CN104374918B - Heat shock protein 47 glioma associated tumor antigen and application - Google Patents
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Abstract
The invention belongs to field of biological pharmacy, relate to heat shock protein 47 glioma associated tumor antigen, it is specifically related to heat shock protein 47 as the glioblastoma specific tumor antigen relevant to prognosis and two CTL epi-positions of described antigen and nonapeptide aminoacid sequence thereof, judge for clinical prognosis with the reaction of described CTL epi-position inducing T cell, and the purposes in preparation glioblastoma immunization therapy vaccine.The present invention passes through qPCR and the method for protein immunoblot, it was demonstrated that heat shock protein 47 high expressed in glioblastoma multiforme；Demonstrate the dependency of CTL epi-position and clinical prognosis simultaneously, the heat shock protein 47 glioma associated tumor antigen of the present invention can prepare the compositions and compound comprising this antigen and the preparation of preparation immunization therapy further, is used for judging glioblastoma prognosis and immunization therapy.
Description
Technical field
The invention belongs to field of biological pharmacy, relate to heat shock protein 47 glioma associated tumor antigen, more specifically, relate to
And heat shock protein 47 is as the glioblastoma specific tumor antigen relevant to prognosis and two CTL tables of described antigen
Position and nonapeptide aminoacid sequence thereof, and the reaction of described CTL epi-position inducing T cell is in order to clinical prognosis judgement, and in preparation
Purposes in glioblastoma immunization therapy vaccine.
Background technology
Prior art discloses glioma (glioma) is the modal tumor of central nervous system, wherein glioblastoma
(WHO III, IV level) cure rate is low, and relapse rate is high, and poor prognosis, especially for the glioblastoma multiforme of IV level
(glioblastoma, GBM) patient.Clinical practice shows, owing to most of gliomas are infiltrative growth, with normal structure without
Substantially boundary, cannot effect a radical cure so depending merely on operation.Currently for GBM proposed standard therapeutic scheme, i.e. Post operation Synchronous chemoradiotherapy
Adding temozolomide (TMZ) chemotherapy of 6 courses for the treatment of, its mean survival time (MST), is only 12-15 month.Therefore, for glioblastoma, seek
Looking for more effective Therapeutic Method is the difficult problem that current urgent clinical needs solve.Immunization therapy at present, kills especially with self T-cell
The tumor cell remained after hindering microsurgery, has obtained paying close attention to the most greatly.
Heat shock protein (heat shock protein, HSP) is the albumen of the upper high conservative of evolution, and its function relates to carefully
The biological various aspects of born of the same parents.HSP is divided into following six classes at present: Hsp100, Hsp90, Hsp70, Hsp60, Hsp40 and little molecule
HSP.For the angle of immunity, HSP itself as antigen, or can assist a ruler in governing a country molecule as antigen presentation.Research display,
HSP can bind antigenic peptides thus be presented to antigen-presenting cell (antigen presentation cell, APC).Cancer is thin
HSP peptide complexes on born of the same parents can excite adaptability and the inherent immunity system of self.International Patent Publication No. W WO2009/
In the vaccination of 117116EN---heat shock protein GP96 and using method thereof, disclose by genetic modification to express coding
The tumor cell of the nucleic acid of heat shock protein (hsp) the gp96 polypeptide of secreted form.This HSP-96 peptide vaccine is used the most safely
In GBM patient clinical treatment (Crane CA, etal.Clin Cancer Res, 2013;19:205-214.).
HSP47, also known as SERPINH1 or colligin2, as the specific molecular chaperones of collagen, it participates in various collagens
Each process of front protein maturation.For glioma, during the document about HSP47 is reported at present, relate to HSP47 in colloid
Expression on tumor blood vessel (Mustafa D, et al.Brain Pathol, 2010;20:50-65.)；HSP47 take part in glioma
New vessels develop (Mustafa DA, et al.Molecular&cellular proteomics:MCP, 2007;6:
1147-1157.).So far, there is not yet HSP47 as glioblastoma related antigen or the report of the related antigen of other tumors
Road.
