CN101675994B - Therapy vaccine preparation - Google Patents
Therapy vaccine preparation Download PDFInfo
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- CN101675994B CN101675994B CN2008101398580A CN200810139858A CN101675994B CN 101675994 B CN101675994 B CN 101675994B CN 2008101398580 A CN2008101398580 A CN 2008101398580A CN 200810139858 A CN200810139858 A CN 200810139858A CN 101675994 B CN101675994 B CN 101675994B
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Abstract
The invention relates to a therapy vaccine preparation. The preparation comprises immunogen with immunological activity, oligo-poly deoxynucleotide as main adjuvant comprising at least one non-methylated CpG dinucleotide, one or various kinds of oil-phase medium and buffer as water-phase medium. The adjuvant for reinforcing vaccine immunity effect is artificial oligo-poly deoxynucleotide (for short CpG-ODN) containing CpG dinucleotide. The adjuvant activates the immune system by the receptor TLR9 on immunological cells, thereby reinforcing specific immune response direct towards vaccine immunogen. The preparation is a water-in-oil type emulsion. The animal experimental result shows that: the therapy vaccine preparation continuously stimulates the immune system to generate high-titer antibody with stronger inhibition capacity to the gastrointestinal tract tumor.
Description
Technical field
The present invention relates to a kind of therapy vaccine preparation, the bacterin preparation that is specifically related to strengthen the vaccine adjuvant of vaccine immunogenicity and is used for oncotherapy belongs to molecular biology and field of immunology.
Background technology
Malignant tumor is one type of serious threat people's life and healthy disease, and the prevention of tumor and treatment have become the vital task of global medical and health work, human to tumor treatment research also always in unremitting effort.Tumor vaccine becomes the tumor therapeuticing method after operation, radiation and chemotherapy; Tumor vaccine is treated as active specific immunotherapy; In clinical, play more and more important effect; People begin to attempt utilizing several different methods to prepare tumor vaccine and utilize it to promote immune response, and from whole cell, molecular level regulation and control body is to the immunity of tumor.
The essence of vaccine is that a certain immunogen component is acted on organism; Thereby excitating organism is to the immunoprotection of this immunogen or immunogenic carrier; Tumor vaccine just according to this immunity principle with the processing of self tumor cell through physical factor; Change or eliminate its oncogenicity, keep its immunogenicity.And normal and adjuvant Combined application strengthen its immunogenicity.This is to be the vaccine on basis with the tumor cell, also is first kind tumor vaccine.
Second type of tumor vaccine is to be the vaccine on basis with the tumor associated antigen, comprises peptide and protein vaccine, recombinant viral vaccine, dna vaccination etc.
The 3rd type of tumor vaccine is to be the vaccine on basis with the BMDC, and it has utilized the strong in vivo antigen presentation ability of BMDC, catches immunogen, and information is passed to T, bone-marrow-derived lymphocyte, thereby cause a series of specific immune response reaction.
In order to strengthen the immunogenicity of tumor vaccine,, thereby cause the effective antitumour immunne response usually with the adjuvant Combined application.Adjuvant mainly can be divided into two kinds: a kind of itself have immunogenicity, like bordetella pertussis, acid-fast bacilli (mycobacterium tuberculosis) and gram negative bacilli etc.; Another kind of non-immunogenicity own is like aluminium hydroxide, calcium phosphate, mineral oil emulsion, surfactant etc.The Freund adjuvant that we know is at present the most frequently used.
In 19th century, the surgeon William Coley in New York notices that the extract of antibacterial has therapeutical effect to tumor, it is found that afterwards effective ingredient wherein is the DNA that contains unmethylated CpG dinucleotide.CpG ODN is the oligodeoxynucleotide of unmethylated CpG motif of containing of synthetic; It can simulate the immune system that prokaryote DNA stimulates body; Activate the panimmunity cell; Produce the various kinds of cell factor, have and induce the ability that activates anti-tumor immune response, application of treatment tumor separately.Simultaneously, CpG ODN also is a kind of effective Th1 type immunological adjuvant, can promote the protide immunogen to bring out specific CTL and reply, and is stronger than Freund's complete adjuvant, therefore, becomes the focus of immunotherapy of tumors research field.
