CN107216385A

CN107216385A - Tumour-specific lepirudin 023 ludon and its production and use

Info

Publication number: CN107216385A
Application number: CN201710362103.6A
Authority: CN
Inventors: 何向锋; 施文
Original assignee: 何向锋
Current assignee: New key (Nanjing) Biotechnology Co., Ltd.
Priority date: 2017-05-19
Filing date: 2017-05-19
Publication date: 2017-09-29

Abstract

The invention discloses a kind of tumour-specific lepirudin 023 ludon, the N-terminal of lepirudin 023 ludon contains the oligopeptide sequence Pro Gly Arg Val Val that can be recognized and be cracked by the plasma urokinase-type plasminogen activator (uPA) of tumor tissues height expression, and the hirudin for restructuring is the hirudin or hirudin fusion protein of hirudin isomers, hirudin mutant, hirudin chimera, the HIRULOG truncated, genetic modification.Correspondingly, present invention also offers the preparation method of tumour-specific lepirudin 023 ludon and its application in the medicine for preparing anti-curing oncoma, the preparation method and purposes of the adoptive immunity cell of expression tumour-specific lepirudin 023 ludon are additionally provided.The lepirudin 023 ludon of the present invention, hirudin anticoagulant activity is set to carry out tumor-targeting release, construction is difficult thrombosed microenvironment, established emboli even is dissolved to reach anti-freezing, thrombolysis, prevent the function of Nasopharyngeal neoplasms, and this just overcomes the risk that wild type hirudin systemic administration causes systemic bleeding.

Description

Tumour-specific lepirudin 023 ludon and its production and use

Technical field

The present invention relates to biological therapy and biomedicine field, more particularly to a kind of tumour-specific lepirudin 023 ludon and its Preparation method and purposes.

Background technology

Hirudin is the activity most strong natural thrombin inhibitor found so far, initially from Hementaria officianalis saliva It is isolated in gland, it is made up of 65~66 amino acid, can be directly with fibrin ferment with l：L (mol ratio) mode combines to form non- Covalent complex, so that fibrin ferment loses the ability of cracking fibrinogen, suppresses the formation of thrombus.Natural hirudin is present More than ten kinds variants, mainly there is referred to as tri- kinds of homologys of HV1, HV2, HV3 very high isomers (Hirudin Variant).

At present, external existing two lepirudin 023 ludon products go through to list：1.Desirudin (trade names：Revasc, Switzerland's Novartis products), 2.Lepirudin (trade names：Refludan, Britain Pharmion and the U.S. BerlexLaboratories products).Only there is fine difference at N- ends in both structures, Desirudin is Val1-Val2, And Lepirudin is Leu1-Tyr2.The prevention and treatment of DVT (DVT) when Desirudin is used to perform the operation, Lepirudin is used for the anticoagulant therapy of heparin-induced thrombocytopenic Disease.In addition, hirudin is preventing and treating unstable Property angina pectoris, disseminated intravascular coagulation, cerebral thrombus, thrombophlebitis and coronary artery thrombosis in terms of all have it is huge latent In clinical value.

High blood coagulation state, such as lung cancer, breast cancer, stomach cancer, cancer of pancreas are there is in Several Kinds of Malignancy patient.Dislike Property tumor patient blood in hypercoagulative state, can not only promote internal thrombosis, and with tumour growth, invasion and attack and shifting It is substantially related.Coagulation function change can cause tumour cell phenotype and activity change, promote tumour cell and blood platelet, endothelium thin Born of the same parents, fibronectin (Fibrinectin) and Von Willebrand factor (vWF) stick, so that tumour is thin Born of the same parents even shift in local multiplication, infiltration to other positions.Thrombotic diseases are the malignant tumor patients for being only second to metastases Second largest lethal factor.Drug intervention is carried out early to the high-risk tumor patient of thrombosis, for extension patient survival, drop Low actual is significant.Research shows that hirudin can also play a role in oncotherapy.Lepirudin 023 ludon is anti-by it Solidifying effect, suppresses fibrinous and is formed, can prevent tumour cell and fibrin or platelet aggregation, make NK cells or its He is played the activity of cytotoxic effector cell.It is proved leech extract for treating and can play the tumour of curative effect to have fibrosarcoma, bone Sarcoma, angiosarcoma, melanoma and leukaemia etc..Hirudin can also coordinate chemotherapy and radiation, by promote blood flow in tumour, Improve anoxia state and heighten the effect of a treatment.

Animal experiment and clinical research show that vein or hypodermic injection hirudin are without obvious toxic-side effects, semilethal agent Amount is easily accepted by much larger than dosage needed for treatment, body, and immunity is weak, and blood platelet, fibrinogen level and blood red egg are not influenceed Bai Hanliang, not easily passs through blood-CSF barrier.No matter acute, subacute toxicity test, hirudin is to breathing, blood pressure, heart rate Do not influence.Hirudin is in vivo without degraded, with active component through kidney excretion.

Hirudin as a kind of efficient, direct, special thrombin inhibitor, with very strong anti-freezing, antithrombotic and A variety of pharmacological activity such as anti-inflammatory.Compared with traditional anticoagulant such as heparin, its therapeutic dose is small, curative effect is high, will not cause Quick reaction.Leech have antigenicity, but its antibody does not do harm to, and prolongs long elimination half-life on the contrary, is that a kind of rare plus effect resists Body.Therefore the application of hirudin will be continuously available developing and promote, and lepirudin 023 ludon will turn into the anti-freezing of a new generation, anti-bolt And anti-cancer agent.But, hirudin can cause blood coagulation relevant parameter again, such as activated partial thromboplastin time (APTT), solidifying Hemase time (TT) and prothrombin time (PT) etc. are significantly raised, so as to cause whole body or systemic bleeding risk.Face On bed, as a preferable anticoagulant, under the conditions of systemic administration, should have clear and definite anti-freezing, anti-bolt effect, but Do not cause bleeding side effect, especially in the clinical practice for the purpose for the treatment of tumour, and its preferable working condition is targeting Property tumor by local play anti-freezing, anti-bolt, antitumor action so that clinical application security improve.In oncotherapy neck How domain, further reduce the side effect of hirudin, and it is the problem of being worth inquiring into increase its oncotherapy targeting.

The content of the invention

It is an object of the invention to provide a kind of tumour-specific lepirudin 023 ludon and expression tumour-specific lepirudin 023 ludon Adoptive immunity cell, at least can solve the problem that one of above mentioned problem.

The key of the guideline of inventive concept is to build hirudin derivative, hirudin anticoagulant activity is carried out tumour Targeting NO release, i.e., under normal operation, the hirudin derivative is without anticoagulating active, and when positioned at knub position, the leech The anticoagulating active of plain derivative is just in tumor by local release, and construction is difficult thrombosed microenvironment, or even dissolves established Emboli is to reach anti-freezing, thrombolysis, prevent the function of Nasopharyngeal neoplasms.This just overcomes wild type hirudin systemic administration Cause the risk of systemic bleeding.

