CN109620952A - A kind of tumor vaccine and preparation method thereof - Google Patents
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention provides a kind of tumor vaccine and preparation method thereof.The tumor vaccine includes the Dendritic Cells for having blocked CD44 signaling pathway molecule.The present invention also provides a kind of methods for preparing the tumor vaccine, including 1) obtaining mononuclearcell by mammalian peripheral blood or Cord blood, it is induced as mature dendritic cell or marrow hemopoiesis precursor is obtained by the marrow of mammal, induced as mature dendritic cell；2) the CD44 signaling pathway molecule of the mature dendritic cell is blocked；3) Dendritic Cells for having blocked CD44 signaling pathway molecule is collected.Tumor vaccine provided by the invention has good antitumor action, while antitumor spectra is extensive, and has good safety in utilization.
Description
Technical field
The present invention relates to a kind of vaccine and preparation method thereof more particularly to a kind of tumor vaccines and preparation method thereof.
Background technique
Dendritic Cells is the professional antigen presenting cell in immune system, the starting of immunocyte, activation, effect and
The stages such as stable state maintenance play a significant role.Since Dendritic Cells is found, antitumor field application just at
For the hot spot of tumour immunity.The dendritic cell tumor vaccine immune activation ability powerful by Dendritic Cells, to receptor
Immune system plays specific for tumour antigen/specific activation effect, reverses before cancer or the inhibition that is formed is immune in tumour
Microenvironment mediates the immunologic cytotoxicity to cancer premutation cell or tumour cell and removing.
2011, Provenge was applied to forefront as first dendritic cell tumor vaccine ratified by FDA listing
The new page of immunotherapy of tumors rapid development in recent years has been opened in gland cancer treatment.But successive treatment process is found, Provenge
Antitumous effect is extremely limited, other dendritic cell-based tumor vaccines, in clinical trial, rests on the clinical I/II phase, seldom more
Number is able to enter the clinical III phase, finally also stagnates because of unsatisfactory curative effect.
CD44 molecule belongs to adhesion molecule, is a kind of wide expression in the I class cross-film single chain glycoprotein of cell surface；This point
Polymorphism is presented in son, and molecular mass changes between 80-200kb；Interaction, cell migration and cell are glutinous between main mediated cell
It is attached, and activation and the signal transduction of lymphocyte gone back to the nest with T lymphocyte are participated in, promote T cell, B cell differentiation and T thin
Born of the same parents' activation.
Existing literature report, CD44 molecule can be with hyaluronic acid, collagen, Laminin lens and fibronectin knot
It closes.The antibody of anti-CD44 is able to suppress the interaction of lymphocyte Yu HEV (Hepatitis E virus), CD44 polyclonal antiserum
It can also inhibit the interaction of lymphocyte Yu peripheral lymph node, synovial membrane and mucosal lymphoid tissue.But it there is no CD44 molecule
Apply the report in immunotherapy of tumors field.
Summary of the invention
The present invention provides a kind of tumor vaccine, the Dendritic Cells comprising having blocked CD44 signaling pathway molecule, described swollen
Tumor vaccine has good immune activation and antitumor action, while antitumor spectra is extensive, and has good safety in utilization.
The present invention also provides the tumor vaccines to prepare the application in the drug for preventing or treating tumour.
The present invention also provides a kind of prevention or treatment tumour composition, comprising the tumor vaccine as effectively at
Point.The composition has the function of preventing and treating tumour simultaneously.
The present invention also provides a kind of method for preparing tumor vaccine, CD44 signaling pathway molecule will have been blocked by realizing
Dendritic Cells prepares tumor vaccine as vaccine entity.
A kind of tumor vaccine provided by the invention, the Dendritic Cells comprising having blocked CD44 signaling pathway molecule.
In the solution of the present invention, the Dendritic Cells of CD44 signaling pathway molecule is blocked (to be expressed as CD44-/-DC is thin
Born of the same parents) can be Dendritic Cells below: 1) on the surface of dendritic cells express MHC-I class molecule, MHC-II class molecule,
CD40, CD80 and CD86, but do not express CD44；2) the CD44 molecule on the surface of dendritic cells is closed by antibody, ligand
Or it is blocked by micromolecular inhibitor.
Further, the ligand includes hyaluronic acid, collagen, Laminin lens, fibronectin etc.；It is described anti-
The antibody of CD44 molecule can be commercially available.
Activation efficiency and intensity and increase Dendritic Cells in a scheme of the invention, for stimulation T cell
Total quantity (because the ratio of dendritic cell vaccine of the invention in blood is smaller) is to extend after feedback dendron shape in blood
The considerations of cellular half-life, the tumor vaccine of the invention allow comprising a certain proportion of CD44+/+DC cell, i.e., do not hinder
The Dendritic Cells of disconnected CD44 signaling pathway molecule.
Specifically, having blocked the dendron shape of CD44 signaling pathway molecule in the tumor vaccine in the solution of the present invention
The ratio of the total Dendritic Cells of cell Zhan be 30% or more, preferably 40% or more, more preferable 50% or more, most preferably 60% with
On.
Further, the total dendron shape of the Dendritic Cells Zhan of CD44 signaling pathway molecule has been blocked in the tumor vaccine
The ratio of cell is 100%.
In the solution of the present invention, the Dendritic Cells is originated from mammal.It is dynamic that the mammal can be health
Object and suffer from cancer animal.Further, the mammal is healthy animal.Further, the mammal can be ox
Section animal, equid, felid, canid, rabbit section animal, porcine animals, camellid, rodent and spirit
One of long class animal is a variety of.Further, the mammal can be ox, horse, dog, goat, sheep, cat, rabbit,
One of pig, camel, alpaca, rat, mouse, cavy, non-human primate (such as ape, monkey, baboon, orangutan) and people or
It is a variety of.
Mammal of the present invention is also possible to common pet.Further, the pet include cat, imperial cat, hamster,
Small white mouse, gerbil jird, chipmunk, Degus, squirrel, flying squirrel, big Siberian chipmunk, one of rabbit, ermine, hedgehog, cavy, pig, dog and monkey
Or it is a variety of.
Tumor vaccine provided by the invention can be any dosage form suitable for clinical application and medication specification, for example, can be with
It is injection.The preparation method of the tumor vaccine further includes that required dosage form is made according to conventional vaccine preparation method,
Such as injection is made, it can be and injection etc. is made by the way that physiological saline is added.
Tumor vaccine provided by the invention can exempt from individual by intravenous injection, abdominal cavity or subcutaneous administrations
Epidemic disease is to inhibit tumour to occur or treated with killing tumor cell.Specifically, the clinical application for 50kg weight human body, institute
Stating tumor vaccine per injection amount can be 1 × 106~1 × 108A cell, preferably 1~10 × 107A cell, the cell are
The Dendritic Cells of CD44 signaling pathway molecule is blocked.
The present invention also provides the tumor vaccines to prepare the application in the drug for preventing or treating tumour.Into
One step, the tumour includes melanoma, lymthoma, colorectal cancer, lung cancer, liver cancer, gastric cancer, breast cancer, cancer of pancreas, forefront
One of gland cancer, the cancer of the esophagus, cervical carcinoma, bladder cancer, oophoroma, carcinoma of endometrium and kidney are a variety of.
