CN103965364B

CN103965364B - A kind of people source PDL2HSA series fusion protein and preparation and application thereof

Info

Publication number: CN103965364B
Application number: CN201410210929.7A
Authority: CN
Inventors: 刘辰; 刘源
Original assignee: YAFEI (SHANGHAI) BIO-PHARMACEUTICAL Co Ltd
Current assignee: Shanghai Helicheng Biomedical Technology Co ltd
Priority date: 2014-05-19
Filing date: 2014-05-19
Publication date: 2016-06-08
Anticipated expiration: 2034-05-19
Also published as: CN103965364A

Abstract

The invention provides a kind of people source PDL2HSA series fusion protein, including the fusion protein that the partial domain of human body PDL2 albumen is directly connected to human albumin, does is sequence SEQ? ID? the aminoacid sequence of N0.1 and SEQ? ID? the nucleotide sequence of N0.2, and the fusion protein that the partial domain of human body PDL2 albumen is connected by small peptide with human albumin. The present inventor is tested at the drug efficacy study of D121 lung cancer tumor immune model by PDL2HSA series fusion protein, and PDLHSA series fusion protein grows the drug efficacy study of model (MMTV-PyVmT) tumor model turning of transfevent mastocarcinoma gene, result display people source PDLHSA series fusion protein has the good drug effect suppressing growth and metastasis of tumours. Therefore, the present invention provides PDLHSA series fusion protein first and may be used for the reagent/test kit of the preparation detection PD-1 reagent combined and the external internal activation of T cell, and prepares antineoplastic immune drug or bacterin preparation. There is provided new effective bio-pharmaceutical for clinical treatment tumour disease and chronic infection, have bigger using value.

Description

A kind of people source PDL2HSA series fusion protein and preparation and application thereof

Technical field

The present invention relates to biological technical field, be specifically related to a kind of human immunity checkpoint modulability fusion protein, particularly relate to a kind of people source PDL2HSA series fusion protein and preparation and application thereof.

Background technology

Existing bibliographical information, programmatic death I (PD-1) mainly expressed in T cell can combine from upper programmatic death ligand II (PD-L2) albumen of different antigen presenting cells (APC). The PD-L2 that antigen presenting cell APC expresses can pass through the connection of PD-1 and suppress t cell activation, thus helping tumor to escape immunne response. (.J.Exp.Med.201:1531 such as Shin). The structure of PD-1 and PD-L2 is had been disclosed at present, and these molecules activate and the signal conduction feature in tolerated and function (Kier etc. (2008) Ann.Rev.Immunol.26:677) at modulating T cell.

The present inventor passes through scientific research and multiple fusion protein expression, it has been found that the protein that dissimilar or small peptide connects is by, after merging with PD-L2 partial domain, producing novel albumen the binding ability of PD-1 is different. Illustrate that its fusion protein is by forming new protein conformation, after changing the intermolecular force at calmodulin binding domain CaM position, the combination of fusion protein PD-1 can be produced material impact. The structure activity relationship new according to fusion protein and PD-1 are in conjunction with screening experiment, and animal model carries out T cell propagation screening, and we obtain current most probable the people source PDL2HSA series fusion protein of therapeutic effect. And be verified in the animal experiment of tumour immunity model and spontaneous tumor model further, the Immunestimulatory effect of this albuminoid is better than PDL2 and antibody crystal region fusion protein (PDL2-FC) and anti-PD-1 antibody, and in the side effect of less autoimmune response, for diseases such as the immunization therapy of clinical treatment tumour disease or chronic infections, reverse the medicine that T cell exhaustion provides new.

Summary of the invention

The technical problem to be solved is in that research design people source PDL2HSA series fusion protein and preparation method thereof and the application in pharmacy.

The invention provides a kind of people source PDL2HSA series fusion protein. Described

People source PDL2HSA series fusion protein includes the fusion protein (fusion protein of PDL2-n-HSA, n=0��12 amino acid short peptides) that the partial domain of fusion protein (PDL2-HSA) that the partial domain of human body PDL2 albumen is directly connected to and human body PDL2 albumen is connected by small peptide with human albumin with human albumin.