Plasma urokinase-type plasminogen activator (uPA) abbreviation urokinase, it is one of primary activation thing of plasminogen. UPA, which is one, the serine protease of strict substrate limitation, cuts off the Cys-Pro-Gly-Arg560- in plasminogen Val1561-Val-Gly-Cys sequences, form fibrinolysin.Discovered in recent years uPA system components are in tumor tissues and circulating In the pathological characteristic of high expression and kinds of tumors, prognosis it is related, uPA be it is a series of tumor-infiltrated with the strong mark shifted, Turn into one of study hotspot of tumor markers.UPA major function is to participate in the decomposition and regeneration of basilar memebrane, with tumour Transfer relationship is close.Its acceptor uPAR can be combined with vitronectin, form α v β 3- vitronectin-uPAR complexs.UPA with UPAR combines rear increased activity.So a large amount of high activity uPA are enriched at tumor cell surface α v β 3.

The single chain polypeptide that hirudin is made up of 65~66 amino acid, its N-terminal can be with catalyzed by thrombin active sites Point is combined, and with anticoagulating active, C-terminal is combined with the substrate recognition site of fibrin ferment, there is very strong specificity parent to fibrin ferment And effect.The present invention devises the measure of a closing hirudin N-terminal, to reach that the anti-freezing for reducing the hirudin derivative is lived Property purpose, while also have the target-seeking function of cancer target.When the hirudin derivative is located at tumor microenvironment, tumour is utilized The characteristics of being rich in uPA enzymes in microenvironment, the hirudin for being closed the hirudin derivative N-terminal is replied as hirudin original again Shape, selectively targeted anticoagulation is played in tumor by local, so that as a class is new, safe and effective tumor-targeting resists Solidifying, anti-bolt, anticancer.

To achieve the above object, according to an aspect of the invention, there is provided a kind of tumour-specific lepirudin 023 ludon, weight The N-terminal of group hirudin contains can be by the oligopeptide sequence that uPA is recognized and is cracked, and the hirudin for restructuring is hirudin isomery Body, hirudin mutant, hirudin chimera, the HIRULOG truncated, the hirudin or hirudin fusion protein of genetic modification.

Further, oligopeptide sequence, which is, to be Pro- by the physiologic substrate small peptide that uPA is recognized and is cracked, amino acid sequence Gly-Arg-Val-Val, i.e. PGRVV；

Or, oligopeptide sequence is can be by the small peptide for the artificial optimization that uPA is recognized and is cracked, and its architectural feature is XnRVV, Xn Represent any number of arbitrary amino acid.

Further, lepirudin 023 ludon is that have oligopeptides PGR in hirudin isomers HV1 N-terminal modification, different with hirudin Structure body HV1 N-terminal sequence VV collectively constitutes the physiologic substrate peptide sequence PGRVV that uPA is recognized and cracked, lepirudin 023 ludon amino acid Sequence is：SEQ ID NO.1, gene order is：SEQ ID NO.2；

Or, lepirudin 023 ludon is that the C-terminal sequence of wild type hirudin HV1 peptide chains is replaced with into wild type hirudin HV3 The C-terminal sequence of peptide chain, then in its N-terminal modification PGR oligopeptides, lepirudin 023 ludon amino acid sequence is：SEQ ID NO.7, gene Sequence is：SEQ ID NO.8；

Or, the hirudin isomers HV1 that lepirudin 023 ludon is human albumin with N-terminal amino acid is VV passes through oligopeptides The fusion protein HPH that PGR is formed by connecting, fusion protein HPH amino acid sequence is：SEQ ID NO.11, gene order is： SEQ ID NO.12；

Or, lepirudin 023 ludon is Fc sections of people's gomphosis immunoglobulin and N-terminal amino acid is VV hirudin isomers The fusion protein F PH that HV1 is formed by connecting by oligopeptides PGR, Fc sections of people's gomphosis immunoglobulin comprising the hinge area of human IgG 4, Fc sections Sequence and IgM cauda sequences, FPH amino acid sequences are：SEQ ID NO.13, gene order is：SEQ ID NO.14.

Tumour cell can build tumor microenvironment, the characteristics of by analyzing tumor microenvironment, can find cancer target The target of property.Research shows α v β 3 in kinds of tumor cells such as lung cancer, breast cancer, prostate cancer, carcinoma of urinary bladder, glioblastoma And the height expression of the surface such as wellability melanoma.The strong expression in tumor tissues neovascular endothelium cell, in ripe blood α v β 3 expression is very low in endothelial cell and most normal structures.In tumor tissues, tumor cell secretion Growth factor activates epithelial cell, and the expression of wherein integrin alpha v beta 3 is very high, and this causes α v β 3 to be controlled as cancer target The preferable site treated.Compared with Normal tissue vascular system, tumor tissues great expression α v β 3, α v β 3 are new in tumor tissues Expression in raw capillary system is higher by much than normal structure.Integrin alpha v beta 3 can recognize extracellular matrix protein, Specifically bound with it, the adhesion and migration of mediate tumor cell, in the growth of tumour, infiltration, shift especially angiogenesis In play a significant role.α v β 3 can be with a variety of protein bindings containing RGD sequence, such as vitronectin, fiber knot Hop protein, fibrin element are former, fibrin ferment is sensitive plain and some simulation RGD sequences endogenous proteins.The antibody of some designs Or the antagonists of α v β 3 of small-molecular-weight, it can suppress viscous between vascular endothelial cell and vitronectin in model in vivo Attached, the angiogenesis of tumour is significantly reduced.

In conventional research, RGD sequence is often by the carrier as targeted drug, by drug targeting to expression integrin alpha v beta 3 Tumor vasculature go.RGD sequence is found as the binding site of fibronectin and cell, and it is recognized substantially Site is made up of tri- amino acid of Arg-Gly-Asp.In RGD derivative, c (RGDfK) often by the carrier as medicine because Lysine residue K be it is optimal can with other chemical substances occur coupling reaction group.People use phage technology It is found that another RGD part that can be specifically bound with α v β 3：RGD sequence (ACDCRGDCFCG) containing two disulfide bond, People are called RGD4C.Its binding ability is 20 times containing a disulfide bond RGD sequence, is the 200 of generally linear RGD sequence Times.RGD4C shortcoming is can to form different cyclic structures, if foring additional monocyclic and bicyclic ring structures, and it is tied Conjunction ability reduces by 10 times.The two is compared, and c (RGDfK) stability is preferable and is easily coupled with other chemical groups, more suitable Cooperate the carrier for chemical coupling.And RGD4C is then suitable as the target medicine carrier designed with recombinant technique.

Therefore, the N-terminal of lepirudin 023 ludon is also associated with the polypeptide for targeted integration element α v β 3, and the polypeptide includes RGD Sequence.The integrin alpha v beta 3 of the more preferable target tumor microenvironment of above-mentioned lepirudin 023 ludon can then be made.When RGD oriented carriers and tumour After cell surface α v β 3 are combined, hirudin digestion is discharged by uPA protease, anti-freezing, anti-bolt and antitumor work is played With.

Further, lepirudin 023 ludon is targeted integration element α v β 3 polypeptide RGD4C and N-terminal amino acid is VV leech The fusion protein RPH that plain isomers HV1 is formed by connecting by Linker and oligopeptides PGR, fusion protein RPH amino acid sequence For：SEQ ID NO.9, gene order is：SEQ ID NO.10.

