CN104211772A - Compound of colorectal cancer antigen peptides and heat shock proteins and use thereof - Google Patents
Compound of colorectal cancer antigen peptides and heat shock proteins and use thereof Download PDFInfo
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Abstract
The invention provides colorectal cancer antigens binding to heat shock protein. The colorectal cancer antigens comprise peptide segments derived from colorectal cancer specific antigens of MU5AC, RNF43 and TOMM34. The invention also provides a compound of the colorectal cancer antigens and heat shock proteins gp96 and HSP78. The compound comprises a compound composed of the antigen peptides and the heat shock proteins gp96 and HSP78 binding to the antigen peptides by non-covalent bonds, and also comprises a fusion protein composed of the antigen peptides and the heat shock proteins gp96 and HSP78 binding to the antigen peptides by a covalent bond. The compound can be used for preparation of a therapeutic vaccine for treating colorectal cancer.
Description
Technical field
The invention belongs to biological technical field, particularly the mixture of colorectal cancer antigenic peptide and heat shock protein(HSP) gp96 and HSP78 and their purposes.
Background technology
Heat shock protein(HSP) (Heat Shock Protein, HSP) is a class high conservative, is extensively present in protein in protokaryon and eukaryote, lacks, can express rising under the stress situation such as bacterium and virus infection at heat-shocked, glucose.Gp96 is the important member in heat shock protein(HSP) HSP90 family, mainly be positioned endoplasmic (Endoplasmic Reticulum), can protein aggregation be stoped, assist protein folding, stretching, extension, assembling and transhipment, suppress the secretion of misfolded proteins matter.Gp96 all has wide expression in healthy tissues and tumour.Gp96 molecule is except possessing the function of molecular chaperones, and gp96 molecule also has the ability that polypeptide combines, can in conjunction with 5-25 in cell 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 also can combine with various small peptide in cell.
Research in recent years finds, gp96, HSP78 albumen of tissue extraction has very strong immunogenicity, can effectively evoke tissue-specific immune response.Also find that the small peptide that this antigenicity is combined by these HSP determined simultaneously.The combining small peptide of HSP, in passing I class MHC molecule, activates specific effect and memory T cell, causes long-term cell immune response.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.
Colorectal cancer is one of most common cancer, and recent domestic sickness rate rises year by year.According to the up-to-date tumour registration annual report delivered in 2012, colorectal cancer annual morbidity reached 29.4/10
5, occupy the 3rd of kinds of tumor.Although the patient of 70% ~ 80% can carry out radical surgery excision when making a definite diagnosis, 5 years overall survival rates still only have 50% ~ 60%.Recurrence or distant metastasis can be there is in about 2/3 patient accepting radical operation, the relapse and metastasis of 85% occurs within postoperative 2 years half, the generation of these relapse and metastasis may with to have there is micrometastasis stove when making a definite diagnosis relevant, and current detection methods also cannot find this micrometastasis.These combined causes cause colorectal cancer annual death rate more than 14/10
5, harm is serious.
Clinical trial confirms, and adjuvant chemotherapy of patients significantly can reduce recurrence rate and lifetime is obviously extended.But because chemotherapy can not kill whole cancer cells in body, cancer whether recur finally depend on body self from steady function and immunologic function, simultaneously the susceptibility of colorectal cancer cells to chemotherapeutics is poor.Therefore chemotherapy bring to body toxic side effect, to kill more than chemotherapy the benefit that part cancer cells brings to body to the strike of body's immunity much bigger.
Clinical trial confirms, and adjuvant chemotherapy of patients significantly can reduce recurrence rate and lifetime is obviously extended, but the adjuvant chemotherapy of patients of II phase colorectal carcinoma still exists many disputes, and suggestion carries out adjuvant chemotherapy of patients to the patient that there is high risk factor usually.
On the other hand, the curative effect of molecular targeted agents combined chemotherapy is still indefinite, but display toxicity increases, and targeted drug combined chemotherapy somewhat expensive.Therefore, how individualized treatment seems that benefit is for important.
Carry out the display of gp96 immunotherapy result to the rectal cancer patient of 29 hepatic metastasess abroad, the patient's overall survival OS and disease free survival rate DFS after 2 years that performs the operation is respectively 79% and 32%, has a significant improvement; Do not observe obvious toxic side effect, for research is from now on laid a good foundation simultaneously.