The invention provides the aminoacid sequence SEQIDN0.1 corresponding to PDL2-HSA fusion protein and nucleotide sequence SEQIDN0.2 that the partial domain of a kind of people source PDL2 albumen and human albumin are directly connected to. Present invention also offers the sequence connecting small peptide corresponding to the PDL2-n-HSA fusion protein of people source and nucleotide sequence and the on position at SEQIDN0.1 and SEQIDN0.2.

The preparation method that it is a further object of the present invention to provide described a kind of people source PDL2HSA series fusion protein, the method comprises the following steps:

(1) nucleic acid molecules of the people source PDL2HSA series fusion protein of preparative separation:

The nucleic acid of the coding PDL2HSA series fused protein separated is obtained by polymerase chain reaction (PCR) technology; Or it is directly synthesized the nucleic acid molecules of PDL2HSA series fused protein; Or obtained the nucleic acid molecules of PDL2HSA series fused protein to expression vector by the nucleic acid molecules of enzyme action connection different fragments.

(2) people source PDL2HSA series fused protein is prepared

Obtained by the restructuring in host cell: with the nuclear transformation of the nucleotide sequence containing coded polypeptide, transduction or transfection antibacterial or eukaryotic host cell, the multiple expression system based on virus is utilized to express PDL2HSA fused protein, preparation people source PDL2HSA fused protein

(3) Purification of Human source PDL2HSA series fused protein.

The eukaryotic host cell of step described in the inventive method (2) is insecticide, yeast or mammalian cell. It is it is known in the art that to include such as mammalian cell such as Chinese hamster ovary cell Chinese hamster ovary celI for expressing and preparing the useful eukaryotic system of polypeptide. For eukaryotic host cell, the available multiple expression system based on virus expresses PDL2-HSA fused protein. Expression system based on virus is it is known in the art that and includes but not limited to baculovirus, simian virus 40 (SV40), retrovirus or the viral vector based on vaccine. The expression vector with suitable regulatory element and selection marker is used to stimulate the mammal cell line of polypeptide altogether to prepare stably express variant, such as, PDL2HSA series fused protein can use glutamine synthetase pressurization screening (GS) eukaryotic expression system of Lonza company, the dihydrofolate reductase (DHFR) of Invitrogen company screens eukaryotic expression system, and Pichia sp. (Pichia) yeast expression system of Invitrogen company carries out expressing and preparing.

The Purification of Human source PDL2HSA series fused protein of step described in the inventive method (3) can use such as chromatography method to separate PD-I antagonist polypeptide, such as DEAE ion exchange, gel filtration and hydroxyapatite chromatography. It is, for example possible to use Protein G post carrys out the common stimulation polypeptide in Dissociated cell culture thing supernatant or Cytoplasmic extract. In some embodiments, it is possible to " engineered " variant stimulates polypeptide to allow that polypeptide is caught in the aminoacid sequence on affinity substrate to contain altogether.It is, for example possible to use label helps peptide purification, such as proto-oncogenes proteins c-myc, hemagglutinin, polyhistidine or FlagTM (Kodak). This type of label may be inserted into any place in polypeptide, arbitrary including carboxyl or aminoterminal. Other fusion come in handy includes the enzyme helping detection polypeptide, such as alkali phosphatase. Immunoaffinity chromatography can also be used to carry out purification and to stimulate polypeptide altogether.

It is yet another object of the invention to provide described a kind of people source PDL2HSA series fusion protein for preparing the relevant reagent/test kit of the detection PD-1 relevant reagent combined and the external internal activation of T cell.

