CN1927881A - Polypeptide, preparation method thereof, pharmaceutical composition and vaccine containing the same and application thereof - Google Patents
Polypeptide, preparation method thereof, pharmaceutical composition and vaccine containing the same and application thereof Download PDFInfo
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Abstract
The present invention relates to one kind of polypeptide, and its preparation process, medicinal salt, analog and derivative, as well as medicine composition and vaccine containing the polypeptide, its medicinal salt, analog or derivative. The polypeptide, its medicinal salt, analog and derivative may be used in preventing and treating EBV virus infection or EBV relevant cancers, especially nasopharyngeal carcinoma.
Description
Technical field:
The present invention relates to a kind of polypeptide, its pharmacologically acceptable salt, analogue or derivative, the preparation method of described polypeptide, contain described polypeptide, its pharmacologically acceptable salt, the pharmaceutical composition of analogue or derivative and vaccine, use described polypeptide, its pharmacologically acceptable salt, analogue or derivative are treated, prevention or improve EBV virus infection or the cancer relevant with EBV, especially nasopharyngeal carcinoma (nasopharyngeal carcinoma, method NPC) and described polypeptide, its pharmacologically acceptable salt, analogue or derivative are in the preparation treatment, purposes in prevention and the medicine that improves EBV virus infection or the cancer relevant with EBV.
Background technology:
Nasopharyngeal carcinoma (NPC) is common tumour in south China, Hong Kong, Xin Jiabo and Taiwan in whole South East Asia especially.Annual morbidity ten thousand/to 5/10000ths (Ref 1-4).Early stage in this disease, radiotherapy is effective means, yet in transition phase, combined utilization cis-platinum and 5 FU 5 fluorouracil (5-Fluoro-uracil; 5-FU) making chemotherapy can only play usually and roughly alleviate effect (Ref5,6).In case these palindromias or deterioration do not have more effective therapy may be utilized, the patient near 85% died of illness in 1 year, almost all death (Ref 1,7) in 3 years.Therefore, pressing for one technically compares safer and effective methods of treatment and handles advanced NPC with comprising radiotherapy with the traditional method of chemotherapy.In addition, also there are the technical needs that healthy individual is carried out vaccine inoculation for the prevention nasopharyngeal carcinoma.
EBV is ubiquitous γ-spiral virus, and it is relevant with some malignant disease, comprises nasopharyngeal carcinoma (Ref 8-10).It is lifelong infection that infection has most of crowds of EBV, CD8+ cytotoxic T cell (CTL) play an important role in keeping a such virus-host's balance (Ref 11).Exist the potential antigen of EBV (Latentantigen) the specific CTL s of One's name is legion in healthy EBV carrier's blood, it can react (Ref 12-13) with the lymph poison cell system that is out of shape with allogeneic virus external.The treatment of carrying out suppressor T cell to transplant patient has increased relevant B cell amplification (the Post-transplant B cell lymphoproliferative disorder of EBV; PTLD) risk can be treated this disease (Ref 14-17) by the reactive EBV-specific CTL of external input s.
Tapes has the nasopharyngeal carcinoma cell of Epstein-Barr virus to express at least three kinds of B viral protein: LMP1, LMP2 and EBNA1 (Ref 18-20) is arranged.Importantly go up several target epi-positions and be firmly established, as HLA-A11, HLA-A24 and HLA-B40 by HLA allelotrope at the LMP2 that is subjected to HLA (human leukocyte antigen) restriction.These allelotrope are ubiquity (HLA-A11,40-60% in the southern china people; HLA-A24,30-35%; HLA-B40,32%) (Ref21-24).Such reaction, though can survey sometimes, patient NPC is more weak than normal healthy controls group.A lot of China's Healthy EBV carrier can detect the CTL to the LMP2 reaction.In vitro study shows that the NPC tumour cell can submission endogenous LMP2 epi-position.Though the ctl response level to unit price immunological effect peptide CLGGLLTMV, YLLEMLWRL, YLQQNWWTL, SSCSSCPLSK, TYGPVFMSL or the IEDPPFNSI of LMP2 (the latent membrane protein 2) epi-position of anti EB virus (EBV) HLA restriction of the spontaneous generation of NPC sufferer is lower than general normal people, but they have comprised and have been subjected to HLA allelotrope (HLA-A11, HLA-A24 and HLA-B40) reaction of restriction, and (Ref23-24) generally appears in these allelotrope usually in the crowd of South Asia.Therefore, the immunotherapy approach based on these epi-positions should have extensive applicability in NPC region occurred frequently.
Be the potential antigen of the assessment EBV-validity of specific CTL immunity on patient NPC, the contriver once used dendritic cell (the Dendritic Cells of patient from body; DCs) (Ref 25), one species-specific antigen presenting cell, with univalent LMP2 specific peptide HLA-A11-restricted " SSCSSCPLSK ", HLA-A24-restricted " TYGPVFMSL " or HLA-B40-restricted " IEDPPFNSI " (Ref 22) suffer from late period NPC patient lymphoglandula (table 1) in 16 in the stimulated in vitro cultivation and carry out the multiple injection dendritic cell with the reaction of generation antitumor CTL behind the dendritic cell of sufferer. and the inductive reaction can detect after injecting for the first time the earliest in 2 weeks in the patient body, and sustainable 3 months, reaction level reduces gradually afterwards, almost returns to the preceding level (Figure 1) of vaccine inoculation in 6 months.After injecting in the lymphoglandula for the second time, the epitope specificity CTLs number in the peripheral blood reaches peak value.
Table 1
| Patient | HLA | Age/gender | Clinical condition | KLH promises |
| 1 | A2402 | 36/M | The original position recurrence | - |
| 2 | A1101 | 43/M | Vertebra shifts | + |
| 3 | A1101 | 45/M | Lung shifts | + |
| 4 | A2402 | 47/MA | The original position recurrence | + |
| 5 | A1101 | 46/M | Hepatic metastases, bilateral Cervical Lymph Node Metastasis | - |
| 6 | A1101 | 48/M | The original position recurrence | + |
| 7 | B40011 | 42/M | Bone shifts | + |
| 8 | A1101 | 56/M | The original position recurrence | + |
| 9 | A2402 | 48/M | Neck and node positive | + |
| 10 | B40011 | 57/M | The original position recurrence | + |
| 11 | B40011 | 46/M | Lung shifts | - |
| 12 | A1101 | 50/M | Lung shifts | - |
| 13 | A2402 | 48/M | The original position recurrence | + |
| 14 | A1101 | 50/M | The original position recurrence | + |
| 15 | B40011 | 47/M | The original position recurrence | + |
| 16 | A2402 | 53/M | The original position recurrence | + |
Body internal dynamics in same clinical experiment studies show that, 3 months the time, compares epitope specificity CTLs appears in A11 responsiveness patient in peripheral blood frequency higher (Ref.26) with A24 responsiveness patient after the vaccine inoculation.With the cell colony test of taking from the abundant enrichment of process in the patient body, these CTLs have epitope specificity cell toxicant function (Ret.26) at once.Two patients (No. 2 and No. 3 patients) that produce the part clinical response after accepting vaccine inoculation are the strong respondent to the A1101 peptide in CTL analyzes.Clinical response comprise in the bone and lung in disappear (table 2 and the Ref.26) of metastatic tumo(u)r, A1101 epitope specificity effector cell that can detected vaccine-induced generation in the blood is soaked into and/or active in the tumor locus performance to tumor locus.