But as malignant tumour serious in Digestive tract, colorectal cancer still can not be cured on the whole.Making slow progress of new chemotherapeutics, targeted drug.Even if the at present autologous gp96 immunotherapy wished of most is because the enough large tumor tissues needed is to prepare vaccine, simultaneously autovaccine prepare disposable, have impact on curative effect and the audient of the method.
The ovalbumin epitope peptide of gp96 and HSP78 molecular energy in conjunction with blister stomatitis virus antigen district peptide, mouse H-2Kb restriction, the antigenic peptide such as leukemia antigen peptide, antigen of hepatitis B virus peptide of La restriction are confirmed at present.But have no the antigenic peptide that isolation identification is combined with HSP from colorectal cancer patients tumor tissues so far.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is the specific peptide section of screening colorectal cancer.Overcome the reduced immunogenicity that tumour cell immunoediting causes, can in colorectal cancer patients or healthy individuals inducing producing specificity cytotoxic T lymphocyte (killer T cell, CTL), and CTL has powerful anti-colorectal carcinoma effect, to normal cell and tissue without destruction.
An object of the present invention is to provide a kind of mixture of the colorectal cancer antigen be combined with gp96 and HSP78.Described colorectal cancer antigen can be the aminoacid sequence of 466-474 position on colorectal cancer tumour antigen MU5AC albumen respectively, and its sequence can be SLDGAQTVV, or its series of variation, colorectal cancer antigen can be the aminoacid sequence of 721-729 position on colorectal cancer tumour antigen MU5AC albumen respectively, and its sequence can be FLDDTGKCV, or its series of variation, colorectal cancer antigen can be the aminoacid sequence of 305-314 position on colorectal cancer tumour antigen MU5AC albumen respectively, its sequence can be KMYATIPEL, or its series of variation, colorectal cancer antigen can be the aminoacid sequence of 631-639 position on colorectal cancer tumour antigen RNF43 albumen respectively, its sequence can be SICPSTSSL, or its series of variation, colorectal cancer antigen can be the aminoacid sequence of 201-209 position on colorectal cancer tumour antigen RNF43 albumen respectively, its sequence can be ILMTVVGTI, or its series of variation, colorectal cancer antigen can be the aminoacid sequence of 30-38 position on colorectal cancer tumour antigen TOMM34 albumen respectively, its sequence can be ALYGRALRV, or its series of variation, colorectal cancer antigen can be the aminoacid sequence of 77-85 position on colorectal cancer tumour antigen TOMM34 albumen respectively, its sequence can be ALALVPFSI, or its series of variation.
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 epitope peptide that the present invention tells can stimulate the positive colorectal cancer patients PBMC secretion of gamma-IFN of HLA-A2 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 positive colorectal cancer patients specific T-cells reaction of HLA-A2 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.
Special 9 peptides are isolated in the polypeptide be combined with heat shock protein(HSP) gp96 from five routine colorectal cancer tumor tissues first, be " SLDGAQTVV " through amino acid sequence analysis, find that this sequence is positioned at the 466-474 position of colorectal cancer specific antigens MU5AC through inquiry, this sequence of synthetic is also assembled with the gp96 albumen of vivoexpression, 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), and the pre-treatment of regulatory T cells inhibitor can strengthen the immune effect more than 2 times of gp96-9 peptide complex.Experimental result shows that gp96-9 peptide complex can be developed into the curative drug into a kind of novel colorectal cancer.
brief Description Of Drawings
Fig. 1. mouse (BALB/c
hLA-A2+) specific CTL reaction.With the cycle immune mouse of gp96-9 peptide complex by 0,1,3 week, last immunity detection specificity lysis rate after 3 days.Effector cell CTL to the cracking percentage of specificity target cell with 4 hours standards
51cr method for releasing measures, effector cell and target cell ratio be respectively 10,25,50 and 100, figure in cleavage rate be the mean value of 10 mouse.
Fig. 2 PC-9 tumor bearing nude mice tumor growth curve.Nude mice by subcutaneous inoculation Human colorectal cancer cells HCT-116, 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 (HBcAg82-90) mixture immunity
+) spleen lymphocyte of mouse.
The change of 9 peptide-specific T-cell after Fig. 3 colorectal cancer patients immunity autologous tumor gp96 mixture.9 peptide-specific T-cell in peripheral blood in patients are detected with the ELISPOT method of IFN-γ.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 colorectal carcinoma
By centrifugal after five parts of Colorectal Carcinomas and a normal bowel 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 α-methylglucoside elution of bound of 8%, glucosides elutriant carries out anion chromatography with Hitrap-Q (GE company), 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 use ultrafiltration process (molecular retention is 30kDa, Millipore company) isolated polypeptide, polypeptide mixture is carried out
mALDI-TOFmass spectrum (ABI 4700) is analyzed, and 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 anti-phase C18 chromatography column (Sephasil peptide C18; 5 um 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 Colorectal Carcinomas have and the peptide peak do not had in normal bowel tissue.