Understood at present, the N-terminal of hirudin molecule is its core, containing three pairs of disulfide bond (Cys6-Cys14, Cys16-Cys28, Cys32-Cys39), make N-terminal peptide chain around dense core cyclic peptide structures are changed into, the catalysis with fibrin ferment is lived Property site combine；And C-terminal is the chain structure of a random stretching, extension, wherein Gln49~Gly54 has space and hydrophobic effect, And Asp55~Pro60 is rich in acidic amino acid, with negative electrical charge, the easily substrate recognition site with fibrin ferment alkalescence is combined.HV1/ HV2 Ser32-Asp33-Gly34-Glu35 (SDGE) or HV3 Ser32-Asn33-Gly34-Lys35 (SNGK) are one prominent For the Loop structures between beta sheet, conformation is free, to activity without obvious effect., will be wild according to the above characteristic of hirudin The Ser32-Asp33-Gly34-Glu35 (SDGE) of type hirudin (HV1) peptide chain replaces with Arg32-Gly33-Asp34-Ser35 Or Arg32-Gly33-Asp34-Met35 (RGDM) (RGDS).RGD sequence is made up of arginine, glycine and aspartic acid, It is present in various kinds of cell epimatrix, can be specifically bound with integrin alpha v beta 3.The restructuring leech modified by RGDS or RGDM Element can more preferable target tumor microenvironment integrin alpha v beta 3, hirudin digestion is discharged by uPA protease, played Anti-freezing, anti-bolt and antitumor action.

Further, lepirudin 023 ludon is to replace the Ser32-Asn33-Gly34-Glu35 of wild type hirudin HV1 peptide chains Arg32-Gly33-Asp34-Ser35 is changed to, then in its N-terminal modification PGR oligopeptides, lepirudin 023 ludon amino acid sequence is：SEQ ID NO.3, gene order is：SEQ ID NO.4；

Or, lepirudin 023 ludon is to replace with the Ser32-Asn33-Gly34-Glu35 of wild type hirudin HV1 peptide chains Arg32-Gly33-Asp34-Met35, then in its N-terminal modification PGR oligopeptides, lepirudin 023 ludon amino acid sequence is：SEQ ID NO.5, gene order is：SEQ ID NO.6.

Correspondingly, present invention also offers the preparation method of tumour-specific lepirudin 023 ludon, comprise the following steps：Chemistry The above-mentioned tumour-specific lepirudin 023 ludon genetic fragment of synthesis, signal peptide is connected in its N-terminal, and in restructuring encoding gene Restriction enzyme site is added at two ends, by Prepare restructuring carrier in restructuring encoding gene insertion genophore, uses the recombinant vector Conversion or transfection host cell, cultivate host cell, and therefrom reclaim and purify the tumour-specific lepirudin 023 ludon, Su Zhuxi Born of the same parents are Escherichia coli, yeast, insect cell or mammalian cell.

Correspondingly, anti-freezing, antithrombotic are being prepared, is preventing and treating swollen present invention also offers above-mentioned tumour-specific lepirudin 023 ludon Application in the medicine of knurl, especially anti-curing oncoma occurs together hypercoagulative state and medicine, health products and the feature of Thrombotic lesion Application in food.

Correspondingly, present invention also offers the adoptive immunity cell preparation method of expression tumour-specific lepirudin 023 ludon, Vitro culture of human source immunocyte, by people source coding sequence of secretory signal peptide encoding gene and above-mentioned tumour-specific lepirudin 023 ludon Encoding gene is connected, and adds restriction enzyme site at the two ends of restructuring encoding gene, will be made in encoding gene insertion vector It is standby to transfect immunocyte into recombination carrier, obtain the adoptive immunity cell of expression tumour-specific lepirudin 023 ludon.

Further, people source coding sequence of secretory signal peptide behaviour amidating enzyme signal peptide, its amino acid sequence is：SEQ ID NO.15, gene order is SEQ ID NO.16.

Further, immunocyte is one kind in α β T, gamma delta T, NKT, NK, DC, CIK, CAR-T, CAR-NK, TCR-T Or a variety of, immunocyte behaviour source immunocyte.

Further, the Membrane surface expression of adoptive immunity cell has target molecules, and target molecules are positioned by cross-film sequence In cell membrane surface.

Further, target molecules are CD19 total length or its truncated segment, or CD20 total length or its truncated segment.

Further, the structure of target molecules and cross-film section is：GM-CSFR signal peptides-Δ CD20-Linker2-CD28 across Film section.Its amino acid sequence is：SEQ ID NO.17, gene order is SEQ ID NO.18.

Using adoptive immunity cell as carrier, the continual and steady expression tumour-specific lepirudin 023 ludon in tumor patient body, The effect in " minicell pharmaceutical factory " is played, both by adoptive cellular immunotherapy technology and the anti-bolt of tumor-targeting lepirudin 023 ludon Anti-cancer function is integrated, and preferably plays antitumor action, can recombinate water by expression tumour-specific steady in a long-term in vivo again Leech element, it is to avoid inject tumour-specific lepirudin 023 ludon repeatedly and cause potential allergic reaction.

Correspondingly, answering present invention also offers the adoptive immunity cell of above-mentioned expression tumour-specific lepirudin 023 ludon With, for anti-freezing, antithrombotic, anti-curing oncoma biological therapy, especially prevent and treat with hypercoagulative state and/or the tumour of thrombus trouble The biological therapy of person.

Beneficial effects of the present invention are：The N-terminal of the tumour-specific lepirudin 023 ludon of the present invention contains and can recognized by uPA And the short peptide sequence cracked, the catalyzed by thrombin avtive spot of hirudin N-terminal can be closed, to reach that reducing the hirudin spreads out The anticoagulating active of biology, the purpose for reducing hemorrhage side effect, while also there is the target-seeking function of cancer target, when the hirudin derives Thing positioned at tumor microenvironment make when, using in tumor microenvironment be rich in uPA enzymes the characteristics of, the specific excision hirudin derivative The blocking peptide of N-terminal, makes the hirudin being closed reply again for hirudin original shape, and playing specific anti-freezing in tumor by local makees With so as to turn into a new class, safe and effective tumor-targeting anti-freezing, anti-bolt and anticancer.The present invention is further carried Supply to express the immunocyte of tumour-specific lepirudin 023 ludon, by using adoptive immunity cell as carrier, lasting table in vivo Up to tumour-specific lepirudin 023 ludon, it is possible to resolve the drawbacks of hirudin half-life short, without repetitively administered in the short time, it can avoid Repetitively administered causes allergy or induction of antibodies to produce, and is more suitable for needing long-term anti-freezing, the tumor patient of anti-bolt.Express tumour special The adoptive immunity cell of different in nature lepirudin 023 ludon can target tumor local expression, improvement tumor patient hypercoagulative state, the micro- blood of dissolving Bolt, prevents metastases, promotes immunocyte to enter inside tumor and plays killing ability, can be used alone or it is clinical with combination/ Radiotherapy is used.

Embodiment

The invention will be further described below.

The present invention tumour-specific lepirudin 023 ludon N-terminal contain can by the oligopeptide sequence that uPA is recognized and is cracked, UPA is the plasma urokinase-type plasminogen activator of tumor tissues height expression, and the hirudin for restructuring is hirudin isomers, water Leech element mutant, hirudin chimera, the HIRULOG truncated, the hirudin or hirudin fusion protein of genetic modification.

