CN110124018A - It is a kind of simulate necrotic tumor cells calcium phosphate-lipid nanometer vaccine and its application - Google Patents
It is a kind of simulate necrotic tumor cells calcium phosphate-lipid nanometer vaccine and its application Download PDFInfo
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Abstract
The invention belongs to vaccines arts, it is related to a kind of bionical nano vaccine preparation, and in particular to a kind of calcium phosphate-lipid nanometer vaccine for simulating necrotic tumor cells and its application.The calcium phosphate of the vaccine internal layer is the hydrophobic nanoparticles for efficiently containing Oligonucleotide substance；Outer layer lipid structure is the material of altitude simulation cell membrane fluidity, can load natural epicyte protein and HSP70 active peptide segment.This nano vaccine preparation can overcome many limitations of traditional tumour vaccine, its Core Superiority is the integration ability of height, by the natural multivalence tumor cell membrane antigen protein (CM) of 3 kinds of active constituents, the heat shock protein A subunit active peptide segment (α HSP70p) of α spiral modification, adjuvant oligonucleotide (CpG) is contained into calcium phosphate nano grain (CaP), it is delivered to antigen presenting cell simultaneously, to activate inherent immunity and adaptive immunity.With higher potential using value and clinical conversion prospect.
Description
Technical field
The invention belongs to vaccines arts, are related to a kind of bionical nano vaccine preparation, and in particular to one kind has nucleocapsid knot
Calcium phosphate-lipidic nanoparticles of structure, especially it is a kind of simulate necrotic tumor cells calcium phosphate-lipid nanometer vaccine and its answer
With.The calcium phosphate of the vaccine internal layer is the hydrophobic nanoparticles that can efficiently contain Oligonucleotide substance；Outer layer
Lipid structure is the material of altitude simulation cell membrane fluidity, can load natural epicyte protein and HSP70 active peptide segment.
Background technique
Data shows, malignant tumour is the number one killer for threatening human health, conventional treatment means include operative treatment,
Radiotherapy, chemotherapy, but practice and show that the affiliated equal effect of therapeutic scheme is limited, basic reason is not inhibiting swollen
The transfer and recurrence of tumor in vivo, while there are a variety of toxic side effects over the course for the treatment of.Studies have reported that immunization therapy work
For a kind of novel antineoplaston mode, the immune system of body itself can be stimulated to realize to the effective clear of tumour cell
It removes, brings new hope for the treatment of malignant tumour.Since 2011, the immunization therapy of tumour have passed through a fast development
Stage, the immunotherapy of tumour is also classified as scientific and technological ten quantum jumps, immunologic test point inhibitor by Science magazine in 2013
PD/PDL-1 antibody also achieves the effect to attract people's attention for the effect of malignant mela noma, significantly improves the existence of patient
Time；However, immunologic test point inhibitor therapy is encountered by the low problem for the treatment of response rate, wherein significant portion is the reason is that tumour
The infiltration of immunocyte caused by Tumor mutations are underload in microenvironment is on the low side, therefore, is constructed by means of other modes new
General treatment measures for improve checkpoint blocking treatment clinical effectiveness be particularly important.
Tumor vaccine is the preferred option of the synergistic treatment of current checkpoint blocking treatment.Tumor vaccine is by directly stimulating
Dendron shape immunocyte, can potentially T cell of the CD4+/CD8+ of activated peripheral etc. tumor locus infiltration, thus
The immune function of the potential human body of upstream activat.Ideally, the CD8+T cell that vaccine evokes has activity well, energy
Polypeptide-mhc class i antigenic peptide complexes on enough tumor cells, have preferably for the Specific marker of tumour cell
Identification.But it is the work challenged very much, including three aspect factors in need of consideration that it, which is designed: (1) selecting suitable tumour related
Antigen, it is intended to cause the response of specific T-cells；(2) a kind of suitable immunostimulation adjuvant is selected, it is intended to activation pattern identification
Receptor enhances inherent immunity；(3) antigen and adjuvant high-efficiency delivery are entered in the same Dendritic Cells simultaneously.Practice is aobvious
Showing, the tumor vaccine of single antigenic component is easiest to realize clinical conversion, and there are many ongoing clinical tests, but because
The height heterogeneity of tumour and the immune influence resisted, not yet realize and break through；The personalized tumor vaccine developed in recent years is more
Concern Tumor Heterogeneity and antigen otherness, achieve some good effects, such as Ott, P.A. etc. and pass through computer
Algorithm and laboratory high-flux sequence have filtered out 20 kinds of neoantigens of patient itself, the 20 kinds of polypeptides and assistant prepared with this
The mixture of agent excites the strong immune response of patient, and personalized tumor vaccine of the preliminary proof based on cancer is feasible
Property；But this method faces some defects, such as preparation process is comparatively laborious, and it is more low that free mix ingredients enters born of the same parents' efficiency.Though
Right Tumor lysate dendritic cell by vitro stimulation tumor vaccine Provenge has been approved for the treatment of prostate cancer, but
It is constrained to ingredient and immune tolerance problem, effect complicated in Tumor lysate still to have much room for improvement, so tumor vaccine
Research and development be a urgent problem to be solved.
