CN104211770A - Compound formed by neuroglioma related peptide and hot shock protein and applications thereof - Google Patents
Compound formed by neuroglioma related peptide and hot shock protein and applications thereof Download PDFInfo
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Abstract
The invention provides neuroglioma antigens capable of binding with hot shock protein, including Survivin-2B antigen peptide, Ku70 antigen peptide, and Ku80 antigen peptide. The invention further provides a compound formed by neuroglioma related peptide with hot shock proteins (gp96 and HSP78) and a preparation method thereof. The compound comprises a complex formed by connecting the gp96 and HSP78 to antigen polypeptide through non-covalent bonds and also comprises fusion proteins which are formed by connecting gp96 and HSP78 to antigen polypeptide through covalent bonds. The compound can be used to prepare curative vaccines for treating neuroglioma.
Description
Technical field
The invention belongs to biological technical field, particularly the mixture of glioma antigen polypeptide and heat shock protein(HSP) gp96 and HSP78 and their purposes.
Background technology
Neurospongioma is most common cancer in neural system, accounts for 32% ~ 60% of intracranial tumors, there is no radical cure method at present.Annual neurospongioma sickness rate is 3-6 people/100,000 people, year death toll reach 30,000 people, and sickness rate is in continuous rising.Neurospongioma is infiltrative growth, and operation cannot be excised completely, must could extend patient's lifetime by the general treatment measures such as combination with radiotherapeutic, chemotherapy.But due to the existence of hemato encephalic barrier and the heterogeneity of tumour and resistance, growth and the transfer of tumour all cannot be effectively eliminated or be controlled in traditional radiotherapy, chemotherapy and associating thereof.
Recent study finds that the heat shock protein(HSP) gp96 of separation and purification from Response in Patients with Gliomas tumor tissues and HSP78 molecular immune sufferers themselves can cause the specific immunity rejection of body generation for self neural glioma, plays the effect postponing tumor recurrence.
Heat shock protein(HSP) (Heat Shock Protein, HSP) is a class high conservative, is extensively present in the protein in protokaryon and eukaryote.Gp96 is the important member in heat shock protein(HSP) HSP90 family, in healthy tissues and tumour, all have wide expression, is mainly positioned endoplasmic (Endoplasmic Reticulum).Gp96 is low expression level in cell under normal circumstances, but heat-shocked, glucose shortage, bacterium and virus infection etc. all can induce it to express increase.Gp96, as molecular chaperones, can stop protein aggregation, assists protein folding, stretching, extension, assembling and transhipment, suppresses the secretion of misfolded proteins matter.Gp96 molecule has the ability of Binding peptide, can in conjunction with 5-25 amino acid whose peptide sequence.Heat shock protein(HSP) HSP78 is the member in tenuigenin in HSP70 family, and molecular weight is about 78kDa.HSP78 molecule can be combined by various small peptide as molecular chaperones in cell.
Research in recent years finds, HSP itself does not have antigenicity, and antigenicity is determined by the small peptide combined.The combining small peptide of HSP, in passing MHC I quasi-molecule, activates specificity and memory t cell, causes Cellular Immunity reaction.Gp96 can also promote the expression of MHC I class and MHC II quasi-molecule and costimulating factor by activating dendritic cells (DC), thus improves immune response.Therefore gp96 can the effective polypeptide that combines of submission, the specific ctl response of efficient activating peptide.The mixture of this gp96-polypeptide and HSP78-polypeptide complex can be used for control autologous tumor and some communicable disease.
Confirm that gp96 and HSP78 molecular energy is in conjunction with plurality of antigens polypeptide at present, comprised vesicular stomatitis virus antigen district peptide, the ovalbumin epitope peptide of mouse H-2Kb restriction, the leukemia epitope peptide, antigen of hepatitis B virus peptide etc. of H-2La restriction.But have no the antigenic peptide that isolation identification is combined with HSP from neurotic knurl specimens so far.
Neurospongioma is as common cancer most in neural system.Although the Progression free survival phase of onset tumour patient can be improved 160% by autologous gp96 immunity and chemoradiotherapy plus application, the overall survival phase was extended to 24 months by 14.6 months.But 2 years Progression free survival rates of all Response in Patients with Gliomas are still no more than 20%, and neurospongioma still can not be cured on the whole.Its major cause be this methods for the treatment of to the requirement of tumor tissues size and vaccine prepare disposable.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is the peptide section of screening Glioma-Specific.Overcome the reduced immunogenicity that tumour cell immunoediting causes, can at healthy individuals and Response in Patients with Gliomas inducing producing specificity cytotoxic T lymphocyte (also known as killer T cell, CTL), and CTL has powerful anti-glioma effect, to normal cell and tissue without destruction.