In the embodiment of the present application 2, therefore PDL2HSA fusion protein directly in conjunction with PD-1, can be used directly for the preparation detection PD-1 relevant reagent/test kit combined. In embodiment 3, PDL2HSA fusion protein can stimulate T cell to increase, therefore PDL2HSA fusion protein may be directly applied to the activation of T cell body activate in reagent/or the test kit relevant with activation, PDL2HSA series fusion protein can also be applied to In vitro culture, activates or proliferative T cell. PDL2 and antibody crystal region fusion protein (PDL2-FC) are because of containing antibody crystal region (FC) part, there is FC recipient cell generally have B cell, kill cell, macrophage, these cells by FC receptor in conjunction with PDL2-FC fusion protein, therefore can still be likely to premunition and suppress signal. And because PDL2-FC fusion protein is in conjunction with sending out after FC receptor endocytosis celliferous, it is possible to reduce the concentration of extracellular PDL2-FC fusion protein, thus reduce the effect being combined with PD-1 and stimulating T cell to breed. The inventors discovered that owing to people source PDL2HSA series albumen can have curative effect in the animal experiment of tumour immunity model and spontaneous tumor model, show that only people source PDL2HSA series albumen can meet and clinical medicine development process needs zooperal drug effect requirement.

It is a still further object of the present invention to provide the application in preparation treatment tumor disease medicine of described a kind of people source PDL2HSA series fusion protein.

In tumour patient, tumor cell, or with the antigen presenting cell (APC) of tumor antigen by conjunction with T cell, and partially or completely suppress the immunologic cytotoxicity power for tumor of host. And PDL2HSA series fused protein can be urged to increase through T cell in embodiment 3, therefore PDL2HSA series fused protein can be used for improving the proliferated specifically for antigen of T cell, tumour patient is applied PDL2HSA series fusion protein can directly treat as medicine or all will urge as auxiliary treatment to breed through T cell, cause the strong immunologic cytotoxicity power for tumor antigen the most at last. The present inventor grows the drug efficacy study (embodiment 4) of model (MMTV-PyVmT) tumor model by people source PDL2HSA series fusion protein turning of transfevent mastocarcinoma gene, result shows and model control group, compared with PDL2-FC group (PDL2 and antibody crystal region fusion protein) and PDL2-transferrin group (PDL2 and transferrin fusion protein) group, PDL2HSA fusion protein treatment group can be greatly improved suppression tumor growth effect, has good medicine for treating tumor effect.

The present inventor by answering employment source PDL2HSA series fusion protein to find in kinds of tumors model, and people source PDL2HSA series fusion protein can stimulate increasing of tumor locus CD8 positive T cell, thus improving the immunologic cytotoxicity power for tumor of host.Because this person source PDL2HSA series fusion protein is the treatment that can operate with various cancers type. Cancer types of the present invention includes but not limited to following: bladder, brain, breast/mammary gland, cervix uteri, colon-rectum, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, uterus, ovary, testis and hematological. Treatable malignant tumor is sorted out according to embryo's origin of the tissue of derivative tumors in this article. Carcinoma (Carcinoma) is the tumor being derived from entoderm or ectodermal histological, the epithelial layer of such as skin or internal organs and gland. Less frequently the sarcoma (Sarcoma) of generation is derived from mesoderm connective tissue, such as bone, fat and cartilage. The malignant tumor of the hematopoietic cell that leukemia (leukemia) and lymphoma (lymphoma) are bone marrow. Leukemia is bred as unicellular, and lymphoma tends to growing as tumor mass. Malignant tumor can reveal to produce cancer at the place of multiple organ or tissue of health.