Natural hirudin has a variety of isomers, wherein three kinds of main isomers are respectively Hirudin variant 1 (HV1)、Hirudin variant 2(HV2)、Hirudin variant 3(HV3)。

Two amino acid of HV1 amino terminal are VV, and uPA physiologic substrate sequence is PGRVV, breakaway poing position after digestion For PGR ↓ VV.

Connecting PGR small peptides, or SGR, GSGR, GGSGR etc. in HV1 N-terminal has the small peptide of equivalent efficacy, with HV1 Two VV of N-terminal collectively constitute uPA substrate sequence PGRVV, after being cracked by uPA between R and V, can completely discharge The anticoagulating active of hirudin.

Further, oligopeptide sequence, which is, to be PGRVV by the physiologic substrate small peptide that uPA is recognized and is cracked, amino acid sequence；

Further, the N-terminal of recombinant tumor specificity hirudin is also associated with the polypeptide for targeted integration element α v β 3, The polypeptide includes RGD sequence.The integrin alpha v beta 3 of the more preferable target tumor microenvironment of above-mentioned hirudin derivative can then be made.Specifically RGD sequence be RGD4C sequences.After RGD4C oriented carriers are combined with tumor cell surface α v β 3, by uPA by hirudin Original shape is discharged, and plays anti-freezing, anti-bolt and antitumor action.

Correspondingly, present invention also offers the preparation method of above-mentioned tumour-specific lepirudin 023 ludon, comprise the following steps： The above-mentioned tumour-specific lepirudin 023 ludon genetic fragment of chemical synthesis, connects signal peptide, and encode base in restructuring in its N-terminal The two ends addition restriction enzyme site of cause, Prepare restructuring carrier in restructuring encoding gene insertion genophore is recombinated with this Carrier is converted or transfection host cell, cultivates host cell, and therefrom reclaim and purify the tumour-specific lepirudin 023 ludon, place Chief cell is Escherichia coli, yeast, insect cell or mammalian cell.

Carrier for restructuring is preferably carrier for expression of eukaryon, preferably pPIC9K.Host cell is preferably first Alcohol auxotype yeast strain GS115.

Correspondingly, present invention also offers the adoptive immunity cell preparation side for expressing above-mentioned tumour-specific lepirudin 023 ludon Method, people source coding sequence of secretory signal peptide encoding gene is connected with the encoding gene of above-mentioned tumour-specific lepirudin 023 ludon, is prepared Into recombination carrier, immunocyte is transfected, the adoptive immunity cell of expression tumour-specific lepirudin 023 ludon is obtained.

The Membrane surface expression of the adoptive immunity cell has target molecules, and target molecules are positioned at cell membrane by cross-film sequence Surface, tumour-specific lepirudin 023 ludon is directly expressed or carries out induced expression by being coupled inducible expression.Target molecules For CD19 total length or its truncated segment, or CD20 total length or its truncated segment.

Correspondingly, the adoptive immunity cell of present invention expression tumour-specific lepirudin 023 ludon, tumour-specific restructuring water Leech element can also be coupled inducible expression and be carried out induced expression with constructive expression.Being coupled inducible expression can be Tetracycline inducible expression, moulting hormone (ecdysone) inducible expression, tacrolimus (tacrolimus, FK506)/ One kind in rapamycin (rapamycin) inducible system or RU486 inducible systems.Immunocyte be α β T, gamma delta T, NKT, NK, One or more in DC, CIK, CAR-T, CAR-NK, TCR-T, immunocyte behaviour source immunocyte.

The adoptive immunity cell of expression tumour-specific lepirudin 023 ludon also expresses suicide gene/prodrug system.Suicide base Cause/prodrug system is cyclic guanosine (HSV-tk/GCV) system of herpes simplex virus type 1 thymidine kinase/penta, herpes zoster disease Flucytosine (the CD/ of malicious thymidine kinase/arabinose methoxypurine (VZV-tk/Ara-M) system, cytosine deaminase/5 5FC) system, Cytochrome P450 microsomal enzyme CYP2B1/ endoxan (CYP2B1/CPA) system；Cytochrome P450 CYP4B1/ amino anthracenes (CYP4B1/2AA) system, deoxycytidine kinase/cytarabine (dCK/Ara-C) system, black glycosides-Huang are fast Purine phosphoribosyl transferase/6- Sulfurs purine (gpt/6TX) system, nitroreductase/CB1954 (NTR/CB1954) system, Purine nucleoside phosphorylase/6- methyl purines deoxyribonucleoside (PNP/6-MeP-dR) system, thymidine phosphorylase/5 '-de- Oxygen -5 FU 5 fluorouracil (TP/5 '-DFUR) system, carboxypeptidase G2/CMDA systems (CPG2/CMDA), carboxy-lesterase/CPT-11 (CE/CPT-11) one kind in system.

The preparation method of the adoptive immunity cell of above-mentioned expression tumour-specific lepirudin 023 ludon, comprises the following steps： The genophore of the specific lepirudin 023 ludon of codes for tumor and membrane-type molecules target is prepared, genophore includes (signal peptide sequence Row-tumour-specific lepirudin 023 ludon sequence) and two sections of (signal peptide sequence-target molecules sequence-Linker- cross-film section sequence) Gene order；The genophore of the specific lepirudin 023 ludon of above-mentioned codes for tumor and target molecules is turned by technique for gene engineering Contaminate immunocyte.

Technique for gene engineering is known technology, and gene transfection system used includes slow-virus transfection system, retrovirus Transfection system, Adenovirus Transfection system, adeno-associated virus transfection system, sleeping beauty transposon stand transfection system, plasmid electrotransfection system System.It is preferred that, using sleeping beauty transposon stand transfection system or electrotransfection system.With transient expression vector, by tumour-specific weight Group hirudin gene imports adoptive immunity cell, its exogenous gene expression amount can be made controllable, it is to avoid the risk of overexpression.

The structure of genophore of codes for tumor specificity lepirudin 023 ludon and membrane-type molecules target is：Signal peptide-tumour Specific lepirudin 023 ludon-IRES- signal peptides-target molecules-Linker- cross-films section, or, signal peptide-target molecules- Linker- cross-films section-P2A- signal peptides-tumour-specific lepirudin 023 ludon.

With reference to embodiment, the present invention is further detailed explanation.

Embodiment 1

The lepirudin 023 ludon of the present embodiment is that have oligopeptides PGR, same hirudin in hirudin isomers HV1 N-terminal modification Isomers HV1 N-terminal sequence VV (valine-valine) is collectively constituted can be by uPA is recognized and is cracked physiologic substrate peptide sequence PGRVV is arranged, lepirudin 023 ludon amino acid sequence is：SEQ ID NO.1, gene order is：SEQ ID NO.2.

Wherein, PGRVV can be replaced by other uPA digestions substrate peptide sequences, and its common feature is includes RV sequences, such as： GSGRVV, GGSGRVV, LGGSGRVV.To ensure after uPA digestions, two amino acid of the hirudin amino terminal of release are VV.Hirudin isomers HV1 can be replaced by other N-terminals for VV hirudin isomers.