The subcellular component that Recent study gradually discloses tumour cell has respective immunogenicity, such as cell
Core, cytoplasm and cell membrane component etc.；Firstly, in certain tumours, such as melanoma, contain a large amount of tumour in cell membrane
Relevant antigen (TumorAssociatedAntigens, TAA) can use it and be used as a kind of potential effective antigen-like material,
And the tumor cell membrane from patient itself may be one of the ideal material precisely treated, but natural tumor cell membrane
Ingredient is not enough to effectively activate Dendritic Cells, and adding suitable immunologic adjuvant ingredient is the key that play therapeutic effect.It grinds
Study carefully the necrosis (necrosis) that mediate tumor cell is capable of in discovery in the acute variation of the chemotherapy of low dosage, radiotherapy and environment,
To release a large amount of substance --- danger signal molecule (the danger associated with strong immunogenic
Molecular patterns, DAMP), such as heat shock protein, GRP78BiP and calprotectin etc., it can be used as new
Immunologic adjuvant material.Heat shock protein A subunit is heat stress proteins matter of the molecule amount in 70kDa or so, is divided in addition to having
Outside the function of sub- companion, its expression is found in kinds of tumors, and induces and enhance antitumor immunity of organism reaction；
The special construction that HSP70 has can form compound with polypeptide, in conjunction with the associated receptor of antigen presenting cell surface, swash simultaneously
Send out inherent immunity and specific immunity；Relevant clinical test for granulocytic leukemia treatment is positive to be carried out, but from patient body
The natural HSP70 polypeptide complex technology inside isolated is complicated, and extraction efficiency is low, and preparation difficulty is higher.
Status based on the prior art；Present inventor intends providing a kind of " necrotic tumor cells of manual simulation "
Nanometer drug delivery platforms, and in particular to a kind of calcium phosphate-lipidic nanoparticles with core-shell structure, the nanometer drug delivery platforms can
Tumour cell memebrane protein and HSP70 active peptide segment are delivered in Dendritic Cells simultaneously, potentially will preferably activate people
The inherent immunity and adaptive immunity of body.
Summary of the invention
It is an object of the invention to based on the prior art status and basis, a kind of phosphoric acid with core-shell structure is provided
Calcium-lipidic nanoparticles, especially a kind of calcium phosphate-lipid nanometer vaccine for simulating necrotic tumor cells and its application.
The present invention provides a kind of nano particle of " necrotic tumor cells of manual simulation " based on bionics principle design
(α HSP70p-CM-CaP) loads natural multivalence tumor antigen protein and HSP70 active peptide segment (assistant in artificial matrix material
Agent), nanoparticle is prepared as kernel as material using calcium phosphate and contains another adjuvant oligonucleotide CpG ODN: on the one hand
Using the neoantigen ingredient in tumour cell memebrane protein, cause antineoplastic specificity immune；On the other hand oligonucleotides are utilized
Two kinds of adjuvant components of sour CpG and α HSP70p, the inherent immunity function of human activin are comprehensive to play antineoplastic action.The present invention
" necrotic tumor cells of manual simulation " (α HSP70p-CM-CaP) nano particle that traditional tumour vaccine will be overcome to face is all
More limitations, Core Superiority is the integration ability of height, by the natural multivalence tumor cell membrane antigen protein of 3 kinds of active constituents
(CM), the heat shock protein A subunit active peptide segment (α HSP70p) of α spiral modification, adjuvant oligonucleotide (CpG) are contained into phosphorus
Sour calcium nanoparticle (CaP), while it being delivered to antigen presenting cell, to activate inherent immunity and adaptive immunity.
Specifically, the present invention provides a kind of calcium phosphate-lipid nanometer vaccine for simulating necrotic tumor cells, the vaccine
The calcium phosphate of internal layer is the hydrophobic nanoparticles that can efficiently contain Oligonucleotide substance；The lipid structure of outer layer is high
The material of degree simulation cell membrane fluidity, can load natural epicyte protein and HSP70 active peptide segment；It, will in the present invention
Natural tumour cell memebrane protein and the HSP70 active peptide segment of α spiral modification are bound to the artificial constructed flowing of one kind
In lipid membrane material, internal calcium phosphate nanoparticles efficiently contain oligonucleotide CpG.
The used calcium phosphate of the present invention constructs nanoparticle kernel, and it is with higher steady to contain Oligonucleotide drug CpG
It is qualitative and contain efficiency；Preparation method is reverse microemulsion method, and for this method for containing siRNA class, the nanoparticle of preparation is uniform
It is dispersed in oily phase, there is certain slow release effect.