Emulsion is meant two kinds of immiscible liquid mixing, and wherein a phase liquid is scattered in the heterogeneous liquid dispersion system that forms in another phase liquid with the drop state.Emulsion is made up of water, oil phase and emulsifying agent, forms oil-in-water or water-in-oil type according to emulsifier type character and phase volume ratio.Big or small Emulsion according to emulsion droplet is divided into common breast, submicron emulsion, nano-emulsion.The dispersion of drop is very big in the Emulsion, and the performance of drug absorption and drug effect is very fast, and bioavailability is high.The oiliness medicine is processed Emulsion can guarantee that dosage is accurate, and easy to use; Oil-in-water emulsion can be covered the bad smell of medicine, and can add correctives; Exterior-applied emulsion can improve the permeability to skin, mucosa, reduces zest; Intravenous injection emulsion injection back distributes comparatively fast, drug effect is high, targeting property is arranged; The important component part of energetic nourishing transfusion during intravenous nutrition Emulsion.At present; There has been multiple vaccine carrying out clinical trial with the Emulsion form; Being with ISA720 like the HIV vaccine TAB9 of people such as He Liou preparation is the emulsion vaccine of oil phase and adjuvant; Carrying out clinical research (He Liou Francesco Toldo etc. at present; Multi-epitope polypeptide associating MontanideISA720 infects the I clinical trial phase among the volunteer as adjuvant at non-HIV-1. vaccine; 19 phases of calendar year 2001,4328-4336 page or leaf .Herio Toledo et al.A phase I clinical trial of a multi-epitope polypeptideTAB9combined with Montanide ISA720adjuvant in non-HIV-1 infected humanvolunteers.Vaccine19 (2001) 4328-4336).The influenza subunit vaccine (Europe listing in 1997) that comprises the water in oil emulsion Hepatitis B virus vaccine Fendrix (2005, the Europe listing) and the Chiron company oil in water emulsion of GlaxoSmithKline PLC company with the vaccine of Emulsion form listing.
U.S. Pat 5023077, US5468494, US5607676, US5609870, US5622702, WO2004004687 disclose through producing anti-gastrin antibody; Be used to control the immunogen and the immunogenic composition of patient G17 and G34 level; These compositionss promptly are the peptide vaccines that originally is the basis with the tumor related immune, and they also are to study with the form of Emulsion.Therapeutic vaccine of the present invention relates to disclosed anti-G17 peptide vaccine among U.S. Pat 5023077, US5468494, US5607676, US5609870, US5622702, the WO2004004687, and therapeutic vaccine of the present invention also relates among the Chinese patent CN101050236 disclosed carrier protein among disclosed anti-G17 peptide vaccine and the Chinese patent CN100999548.
Summary of the invention
The object of the present invention is to provide a kind of bacterin preparation that is used for oncotherapy.
Bacterin preparation of the present invention comprises a kind ofly itself having immunogenic immunogenic substances, have the active vaccine for man adjuvant of immunostimulant, be suitable for the oil-phase medium of water in oil emulsion and as the buffer of water.Described have a cross-linking agent that immunogenic immunogenic substances is polypeptide immunogen or polypeptide immunogen and high molecular weight protein.
The cross-linking agent of aforementioned polypeptides immunogen or polypeptide immunogen and high molecular weight protein can adopt prior art, for example disclosed immunogen, immunogen and proteic cross-linking agent among U.S. Pat 5023077, US5468494, US5607676, US5609870, US5622702, WO2004004687, the Chinese patent CN101050236.
It is above-mentioned that to have the active vaccine for man adjuvant of immunostimulant be the oligodeoxynucleotide that contains at least one non-methylated CpG dinucleotide; This oligodeoxynucleotide length is 15-35 nucleotide, and preferred length is a 20-30 nucleotide, and more preferably length is 20-25 nucleotide.