The present invention also provides a kind of prevention or treatment tumour composition, comprising the tumor vaccine as effectively at
Point.Further, the tumour includes melanoma, lymthoma, colorectal cancer, lung cancer, liver cancer, gastric cancer, breast cancer, pancreas
One of cancer, prostate cancer, the cancer of the esophagus, cervical carcinoma, bladder cancer, oophoroma, carcinoma of endometrium and kidney are a variety of.
It includes tumor vaccine of the invention as main active that prevention of the invention or treatment tumour, which use composition,.Into
One step, it is described prevention or treatment tumour composition in can also containing it is other with therapeutical uses biomolecule (for example,
Protein, nucleic acid, antibody etc.) and/or chemicals (for example, chemical synthetic drug).
Further, the treatment of the therapeutical uses of the biomolecule and/or chemicals and the tumor vaccine is used
Way can be identical, for example, being used to antitumor.Further, the therapeutical uses of the biomolecule and/or chemicals with
The therapeutical uses of the tumor vaccine can also be different, and apply for example, the biomolecule and/or chemicals can play antagonism
With the adverse reaction or side effect after the tumor vaccine, and for treating other tumor complications or complication (for example, hair
Heat, shiver with cold, anxiety；Ascites, Nausea and vomiting, abdominal distension, diarrhea, obstruction, heart failure, arrhythmia cordis, dry cough, cough, breathing are tired
Hardly possible, oliguresis, diuresis, albuminuria, blood urine, pain, skin allergy, erythema, pruitus etc.) effect.
When the composition of the invention is comprising the tumor vaccine and treatment biomolecule and/or chemicals
(combining form can be the form of mixture to combining form, be also possible to separate the form of independent packaging, in the present invention
It is unrestricted) when, then the tumor vaccine of the invention by weight account for therapeutic composition of the invention activity at
Point (that is, total amount of tumor vaccine and treatment biomolecule and/or chemicals) 30%, 40%, 50%, 60%, 70%,
80%, 90% or 95% or more or even 100%, preferably 50% or more, more preferable 60% or more, most preferably 70% or more.
In a preferred embodiment, the composition of the invention can also include pharmaceutically acceptable or physiology
Acceptable pharmaceutic adjuvant, carrier, stabilizer, diluent, excipient, buffer, isotonic agent and/or additive etc..
In a further preferred embodiment, the composition of the invention can also be comprising conventionally used for lymphocyte
The anti frozen liquid (for example, ZENOAQ company CELLBANKER series of cell frozen stock solution) of cryo-conservation, so as to carry out profound hypothermia
(- 80 DEG C hereinafter, for example, in liquid nitrogen) saves.
The present invention also provides a kind of methods for preparing the tumor vaccine, comprising the following steps:
1) mononuclearcell is obtained by mammalian peripheral blood or/Cord blood, and being induced is mature dendritic cell,
Or marrow hemopoiesis precursor is obtained by the marrow of mammal, and induced as mature dendritic cell；
2) the CD44 signaling pathway molecule of the mature dendritic cell is blocked；
3) Dendritic Cells for having blocked CD44 signaling pathway molecule is collected.
Further, mature dendritic cell preferably originates from pair that tumor vaccine of the present invention will prevent or treat in the present invention
As, and blocked the dendron shape of CD44 signal path thin by mature dendritic cell acquisition according to method recorded in the present invention
Born of the same parents.Further, the mature dendritic cell can be originated from the object half that prevent or treat with tumor vaccine of the present invention
The individual (such as parent and children) being harmonious.Further, the mature dendritic cell can be originated from tumour epidemic disease of the present invention
Seedling to prevent or the individual of the object allogeneic treated (different genotype individual i.e. of the same race).
In the specific embodiment of the present invention, the resistance of the CD44 signaling pathway molecule of the mature dendritic cell
It is open close cross a) genetic engineering means knock out CD44 molecular gene in the mature dendritic cell (such as Crisper/Cas9,
The methods of TALEN, RNA interference), or pass through b) using can be with antibody, ligand or the micromolecular inhibitor in conjunction with CD44 molecule
It realizes.
Further, CD44 molecular gene in the mature dendritic cell is knocked out into packet by genetic engineering means
It includes: will be described in virus (being also possible to the common carriers such as the adenovirus, slow virus) transfection containing the carrier for knocking out CD44 molecular gene
The mature dendritic cell that step 1) obtains is collected and is transferred to the mature dendritic cell containing the carrier for knocking out CD44 gene, that is, obtains
The Dendritic Cells of CD44 signaling pathway molecule must have been blocked.
It, can be by mature dendritic cell and targeting knockout CD44 gene in the specific embodiment of the present invention
Slow virus realizes the knockout to CD44 gene with 2:1-1:50 quantity than mixing.Further, the mature dendron shape is thin
The slow virus quantity ratio of born of the same parents and targeting knockout CD44 gene is 1:10.Those skilled in the art can according to need in above range
The ratio of the slow virus of the mature dendritic cell and targeting knockout CD44 gene is selected, as long as can be realized to CD44 gene
Knockout, what this can be achieved on to those skilled in the art.
Further, it can will contain Dendritic Cells culture medium (2 × 106/ mL) and anti-mouse CD44 antibody (concentration
It 0.5mg/mL) is mixed with the volume ratio of 1:10-1000 to realize the closing to CD44.Further, the shape containing dendron is thin
The volume ratio of born of the same parents' culture medium and the anti-mouse CD44 antibody is 1:100.Those skilled in the art can according to need above-mentioned
Range adjusts the usage amount of anti-mouse CD44 antibody, as long as CD44 is enabled to close, this is for those skilled in the art
For can be achieved on.
In the preparation process in accordance with the present invention, it is thin to separate single core for acquisition mammalian (such as peripheral blood or Cord blood)
Born of the same parents, for example, (it is, for example, possible to use people's mononuclearcell seperator, circulations 500 for the method or equipment that can use extracorporal circulatory system
~6000mL peripheral blood or Cord blood or syringe extract peripheral blood or 10~500mL of Cord blood).Acquire mammal marrow
The method of separation marrow hemopoiesis precursor is not also particularly limited, and can use method as known in the art.
Used Dendritic Cells culture technique is well known in the art in the preparation process in accordance with the present invention, can be with
It is completed using method or equipment commonly used in the art, it is unrestricted in the present invention.As long as can cultivate and lure
The technology for leading Dendritic Cells directed differentiation can be used in the present invention.For example, the culture to mouse dcs is more
Using the method for taking in vitro culture after marrow hemopoiesis precursor；And it obtains human dendritic cell and mostly uses peripheral blood or Cord blood
The method of attached cell in vitro culture is taken after mononuclearcell is adherent.The above method can be found in many scientific literatures, or
Implement according to the specification or recommendation experiment flow of related reagent production firm.Those skilled in the art have the ability to obtain completely
These specific embodiments or experiment flow.