The non-methylation oligodeoxynucleotide of adjuvant component (abbreviation CpG ODN) of the present invention is a kind of artificial
The DNA sequence dna of the CpG oligonucleotide containing non-methylation with immunostimulation of synthesis；CpG ODN by with Toll
Sample receptor -9 (TLR9) combines, and can directly activating B cell and dendritic cells to cause direct Th1 type immune response, (CpG ODN makees
Universal approval is obtained for the immunologic adjuvant of vaccine, will realize its commercial applications), it is contained in above-mentioned nano-carrier
In, it can be realized certain slow releasing function, additionally it is possible to synergistic effect is realized with α HSP70p active peptide segment, it is jointly thin by dendron shape
Born of the same parents effectively absorb.
Particle construction method of the present invention is specific as follows: the phosphorus of CpG ODN is 1. contained with reverse microemulsion method preparation
Sour calcium nanoparticle core, reverse microemulsion be by aqueous dispersion into the hexamethylene oil-phase solution containing nonylphenol polyoxyethylene ether
It is prepared；2. the B16OVA tumour cell of in vitro culture obtains tumor cell membrane by ultracentrifugation, pass through suitable de-sludging
Agent is by epicyte protein separated and dissolved, and then further ultracentrifugation obtains epicyte protein solution；3. prepared by film hydration method
After previously prepared calcium phosphate nano core, lipid membrane material form a film jointly, memebrane protein and α HSP70p polypeptide is added in outer layer
Mixed liquor carries out constant temperature aquation；To which calcium phosphate-lipidic nanoparticles of self assembly be made.
In the present invention, the matrix material includes the lipid and cholesterol of a kind of phosphocholine structure, according to a certain percentage
Addition, i.e. DPPC:DSPC:DOPC: cholesterol 5:1:3:1.
The sequence of the α HSP70 active peptides are as follows:
AC-FAEKFKEAVKDYFAKFWD-GSG-TKDNNLLGRFELSG adds one section of α spiral sequence AC-
FAEKFKEAVKDYFAKFWD can enhance stability of the biomaterial adjuvant HSP70p active peptide segment in artificial matrix material and
Drugloading rate can be used artificial synthesized mode and construct.
In the present invention, the nanoparticle kernel and lipid layer of the calcium phosphate building are combined by hydrophobic effect, form core
The bionical particle of the nanoscale of shell structure；
The calcium phosphate nano kernel average grain diameter is 30-40nm, and particle ultimate size is 50nm or so, compared to micron
The cell of scale, specific surface area is larger, can more efficiently play the effect of membrane structure.
The present invention uses a variety of inside and outside technologies and evaluation method, is with the Dendritic Cells (BMDC) of bone marrow derived
Its external evoked DC cell of model evaluation enters its mature function of born of the same parents' characteristic and induction, is resisted using imaging streaming technology quantitative assessment
The expression of original-MHC compound, above two evaluation method are art-recognized and known.
The present invention describes the drug and passs the preparation method of release system, and provides the knot of the system inside and outside immune activation
Fruit and pharmacodynamic evaluation.
The present invention is used as model by using bone marrow derived Dendritic Cells (BMDC), and evaluation preparation is thin in dendron shape
The intake of born of the same parents and the function of inducing it mature, and being capable of Th1 type cellular immunity in significant inductor；Streaming the results show
Said preparation can significantly improve IFN γ in Mice Body+specific C D8+T cell content；Lung metastases model nude mice living imaging
It is demonstrated experimentally that said preparation can specifically be accumulated to draining lymph node；Internal pharmacodynamic evaluation monitoring display, compared to free
Mixture, the delivery system can significantly inhibit the formation of metastatic lesion；It is thin that Memorability T can be obviously improved after preparation is immune
The content of born of the same parents；With the antibody combined application of PD-1, the progress of subcutaneous tumors is significantly inhibited, big graded effect is able to detect that after treatment end
The presence of type memory T cell, synthesis result prove that bionical nano vaccine preparation of the present invention has significant antitumor
Effect, and it effectively prevent the recurrence of tumour.
In the present invention, the α HSP70p-CM-CaP partial size of preparation can effectively activate mouse bone marrow cells in 30nm or so in vitro
The maturation of dendritic cells derived (BMDC) and offering for promotion antigen；Small animal living body imaging test is shown, is subcutaneously injected
Nano particle in Mice Body selectively accumulation in draining lymph node, holding time more than 24 hours；It is small in priming experiments
T cell and killer T cell of the mouse vivo detection to a variety of antigenic components of identification；Lung metastases and subcutaneous tumors pharmacophore model are commented
α HSP70p-CM-CaP and PD-1 monoclonal antibody combined treatment have obvious inhibitory effect to valence as the result is shown, and after treatment end
A period of time continues to monitor the presence of memory t cell.Further, the bright vaccine can with the antibody combined application of PD-1, and
And clinical conversion prospect with higher.