Preferably, the nucleotides sequence of said oligodeoxynucleotide is classified as one of following:
5’-TCGTACGTACGTACGTACGTACGTG-3’
5’-TCGTACGTACGTACGTACGTACGTACGTG-3’
5’-TCGTATCGTATCGTATCGTG-3’
5’-TCGTATCGTATCGTATCGTATCGTG-3’
5’-TCGTTCGTTCGTTCGTTCGTT-3’
5’-TCGTATCGTATCGTATCGTATCGTATCGTG-3’
5’-TCGATCGATCGATCGATCGATCGTA-3’
5’-TCGATCGATCGATCGATCGATCGATCGTA-3’
5’-TCGATTCGATTCGATTCGTA-3’
5’-TCGATTCGATTCGATTCGATTCGTA-3’
5’-TCGATTCGATTCGATTCGATTCGATTCGTA-3’
More preferably, the nucleotides sequence of said oligodeoxynucleotide is classified as one of following:
5’-TCGTATCGTATCGTATCGTG-3’
5’-TCGTTCGTTCGTTCGTTCGTT-3’
5’-TCGATTCGATTCGATTCGTA-3’
Wherein preferred, said oligodeoxynucleotide is a thio-modification.
The method for preparing of above-mentioned sulpho-oligodeoxynucleotidewith is well-known to those skilled in the art, for example can adopt the chemosynthesis of solid phase phosphoramidite triester method.
Therapy vaccine preparation involved in the present invention also comprises other adjuvant, like ISA720 (French SEPPIC company), MF59 (Novartis Co.,Ltd), aluminium hydroxide, aluminum phosphate, unformed aluminum or other any adjuvant that is applicable to vaccine for man.
Among the present invention related immunogenic substances specifically be a kind of have stimulate the active polypeptide immunogen of tumor growth or this polypeptide immunogen through the spacer peptide formed by 7 aminoacid cross-linking products, this cross-linking products such as G17CRM197, G17DT, G17BSA, G17P64K etc. by means of bi-functional cross-linking agent and carrier protein.Wherein, the aminoacid sequence of described polypeptide immunogen is following:
pGlu-Gly-Pro-Trp-Leu-Glu-G1u-Glu-Glu
Wherein, said spacer peptide aminoacid sequence is following:
Ser-Ser-Pro-Pro-Pro-Pro-Cys
Said bi-functional cross-linking agent is ε-dimaleoyl imino caproic acid N-hydroxy-succinamide ester (EMCS);
Said carrier protein comprises diphtheria toxoid (DT), tetanus toxin (TT), bovine serum albumin (BSA), human serum albumin (HAS), diphtheria toxin muton CRM 197 and meningococcus P64K albumen; Wherein preferred diphtheria toxoid and CRM197.
Preferred G17CRM197 of cross-linking agent of the present invention and G17DT adopt the preparation of method described in one Chinese patent application CN101050236 (200610043442.X) and the U.S. Patent application US5468494.
The oil-phase medium that is suitable for water in oil emulsion of the present invention is selected from injection soybean oil, Squalene, ISA720 (French SEPPIC company), ISA51 (French SEPPIC company), wherein preferred ISA720 (French SEPPIC company).
Preferably, therapy vaccine preparation of the present invention is a kind of water in oil emulsion, and every vaccinating agent (250ul is a potion) contains and has immunocompetent immunogen 10ug-1mg, preferred 50ug-0.5mg; Contain oligodeoxynucleotide adjuvant 10ug-10mg, preferred 25ug-5mg; Containing oil-phase medium ISA720 is 0.05-1ml, preferred 0.1-0.5ml.
Wherein, also contain buffer as aqueous media in the above-mentioned preferred water in oil emulsion, these buffer comprise physiologically acceptable aqueous medias such as phosphate buffer, Tris salt buffer, acetate buffer.
Preparation according to the invention prepares through following key step:
(1) a certain amount of immunogen and immunological adjuvant are added to the immunogen assist agent solution of processing suitable concentration in the buffer;
(2) oil-phase medium that takes by weighing recipe quantity is put in the appropriate vessel as oil phase;
(3) the immunogen assist agent solution of getting recipe quantity slowly adds in the oil phase, to carry out homogenizing than the slow-speed of revolution, again with the higher rotation speed homogenizing, processes homogeneous, stable water in oil emulsion;
(4) with particle size analyzer determination Emulsion particle diameter.
Adopt the method for animal immune test to measure the situation that prepared Emulsion stimulates animal generation antibody, carry out immunity, adopt enzyme linked immunosorbent assay (ELISA) to detect the antibody titer that produces according to the method for routine.