In above-mentioned Process of in vitro, the condition of culture of Dendritic Cells of the invention is not particularly limited.For training
Base is supported, it can be using the culture medium conventionally used for lymphocyte culture, such as RPMI-1640 culture medium.It, can be with for condition of culture
Using the typical conditions of this field medium size lymphocyte culture, such as 37 DEG C of temperature, CO2Concentration 5v/v%, replacement one in every 3~5 days
Subculture.It, can be by adding some cell factors, example for the external evoked of the tumor vaccine of the invention and preparation
Such as: one of GM-CSF, IL-2, IL-4, IL-5, IFN-γ and TNF-α a variety of are realized.
For incubation time, those skilled in the art can specifically determine according to the phenotype and form of cell in induction, can
To carry out the phenotype of flow cytometry cell by periodically sampling, to obtain the cell of desired state and function.
In the specific embodiment of the present invention, the method for preparing the tumor vaccine, comprising:
1) it by the peripheral blood of subject or umbilical cord blood collection into anticoagulant test tube, is removed by the method for density gradient centrifugation
Red blood cell；
2) mononuclearcell is separated from the peripheral blood or Cord blood for removed red blood cell, in complete medium, is placed in
37 DEG C, 5v/v%CO2In vitro culture under environment obtains monocyte in peripheral blood or Cord blood using adherent method, and adds choosing
From one or more directional inductions of GM-CSF, IL-2, IL-4, IL-5, IFN-γ, TNF-α etc. and dendritic cell activated
Cell factor；Incubation time is 3~14 days, and preferably 4~10 days, more preferable 5~8 days, during which periodically sampling passed through fluidic cell
Instrument detects cell, obtains mature dendritic cell；
3) the CD44 signal path of the mature Dendritic Cells of acquisition is blocked, including passes through genetic engineering means
It knocks out CD44 molecular gene in the mature dendritic cell (such as the means such as Crisper/Cas9, TALEN, RNA interference), or
Person with antibody, ligand or the micromolecular inhibitor in conjunction with CD44 molecule by using can block；
4) Dendritic Cells for having blocked CD44 signal path is collected.
Scheme provided by the invention has the advantage that
(1) tumor vaccine provided by the invention, the Dendritic Cells comprising having blocked CD44 signaling pathway molecule, can swash
The intracorporal responsiveness immunocyte of Recipient mice living, the latter is directly related with anti-tumor effect, can pass through direct killing and improvement
The antineoplastic immune ability of the various ways such as antineoplastic immune microenvironment raising Recipient mice.
(2) tumor vaccine provided by the invention, antitumor spectra is extensive, can effectively kill to kinds of tumor cells, and to machine
The toxic side effect of body is small, and use is safe.
(3) tumor vaccine provided by the invention, can be used as therapeutic vaccine can also be used as preventative vaccine, in difference
The oncotherapy stage use, by activating body immune system, the generation of pre- preventing tumor or have to existing tumour cell
Effect killing.
(4) by tumor vaccine of the invention be prepared into prevention or treatment tumour composition, can with long-term preservation in case with
When use.
Detailed description of the invention
Figure 1A shows the microscope photo for not knocking out the mature dendritic cell of CD44 gene.Figure 1B, which is shown, not to be had
(first peak is intensity less than 10 to the flow cytomery result of the mature dendritic cell of knockout CD44 gene in Figure 1B3
Peak represents the Isotype control of CD44, and second peak intensity is 103To 104Represent CD44).Fig. 1 C, which is shown, knocks out CD44 gene
The microscope photo of Dendritic Cells.Fig. 1 D shows the flow cytomery result for knocking out the Dendritic Cells of CD44 gene
(only have because CD44 gene is knocked, in Fig. 1 D first peak be represent Isotype control less than 103Peak).
Fig. 2A shows the microscope photo of the Dendritic Cells in conjunction with anti-mouse CD44 antibody.Fig. 2 B is shown in conjunction with anti-
The flow cytomery result of the Dendritic Cells of mouse CD44 antibody (only has first peak to represent Isotype control in Fig. 2 B
Less than 103Peak).
Fig. 3 shows mature dendritic cell (CD44+/+DC) and knock out CD44 gene Dendritic Cells (CD44-/-DC)
Phenotypic difference.Wherein-feminine gender is represented ,+low expression is represented, ++ represent high expression.
Fig. 4 A shows mature dendritic cell (CD44+/+DC) and knock out CD44 gene Dendritic Cells (CD44-/-
DC the difference of cell factor) is generated after injection mouse.Fig. 4 B shows conventional dendritic shape Cell vaccine (CD44+/+DC) He Benfa
Bright tumor vaccine (the CD44-/-DC the proportional difference of cd8 t cell) is generated after injection mouse.Fig. 4 C shows conventional dendritic
Shape Cell vaccine (CD44+/+) and the tumor vaccine (CD44 of the invention DC-/-DC cd8 t cell is generated after) injecting mouse
The difference of quantity.
Fig. 5 A, which is shown, injects EL4 cell using the immune mouse of the application tumor vaccine and conventional dendritic shape Cell vaccine
The difference of EL4 tumor size afterwards.Fig. 5 B is shown using immune small of the application tumor vaccine and conventional dendritic shape Cell vaccine
Mouse injects the difference of melanoma Pulmonary metastasis focuses after B16-F10 melanoma cells.
Fig. 6 A shows the lotus EL4 mice with tumor using the application tumor vaccine and the treatment of conventional dendritic shape Cell vaccine
The difference of EL4 tumor size.Fig. 6 B shows the lotus using the application tumor vaccine and the treatment of conventional dendritic shape Cell vaccine
The difference of B16-F10 melanoma murine melanoma Pulmonary metastasis focuses.
Fig. 7 A shows lotus MC38 colon cancer cell mouse respectively through conventional dendritic cell-based tumor vaccines and the application tumour epidemic disease
The difference of tumor size after seedling treatment.Fig. 7 B shows lotus Lewis lung carcinoma cell mouse respectively through conventional dendritic cell-based tumor vaccines
With the difference of tumor size after the application tumor vaccine therapy.Fig. 7 C shows lotus H22 liver cancer cells mouse respectively through conventional tree
The difference of tumor size after prominent shape Cell vaccine and the application tumor vaccine therapy.Fig. 7 D shows lotus MFC stomach cancer cell mouse
Respectively after conventional dendritic cell-based tumor vaccines and the application tumor vaccine therapy tumor size difference.Fig. 7 E shows lotus 4T1
Breast cancer cell mouse respectively after conventional dendritic cell-based tumor vaccines and the application tumor vaccine therapy tumor size difference.Figure
7F shows the lotus Renca kidney cancer cell mouse tumour after conventional dendritic cell-based tumor vaccines and the application tumor vaccine therapy respectively
The difference of size.Fig. 7 G shows that lotus Pan02 pancreatic cancer cell mouse is swollen through conventional dendritic cell-based tumor vaccines and the application respectively
The difference of tumor size after tumor vaccine therapy.Fig. 7 H shows that lotus RM-1 prostate gland cancer cell mouse is thin through conventional dendritic shape respectively
The difference of tumor size after born of the same parents' tumor seedling and the application tumor vaccine therapy.Fig. 7 I shows that lotus AKR esophageal cancer cell mouse is distinguished
The difference of tumor size after conventional dendritic cell-based tumor vaccines and the application tumor vaccine therapy.Fig. 7 J shows lotus U14 uterine neck
Cancer cell mouse respectively after conventional dendritic cell-based tumor vaccines and the application tumor vaccine therapy tumor size difference.Fig. 7 K is aobvious
Having shown lotus MB49 bladder cancer cell mouse, tumour is big after conventional dendritic cell-based tumor vaccines and the application tumor vaccine therapy respectively
Small difference.Fig. 7 L shows lotus ID8 ovarian cancer cell mouse respectively through conventional dendritic cell-based tumor vaccines and the application tumour epidemic disease
The difference of tumor size after seedling treatment.