Embodiment 4. polypeptide microsequencing and sequential analysis
Collect this special peptide peak MALDI-TOF mass spectrum (PE company Voyager-DE system) and identify its purity, only find that molecular weight is the simple spike of 889 Da.To this peptide microsequencing (Procise 491. Protein Sequencer, ABI company), its aminoacid sequence is " SLDGAQTVV ", 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 466-474 position of MU5AC albumen.
The external rapid-assembling of polypeptide of embodiment 5. gp96 albumen and synthetic
Synthetic 9 peptide " SLDGAQTVV " (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, 138mmo1/L NaCl, 10% (V/V) glycerine, 3.0 mmol/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.
Embodiment 6 expresses the fusion rotein of colorectal cancer antigen peptide 1 and heat shock protein(HSP) gp96
This example provides colorectal cancer antigen peptide 1(SLDGAQTVV) method of fusion rotein that formed with covalent linkage with heat shock protein(HSP) gp96.Our synthetic corresponds to the nucleotide sequence (AGT TTA GAT GGT GCT CAA ACT GTT GTT) of this peptide, 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 EcoR I and Xho I of product, to be connected in expression vector pPICZ α A 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 100uL.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 being dissolved in damping fluid (90% PBS 10% DMSO 0.1% TFA) gp96-9 peptide complex is dissolved in PBS, thermal agitation 1 minute before injection, every mouse immune dosage 9 peptide is respectively 0.2 nmol, 2 nmol and 20 nmol, by immune mouse after peptide and freund's adjuvant emulsification.Gp96-9 peptide complex immunizing dose is 0.01 nmol respectively, 0.05 nmol, 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 (CTL) is analyzed
Mouse booster immunization, after 3 days, gathers in the crops to obtain about 3 X 10 from every mouse
7splenocyte is suspended from containing 10 mM HEPES damping fluids, 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 1ug/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. InternationalImmunology, 9 (8): 1203-1212) measures cellular cytoxicity activity.In brief, target cell 10ug/mL target peptide or irrelevant peptide add the effector cell of different quantities in 37 DEG C of sensitization after 30 minutes, reaction system is the perfect medium of 100 uL.37 DEG C of Dual culture 4 h before harvest supernatants measure specific lysis rate.
From
51cr release experiment can find out that gp96-9 peptide complex can stimulate mouse to produce specific cytotoxic t lymphocytes, the gp96-9 peptide complex of every mouse immune 0.1 nmol (about 10ug) can bring out body and produce strong cell immune response, the cleavage rate of cytotoxic assay target cell is more than 40%, significantly can improve the immune effect of gp96-9 peptide complex by injecting the pre-treatment of endoxan, and this cellulotoxic effect be epitope peptide specific (Fig. 1).Experimental result shows that gp96-9 peptide complex can be developed into the medicine into a kind of novel colorectal cancer.
Embodiment 9. cell transfusions tumor inhibition
With HLA-A2 positive human colorectal cancer cell HCT-116 subcutaneous injection nude mice.Transfer the spleen lymphocyte (n=20), the gp96-that obtain from gp96-colorectal cancer 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-LuCA 9 peptide 1 mixture immune mouse obviously can suppress colorectal cancer cell.
The autologous gp96 complex therapies tumour of embodiment 10 colorectal cancer patients immunity
Enter the III of the gp96 immunotherapy of group in this research, the postoperative gp96 that accepts of IV phase colorectal cancer patients treats and doctor's conventional treatment (chemicotherapy).Within one week, be a course for the treatment of, each injection course for the treatment of once.The basis of test group 8 weeks inherent conventional treatmenies after surgery starts autologous gp96 immunotherapy.
Operating process
N chooses III, the colorectal cancer patients of IV phase of the operation of plan row;
N obtains " Informed Consent Form " and " Operation Agreement Letters " that patient signs voluntarily;
The capable preoperative comprehensive inspection of n is with clear and definite radical surgery feasibility;
N performs the operation excised tumor tissue;
L by organize in sterile tube;
The sterile tube that l has a tumor tissues was delivered to GMP and is prepared workshop under 0 DEG C of condition in 2 hours.
