFRO KD-A-BD2-A-mut 41 SEQ ID 43 YPEYCTANASTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLRCFRO KD-A-BD2-A-mut 42 SEQ ID NO: 44 YEEYCTANASTGPCRASFPRWYFDVERNSClSiNFIYGGCRGNKNNFRSEEACMLRCFRO KD-A-BD2-A-mut 43 SEQ ID NO: 45 AEDFCTEPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCGGA KD-A-BD2-A-mut 44 SEQ ID NO: 46 AEDFCLAPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCGGA KD-A-BD2-A- Mut 45 SEQ ID NO: 47 AEDFCTAPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCGGA KD-A-BD2-A-mut 46 29 20 200846356 SEQ ID NO: 48 AEEFCLEPPVTGPCRAS FPRWYFDVERNS CNNFIYGGCRGNKNNFRSEEACMLTCGGA KD-A-BD2-A-inut 47 One Serial Number 49 ^JEDYCLE PPVTGPCRAS FPRWYFDVERNS CNNFIYGGCRGNKNNFRSEEACMLTCGGA KD-A-BD2- A-mut 48 SEQ ID NO: 50 AEEYCLEPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGMCNNFRSEEACMLTCGGA K D=A-BD2-A-mut 49 SEQ ID NO: 51 AEMCTAPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCGGA KD-A-BD2-A-mut 50 10 15

Serial No. 52 AEDFCLEPPVTGPCRASFPRWYFD \ nERNSCNNFIYGGCRGNKNNFRSEEACMLTCFRO KD-A-BD2-A-mut 51 SEQ ID NO 53 AEEYCTAPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCFRO KD-A-BD2-A-mut 52 SEQ ID NO 54 AEDFCLEPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMRTCGGA KD-A-BD2-A-mut 53 SEQ ID NO 55 AEDFCLEPFSTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNPRSEEACMRTCGGA KD- A-BD2-A-mut 54 SEQ ID NO: 56 AEDFCLEPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCGGA KD-A-BD2-A-mut 55 SEQ ID NO: 57 AEDFCLEPPSTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCGGA KD-A-BD2-A-mut 56 SEQ ID NO: 58 AEDFCLEPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCFAO KD-A-BD2-A-mut 57 SEQ ID NO 59 AEDFCLEPPVTGFCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCFGO KD-A-BD2-A-mut 58 30 20 200846356 serial No. 60 AEEYCTAPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCFAO KD-A-BD2-A-mut 59 SEQ ID NO 61 AEEYCTAPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCTGO KB-A-BD2-A-nmt 60 SEQ ID NO 62 MDFCLEPPVTGPCgASFPRWYFDVERNSaWFIYGGCRGNGNNFRSEEACMLTCGGA KD -A-BD2-A-mu t 61

10 15

SEQ ID NO 63 AEDFCLEPPSTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCFAO KD-A-BD2-A-mut 62 SEQ ID NO 04 AEDFCLEPPSTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCFGO KD-A-BD2-A-mut 63 SEQ ID NO 65 AE-FCLEPPVTGPCRASFKRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCGGA KD-A-BD2-A-mut 64 SEQ ID NO 66 AE-FCLEPPVTGPCEASFKRWYFDVERNSCNNFIYGGCRGNKNNFRSLEACMLTCGGA KD-A-BD2-A-mut 65 SEQ ID NO: 67 AE-FCLEPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSLEACMLTCGGA KD-A-BD2-A-mut 66 SEQ ID NO: 68 AD-FCLEPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCGGA KD-A-BD2-A-mut 67 SEQ ID NO: 69 AS -FCLEPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCGGA KD- A-BD2-A-mut 68 SEQ ID NO: 70 AE-FCLEPPVTGFCRASFPRWYFDVERNSCNNFIYGGCSGNKNNFRSEEACMLTCGGA KD-A-BD2-A-mut 69 SEQ ID NO: 71 AE-FCLEPPVTGPCRASFPRWYFDVERNSCNNFIYGGCKGNKNNFRSEEACMLTCGGA KD-A-BD2-A-mut 70 31 20 200846356 Serial Number 72 Μ -Ρ0ΧΕΡΡγΤΟΡεΕΑ3Π) Ι^ΥΡ〇νΕΚΝ8ΟΝΝΠΥΟ〇σΐ2ϊίΚΝΝΡΕ3υΕ4€Μυ'(:(3ΟΑ KD-A-BD2-A-mut 71 SEQ ID NO: 73 AE-FCLEPPVTGPCRASF PRWYFDVERNSCNNFIYGGCRGNKNNFESEEACMLTCGGA KD-A-BD2-A-mut 72 5

10 SEQ ID NO: 74 ^FCLEPPVTGPCRASFPRYYFDVKRGSCNOTIYGGCRGNKNNFRSEEACMLTCGGA KD-A-BD2-A-mut 73 SEQ ID NO: 75 AE-FCLEPPVTGPCKASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCGGA KD-A-BD2-A-mut 74 SEQ ID NO: 76 AEEYCTAPPVTGPCKASIFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCFRO KD-A-BD2-A-mut 75 Example 7 Preparation of Others Inflammatory protein " In principle, the technique described in Example 2 is suitable for the preparation of a large number of different 15 sandwich molecules, namely natural and non-natural variants of the Kunitz function • sites from different sources (human and other species). Here, a defined midsection of the Kunitz functional site is combined with the different N- and C-termini of other Kunitz functional sites. This produces a large number of different Kunitz functional sites whose inhibitory properties can be analyzed and further altered by subsequent mutations.