Fig. 8 shows the safety evaluatio result of the application tumor vaccine.
Fig. 9 shows the single-dose oncogenicity evaluation result of the application tumor vaccine.
Figure 10 shows the multiple dosing oncogenicity evaluation result of the application tumor vaccine.
Figure 11 shows the Vascular stimulation experimental result of the application tumor vaccine.
Specific embodiment
Unless otherwise defined, whole technical and scientific terms used herein have and one in fields of the present invention
As the normally understood identical meaning of technical staff.Although with similar or equivalent any method and material those of described herein
Material can be used for implementing or test the present invention, but preferred method and material will now be described.The whole of application cited herein
It is whole incorporated herein by reference to publish publication and patent application.It can be interpreted to hold without any content herein
Recognize the present invention to have no right to authorize the date earlier than present disclosure and formerly inventing.
It has to be noticed that in this article in appended claims when, singular "one", " some " and "the" include
Plural object, unless otherwise clearly specified in the context.
" Dendritic Cells ": the antigen presenting cell of one kind sole duty in immunocyte is gained the name because shape is like dendron.It should
Cell function is identification antigen and processes, handles antigen, by the highly expressed MHC-I class molecule in its surface and MHC-II class molecule
By antigen fragment difference submission to cd8 t cell and cd4 t cell, the activation and proliferation of mediate T cell.Dendritic Cells is immune
The key link of response is the bridge for connecting congenital immunity and adaptive immunity.
" tumor seedling ": tumor seedling, that is, tumor vaccine abbreviation has broad sense and chivalrous two kinds of definition.The tumor vaccine of broad sense prevents
The general name of property tumor vaccine and therapeutic tumor vaccine；Chivalrous tumor vaccine list refers to preventative tumor vaccine.In the present invention,
It is indicated if be not known, the tumor vaccine of broad sense is referred both to when referring to tumor vaccine.
“CD44+/+DC ": mature dendritic cell (or Dendritic Cells without blocking CD44 signaling pathway molecule).
“CD44-/-DC ": knocking out the Dendritic Cells of CD44 gene, (or other means have blocked the tree of CD44 signal path
Prominent shape cell).
Following embodiment further describes the present invention, but the embodiment is merely to illustrate the present invention, but rather than to limit
The scope of the present invention processed.When reading specification and with reference to common knowledge, other embodiments are for a person skilled in the art
It will be apparent.
Experimental method in following embodiments is unless otherwise specified conventional method.Reality as used in the following examples
Material is tested, is to be commercially available from conventional reagent company unless otherwise specified.
Embodiment 1
The preparation of the application tumor vaccine
1. experimental material and reagent
Experiment with C57BL/6 mouse be purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., the age 6-8 weeks, weight
18g or so.
Fetal calf serum (FCS): it is purchased from PAN company；
RPMI-1640 culture medium: it is purchased from PAN company；
GM-CSF, IL-4 are purchased from Peprotech company, the U.S.；
2. experimental procedure
(1) separating mouse femur bone marrow hemopoietic forebody cell；
(2) by isolated mouse bone marrow cells hemopoietic forebody cell with 2 × 106The density of/mL is incubated at addition containing 50ng/mL
GM-CSF, 50ng/mL IL-4, in the RPMI-1640 culture medium of 10% fetal calf serum (FCS), 37 DEG C of temperature, CO2Concentration 5v/
V% is induced 7 days, collects cell precipitation, smear after sampling is resuspended with physiological saline is observed under the microscope, as a result such as Figure 1A institute
Show；It samples simultaneously using flow cytomery, as a result as shown in Figure 1B, ordinate represents quantity, and it is strong that abscissa represents fluorescence
Degree, first peak are less than 103Peak represents Isotype control, and second peak intensity is 103To 104Peak represents CD44.
(3) step (2) mature dendritic cell obtained is precipitated, washs removal fetal calf serum (FCS) with PBS, so
Afterwards with 2 × 106/ mL density is resuspended in training of the acquisition containing mature dendritic cell in the RPMI-1640 culture medium without 10%FCS
Base is supported, in the slow virus that targeting knockout CD44 gene is wherein added, contral ripening Dendritic Cells and targeting knockout CD44 gene
Slow virus quantity ratio be 1:10, at 37 DEG C, infect 2h after, collect cell precipitation, smear after sampling is resuspended with physiological saline,
It observes under the microscope, as a result as shown in Figure 1 C；It samples using flow cytomery, as a result as shown in figure iD simultaneously.
3, experimental result
Figure 1A shows the microscope photo for not knocking out the mature dendritic cell of CD44 gene.Figure 1B, which is shown, not to be had
Knock out the flow cytomery result of the mature dendritic cell of CD44 gene, it can be seen that surface of dendritic cells expression
CD44 molecule.Fig. 1 C shows the microscope photo for knocking out the Dendritic Cells of CD44 gene, it can be seen that has phase with Figure 1A
As form, that is, there is mature Dendritic Cells form.Fig. 1 D shows the streaming for knocking out the Dendritic Cells of CD44 gene
Cell instrument testing result, it can be seen that the surface of dendritic cells does not express CD44 molecule, illustrates to transfect successfully, that is, is knocked out
The Dendritic Cells of CD44 gene.
Embodiment 2
The preparation of the application tumor vaccine
1, experimental material and reagent
Experiment with C57BL/6 mouse be purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., the age 6-8 weeks, weight
18g or so.
Fetal calf serum (FCS): it is purchased from PAN company；
RPMI-1640 culture medium: it is purchased from PAN company；
GM-CSF, IL-4 are purchased from Peprotech company, the U.S.；
Anti-mouse CD44 antibody is purchased from U.S. Biolegend company, article No. 156002.
2, experimental procedure
(1) separating mouse femur bone marrow hemopoietic forebody cell；
(2) by isolated mouse bone marrow cells hemopoietic forebody cell with 2 × 106The density of/mL is incubated at addition containing 50ng/mL
In the RPMI-1640 culture medium of GM-CSF, 50ng/mL IL-4,10%FCS, 37 DEG C of temperature, CO2Concentration 5v/v%, induction 7
It, collects cell precipitation, and smear after sampling is resuspended with physiological saline is observed under the microscope；It samples simultaneously using fluidic cell
Instrument detection.