N identifies tumor tissues quality, confirms to extract enough heat shock protein(HSP) gp96-polypeptide complexes;
Heat shock protein(HSP) gp96-polypeptide complex isolation and determination;
Gp96 treatment and conventional treatment;
Heat shock protein(HSP) gp96-polypeptide complex subcutaneous injection flow process:
1st injection first 3 days domestic demands check routine blood test, blood biochemistry and electrocardiogram(ECG.
Injection pre-treatment: each gp96 injects first 1-3 days to patient's intravenous injection endoxan.
Carry out gp96 protein injection by research nurse, formulate dosage according to the past foreign study data, add the rear upper arm of physiological saline mixing and the subcutaneous injection of thigh left and right, 1 time weekly.
1st, after the injection of 2 immune proteins, need remain in a hospital under observation 24 hours, after need observation 1 hour after injection several times, can leave after having no adverse reaction.
2nd time and the 8th injection latter 3 days domestic demand inspections routine blood test, blood biochemistry and electrocardiogram(ECGs.
Before immunotherapy and after immunity terminates, gather 10ml blood samples of patients, carry out the mensuration of amynologic index.
If palindromia during patient treatment, by investigator's well-documented history patient disease recurrence, and give corresponding treatment according to clinic diagnosis specification;
Follow up a case by regular visits to;
In postoperative 2 years, per March makes a house call 1 time, and within 2 years, make a house call 1 time per June afterwards, until patient disease recurrence or dead, content of specifically making a house call is as follows:
1. within 2 years every 3 months, check 1 time:
A) the 3rd, 9,15,21 month check content:
Tumor markers (CEA, CA19-9, AFP);
Abdomen, Visual quality;
Chest x-ray.
B) the 6th, 12,18,24 month check content:
Tumor markers (CEA, CA19-9, AFP);
Chest, abdomen, pelvic cavity enhanced CT;
Sigmoidoscope.
2. within after 2 years every 6 months, check 1 time, check content:
Tumor markers (CEA, CA19-9, AFP);
Chest, abdomen, pelvic cavity enhanced CT;
Sigmoidoscope.
If palindromia during follow-up of patients, by investigator's well-documented history patient disease recurrence, and give corresponding treatment according to clinic diagnosis specification (as NCCN guide etc.).
Embodiment 11 ELISPOT method detection specificity T cell
ELISPOT detects epitope specificity CTL in colorectal cancer patients 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 100uL and directly cultivate 7 days with fresh PBMC or by PBMC amplification in vitro containing the PBMC(of people of 3 X 105). add the peptide 1 of 10ug/mL in experimental group, PHA that positive control adds 4ug/mL stimulates, negative control adds irrelevant peptide.Cultivate 26-36 hour in cell culture incubator after, the formation of detection specificity spot, and carry out statistical study.We choose the colorectal cancer patients of the HLA-A2 positive as study group, choose HLA-A2 negative patient as a control 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 positive colorectal cancer patients of HLA-A2 of autologous gp96 mixture immunity, all detect high-frequency RTDEGDNRV specific CTL in 3 examples, and increase significantly than before immunity, irrelevant peptide HBcAg do not detected simultaneously
82-90(negative control) specific T-cells, illustrates that ELISPOT result is epi-position special (Fig. 2).
The immunocompetence of other gp96-polypeptide complex of embodiment 12 measures
Except above-mentioned antigen 9 peptide, we choose again other 6 colorectal cancer antigenic peptides and gp96 assembled in vitro and measure its immunocompetence, these 4 antigenic peptides are 1 respectively) MU5AC antigenic peptide " FLDDTGKCV ", 2) MU5AC antigenic peptide " KMYATIPEL ", 3) RNF43 antigenic peptide " SICPSTSSL ", 4) RNF43 antigenic peptide " ILMTVVGTI ", 5) TOMM34 antigenic peptide " ALYGRALRV ", 6) TOMM34 antigenic peptide " ALALVPFSI ".