People source of the present invention PDL2HSA fusion protein can be independent be used in promote and improve host produce lasting immunologic cytotoxicity power for tumor, and antineoplastic chemotherapy medicine can directly kill tumor cell, directly kill and pass through the short treatment not contradiction killed through immunocyte. Because this person source PDL2HSA fusion protein can apply to auxiliary treatment and the composition of medicine of antineoplastic chemotherapy medicine. Representational antineoplastic chemotherapy medicine includes but not limited to amsacrine (amsacrine), bleomycin (bleomycin), busulfan (busulfan), capecitabine (capecitabine), card molybdenum (carboplatin), carmustine (carmustine), chlorambucil (chlorambucil), suitable molybdenum (cisplatin), cladribine (cladribine) ^0,3/4^*3/4 (clofarabine) > crisantaspase > �� ^1^1:1³/₄(cyclophosphamide) > cytosine arabinoside (cytarabine), dacarbazine (dacarbazine), actinomycin D (dactinomycin), soft red clouds element (daunorubicin), docetaxel (docetaxel), doxorubicin (doxorubicin), epirubicin (print irubicin), etoposide (etoposide), fludarabine (fludarabine), fluorouracil (fluorouracil), gemcitabine (gemcitabine), light base urea (hydroxycarbamide), idarubicin (idarubicin), ifosfamide (ifosfamide), irinotecan (irinotecan), folinic acid (Ieucovorin), doxorubicin moon purport plastid (liposomaldoxorubicin), soft red clouds bright and clear moon purport plastid (liposomaldaunorubicin), lomustine (Iomustine), melphalan (melphalan), mercaptopurine (mercaptopurine), mesna (mesna), first atmosphere butterfly cry of certain animals (methotrexate), silk splits clouds element (mitomycin), meter Tuo Hui elder brother at the tenth of the twelve Earthly Branches (mitoxantrone), Ao Shali molybdenum (oxaliplatin), middle Bai Litasai (paclitaxel), pemetrexed (pemetrexed), his T (pentostatin) of spray department, third kappa (procarbazine) > Raltitrexed (raltitrexed), husky fearness (satraplatin), chain azoles star (str prints tozocin), UFT (tegafur-uracil), temozolomide (temozolomide), (teniposide) is moored former times for Buddhist nun, phosphinothioylidynetrisaziridine (thiot prints a), sulfur L bird mouth ticket cry of certain animals (tioguanine), hycamtin (topotecan), treosulfan (treosulfan), vinblastine (vinblastine), vincristine (vincristine), vindesine (vindesine), vinorelbine (vinorelbine) or its combination.Representational short apoptosis agent includes but not limited to fludarabine (fludarabine), Calculus Bovis this smooth (taurosporine), cycloheximide (cycloheximide), actinomycin D (actinomycinD), lactosylceramide (Iactosylceramide).

Antineoplastic immune drug of the present invention or bacterin preparation are made up as active component and pharmaceutical carrier according to a conventional method of people source PDL2HSA series fusion protein.

People source of the present invention PDL2HSA series fusion protein immunization medicine can be directly used for improving the immunity for cancer of patient, and thus treats cancer. Embodiment 3 is the development test in D121 lung cancer tumor immune model of the people source PDL2HSA series fusion protein. The tumor cell of fractional injection death carries out antineoplastic immune or vaccine. And in the tumor cell same period of fractional injection death, injection people source PDL2-HSA fusion protein, it is possible to the short antineoplastic immune through host or vaccine, cause the stronger lethality for tumor. Result shows and compares immune group and PDL2-FC immune group (PDL2 and antibody crystal region fusion protein) and PDL2-transferrin immune group (PDL2 and transferrin fusion protein), PD-1 antibody group is compared, it is greatly improved at the tumor growth inhibitory effect of C57 mice containing PDL2HSA amalgamation protein vaccine group, illustrates that it has good antineoplastic immunization therapy or vaccine effect. PD-1 antibody group curative effect is also bad, and its possible cause is that PD-1 antibody can make kinds of tumors produce immunoreation, thus producing the antibody for PD-1 antibody, vivo immuning system can suppress or remove the effect of PD-1 antibody. PD-1 Antybody therapy has been in the news and dependency Adverse Event can occur, produce cause immunity enteritis and pulmonary toxicity damage for PD-1 antibody mediated immunity reaction, and PDL2HSA series fusion protein is because being all derived from the albumen of human body itself, initiation immunity side effect will not be stimulated.

Additionally the nucleic acid of abduction delivering coding PDL2HSA fusion protein in host, can produce the immunization therapy close with injecting PDL2HSA fusion protein or vaccine effect equally. Such as can engineered cell with carry coding PDL2HSA fusion protein nucleic acid, allow human body spontaneous express PDL2HSA fusion protein, then experimenter is implemented the immunization therapy of tumor.