(3) step (2) mature dendritic cell obtained is precipitated, with 2 × 106/ mL density is resuspended in containing 10%FCS
RPMI-1640 culture medium in obtain culture solution containing Dendritic Cells, Dendritic Cells culture medium will be contained and resisted with anti-mouse CD44
Body (purchase concentration 0.5mg/mL) is mixed with the volume ratio of 1:1000, after being incubated for 30 minutes at 4 DEG C, is collected cell precipitation, is taken
Smear after sample is resuspended with physiological saline, is observed under the microscope, as a result as shown in Figure 2 A；It samples and is examined using flow cytometer simultaneously
It surveys, as a result as shown in Figure 2 B.
3, experimental result
Fig. 2A shows the microscope photo of the Dendritic Cells in conjunction with anti-mouse CD44 antibody, it can be seen that has with Figure 1A
There is similar form, that is, there is mature Dendritic Cells form.Fig. 2 B shows the dendron shape in conjunction with anti-mouse CD44 antibody
The flow cytomery of cell results, it can be seen that the CD44 molecule of the surface of dendritic cells by anti-mouse CD44 antibody
Closing obtains the Dendritic Cells for having blocked CD44 signal path.
Embodiment 3:
The application tumor vaccine (knocks out the Dendritic Cells (CD44 of CD44 gene-/-DC)) with conventional dendritic shape cell
Tumor seedling (i.e. mature dendritic cell (CD44+/+DC)) ability of activated receptor immune system compares.
1, experimental material and reagent
Experiment with C57BL/6 mouse be purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., the age 6-8 weeks, weight
18g or so.
It detects Cytokine of Serum and uses Biolegend company cytokine detection kits (article No. 740005).
Anti-mouse H2Kb,Iab, CD40, CD80, the fluorescence antibody of CD86, CD44 is purchased from Biolegend company, the U.S., goods
Number be respectively H2Kb(114605),Iab(116410),CD40(124607),CD80(104705),CD86(105005),CD44
(156002)。
The anti-CD8 antibody (anti that label mouse peripheral blood cd8 cell uses antibody to mark for Biolegend company PE
Mouse CD8a antibody, article No. 100708).Sample application BD company FACSAriaII flow cytometer after label is examined
It surveys.
2, experimentation
Mature dendritic cell (CD44 is obtained according to the method for embodiment 1+/+DC) and the dendron shape of knockout CD44 gene is thin
Born of the same parents (CD44-/-DC) (after being resuspended with physiological saline, that is, conventional dendritic shape Cell vaccine and tumor vaccine of the present invention are obtained),
Two groups are classified as, following experiment is carried out.
The injection volume of tumor vaccine of the present invention is to have blocked the quantity of the Dendritic Cells of CD44 signaling pathway molecule
Meter, the injection volume of the conventional dendritic shape Cell vaccine is with the quantity of no Dendritic Cells for blocking CD44 signaling pathway molecule
It counts, injection volume is identical with this in following embodiment.
2.1H2Kb,Iab, CD40, CD80, CD86, the detection of CD44 molecule
By first group of mature dendritic cell (CD44+/+) and the Dendritic Cells of first group of knockout CD44 gene DC
(CD44-/-DC), anti-mouse H is marked respectively2Kb,Iab, CD40, CD80, the fluorescence antibody of CD86, CD44, using flow cytometer
Two groups of surface of dendritic cells fluorescence antibody label situations are detected, as a result as shown in Figure 3.
2.2 cell factor IL-2, IFN-γ, the detection of TNF-α and the detection of the quantity of cd8 t cell and ratio
By second group of mature dendritic cell (CD44+/+) and the Dendritic Cells of second group of knockout CD44 gene DC
(CD44-/-DC following tests) is carried out:
1st day, 1 (CD44 of experimental group+/+DC): to C57BL/6 mouse with 2 × 106It is thin that/amount only injects mature dendron shape
Born of the same parents (CD44+/+DC)；2 (CD44 of experimental group-/-DC): to C57BL/6 mouse with 2 × 106/ amount injection only knocks out CD44 gene
Dendritic Cells (CD44-/-DC).Control group (PBS): to the injection of C57BL/6 mouse and the isometric physiological saline of experimental group.
8th day, experimental group and control group mice peripheral blood serum are taken, (Biolegend is public by multiple-factor detection kit
Take charge of LEGENDplexTMMouse Th Cytokine Panel (13-plex) kit, article No. 740005) detection cell factor
IL-2, IFN-γ, TNF-α, as a result as shown in Figure 4 A.
Mouse peripheral blood mononuclearcell is acquired while taking above-mentioned experimental group and control group mice peripheral blood serum
(PBMC), and anti-CD8 antibody is marked, using the quantity and ratio of flow cytomery cd8 t cell, as a result such as Fig. 4 B- Fig. 4 C
It is shown.
3, experimental result
Fig. 3 shows mature dendritic cell (CD44+/+DC) and knock out CD44 gene Dendritic Cells (CD44-/-DC)
Phenotypic difference.From figure 3, it can be seen that Dendritic Cells (the CD44 of the knockout CD44 gene in mouse source-/-) and adult tree DC
Prominent shape cell (CD44+/+DC) high expression MHC-I class molecule (H2Kb), MHC-II class molecule (Iab), CD40, CD80 and CD86,
CD44 molecule is not expressed.The expression of activated dendritic cell mark CD40, CD80 and CD86 show knockout CD44 base of the invention
Dendritic Cells (the CD44 of cause-/-It DC is) Dendritic Cells of activation, the ability with dendritic cell ciita immune response.
Fig. 4 A shows mature dendritic cell (CD44+/+DC) and knock out CD44 gene Dendritic Cells (CD44-/-
DC the difference of cell factor) is generated after injection mouse.As can be seen that tumor vaccine (the CD44 of the invention-/-DC it) is immunized small
Mouse can activated receptor immune system, generate compared with conventional dendritic shape Cell vaccine (CD44+/+DC the proinflammatory that) conspicuousness increases is thin
Intracellular cytokine IL-2, IFN-γ and TNF-α.
Fig. 4 B shows conventional dendritic shape Cell vaccine (CD44+/+) and the tumor vaccine (CD44 of the invention DC-/-
DC the proportional difference of cd8 t cell) is generated after injection mouse.Fig. 4 C shows conventional dendritic shape Cell vaccine (CD44+/+DC) and
Tumor vaccine (the CD44 of the invention-/-DC the difference of the quantity of cd8 t cell) is generated after injection mouse.As can be seen that this
The tumor vaccine of invention is immunized mouse and can generate compared with the more responsiveness cd8 t cells of conventional dendritic shape Cell vaccine, energy
Mediate more effective anti-tumor effect.
To sum up, the tumor vaccine of the invention can generate more Cytokines and effector cell,
Illustrate it with stronger antitumous effect.
Embodiment 4
The application tumor vaccine has the effect of pre- preventing tumor
1, experimental material and reagent
(C57BL/6 loses by mouse melanin tumor cell system B16 (C57BL/6 genetic background), mouse lymphoma cell system EL4
Pass background) it is the U.S. source ATCC, experiment ties up the limited public affairs of tonneau China experimental animal technology purchased from Beijing with C57BL/6 mouse
Department, the age 6-8 weeks, weight 18g or so.