Respectively this 6 peptide species of synthetic, adopt the reaction system described in embodiment 5 to be combined with gp96 in vitro, this 6 peptide species and gp96 all have higher avidity, and by mensuration association reaction equilibrium constant K, in 6 kinds of peptides and gp96 association reaction, K value is all more than 4.The mixture that the method described in embodiment 7 of employing is formed with above-mentioned 6 kinds of colorectal cancer antigenic peptides and gp96, with the fusion rotein immune mouse respectively of above-mentioned gp96 and 6 kind of colorectal cancer antigenic peptide, and adopt the method described in embodiment 7 to carry out CTL analysis to these 6 kinds of mixtures respectively, from
51cr release experiment can find out that the mixture that these 4 kinds of colorectal cancer antigenic peptides and gp96 are formed all can stimulate HLA-A2 transgenic mice to produce specific cytotoxic t lymphocytes, the immunocompetence of the mixture that 6 peptide species are formed with gp96 than independent polypeptide height 100-200 doubly, and can improve immune effect further by the pre-treatment of Treg cytostatics.Every mouse immune dosage can bring out body when being 0.1 nmol (about 10ug) and produce strong cell immune response, finds that the cleavage rate of target cell is between 40%-65% by the cytotoxic assay of the mixture formed this 6 peptide species and gp96.
Above experimental result shows that mixture that gp96 and colorectal cancer antigenic peptide assembled in vitro are synthesized can be developed into treatment into a kind of novel colorectal cancer or prophylactic agent.Because experiment condition limits patent of the present invention can not carry out assembled in vitro and immunocompetence mensuration to each colorectal cancer antigenic peptide, but we make research object by choosing 7 kinds of representational tumour antigens, 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, can be developed into as a kind of novel therapeutic vaccine.Therefore, except above-mentioned 7 kinds of antigenic peptides, other any colorectal cancer antigenic peptide and gp96 are combined as new generation vaccine also should within protection scope of the present invention.
Embodiment 13. HSP78-immunogenicity of polypeptides measures
By the mixture that HSP78 and 7 peptide species assembled in vitro are formed, the same gp96 of reaction system (see embodiment 5), by fusion rotein (see the 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().From
51cr release experiment can find out that HSP78-9 peptide complex can stimulate mouse to produce specific cytotoxic t lymphocytes, the immunogenicity of HSP78-9 peptide complex is more than 150 times of independent 9 peptides, every mouse immune 0.1 nmol (about 10ug) 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 novel colorectal cancer.
Kang Ernuo Bioisystech Co., Ltd of <110> Shenzhen
The mixture of <120> colorectal cancer antigen peptide and heat shock protein(HSP) and application thereof
<130>
<160> 7
<170> PatentIn version 3.3
<210> 1
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<212> PRT
<213> artificial sequence
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<223> epitope peptide 1
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Ser Leu Asp Gly Ala Gln Thr Val Val
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Phe Leu Asp Asp Thr Gly Lys Cys Val
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Lys Met Tyr Ala Thr Ile Pro Glu Leu
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Ser Ile Cys Pro Ser Thr Ser Ser Leu
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Ile Leu Met Thr Val Val Gly Thr Ile
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Ala Leu Tyr Gly Arg Ala Leu Arg Val
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Claims (10)
1. the epitope peptide SLDGAQTVV 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 FLDDTGKCV 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 KMYATIPEL 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 SICPSTSSL 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 ILMTVVGTI 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 epitope peptide ALYGRALRV be combined with heat shock protein(HSP), is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.6 in sequence table.
7. the epitope peptide ALALVPFSI be combined with heat shock protein(HSP), is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.7 in sequence table.
8. the mixture be covalently or non-covalently connected of the epitope peptide as described in any one of claim 1-7 and gp96 or HSP78.
9. the epitope peptide as described in any one of claim 1-7 forms the purposes of mixture as claimed in claim 8 separately or in co-immunization human body inductive formation killer T cell separately or with heat shock protein(HSP) in vitro.
10. as right wants the purposes of immune induction killer T cell in prevention or treatment colorectal cancer as described in 9.
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| CN105176957A (en) * | 2015-10-09 | 2015-12-23 | 深圳市康尔诺生物技术有限公司 | Complexes formed by RCC (renal cell carcinoma) related peptide and HSPs (heat shock proteins) as well as applications of complexes |
| CN106884005A (en) * | 2017-01-19 | 2017-06-23 | 樊克兴 | A kind of preparation method of colorectal cancer T cells with antigenic specificity |
| CN107090432A (en) * | 2017-01-19 | 2017-08-25 | 上海市东方医院 | A kind of method for preparing colorectal cancer T cells with antigenic specificity |
| CN114409760A (en) * | 2022-02-13 | 2022-04-29 | 奥明(杭州)生物医药有限公司 | Cyclic mRNA tumor immunity medicine for colorectal cancer |
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| CN114409760B (en) * | 2022-02-13 | 2024-05-28 | 奥明(杭州)生物医药有限公司 | Annular mRNA tumor immunity medicine for colorectal cancer |
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