The present inventor passes through large-scale expressing fusion protein and screening operation, obtain first and have and PD-I high affinity and the people source PDL2HSA series fusion protein promoting T cell propagation, and in the animal experiment of spontaneous tumor model, obtain more effective therapeutic outcome, this especially new fusion protein can apply in tumor vaccine or immunotherapy of tumors, there is provided new effective bio-pharmaceutical for clinical treatment tumour disease, have bigger using value.

Accompanying drawing explanation

The confirmation of Fig. 1 people source PDL2-HSA fusion protein.

Left figure carries out protein immunoblot testing result with anti-PD-L2 antibody. Right figure carries out protein immunoblot testing result with anti-HSA antibody. The electrophoresis loading of 1,4 is the PDL2-HAS culture supernatant converting cell. 2,3 is the molecular marker of electrophoresis.

Fig. 2 people source PDL2HSA series fusion protein is in conjunction with PD-I

The PD-1 being assessed the people source PDL2HSA series fusion protein that embodiment 1 obtains by ELISA combines activity. Abscissa is the OD value of 450nm detection. Vertical coordinate is followed successively by conjunction with 1:PDL2-HSA fusion protein, 2:PDL2-GSGS-HSA fusion protein, 3:PDL2-GSGSGSGS-HSA fusion protein, 4:PDL2-GSGSGSGSGSGS-HSA fusion protein 5:PDL2-GSGSGSGSGSGSGSGS-HAS fusion protein 6:PDL2-FC fusion protein 7:PDL2-tranferin fusion protein

The abscissa that affects of CD8 positive T cell is the percentage ratio of CD8 positive T cell in CD45 positive leukocytes at tumour immunity model by Fig. 3 people source PDL2HSA series fusion protein.Vertical coordinate is followed successively by treatment group tumors tissue 1:PDL2-HSA, 2:PDL2-GSGS-HSA, 3:PDL2-GSGSGSGS-HSA, 4:PDL2-GSGSGSGSGSGS-HSA5:PDL2-GSGSGSGSGSGSGSGS-HAS6:PDL2-FC7:PDL2-tranferin treatment group

Detailed description of the invention

The expression of embodiment 1 people source PDL2-HSA fusion protein and confirmation

The dihydrofolate reductase (DHFR) expressing use Invitrogen company of the people source PDL2-HSA fusion protein of cell exocrine screens eukaryotic expression system and expresses. Transfection procedure: 1. first 24 hours of transfection, CHO-S cell is with 5 �� 10⁵��6 �� 10⁵Cell/mL density goes down to posterity fresh CDFortiCHO culture medium. (Invitrogen) 130��150rpm, 37 DEG C, 8%CO₂Incubator is cultivated. 2., during transfection, it is 1 �� 10 that cell CDFortiCHO culture medium is diluted to cell density⁶Cells/ml, 125mL bottle (Flask) fills 30mL Cell sap. 3. take 50ug plasmid in 1.5mLOptiPROSFM culture medium (Invitrogen), mix gently. 4. take 50uL conversion reagent (FreeStyleMAXReagent, Invitrogen) in 1.45mLOptiPROSFM culture medium (Invitrogen), mix gently. 5. being mixed with FreeStyleMAXReagent solution by plasmid solution, room temperature is placed 10 minutes. Addition 125mL shaking flask slowly, 130��150rpm, 37 DEG C, 8%CO₂Incubator is cultivated. 6. carry out feed-batch culture: the 3rd day, add 4g/L glucose, 5th day, add 4g/L glucose, 7th day, add 6g/L glucose. purification step: 1.20mM phosphate buffer, 100mMNaCl, pH7.2 balances blue-sepharose PhastGel (BlueSepharoseFastFlow). 2. the supernatant of expressing collected crosses post with the speed of 1mL/min. 3. elution buffer: 20mM phosphate buffer, 2MNaCl, pH7.2. final often going up can obtain clearly more than 400mg albumen. collect the expression-secretion liquid of Chinese hamster ovary cell (Chinese hamster ovary celI), it is concentrated into 1/5 volume, loading carries out gel electrophoresis of protein (model Tanon company VE-180 miniature vertical electric current groove, condition 150V, 1 hour 30 minutes), then (concrete grammar takes the wet method turned to transferring film, transferring film buffer 1L formula: Tris3.03g, Glycine14.4g, SDS0.1g, condition 200mA, 2 hours), utilize anti-PD-L2 antibody (RabbitmonoclonalantibodytoHumanPD-L2, 1ug/mL Sino Biological Inc.) and anti-HSA antibody (RabbitantiHSAIgG, 1ug/mL Shanghai Zu Rui Bioisystech Co., Ltd) carry out protein immunoblotting detection (concrete grammar, condition film in 5% defatted milk powder solution incubated at room 1 hour with the non-specific binding on closing membrane. the film closed adds primary antibodie incubated at room 1.5 hours, and antigen-antibody combines. then film, 5min �� 4 time are washed with phosphoric acid Tween buffer (PBST). add two anti-(by 1:3000 dilution ratio 5% defatted milk powder solution dilutions) of horseradish peroxidase, steadily shake, room temperature 2h. abandon two to resist, wash film, 5min �� 4 time with PBST. adding nitrite ion, lucifuge colour developing is to putting into termination reaction in distilled water when there is band. ). result illustrates that the people source PDL2-HSA fusion protein that anti-PD-L2 antibody test obtains has the binding domain of anti-PD-L2 antibody, and anti-HSA antibody test illustrates that fusion protein also has the binding domain of anti-HSA antibody simultaneously. see Fig. 1