20 The following sandwich proteins are examples: hTFPll-Dl-hBikD2-BPTI 1 10 20 30 40 50 58 MHSFCAFKADDGPCKASFPRWYFDVHRNSCNNFIYGGCRAKRNNFKSAEDCMRTCGGA hTFPIl-Dl-

BikD2-BPTI 32 200846356 aa 1~15 is a Kumtz functional part derived from the human tissue factor pathway inhibitor 丨 functional site j, and aa 16~38 is derived from the Kunitz functional part of the human placenta bikunin functional site 2 (bottom line), (10) 39 ~58 is a Kunitz functional site derived from bovine inhibitory enzyme (BPTI). hTFPIl-Dl^hBikD2-hTFPIl-Dl

1〇 20 30 40 50 mhsfcafkaqing PCK New Zealand yFD Swallow Shun Sister Qingcai (10) is similar to Hongli·BikD2-BPTI 58 hTFPIl-Dl- 10 15

Aa 1~15 is derived from the Kunitz functional part of the human tissue factor pathway inhibitor 丨 functional site i, and aa 16~38 is derived from the Kunitz functional part of the human placenta bikunin functional site 2 (bottom line), aa 39~58 source The Kunitz functional site of the functional site of the human tissue factor pathway inhibitor 1 。.

hTFPI2-Dl-BikD2-BPTI 1 10 20 30 40 50 57

CLLpLDYGPCR 趟 is DVERN-sc_FIXagcRAKRNNFKSA TCCGGA hTFPI2-Dl-

BikD2-BPTI aa 1~14 is derived from the Kunitz functional part of the functional site i of human tissue factor pathway inhibitor 2, and aa 15~37 is derived from the κ dishitz functional part of human placental Wkunin functional site 2 (bottom line), (10) 38 ~57 is a Kunitz functional site derived from bovine aprotinin (BPTI). 33 200846356 hTFPI2-Dl-BikD2*hTFPI2-Dl 1 10 20 30 40 50 57 AEICLLPLDYGPCRASFPRWYFDVERNSCNNFIYGGCEGNANNFYTWEACDDACWRI hTFPI2-Dl.

BikD2-hTFPI2-Dl 5 (10) 1~14 is derived from human tissue factor pathway inhibitor 2 functional site 1

Kunitz power month b part 'aa 15~37 series derived from human placenta】Kunitz functional part of functional part 2 (bottom line), aa 38~57 series derived from Kunitz functional part of human lure tissue factor pathway inhibitor 2 functional part i . 1〇Example 8 Transformation of Saccharomyces cerevisiae • Growth of yeast cells such as JC34.4D (MATa, ura3~52, suc2) • 1 liter of YEPD (2% glucose, 2% protein gland, 1 〇/〇 Difco) Within the yeast extract), then collect 〇D6(8) from 0.6 to 〇·8. Rinse the cells with 5 ml of Solution A (! mg molecular sorbitol; milligrams of hydrazine, bismuth-bis(2-hydroxyethyl) • glycoline (8.3.3%; 3% ethylene glycol). Resuspend in 2 ml of solution A and store at _7 ° °c. Plasmid DNA (5 μg) and vector DNA (50 μg DNA taken from sputum sperm) were added to lyophilized cells. The cells were then thawed by shaking at 37 ° C for 5 20 minutes. After adding L5 soaring solution B (40% PEG 1000; 200 bic bic bicin pH 8.35), the cells were cultured at 3 〇t: 6 〇ϋ, and after centrifugation, 1.5 liters of solution c (〇15 mg molecule) NaC1; 1 〇 molecular molecule bidne pH 8.35) was rinsed and then suspended in 1 〇〇 microliter of solution 〇. Isolation was performed on selection medium containing 2% agar. At 3 〇. 〇Cultivate 200846356 After 3 days, the transformed strain was obtained. Example 9 Fermentation nutrient solution of yeast cells Fermentation of yeast cells expressing aprotinin or KTPI variants with modified amino acid sequences, using the following nutrient solution: ίο SL4 solution: nutrient solution SD2 SC5 Bacto yeast Nitrogen source 6.7 g / liter - Difco Bacto yeast extract - 20.0 g / liter of glucose 20.0 g / liter 20.0 g / liter KH2P 〇 4 6.7 g / liter 6.7 g / liter (NH4) 2S 〇 4 - 2 · 0 g / liter MgS04x7 Π20 - 1.0 g / liter of trace element solution 4 - 1 · 0 ml / liter of NaOH after titration pH 6 6