The present invention solves the problems of the technologies described above one of adopted technical scheme: a kind of epitope peptide, it is characterized in that epitope peptide derives from tumour antigen SURVIVIN.Described epitope peptide is from the 87th amino acids of SURVIVIN-2B, and peptide section is made up of 9 aminoacid sequences, and the amino acid of described epitope peptide is TLGGRGGRI, as shown in SEQ ID NO.1 in sequence table.
In the present invention, described epitope peptide can form mixture with heat shock protein(HSP) gp96 or HSP78.
Mixture of the present invention had both comprised heat shock protein(HSP) and had been combined with polypeptide with non covalent bond, comprised again heat shock protein(HSP) and was combined with polypeptide with covalent linkage.The invention provides the method for the mixture of preparation antigenic peptide described above of the present invention and heat shock protein(HSP) gp96 and HSP78.
The epitope peptide that the present invention tells can separately or with heat shock protein(HSP) composite form specific CTL of induced synthesis in HLA-A2 transgenic mice.
The specific CTL of formation of the present invention can transfer the significant growth suppressing the neuroglial cytoma U251 of the HLA-A2 positive in experiment.
The epitope peptide that the present invention tells can stimulate HLA-A2 positive neurons patients with gliomas PBMC to secrete IFN-g separately or with heat shock protein(HSP) composite form in vitro, and thisly acts on the immunity of autologous gp96 mixture and significantly strengthen afterwards.
The epitope peptide that the present invention tells can improve the reaction of HLA-A2 positive neurons patients with gliomas specific T-cells separately or with heat shock protein(HSP) composite form.
The induction of specific CTL of the present invention can be strengthened by the inhibitor of Treg cell.
The present invention solves the problems of the technologies described above two of adopted technical scheme: a kind of neurospongioma epitope peptide, it is characterized in that epitope peptide derives from tumour antigen SURVIVIN.Described epitope peptide is from the 74th amino acids of SURVIVIN-2B, and peptide section is made up of 9 aminoacid sequences, and the amino acid of described epitope peptide is IGPGTVAYA, as shown in SEQ ID NO.2 in sequence table.
In the present invention, described epitope peptide can form mixture with heat shock protein(HSP) gp96 or HSP78.
Mixture of the present invention had both comprised heat shock protein(HSP) and had been combined with polypeptide with non covalent bond, comprised again heat shock protein(HSP) and was combined with polypeptide with covalent linkage.The invention provides the method for the mixture of preparation antigenic peptide described above of the present invention and heat shock protein(HSP) gp96 and HSP78.
The epitope peptide that the present invention tells can separately or with heat shock protein(HSP) composite form specific CTL of induced synthesis in HLA-A2 transgenic mice.
The specific CTL of formation of the present invention can transfer the significant growth suppressing the neuroglial cytoma U251 of the HLA-A2 positive in experiment.
The epitope peptide that the present invention tells can stimulate HLA-A2 positive neurons patients with gliomas PBMC to secrete IFN-g separately or with heat shock protein(HSP) composite form in vitro, and thisly acts on the immunity of autologous gp96 mixture and significantly strengthen afterwards.
The epitope peptide that the present invention tells can improve the reaction of HLA-A2 positive neurons patients with gliomas specific T-cells separately or with heat shock protein(HSP) composite form.
The induction of specific CTL of the present invention can be strengthened by the inhibitor of Treg cell.
The present invention solves the problems of the technologies described above three of adopted technical scheme: a kind of neurospongioma epitope peptide, it is characterized in that epitope peptide derives from tumour antigen SURVIVIN.Described epitope peptide is from the 77th amino acids of SURVIVIN-2B, and peptide section is made up of 9 aminoacid sequences, and the amino acid of described epitope peptide is GTVAYACNT, as shown in SEQ ID NO.3 in sequence table.
In the present invention, described epitope peptide can form mixture with heat shock protein(HSP) gp96 or HSP78.
Mixture of the present invention had both comprised heat shock protein(HSP) and had been combined with polypeptide with non covalent bond, comprised again heat shock protein(HSP) and was combined with polypeptide with covalent linkage.The invention provides the method for the mixture of preparation antigenic peptide described above of the present invention and heat shock protein(HSP) gp96 and HSP78.