PDL2-GSGS-HSA,PDL2-GSGSGSGS-HSA,PDL2-GSGSGSGSGSGS-HSA

The expression and purification of PDL2-GSGSGSGSGSGSGSGS-HSA and PDL2-GSGSGSGSGSGSGSGS-HSA fusion protein is in consistent with PDL2-HSA fusion protein method, and the nucleotide sequence of the simply insertion when synthetic nucleic acid sequence is different.See following table

Embodiment 2: people source PDL2HSA series fusion protein (embodiment 1 obtains) is analyzed in conjunction with PD-I

The PD-1 being assessed the people source PDL2HSA series fusion protein that embodiment 1 obtains by ELISA combines activity.

96 hole elisa plates are used in the 100 �� L1 �� g/mL recombined human PD-I (self-control of dilution in carbonate/bicarbonate pH9.4 buffer (Pierce), mammalian CHO cells expression system secreting, expressing purification obtains) it is coated 2 hours, then close 90-120 minute with BSA (bovine serum albumin) solution. People source PDL2HSA fusion protein and reference protein (concentration 10ug/mL) were in conjunction with 90 minutes. Use the 100 �� anti-human PD-L2 antibody (RabbitmonoclonalantibodytoHumanPD-L2 of L0.5 �� g/mL rabbit, 1ug/mL Sino Biological Inc.) detection, HRP followed by 1:500 dilution marks anti-(Ge Langrui bio tech ltd, Hangzhou) and the TMB (Chinese 3 of goat-anti rabbit two, 3 ', 5,5 '-tetramethyl benzidine) substrate. Microplate reader (model BioTekElx800) is used to read the absorbance that diaminobenzidine (DAB) substrate at 450nm place produces. Experiments show that PDL2-HSA, PDL2-GSGS-HSAPDL2-GSGSGSGS-HSA and PDL2-GSGSGSGSGSGS-HSA fusion protein has the stronger combination activity in conjunction with PD-I, and PDL2-GSGSGSGSGSGSGSGS-HSA, PDL2-FC (PDL2 and antibody crystal region fusion protein, laboratory is expressed and is obtained) and PDL2-transferrin (PDL2 and transferrin fusion protein, laboratory expression obtain) in conjunction with activity relatively low (see Fig. 2).

Embodiment 3: people source PDL2-HSA fusion protein (embodiment 1 obtains) is at the drug efficacy study of D121 tumour immunity model

Test objective: treat model by D121 lung cancer tumor immune model, understands the antitumor drug effect of PDL2-HSA fusion protein.

Animal: C57 mice, 6-8 week old, is female entirely.