Epi-position is can be combined with CTL, Th and B cell surface receptor in antigen molecule, inducing specific cellular immunization and body
Liquid immunity, and then produce one section of aminoacid sequence of immanoprotection action.Research and propose, by screening rich in multiple CTL, Th
It is antigen with the aminoacid sequence of B cell Dominant Epitopes, can reach and immunne response can be induced can to reduce again immunopathogenesis reaction
Purpose；Can effectively strengthen the efficiency that epi-position is offered, solve the weak purpose of immunogenicity of epiposition vaccine.Glioma-associated antigen
T cell epitope energy inducing T cell reaction thus killing tumor cell, in the glioma-associated antigen reported, relate to T thin
Born of the same parents' epi-position have a following gene: SART-1, SART-3, IL-13receptora2chain, ARF4L, GALT3, AIM-2,
EphA2, EGFRvIII, HER-2, gp100, MAGE-1, gBK, TRP-2, Sox2, Sox11, Sox6, HSD3B7 etc..It is corresponding
T cell epitope is as shown in table 1.
The epitope peptide of table 1 glioma-associated antigen
Author Year Gene name HLA classification Amino Acid position Sequence
Research display, the epitope peptide of above-mentioned glioma-associated antigen remains at following Railway Project: 1. incomplete antigen is just
Often there is high expressed, such as ARF4L, GALT3 etc. in cerebral tissue；The ctl response of the most above-mentioned antigenic peptides induction is the most relevant with prognosis still
Unclear.
For the deficiencies in the prior art, present inventor intends providing a kind of brand-new evil relevant to prognosis
Property glioma specific tumor antigen and 9 ctl epitope peptide positions of two protectiveness thereof, and the T cell immunne response of induction in order to
Clinical prognosis judges.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of brand-new heat shock protein 47 glue
Matter tumor associated tumor antigen, is specifically related to heat shock protein 47 as the glioblastoma specific tumor antigen relevant to prognosis
And two protectiveness CTL epi-positions of described antigen and nonapeptide aminoacid sequence thereof, and described CTL epi-position inducing T cell exempts from
Epidemic disease response judges in order to clinical prognosis, and the purposes in preparation glioblastoma immunization therapy vaccine.
In a first aspect of the present invention, it is provided that a kind of specific tumor antigen of high expressed on glioma blastoma.
In a second aspect of the present invention, it is provided that protectiveness CTL epitope peptide that a kind of described antigen is relevant and aminoacid sequence thereof
Row, its aminoacid sequence contains:
(a) HSP47 upper 184-192 amino acids sequence KLPEVTKDV；With
(b) HSP47 upper 3-11 amino acids sequence LLLLSAFCL.
In a third aspect of the present invention, the T cell immunne response of above-mentioned CTL epi-position induction is in order to the prognosis of glioblastoma
Judge.
The present invention passes through qPCR and the method for protein immunoblot, it was demonstrated that HSP47 is high expressed in glioblastoma multiforme；With
Time, in the present invention, based on the HSP47 albumen of total length, it was predicted that and demonstrate the aminoacid sequence of 2 CTL epi-positions:
HSP47184-192And HSP47 (KLPEVTKDV)3-11(LLLLSAFCL), described epitope peptide is detected its ctl response, analyze its with
The dependency of clinical prognosis, result shows, described small peptide can efficiently be attached to HLA-0201 molecule, and can specificity induction dislike
The ctl response of property glioma, its positive rate of CTL in glioblastoma is 26.9%.Meanwhile, above-mentioned ctl response and patient
Prognosis relevant, reactor's tumor Progression free survival and total life span significantly extend.
The heat shock protein 47 glioma associated tumor antigen of the present invention can prepare the compositions comprising this antigen further
With compound and the preparation of preparation immunization therapy, preparation judges glioblastoma prognosis and is used for swelling glioblastoma specificity
The prognosis judgement of tumor and immunization therapy.
In the present invention, being judged the prognosis of glioblastoma by detection HSP47 Specific T cell immunity response, T cell is exempted from
Epidemic disease response positive good prognosis, negative patient poor prognosis.In embodiments of the invention, the partial sequence for HSP47 include but not
It is limited to (a) HSP47 upper 184-192 amino acids sequence KLPEVTKDV；(b) HSP47 upper 3-11 amino acids sequence
LLLLSAFCL, detects T cell immunne response；
In the present invention, for detect the method for T cell immunne response be ELISPOT method based on IFN-gamma, based on stream
The IFN-gamma intracellular staining assays of formula technology, Tetramer flow cytometer detection method and the quantitative square of other T cell immunne response
Method；Judge that the positive of T cell immunne response is with negative by the particular requirement of every kind of described method.
Heat shock protein 47 glioma associated tumor antigen and the described CTL epitope peptide of the present invention can prepare use further
Compositions in glioblastoma therapeutic vaccine.