Table 2
| Patient | HLA | ELISPOT | CD8+/IFN + | Cytotoxicity | Clinical response (following the trail of in 1 year) |
| 1 | A2402 | - | - | - | PD |
| 2 | A1101 | + | + | + | PR |
| 3 | A1101 | + | + | + | PR- |
| 4 | A2402 | + | + | - | PD |
| 5 | A1101 | - | - | - | PD |
| 6 | A1101 | + | + | + | PD |
| 7 | B40011 | - | - | - | PD |
| 8 | A1101 | + | + | + | PD |
| 9 | A2402 | + | + | - | PD |
| 10 | B40011 | - | - | - | PD |
| 11 | B40011 | - | - | - | PD |
| 12 | A1101 | - | - | - | PD |
| 13 | A2402 | + | + | - | PD |
| 14 | A1101 | + | + | + | PD |
| 15 | B40011 | - | - | - | PD |
| 16 | A2402 | + | + | - | PD |
Table 2 is summaries of inoculation back immune analysis and clinical response.After the DC inoculation, patient 2,3,6,8 and 14 (HLA-A1101) and patient 4,9,13 and 16 (HLA-A2402) have epitope specificity t cell response frequency to increase in ELISPLT analyzes, and the frequency of CD8+/IFN γ CTLs also increases.Patient 2,3,6,8 T cytological effect is increased by the dissolving power of target cell the epi-position bag is arranged.Patient 2 has clinical can detected tumour shrinking back.Patient 3 begins to have the part clinical response, but next tumour is long again.
*PR: part is replied PD: carrying out property clinical disease
# (+): quantity or effect increase, (-): not change
In a first phase human trial, the contriver uses HLA-A11, HLA-A24, and the LMP2 epi-position monomeric peptide of HLA-B40 restriction (is SSC-, TYG-, with the IED peptide) stimulate and to be injected into again behind body dendritic cell (Dendritic Cells) that these 16 patients of ， Fa Now do not produce any great side effect in 16 latter stage nasopharyngeal cancer patient bodies.In addition, 9 patients are arranged after having injected 3 months, the specific meaningful remarkable rising of frequency of killing the type cell of LMP2 peptide epitopes in the peripheral blood, the gross tumor volume that wherein has 2 patients' nasopharyngeal carcinoma to transfer to lumbar vertebrae and lung obviously dwindle (Lin CL et al.Cancer Research 2002,62:6952-6958).These results show and utilize LMP2 specificity epitope peptide, can be used for really strengthening human body functional LMP2 epitope peptide specificity t cell responses ， And and do not have any side effect.
Summary of the invention:
By The above results as can be known, existing non-all sufferers of Mian epidemic disease Liao Fa And with the single aggressiveness stimulation of LMP2 human immune system all have tangible immune response.Enough is not strong for the immunity (Immunogenecity) of the single aggressiveness of the LMP2 that one of them reason is to use itself.According to immunology principle, if antigen protein or antigen win peptide and exist with the polymer pattern, it is much higher that the immunity that immune pungency or itself are had generally can single poly-pattern, the present invention's purpose promptly is exploitation LMP2HLA-A11,-A24, this type of polymer of polymer ， And good authentication that-B40 epi-position wins peptide has the immunostimulating higher than monomer really.
Therefore, the present invention relates to a kind of polypeptide, its pharmacologically acceptable salt, analogue or derivative, wherein said polypeptide derives from the proteic fragment SSCSSCPLSK of the distinctive LMP2 of Epstein-Barr virus (being abbreviated as SSC) for being selected from; TYGPVFMSL (being abbreviated as TYG); Or two-eight aggressiveness of IEDPPFNSI (being abbreviated as IED), described polymer is connected by being selected from following joint by corresponding polypeptide fragment:
Aminoterminal and fragment SSCSSCPLSK among "-" expression K in the wherein above-mentioned joint; TYGPVFMSL; Or the key of IEDPPFNSI carboxyl terminal connection, " K ", " A " and " C " are amino acid whose abbreviation.
In above-mentioned joint, be preferably selected from following joint:
The structure of joint illustrates as follows:
All above-mentioned joints all are known in the art, and can be commercially available from for example AdvancedChemtech company (USA).
In polypeptide of the present invention, its pharmacologically acceptable salt, analogue or derivative, preferred fragment SSCSSCPLSK; TYGPVFMSL; Or four-eight aggressiveness of IEDPPFNSI, more preferably four or eight aggressiveness, the more preferably tetramers.The most preferred tetramer is selected from:
The invention still further relates to the preparation method of polypeptide of the present invention, its pharmacologically acceptable salt, analogue or derivative; comprise the peptide solid phase synthesis process of selecting Fmoc amino acid strategy for use; by using precoating to have the Wang resin of Methionin nuclear to go protection and the next amino acid of coupling to make the peptide chainpropagation by alternately making amino, and the step of cleavage of peptide from the resin at last.
In addition, the invention still further relates to a kind of pharmaceutical composition, wherein comprise at least a polypeptide of the present invention, its pharmacologically acceptable salt, analogue or derivative, and conventional pharmaceutical carrier or excipient.The present composition is also optional to comprise another kind of active ingredient, for example another kind of EBV specific proteins (for example EBNA (Epstein Barr Nuclear Antigen-1) etc.) and cytokine (for example GM-CSF (granulocyte/macrophage colony stimulating factor) etc.).
The invention still further relates to a kind of treatment and prevention Epstein Barr virus infection or the method for cancer viral relevant, comprise to being executed individuality and directly use polypeptide of the present invention, its pharmacologically acceptable salt, analogue or derivative with EpsteinBarr; Perhaps inject polypeptide of the present invention, its pharmacologically acceptable salt, analogue or the post-stimulatory dendritic cell of derivative, preferably come from from body or allochthonous peripheral blood monocyte; Or injection is subjected to the T cell of polypeptide of the present invention, its pharmacologically acceptable salt, analogue or derivative stimulation back amplification in vitro, the lymphocytic step of preferred cell poison T.Comprise cancer of the stomach, mammary cancer, B cell lymphatic cancer, transplant back B cell amplification pathology and nasopharyngeal carcinoma with the medicable cancer of the inventive method, be preferred for treating nasopharyngeal carcinoma.