The epitope peptide that the present invention tells can separately or with heat shock protein(HSP) composite form specific CTL of induced synthesis in HLA-A2 transgenic mice.
The specific CTL of formation of the present invention can transfer the significant growth suppressing the neuroglial cytoma U251 of the HLA-A2 positive in experiment.
The epitope peptide that the present invention tells can stimulate HLA-A2 positive neurons patients with gliomas PBMC to secrete IFN-g separately or with heat shock protein(HSP) composite form in vitro, and thisly acts on the immunity of autologous gp96 mixture and significantly strengthen afterwards.
The epitope peptide that the present invention tells can improve the reaction of HLA-A2 positive neurons patients with gliomas specific T-cells separately or with heat shock protein(HSP) composite form.
The induction of specific CTL of the present invention can be strengthened by the inhibitor of Treg cell.
The present invention solves the problems of the technologies described above four of adopted technical scheme: a kind of neurospongioma epitope peptide, it is characterized in that epitope peptide derives from tumour antigen Ku70.Described epitope peptide is from the 373rd amino acids of Ku70, and peptide section is made up of 9 aminoacid sequences, and the amino acid of described epitope peptide is SLVIGSSTL, as shown in SEQ ID NO.4 in sequence table.
In the present invention, described epitope peptide can form mixture with heat shock protein(HSP) gp96 or HSP78.
Mixture of the present invention had both comprised heat shock protein(HSP) and had been combined with polypeptide with non covalent bond, comprised again heat shock protein(HSP) and was combined with polypeptide with covalent linkage.The invention provides the method for the mixture of preparation antigenic peptide described above of the present invention and heat shock protein(HSP) gp96 and HSP78.
The epitope peptide that the present invention tells can separately or with heat shock protein(HSP) composite form specific CTL of induced synthesis in HLA-A2 transgenic mice.
The specific CTL of formation of the present invention can transfer the significant growth suppressing the neuroglial cytoma U251 of the HLA-A2 positive in experiment.
The epitope peptide that the present invention tells can stimulate HLA-A2 positive neurons patients with gliomas PBMC to secrete IFN-g separately or with heat shock protein(HSP) composite form in vitro, and thisly acts on the immunity of autologous gp96 mixture and significantly strengthen afterwards.
The epitope peptide that the present invention tells can improve the reaction of HLA-A2 positive neurons patients with gliomas specific T-cells separately or with heat shock protein(HSP) composite form.
The induction of specific CTL of the present invention can be strengthened by the inhibitor of Treg cell.
The present invention solves the problems of the technologies described above five of adopted technical scheme: a kind of neurospongioma epitope peptide, it is characterized in that epitope peptide derives from tumour antigen Ku80.Described epitope peptide is from the 356th amino acids of Ku80, and peptide section is made up of 9 aminoacid sequences, and the amino acid of described epitope peptide is FMGNQVLKV, as shown in SEQ ID NO.5 in sequence table.
In the present invention, described epitope peptide can form mixture with heat shock protein(HSP) gp96 or HSP78.
Mixture of the present invention had both comprised heat shock protein(HSP) and had been combined with polypeptide with non covalent bond, comprised again heat shock protein(HSP) and was combined with polypeptide with covalent linkage.The invention provides the method for the mixture of preparation antigenic peptide described above of the present invention and heat shock protein(HSP) gp96 and HSP78.
The epitope peptide that the present invention tells can separately or with heat shock protein(HSP) composite form specific CTL of induced synthesis in HLA-A2 transgenic mice.
The specific CTL of formation of the present invention can transfer the significant growth suppressing the neuroglial cytoma U251 of the HLA-A2 positive in experiment.
The epitope peptide that the present invention tells can stimulate HLA-A2 positive neurons patients with gliomas PBMC to secrete IFN-g separately or with heat shock protein(HSP) composite form in vitro, and thisly acts on the immunity of autologous gp96 mixture and significantly strengthen afterwards.
The epitope peptide that the present invention tells can improve the reaction of HLA-A2 positive neurons patients with gliomas specific T-cells separately or with heat shock protein(HSP) composite form.
The induction of specific CTL of the present invention can be strengthened by the inhibitor of Treg cell.
The present invention solves the problems of the technologies described above six of adopted technical scheme: provide the method preparing glioma antigen peptide and heat shock protein(HSP) formation mixture.