Titriplex III (Merck 8418) 5 g FeS04 · 7H20 (Merck 3965) 2 g ZnS04 · 7H20 (Merck 8883) 〇·1 g MnCl2 · 4H20 (Merck 5927) 30 mg 35 200846356 h3b〇3 (Merck 165) 0.3 g C0CI2 · 6H2O (Merck 2533) 〇·2g CuC12 · 2H20 (Merck 2733) 10 mg NiCl2 · 6H20 (Merck 6717) 20 mg Na2Mo04 · 2H20 (Merck 6521) 30 mg (dissolved in 1000 ml of distilled water, adjusted to pH with NaOH 3 to 4. Place in up to 12 containers in batches and store at _18 ° C; use defrosted individual containers for up to one month). This component was prepared in deionized water and the pH was adjusted to 5·5. Sterilize at 121 C for 20 minutes. Glucose was dissolved in 1/5 of the desired grain product in deionized water, sterilized separately, and the remaining nutrient solution was added after cooling. Stock solution The preparation of the stock solution of all yeast transformed plants was stored in a mixture of 丨 milliliters of pupa and 1 milligram of 8 g% glycerol solution. C. The pre-cultivation % was filled with 10 or 100 亳 Di _ flasks (small amount of main culture) or i liters of LV, 503⁄4 liters of shaking culture fermentation. Pre-existing with stock solution or medium amount, inoculation with strains from the SD2 agar plate 36 20 200846356 strain. The medium was incubated for 2 to 3 days under continuous shaking (240 rpm) at 28 to 30 ° C. 0 Fermentation fermentation The main culture fermentation was carried out on a small scale using a 1 liter shake flask filled with 100 ml of SC5 nutrient solution. Usually, the above 3 ml of the preculture solution is used to carry out the selection. Next, the medium was cultured for 4 days under continuous shaking (240 ipm) at 28 to 30 °C. 1〇 The Wave Biotech (Tagelswangen, CH) bioreactor was used in medium or large-scale main culture fermentation. Specifically, inoculate 1 〇〇〇 or 1 〇〇〇〇 ml of 805 medium with 3 〇 or 300 ml of preculture, and then incubate for 4 days at 30 ° C in a Wavebag with a 32/min oscillation frequency (angle · 10 °) Air supply: 0·25 l/min). The pH of the culture was checked on days 1 to 3 and the pH was adjusted to ρΗ _ 5 to 6 using NaOH as needed. This medium was added on days 1 to 3, i.e., 1 ml of 50% strength yeast extract and 4 ml of 4 g glucose solution were added to 1 ml of the medium. Therefore, 10 or 100 volumes of the filling solution were continuously added over 1000 days in 1000 and 10000 ml of the medium. The 〇D_ of the culture medium was measured at various day-to-day patencies to check its growth. 2〇 After four days, cell-free supernatants were collected by centrifugation (centrifugation at 6000 rpm for 15 minutes in a JA14 roller). 37 200846356 Example ίο Purification of hBik D2 variant (SEQ ID NO: 35, KI>-A-BD2-A-miit34) from cell supernatant of fermentation yeast • Treatment of master culture containing SEQ ID NO: 35 with 1 gram of NaOH Fermentation, cell free supernatant prepared in 5 liquids until its pH was 7.8. The suspended particles in the supernatant were sedimented by centrifugation at 2000 rpm (15 minutes; Beckman-Allegra 6KR) at 4 °C. The supernatant was passed through a 10 ml Φ l trypsin-3⁄4 lipid column (Sigma-T1763) at 1 ml/min. Next, the column was washed with 70 ml of 50 mg of tris pH 7·8, 250 mg of NaCl, and 50 ml of 50 mg of tris pH 7·8, 600 mg of NaCl. Then, serial number 35 was eluted by using 180 ml of 50 mg of KC1/10 mg of molecule • HC1PH 2.0 and using 1 ml of 5 mg of KC1/250 mg of the molecule HC1 pH 0·9. Two milliliters of the fraction containing 500 microliters of 200 milligrams of tris pH 7.6, 2 moles of NaCl for 15 neutralization, was taken from the test tube. It was identified by the following trypsin inhibition assay: SEQ ID NO:35. The trypsin-suppressed fractions were pooled, mixed with the same volume of 0.1% TFA and passed through a Source 15 RPC column (GE Healthcare). The column was washed with 6 ml of 0.1% TFA (buffer HPLC_A), followed by a 25 ml gradient of 20 5 % buffer HPLC-B (〇.1〇/0 TFA, 60% acetonitrile) and further to 5 100°/ml gradient. Buffer HPLC-Β elution sequence number 35. The eluate containing SEQ ID NO: 35 was lyophilized and the lyophilizate was extracted in 250 μl of 120 mg of NaCl, 5 mg of citrate pH 3.1. 38 200846356 Example 11 Molecular Weight of the Protein of the Sigma Sequence Number 35 (KD_A-BD2-A-mut34) by the Proteoglycan Peptide and Molecular Qualitative Sequence Number 35 (KD_A-BD2-A-mut34) The purified protein of the TFA solution was diluted to 2 picomoles per microliter and then acidified simultaneously. After separation on Gr〇mSil 12〇 〇DS_4 HE (3 μl, 250×0.2 halo), the sample was analyzed by mass spectrometry. The molecular weight of SEQ ID NO: 35 (6381.2 迢) is clearly identified as 〇·7 Dalton by delta by the aid of the multiplicity of charged ions. In order to more accurately determine the amino acid sequence, the protein was cleaved by trypsin after denaturation with guanidine hydrochloride, which was reduced with dithiotreitol and derivatized with acetylacetate. The peptides obtained by mass spectrometry analysis as well as the detection of peptide fragment mass and MS/MS mass spectrometry can be used to establish accurate protein sequences.

[M+H]+=1014,42 : AEFCLEPPVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNNFRSEEACMLTCGGA WYFDVER - [M+H]+=1572,75 : ASFPRWYFDVER [M+H]+=1632,74 : AEFCLHPPVTGPCR [M+H]+=1816,74 : NNFRSEEACMLTCGGA [M+H]+=2115,93 : GNKNNFRSEEACMLTCGGA [M+H]+=2191,09 : AEFCLEPPVTGPCRASFPR [M+H]+=2292,10 : NSCNNFIYGGCRGNKNNFR [Μ+Η]+=3186,46 : AEFCLEPPVTGPCRASFPRWYFDVER [M +H]+=3558,42 : NSOTNFIYGGCRGNKNNFRSEEACMLTCGGA 15

The disclosed sequence of SEQ ID NO: 35 (KD-A-BD2-A-mut34) can be used to identify all changes in the amino acid sequence by means of the peptide. 39 200846356 Example 12 Determination of ICS0 values against trypsin, fibrin, plasma kallikrein The hBikD2 variant was determined by biochemical assay in a white 384-well microtiter plate against fluorescent trypsin, fibrin, Inhibitory strength of the activity of plasma kallikrein. The composition of the assay buffer was 50 mg of tris/Cl, pH 7.4, 100 ml of NaCl, 5 mg of molecularlyzed ClCl2, and 〇·〇8% (w/v) of BSA. The measurement conditions are as follows: The final concentration of the enzyme enzyme is determined. The final concentration of the matrix matrix is determined. Trypsin 5 Ng/ml T-6424 (Sigma) Boc-Ile-Glu-Gly-Arg-AMC 5 μmol 1-1100 (Bachem) Fibrin 20 ng/ml Hplas (Enzyme Research) MeOSuc-Ala-Phe-Lys-AMC 50 μg 1-1275 (Bachem) Plasma kallikrein 1 Nike 420307 (Calbiochem) Boc-Val-Pro- Arg- AMC 80 microgram ES-011 (R&D)