Bionical particle platform of the present invention has its unique advantages in terms of anti-tumor immunotherapy:
1. building the nanometer precisely treated, to pass medicine flat with the tumor associated antigen of high degree of immunogenicity on enrichment of cell film
Platform；2. constructing α HSP70p using a kind of alpha helical peptides integration technology, lipid membrane structure is improved to the load capacity of HSP70 polypeptide,
To realize the concertedness Th1 response to two kinds of immunologic adjuvants；3. calcium phosphate molecule can be with the DNA framework ingredient of CpG ODN
Height combines, and improves calcium phosphate nanoparticles and contains ability to CpG ODN；4. nanoparticle size effect is obvious, average
The partial size of 50nm or so can be improved ingestion efficiency of the particle in Dendritic Cells, and realize a variety of active ingredients same
Delivering in a Dendritic Cells.
Detailed description of the invention
The characterization of Fig. 1 α HSP70p-CM-CaP, wherein
(A) calcium phosphate-lipidic nanoparticles (α HSP70p-CM-CaP) ideograph, artificial lipid membrane structure therein is one
The lipid and cholesterol of cholinomimetic phosphoric acid structure, are added, i.e. DPPC:DSPC:DOPC according to a certain percentage: cholesterol 5:1:3:
1；
(B) transmission electron microscope photo of four kinds of nano particles, wherein the calcium phosphate nanoparticles pair of (a) artificial lipid membrane cladding
According to group (without CM and α HSP70p), referred to as LCP, (b) α HSP70p-CM-CaP (c) have contained the artificial lipid membrane cladding of CM
Calcium phosphate nanoparticles control group (CM-CaP), (d) B16OVA tumour cell membrane vesicle control group (CMV)；
(C) partial size and zeta of four kinds of nano particle LCP, α HSP70p-CM-CaP, CM-CaP, CMV being measured in solution
Potential；
(D) formulation optimization is tested, and α HSP70p, CM and CpG are added to nanometer formulation particle according to different proportion, with most
The encapsulation rate (%) of several active constituents is used as evaluation index (n=3) eventually, selects optimal as subsequent experimental prescription；
(E) coomassie brilliant blue staining experimental result；
(F) the Westernblot result of the immune activation molecule of several keys.
Fig. 2 α HSP70p-CM-CaP induces BMDC mature and promotes the presenting function of antigen, wherein
(A) external evoked effect of the α HSP70p-CM-CaP to BMDC maturation；
(B) quantitative analysis results of a few class cells in figure A；
(C) antigen presentation activation of the α HSP70p-CM-CaP for BMDC cell；
MHC- antigenic peptide complexes (the SIINFEKL-H-2K on the surface BMDC in (D, E) figure Cb, it is red) signal it is quantitative
Analysis as a result, be fluorogram and semi-quantitative analysis, data are analyzed with average value ± SD (n=3), are had significant as shown in the figure
Sex differernce, p < 0.001 * * *.
Selectivity of Fig. 3 nano particle in Mice Body in draining lymph node is accumulated and for not isophenic T cell
Sensitization, wherein
(A) small animal living body imaging experiment result: (a) α HSP70p-CM-CaP-Cy5, (b) α HSP70p-
CM(Fluorescein)-CaP；
(B) CD8 of α HSP70p-CM-CaP nano particle antigenic phenotypes different for identification+The activation of T cell；
(C) CD8 in figure B+The quantitative analysis results of the activation of T cell, data are with average value ± SD (n=3) progress
Analysis has significant difference, p < 0.001 * p < 0.05, * * * compared with the mouse of injection α HSP70p-CM-CaP.
Fig. 4 α HSP70p-CM-CaP mediates the activation of adaptive immune system in C57BL/6 Mice Body, wherein (A) α
The IFN-γ in C57BL/6 mouse spleen after HSP70p-CM-CaP is immune+CD8+T cell fluidic cell figure；
(B) CD8 of the secretion of gamma-IFN of the OVA specificity in figure A+T cell measures quantitative result；
(C) cell factor in C57BL/6 mouse peripheral blood (IFN-γ, IL-4, IL-2, IL-10) ELISA as a result,
Data are analyzed with average value ± SD (n=3), compared with the mouse of injection α HSP70p-CM-CaP, have conspicuousness poor
It is different, p < 0.001 * * p < 0.01, * * *.