In addition, the present invention relates to a kind of vaccine, wherein comprise polypeptide of the present invention, its pharmacologically acceptable salt, analogue or derivative.Randomly, vaccine of the present invention also comprises other activeconstituents together.
The invention still further relates to the purposes that polypeptide of the present invention, its pharmacologically acceptable salt, analogue or derivative are used to prepare the medicine of treatment and prevention Epstein Barr virus infection or the cancer relevant with Epstein Barr virus or are used to prepare vaccine.The described cancer relevant with Epstein Barr virus is selected from cancer of the stomach, mammary cancer, B cell lymphatic cancer, transplants back B cell amplification pathology and nasopharyngeal carcinoma.Be preferred for preparing the medicine for the treatment of nasopharyngeal carcinoma.
Polypeptide of the present invention
Correspond respectively to following molecular structure:
Four poly-peptides of the fan-shaped HLA-A11 epi-position of LMP2
Four poly-peptides of the fan-shaped HLA-A24 epi-position of LMP2; With
Four poly-peptides of the fan-shaped HLA-B40 epi-position of LMP2.
Illustrate the preparation route of polypeptide of the present invention below:
Reaction scheme 1 and 2 the expression be with
With
Be example explanation solid-phase peptide synthetic reaction scheme.
Reaction scheme 1
The amino acid that is used for described solid-phase peptide synthetic Fmoc and side chain protected is as follows: Fmoc-K (BOC), Fmoc-S (tBu), Fmoc-L, Fmoc-P, Fmoc-C (Trt).The amino acid of side chain protected indicates with underscore in the sequence.The chainpropagation step is undertaken by the Fmoc amino acid peptide synthesis strategy of routine.
1.Activation of the protected peptide by formation of the peptide succinimidyl carboxylate
2.Coupling of polylysine backbone with activated protected peptide
Reaction scheme 2
aIt is synthetic that the amino acid [Fmoc-K (BOC), Fmoc-S (tBu), Fmoc-L, Fmoc-P, Fmoc-C (Trt)] of Fmoc-protection is used for solid-phase peptide, with the peptide of preparation side chain protected.The amino acid of side chain protected indicates with underscore in the sequence.Under the not de-protected condition of side chain with described peptide from the solid carrier under the cracking.
Reaction conditions: the carboxylic acid of the peptide of described protection at room temperature, in DMF (1mL), by adding 1; 3-di-isopropyl carbodiimide (DIC; 1 equivalent), I-hydroxybenzotriazole (HOBT, 1 equivalent) and N-hydroxy-succinamide (HOSu, 1 equivalent) and stirred 4 hours and activate.To the four polylysine skeletons (0.2 equivalent) that wherein add Fmoc protection and stirred 16 hours.Add piperidines (0.25mL) and at room temperature stir 20 minutes to remove the Fmoc protection.Reduce pressure out and desolvate.At room temperature resistates is suspended in again 15mL contain trifluoroacetic acid, phenol, TIS, water, DTT and thioanisole mixture (88: 5: 2: 5: 0.05: 0.5, v/v) in and stir 2 hours to remove the Side chain protective group of four poly-peptides.End product is by being the permeable membrane dialysis purification of 1.2kDa with cutoff value.
Such peptide solid phase synthesis process is that prior art is known, for example with reference to reference 1 and 2.
The invention still further relates to application polypeptide of the present invention, its derivative or analogue and handle the patient, amplification in vitro toxicity killer type T cell (cytotoxic T cells) for example, and then in the input patient, or use polypeptide of the present invention, its derivative or analogue and be used for stimulating dendritic cell, and then inject back sufferer, or directly use polypeptide of the present invention, its derivative or analogue, be used for the treatment of, prevent and improve patient's EBV related neoplasms or EBV virus infection.The inventive method also can be treated EBV associated cancer or EBV virus infection with other methods of treatment.
The purpose of this invention is to provide the polypeptide or their analogue that use EBV specificity LMP2 protein fragments stimulates the dendritic cell later or the specific cytotoxic t lymphocytes immunity of amplification in vitro to import in the patient by injecting these peptides again, treats and prevent EBV virus infection and EBV associated cancer (as nasopharyngeal carcinoma).In the present invention, the four poly-peptides of using the LMP2 HLA-A11 epi-position of HLA restriction come stimulated in vitro to cultivate the DC that derives from sufferer, inject patient's NPC inguinal lymph nodes in late period (lymph nodes) then.Polypeptide of the present invention is used on the person, treats or preventing cancer or virus infection.The result shows, the four poly-peptides that the present invention describes have than the unit price peptide t cell responses of Strong more through experiment confirm, and causing or strengthened in the peripheral blood can detected epitope specificity CD8+T cell response, therefore can apply to the immunotherapy of EBV related neoplasms.In this methods of treatment, the dynamic variation situation of the intravital functional epitope specificity CTLs of patient can be adjusted by the progress of vaccine inoculation.The special antigenic structure molecule that the invention provides EBV specificity LMP2 is used for inducing the persistence t cell responses of going up the EBV epi-position of expressing at NPC.
The present invention has been contained with Methionin or other and can be used for connecting monomeric peptide SSCSSCPLSK, TYGPVFMSL or IEDPPFNSI become the physics or the chemical process of polymer, three kinds of EBV virus-specifics, HLA-A11, the restrictive LMP2 peptide of HLA-A24 and HLA-B40 synthesizes polymer.Monomolecular aminoacid sequence is respectively SSCSSCPLSK, TYGPVFMSL and IEDPPFNSI.In specification sheets of the present invention and claim
S represents Serine, Serine
C represents Cysteine, halfcystine
P represents Proline, proline(Pro)
L represents Leucine, leucine
K represents Lysine, Methionin
T represents Threonine, Threonine
Y represents Tyrosine, tyrosine
G represents Glycine, glycine
V represents Valine, Xie Ansuan
F represents Phenylalanine, phenylalanine
M represents Methionine, methionine(Met)
I represents Isoleucine, Isoleucine
E represents Glutamic acid, L-glutamic acid
D represents Asparatic acid, aspartic acid
N represents Asparagine, arginine.
Being meant of polypeptide analogue of the present invention or derivative carried out the product that the similar functions that obtains was replaced or end group was modified or protected to discrete amino acid to polypeptide of the present invention.
The invention provides by using, especially the polypeptide of the present invention of injection for curing dosage or preventive dose is treated or the method for preventing cancer, described cancer includes but not limited to neoplasms, and metastatic tumo(u)r or other are with cell disease or the dysfunction that is grown to feature out of control.The cancer that treats and/or prevents of indication includes but not limited to, alleviates the relevant symptom of cancer, suppresses the process of cancer, promotes cancer to lapse to and the enhancing immunity reaction.