Produce tumor model:

1) D121 lung cancer tumor is bought from American Type Culture Collection ATCC, and cell uses containing 10% hyclone DMEM culture fluid at 37 DEG C, cultivates under the carbon dioxide conditions of 5%. Within every 3 days, go down to posterity once, within cell was used in for 15 generations.

2) tumour immunity, mouse peritoneal injection 5x10⁵Through the D121 lung carcinoma cell (purchased from American Type culture collection institute) of radiation death, inoculation 3 times, every minor tick 2 weeks. Immunization therapy process: (embodiment 1 obtains PDL2HSA series fusion protein, 10mg/kg), PDL2-FC (PDL2 and antibody crystal region fusion protein, laboratory is expressed and is obtained, 10mg/kg) with PDL2-transferrin (PDL2-transferrin fusion protein, 10mg/kg) with PDL1-antibody (Beijing justice is stuck up), with normal saline dilution to respective concentration (1mg/ml), intravenous injection (200 microlitre/20 gram), it is administered when immunity starts, weekly administration, totally 7 weeks.

3) tumor produces: at the 32nd day, by 10⁶The D121 lung cancer tumor cell subcutaneous injection lived, to the C57 mouse back of tumour immunity, starts treatment when tumor length to about 0.3��0.4cm.

4) tumor CD8+T cell analysis. Tumor tissues is through homogenate, filter to isolate individual cells in tumor, twice is washed with buffer, the antibody of leukocyte common antigen CD45-PE and CD8-FITC labelling combines in room temperature for 1 hour, cell is washed twice with comprising 1% hyclone phosphate buffer PBS, then by the ratio (see Fig. 3) of T lymphocyte antigen (CD8) positive cell in flow cytometry analysis leukocyte common antigen (CD45) positive cell.

5) the side effect detection of immunization therapy, PDL2-FC and PDL1-antibody group there is some animals to have archorrhagia phenomenon, detect hemorrhage mouse by pathological section and be confirmed whether as immunity enteritis, finally determine the incidence of side effects of enteritis, PDL2-HSA, the immunity enteritis sickness rate of PDL2-FC and PDL1-antibody group is followed successively by 3.3%, 23.3% and 16.7% result and shows that the PDL2-HAS immunity enteritis side effect caused is minimum.

6) result and discussion: compared with immunized controls group and other treatment matched group, people source PDL2HSA series fusion protein is greatly improved at the immunotherapeutic effects of C57 mice, illustrate that PDL2HSA series fusion protein has good raising immune effect effect, it is possible to by improving the growth of immunosuppressant tumor. See following table:

Embodiment 4:PDL2-HSA fusion protein grows the drug efficacy study of model (MMTV-PyVmT) tumor model in turning of transfevent breast cancer gene

Test objective: grow model (MMTV-PyVmT) tumor model treatment model by turning of transfevent breast cancer gene, understand the antitumor drug effect of PDL2-HSA fusion protein (embodiment 1 obtains).

Animal: model (MMTV-PyVmT) mice is grown in turning of transfevent breast cancer gene, and (autotrophy) is female entirely.

Producing tumor model, turning of transfevent breast cancer gene grows model M TV-PyMT Mouse feeder in SPF level environment, and that observes mice breeds situation. Mammary gland of mouse can touch tumor for 9 weeks, and along with the increase of week old, the number of tumor is gradually increased, and volume is gradually increased, at most up to 8; There is Lung metastases in 12 weeks in major part; Generally the longest life span is 16 weeks.

Therapeutic process, chooses gross tumor volume 100��200mm³The mouse of size, carries out intravenous medical treatment with PDL2-HSA fusion protein (embodiment 1), PD-L2-Fc fusion protein and solvent (normal saline) comparison successively respectively, weekly administration (15mg/kg), totally 6 weeks.