Application of the tumour-specific lepirudin 023 ludon in anti-freezing, anti-bolt, antineoplastic is prepared, is especially prevented and treated swollen Knurl occur together hypercoagulative state and Thrombotic lesion medicine in application.

Embodiment 2

The lepirudin 023 ludon of the present embodiment is by the Ser32-Asn33-Gly34-Glu35 of wild type hirudin HV1 peptide chains Arg32-Gly33-Asp34-Ser35 is replaced with, then in its N-terminal modification PGR oligopeptides, lepirudin 023 ludon amino acid sequence is： SEQ ID NO.3, gene order is：SEQ ID NO.4.

Embodiment 3

The lepirudin 023 ludon of the present embodiment is that the C-terminal sequence of wild type hirudin HV1 peptide chains is replaced with into wild type leech The C-terminal sequence of plain HV3 peptide chains, then in its N-terminal modification PGR oligopeptides, lepirudin 023 ludon amino acid sequence is：SEQ ID NO.7, Gene order is：SEQ ID NO.8.

Embodiment 4

The lepirudin 023 ludon of the present embodiment is targeted integration element α v β 3 polypeptide RGD4C and N-terminal amino acid is VV (figured silk fabrics ammonia Acid-valine) the fusion protein RPH (RGD4C- that are formed by connecting by Linker and oligopeptides PGR of hirudin isomers HV1 PGR-HV1), fusion protein RPH amino acid sequence is：SEQ ID NO.9, gene order is：SEQ ID NO.10.

Embodiment 5

The hirudin isomers HV1 that the lepirudin 023 ludon of the present embodiment is human albumin with N-terminal amino acid is VV passes through The fusion protein HPH that oligopeptides PGR is formed by connecting, fusion protein HPH (HSA-PGR-HV1) amino acid sequence is：SEQ ID NO.11, gene order is：SEQ ID NO.12.

Embodiment 6

The lepirudin 023 ludon of the present embodiment is Fc sections of people's gomphosis immunoglobulin and N-terminal amino acid is VV hirudin isomery The fusion protein F PH (HSA-PGR-HV1) that body HV1 is formed by connecting by oligopeptides PGR, Fc sections of people's gomphosis immunoglobulin includes people IgG4 hinge areas, Fc section sequence and IgM cauda sequences, FPH amino acid sequences are：SEQ ID NO.13, gene order is：SEQ ID NO.14。

Embodiment 7

The preparation method of the tumour-specific lepirudin 023 ludon of the present embodiment, comprises the following steps：The above-mentioned reality of chemical synthesis Any tumour-specific lepirudin 023 ludon genetic fragment of example 1~6 is applied, and connects into encoding gene, encoding gene is inserted Enter Prepare restructuring carrier in carrier, with the recombinant vector conversion or transfection host cell, host cell be Escherichia coli, yeast, Insect cell or mammalian cell, cultivate host cell, supernatant is collected by centrifugation afterwards, through ultrafiltration concentration, gel filtration, from Son exchanges three-step approach purifying and obtains tumour-specific lepirudin 023 ludon.

The carrier that the present embodiment is used to recombinate is preferably carrier for expression of eukaryon, using pPIC9K.Host cell is methanol Auxotype yeast strain GS115.

Host cell GS115 fermentation process is：Engineering bacteria is expanded with less salt culture medium, with containing trace element before induction Glycerite is carried out after feed supplement, culture propagation, carries out induced expression with the methanol solution containing trace element, fermentation parameter is：Temperature 30 DEG C of degree, oxygen capacity control are interlocked in 35 ± 5%, pH=5, mixing speed and DO.

Embodiment 8

The adoptive immunity cell preparation method of the expression tumour-specific lepirudin 023 ludon of the present embodiment is to secrete people source The encoding gene of the type signal DNA encoding peptide tumour-specific lepirudin 023 ludon any with above-described embodiment 1~6 is connected, and prepares Into recombination carrier, transfection immunocyte obtains the adoptive immunity cell of expression tumour-specific lepirudin 023 ludon.

Immunocyte is the one or more in α β T, gamma delta T, NKT, NK, DC, CIK, CAR-T, CAR-NK, TCR-T, is exempted from Epidemic disease cell behaviour source immunocyte.The present embodiment uses CIK cell.

The Membrane surface expression of adoptive immunity cell has target molecules, and target molecules are positioned at cell membrane table by cross-film sequence Face.Total length or phase truncated segment that target molecules are CD19, or CD20 total length or its truncated segment.

The present embodiment expression tumour-specific lepirudin 023 ludon adoptive immunity cell preparation method include step S1~ S3, it is specific as follows.

S1, vitro culture of human source CIK cell, including step A PMNC (PBMC) are gathered with step B's CIK cell culture.

A, the collection of PMNC have steps of：

A1, the extraction peripheral blood 50-100ml from patient's body；

PBMC is further purified in A2, lymphocyte separation medium density-gradient centrifugation method；

A3, serum-free medium are washed 2 times, obtain PBMC of the purity more than 90%.

B, CIK cell culture：

B1, by PBMC press 1~2 × 10⁶/ ml concentration is suspended in serum-free medium, adds 1,000U/ml restructuring People's IFN-γ, 37 DEG C, 5%CO₂Cultivated in incubator；

50ng/ml CD3 monoclonal antibodies and 300U/ml recombinant human il-2 are added after B2,24h, CIK cell is stimulated Growth and propagation；

Note：100U/ml recombined human IL-1 α now can be also added simultaneously.

B3, every 3 days half amounts change liquid or expand bottle once, and add recombinant human il-2 300U/ml；

B4, the 14d in culture, harvest CIK cell.

Wherein, 5%~20% autoserum can be also added in cell culture medium.

S2, Prepare restructuring genophore

This implementation row use tumour-specific lepirudin 023 ludon RPH (RGD4C-PGR-HV1) gene order, fusion protein RPH amino acid sequence is：SEQ ID NO.9, gene order is：SEQ ID NO.10.Leech is recombinated according to tumour-specific Plain RPH gene order, people's amidating enzyme signal peptide sequence is added in its N-terminal, and its amino acid sequence is：SEQ ID NO.15, gene order is SEQ ID NO.16.Chemical synthesis genetic fragment, connects into restructuring encoding gene, N-terminal addition The restriction enzyme sites of Nhe I, the C-terminal addition restriction enzyme sites of EcoR I.

This implementation row use target molecules structure for：GM-CSFR signal peptides-Δ CD20-Linker2-CD28 cross-films section, its Amino acid sequence is：SEQ ID NO.17, gene order is SEQ ID NO.18.Chemical synthesis genetic fragment, N-terminal addition BamH I restriction enzyme site, the C-terminal addition restriction enzyme sites of Not I.

The genophore of the specific lepirudin 023 ludon of codes for tumor and membrane-type molecules target is prepared using technique for gene engineering, By signal peptide sequence-tumour-specific lepirudin 023 ludon sequence and signal peptide sequence-target molecules sequence-Linker- cross-films section Two sections of genes of sequence are cloned into two cloning sites of pIRES plasmids respectively.Technique for gene engineering used is known technology, summary It is as follows：

The digestion with restriction enzyme system of standard is set up according to existing method, is then handled using Nhe I and EcoR I PIRES plasmids, reclaim large fragment；Handled using identical restriction enzyme Nhe I and EcoR I and to reclaim recombinant tumor special Property hirudin RPH genetic fragments, then the carrier pIRES large fragment good with above-mentioned digestion be connected, construct recombinant tumor special Property hirudin expression Plasmid pIRES-RPH；Convert after bacillus coli DH 5 alpha, filter out positive colony and expand.