Polypeptide of the present invention can use separately or with other cancer treatment methods (as, radiotherapy, chemotherapy, hormonotherapy, immunotherapy and anti-tumor agent comprising salmosin) unite use.The example of anti-tumor agent comprising salmosin includes but not limited to cis-platinum, ifosfamide, taxol (paclitaxel), taxanes (taxanes), topological enzyme I inhibition (as CPT-11, topotecan (camptothecin derivative), 9-AC (anthracene-9-carboxylic acid) and GG-211), gemcitabine (gemcitabine), Vinorelbine (vinorelbine), oxaliplatin (oxaliplatin), 5 FU 5 fluorouracil (5-FU), formyl tetrahydrofolic acid, Temozolomide (temodal) and taxol (taxol).In one application, in the polypeptide of the present invention animal body of cancer that has been expelled to surgical resection, this animal is preferably Mammals, most preferably is the people.In another kind is used, polypeptide of the present invention to animals administer, is united use with chemotherapy or radiotherapy, this animal is preferably Mammals, most preferably is the people.In another kind is used, in order to prevent or treat cancer, with polypeptide of the present invention before antiserum(antisera) (as, 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or before 1 week) or afterwards (as, 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or after 1 week) to animals administer, this animal is preferably Mammals, most preferably is the people.
In a kind of preferred application of the present invention, be prevention or treatment cancer, before or after IgG, IgM and/or one or more complement components or simultaneously to animals administer, this animal is preferably Mammals with polypeptide of the present invention, most preferably is the people.In another kind of the present invention is preferably used, before or after the antibody of anti-one or more cancer cell antigens of specificity or simultaneously to animals administer, this animal is preferably Mammals, most preferably be the people with polypeptide of the present invention.In another advantageous applications of the present invention, be prevention or treatment cancer, before or after the antibody that is used for cancer therapy at present or simultaneously to animals administer, this animal is preferably Mammals with polypeptide of the present invention, most preferably is the people.
In advantageous applications more of the present invention, duplicate injection polypeptide of the present invention after after a while.In some applications, per 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months are duplicate injection polypeptide once of the present invention at least.In other are used, with polypeptide duplicate injection of the present invention 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times.
The invention provides the method for treatment or the intravital virus infection of prevention animal, this animal is preferably Mammals, most preferably is the people.Described method comprises the polypeptide of the present invention of injection for curing dosage or preventive dose.The example of the virus infection that can treat or prevent according to the present invention includes but not limited to the virus infection that Epstein-Barr virus causes.Treating and/or preventing of virus infection included but not limited to, alleviate the symptom relevant, suppress virus replication wholly or in part with said infection, and the enhancing immunity reaction.
Polypeptide of the present invention can use separately or unite use with the Anti-virus agent of other types.The example of Anti-virus agent includes but not limited to: cytokine (as, IFN-α, IFN-β, IFN-γ); Reverse transcriptase inhibitors (as, AZT, 3TC, D4T, ddC, ddI, d4T, 3TC, lamivudine (adefovir), Stocrin (efavirenz), ground La Weiding (delavirdine), nevirapine (nevirapine), Abacavir (abacavir), and other dideoxy nucleotides or dideoxyfluoronucleosides); The reagent that suppresses virus mRNA is as three (nitrogen) azoles nucleosides; Protease inhibitor such as hiv protease inhibition (as, amprenavir (amprenavir), Crixivan (indinavir), viracept see nelfinaivr (nelfinavir), ritonavir (ritonavir), and saquinavir (saquinavir)); Amphotericin B; The castanospermine (castanospermine) that suppresses glycoprotein processing; Neural ammonia (sugar) neuraminidase inhibition such as neural ammonia (sugar) neuraminidase of influenza virus inhibition (as, Zha Namiwei (zanamivir) and Tamiflu capsule (oseltamivir)); Topological enzyme I inhibition (as, camptothecine (camptothecins) and pharmacologically acceptable salt thereof, analogue); Buddha's warrior attendant (alkane) amine and Rimantadine (rimantadine).In one application, be prevention or treatment virus infection, before or after antiserum(antisera) or simultaneously to animals administer, this animal is preferably Mammals with polypeptide of the present invention, most preferably is the people.
In a kind of preferred application of the present invention, be prevention or treatment virus infection, at IgG, before or after IgM and/or one or more complement components or simultaneously to animals administer, this animal is preferably Mammals, most preferably is the people with polypeptide of the present invention.In another kind of application the of the present invention, be prevention or treatment virus infection, before or after the antibody of anti-one or more virus antigens of specificity or simultaneously to animals administer, this animal is preferably Mammals, most preferably be the people with polypeptide of the present invention.The example of the antiviral antigenic antibody of immunologic opsonin includes but not limited to, Synagis , PRO542, Ostavir, and Protovir.
Can detect it to polypeptide to be measured and reduce the ability that tumour patient (being animal) in-vivo tumour forms; Can also detect the ability that it reduces intravital virus of infectious diseases patient or bacterial number; Can also detect the ability that it alleviates one or more relevant symptoms of cancer or virus infection; Can also detect the ability that it shortens the infectious diseases course of disease.Further, can detect the ability that polypeptide to be measured prolongs cancer patients or infectious diseases (as, virus infection) patient's survival time.Can use method known to those skilled in the art and detect polypeptide to be measured at the intravital functional level of patient.
In the different application of the present invention, analyzed in vitro can be used for determining whether a kind of specific polypeptide of injection demonstrates effect, analytical procedure comprises that cell cultures detects, promptly, patient's tissue samples is carried out cell cultures, make it contact this polypeptide then, perhaps this polypeptide of injection in sample is observed the action effect of polypeptide to tissue samples then.
Before test on the human body, the polypeptide that is used for the treatment of can be tested in the appropriate animal model system earlier, and animal model includes but not limited to mouse, rat, and chicken, ox, monkey, dog, cat, pig, rabbit, or the like.For in vivo test, in being expelled to human body before, can be earlier any to solve this polypeptide on probation on the very thorough animal model system.
The invention provides by to animal (as ox, pig, horse, chicken, cat, dog, the people, etc.) polypeptide of the present invention of injection effective dose prevents and treats cancer, the method for viral infection and infected by microbes in the body.
Polypeptide of the present invention can be used as vaccine, be used to improve needs are arranged the experimenter to EBV specific peptide and derivative thereof, analogue or its segmental immunological competence.Vaccine of the present invention and method both can be used for preventing disease or disorder, can be used for treating specific disease or functional disorder again.