In the present invention, the preferably preparation of immunization therapy is the therapeutic vaccine of immunotherapy of malignant glioma, and described controls
The property treated vaccine can be DNA, viral vector, bacteria carrier or protein subunit vaccine, including but not limited to following sequence:
(a) HSP47 upper 184-192 amino acids sequence KLPEVTKDV；
(b) HSP47 upper 3-11 amino acids sequence LLLLSAFCL.
Further, heat shock protein 47 glioma associated tumor antigen and the described CTL epitope peptide of the present invention can
Preparation is for preventing or treat the compositions of other associated malignancies.
By concrete accompanying drawing, embodiment, the present invention will be described in detail in order to make it easy to understand, following.Need spy
Not it is noted that these describe the description being merely exemplary, it is not intended that limitation of the scope of the invention.According to this specification
Discussion, many changes of the present invention, change and will be apparent from for one of ordinary skill in the art.It addition, the present invention
Refer to open source literature, these documents are to more clearly describe the present invention, and their entire contents is all included in and carried out herein
Reference, just look like they full text repeated description the most in this article excessively as.
Accompanying drawing explanation
Fig. 1 .HSP47mRNA specificity overexpression in GBM,
Wherein show, in 9 example check samples (cerebral tissue around contusion and laceration of brain) and 50 examples each rank glioma (one-level 6
Example, two grade of 8 example, three grade of 17 example, level Four 19 example) mRNA level in-site confirm the HSP47 high expressed at GBM, * * P < 0.01.
Fig. 2 .HSP47 albumen high expressed in glioblastoma multiforme,
Wherein A:17 example GBM and the westernblot result of 10 examples comparison cerebral tissue (cerebral tissue around contusion and laceration of brain)；
B: statistical result shows HSP47 albumen significance high expressed (P=0.018) on GBM.
Fig. 3 .HSP47 location in tumor cell.
Wherein, paraffin-embedded GBM section fixed by formalin, dyes HSP47 by the method for immunofluorescence, aobvious
Show that it is expressed in tumor vessel (single arrow) and the bag slurry of tumor cell (double-head arrow).
The HLA-A0201+T2 cell binding force test of Fig. 4 .HSP47 antigenic peptides,
Wherein A:HSP47184-192(KLPEVTKDV)、HSP473-11And the T2 cell binding force of hybrid peptide (LLLLSAFCL)
Liu Shi testing result；
The percentage ratio that the Mean Fluorescent Index of B: above-mentioned three peptides increases is respectively 78.3%, 84.5% and 92.8%.
Fig. 5 .HSP47 antigenic peptides mixture induction ctl response
Wherein left: 5 example normal persons and 39 example tumor patients (benign brain tumors 6 example, 2 grade of 7 example of glioma, glioma 3 grade 10
Example, glioma 4 grades be GBM16 example) peripheral blood lymphocytes (PBMC), through peptide complexes stimulate after with ELISpot method examine
Surveying IFN-γ secretion, wherein 7 example patients occur that ELISpot positive reaction, 2 examples are glioma 3 grades, and 5 examples are GBM patient；
Right: the speckle number of 7 example ELISpot reactors.
The prognostic analysis of Fig. 6 .16 example GBM patient,
Kaplan-Meier survival analysis shows, 5 example ELISpot reactors, and its PFS relatively 11 example negative patients significantly prolong
Long, difference statistically significant (P=0.04).
Detailed description of the invention
Embodiment 1, HSP47 expression in glioma and location
1.HSP47 high expressed in glioblastoma multiforme
The present invention to 9 example check samples (cerebral tissue around contusion and laceration of brain) and 50 examples each rank glioma (one-level 6 example,
Two grade of 8 example, three grade of 17 example, level Four 19 example) extract RNA(through the agreement of Huashan Hospital Affiliated To Fudan Univ Ethics Committee), to swollen
Tumor sample and check sample use Trizol one-step method to extract total serum IgE, after Purification Kit RNA, use NanoDrop
Concentration and the purity of RNA are detected by micro-spectrophotometer；The RNA taking equivalent carries out reverse transcription, and reverse transcription uses
Invitrogen company SuperScriptTMII reverse transcription and random primer, with total serum IgE for templated synthesis cDNA；In
On Applied Biosystems7900Real-time PCR Detection System, with genes of interest and reference gene it is
Sample, taking equivalent cDNA is that template carries out real-time fluorescence quantitative PCR reaction (final volume is 20ul), and reaction condition is as follows: 95 DEG C
After heat treatment 10 minutes, with 95 DEG C 15 seconds, 60 DEG C 60 seconds, 72 DEG C of 30s react 40 circulations, test after terminating, with 2-ΔΔCtAt method
Reason data；Experiment primer strand is as follows: HSP47 (forward): 5 '-CACCGCCTTTGAGTTGGACAC-3 '；HSP47
(reverse):5’-GGCGCCCAATGAATAGCAG-3’；Result is as it is shown in figure 1, HSP47mRNA specificity height table in GBM
Reach.