Ten microliters of the serially diluted test substance was placed in each well and then preincubated with 20 μl of enzyme at room temperature for 5 minutes. Next, the reaction was initiated by the addition of 2 Torr of the substrate. The measurement was carried out in a Tecan interpreter of 360 nm excitation and 465 nm emission after 60 to 90 minutes. The dose-effect curve and the half-maximum inhibition constant (IC5 〇 value) were measured by GraphPad Prism software (version 4.02). Inhibition of fibrinolysis by SEQ ID NO: 35 (hBikD2-nmt34) 15 200846356 The effect of hBikD2-mut34 was determined in an in vitro fibrinolysis mode and compared to Trasylol (aprotinin). Treatment with 〇13 picogram tissue factor (TF) and 164 units/ml tissue plasminogen activator (τρΑ) and also with various concentrations (0.1 to 10 μmol) of hBikD2-mut34 or aprotinin 5 Human citrate plasma was then incubated at 37 for 40 minutes. A physiological saline solution was used instead of the Kunitz functional site as a control. Tecan Safire was used to measure the optical density (〇〇4〇5 nm) to determine the lumps formed by TF and the clot lysis of TPA. Fibrinolysis is defined as the relative increase in OD after the formation of a depletion block. The anti-fibrinolytic activity was determined by using a relative increase in inhibition of 〇D. The 1〇5〇 value of hBikD2-mut34 for inhibiting fibrin dissolution is ι·〇±〇.2 μg. In this mode, the IC5G value of Trasyl〇1 (aprotinin) for inhibiting fibrinolysis is 〇·8±〇·2 μg. • Example 14 15 Inhibition of coagulation by SEQ ID NO: 35 (hBikD2-nmt34) _ The effect of hBikD2-mut34 was determined in an in vitro coagulation mode and compared to T^asylol (aprotinin). The human bark acid salt slurry was treated with 12 mg of CaCl2 and also with various / Chen (1 to micromolecule) hBikD2-mul34 or aprotinin to induce a blood coagulation reaction. The Kunitz functional site was used as a control using a physiological saline solution. During the incubation at 37 ° C for 90 minutes, an increase in 〇 d indicating coagulation was measured at 405 nm. According to this. Ten # out of its longest clotting time. The extension of the semi-longest condensate time indicates that the condensate is also suppressed. WBikD2-mut34 inhibits coagulation cT2 (twice the half-longest time) of 0.16 ± 0.02 micrograms. · 41 200846356 Example 15 - Hemostasis of SEQ ID NO: 35 (hBikD2-mut34) in rat withdrawal model The hemostatic effect of hBikD2-mut34 in rat hemorrhagic mode was investigated. In the anesthetized large breast, the jugular veins on both sides were inserted using a polyethylene sputum catheter. For the prolonged normal bleeding time, tissue plasminogen activator (ΤΡΑ) (8 mg/kg/hr) was injected into the rats during the entire test period. After ί minutes of injecting μ, the animals were treated with mBikD2-mut34 or aprotinin by high-dose injection and subsequent spotting. The dose used is 5 g/kg (high dose) and 3 mg/kg/hr (drip) to $mg/m = and 1 mg/kg/hr. A physiological saline solution was used as a pair. After the start point _ B resection, the tail end is immersed in the 37t: warm physiological food wood) and the tail sputum-water solution is then measured for bleeding time. Flow 15 Γ:: ί When the end of the blood flow begins to appear at the end of the hB.ldJ 〇 The bleeding time will be shortened when the woman stops bleeding. When bleeding is inhibited by hBlkm-nrnm (a large dose of butterfly and 6 mg / kg / small mile is 3 g / kg, similar effects can be seen after treatment, Λ). Example 16 of aprotinin was a rat model of arteriovenous (A V) shunting in the arteriovenous (A V) shunt mode], and squash 35 (hBikD2-mnt34) in the large arteriovenous shunt mode. In the anesthetized rats, the anti-blood of the ship was used to conduct the carotid artery and the cannula of the veins and the small catheter (shunt) to connect the catheters. Tube 甬t:!!. is a rough nylon ring for the surface of the thrombus. Extracorporeal blood circulation ^4 (4) The monitoring of thrombus formation began 15 minutes later. After that, the thrombus from 10 is then measured for the weight of the plug. The rats were continuously treated with hBikD2_mum or inhibitory enzyme by bolus injection and post-drip injection. The doses used were 〇15 mg/kg (large dose) and 〇3, gram/kg/hour (drip) to 5 mg/kg and 1 〇mg cold jin/sm%. Using physiological saline solution as a control. The antithrombotic effect was measured by the decrease in blood test weight. The ed5〇 formed by hBikD2_mut34 inhibition was 2.5 ng/kg (high dose) and 5 mg/kg/hr (continuous drip). TraSyl01 is resistant to ash at 4,9 mg/kg and 9.8 g/kg/hr ED5〇. Example 17 Cross-Reactivity Study by Competitive ELISA The cross-reactivity of the hBikD2 variant sequence number 35 with the aprotinin-treated patient serum was assessed using a competitive ELISA. Therefore, a commercially available ELISA method was developed to detect human serum anti-apoptinase antibodies (CdlTrend, Luckenwalde) to enable high sensitivity detection of variant and aprotinin competition for binding to anti-apoptinase antibodies. Sex. The anti-apoptinase-containing antiserum is obtained from a patient treated with aprotinin to produce a high antibody titer against the protein. In detail, the antiserum was preincubated for 1 hour at room temperature with slow shaking at a dilution buffer of 300 μl total volume and a test aprotinin or hBikD2 variant. Here, depending on the antiserum used, the dilution factor is 1:3〇00 to 1:75〇0. Each protein was assayed at three different concentrations (Bu 〇, 1 〇〇 Nike molecule). A blank group without aprotinin or hBikD2 variant was used as a control. After pre-incubation, it was displaced into the wells of the microtiter plates pre-coated with aprotinin in the kit. After further incubation for 1 hour at room temperature, the roots were rinsed according to the manufacturer's instructions and thereafter a photochromic reaction was generated by the addition of a peroxide to couple the first antibody. The optical density was measured in a Tecan interpreter at 450 nm (reference wavelength: 620 nm). At the time of measurement, the measured protein concentration was measured as a percentage of its relative reference empty ε white value (100%). Example 18 - Analysis of T cell epitopes (immunogenicity analysis) - Using Epibase® platform 15 from Algo Nomics NV, Belgium (Desmet [1992], Desmet [1997], Desmet|; 2002], Desmet [; 2005 ]) To study the HLA class II epitopes of aprotinin and hBikD2 variants, which are also referred to as TH epitopes.