Antitumous effect in Mice Body after Fig. 5 α HSP70p-CM-CaP is immune, wherein
(A) inhibition assay result of the preparation on mouse lung metastasis model；
(B) quantitative analysis of mouse lung metastatic lesion, data are analyzed with average value ± SD (n=3), with injection α
The mouse of HSP70p-CM-CaP is compared, and has significant difference, p < 0.001 * * p < 0.01, * * *；
It (C) is as shown in the figure the dosage regimen of B16OVA mouse subcutaneous tumors model；
(D) tumor growth curve after treating, data are analyzed with average value ± SD (n=6), with injection α HSP70p-
The mouse of CM-CaP+PD-1 monoclonal antibody is compared, and has significant difference, p < 0.01 * * * p < 0.001, * *；
(E) the mouse lymph nodes internal effect memory T cell subsets (T after being immunized 40 daysEM,CD3+CD8+CD62L–CD44+)；
(F) T in figure EEMQuantitative analysis results have aobvious compared with the mouse of injection α HSP70p-CM-CaP+PD-1 monoclonal antibody
Write sex differernce, p < 0.001 * *.
Specific embodiment
Embodiment 1
The calcium phosphate nanoparticles control group of artificial lipid membrane cladding is prepared by the method in claims of the present invention
(LCP), α HSP70p-CM-CaP has contained the calcium phosphate nanoparticles control group (CM-CaP) of the artificial lipid membrane cladding of CM,
B16OVA tumour cell membrane vesicle control group (CMV), using the partial size of Malvern particle size determination instrument measurement nanoparticle, transmitted electron
Its form of micro- sem observation；
As the result is shown: to contain the calcium phosphate nanoparticles of CpG as kernel, outside has been coated embedded with α HSP70p polypeptide
It is scattered in aqueous solution after successfully being prepared with the nano particle of " artificial lipid membrane " of epicyte protein (CM), DLS is as the result is shown
The average grain diameter of LCP, α HSP70p-CM-CaP and CMV are respectively 18.7nm, 31.4nm, 24.7nm and 196.7nm.α
Compared with LCP, partial size has increased slightly HSP70p-CM-CaP.The potential of LCP, α HSP70p-CM-CaP and CMV are -17mV, -
22mV, and -32.3mV.
Electron microscope shows that polymer nanoparticle surface covers one layer of lipid components, shows that α HSP70p-CM-CaP is built into
Function.Four kinds of nanoparticle sizes are uniform, form rounding；
Using different dyestuff NHS- fluoresceins, FITC, Cy5 mark CM respectively, after α HSP70p and CpG, measure 3 kinds
After the encapsulation rate of active constituent, three kinds of active constituents when optimal prescription α HSP70p:CM:CpG is 5:50:2 weight ratio are obtained
Encapsulation rate is respectively 31.4%, 42.5%, 35.6%；
By B16OVA tumour cell, CMV, several groups of samples of CM and α HSP70p-CM-CaP use equivalent loading, electrophoresis knot
It is handled after beam using coomassie brilliant blue staining liquid, polyacrylamide gel electrophoresis figure (SDS-PAGE) shows several groups of samples
Western blot is almost the same, shows that epicyte protein has been successfully moved to nanometer formulation surface；
Western blot is the results show that people's chromoma related antigen (gp100), melanocyte tyrosinase are related
Albumen -2 (Trp-2), the critical immunes activating molecules such as molecular chaperones (GRP78BiP, calreticulin) on B16OVA cell,
Completely retain on several samples of CMV, CM and α HSP70p-CM-CaP.
Embodiment 2
Immature BMDC is extracted in Mice Body with 1 × 106The density of cell is inoculated in 12 orifice plates and cultivates 7 days for 37 DEG C,
And liquid is changed every other day.Subsequent cell culture fluid is mixed with the CM of the CpG and 1 μ g/mL of α HSP70p, 40ng/mL containing 100ng
The LPS solution of solution and 0.5 μ g/mL are respectively 12,24 and 48 small respectively as negative and positive controls, incubation time
When, 37 DEG C.Then, BMDC is incubated at room temperature CD16 antibody, is then incubated for CD11c on ice, CD80, CD86 and
MHCII antibody, cell use Flow cytometry experiments to CD11c+CD80+, CD11c+CD86+, CD11c+ after being washed one time with PBS
II+cell of MHC is analyzed.In order to further verify the antigen presentation of BMDC, the BMDC after each group preparation is incubated for is used
SIINFEKL/H-2KbAntibody incubation is analyzed using processing scheme ibid；
As the result is shown: Fig. 2A has been incubated for BMDC the surface C D80, CD86 point of α HSP70p-CM-CaP compared with CM-CaP
The expression quantity of son, MHCII molecule is obviously improved, illustrate α HSP70p-CM-CaP can improve to conspicuousness BMDC cell at
Ripe, the quantitative result in B similarly proves the conclusion.Scheme shown in B, data are divided with average value ± SD (n=3)
Analysis, * * * represent p < 0.001, and * * represents p < 0.01, and * represents p < 0.05；
In order to further prove whether OVA antigenic component is rendered, marked using fluorescent monoclonal antibody APC-25-D1.16
Remember SIINFEKL-H-2KbCompound, for imaging streaming as a result, BMDC and fluorescein Fluorescein (green) shown in Fig. 2 C
A few group reagent α HSP70p-CM of label(Fluorescein)-CaP,CM(Fluorescein)V and free CM(Fluorescein)+ CpG is incubated
After educating 48 hours, MHC- antigenic peptide complexes (SIINFEKL-H-2K of the different component for the surface BMDCb, it is red) table
Up to inducing action.It has been incubated for α HSP70p-CM(Fluorescein)Fluorescein and APC letter in the BMDC cell of the reagent of-CaP
Number most strong, quantitative result is shown in 2D, with CM(Fluorescein)-CaP,CM(Fluorescein)V, and free CM(Fluorescein)+
CpG ingredient is compared, α HSP70p-CM(Fluorescein)The BMDC complex fluorescence intensity of-CaP induction respectively to improve
62.3%, 128%, and 116%.Illustrate containing so that the intake of nanometer formulation is reinforced due to active constituent, and MHC- is anti-
Former peptide compound expresses highest；
The experimental results showed that nano particle can be significantly increased by simulating the albumen of necrotic tumor cells and polypeptide active ingredient
Intake with stimulation BMDC maturation.