PIRES-RPH plasmids are purified, then using BamH I and Not I processing pIRES-RPH plasmids, large fragment are reclaimed；Profit Handled with identical restriction enzyme BamH I and Not I and reclaim leukine R signal peptide-Δ CD20-Linker2- CD28 cross-films section genetic fragment, then the pIRES-RPH carrier large fragment good with above-mentioned digestion be connected, construct while expression is weighed The double expression plasmid pIRES-RPH/CD20 of group tumour-specific hirudin and membrane-type molecules target CD20.Convert Escherichia coli After DH5 α, filter out positive colony and expand, purify pIRES-RPH/CD20 plasmids.

The pIRES-RPH/CD20 recombinant plasmids that S3, purifying are obtained through step S2, with the recombinant vector with plasmid electrotransfection The CIK cell that method transfection procedure S1 is obtained, carries out more than cell culture 36h afterwards, and the CIK for obtaining expressing lepirudin 023 ludon is thin Born of the same parents, transfection efficiency is obtained through flow cytomery, is needed positive cell quantity needed for calculating according to clinic, is fed back tumor patient.

The CIK cell of the expression tumour-specific lepirudin 023 ludon, the biology for anti-freezing, antithrombotic, anti-curing oncoma is controlled Treat, especially prevent and treat the biological therapy of the tumor patient with hypercoagulative state and/or thrombus.

Above-described is only some embodiments of the present invention.For the person of ordinary skill of the art, not On the premise of departing from the invention design, various modifications and improvements can be made, these belong to the protection model of the present invention Enclose.