The invention provides a kind of method preparing the non-covalent complex of glioma antigen peptide and heat shock protein(HSP) gp96.In the method for the invention, at the DPBS damping fluid of pH 7.4, temperature of reaction 60 DEG C, 10 minutes reaction times, the concentration of gp96 albumen is 0.421 mmol/L, effectively can form the mixture of gp96-antigen peptide when the concentration of 9 peptide antigens is 3.0 mmol/L in vitro.
Present invention also offers the method for the fusion rotein that glioma antigen peptide and heat shock protein(HSP) gp96 are formed with covalent linkage.Synthetic corresponds to the nucleotide sequence of this peptide, adopts the conventional method of molecular biology this sequence to be connected to gp96 gene 5 ' end, then amalgamation and expression in debaryomyces hansenii.Such as, introduce restriction enzyme site Bgl II the nucleotide sequence of this 9 peptide is connected with gp96 gene 5 ' end T4 ligase enzyme, connect 5 ' and 3 ' end introducing, 2 restriction enzyme site EcoRI and XhoI of product, to be connected in expression vector pPICZaA secreting, expressing in pichia spp, expression product is the fusion rotein of gp96 and 9 peptides.
Special 9 peptides are isolated in the polypeptide be combined with heat shock protein(HSP) gp96 from five routine human gliomas first, be " TLGGRGGRI " through amino acid sequence analysis, find that this sequence is positioned at the 87-95 position of Glioma-Specific antigen SURVIVIN-2B through inquiry, this sequence of synthetic is also assembled with recombinant expressed gp96 albumen, external synthesis gp96-9 peptide complex, by 9 peptides and gp96-9 peptide complex immunity HLA-A2 transgenic mice, equal can stimulate mouse produce specific cytotoxic t lymphocytes (CTL), it is high more than 100 times that gp96-9 peptide complex immunogenicity ratio is used alone peptide.Experimental result shows that gp96-9 peptide complex can be developed into the curative drug into a kind of new type nerve glioma.
brief Description Of Drawings
Fig. 1 is with 9 peptides and gp96-9 peptide complex (0,1,3 weeks) immune mouse BALB/c (HLA-A2
+) cause afterwards specific CTL reaction.Effector cell CTL to the cracking percentage of specificity target cell with 4 hours standards
51cr discharges mensuration, and effector cell and target cell ratio are that in 10,25,50 and 100, figure, cleavage rate is 10 mouse mean values respectively.
Fig. 2 U251 tumor bearing nude mice tumor growth curve.Nude mice by subcutaneous inoculation human glioma cell U251, lotus knurl transfers the mouse spleen lymphocyte of different sources after 7 days.The acceptance of peptide section 1 group (◆ n=20) is through the BALB/c (HLA-A2 of gp96-peptide section 1 mixture immunity
+) spleen lymphocyte of mouse; Irrelevant peptide group (▲ n=20) accepts to have nothing to do through gp96-the BALB/c (HLA-A2 of peptide (HBcAg80-92) mixture immunity
+) spleen lymphocyte of mouse.
The change of 9 peptide-specific T-cell after Fig. 3 Response in Patients with Gliomas immunity autologous tumor gp96 mixture.9 peptide-specific T-cell in peripheral blood in patients are detected with the ELISPOT method of IFN-g.Irrelevant peptide HBcAg
82-90peptide is negative control.
embodiment:
Further illustrate the present invention by embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that manufacturer advises.
Embodiment 1. is purifying gp96 albumen from cerebral tissue
By centrifugal after five parts of human gliomas and a brain tissue homogenate, with 30%/70% saturation ratio (NH
4)
2sO
3precipitation, ConA Sepharose(GE company is adopted after resolution of precipitate) carry out affinity chromatography, with the albumen of the a-methyl glucoside elution of bound of 10%, glucosides elutriant carries out anion chromatography with Hitrap-Q (GE company chromatographic system), obtains the gp96 albumen of >95% purity through this three steps purifying.Gp96 albumen gp96/grp94 monoclonal antibody (Santa Cruz company) carries out Western qualification.Its purity SDS-PAGE, silver dye and reversed-phase HPLC qualification.
Embodiment 2. is from the polypeptide of gp96 protein delivery Non-covalent binding
The gp96 albumen of purifying being added trifluoroacetic acid (TFA) makes its final concentration reach 0.2%(pH about 2.0), then (molecular weight cut-off is 30kDa to use ultrafiltration process, Millipore company) isolated polypeptide, polypeptide mixture is carried out MALDI-TOF mass spectrum (ABI 4700) analysis, polypeptide molecular weight is mostly between 600-1100 Da.