2, experimental procedure
(1) mature dendritic cell (CD44 is prepared according to the method that embodiment 1 describes+/+DC) and CD44 gene is knocked out
Dendritic Cells (CD44-/-DC), after being resuspended respectively using physiological saline, that is, conventional dendritic shape Cell vaccine and the application are obtained
Tumor vaccine.
1st day, 1 (CD44 of experimental group+/+DC): to C57BL/6 mouse with 2 × 106It is thin that/amount only injects mature dendron shape
Born of the same parents (CD44+/+DC)；2 (CD44 of experimental group-/-DC): to C57BL/6 mouse with 2 × 106/ amount injection only knocks out CD44 gene
Dendritic Cells (CD44-/-DC)；It is injected again with same dose within 8th day.Control group (PBS): exist respectively to C57BL/6 mouse
The physiological saline of injection and experimental group equivalent in 1st day and the 8th day.
(2) the 11st days, the mouse of the experimental group 1-2 after above-mentioned be immunized is respectively divided into two groups, one group of tail vein injection
B16-F10 melanoma cells 2 × 105；Another group of subcutaneous injection EL4 cell 2 × 106.By the small of the control group after above-mentioned be immunized
Mouse is divided into two groups, one group of tail vein injection B16-F10 melanoma cells 2 × 105；Another group of subcutaneous injection EL4 cell 2 ×
106。
(3) it is shown for injecting the experimental group 1-2 and control group mice of EL4 cell in the 32nd day measurement EL4 tumor size
Major diameter (mm) is shown as multiplied by the numerical value (mm of minor axis (mm)2), as a result as shown in Figure 5A；
(4) it for the experimental group 1-2 and control group mice of injection B16-F10 melanoma cells, is put to death when the 32nd day
Mouse, isolated lung tissue count the macroscopic melanoma Pulmonary metastasis focuses of each group, shown in result figure 5B.
3, experimental result
Fig. 5 A, which is shown, injects EL4 cell using the immune mouse of the application tumor vaccine and conventional dendritic shape Cell vaccine
The difference of EL4 tumor size afterwards, it can be seen that the size using the immune mouse EL4 tumour of the application tumor vaccine is significantly small
In the mouse being immunized using conventional dendritic shape Cell vaccine.
Fig. 5 B, which is shown, injects B16-F10 using the immune mouse of the application tumor vaccine and conventional dendritic shape Cell vaccine
The difference of melanoma Pulmonary metastasis focuses after melanoma cells, it can be seen that the mouse EL4 being immunized using the application tumor vaccine
The Pulmonary metastasis focuses of tumour are almost without the mouse Pulmonary metastasis focuses being immunized using conventional dendritic shape Cell vaccine are compared to injection physiology
The control group of salt water is not significantly different.
To sum up, the application tumor vaccine is capable of providing the antineoplastic immune protection for being significantly better than conventional dendritic shape Cell vaccine
Effect.
Embodiment 5
The application tumor vaccine has the effect for the treatment of tumour
1, experimental material and reagent
(C57BL/6 loses by mouse melanin tumor cell system B16 (C57BL/6 genetic background), mouse lymphoma cell system EL4
Pass background) it is the U.S. source ATCC, experiment ties up the limited public affairs of tonneau China experimental animal technology purchased from Beijing with C57BL/6 mouse
Department, the age 6-8 weeks, weight 18g or so.
2, experimental procedure
(1) mature dendritic cell (CD44 is prepared according to the method that embodiment 1 describes+/+DC) and CD44 gene is knocked out
Dendritic Cells (CD44-/-DC), after being resuspended respectively using physiological saline, that is, conventional dendritic shape Cell vaccine and the application are obtained
Tumor vaccine.
(2) the 0th days, mouse is divided into two groups, one group of tail vein injection B16-F10 melanoma cells 2 × 105；It is another
Group subcutaneous injection EL4 cell 2 × 106。
(3) the 1st days, lotus B16-F10 melanoma mouse is divided into experimental group and control group, 1 (CD44 of experimental group+/+DC):
To lotus B16-F10 melanoma mouse with 2 × 106/ only amount inject mature dendritic cell (CD44+/+DC)；Experimental group 2
(CD44-/-DC): to lotus B16-F10 melanoma mouse with 2 × 106/ only amount injection knock out CD44 gene Dendritic Cells
(CD44-/-DC)；It 8th day, is injected again with same dose.Control group (PBS): it injected and tested with the 8th day on day 1 respectively
The physiological saline of group equivalent.
1st day, lotus EL4 mice with tumor is divided into experimental group and control group, 1 (CD44 of experimental group+/+DC): to lotus EL4 tumour
Mouse is with 2 × 106/ only amount inject mature dendritic cell (CD44+/+DC)；2 (CD44 of experimental group-/-DC): to lotus EL4 tumour
Mouse is with 2 × 106/ only amount injection knock out CD44 gene Dendritic Cells (CD44-/-DC)；8th day, again with identical dose
Amount injection.Control group (PBS): distinguishing on day 1 and injects for the 8th day and the physiological saline of experimental group equivalent.
(3) at the 21st day, for lotus EL4 mice with tumor, EL4 tumor size is measured, is shown as major diameter (mm) multiplied by minor axis
(mm) numerical value (mm2), shown in result figure 6A；
(4) it for lotus B16-F10 melanoma mouse, was put to death at the 21st day, isolated lung tissue, counting each group naked eyes can
The melanoma metastasis stove seen, shown in result figure 6B.
3, experimental result
Fig. 6 A shows the lotus EL4 mice with tumor using the application tumor vaccine and the treatment of conventional dendritic shape Cell vaccine
The difference of EL4 tumor size, it can be seen that using the application tumor vaccine therapy mouse EL4 tumour size significantly less than
The mouse treated using conventional dendritic shape Cell vaccine, tumour growth are significantly suppressed.
Fig. 6 B shows the lotus B16-F10 melanin using the application tumor vaccine and the treatment of conventional dendritic shape Cell vaccine
The difference of tumor murine melanoma Pulmonary metastasis focuses, it can be seen that use the melanoma of the mouse of the application tumor vaccine therapy
Pulmonary metastasis focuses are substantially reduced compared to the mouse for using conventional dendritic shape Cell vaccine to treat.
To sum up, the application tumor vaccine is capable of providing the treatment tumor effect for being significantly better than conventional dendritic shape Cell vaccine.
Embodiment 6
The application tumor vaccine to melanoma, lymthoma outside other tumor types it is equally effective
1. experimental material and reagent
Mouse colonic cell system MC38 (C57BL/6 genetic background), mice lung cancer cell line Lewis (C57BL/6 heredity
Background), Mouse hepatoma H22 (C57BL/6 genetic background), Mouse Gastric Cancer cell line MFC (C57BL/6 genetic background),
Mouse mammary carcinoma cell line 4T1 (Balb/c genetic background), mouse renal carcinoma cell line Renca (Balb/c genetic background), mouse
It is pancreatic carcinoma Pan02 (C57BL/6 genetic background), mouse prostate cancer cell system RM-1 (C57BL/6 genetic background), small
Mouse esophageal carcinoma cell line AKR (C57BL/6 genetic background), mouse cervix cancerous cell line U14 (C57BL/6 genetic background), mouse
Bladder cancer cell lines MB49 (C57BL/6 genetic background), mouse ovarian cancerous cell line ID8 (C57BL/6 genetic background) are beauty
The source state ATCC, experiment are purchased from Beijing dimension limited public affairs of tonneau China experimental animal technology with C57BL/6 mouse and Balb/c mouse
Department, the age 6-8 weeks, weight 18g or so.