Tumor therapeutic vaccine preparation of the present invention is as the application of the medicine of anti-alimentary tract tumor.
Tumor therapeutic vaccine preparation of the present invention is suitable for intramuscular injection or subcutaneous injection.
With prepared Emulsion immune mouse, regularly gather antiserum.The serum of being gathered is carried out purification obtain antibody, and resulting antibody is carried out external pharmacodynamics evaluation.The tumor cell that experiment in vitro adopted is stomach cancer cell MKN45, BGC823 and colorectal cancer cells LOVO, SW480.Experiment is carried out with tumor-bearing rat in the body, and an amount of Emulsion is injected in the rat body, estimates the effect of therapy vaccine preparation of the present invention to tumor through the size that detects tumor.Through pressing down tumor experiment proof therapy vaccine preparation of the present invention in tumor cell in vitro cultivation and the rat body gastric cancer and colorectal cancer there is the obvious treatment effect.Particular content is with further explain in an embodiment.
The Emulsion of processing as the effective ingredient of therapeutic vaccine with the compositions of immunogen of the present invention and adjuvant can directly be used for the human injection.This immunogen can produce corresponding anti-gastrin antibody by stimulating immune system in human body, this antibody can in the Infusion in Patients with Digestive body in too much gastrin, thereby reach the treatment tumor purpose.
Be used to strengthen the oligodeoxynucleotide (be called for short CpG-ODN) of the adjuvant of immune effect of vaccine among the present invention for the artificial synthetic CpG of containing dinucleotide; This adjuvant is through the receptor TLR9 activating immune system on the immunocyte, thereby enhancing is to the specific immune response of vaccine immunogens.Immunogen in this preparation is compared with the preparation that does not add adjuvant or add other adjuvants under the situation that described adjuvant CpG-ODN exists, and can stimulate the higher titer antibody of body generation, and is more obvious to the inhibition effect of transplanted tumor model in the body.In addition, because the adding of oil-phase medium in this preparation makes the water-in-oil emulsion for preparing with method provided by the present invention to stimulate body to produce high titer antibody in vivo continuously.
Compared with prior art, use bacterin preparation of the present invention and come immune animal, the antibody titer that is produced does not add the antibody titer of the vaccine of adjuvant far above independent application, and the immunogen promptly involved in the present invention and the compositions of adjuvant have higher immunogenicity.Prove that through zoopery the immunogen involved in the present invention and the compositions of adjuvant have more significantly tumor suppression effect than independent use vaccine.
Description of drawings
Fig. 1 is an animal immune experimental result of utilizing the prepared therapy vaccine preparation of the present invention, shows with CpG to be the adjuvant former immunogenicity of enhance immunity greatly.The A group: the blank group, only inject phosphate buffer; B group: CpG adjuvant group (G17CRM197 is an immunogen, and CpG-ODN is an adjuvant, and ISA720 is an oil phase, and phosphate buffer is a water); C group: aluminium adjuvant group (G17CRM197 is an immunogen, and aluminium hydroxide is an adjuvant, and ISA720 is an oil phase, and phosphate buffer is a water); The D group: no adjuvant group G17CRM197 is an immunogen, and ISA720 is an oil phase, and phosphate buffer is a water).
Fig. 2 is that different CpG-ODN contrast the immunogenic immunostimulant ability of G17CRM197, shows that different CpG-ODN can strengthen the immunne response of rabbit to the G17CRM197 vaccine.
The specific embodiment
Further specify the present invention below in conjunction with embodiment, but be not limited to practical range.Those skilled in the art can make various modifications or improvement according to basic thought of the present invention, but only otherwise break away from basic thought of the present invention, all within scope of the present invention.
Embodiment 1: the preparation of therapy vaccine preparation of the present invention
1.1 precision takes by weighing 2mg immunogen G17CRM197 and is dissolved in the saline of 3.3ml phosphate-buffered with 1mg immunological adjuvant CpG, processes the immunogen assist agent solution.Precision is measured 6.7ml oil-phase medium ISA720 and is put in the appropriate vessel as oil phase.Under the 5000rpm rotating speed, the immunogen assist agent solution is slowly added in the oil phase, homogenizing 2 minutes again with 10000rpm rotating speed emulsifying 3 minutes, is processed outward appearance homogeneous, stable water in oil emulsion.