Embodiment 3. analysed by reverse phase HPLC polypeptide
Be dissolved in solution A (0.065% TFA, 2% acetonitrile) after polypeptide mixture freeze-drying, be splined on C18 reversed-phase column (Sephasil peptide C18; 5 mm granularities; 4.6 X 250 mm, GE company), carry out gradient elution from 0 to 65% solution B (0.05% TFA, 100% acetonitrile), flow velocity is 1 mL/min, uses 214nm wavelength detecting.Comparison of tumor tissue and healthy tissues HPLC collection of illustrative plates, find that five parts of neurospongioma tumor tissues have and the peptide peak do not had in healthy tissues.
Embodiment 4. polypeptide microsequencing and sequential analysis
Collect this special peptide peak, identify its purity with MALDI-TOF mass spectrum (PE company Voyager-DE system), only find that molecular weight is the simple spike of 1033 Da.To this peptide microsequencing (ABI, Procise 491 Protein Sequencer), its aminoacid sequence is " TLGGRGGRI ", and the sequence that 5 parts of tumor tissues obtain is consistent.By this sequence in online albumen database (NCBI) inquiry, find that it is positioned at the 87-95 position of SURVIVIN-2B albumen.
The external rapid-assembling of polypeptide of embodiment 5. gp96 albumen and synthetic
Synthetic 9 peptide " TLGGRGGRI " (entrusting the synthesis of gill biochemical company limited), carries out assembled in vitro by gp96 albumen and polypeptide, external combination anchor:
2.7 mmol/L KCl, 1.47 mmol/L KH
2pO
4, 8.1 mmol/L Na
2hPO
4, 138 mmo1/L NaCl, 10% (V/V) glycerine, 3.0 mol/L 9 peptides, 0.421 mmol/L gp96 albumen, 60 DEG C are reacted 10 minutes, and ultrafiltration process (molecular retention 30 KDa, Millipore company) removes unconjugated polypeptide.
The fusion rotein of secreting, expressing glioma antigen peptide 1 and heat shock protein(HSP) gp96 in embodiment 6 Bichi yeast system
This example provides antigen peptide 1(TLGGRGGRI) method of fusion rotein that formed with covalent linkage with heat shock protein(HSP) gp96.We have done the optimization for Pichia anomala expression to the nucleotide sequence corresponding to this peptide, and synthetic this sequence (ACT TTG GGT GGT CGT GGT GGT CGT ATT), introduce restriction enzyme site Bgl II the nucleotide sequence of this 9 peptide is connected with the people gp96 gene 5 ' end T4 ligase enzyme optimized for pichia spp preferred codons, connect 5 ' and 3 ' end introducing 2 restriction enzyme site EcoR I and Xho I of product, to be connected in expression vector pPICZaA secreting, expressing in pichia spp, expression product is the fusion rotein of gp96 and this 9 peptide.
Embodiment 7. immune mouse
Select the genetically modified BALB/c mouse (HLA-A2 of HLA-A2 in growth 8-10 week
+) for this experiment.
Immunization ways adopts nape subcutaneous injection in PBS sample being dissolved in 100mL.Subcutaneous injection operation is relatively simple, requires that immunizing dose is moderate, therefore adopts this kind of immunization ways.Simultaneously at the endoxan of each immunity first 1 day abdominal injection 0.4 mg.Low-dose cyclophosphamide can suppress in gp96 immunity, play inhibiting Treg cell thus can strengthen immune effect.
The suitableeest immunizing dose of gp96 albumen-9 peptide complex is 0.1 nmol.
Immunization time is 0,1,3 week reinforced immunological carrying out three times, is better than immunity once or the effect of secondary.
Therefore subcutaneous injection immunity is adopted, 9 peptides are dissolved in damping fluid (90% PBS 10% DMSO 0.1% TFA), gp96-peptide 1 mixture is dissolved in PBS, thermal agitation 1 minute before injection, every mouse immune dosage peptide section 1 is respectively 1 nmol and 10 nmol, by immune mouse after peptide and freund's adjuvant emulsification.Gp96-peptide section 1 mixture immunizing dose is 0.05 nmol respectively, 0.10 nmol and 0.50 nmol, adopts nape subcutaneous injection, and carry out second time immunity after immune 1 week of first time, booster immunization after 2 weeks, carried out cytotoxicity analysis after 3 days.Each process use 10 mouse.