Embodiment 3
In small animal living body imaging test, C57BL/6 mouse is randomly divided into two groups, and one group in advance in C57BL/6 mouse tail
Portion subcutaneously carries out inoculation nano particle α HSP70p-CM(Fluorescein)- CaP is (average often only according to 25ng α HSP70p, 250ng
The amount of CM, 10ng CpG are administered), another set subcutaneously carries out inoculation nano particle α HSP70p- in mouse tail in advance
CM-CaP-Cy5.Fluorescence signal is detected in small animal living body imaging system, in vitro study, by mouse lymph nodes
It isolates, takes pictures；
As the result is shown: as shown in Figure 3, mouse injects α HSP70p-CM respectively(Fluorescein)- CaP and α HSP70p-CM-
Occurs signal after CaP-Cy56 hours in draining lymph node, signal is remarkably reinforced after 24 hours, illustrates that nano particle can
Significantly to extend CM and depot accumulation time of the CpG active constituent in lymphatic system, and the preparation group of free active constituent is injected
Clear signal is not detected within two periods.Lymph node is taken out after 6,24 hours respectively and carries out sxemiquantitative point
Analysis, it is small to be significantly higher than 6 in 24 hours for the fluorescence signal value of tumour antigen ingredient and CpG content in lymph node as the result is shown
When.
Embodiment 4
In T cell body in priming experiments, the HSP70p-CM-CaP of C57BL/6 mouse hypodermic inoculation equivalent, dissociate CM+
CpG, LCP, CM-CaP, CMV and PBS 3 times.Take animal peripheral blood flow cytometer interpretation of result PE-H-2KbOVA and
PE-H-2KbThe CD8 α of the various hypotypes of Trp2 tetramer label+T cell quantity；
As the result is shown: mouse intracorporal OVA and the Trp2 specificity after α HSP70p-CM-CaP and CM-CaP are immune
CD8+T cell content is significantly higher than CMV group, LCP group and the free mouse organized after being immunized, and illustrates while delivering CM and CpG
Adaptive immunity can be activated to lymphatic system, specific T-cells content increases, wherein after immune α HSP70p-CM-CaP
The T cell content of OVA specificity is 5 times of free group in Mice Body, and the T cell content of Trp-2 specificity is free group
7.2 times, α HSP70p-CM-CaP greatly improved the antigen presentation function of DC cell, and then the subsequent adaptability of efficient activation
It is immune.
Embodiment 5
In the experiment of OVA specific killing t cell activation, take out in the C57BL/6 mouse kidney after each group preparation is immunized
T cell, according to 1 × 106The density in a/hole is inoculated in orifice plate, is incubated for 20 μM of antigen OVA polypeptides after 24 hours, further
Be incubated for 6 hours with the uptake inhibitor containing BFA, be marked later using CD3e-Percp-Cy5.5 antibody, is measured
The IFN-γ of OVA specificity+CD8+T cell content (every mouse according to the α HSP70p, 250ng of about 25ng CM, 10ng's
CpG is administered).The blood for taking same mouse, is handled using ELISA method, detection IFN-γ, IL-4, IL-2,
The content of IL-10；
As the result is shown: being vaccinated with the CD8 that secretion of gamma-IFN is directly measured in the C57BL/6 Mice Body of α HSP70p-CM-CaP+T cell content is free CM+CpG solution group, 5.51,0.37 and 10.29 times of CM-CaP group and LCP group.Illustrate T cell point
Secrete the ability of IFN-γ the strongest in the case where antigen, α HSP70p and CpG three deliver simultaneously, indirect proof α
There are certain facilitations for the activation of HSP70p contained for whole fragmentation effect.The activation of downstream killer T cell from
Auxiliary type Th1 and Th2 type cell are not opened, and several levels of cytokine secretion are able to reflect the activation level of auxiliary type T cell, real
Display is tested, the mouse IL-2 and IFN-γ level after α HSP70p-CM-CaP is immune are 3.9 and 123 times of PBS group respectively, and
IL-10 and IL-4 level does not have significant difference compared with PBS group, illustrates that the nano particle is promoted by causing the reaction of Th1 type
The differentiation of downstream killer T cell, IL-2 and the secretion level of IFN-γ are better than free in the Mice Body after CM-CaP is immune
CM+CpG shows combining delivering and capable of inducing the immune response of Th1 type to a certain extent for CM and CpG.