Method of the present invention and vaccine can be used for inducing the anti-EBV specific peptide of generation and derivative, analogue or its segmental body fluid and/or cell-mediated immune response in the subject.In a kind of concrete application, produce anti-EBV specific peptide and derivative, analogue or its segmental humoral response in method of the present invention and the vaccine-induced subject.In the concrete application of another kind, produce anti-EBV specific peptide and derivative, analogue or its segmental cell-mediated immune response in method of the present invention and the vaccine-induced subject.In a kind of advantageous applications that the present invention recommends, produce body fluid and cell-mediated immune response in method of the present invention and the vaccine-induced subject simultaneously.
Known multiple transfer system can be used for giving polypeptide of the present invention to the patient, as liposome, particulate, micro-capsule, can express the reconstitution cell of this mixture, receptor-mediated endocytosis (referring to, as, Wu and Wu, 1987, J.Biol.Chem.262:4429-4432), make up the part of nucleic acid fragment as retrovirus or other carriers, or the like.Import intravital method and include but not limited to, intracutaneous, intramuscular, intraperitoneal, intravenously, subcutaneous, in the nose, on the endocranium and oral cavity route.Polypeptide can give the patient with any approach easily, for example, inculcates or the bolus injection, the absorption of epithelium or mucous membrane and skin inner layer (as, oral mucosa, rectum and intestinal mucosa, etc.), also can together give with the other biological active agent.Administration can be systematized or partial.And, polypeptide of the present invention can be imported in the central nervous system by suitable approach, comprise in the ventricle and intrathecal injection; Injection can be undertaken by the ventricle inner catheter in the ventricle.Also can adopt pulmonary administration, as, use sucker or atomizer, then add a kind of atomized reagent in the prescription.
In a kind of concrete application, the position topical of polypeptide of the present invention in the needs treatment then can be passed through if wish, realize such as but not limited to following method, as, carry out the part in the surgical operation and inculcate, local surfaces is used as applying after the surgical operation on wound, binds up a wound injection then, use conduit, suppository, or implant, the implant of indication is to contain hole, solid or gelatinous material, comprise film, as the sialastic film, or fiber.
In another kind was used, polypeptide of the present invention can be rolled in the particle, especially in the liposome, transmits.(referring to Langer, 1990, Science 249:1527-1533; Treat et al., inLiposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestcinand Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestcin, ibid., pp.317-327; See generally ibid.)
In another was used, polypeptide of the present invention can transmit a controlled release or in the system that continues to discharge.Have a kind of application can use pump (referring to, Langer, supra; Sefton, 1987, CRC Crit.Ref.Biomed.Eng.14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N.Engl.J.Med.321:574).In another kind is used, controlled release system can use polymer material (referring to, Medical Applications of ControlledRelease, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailablility, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol.Sci.Rev.Macromol.Chem.23:61; Levy et al., 1985, Science 228:190; During et al., 1989, Ann.Neurol.25:351; Howard et al., 1989, J.Neurosurg.71:105).Other controlled release system (1990, Science 249:1527-1533) have been discussed in the summary of Langer, have been introduced into this paper as a part disclosed by the invention.
The present invention also provides a kind of pharmaceutical composition, comprises acceptable carrier on the polypeptide of the present invention of effective preventive dose or therapeutic dose and the pharmacopedics." carrier " is meant diluent, adjuvant, excipient, or the vehicle of administration.These pharmaceutical carriers can be sterile liquids, and Ru Shui and oil comprise from oil, animal, plant or synthetic oils, peanut oil for example, soybean oil, mineral oil, sesame oil and so on.When medicine component will pass through intravenous administration, water was first-selected carrier.Salts solution, the aqueous solution of glucose and glycerine also can be used as liquid vehicle, especially is suitable for as injection solution.Suitable medicament excipient comprises starch, glucose, lactose, sucrose, gelatinum, Fructus Hordei Germinatus, ground rice, flour, chalk, silica gel, sodium stearate.If necessary, also can comprise a spot of wetting agent, emulsifying agent or pH buffer reagent in the composition.Composition of the present invention can be a solution, suspension, and emulsion, tablet, pill, capsule, pulvis, the lasting formulation that discharges, or the like.Also the present composition suppository be can be made, traditional matrix such as triglyceride level for example adopted.Oral preparations can comprise the carrier of standard such as the N.F,USP MANNITOL of pharmaceutical grade, lactose, and starch, Magnesium Stearate, soluble saccharin, Mierocrystalline cellulose, magnesiumcarbonate, or the like.E.W.Martin has described the example of suitable medicament carrier in its " Remington ' s PharmaceuticalSciences " literary composition, be introduced into this paper as a part disclosed by the invention.These preparations comprise the polypeptide of the present invention for the treatment of significant quantity, and an amount of carrier is to provide suitable form of medication.The prescription of preparation should adapt with administering mode.
In a kind of preferred application, polypeptide of the present invention made be suitable for the medicament that approach is routinely given the venous patient administration.The medicine component of typical intravenously administrable is a solution of making solvent with sterile isotonic aqua damping fluid.If needed, healing potion can also comprise the pain that a kind of solubilizing agent and a kind of local anesthetic such as lignocaine are alleviated the injection site.In general, these added ingredientss can separately provide or mix mutually with unit dosage form, for example, are contained in the lyophilisate in the band graduated vessels of sealing or do not have aqueous concentrate, and container can be ampoule or sachette, can indicate the amount of the active agent of adorning.Transfusion is during administration, just it can be added in the infusion bottle of the sterilized water that fills pharmaceutical grade or salts solution.If adopt drug administration by injection, the Injectable sterile water or the salts solution of an ampoule can be provided, before administration, each composition can be mixed like this.
Polypeptide of the present invention can be the form of neutral form or its pharmacologically acceptable salt.Acceptable salt form comprises anion salt example hydrochloric acid salt, phosphoric acid salt, acetate on the pharmacopedics, oxalate, tartrate etc., and cationic salts such as sodium salt, sylvite, ammonium salt, calcium salt and and Isopropylamine, triethylamine, the 2-ethyl amido alcohol, Histidine, the salt of PROCAINE HCL, PHARMA GRADE (a kind of local anesthetic), or the like.
The present invention also provides the vaccine composition that is used for therapy of the present invention, and this vaccine composition is suitable for protective immunity (body fluid or the cell-mediated) reaction of administration to cause anti-specific antigen, and purpose is treatment or preventing disease, etc.