The Epibase® platform analyzes the possible peptide fragments of all 10 amino acid residues that bind to 48 HLA class II receptors (the allotypes are 20xDRB1, 7xDRB3/4/5, 14xDQ, and 7xDP) 20 The sequence of the target. The binding free energy is calculated and the dissociation constant (Kd) is determined therefrom. Individual peptides are classified as strong (S), medium (M), and weak or no (N) binding agents. The following critical values were used: S: strong binding agent, Kd < 0.1 microgram; Μ: medium binding agent 'Kd< 微. 8 micrograms; Ν: weak or no binding agent, 0.8 micrograms 44 200846356 sub$^. In the antigen-induced humoral response, the observed D-cell activity/increase was considered to have DRB1-specificity. However, it cannot be excluded from DRB3/4 /5, DQ and DP. In the consideration of key epitopes, these genes have a lower expression rate than DRB1 and thus exclude strong binding agents with DRB3/4/5, DQ and DP. Its key epitopes, therefore, include strong binding agents for DRB1, DRB3/4/5 and DP, and medium binding agents for DRB1. To indicate immunogenicity, the number of binding peptide fragments of the HLA class II receptor of the DRB1, DRB3/4/5, DQ and DP genes was calculated for the target sequence. Only many isoforms that bind to the same group are counted once. Example 1 g Quantification of test substance in renal homogenate Wistar rats (180 to 180 g) were anesthetized with thiopental Na (100 mg/kg, intraperitoneal injection). The test substance (1 μg/kg) was administered via a venous catheter in a large dose. Two kidneys were removed after 6 minutes and stored at _2 °C before further analysis. The kidneys of the test substance or mock-treated rats were homogenized in 6 ml of buffer containing 0.9% NaCl/0.05% Triton-xi00 with the help of ultrathurrax. The homogenate was then further diluted 1 · 1 在 in a single buffer and then 100 μl of each serial dilution was filled into a dish of a microtiter plate. Thereafter, 100 μl of trypsin solution (trypsin from Merck's porcine pancreas; 5 μg/ml in 0.001 equivalent of HC1/45 200846356 0.05% Tween 80) was added and pre-incubated for 30 minutes at room temperature. The reaction was initiated by the addition of 100 microliters of L-BAPA matrix solution (sigma B3279., 1 mg/ml dissolved in water) and the process was monitored for 60 minutes at a temperature of 405 nm wavelength in a Tecan interpreter. The kidney homogenate of the control animals was taken as a reference sample, treated with the respective test substances and measured in the above series of diluents. The concentration of the unknown substance in the homogeneous liquid and its relationship with the total dose administered per animal were calculated from the obtained standard straight line.

Example 20 10 Cell migration analysis Human coronary vascular smooth muscle cells (hCAVSMC, 1.5×l〇5 cells/well, TEBU, Offenbach, Germany) were inoculated into 6-well plates and then at 37° (:/5% 032 of M231) The culture medium (growth medium, TEBU, Offenbach, Germany) was cultured for 48 hours. Pre-coated with vitronectin (5 〇Nike / 15 cm 2 ) (Gibco/Invitrogen, Karlsruhe, Germany). After the culture period 'Remove half of the monolayer cells. About 50% of the vitronectin coating remains in the cell-free zone of the well. To test the culture medium MCDB-131/0.2% BSA (Molecular Cell and Developmental Biology, MCDB; basal medium (BSA), Gibco/Invitrogen, Karlsruhe, Germany) 20 Instead of growing culture medium, the cells were stimulated with 10 ng PDGF-BB (R&D, Wiesbaden-Nordenstadt, Germany). Test substance. After 24 and 48 hours of culture, the migration distance of the cells in the non-porous area was determined by microscopy. Each measurement point represents the average of the four assay areas and at least three independent experiments. Calculation and PDGF-induced 46 200846356 Row distance (=100%) related migration distance. Example 21 - Chemical tropism analysis Separation of neutrophils from blood according to standard methods. Chemical tropism analysis of neutrophils was performed in a two-compartment system. HUVEC monolayer cells were cultured on membrane (3 micron pore size, polycarbonate, Falcon) for 24 hours. #RPMI 1640 culture medium was prefilled with fluorochrome-loaded ιχ1〇5 neutrophils into the upper chamber. A thorny agonist (IL-8: 5 ng or C5a: 10 ng) containing various concentrations or concentrations of sirolimus, and various concentrations of the test substance. The substance to be analyzed is placed in the above two chambers. It was incubated at 37 ° C and 5% C 〇 2 for 45 minutes. After the culture, cells that had migrated into the lower chamber (fluorescence measurement, counting) were measured. is B 彡 permeability analysis in a two-compartment system ( Within Falcon), monolayers of human umbilical vein endothelial cells (HUVEC) are cultured on the membrane of the insert. Based on this, 2xl〇5 cells/inserts are inoculated and cultured for 18 to 20 hours (37 °C/ 5% C02). 20 by trypan blue before starting the experiment The albumin solution was used to examine the thickness of the monolayer cells. Then CAP37 (5 μmol) was added to the upper chamber. The change in permeability was measured during the 3-hour period of various aprotinin concentrations (1 〇 ~ 〇. 1 μmol). The efflux of the insect blue-conjugated albumin solution is here used as an indicator of the change in the infiltration 47 200846356. The 〇D measurement was performed at 590 nm. References - Apeler et al; 2004, 54, 483 ~ 497 5 Apeler Yu: J. Knablein (editor), Hyundai Cha Leqin, WLEY-VHC,

Weinheim, 2005, pp. 1021 ~ 1032 Asimakopoulos et al; 2000 ‘J. TTiorac· Cflnizoviwe· iSWg, 120, 361~369