Embodiment 6
It is 3 times (every small using the immune mouse of each group preparation in 20 days in the evaluation test on metastases lesion model
According to the CM of the α HSP70p, 250ng of about 25ng, the CpG of 10ng is administered mouse).After 20 days, every mouse is according to 5 × 105
A B16OVA cell is injected, and the metastatic lesion model in mouse lung metastasis model is established.Taken after 20 days again mouse lung into
Row observation, detects the quantity of Lung metastases lesion.In order to evaluate the combination therapy effect of vaccine and PD-1, press down in mouse subcutaneous tumors
In system experiment, at the 0th day, 2 × 10 are subcutaneously injected in advance5A B16OVA cell is distinguished using same preparation and dosage later
It is immune every time to inject PD-1 antibody (every mouse gives 100 μ g) later at the 10th, 17,23 day immune mouse 3 times, remember every other day
The size for recording tumour, using vernier caliper measurement, and calculates tumorous size:
As the result is shown: as shown in Figure 5A, in mouse lung shift experiment, after α HSP70p-CM-CaP and CM-CaP is immune
Mouse receives tumor cell injection again, and only micro metastatic lesion generates, and other PBS groups, preparation group of dissociating, CMV group
And LCP group has macroscopic transfer stove, the shape of mouse lung metastatic lesion may be able to suppress by illustrating both preparations
At by finding that α HSP70p-CM-CaP effect is better than CM-CaP group after artificial counting, lesion quantity reduces 77%.Explanation
The carrier band of α HSP70p can obviously inhibit the formation of final metastatic lesion, and the thinking of this simulation necrotic signal is the pre- of tumour
It is anti-to provide new approach, it is shown that the superiority for the treatment of, this experiment continue to measure preparation using subcutaneous tumors treatment model mice
Therapeutic effect, as shown in Figure 5 D, free preparation group, which is compared to PBS group, cannot obviously slow down the growth of tumour, combined PD-
After 1 antibody is treated, the group+PD-1 monoclonal antibody combination that finally dissociates can reduce 46.2% tumour growth, especially, α
After HSP70p-CM-CaP and the combination therapy of PD-1 monoclonal antibody, tumor control rate 89%.The result illustrates that this bionical is received well
Metric system agent has very strong inhibiting effect for melanoma.
Embodiment 7
In the detection test that memory t cell generates, after PD-1 monoclonal antibody combination therapy off-test 40 days, mouse lymph is taken
It ties, removal connective tissue is lightly ground on cell filter membrane, lymphocyte is extracted after PBS washing, passes through anti-CD3e-
Percp-Cy5.5(eBioscience,48-0031-82),anti-CD8a-Alexa 647(Biolegend,
100724), anti-CD44-FITC (ebioscience, 11-0441-82) and anti-CD62L (L-Selectin)-PE
(ebioscience, 12-0621-82) antibody is incubated for and is marked, machine testing on flow cytometer；
As the result is shown: CD8 in the Mice Body after α HSP70p-CM-CaP is immune+Two class memory t cell content situations
It is different, such as Fig. 5 E&F, effect type memory T cell (TEMCell, CD3+CD8+CD62L–CD44+) content highest, account for lymph node
Immunocyte CD8+The 40% of T cell quantity, hence it is evident that be higher than free preparation group；Central type memory T cell (TCMCell, CD3+CD8+
CD62L+CD44+) content is minimum, effect of such cell in digital preservation function is simultaneously not so good as TEMCell is important, illustrates α
HSP70p-CM-CaP has Long-Term Protection, prevents the recurrence of tumour.