The present embodiment is collected the specimen of 17 examples GBM and 10 examples comparison cerebral tissue further and is western blot to contrast two
The expression of results of person, collection is organized after processing, and adds in 4 DEG C of pre-cooling cell pyrolysis liquids of the final concentration of 1mmol/L of PMSF, on ice
After hatching 30 minutes, 4 DEG C, 10000g, centrifugal 15 minutes, take supernatant and carry out BCA protein quantification, 100 DEG C of heating make egg in 15 minutes
Leucismus, the separation gel of preparation 12% and the concentration glue of 5%, take 30-40ug albumen sample in loading wells, voltage 80V, electrophoresis 30
Voltage 120V after minute, electrophoresis makes Protein Separation in 60 minutes, and wet turn 120 minutes under 170mA constant current make protein delivery extremely
The pvdf membrane of 0.22um, takes out in the skim milk that film puts into 5%, and room temperature is closed 90 minutes, removes skim milk, and TBST washs
Three times, film is put in the anti-diluent prepared with 5%BSA, 4 DEG C of overnight incubation, remove one and resist, after TBST washs three times
Adding the two of the horseradish peroxidase-labeled that 1:2000 dilutes to resist, incubated at room temperature 60 minutes, film for several times, is put into by washing
In ECL luminescent solution, develop the color in LAS-4000 imager imaging (as shown in Figure 2), 17 examples GBM and 10 examples comparison cerebral tissue statistics
Result shows, GBM upper HSP47 albumen significance high expressed (as in figure 2 it is shown, P=0.018).
The location that 2.HSP47 expresses in cell
Formalin is fixed paraffin-embedded GBM section and is done immunofluorescence dyeing, described in method reference literature (JinK,
etal.Proc Natl Acad Sci U S A,2006;103:13198-13202.)；Rabbit source anti-HSP47 (Santa
Crus, 1:50) carry out immunofluorescence dyeing, show that it is in tumor vessel (single arrow) and the bag slurry of tumor cell (double-head arrow)
Express (as shown in Figure 3).
Embodiment 2, the prediction of HSP47CTL epi-position and affinity test
Application network resource database (Genbank, Pubmed) obtains the heat shock protein 47(HSP47 in people source) total length
Aminoacid sequence；It is HLA-A*0201 gene (Song Yonghong, Ma Chunhong, Lv Hong according to Chinese han population modal HLA gene
Beautiful etc.；Han People of North China HLA Study on gene polymorphism [J]. journal of Shandong university (medicine), 2007 (6): 546-
553.) feature, utilize online software (http://www-bimas.cit.nih.gov/molbio/hla_bind/, BIMAS)
The aminoacid sequence of HSP47 is carried out the prediction of above-mentioned HLA gene restricted CTL epitope, two Dominant Epitopes that selection score value is high:
HSP47184-192And HSP47 (KLPEVTKDV)3-11(LLLLSAFCL), as shown in table 2.
The HLA-A0201 prediction binding score value of form 2HSP47
Peptide to be measured (the 50 μ g/ml) addition serum-free RPMI1640 culture medium containing β 2 microsphere egg (3 μ g/ml) is incubated for 37 DEG C altogether
After educating 18h-24h, cold PBA washs 3 times, and add that 100 μ l dilution factors are 1:200 one is anti-, and (T2A2 cell adds monoclonal antibody
BB7.2, T2A3 cell adds monoclonal antibody mouse-anti people β 2-M), place 30min for 4 DEG C.After cold PBA washs 3 times, add 50 μ l dilutions
Degree is the FITC-sheep anti-mouse igg solution of 1:50, places 30min for 4 DEG C, and in flow cytomery after washing, PBS is as feminine gender
Comparison, result represents with following mean fluorecence coefficient (MFI):
Mean fluorecence coefficient (MFI%)=(epitope peptide average fluorescent strength-background average fluorescent strength)/background increased is put down
All fluorescence intensity × 100%；
The HLA-A0201+T2 cell binding force result of the test of HSP47 antigenic peptides shows: HSP47184-192
(KLPEVTKDV)、HSP473-11(LLLLSAFCL) and the percentage ratio that increases of the Mean Fluorescent Index of hybrid peptide is respectively 78.3%,
84.5% and 92.8%(as shown in Figure 4).