Embodiment 8. cytotoxicity analysis
Mouse booster immunization is after 3 days, from every mouse gather in the crops about 3 × 10
7splenocyte is suspended from containing 10 mM HEPES damping fluids, 5 × 10
-5m coloured glaze base ethanol, in microbiotic and 10% (V/V) FCS nutrient solution, in culturing bottle with penetrate through width the T2 cell (3:1) of (4500 Rad) and 1 mg/mL peptide in perfect medium 37 DEG C cultivate.Within 6 days, collect splenocyte afterwards and carry out 4 hours standards
51cr release experiment (concrete grammar is shown in Kuhrober, A, et al. 1997. International Immunology, 9 (8): 1203-1212) measures cellular cytoxicity activity.In brief, target cell adds the effector cell of different quantities after 30 minutes in 37 DEG C of sensitization with 10mg/mL peptide, reaction system is the perfect medium of 100 mL.37 DEG C of Dual culture 4 h before harvest supernatants measure specific lysis rate.
From
51cr release experiment can find out that gp96-peptide 1 mixture and independent peptide section 1 all can stimulate mouse to produce specific cytotoxic t lymphocytes, but the immune effect of gp96 mixture makes more than 100 times of independent 9 peptides.Gp96-peptide 1 mixture of every mouse immune 0.1 nmol (about 10mg) can bring out body and produce strong cell immune response, the cleavage rate of cytotoxic assay target cell is more than 50%, and the pre-treatment of endoxan can strengthen this cellulotoxic effect (Fig. 1) further.Experimental result shows that gp96-peptide 1 mixture can be developed into the medicine into a kind of new type nerve glioma.
Embodiment 9 cell transfusions tumor inhibition
With HLA-A2 positive human neuroglial cytoma U251 subcutaneous injection nude mice.Transfer the spleen lymphocyte (n=20), the gp96-that obtain from gp96-glioma antigen 9 peptide 1 mixture immunity HLA-A2 transgenic mice after 7 days to have nothing to do peptide complex immune mouse lymphocyte (n=20), transfer weekly once, totally 3 weeks.Detect the growth (Fig. 2) that lymphocyte that tumor size can find only to transfer gp96-glioma antigen 9 peptide 1 mixture immune mouse obviously can suppress neuroglial cytoma.
The autologous gp96 complex therapies tumour of embodiment 10 Response in Patients with Gliomas immunity
The Response in Patients with Gliomas entering the gp96 immunotherapy of group in this research completes the treatment of 6 courses for the treatment of.Within one week, be a course for the treatment of, each injection course for the treatment of once.Test group carries out conventional treatment and gp96 autologous immunotherapy after 12 weeks after surgery.
Operating process
I patient selection: onset curtain epineural glioma, accept radical operation, resection rate is greater than 80%.
The purifying of gp96-tumour antigen mixture in ii tumor tissues
Three-step approach (the Meng S delivered according to Meng Songdong etc., Song J, Rao Z, Tien P, Gao G. 2002. Three-step purification of gp96 from human liver tumor tissues suitable for isolation of gp96-bound peptides. Journal of Immunological Methods, 264 (1-2): 29-35.) extraction purification is carried out to gp96 albumen in tumor tissues, obtain purity more than 95% gp96 albumen by ConA post affinity chromatography, Hitrap Q post ion-exchange purification.
Xylene Brilliant Cyanine G determination of color protein concentration, protein extraction amount enough should complete 6 courses for the treatment of.
Sample should meet the Microbiological requirements of vaccine application.
Sample should meet the endotoxin content requirement of vaccine application.
Gp96 sample should have biological activity.
The use of iii gp96-tumour antigen mixture
Patient accepts first time immunotherapy after 12 weeks after surgery.First 1 day of each immunity, intravenous injection endoxan, the then subcutaneous or autologous gp96 mixture of abdominal part hypodermic in deltoid muscle position.1 time weekly, inject 6 times altogether.
Iv amynologic index checks
With autologous tumor lysate and autologous gp96 compound as antigen for stimulator detects autologous tumor specific T-cells.Relatively specific T-cells before and after Response in Patients with Gliomas immunity, finds that the immunity of autologous gp96 mixture significantly have activated patient-specific T cell, predictive of good prognosis.
V follows up a case by regular visits to
After having injected, respectively made a house call 1 time in 3 months, 6 months, 1 year, 1 year half, 2 years time; Make a house call every half a year afterwards 1 time, until Patients on Recurrence or death.
Check content:
1. imaging examination: head nucleus magnetic resonance (MRI) and head strengthening MRI;
2. clinical neural function detects: sensation, motion, vision, KPS scoring;
3. injection site anaphylaxis detects: observe injection site skin peptide situation;
4. autoimmune thyroiditis detects: when the clinical symptom of autoimmune thyroiditis appears in patient, carry out Tiroidina detection.