Sequence table

<110>He Xiangfeng

<120>Tumour-specific lepirudin 023 ludon and its production and use

<160>18

<210>1

<211>68

<212>PRT

<213>Artificial sequence

<400>1

Pro Gly Arg Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn

5 10 15

Leu Cys Leu Cys Glu Asp Ser Asn Val Cys Gly Gln Gly Asn Lys

20 25 30

Cys Ile Leu Gly Ser Asn Gly Glu Lys Asn Gln Cys Val Thr Gly

35 40 45

Glu Gly Thr Pro Lys Pro Gln Ser His Asn Asp Gly Asp Phe Glu

50 55 60

Glu Ile Pro Glu Glu Tyr Leu Gln

65

<210>2

<211>207

<212>DNA

<213>Artificial sequence

<400>2

ccaggcaggg ttgtttacac ggattgtaca gaatcgggtc aaaatttgtg cctctgcgag 60

gatagcaatg tttgcggtca aggcaataag tgcatattgg gttctaatgg agagaaaaac 120

caatgtgtca ctggcgaagg tacaccgaag cctcaaagcc ataatgacgg cgatttcgaa 180

gaaattccag aagaatattt acaatag 207

<210>3

<211>68

<212> PRT

<213>Artificial sequence

<400>3

Pro Gly Arg Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn

5 10 15

Leu Cys Leu Cys Glu Asp Ser Asn Val Cys Gly Gln Gly Asn Lys

20 25 30

Cys Ile Leu Gly Arg Gly Asp Ser Lys Asn Gln Cys Val Thr Gly

35 40 45

Glu Gly Thr Pro Lys Pro Gln Ser His Asn Asp Gly Asp Phe Glu

50 55 60

Glu Ile Pro Glu Glu Tyr Leu Gln

65

<210>4

<211>207

<212> DNA

<213>Artificial sequence

<400>4

ccaggcaggg ttgtttacac ggattgtaca gaatcgggtc aaaatttgtg cctctgcgag 60

gatagcaatg tttgcggtca aggcaataag tgcatattgg gtcgcggaga ttctaaaaac 120

caatgtgtca ctggcgaagg tacaccgaag cctcaaagcc ataatgacgg cgatttcgaa 180

gaaattccag aagaatattt acaatag 207

<210>5

<211>68

<212> PRT

<213>Artificial sequence

<400>5

Pro Gly Arg Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn

5 10 15

Leu Cys Leu Cys Glu Asp Ser Asn Val Cys Gly Gln Gly Asn Lys

20 25 30

Cys Ile Leu Gly Arg Gly Asp Met Lys Asn Gln Cys Val Thr Gly

35 40 45

Glu Gly Thr Pro Lys Pro Gln Ser His Asn Asp Gly Asp Phe Glu

50 55 60

Glu Ile Pro Glu Glu Tyr Leu Gln

65

<210>6

<211>207

<212> DNA

<213>Artificial sequence

<400>6

ccaggcaggg ttgtttacac ggattgtaca gaatcgggtc aaaatttgtg cctctgcgag 60

gatagcaatgt ttgcggtcaa ggcaataagt gcatattggg tcgcggagat atgaaaaac 120

caatgtgtca ctggcgaagg tacaccgaag cctcaaagcc ataatgacgg cgatttcgaa 180

gaaattccaga agaatattta caatag 207

<210>7

<211> 70

<212> PRT

<213>Artificial sequence

<400>7

Pro Gly Arg Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn

5 10 15

Leu Cys Leu Cys Glu Asp Ser Asn Val Cys Gly Gln Gly Asn Lys

20 25 30

Cys Ile Leu Gly Ser Asn Gly Glu Lys Asn Gln Cys Val Thr Gly

35 40 45

Glu Gly Thr Pro Lys Pro Gln Ser His Asn Gln Gly Asp Phe Glu

50 55 60

Pro Ile Pro Glu Asp Ala Tyr Asp Glu Lys

65 70

<210>8

<211>213

<212> DNA

<213>Artificial sequence

<400>8

ccaggcaggg ttgtttacac ggattgtaca gaatcgggtc aaaatttgtg cctctgcgag 60

gatagcaatg tttgcggtca aggcaataag tgcatattgg gttctaatgg agagaaaaac 120

caatgtgtca ctggcgaagg tacaccgaag cctcaaagcc ataatcaagg cgatttcgaa 180

ccaattccag aagacgctta tgatgaaaaa tga 213

<210>9

<211>90

<212> PRT

<213>Artificial sequence

<400>9

Ala Cys Asp Cys Arg Gly Asp Cys Phe Cys Gly Ser Gly Gly

5 10

Gly Gly Ser Gly Gly Gly Gly Ser Pro Gly Arg Val Val Tyr Thr Asp

15 20 25 30

Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Asp Ser Asn

35 40 45

Val Cys Gly Gln Gly Asn Lys Cys Ile Leu Gly Ser Asn Gly Glu

50 55 60

Lys Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser

65 70 75

His Asn Asp Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Gln

80 85 90

<210>10

<211>273

<212> DNA

<213>Artificial sequence

<400>10

gcatgtgatt gtaggggaga ttgtttttgt ggtagcggtg gcggtggctc tggtggtggt 60

ggcagcccag gcagggttgt ttacacggat tgtacagaat cgggtcaaaa tttgtgcctc 120

tgcgaggata gcaatgtttg cggtcaaggc aataagtgca tattgggttc taatggagag 180

aaaaaccaat gtgtcactgg cgaaggtaca ccgaagcctc aaagccataa tgacggcgat 240

ttcgaagaaa ttccagaaga atatttacaa tag 273

<210>11

<211>653

<212> PRT

<213>Artificial sequence

<400>11

Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly

5 10 15

Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr

20 25 30

Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu

35 40 45

Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu

50 55 60

Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys

65 70 75

Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys

80 85 90

Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His

95 100 105

Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val

110 115 120

Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu

125 130 135

Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr

140 145 150

Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe

155 160 175

Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro

180 185 190

Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys

195 200 205

Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala

210 215 220

Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys

225 230 235

Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys

240 245 250

Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp

255 260 265

Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser

270 275 280

Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu

285 290 295

Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala

300 305 310

Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val

315 320 325

Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe

330 335 340

Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu

345 350 355

Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys

360 365 370

Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp

375 380 385

Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln

390 395 400

Asn Cys Glu Leu Phe Lys Gln Leu Gly Glu Tyr Lys Phe Gln Asn

405 410 415

Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr

420 425 430

Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser

435 440 445

Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu

450 455 450

Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu

455 460 465

Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser

470 475 480

Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu

485 490 495

Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His

500 505 510

Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys

515 520 525

Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr

530 535 540

Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val

545 550 555

Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu

560 565 570

Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu

575 580 585

Pro Gly Arg Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn

590 595 600

Leu Cys Leu Cys Glu Asp Ser Asn Val Cys Gly Gln Gly Asn Lys

605 610 615

Cys Ile Leu Gly Ser Asn Gly Glu Lys Asn Gln Cys Val Thr Gly

620 625 630

Glu Gly Thr Pro Lys Pro Gln Ser His Asn Asp Gly Asp Phe Glu

635 640 645

Glu Ile Pro Glu Glu Tyr Leu Gln

650

<210>12

<211>1962

<212> DNA

<213>Artificial sequence

<400>12

gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60

gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120

aaattagtga atgaagtaac tgaatttgca aaaacatgtg tagctgatga gtcagctgaa 180

aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240

cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300

tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360

gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420

gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480

tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540

aagctcgatg aacttcggga tgaagggaag gcttcgtctg ccaaacagag actcaaatgt 600

gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtggc tcgcctgagc 660

cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720

gtccacacgg aatgctgcca tggagatctg cttgaatgtg ctgatgacag ggcggacctt 780

gccaagtata tctgtgaaaa tcaggattcg atctccagta aactgaagga atgctgtgaa 840

aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900

gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960

gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020

tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080

tgtgccgctg cagatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140

gtggaagagc ctcagaattt aatcaaacaa aactgtgagc tttttaagca gcttggagag 1200

tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260

ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gcagcaaatg ttgtaaacat 1320

cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380

tgtgtgttgc atgagaaaac gccagtaagt gacagagtca caaaatgctg cacagagtcc 1440

ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg atgaaacata cgttcccaaa 1500

gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560

agacaaatca agaaacaaac tgcacttgtt gagcttgtga aacacaagcc caaggcaaca 1620

aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680

gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740

gctgccttag gcttaccagg cagggttgtt tacacggatt gtacagaatc gggtcaaaat 1800

ttgtgcctct gcgaggatag caatgtttgc ggtcaaggca ataagtgcat attgggttct 1860

aatggagaga aaaaccaatg tgtcactggc gaaggtacac cgaagcctca aagccataat 1920

gacggcgatt tcgaagaaat tccagaagaa tatttacaat ag 1962

<210>13

<211>313

<212> PRT

<213>Artificial sequence

<400>13

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

5 10 15

Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

20 25 30

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

65 70 75

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

80 85 90

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

95 100 105

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

110 115 120

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

125 130 135

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

140 145 150

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

155 160 165

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

170 175 180

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

185 190 195

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

200 205 200

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

215 220 225

Ser Leu Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser

230 235 240

Asp Thr Ala Gly Thr Pro Gly Arg Val Val Tyr Thr Asp Cys Thr

245 250 255

Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Asp Ser Asn Val Cys

260 265 270

Gly Gln Gly Asn Lys Cys Ile Leu Gly Ser Asn Gly Glu Lys Asn

275 280 285

Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn

290 295 300

Asp Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Gln

305 310

<210>14

<211>942

<212>DNA

<213>Artificial sequence

<400>14

gaatctaagt acggccctcc ctgcccaccc tgtcctgctc cagagtttct gggcggaccc 60

tccgtgttcc tgttcccccc aaagcccaag gacaccctga tgatctcccg gacccccgaa 120

gtgacctgcg tggtggtgga cgtgtcccag gaagatcccg aggtccagtt caattggtac 180

gtggacggcg tggaagtgca caacgccaag accaagccca gagaggaaca gttcaactcc 240

acctaccggg tggtgtctgt gctgaccgtg ctgcaccagg actggctgaa cggcaaagag 300

tacaagtgca aggtctccaa caagggcctg ccctccagca tcgaaaagac catctccaag 360

gccaagggcc agccccgcga gcctcaggtg tacacactgc cccctagcca agaagagatg 420

accaagaacc aggtgtccct gacatgcctg gtgaagggct tctacccctc cgatatcgcc 480

gtggaatggg agtccaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg 540

gactccgacg gctccttctt cctgtactct cggctgaccg tggacaagtc ccggtggcag 600

gaaggcaacg tcttctcctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 660

aagtctctga gcctgtccct gggcaagccc accctgtaca acgtgtccct ggtcatgtcc 720

gacacagctg gcaccccagg cagggttgtt tacacggatt gtacagaatc gggtcaaaat 780

ttgtgcctct gcgaggatag caatgtttgc ggtcaaggca ataagtgcat attgggttct 840

aatggagaga aaaaccaatg tgtcactggc gaaggtacac cgaagcctca aagccataat 900

gacggcgatt tcgaagaaat tccagaagaa tatttacaat ag 942

<210>15

<211>20

<212> PRT

<213>Artificial sequence

<400>15

Met Ala Gly Arg Val Pro Ser Leu Leu Val Leu Leu Val Phe Pro

5 10 15

Ser Ser Cys Leu Ala

20

<210>16

<211>60

<212>DNA

<213>Artificial sequence

<400>16

atggctggcc gcgtccctag cctgctagtt ctccttgttt ttccaagcag ctgtttggct 60

<210>17

<211>83

<212> PRT

<213>Artificial sequence

<400>17

Met Val Leu Ala Gln Gly Leu Leu Ser Met Ala Leu Leu Ala Leu

5 10 15

Cys Trp Glu Arg Ser Leu Ala Asn Ile Tyr Asn Cys Glu Pro Ala

20 25 30

Asn Pro Ser Glu Lys Asn Ser Pro Ser Thr Gln Tyr Cys Tyr Ser

35 40 45

Ile Gln Ser Gly Gly Ser Gly Gly Pro Phe Trp Val Leu Val Val

50 55 60

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala

65 70 75

Phe Ile Ile Phe Trp Val Arg Ser

80

<210>18

<211>252

<212> DNA

<213>Artificial sequence

<400>18

atggtgctgg cccaggggct gctctccatg gccctgctgg ccctgtgctg ggagcgcagc 60

ctggcaaaca tatacaactg tgaaccagct aatccctctg agaaaaactc cccatctacc 120

caatactgtt acagcataca atctggcgga agcggaggcc ccttttgggt gctggtggtg 180

gttggtggag tcctggcttg ctatagcttg ctagtaacag tggcctttat tattttctgg 240

gtgaggagtt aa 252

Claims

1. tumour-specific lepirudin 023 ludon, it is characterised in that the N-terminal of the lepirudin 023 ludon contains can be by uPA identifications simultaneously The oligopeptide sequence of cracking, uPA is the plasma urokinase-type plasminogen activator of tumor tissues height expression, and the hirudin for restructuring is Hirudin isomers, hirudin mutant, hirudin chimera, the HIRULOG truncated, the hirudin or hirudin of genetic modification Fusion protein.