1.2 precision takes by weighing 10mg immunogen G17DT and is dissolved in the saline of 3.3ml phosphate-buffered with 15mg immunological adjuvant CpG, processes the immunogen assist agent solution.Precision is measured 6.7ml oil-phase medium ISA720 and is put in the appropriate vessel as oil phase.Under the 5000rpm rotating speed, the immunogen assist agent solution is slowly added in the oil phase, homogenizing 2 minutes again with 10000rpm rotating speed emulsifying 3 minutes, is processed outward appearance homogeneous, stable water in oil emulsion.
1.3 precision takes by weighing 40mg immunogen G17BSA and is dissolved in the saline of 10ml phosphate-buffered with 100mg immunological adjuvant CpG, processes the immunogen assist agent solution.Precision is measured 20ml oil-phase medium ISA720 and is put in the appropriate vessel as oil phase.Under the 5000rpm rotating speed, the immunogen assist agent solution is slowly added in the oil phase, homogenizing 2 minutes again with 10000rpm rotating speed emulsifying 3 minutes, is processed outward appearance homogeneous, stable water in oil emulsion.
1.4 precision takes by weighing 2.5mg immunogen G17CRM197 and is dissolved in the saline of 3.3ml phosphate-buffered with 0.5mg immunological adjuvant MF59, processes the immunogen assist agent solution.Precision is measured 6.7ml oil-phase medium ISA720 and is put in the appropriate vessel as oil phase.Under the 5000rpm rotating speed, the immunogen assist agent solution is slowly added in the oil phase, homogenizing 2 minutes again with 10000rpm rotating speed emulsifying 3 minutes, is processed outward appearance homogeneous, stable water in oil emulsion.
1.5 precision takes by weighing 4mg immunogen G17CRM197 and is dissolved in the saline of 3.3ml phosphate-buffered with 10mg immunological adjuvant aluminium hydroxide, processes the immunogen assist agent solution.Precision is measured 6.7ml oil-phase medium ISA720 and is put in the appropriate vessel as oil phase.Under the 5000rpm rotating speed, the immunogen assist agent solution is slowly added in the oil phase, homogenizing 2 minutes again with 10000rpm rotating speed emulsifying 3 minutes, is processed outward appearance homogeneous, stable water in oil emulsion.
1.6 precision takes by weighing 5mg immunogen G17CRM197 and is dissolved in the saline of 3ml phosphate-buffered, processes the immunogen assist agent solution.Precision is measured 7ml oil-phase medium ISA720 and is put in the appropriate vessel as oil phase.Under the 5000rpm rotating speed, the immunogen assist agent solution is slowly added in the oil phase, homogenizing 2 minutes again with 10000rpm rotating speed emulsifying 3 minutes, is processed outward appearance homogeneous, stable water in oil emulsion.
1.7 precision takes by weighing 2.5mg immunogen G17CRM197 and is dissolved in the saline of 3ml phosphate-buffered with 0.5mg immunological adjuvant CpG, processes the immunogen assist agent solution.Precision is measured 7ml oil-phase medium ISA51 and is put in the appropriate vessel as oil phase.Under the 5000rpm rotating speed, the immunogen assist agent solution is slowly added in the oil phase, homogenizing 2 minutes again with 10000rpm rotating speed emulsifying 3 minutes, is processed outward appearance homogeneous, stable water in oil emulsion.
1.8 precision takes by weighing 2.5mg immunogen G17CRM197 and is dissolved in the saline of 1ml phosphate-buffered with 0.5mg immunological adjuvant CpG, processes the immunogen assist agent solution.Precision is measured 9ml injection soybean oil and is put in the appropriate vessel as oil phase.Under the 5000rpm rotating speed, the immunogen assist agent solution is slowly added in the oil phase, homogenizing 2 minutes again with 10000rpm rotating speed emulsifying 3 minutes, is processed outward appearance homogeneous, stable water in oil emulsion.