Embodiment 11. ELISPOT method detection specificity T cell
ELISPOT detects epitope specificity CTL in Response in Patients with Gliomas PBMC.Operate according to the specification sheets of ELISPOT test kit.First close the pre-coated plate of ELISPOT one hour with PRMI 1640 substratum containing 10% foetal calf serum, outwell confining liquid before use, add 100mL containing 1 × 10
5the PBMC(of people directly cultivate 7 days with fresh PBMC or by PBMC amplification in vitro).Add 9 peptides 1 of 10mg/mL in experimental group, PHA that positive control adds 4mg/mL stimulates, negative control adds irrelevant peptide (HBcAg
82-90).Cultivate 26-36 hour in cell culture incubator after, the formation of detection specificity spot, and carry out statistical study.We choose the Response in Patients with Gliomas of the HLA-A2 positive as study group, detect epitope specificity CTL in patient's fresh blood by ELISPOT method, and the frequency of specific CTL is determined by counting amount of speckle.In the HLA-A2 positive neurons patients with gliomas of autologous gp96 mixture immunity, high-frequency TLGGRGGRI specific CTL is all detected in 4 examples, and increase significantly than before immunity, irrelevant peptide HBcAg82-90(negative control do not detected simultaneously) specific T-cells, illustrate that ELISPOT result is epi-position special (Fig. 3).
The immunocompetence of other gp96 mono-polypeptide complex of embodiment 12. measures
Except above-mentioned antigen 9 peptide, we choose again other 4 glioma antigen polypeptide and gp96 assembled in vitro and measure its immunocompetence, these 3 antigenic peptides are 1 respectively) Survivin-2B antigenic peptide " IGPGTVAYA ", 2) Survivin-2B antigenic peptide " GTVAYACNT ", 3) Ku70 antigenic peptide " SLVIGSSTL ", 4) Ku80 antigenic peptide " FMGNQVLKV ".
Respectively this 4 peptide species of synthetic, adopt the reaction system described in embodiment 7 to be combined with gp96 in vitro, this 4 peptide species and gp96 all have higher avidity, and by mensuration association reaction equilibrium constant K, in 4 kinds of peptides and gp96 association reaction, K value is all more than 5.The mixture that the method described in embodiment 5 of employing is formed with above-mentioned 4 kinds of glioma antigen polypeptide and gp96, with the fusion rotein immune mouse respectively of above-mentioned gp96 and 4 kind of glioma antigen polypeptide, and adopt the method described in embodiment 7 to carry out CTL analysis to these 4 kinds of mixtures respectively, from
51cr release experiment can find out that the mixture that these 4 kinds of glioma antigen polypeptide and gp96 are formed all can stimulate HLA-A2 transgenic mice to produce specific cytotoxic t lymphocytes, and the immunocompetence of the mixture that 4 peptide species are formed with gp96 than independent polypeptide height 100-200 doubly.Every mouse immune dosage can bring out body when being 0.1 nmol (about 10mg) and produce strong cell immune response, finds that the cleavage rate of target cell is between 50%-75% by the cytotoxic assay of the mixture formed this 4 peptide species and gp96.
Above experimental result shows that mixture that gp96 and glioma antigen polypeptide assembled in vitro are synthesized can be developed into the medicine into a kind of new type nerve glioma.Because experiment condition limits patent of the present invention can not carry out assembled in vitro and immunocompetence mensuration to each glioma antigen polypeptide, but we make research object by choosing 5 kinds of representational tumor antigen peptides, be combined with gp96 in vitro and carry out immunocompetence mensuration, great many of experiments shows that the mixture that gp96 and these antigenic peptides are formed all can stimulate mouse to produce strong immune response, gp96, as the novel good adjuvant of antigenic peptide, can be developed into as a kind of novel therapeutic vaccine.Therefore, except above-mentioned 5 kinds of antigenic peptides, other any glioma antigen polypeptide and gp96 are combined as new generation vaccine also should within protection scope of the present invention.