Beierlein et al; 2005, such as "· TTzon Swrg 79,741 ~ 748 10 Blauhut et al; 1991, /· TTzorac. iSWg· 101, 958~967

Bode and Huber, 1992, five wr· J. 204, 433~451

Chun et al; 1990, EMSO/owrwa/ 9,385~393 Claus et al; 10,000/ocAem/owr and /368,233~242 15 Dennis et al; 1995, J. 〇/· CAem· 270,25411 ~ 25417 • Dietrich et al; 1995, 83, 679~689

Dietrich et al; 2001, on /ze57\9A9gj; 95, 64~71 Delariaeta Bu 1997, J. (?/. 272,12209~12214

Fritz and Wunderer, 1983 Jane 33, 479~494 20 Gautam et al; 2001, TVfliwre Med· 7,1123~1127

Glasser et al.; 1972, in ''Verhandlungen der Deutschen Gesellschaft fur Innere Medizin, 78th meeting, Bergmann Munich Hess Jr., 2005 'J· /fea/i/z· 办 · P /mrm 62 (18 Supplement 4) ·· 6~9 48 200846356

Kawaguchi et al; 1997, J. 5ζ·ο/· CAem·272, 27558~27564 Krowarsch et al; 2005, Ze仏12, 403~407 Laskowski and Kato, 1980, d-shaped.i^ev. million/c^ /zem 49,593~626 ΙΛ 九, 1998, American Journal of Alzheimer Disease, 9/10 May, 236~244 B Markland et al; 1996a, 10,000 zoc/zewz·for rj; 35,8045~8057

Markland et al; 1996b, 0c/zew/5ir3; 35, 8058~8067, Marlor et al; 1997, J. 〇/· Ckm· 272, 12202~12208

Muller-Pillasch et al; 1998, 5/oe/n·claw·dcia 1395, ίο 88~95

Otiewski et al; 2001, dcia. 5iocAz_m. corpse) /. 48, 419~428 Parente et al; 1991, Proc·iVai·dcad.iSW·88, 6931~6935 • ?6 generation ^^ et al; 1995, */.1^ with moxibustion (: > ^ by < 9 people 57, 805~812 - Petersen et al; 1994, said 338, 53~57 15 Ponte et al; 1988, TVa / wre 331, 525~ 527

Richardson et al; 2001, Ge-like 270, 93~102 _ Royston, 1992, J. Fase. dwesi/z 6, 76~100

Sambrook et al.; 1989, Mo/ecw/flr C7<9«z>2g· Co/i/ Factory/Shrimp Harbor, % Second Edition 20 Schechter Berger? 196Ί 9 Biochem. Biophys. Res. Commun. 27 , 157~ 162

Shimomura et al.; 1997, J. 〇/· CAem. 272, 6370~6376 Sprecher et al; 1994, Proc. #加·dcm/Jcz·· 91,3353~3357 Trexler et al; 2001, Proc·W, · dcfl times 98,3705~3709 49 200846356

Trexler et al.; 2002, 〇/. CTzem.383, 223~228 Vetrand Gebhard 5 1990 ? Biol. Chem. Hoppe-Seyler 371 5 1185~1196

Wun et al.; 1988, 10,000/〇/. CAern·263,6001~6004 5 Patent US 6010880 4/2000 » US 5795865 8/1998 10 WO 92/06111 7/1997 - US 5777568 7/1998 15 • US 6482798 11/2002 EP 0 238993 8/1990 20 EP 0 307592 7/1988 EP 0821007 7/1997

Markland and Ladner; inhibitors of human plasmids derived from the kunitz functional site (Dyax). Markland and Ladner; kallikrein inhibits "kunitz functional site" protein and its analogues (Dyax)

Schroder et al; aprotinin variants with improved properties

Schroder et al; Bovine trypsin inhibitors prepared by recombinant DNA technology, expression vectors and recombinant hosts, and pharmaceutical uses

Noiris et al; aprotinin variants with improved properties

Ebbers et al; Recombinant aprotinin variants for homogenization of genetically engineered aprotinin variants and therapeutic uses thereof

Auerswald et al; bovine trypsin inhibitor variant' preparation method and use thereof

Schroder #人; aprotinin with improved properties 200846356 Enzyme variant

Us 5863893 3/J995 Dennis and Lazarus; Vila factor from kunitz functional site protein 5 US 5914315 6/1995 Sprecher et al; human Kunitz-type inhibitor and its composition US 7019123 10/2001 Tamburini et al; human bikunin • Brief description of the formula] 10 DNA sequence of plasmid pPCRscript; its hBik D2 synthetic gene and the flanking sequence of the bottom line; Αχί. Preparation of the hBikD2 variant "sandwich protein, schematic diagram" - Figure 3 contains the selective sandwich structure Kd-A DNA sequence of plasmid plVEX2.3d of BD2-A; 4th DNA sequence of yeast secretion vector 15 plUlO.lO.W containing selection of sandwich structure Kd-A-BD2-A; • Figure 5 contains secondary selection The DNA sequence of the yeast secretion vector plU3.12.lVt of the Kd-A-BD2-A; the codons of the beginning and the end of its expression in bold; Figure 6 The fibrinolysis in human plasma; 20 Figure 7 Coagulation in human plasma; bleeding time in rats in Figure 18; anti-i-plug effect of Tmsylol in rat hemodialysis tubing mode in Figure 9; anti-i plug effect in hBikD2-mut 34 in hemodialysis tubing mode in rats Thrombosis effect; Figure 11 shows horizontal migration of hCASMC; Figure 12 shows IL-8 and C5a-induced chemotaxis of neutrophils; Figure 13 shows inhibition of induced permeability increase by CAP 37; sequence A of Figure 14 , B and C and amino acid sequences of SEQ ID NOs: 1 to 76; Figure 15 Human trypsin, fibrin, plasma kallikrein