Sequence table
<110>Fudan University
<120>a kind of calcium phosphate-lipid nanometer vaccine for simulating necrotic tumor cells and its application
<130> 201802
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<211> 20
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tccatgacgt tcctgacgtt 20
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<212> PRT
<213>the HSP70 active peptide segment (α HSP70) of α spiral modification
<400> 2
Ala Cys Phe Ala Glu Lys Phe Lys Glu Ala Val Lys Asp Tyr Phe Ala
1 5 10 15
Lys Phe Trp Asp Gly Ser Gly Thr Lys Asp Asn Asn Leu Leu Gly Arg
20 25 30
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<213>α spiral sequence (α helix sequence)
<400> 3
Ala Cys Phe Ala Glu Lys Phe Lys Glu Ala Val Lys Asp Tyr Phe Ala
1 5 10 15
Lys Phe Trp Asp
20
Claims (12)
1. a kind of calcium phosphate-lipid nanometer vaccine for simulating necrotic tumor cells, which is characterized in that be the nanoscale of core-shell structure
Bionical particle, the nano inner core be built by calcium phosphate and artificial synthesized lipid bilayer shell pass through hydrophobic effect phase
In conjunction with composition, the calcium phosphate of the kernel is the hydrophobic nanoparticles for efficiently containing Oligonucleotide substance, and shell is
The artificial matrix material of altitude simulation cell membrane fluidity loads natural epicyte protein and HSP70 active peptide segment.
2. calcium phosphate-lipid nanometer vaccine of simulation necrotic tumor cells according to claim 1, which is characterized in that described
The artificial matrix material of shell loads the HSP70 active peptide of natural multivalence tumor cell membrane antigen protein and the modification of α spiral
Section (α HSP70p)；The kernel prepares nanoparticle with calcium phosphate (CaP) for material and contains adjuvant oligonucleotide CpG；
The vaccine is using oligonucleotide CpG and HSP70p as adjuvant component.
3. calcium phosphate-lipid nanometer vaccine of simulation necrotic tumor cells according to claim 1, which is characterized in that described
Calcium phosphate kernel used by the non-methylation oligodeoxynucleotide (abbreviation CpG ODN) of adjuvant component be artificial synthesized tool
There is the DNA sequence dna of the CpG oligonucleotide containing non-methylation of immunostimulation, the CpG ODN sequence is CpG ODN
1826(5’-TCCATGACGTTCCTGACGTT-3’)。
4. calcium phosphate-lipid nanometer vaccine of simulation necrotic tumor cells according to claim 1, which is characterized in that described
Shell lipid in addition adjuvant component be α spiral modification HSP70 active peptide segment (α HSP70p).
5. calcium phosphate-lipid nanometer vaccine of simulation necrotic tumor cells according to claim 1, which is characterized in that described
Epicyte protein be multivalence tumor cell membrane protein ingredient, be selected from mouse source B16OVA cell, for by expanding in vitro
Increase and extract the mouse melanin tumor cell system for stablizing expression OVA model antigen of the genetic engineering transformation obtained.
6. calcium phosphate-lipid nanometer vaccine of simulation necrotic tumor cells according to claim 1, which is characterized in that described
Artificial lipid membrane material be phosphocholine structure lipid and cholesterol, including DPPC, DSPC, DOPC and cholesterol.
7. calcium phosphate-lipid nanometer vaccine of simulation necrotic tumor cells as described in claim 2, which is characterized in that described
Kernel contain nucleic acid drug, be selected from adjuvant oligonucleotide CpG ODN, siRNA, DNA.
8. calcium phosphate-lipid nanometer vaccine of simulation necrotic tumor cells according to claim 3, which is characterized in that described
Adjuvant component CpG ODN, have strong inherent immunity stimulation, by TLR9 mediate directly activate inherent immunity system with
And activation panimmunity cell causes B cell differentiation and T cell differentiation.
9. calcium phosphate-lipid nanometer vaccine of simulation necrotic tumor cells according to claim 4, which is characterized in that described
α spiral modification HSP70 active peptide segment (α HSP70), sequence AC-FAEKFKEAVKDYFAKFWD-GSG-
TKDNNLLGRFELSG is inserted into technology by lipid, α spiral sequence is added outside HSP70 active peptide segment TKDNNLLGRFELSG
AC-FAEKFKEAVKDYFAKFWD is made.
10. calcium phosphate-lipid nanometer vaccine of simulation necrotic tumor cells according to claim 4, which is characterized in that described
The HSP70 active peptide segment (α HSP70) of α spiral modification be active constituent in heat shock protein A subunit, can directly activate tree
CD49 molecule on prominent shape cell and natural kill (NK) cell.
11. calcium phosphate-lipid nanometer vaccine of simulation necrotic tumor cells according to claim 1, which is characterized in that described
The bionical particle of the nanoscale of core-shell structure, calcium phosphate nano kernel average grain diameter are 30-40nm, are coated with after artificial lipid membrane most
Whole partial size is in 50nm or so.
12. calcium phosphate-lipid nanometer vaccine of simulation necrotic tumor cells described in claim 1 is in preparation for inhibiting tumour
The purposes in growth and transfer preparation in vivo, the preparation are locally targeted after mouse is immunized by transdermal delivery mechanisms
Dendritic Cells into draining lymph node generates the Immunestimulatory effect of series of downstream after vivo activation, induces Th1 type cell
Factor reaction, the generation of lethal CD8+T cell (CTL) and the generation of complementary CD4+ cell.
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