In a preferred application, Shi Yi vaccine composition comprises injectable solution or suspension; Dissolving or be suspended in solid form in the liquid before being suitable for injecting.The all right emulsification of composition forms capsule shape in the liposome of maybe this polypeptide being packed into.Acceptable and can mix by the excipient compatible on the normal and pharmacopedics of active immne ultimate constituent with activeconstituents.Suitable excipient has: water, and salt, phosphate buffered saline buffer, glucose, glycerine, ethanol, sterile isotonic aqua damping fluid or the like, and composition thereof.In addition, in needs, vaccine composition can also comprise a spot of supplementary component such as wetting or emulsification reagent, pH buffer reagent, and/or the adjuvant of promotion vaccine efficacy.
Effectively the example of adjuvant includes but not limited to: aluminium hydroxide; N-ethanoyl-born of the same parents' ancient piece of jade, round, flat and with a hole in its centre acyl group-L-threonyl-D-isoglutamine (thr-MDP); N-ethanoyl-fall-born of the same parents' ancient piece of jade, round, flat and with a hole in its centre acyl group-L-alanyl-D-isoglutamine; N-ethanoyl-born of the same parents' ancient piece of jade, round, flat and with a hole in its centre acyl group-L-alanyl-D-isoglutamine acyl-L-L-Ala-2-(1 ', 2 '-two palmityls-sn-glyceryl-3-hydroxyl phosphorus acyloxy)-ethamine.
Composition of the present invention or vaccine can be determined by the clinical technology of standard the effective treatment or the preventive dose of cancer or virus infection.In addition, also can adopt in vitro tests to help determine the optimal dose scope.Exact dosage desired used in the composition is different and different with the degree of disease or disorder with route of administration, also will determine according to doctor's judgement and patient's particular case.
The present invention also provides a kind of Drug packing or test kit, comprises one or more containers, and polypeptide of the present invention or its pharmaceutical composition wherein are housed, optional other active ingredient and specification sheets.
The present invention has measured monomeric peptide SSCSSCPLSK by enzyme linked immunological spot analysis (ELISPOT); TYGPVFMSL; IEDPPFNSI; Four poly-peptides of the fan-shaped HLA-A11 epi-position of LMP2 of the present invention, eight poly-peptides; Four poly-peptides of the fan-shaped HLA-A24 epi-position of LMP2, eight poly-peptides; Four poly-peptides of the fan-shaped HLA-B40 epi-position of LMP2, eight poly-peptides; Four poly-peptides of line style LMP2 HLA-A11 epi-position, eight poly-peptides; Four poly-peptides of line style LMP2 HLA-A24 epi-position, eight poly-peptides; Four poly-peptides of line style LMP2 HLA-B40 epi-position, eight poly-peptides; And the activity of irrelevant peptide (seeing accompanying drawing 1A-1C and 2A-2C).The result shows, no matter be the A11 SSC that the joint with what shape connects, the four poly-peptides that A24 TYG or B40 IED obtain all more can effective stimulus than eight poly-peptides produce the toxicity T cell (Cytotoxic T Cells) of the peptide specific of secretion IFN-gamma.And eight poly-peptides are more effective than monomeric peptide.This illustrates that polypeptide of the present invention is more effective than monomeric peptide.
Description of drawings:
Fig. 1. by ELISPOT assay setting-out line type LMP2 HLA-A11 (figure A),-A24 (figure B), the active result of-B40 (figure C) .The epi-position tetramer after 24 hours, induces the LMP2 specific T cell ， a little T cells that produce higher frequency can make IFN-r than monomer and octamer at stimulated in vitro T cell.Irrelevant peptide is as negative control.
Fig. 2. by the fan-shaped LMP2 HLA-A11 (figure A) that ELISPOT assay measures,-A24 (figure B), the active result of-B40 (figure C) .The epi-position tetramer after 24 hours, induces the LMP2 specific T cell ， a little T cells that produce higher frequency can make IFN-r than monomer and octamer at stimulated in vitro T cell.Irrelevant peptide is as negative control.
Conclusion: no matter be the A11 SSC that fan-shaped (Fan shaped) or linear (Linear Shaped) connects, A24 TYG or the B40 IED tetramer more can effective stimulus than eight aggressiveness produce the toxicity T cell (Cytotoxic T cells) of the victory peptide specific of secreting IFN-gamma. And eight aggressiveness are more effective than monomer.
Embodiment
The following examples are in order to further specifying the inventive method, but this invention is not subject to these embodiment.
Material and experiment
All solvents of material are AR level or solvent at the same level.Use the dimethyl formamide (DMF) of the synthetic level of peptide quality.Distillation in the presence of hydrolith before methylene dichloride (DCM) uses.Distillation newly in the presence of granular potassium hydroxide before piperidines uses.Use HPLC level solvent to carry out HPLC and analyze, described HPLC level solvent is earlier the nylon membrane filtration of 0.45 μ m before use through hole.Buy amino acid, Wang resin, the trityl chlorine resin of Fmoc-protection and have the Wang resin of Methionin nuclear from AdvancedChemTech Inc. (its address is 5609 Fern Valley Road, Louisville, KY 40228).Buy all the other chemical reagent from Aldrich Chemical Company Ltd. (its address is 1001 WestStreet, Paul Avenue, Milwaukee, WI 53233).Use ChemSpeed WSM 500 parallel synthesizers to carry out peptide solid phase synthesis (SPPS).Use SpeedvacSC 250DDA (Thermo Savant) that the synthetic peptide is carried out freeze-drying.
The Fmoc amino acid strategy of the conventional peptide solid phase synthesis of synthetic employing of fan-shaped polypeptide synthesizes polypeptide according to literature method
26-27To be coated with the Wang resin (referring to reaction scheme 1) of Methionin nuclear in advance as solid carrier.The growth of peptide chain is finished by repeating following go protection (DeFmoc step) and coupling (Coupling step) step:
The DeFmoc step under room temperature, with freshly prepd 20% (v/v) piperidine solution (the 0.6mL piperidines is dissolved in the 2.4mL dimethyl formamide) be added to the Wang resin (0.2mmol/g, 100mg) in, and stirred 20 minutes.Filter resin, use DMF: thioanisole mixture (100: 0.5, each 3mL, 3 times) and methylene dichloride (DCM, each 2mL, 3 times) clean, then at N
2Following dry.Test by triketohydrindene hydrate
28Determine that Fmoc goes protection whether successful.
Coupling step will be dissolved in DMF-thioanisole mixture (100: 0.5; amino acid (1mmol), the I-hydroxybenzotriazole hydrate (HOBT of the Fmoc protection 2mL); 1.0mmol) and 1; (DIC, solution 1.0mmol) are added in the aforementioned de-protected resin 3-di-isopropyl carbodiimide.Stirred reaction mixture is 2 hours under room temperature.Filter resin, use DMF: thioanisole mixture (100: 0.5, each 3mL, 3 times) and methylene dichloride (DCM, each 2mL, 3 times) clean, then at N
2Following dry.Whether the coupling that detects the amino acid of Fmoc-protection and resin with ninhydrin reaction is complete.By the beginning of C-end, repeat to protect the peptide that obtains the hope of following concrete aminoacid sequence with coupling step.