Embodiment 2: the rabbit immunization experiment of therapy vaccine preparation of the present invention
The immunogen of using is G17CRM197 in the present embodiment, also can select other immunogen such as G17DT, G17BSA, G17P64K, G17TT.Method according to described in the patent CN101050236 prepares G17CRM197, prepares bacterin preparation according to the method for the embodiment of the invention 1.4.Wherein, employed CpG-ODN sequence is following in the present embodiment: 5 '-TCGTTCGTTCGTTCGTTCGTT-3 ', carry out synthetic by the method that those skilled in the art are familiar with, and carry out full chain thio-modification.
The immune animal that present embodiment uses is new zealand white rabbit.Rabbit is divided into 4 groups, 5 every group.4 groups are respectively A: the blank group, only inject phosphate buffer; B:CpG adjuvant group (G17CRM197 is an immunogen, and CpG-ODN is an adjuvant, and ISA720 is an oil phase, and phosphate buffer is a water); C: aluminium adjuvant group (G17CRM197 is an immunogen, and aluminium hydroxide is an adjuvant, and ISA720 is an oil phase, and phosphate buffer is a water); D: no adjuvant group G17CRM197 is an immunogen, and ISA720 is an oil phase, and phosphate buffer is a water).
The animal immune experiment is carried out according to following process:
(1) with the preparation bacterin preparation respectively at 0,21,42 day immune new zealand white rabbit.Immunization method adopts the intramuscular injection of rabbit back leg, injects right lower limb for the first time, injects left lower limb for the second time, injects right lower limb for the third time, and injection point is a little more than the injection point first time.Immunizing dose is 1mg for the first time, and the immunizing dose that the back is twice is 0.5mg.
(2) gather Sanguis Leporis seu oryctolagi respectively at the 14th, 35,56 day and carry out the antibody titer detection.Wherein preceding twice is the rabbit ear edge vein exploitating blood, is the carotid artery blood sampling for the third time, and the serum room temperature held of collection 1 hour is solidified rearmounted 4 ℃ and spent the night, and separates out serum, and 2000rpm is centrifugal, separation of serum.Serum is stored in-4 ℃, and is subsequent use in 2 weeks.
Antiserum titre detection method used in the present invention is the ELISA method.Be specially: with immunogen G17BSA with coating buffer (Na2C03-NaHCO3 pH9.6) is diluted to 0.1ug/ml, encapsulates polystyrene 96 orifice plates, the 50ul/ hole, 4 ℃ of cultivations are spent the night.Wash behind the plate three times with confining liquid (2%BSA-PBS) sealing, 50ul/ hole, 37 ℃, 2 hours.Add antiserum, the antiserum of gathering is done following multiple dilution: 2000,4000,8000,16000,32000,64000,128000,256000,512000, every hole 50ul cultivated 2 hours for 37 ℃.With the negative contrast of the rabbit anteserum of injecting normal saline,, compare with the antiserum that produces with the rabbit of injecting G17DT simultaneously with the positive contrast of commodity diphtheria toxin antibody.Wash after three times and to add the two anti-of horseradish peroxidase-labeled, the 1:4000 dilution was hatched 1 hour for 37 ℃.Add the substrate colour developing after washing plate 10 times, hatch cessation reaction after 40 minutes for 37 ℃, measure the A491 value.
Experimental result is as shown in Figure 1.The result can find out from figure, behind first time animal immune, can detect anti-G17 antibody on the 14th day to produce, and antibody titer reaches peak in the time of the 56th day.Can find out simultaneously that utilize the prepared bacterin preparation of the present invention (B group) antibody titer in three times are detected all to be much higher than C group and D group, C group antibody titer is lower than the B group but is higher than the D group.This explanation, the preparation that contains the CpG adjuvant that the present invention is prepared is the immunogenicity of the former G17CRM197 of enhance immunity greatly not only, and its immunostimulant ability is higher than other adjuvants.
Embodiment 3: different CpG-ODN can strengthen rabbit to the immunogenic immunne response of G17CRM197
According to new zealand white rabbit being carried out immunity and detects anti-G17 antibody titer with embodiment 2 identical methods; To measure the influence to the immune effect of G17CRM197 vaccine in rabbit of different CpG-ODN sequences, difference is to use the CpG-ODN sequence shown in the following C1-C6.