10 15

IC50 value, expression level and theoretical pi of inhibition of XIa factor; Figure 16 Quantification of test substance in renal homogenate (data is based on the total amount of a kidney); Figure 17 is a one-time calculation for each HLA gene Sequence A (arotinin/BPTI), sequence 歹丨J Nos. 35 and 53 (hBik D2 variant) bind to the number of TH epitopes of many HLAs (DRB 1, DRB3/4/5, DQ and DP) of the same gene, Does not consider the autoantigen determination site. [Main component symbol description] None 52 20

Claims (1)

200846356 10 15 Patent Application Range: 1. A non-natural Kunitz-type protease inhibitor having a length of about 58 amino acids of the formula NH2 (amino terminal)-ABC-COOH (mercapto end); the A has at least 5 The length of the amino acid, which has a length of at least 10 amino acids. 2. The non-native protease inhibitor of claim 1, wherein the A is derived from any desired protease inhibitor of the Kunitz type. 3. A non-natural protease inhibitor according to any one of claims 1 or 2, wherein the A can be a human source. 4. In the scope of the patent range i or 3 a non-native protease inhibitor, wherein the A line is derived from the kunitz functional site selected from the group consisting of a translation product of the following human genes: a. Dian powder-like y5 (A4) precursor protein (peptidase is pure, A Zhaimer's disease) b. serine peptidase inhibitor-like, with (6) pool and wAp functional site l (eppin) C. serine peptidase inhibitor, kunitz type 2, SPINT2 (placenta bikunm) functional part value id · Serine acid enzyme inhibition 丨I v , 卩 remaining sword, kumtz type 2, SPINT2 (placenta bikunin) functional part 2 e·tissue factor slave inhibitor 2 precursor functional site 1 f·tissue factor posterior inhibitor 2 precursor (TFpU), functional site 2 53 20 200846356 g·tissue factor pathway inhibitor 2 precursor (TFH-2 ), functional site 3 h · tissue factor pathway inhibitor, TFPI (fat: protein-associated coagulation inhibitor), functional site 1 1. tissue factor pathway inhibitor, TFPI (lipoprotein-associated coagulation inhibitor), functional site 2 j· tissue factor pathway inhibitor, TFPI (lipoprotein-associated coagulation inhibitor), functional site 3 k. amyloid/3 (A4) precursor-like protein 2 (APLP2) 1· a -1 _ microglobulin/bukunin precursor (AMBP) functional site 1 10 m. a -1 - microglobulin/bukimin precursor (AMBP) functional site 2 η. hepatocyte growth factor activation inhibitor type 1 (ΗΑΙ-1, -SPINT1), functional site 1 - 0. Hepatocyte growth factor activation inhibitor type 1 (ΗΑΙ-1, SPINT1), functional site 2 15 Ρ · Collagen type VI, a3 (COL6A3) # q· Collagen type VII, a 1 ( Malnutrition bullous epidermis release, dominant and recessive, COL7A1) r. Kunitz-type protease Formulation 3 precursor (HKIB9, SPIT3) s. WAP tetra-disulfide nuclear functional site 8 (WFDC8) 20 t. WAP, follistatin/kazal immunoglobulin, containing kunitz and netrin functional site (WFIKKN), functional site 1 u. WAP, follistatin/kazal immunoglobulin, containing kunitz and netrin functional sites (WFIKKN), functional site 2 54 200846356 ν· WAP, follistatin/kazal immunoglobulin, containing kunitz and netrin functional site 2 (WFIKKN2 ), functional site 1 w· WAP, follistatin/kazal immunoglobulin, containing kunitz and netrin functional site 2 (WFIKKN2), functional site 2 X·serine peptidase inhibitor, kunitz type 4 (SPINT4) y· Lactokinin, proteoglycan-like decanoylated glycoprotein (PAPLN) ζ·Q9BQP5-QTTHUMP00000031164 (fragment); and • 5. aa·LOC285929, similar to matrinin 2 precursor, such as non-native protease inhibition in claim 1 Agent, wherein 10 of the B lines are derived from the amino acid sequence of any desired protease inhibitor of the Kunitz type. - 6. A non-native protease inhibitor as claimed in claim 5, wherein the B may be of human origin. VIII 15 10 7. If applying for a non-native protease inhibitor of any of items 5 to 6 of the special (4); wherein B_ is from the kunitz functional part of the transgenic product selected from the group consisting of the following human genes: a .Amyloid record 4) precursor protein (peptidase job (four), Azhai disease) 20 b · serine peptidase inhibition sword, jl ancient 2 „ | towel 4剜铋 with kunitz and WAP functional part l (eppin) C. Serine peptidase inhibitor, kunitz type 2, SPINT2 (placental bikunin) functional part value} d·serine peptidase inhibition fast, 1. I don d kunitz type 2, SpINT2 (placenta 55 200846356 bikunin) Functional site 2 e· Tissue factor pathway inhibitor 2 precursor (TFPI-2), functional site 5 f· Tissue factor pathway inhibitor 2 precursor (TFPI-2), functional site 2 Η g· tissue factor pathway Inhibitor 2 precursor (TFPI-2), functional site 3 h. Tissue factor pathway inhibitor, TFPI (lipoprotein-associated coagulation inhibitor), functional site 1 10 i. Tissue factor pathway inhibitor, TFPI (lipoprotein) Related coagulation inhibitors), functional site 2 - j · tissue factor pathway inhibitor, TFI > I (fat egg) Related coagulation inhibitors), functional site 3 k. amyloid A (A4) precursor-like protein 2 (APLP2) 15 • 1. m· α -1 - microglobulin/bukimin precursor (AMBP) functional site 1 α -1 - Microglobulin/buk dish in precursor (AMBP) functional site 2 n. 0. Hepatocyte growth factor activation inhibitor type 1 (HAI-1, SPINT1), functional site 1 hepatocyte growth factor activation inhibitor 1 Type (HAI-1, 20 SPINT1), functional part 2 P· Collagen type VI, a3 (COL6A3) q. Collagen type