2. tumour-specific lepirudin 023 ludon according to claim 1, it is characterised in that the oligopeptide sequence is can quilt The physiologic substrate small peptide that uPA is recognized and cracked, amino acid sequence is Pro-Gly-Arg-Val-Val, i.e. PGRVV；

Or, the oligopeptide sequence is can be by the small peptide for the artificial optimization that uPA is recognized and is cracked, and its architectural feature is XnRVV, Xn Represent any number of arbitrary amino acid.

3. tumour-specific lepirudin 023 ludon according to claim 2, it is characterised in that the lepirudin 023 ludon is in water Leech element isomers HV1 N-terminal is modified with oligopeptides PGR, and the N-terminal sequence VV with hirudin isomers HV1 is collectively constituted and can known by uPA Not and the physiologic substrate peptide sequence PGRVV that cracks, the lepirudin 023 ludon amino acid sequence is：SEQ ID NO.1, gene order For：SEQ ID NO.2；

Or, the lepirudin 023 ludon is to replace with the Ser32-Asn33-Gly34-Glu35 of wild type hirudin HV1 peptide chains Arg32-Gly33-Asp34-Ser35, then in its N-terminal modification PGR oligopeptides, the lepirudin 023 ludon amino acid sequence is：SEQ ID NO.3, gene order is：SEQ ID NO.4；

Or, the lepirudin 023 ludon is to replace with the Ser32-Asn33-Gly34-Glu35 of wild type hirudin HV1 peptide chains Arg32-Gly33-Asp34-Met35, then in its N-terminal modification PGR oligopeptides, the lepirudin 023 ludon amino acid sequence is：SEQ ID NO.5, gene order is：SEQ ID NO.6；

Or, the lepirudin 023 ludon is that the C-terminal sequence of wild type hirudin HV1 peptide chains is replaced with into wild type hirudin HV-3 The C-terminal sequence of peptide chain, then in its N-terminal modification PGR oligopeptides, the lepirudin 023 ludon amino acid sequence is：SEQ ID NO.7, Gene order is：SEQ ID NO.8；

Or, the lepirudin 023 ludon be targeted integration element α v β 3 polypeptide RGD4C and N-terminal amino acid be VV hirudin it is different The fusion protein RPH that structure body HV1 is formed by connecting by Linker and oligopeptides PGR, fusion protein RPH amino acid sequence is：SEQ ID NO.9, gene order is：SEQ ID NO.10；

Or, the hirudin isomers HV1 that the lepirudin 023 ludon is human albumin with N-terminal amino acid is VV passes through oligopeptides The fusion protein HPH that PGR is formed by connecting, fusion protein HPH amino acid sequence is：SEQ ID NO.11, gene order is： SEQ ID NO.12；

Or, the lepirudin 023 ludon is Fc sections of people's gomphosis immunoglobulin and N-terminal amino acid is VV hirudin isomers The fusion protein F PH that HV1 is formed by connecting by oligopeptides PGR, Fc sections of people's gomphosis immunoglobulin comprising the hinge area of human IgG 4, Fc sections Sequence and IgM cauda sequences, FPH amino acid sequences are：SEQ ID NO.13, gene order is：SEQ ID NO.14.

4. the preparation method of tumour-specific lepirudin 023 ludon, it is characterised in that comprise the following steps：Chemical synthesis claim Any one of 1~3 tumour-specific lepirudin 023 ludon genetic fragment, signal peptide is connected in its N-terminal, and in restructuring encoding gene Two ends addition restriction enzyme site；Restructuring encoding gene is inserted into Prepare restructuring carrier in genophore；Carried with the restructuring Body is converted or transfection host cell；Host cell is cultivated, and therefrom reclaims and purify the tumour-specific lepirudin 023 ludon, host Cell is Escherichia coli, yeast, insect cell or mammalian cell.

5. the tumour-specific lepirudin 023 ludon described in any one of claims 1 to 3 is preparing anti-freezing, antithrombotic, anti-curing oncoma Medicine in application, especially anti-curing oncoma occurs together medicine, health products and the feature food of hypercoagulative state and Thrombotic lesion Application in product.

6. express the adoptive immunity cell preparation method of tumour-specific lepirudin 023 ludon, it is characterised in that vitro culture of human source Immunocyte, by the tumour-specific lepirudin 023 ludon of any one of people source coding sequence of secretory signal peptide encoding gene and claims 1 to 3 Encoding gene be connected, and restructuring encoding gene two ends add restriction enzyme site, by encoding gene insertion vector Recombination carrier is prepared into, transfection immunocyte obtains the adoptive immunity cell of expression tumour-specific lepirudin 023 ludon.

7. the adoptive immunity cell preparation method of expression tumour-specific lepirudin 023 ludon according to claim 6, it is special Levy and be, the people source coding sequence of secretory signal peptide behaviour amidating enzyme signal peptide, its amino acid sequence is：SEQ ID NO.15, base Because sequence is SEQ ID NO.16.

8. the adoptive immunity cell preparation method of expression tumour-specific lepirudin 023 ludon according to claim 6, it is special Levy and be, the immunocyte is the one or more in α β T, gamma delta T, NKT, NK, DC, CIK, CAR-T, CAR-NK, TCR-T, The immunocyte behaviour source immunocyte.

9. the adoptive immunity cell preparation method of expression tumour-specific lepirudin 023 ludon according to claim 6, it is special Levy and be, the Membrane surface expression of the adoptive immunity cell there are target molecules, and target molecules are positioned at cell by cross-film sequence Film surface.

10. the adoptive immunity cell preparation method of expression tumour-specific lepirudin 023 ludon according to claim 9, it is special Levy and be, total length or its truncated segment that the target molecules are CD19, or CD20 total length or its truncated segment.

11. the adoptive immunity cell preparation method of expression tumour-specific lepirudin 023 ludon according to claim 10, its It is characterised by, the structure of target molecules and the cross-film section is：GM-CSFR signal peptides-Δ CD20-Linker2-CD28 cross-films Section, its amino acid sequence is：SEQ ID NO.17, gene order is SEQ ID NO.18.

12. described in claim 6 expression tumour-specific lepirudin 023 ludon adoptive immunity cell, for anti-freezing, antithrombotic, Tumor biotherapy, especially prevents and treats the biological therapy of the tumor patient with hypercoagulative state and/or thrombus.