C1：5’-TCGTATCGTATCGTATCGTG-3’
C2：5’-TCGTTCGTTCGTTCGTTCGTT-3’
C3：5’-TCGATTCGATTCGATTCGTA-3’
C4：5’-TCGATCGATCGATCGATCGATCGTA-3’
C5：5’-TCGTATCGTATCGTATCGTATCGTG-3’
C6：5’-TCGATTCGATTCGATTCGATTCGTA-3’
Shown in Figure 2 is back 56 days detected anti-G17 antibody titers of blood sampling of immunity, compares with the matched group that uses aluminum hydroxide adjuvant, and above-mentioned any one CpG-ODN all can make the specific antibody titre increase considerably.Explain the CpG-ODN adjuvant that uses above-mentioned sequence and other adjuvants mutually specific energy improve the immunogenicity of bacterin preparation greatly.
Embodiment 4: the mouse immune contrast experiment of therapy vaccine preparation of the present invention
G17CRM197 bacterin preparation mouse immune contrast experiment
Laboratory animal: BALB/c mouse; Regular grade; Age in age 6-8 week; The male and female dual-purpose; Body weight 20g ± 2g.Conventional environment, 18-26 ℃ of temperature, relative humidity 40-70%.Cage for rearing poultry is a plastics Mus box, and the cage tool is replaced weekly 2 times.
Grouping situation: be divided into three groups, every group of 10 mices.First group is immunogen with G17CRM197, is adjuvant with the Freund adjuvant, is oil phase with ISA720, is water with the phosphate buffer; Second group to be immunogen with G17CRM197, do not add adjuvant, is oil phase with ISA720, is water with the phosphate buffer; The 3rd group is immunogen with G17CRM197, is adjuvant with CpG-ODN, is oil phase with ISA720, is water with the phosphate buffer.
The immunity of animal: dosage: G17CRM197 immunity commercial weight is 50ug/, and the CpG-ODN adjuvant that the present invention relates to is 10ug/; Immunization route is intramuscular injection; The immunity time is the 0th, 21 day; Immunization method is 2 injections of back leg muscle.
Antiserum Preparation and antibody purification are undertaken by following operation:
(1) blood sampling time: 21st, 42 days (immunity blood sampling back immunity earlier on the same day).
(2) preparation serum: adopt rathole socket of the eye venous blood collection, get 0.25ml approximately, will gather blood and place centrifuge tube static 30 minutes, 4000 rev/mins of centrifugal 10min, separation of serum.
(3) preservation of serum: place-20 ℃ of refrigerators to preserve.
(4) purifying antibody: adopt the method antagonistic Serum that those skilled in the art were familiar with to carry out purification, like ammonium sulfate precipitation method, the caprylic acid sedimentation method and Protein A affinity chromatography.Prepared antibody detects purity greater than 97% through SDS-PAGE.
The detection of antibody titer
Carrying out antibody titer according to the embodiment of the invention 2 said methods detects.
It is as shown in the table that ELISA detects the antibody titer result:
Table 1 immunity blood sampling in back 21 days for the second time testing result
| First group | Second group | The 3rd group | |
| Mus 1 | 256000 | 128000 | 2048000 |
| Mus 2 | 256000 | 256000 | 512000 |
| Mus 3 | 128000 | 1024000 | 512000 |
| Mus 4 | 128000 | 1024000 | 512000 |
| Mus 5 | 1024000 | 64000 | 512000 |
| Mus 6 | 64000 | 128000 | 1024000 |
| Mus 7 | 64000 | 256000 | 512000 |
| Mus 8 | 64000 | 256000 | 512000 |
| Mus 9 | 64000 | 512000 | 512000 |
| Mus 10 | 128000 | 256000 | 512000 |
| Geometric mean titer | 137187 | 276495 | 630346 |
Visible by last table result, bacterin preparation according to the invention can stimulate mice to produce high titre antibody.Antibody titer not only is higher than no adjuvant group, and far above traditional Freund adjuvant group.Prove that bacterin preparation according to the invention has higher immunogenicity.
G17DT bacterin preparation mouse immune contrast experiment
Carry out the mouse immune contrast experiment according to this patent embodiment 4.2 described methods.This experiment adopts G17DT to replace the G17CRM197 among the embodiment 4.2.