Embodiment 3, the qualification of HSP47 antigens c TL epitope peptide
In order to confirm whether above-mentioned peptide has immunogenicity, can the present embodiment induce by ELISpot detection antigenic peptides
IFN-γ secretion.It is coated with the Purified Rat Anti-Mouse INF-γ Monoclonal Antibody of 1:100
Multiscreen96-wellFiltration Plate, 4 DEG C are overnight, topple over next day and are coated liquid, and complete medium washed once,
Drying in aseptic absorbent paper, close 2 hours with complete medium 37 DEG C, topple over confining liquid, counting processes cell, according to experiment
Design addition 1 × 106 power detection cell and the antigen peptide fragment of final concentration of 4ug/ml, carry out cell by 37 DEG C under the conditions of 5%CO2
Cultivating 2-24 hour, remove culture medium, every hole 200ul ice deionized water, wash three times, each 5 minutes, cell was removed in cracking；
Respectively washing with PBST and PBS three times, adding concentration is 1.3ug/ml Biotinylates Rat Anti-Mouse INF-γ again
Monoclonal Antibody, hatches 2 hours for 37 DEG C；Topple over ELIAS secondary antibody, respectively wash with PBST and PBS three times, in absorbent paper
Dry, add AP-Streptavidin Conjugate, the 100ul/ hole that concentration is 2.0ug/ml, act on 60 minutes under room temperature；
Again topple over liquid in plate, respectively wash with PBST and PBS three times, sterilizing absorbent paper dries, takes BDTMAEC Substrate
Reagent Set, every hole adds 100ul, lucifuge, after acting on 5-60 minute, is washed with deionized and terminates for several times showing under room temperature
Color；Take off protective layer, put ventilation natural air drying, in ChampSpot IV Bioreader reads plate instrument automatically, carry out speckle meter
Number, and record the various parameters of speckle, carry out statistical analysis；The ELISpot positive is defined as: speckle number more than 50/1 ×
106PBMCs and more than without peptide stimulation negative control twice more than (BredenbeckA, etla.JImmunol, 2005;174:
6716-6724.Hakamada T,et al.Journal of general virology,2004;85:1521-1531.).
By the 5 example normal persons obtained and 39 example tumor patients (benign brain tumors 6 example, 2 grade of 7 example of glioma, glioma 3 grades
10 examples, glioma 4 grades be GBM16 example) peripheral blood lymphocytes (PBMC), through above-mentioned two peptides complex stimulate after use
Stating ELISpot method detection IFN-γ secretion, result shows: ELISpot positive reaction occur in 7 example patients, speckle number such as figure Fig. 5
Shown in, wherein 2 examples are glioma 3 grades, and 5 examples are GBM patient.
Embodiment 4, ctl response and clinical prognosis correlation research
16 examples GBM are followed up a case by regular visits to, observes the relation of its ctl response and clinical prognosis.3 are all accepted after all operation in patients
The Therapeutic Effect of Temozolomide more than course for the treatment of, accepts radiotherapy 12 example, follwing-up in average 11.3 ± 2.8 months the most simultaneously；ELISpot in 16 examples
Positive 5 examples, negative 11 examples (as shown in table 3), result shows, the tumor Progression free survival (PFS) of two groups is respectively positive group
12.6 ± 1.3 months, negative group 8.1 ± 3.2 months, difference statistically significant (P=0.01)；Kaplan-Meier survival curve shows
Show the PFS of two groups exist significant difference (P=0.04, as shown in Figure 6)；The overall survival (OS) of two groups is respectively positive group
13.4 ± 1.3 months, negative group 10.4 ± 2.7 months, there is notable significant difference (P=0.035) between two groups.
Table 3: patient information and survival state
Claims (2)
1. the heat shock protein 47 (HSP47) purposes in preparation judges the preparation of glioblastoma prognosis；
Wherein, for glioblastoma prognosis judgement is detection HSP47 Specific T cell immunity response, T cell immunne response
Positive good prognosis, negative patient poor prognosis；
Wherein, detection T cell immunne response is the partial sequence for HSP47, including:
(a) HSP47 upper 184-192 amino acids sequence KLPEVTKDV；
(b) HSP47 upper 3-11 amino acids sequence LLLLSAFCL.
2. the purposes as described in claim 1, the method being wherein used for detecting T cell immunne response is based on IFN-gamma
ELISPOT method, IFN-gamma intracellular staining assays based on streaming technology, Tetramer flow cytometer detection method and other T cell
The quantitative approach of immunne response；Judge that the positive of T cell immunne response is with negative by the particular requirement of every kind of described method.
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