Cleavage of peptide is for the described peptide of cracking from the Wang resin from the resin; at first make polypeptide go protection by the DeFmoc step; then at room temperature, the de-protected peptide that will be connected with resin is added to 4mL and contains (TFA: phenol: TIS: H in the mixture of trifluoroacetic acid, phenol, triisopropyl silicomethane (TIS), water, dithiothreitol (DTT) (DTT) and thioanisole
2O: DTT: thioanisole, 88: 5: 2: 5: 0.05: 0.5, v/v) and stirred 2 hours.Filtering mixt is removed resin, cleans with trifluoroacetic acid (3mL, 3 times).Filtrate is merged, by decompression filtrate is concentrated into about 5mL then.The solution of remnants dropwise is added to makes the peptide precipitation in the t-butyl methyl ether (40mL).With the suspensoid that obtains remain on-20 ℃ 24 hours so that precipitation is able to fully.The centrifugal throw out (2500rpm/ minute, 15 minutes) of collecting white, decant suspends again with t-butyl methyl ether (7mL).Repeated centrifugation-resuspending step 3 time by standing over night under vacuum, is removed trace solvent.Use cutoff value (cutoff) as the permeable membrane of 1.2kDa and dialysis in the deionized water solution of 10% acetate (2 * 3L), use deionized water dialysis (3 * 3L) the light yellow or white solids that obtain then.With the solution freeze-drying of gained, obtain solid (29.3mg, 6.41 * 10 of white
-3Mmol), i.e. four of the fan-shaped HLA-A11 epi-position of LMP2 poly-peptides.Resulting polypeptide characterizes by ESI-MS and amino acid sequence analysis.
The synthetic line style polypeptide of linear polypeptide synthesizes (referring to reaction scheme 2) by the peptide and the low polylysine skeleton of coupling side chain protected.
Synthesizing of low polylysine skeleton
Four polylysine skeletons of Fmoc-protection are prepared by the solid-phase peptide synthetic method.
Peptide synthetic with Side chain protective group
A11 (SSCSSCPLSK), A24 (TYGPVFMSL) and B40 (IEDPPFNSI) peptide are to use trityl chlorine resin to prepare by conventional solid-phase peptide synthetic method as solid carrier.The cracking condition of peptide is as follows: at room temperature, the peptide that will be connected with resin is added to 4mL and contains (TFA: phenol: TIS: H in the mixture of trifluoroacetic acid, phenol, triisopropyl silicomethane, water, dithiothreitol (DTT), thioanisole and acetate
2O: DTT: thioanisole, 1: 5: 2: 5: 0.05: 0.5: 87, v/v) and stirred 2 hours.Filtering mixt is removed resin, cleans with acetate (3mL, 3 times).Filtrate is merged, by decompression filtrate is concentrated into about 5mL then.
The peptide of protection is connected with low polylysine skeleton
The peptide of protection is dissolved among the DMF, and stirred together 4 hours with N-hydroxy-succinamide (HOSu, 1 equivalent), HOBT (1 equivalent) and DIC (1 equivalent).By adding ethanol reaction is stopped then.The solvent in the reaction mixture is removed in decompression.To wherein adding low polylysine skeleton (0.2 equivalent) and at room temperature being dissolved among the DMF (1mL) again and stirring 16 hours.Add piperidines (0.25mL) to remove Fmoc protecting group and standing and reacting mixture 20 minutes.Post-processing step is as follows: reduce pressure out and desolvate, at room temperature resistates is dissolved in again the mixture that 15mL contains trifluoroacetic acid, phenol, TIS, water, DTT and thioanisole (88: 5: 2: 5: 0.05: 0.5, v/v) in and stirred 2 hours.By decompression reaction mixture is concentrated into about 5mL.The solution of remnants dropwise is added to makes the peptide precipitation in the t-butyl methyl ether (40mL).With the suspensoid that obtains remain on-20 ℃ 24 hours so that precipitation is able to fully.The centrifugal throw out (2500rpm/ minute, 15 minutes) of collecting white, decant suspends again with t-butyl methyl ether (7mL).Repeated centrifugation-resuspending step 3 time by standing over night under vacuum, is removed trace solvent.Use cutoff value as the permeable membrane of 1.2kDa and dialysis in the deionized water solution of 10% acetate (2 * 3L), use deionized water dialysis (3 * 3L) the light yellow or white solids that obtain then.With the solution freeze-drying of gained, obtain solid (22mg, 5.0 * 10 of white
-3Mmol), i.e. four of the linear HLA-A11 epi-position of LMP2 poly-peptides.Resulting polypeptide characterizes by ESI-MS and amino acid sequence analysis.
Similarly, synthesized eight poly-peptides, four poly-peptides of fan type LMP2 HLA-A24 epi-position, eight poly-peptides, four poly-peptides of fan type LMP2 HLA-B40 epi-position, the eight poly-peptides of fan type LMP2 HLA-A11 epi-position by above-mentioned steps, and eight poly-peptides of line style LMP2 HLA-A11 epi-position, four poly-peptides of line style LMP2HLA-A24 epi-position, eight poly-peptides, four poly-peptides of line style LMP2 HLA-B40 epi-position, eight poly-peptides.
The aminoacid sequence of above-mentioned synthetic peptide and molecular weight (analyzing with Mass Spectrometry) are respectively
Four poly-peptides of line style LMP2 HLA-A11 epi-position, molecular weight 4450kDa
Eight poly-peptides of line style LMP2 HLA-A11 epi-position, molecular weight 8880kDa
Four poly-peptides of line style LMP2 HLA-A24 epi-position, molecular weight 4512kDa
Eight poly-peptides of line style LMP2 HLA-A24 epi-position, molecular weight 9006kDa
Four poly-peptides of line style LMP2 HLA-B40 epi-position, molecular weight 4580kDa
Eight poly-peptides of line style LMP2 HLA-B40 epi-position, molecular weight 9140kDa
Four poly-peptides of fan type LMP2 HLA-A11 epi-position, molecular weight 4568kDa
Eight poly-peptides of fan type LMP2 HLA-A11 epi-position, molecular weight 9000kDa
Four poly-peptides of fan type LMP2 HLA-A24 epi-position, molecular weight 4635kDa
Eight poly-peptides of fan type LMP2 HLA-A24 epi-position, molecular weight 9120kDa
Four poly-peptides of fan type LMP2 HLA-B40 epi-position, molecular weight 4701kDa
Eight poly-peptides of fan type LMP2 HLA-B40 epi-position, molecular weight 9260kDa.