VII, a 1 (nutritive bullous epidermis release, dominant and hidden) Sex, COL7A1) 56 200846356 Γ. S· t 5 UV • W· 10 X. - yz aa 15 Kunitz-type protease inhibitor 3 precursor (HKIB9, SPIT3) WAP four-disulfide nuclear functional part 8 (WFDC8) WAP ' Inhibitor/kazal immunoglobulin, containing kunitz and netrin functional sites (WFIKKN), functional site 1 WAP, follistatin/kazal immunoglobulin, containing kunitz and netrin functional sites (WFIKKN), functional site 2 WAP, follicles Inhibin/kazal immunoglobulin containing kunitz and netrin functional site 2 (WFIKKN2), functional site 1 WAP , follistatin/kazal immunoglobulin, containing kunitz and netrin functional site 2 (WFIKKN2), functional site 2 serine peptidase inhibitor, kunitz type 4 (SPINT4) papillin, proteoglycan-like sulfated sugar Protein (PApLN) Q9BQP5-QTTHUMP00000 (mi64 (fragment); and 8. The non-native protease inhibitor of claim 1, wherein the C line is derived from the amino acid sequence of any desired protease inhibitor of the Kunitz type . 9. A non-native protease inhibitor as claimed in claim 8 wherein the C is human. The non-native protease inhibitor according to any one of claims 5 to 6, wherein the B line is derived from a leunitz functional value selected from the group consisting of a translation product of the group of human genes: a. Powder-like/5 (A4) precursor protein (peptidase nexin_; Q, Alzheimer 57 200846356 5 10 15 Disease) b-serine acid enzyme inhibitor-like, with kunitz and WAP functional site l (eppin) c. serine peptidase inhibitor, kunitz type 2, SPINT2 (placental bikunin) functional site 丨d·serine Peptidase inhibitor, kunitz type 2, SPINT2 (placental bikunin) functional site 2 e·tissue factor pathway inhibitor 2 precursor (TFPI-2), functional site 1 f· tissue factor pathway inhibitor 2 precursor (TFH -2), functional site 2 g · tissue factor pathway inhibitor 2 precursor (TFPI-2), functional site 3 h · tissue factor pathway inhibitor, TFPI (lipoprotein-associated coagulation inhibitor), functional site 1 i · Tissue factor pathway inhibitor, TFPI (lipoprotein-associated coagulation inhibitor), functional site 2 j · tissue factor pathway inhibitor, TFPI (lipoprotein-associated coagulation inhibitor), functional site 3 k · amyloid beta (Α4 Precursor-like protein 2 (APLP2) 1· α-1-microglobulin/bukunin precursor (AMBP) functional site 1 m·α-1-microglobulin/bukunin precursor (AMBP) functional site 2 η· hepatocyte Growth factor activation inhibitor type 1 (ΗΑΙ-1, SPINT1), functional site 1 〇 · hepatocyte growth Sub-activation inhibitor type 1 (ΗΑΙ-1, SPINT1), functional site 2 58 20 200846356 ρ·collagen type VI, a3 (COL6A3) q· Collagen type VII, α 1 (dystrophic bullous Epidermis release, dominant and recessive, COL7A1) 5 10 15 r· Kunitz-type protease inhibitor 3 precursor (HKIB9, SPIT3) s· WAP tetra-disulfide nuclear functional site 8 (WFDC8) t· WAP 'Focustin/kazal immunoglobulin containing kunitz and netrin functional sites (WFIKKN ), functional part 1 u· WAP 'follistatin/kazal immunoglobulin, containing kunitz and netrin functional part (WFIKKN), functional part 2 V· 'follistatin/kazal immunoglobulin, containing kunitz and netrin functional part 2 (WFIKKN2), functional site 1 w· WAP, follistatin/kazal immunoglobulin, containing kunitz and netrin functional site 2 (WFIKKN2), functional site 2 X·serine peptidase inhibitor, kunitz type 4 (SPINT4) y· mastoidin, proteoglycan-like sulfated glycoprotein (papln) ζ·Q9BQP5-QTTHUMP00000031164 (fragment); and aa·LOC285929, similar to matrilin 2 precursor 11 • Non-native protease as claimed in claim 1 Inhibitor, wherein the B has the sequence ASFPRWYFDVERNSCNNFIYGGC. 12. A non-native protease inhibitor as claimed in claim 1 wherein the A has the sequence RPDFCLEPPYTGPCK. 13. A non-native protease inhibitor as claimed in claim 1 wherein the C has the sequence rakRNNFKSAEDCMRTCGGA. 59 20 200846356 14. The non-native protease inhibitor of claim 1, wherein the A, B and C lines are selected from SEQ ID Nos. 1 to 76 (Example 6). 15. A non-native protease inhibitor as claimed in claim 1 which comprises SEQ ID NO: 35 (Example 6). 5 16. A nucleic acid encoding a non-native proteinase inhibitor as claimed in claims 1 to 15. 17. A non-natural egg p-white enzyme inhibitor as a drug, as in claims 1 to 15. 18. A nucleic acid as a drug as claimed in claim 16. ί 19 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 20) using a non-native protease inhibitor as described in claims 1 to 15 or a nucleic acid manufacturing drug as claimed in claim 16 to treat 15 blood loss and thromboembolism during surgery with high bleeding risk Disease treatment, shock, multiple trauma, sepsis, diffuse intravascular blood clot (DIC), multiple organ failure (MOF), unstable angina, myocardial infarction, stroke, embolism, deep vein thrombosis, inflammatory disease, Invasive tumor growth and metastasis, pain and edema treatment, cerebral edema, 20 spinal edema, prevention of hemostasis during dialysis treatment, treatment of symptoms of skin aging, elastic tissue degeneration, atrophy, wrinkle formation, hemodynamic changes, pigmentation, Solar keratosis, blackheads, cystice, wound healing, skin cancer, treatment of skin cancer, solar horn 60 200846356 syndrome, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, multiple sclerosis, fiber Degeneration, cerebral hemorrhage, inflammation of the brain and spinal cord, brain infections, and diseases caused by tendinopathy Method state. 61