Result and discussion: compared with matched group, the treatment of PDL2HSA series fusion protein treatment group is greatly improved the suppression ratio of tumor growth. Neoplasm metastasis is completely different with the mechanism of neoplasm in situ growth, and neoplasm metastasis is only the key that patient is lethal, and PDL2HSA series fusion protein can effectively suppress neoplasm metastasis, and the drug effect with good treatment tumor is described. See following table

Embodiment 5: people source PDL2-HSA fusion protein lymphocytic choriomeningitis virus

The drug efficacy study of infection model (LCMV)

Test objective: infected by the lymphocytic choriomeningitis virus (LCMV) of central nervous system of mice, understands the antiviral effect of PDL2-HSA fusion protein.

Animal: C57 mice, 5-8 week old, is female entirely.

Produce tumor model:

1) mouse infection 1 �� 106p.f.u.LCMV clones 13 (Oldstoma laboratory is given). The dead symptom with disease of record every day after infection. Severe disease symptoms was to maintain the posture of hunch more than 24 hours, and did not move and maybe can only tremble.

2) Drug therapy: at the 0,1,2,3rd day, gives 10 mgs/kg of lumbar injection treatments.

3) CD8+T cell analysis. Spleen tissue is through homogenate, filter to isolate wherein individual cells, twice is washed with buffer, the antibody of leukocyte common antigen CD44-PE and CD8-FITC labelling combines in room temperature for 1 hour, cell is washed twice with comprising 1%FCSPBS, then by the ratio of CD8 positive cell in flow cytometry analysis CD44 positive cell.

4) result and discussion: compared with immunized controls group and other treatment matched group, the PDL2HSA series fusion protein treatment group death of people source reduces, cd8 t cell not exhaustion, antiviral immunity therapeutic effect C57 mice is greatly improved, and illustrates that PDL2HSA series fusion protein has good antiviral effect.See following table:

Claims

1. a people source PDL2HSA fusion protein, it is characterized in that, the aminoacid sequence of described people source PDL2HSA fusion protein is such as shown in SEQIDNO.1, or is made up of the 220th amino acids place insertion connection small peptide GSGS, GSGSGSGS, GSGSGSGSGSGS, GSGSGSGSGSGSGSGS at the aminoacid sequence shown in SEQIDNO.1.

2. a kind of people source according to claim 1 PDL2HSA series fusion protein, it is characterised in that the aminoacid sequence of described a kind of people source PDL2HSA fusion protein is such as shown in SEQIDNO.1, and its nucleotide sequence is such as shown in SEQIDNO.2.

3. the method preparing a kind of people source as claimed in claim 1 PDL2HSA fusion protein, it is characterised in that the method comprises the following steps:

(1) nucleic acid molecules of the encoding human source PDL2HSA fusion protein of preparative separation:

The nucleic acid of the coding PDL2HSA fused protein separated is obtained with polymerase chain reaction technology; Or it is directly synthesized the nucleic acid molecules of coding PDL2HSA fused protein; Or the nucleic acid molecules of PDL2HSA fused protein is obtained by the nucleic acid molecules of enzyme action connection different fragments;

(2) people source PDL2HSA fused protein is prepared

Obtained by the restructuring in host cell: with the nuclear transformation of encoding said fusion protein, transduction or transfection antibacterial, insecticide, yeast or mammalian cell, the expression system based on virus is utilized to express PDL2HSA fused protein, preparation people source PDL2HSA fused protein;

(3) Purification of Human source PDL2HSA fused protein.

4. a kind of people source as claimed in claim 1 PDL2HSA fusion protein application in the reagent/test kit of the preparation detection PD-1 reagent combined and the external internal activation of T cell.

5. a kind of people source as claimed in claim 1 PDL2HSA fusion protein application in preparing antitumor drug or bacterin preparation, described tumor is pulmonary carcinoma or breast carcinoma.

6. application according to claim 5, it is characterised in that described antitumor drug or bacterin preparation are made up of as active component and pharmaceutical carrier people source PDL2HSA fusion protein.

7. a kind of people source PDL2HSA fusion protein application in the medicine preparing antilymphocyte choriomeningitis virus (LCMV) or bacterin preparation as claimed in claim 1.

8. application according to claim 7, it is characterised in that described medicine or bacterin preparation are made up of as active component and pharmaceutical carrier people source PDL2HSA fusion protein.