Enzyme linked immunological spot analysis (ELISPOT)
The special IFN-γ of LMP2 epi-position produces cell among the PBMC for preparing in order to detect, and carries out the ELISPLT operation.Briefly, (Millipore MA) uses resisting-IFN-γ (Endogen, MA, USA) monoclonal antibody bag quilt of 15ug/ml to 96 orifice plates in advance.The PBMCs that gathers with the back before inoculation is with 1 * 10
6/ Kong Jiasan parallel hole adds corresponding peptide (2 μ g/ml) then.This dull and stereotyped night incubation (37 ℃, 5%CO2).(Endogen, MA USA), put room temperature 2-3 hour then, and (Serotec USA) is put two hours more then to add the alkaline phosphatase of avidin mark to add the anti-IFN-γ monoclonal antibody of 1 μ g/ml vitamin H acyl groupization after washing plate in second day.Discrete IFN-γ produces cell detection to being stain after acting on 30 minutes with substrate (5-bromo-4-chloro-3-iodine phosphoric acid salt and nitro tetrazolium drone blue).This spot is counted at the analysis microscopically.Have that specificity replys the T cell count calculated deducting feminine gender (with the hole of DMSO solvent control) contrast back.
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Claims (19)
1. a polypeptide or its pharmacologically acceptable salt, wherein said polypeptide derives from the proteic fragment SSCSSCPLSK of the distinctive LMP2 of Epstein-Barr virus for being selected from; TYGPVFMSL; Or two-eight aggressiveness of IEDPPFNSI, described polymer is connected by being selected from following joint by corresponding polypeptide fragment:
Aminoterminal and fragment SSCSSCPLSK among "-" expression K in the wherein above-mentioned joint; TYGPVFMSL; Or the key of IEDPPFNSI carboxyl terminal connection, " K ", " A " and " C " are amino acid whose abbreviation.
2. the polypeptide of claim 1 or its pharmacologically acceptable salt, joint wherein is selected from:
3. the polypeptide of claim 1 or its pharmacologically acceptable salt, wherein said polypeptide is fragment SSCSSCPLSK; TYGPVFMSL; Or four-eight aggressiveness of IEDPPFNSI.
4. the polypeptide of claim 3 or its pharmacologically acceptable salt, wherein said polypeptide is fragment SSCSSCPLSK; TYGPVFMSL; Or four or eight aggressiveness of IEDPPFNSI.
5. the polypeptide of claim 4 or its pharmacologically acceptable salt, wherein said polypeptide is fragment SSCSSCPLSK; TYGPVFMSL; Or the tetramer of IEDPPFNSI.
6. the polypeptide of claim 5 or its pharmacologically acceptable salt, the wherein said tetramer is selected from:
Claim 1-6 each polypeptide or the preparation method of its pharmacologically acceptable salt; comprise the peptide solid phase synthesis process of selecting Fmoc amino acid strategy for use; by using precoating to have the Wang resin of Methionin nuclear to go protection and the next amino acid of coupling to make the peptide chainpropagation by alternately making amino, and the step of cleavage of peptide from the resin at last.
8. pharmaceutical composition wherein comprises each polypeptide or its pharmacologically acceptable salt of claim 1-6, and conventional pharmaceutical carrier or excipient.
9. the composition of claim 8 wherein also comprises a kind of other active ingredient.
10. the composition of claim 9, wherein other active ingredient is selected from another kind of EBV specific proteins and cytokine.
11. a treatment and prevention Epstein Barr virus infection or the method for cancer relevant with Epstein Barr virus comprise to being executed individuality and directly use each polypeptide or its pharmacologically acceptable salt of claim 1-6; Perhaps inject each polypeptide or the post-stimulatory dendritic cell of its pharmacologically acceptable salt of claim 1-6; Or injection is subjected to each polypeptide or its pharmacologically acceptable salt step of stimulating the T cell of back amplification in vitro of claim 1-6.
12. the methods of treatment of claim 11, the wherein said cancer relevant with Epstein Barr virus is selected from cancer of the stomach, mammary cancer, B cell lymphatic cancer, transplants back B cell amplification pathology and nasopharyngeal carcinoma.
13. the methods of treatment of claim 12, the wherein said cancer relevant with Epstein Barr virus is nasopharyngeal carcinoma.
14. the methods of treatment of claim 11, wherein said T cell is a cytotoxic T cell.
15. the methods of treatment of claim 11, wherein said dendritic cell are to come from from body or allochthonous peripheral blood monocyte.
16. a vaccine wherein comprises each polypeptide or its pharmacologically acceptable salt of claim 1-6.
17. each polypeptide or its pharmacologically acceptable salt purposes of being used to prepare the medicine of treatment and prevention Epstein Barr virus infection or the cancer relevant or being used to prepare vaccine of claim 1-6 with Epstein Barr virus.
18. the purposes of claim 17, the wherein said cancer relevant with Epstein Barr virus is selected from cancer of the stomach, mammary cancer, B cell lymphatic cancer, transplants back B cell amplification pathology and nasopharyngeal carcinoma.
19. claim 18 purposes, the cancer that wherein said and Epstein Barr virus is relevant is a nasopharyngeal carcinoma.
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| CN 200510104019 CN1927881A (en) | 2005-09-09 | 2005-09-09 | Polypeptide, preparation method thereof, pharmaceutical composition and vaccine containing the same and application thereof |
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| CN 200510104019 CN1927881A (en) | 2005-09-09 | 2005-09-09 | Polypeptide, preparation method thereof, pharmaceutical composition and vaccine containing the same and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016095783A1 (en) * | 2014-12-17 | 2016-06-23 | 中国科学院广州生物医药与健康研究院 | T cell receptor for identifying eb virus short peptide |
| CN113943783A (en) * | 2021-06-21 | 2022-01-18 | 重庆天科雅生物科技有限公司 | Fluorescent PCR primer group for specifically recognizing TYGPVFMSL peptide fragment and application thereof |
-
2005
- 2005-09-09 CN CN 200510104019 patent/CN1927881A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016095783A1 (en) * | 2014-12-17 | 2016-06-23 | 中国科学院广州生物医药与健康研究院 | T cell receptor for identifying eb virus short peptide |
| CN107001444A (en) * | 2014-12-17 | 2017-08-01 | 中国科学院广州生物医药与健康研究院 | Recognize the φt cell receptor of Epstein-Barr virus small peptide |
| CN107001444B (en) * | 2014-12-17 | 2020-11-27 | 中国科学院广州生物医药与健康研究院 | T cell receptor for identifying EB virus short peptide |
| CN113943783A (en) * | 2021-06-21 | 2022-01-18 | 重庆天科雅生物科技有限公司 | Fluorescent PCR primer group for specifically recognizing TYGPVFMSL peptide fragment and application thereof |
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