2. experimental procedure
(1) method described according to embodiment 1, the mature dendron shape for preparing C57BL/6 mouse, Balb/c mouse and people are thin
Born of the same parents (CD44+/+DC cell) and knock out CD44 gene Dendritic Cells (CD44-/-DC cell), it is resuspended respectively using physiological saline
Afterwards, that is, conventional dendritic shape Cell vaccine and the application tumor vaccine are obtained；
(2) the 0th days:
15 receptor C57BL/6 mouse subcutaneous injection MC38 colon carcinoma cell lines 5 × 105；
15 receptor C57BL/6 mouse subcutaneous injection Lewis lung cancer cell lines 2 × 105；
15 receptor C57BL/6 mouse subcutaneous injection H22 liver cancer cell lines 1 × 106；
15 receptor C57BL/6 mouse subcutaneous injection MFC gastric carcinoma cell lines 1 × 106；
15 receptor Balb/c mouse subcutaneous injection 4T1 breast cancer cell lines 5 × 105；
15 receptor Balb/c mouse subcutaneous injection Renca renal carcinoma cell lines 1 × 106；
15 receptor C57BL/6 mouse subcutaneous injection Pan02 pancreatic carcinomas 1 × 106；
15 receptor C57BL/6 mouse subcutaneous injection RM-1 prostate cancer cell lines 2 × 105；
15 receptor C57BL/6 mouse subcutaneous injection AKR esophageal carcinoma cell lines 1 × 106；
15 receptor C57BL/6 mouse subcutaneous injection U14 cervical cancer tumer lines 1 × 106；
15 receptor C57BL/6 mouse subcutaneous injection MB49 bladder cancer cell lines 5 × 105；
15 receptor C57BL/6 mouse subcutaneous injection ID8 ovarian cancer cell lines 2 × 105；
(3) illustrate that experimentation, the experimentation of other tumor-bearing mices are same by taking lotus MC38 colon cancer mouse as an example.
1st day: lotus MC38 colon cancer mouse is divided into experimental group and control group, 1 (CD44 of experimental group+/+DC): to lotus MC38
Colon cancer mouse is with 2 × 106/ only amount inject mature dendritic cell (CD44+/+DC)；2 (CD44 of experimental group-/-DC): to lotus
MC38 colon cancer mouse is with 2 × 106/ only amount injection knock out CD44 gene Dendritic Cells (CD44-/-DC).8th day, then
It is secondary to be injected with same dose.Control group (PBS): distinguishing on day 1 and injects for the 8th day and the physiological saline of experimental group equivalent.
21st day, tumor size is measured, is shown as major diameter (mm) multiplied by the numerical value (mm of minor axis (mm)2)。
3, experimental result
Fig. 7 A- Fig. 7 L shows each tumor-bearing mice respectively through conventional dendritic cell-based tumor vaccines and the application tumor vaccine therapy
The difference of tumor size afterwards.Fig. 7 A shows lotus MC38 colon cancer cell mouse respectively through conventional dendritic cell-based tumor vaccines and this Shen
Please after tumor vaccine therapy tumor size difference.Fig. 7 B shows lotus Lewis lung carcinoma cell mouse respectively through conventional dendritic shape
The difference of tumor size after Cell vaccine and the application tumor vaccine therapy.Fig. 7 C shows that lotus H22 liver cancer cells mouse is distinguished
The difference of tumor size after conventional dendritic cell-based tumor vaccines and the application tumor vaccine therapy.Fig. 7 D shows lotus MFC gastric cancer
Cell mouse respectively after conventional dendritic cell-based tumor vaccines and the application tumor vaccine therapy tumor size difference.Fig. 7 E is shown
Lotus 4T1 breast cancer cell mouse after conventional dendritic cell-based tumor vaccines and the application tumor vaccine therapy tumor size respectively
Difference.Fig. 7 F shows that lotus Renca kidney cancer cell mouse is controlled through conventional dendritic cell-based tumor vaccines and the application tumor vaccine respectively
The difference of tumor size after treatment.Fig. 7 G show lotus Pan02 pancreatic cancer cell mouse respectively through conventional dendritic cell-based tumor vaccines and
The difference of tumor size after the application tumor vaccine therapy.Fig. 7 H shows lotus RM-1 prostate gland cancer cell mouse respectively through routine
The difference of tumor size after dendritic cell-based tumor vaccines and the application tumor vaccine therapy.Fig. 7 I shows lotus AKR esophageal cancer cell
Mouse respectively after conventional dendritic cell-based tumor vaccines and the application tumor vaccine therapy tumor size difference.Fig. 7 J shows lotus
U14 cervical cancer cell mouse respectively after conventional dendritic cell-based tumor vaccines and the application tumor vaccine therapy tumor size difference
It is different.Fig. 7 K shows lotus MB49 bladder cancer cell mouse respectively through conventional dendritic cell-based tumor vaccines and the application tumor vaccine therapy
The difference of tumor size afterwards.Fig. 7 L shows lotus ID8 ovarian cancer cell mouse respectively through conventional dendritic cell-based tumor vaccines and this Shen
Please after tumor vaccine therapy tumor size difference.
As can be seen that applying the application tumor vaccine, various Mice bearing cancers compared to application conventional dendritic shape Cell vaccine
Subcutaneous Tumor Growth be suppressed significantly, tumor size is substantially reduced, illustrate the tumor vaccine of the invention have wide spectrum
The effect for treating tumour.
Embodiment 7
The safety evaluatio of the application tumor vaccine
1, experimental material and reagent
Experiment with C57BL/6 mouse be purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., the age 6-8 weeks, weight
18g or so.
2, experimental procedure
(1) Dendritic Cells (CD44 of CD44 gene is knocked out according to the method preparation of step 1-/-DC cell), use physiology
After salt water is resuspended, that is, obtain the application tumor vaccine；
(2) C57BL/6 mouse is divided into experimental group and control group, experimental group 1 injects the application tumour epidemic disease of routine dose
Seedling (2 × 106/ only)；Experimental group 2 injects the application tumor vaccine (4 × 10 of 20 multiple dose of routine dose7/ only)；Control group:
The PBS of injection and experimental group equivalent.
(3) after injecting in 30 days, whether continuous observation mouse is dead, whether hair color is smooth, whether weight declines and live
Whether kinetic force is abnormal, as a result as shown in Figure 8.
3, test result
Fig. 8 shows the safety evaluatio of the application tumor vaccine results, it can be seen that even if according to 20 times of routine dose
Injection, C57BL/6 mouse do not occur death, and the indices such as hair color, weight, mobility are normal, show the application tumour
Vaccine has good safety in utilization.