Embodiment 12. HSP78-immunogenicity of polypeptides measures
By the mixture that HSP78 and 5 peptide species assembled in vitro are formed, the same gp96 of reaction system (see embodiment 5), by fusion rotein (embodiment 6) immune mouse of the mixture of external synthesis and HSP78 and 5 peptide species.The same gp96 of mouse immune mode, immunizing dose and immune programme for children (see embodiment 7) cytotoxicity (CTL) analytical procedure is shown in embodiment 8 with gp96().Can find out that HSP78-peptide complex can stimulate mouse to produce specific cytotoxic t lymphocytes from 51Cr release experiment, the immunogenicity of HSP78-peptide complex is more than 100 times of antigen alone peptide, every mouse immune 0.1 nmol (about 10mg) can be brought out body and be produced strong cell immune response, and the cleavage rate of cytotoxic assay target cell is more than 50%.Experiment shows that HSP78-9 peptide complex can be developed into the medicine into a kind of new type nerve glioma.
sequence table
Kang Ernuo Bioisystech Co., Ltd of <110> Shenzhen
The mixture that <120> neurospongioma related peptides and heat shock protein(HSP) are formed and application thereof
<130>
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 9
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<213> artificial sequence
<220>
<223> epitope peptide 1
<400> 1
Thr Leu Gly Gly Arg Gly Gly Arg Ile
1 5
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<212> PRT
<213> artificial sequence
<220>
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<400> 2
Ile Gly Pro Gly Thr Val Ala Tyr Ala
1 5
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<211> 9
<212> PRT
<213> artificial sequence
<220>
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<400> 3
Gly Thr Val Ala Tyr Ala Cys Asn Thr
1 5
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<211> 9
<212> PRT
<213> artificial sequence
<220>
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<400> 4
Ser Leu Val Ile Gly Ser Ser Thr Leu
1 5
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<212> PRT
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Phe Met Gly Asn Gln Val Leu Lys Val
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Claims (8)
1. the epitope peptide TLGGRGGRI be combined with heat shock protein(HSP), is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.1 in sequence table.
2. the epitope peptide IGPGTVAYA be combined with heat shock protein(HSP), is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.2 in sequence table.
3. the epitope peptide GTVAYACNT be combined with heat shock protein(HSP), is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.3 in sequence table.
4. the epitope peptide SLVIGSSTL be combined with heat shock protein(HSP), is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.4 in sequence table.
5. the epitope peptide FMGNQVLKV be combined with heat shock protein(HSP), is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.5 in sequence table.
6. the mixture be covalently or non-covalently connected of the epitope peptide as described in any one of claim 1-5 and gp96 or HSP78.
7. the epitope peptide as described in any one of claim 1-5 forms mixture as claimed in claim 6 purposes separately or in co-immunization human body inductive formation killer T cell separately or with heat shock protein(HSP) in vitro.
8. as right wants the purposes of immune induction killer T cell in prevention or treatment neurospongioma as described in 7.
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| CN201310550993.5A CN104211770A (en) | 2013-11-09 | 2013-11-09 | Compound formed by neuroglioma related peptide and hot shock protein and applications thereof |
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| CN201310550993.5A CN104211770A (en) | 2013-11-09 | 2013-11-09 | Compound formed by neuroglioma related peptide and hot shock protein and applications thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109796536A (en) * | 2019-02-22 | 2019-05-24 | 上海尚泰生物技术有限公司 | A kind of preparation method for the CTL targeting a variety of epitopes of glioblastoma |
| CN112891513A (en) * | 2021-01-06 | 2021-06-04 | 重庆医科大学附属永川医院 | Cationic polypeptide-heat shock protein-miRNA (micro ribonucleic acid) gene complex as well as preparation method and application thereof |
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| CN101103108A (en) * | 2005-01-25 | 2008-01-09 | 日本电气株式会社 | HLA-binding peptides, DNA fragments encoding the same and recombinant vectors |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101103108A (en) * | 2005-01-25 | 2008-01-09 | 日本电气株式会社 | HLA-binding peptides, DNA fragments encoding the same and recombinant vectors |
Non-Patent Citations (2)
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| 李文斌等: "脑胶质瘤的免疫治疗", 《第十三届中国科协年会第18分会场-癌症流行病趋势和防控策略研讨会论文集》 * |
| 陈才伟等: "Gp96蛋白的免疫学及其临床应用研究进展", 《生物工程学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109796536A (en) * | 2019-02-22 | 2019-05-24 | 上海尚泰生物技术有限公司 | A kind of preparation method for the CTL targeting a variety of epitopes of glioblastoma |
| CN112891513A (en) * | 2021-01-06 | 2021-06-04 | 重庆医科大学附属永川医院 | Cationic polypeptide-heat shock protein-miRNA (micro ribonucleic acid) gene complex as well as preparation method and application thereof |
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Application publication date: 20141217 |