Embodiment 8
The single-dose oncogenicity of the application tumor vaccine is evaluated
1, experimental material and reagent
Experiment with nude mice be purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., the age 6-8 weeks, weight 18g or so.
2, experimental procedure
(1) Dendritic Cells (CD44 of CD44 gene is knocked out according to the method preparation of step 1-/-DC cell), use physiology
After salt water is resuspended, that is, obtain the application tumor vaccine；
(2) nude mice is divided into experimental group and control group, experimental group 1, inject routine dose the application tumor vaccine (2 ×
106/ only)；Experimental group 2 injects the application tumor vaccine (4 × 10 of 20 multiple dose of routine dose7/ only)；Control group: injection with
The PBS of experimental group equivalent.
(3) after injecting in 90 days, whether continuous observation mouse there is tumour, if death occurs, as a result as shown in Figure 9.
3, experimental result
Fig. 9 shows the single-dose oncogenicity evaluation result of the application tumor vaccine, it can be seen that each group nude mice is not
There is tumour or death, shows the application tumor vaccine within the observation period without single-dose oncogenicity.
Embodiment 9
The multiple dosing oncogenicity of the application tumor vaccine is evaluated
1, experimental material and reagent
Experiment with nude mice be purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., the age 6-8 weeks, weight 18g or so.
2, experimental procedure
(1) Dendritic Cells (CD44 of CD44 gene is knocked out according to the method preparation of step 1-/-DC cell), use physiology
After salt water is resuspended, that is, obtain the application tumor vaccine；
(2) nude mice is divided into experimental group and control group, experimental group 1, inject routine dose the application tumor vaccine (2 ×
106/ only)；Experimental group 2 injects the application tumor vaccine (4 × 10 of 20 multiple dose of routine dose7/ only)；Control group: injection with
The PBS of experimental group equivalent.
(3) it to experimental group and control group mice, 1 times a week, successive administration 4 weeks, is administered 5 times, convalescence 4 weeks altogether.Injection
Afterwards in 90 days, whether continuous observation mouse there is tumour, if death occurs, the results are shown in Figure 10.
3, experimental result
Figure 10 shows the multiple dosing oncogenicity evaluation result of the application tumor vaccine, it can be seen that each group nude mice is equal
Do not occur tumour or death, show the application tumor vaccine observation the period in multiple dosing without oncogenicity.
Embodiment 10
The local stimulation of the application tumor vaccine is tested
1, experimental material and reagent
It tests and uses rabbit, the age 6-8 weeks, weight 1000-2000g or so；Physiological saline, water for injection,
2, experimental procedure
Dendritic Cells (the CD44 for knocking out CD44 gene is prepared according to the method for embodiment 1-/-DC cell), use physiology
Salt water is resuspended to 2 × 106After/mL, the application tumor vaccine is obtained.
Rabbit quadriceps muscle of thigh method: a certain amount of test sample is injected in rabbit quadriceps muscle of thigh, at the appointed time, portion opens flesh
The case where meat observation test sample irritates reaction to local muscle, to judge whether the local stimulation test of test sample meets regulation.
Test method: take health without wound, weight 2.0kg or more rabbit (male and female are uniform) 2, respectively in rear left and right
Test sample (the application tumor vaccine or PBS physiological saline) 1ml is respectively injected in two leg musculus quadriceps, puts to death animal after 48 hours,
Quadriceps muscle of thigh is taken out, is longitudinally splitted, observation part irritates reaction.And according to the form below record irritates intensity accordingly, then calculates 4 pieces
The summation of quadriceps muscle of thigh order of reaction.When being greater than 2 such as the highest of each stock four-head reaction and the difference of lowermost level, it should separately take 2 rabbit multiple
Examination, method are same as above.
It irritates intensity and reacts the table of comparisons with irritating
Irritate the case where intensity irritates reaction
0 grade without significant change
1 grade of mild hyperaemia: range is in 0.5 × 1.0cm or less
2 grades of moderate hyperemia: range is in 0.5 × 1.0cm or more
3 grades of severe hyperemia: change colour with muscle
4 grades of appearance necrosis: colour fading is denaturalized
5 grades there is popularity necrosis
As a result judge
Sample is thought below 10 in the sum of the order of reaction that 2 rabbit, 4 pieces of quadricepss muscle of thigh of preliminary examination or retrial irritate intensity
Local stimulation test meet regulation.
3, experimental result
Figure 11 show the local stimulation experimental evaluation of the application tumor vaccine results, it can be seen that each experimental group not
There is significant change, local stimulation is feminine gender.
Although specific embodiments of the present invention have had been illustrated and described, those skilled in the art are come
It says it is readily apparent that various other changes and modification can be made in the case where not departing from spirit and scope of the present invention.Cause
This, includes all these changes and the modification belonged in the scope of the invention in appended claims.
Claims (10)
1. a kind of tumor vaccine, which is characterized in that the Dendritic Cells comprising having blocked CD44 signaling pathway molecule.
2. tumor vaccine according to claim 1, which is characterized in that blocked CD44 molecular signal in the tumor vaccine
The ratio of the total Dendritic Cells of Dendritic Cells Zhan of access is 30% or more, preferably 40% or more, more preferable 50% or more, most
It is preferred that 60% or more.
3. tumor vaccine according to claim 2, which is characterized in that blocked CD44 molecular signal in the tumor vaccine
The ratio of the total Dendritic Cells of Dendritic Cells Zhan of access is 100%.
4. tumor vaccine according to any one of claim 1-3, which is characterized in that wherein the Dendritic Cells is originated from
Mammal.
5. tumor vaccine of any of claims 1-4 is preparing answering in the drug for preventing or treating tumour
With.
6. application according to claim 5, which is characterized in that the tumour includes melanoma, lymthoma, Colon and rectum
Cancer, lung cancer, liver cancer, gastric cancer, breast cancer, cancer of pancreas, prostate cancer, the cancer of the esophagus, cervical carcinoma, bladder cancer, oophoroma, endometrium
One of cancer and kidney are a variety of.
7. a kind of prevention or treatment tumour composition, which is characterized in that include tumour of any of claims 1-4
Vaccine is as effective component.
8. prevention according to claim 7 or treatment tumour composition, which is characterized in that the tumour includes melanin
Tumor, lymthoma, colorectal cancer, lung cancer, liver cancer, gastric cancer, breast cancer, cancer of pancreas, prostate cancer, the cancer of the esophagus, cervical carcinoma, bladder
One of cancer, oophoroma, carcinoma of endometrium and kidney are a variety of.
9. prevention according to claim 7 or treatment tumour composition, which is characterized in that the prevention or treatment tumour
It is ejection preparation with composition.
10. a kind of method for preparing tumor vaccine described in any one of claim 1-4, which comprises the following steps:
1) mononuclearcell is obtained by mammalian peripheral blood or Cord blood, and being induced is mature dendritic cell, or
Marrow hemopoiesis precursor is obtained by the marrow of mammal, and is induced as mature dendritic cell；
2) the CD44 signaling pathway molecule of the mature dendritic cell is blocked；
3) Dendritic Cells for having blocked CD44 signaling pathway molecule is collected.
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