Grouping situation: be divided into three groups, every group of 10 mices.First group is immunogen with G17DT, is adjuvant with the Freund adjuvant, is oil phase with ISA720, is water with the phosphate buffer; Second group to be immunogen with G17DT, do not add adjuvant, is oil phase with ISA720, is water with the phosphate buffer; The 3rd group is immunogen with G17DT, is adjuvant with CpG-ODN, is oil phase with ISA720, is water with the phosphate buffer.
It is as shown in the table that ELISA detects the antibody titer result:
Table 2 immunity blood sampling in back 21 days for the second time testing result
| First group | Second group | The 3rd group | |
| Mus 1 | 256000 | 256000 | 2048000 |
| Mus 2 | 256000 | 256000 | 512000 |
| Mus 3 | 128000 | 512000 | 512000 |
| Mus 4 | 128000 | 1024000 | 512000 |
| Mus 5 | 1024000 | 64000 | 256000 |
| Mus 6 | 64000 | 128000 | 256000 |
| Mus 7 | 128000 | 128000 | 512000 |
| Mus 8 | 128000 | 256000 | 512000 |
| Mus 9 | 64000 | 256000 | 512000 |
| Mus 10 | 128000 | 512000 | 512000 |
| Geometric mean titer | 157586 | 256000 | 512000 |
Can find out equally that by last table result bacterin preparation according to the invention can stimulate mice to produce high titre antibody.Antibody titer not only is higher than no adjuvant group, and far above traditional Freund adjuvant group.Prove that bacterin preparation according to the invention has higher immunogenicity.
Embodiment 5
Present embodiment be intended to illustrate with behind the bacterin preparation immune mouse of the present invention isolated anti-G17 antibody in external growth inhibited effect to tumor cell.
In well-grown tumor cell, add prepared anti-G17 antibody among the embodiment 4, measure the cell viability of different disposal group (add 17 groups of gastrins, add the antibody group, blank group) through mtt assay, to confirm the inhibitory action of antibody to tumor cell.The tumor cell of being selected for use is stomach cancer cell MKN45 and colorectal cancer cells SW480.
The tumor cell line cell in vitro is cultivated, and adds aseptic G17 and anti-G17 antibody in the 10% calf serum RPMI1640 culture fluid respectively, is divided into three groups: blank group, gastrin 17 groups of (25ug/ml) antibody group (30ug/ml).
The mensuration of cell viability: use blue (MTT) colorimetric analysis of thiophene nitrogen azoles and measure cell viability, get the celliferous culture fluid of 100ul (cell concentration is 105/ml), be inoculated in 96 well culture plates, culture fluid was removed in suction after cell pasted an ancient piece of jade, round, flat and with a hole in its centre; Add culture fluid, G17 and antibody respectively by above-mentioned grouping; Each group is all established 8 multiple holes, cultivates that every hole adds MTT (5mg/ml) 20ul after 66 hours, continues cultivation 6 hours; The centrifugal supernatant of abandoning; Every hole adds 20%SDS100ul, shakes 5 minutes, puts room temperature is measured the 570nm wavelength on ELIASA after half an hour absorbance value.
Each organizes indirectly reflection living cells quantity of A570 value, is stomach cancer cell MKN45 or colorectal cancer cells SW480 no matter the result shows, gastrin group viable count is apparently higher than matched group (P < 0.01), and anti-G17 antibody group cell number obviously descend (P < 0.01).
The anti-G17 antibody of table 3 is to the growth inhibited situation of tumor cell
| Divide into groups | MKN45 cell A570 | SW480 cell A570 |
| 17 groups of antibody groups of blank group gastrin | 0.452±0.0480.623±0.0360.258±0.030 | 0.364±0.0390.543±0.0430.198±0.039 |
Embodiment 6
Present embodiment is intended to illustrate with behind the bacterin preparation immunity tumor-bearing mice according to the invention, the growing state of mouse interior tumor.
The foundation of animal model for tumour: get the culture fluid 70ul (cell number 107) that contains MKN45 cell and SW480 cell; Be inoculated in one-tenth tumor in the right axil back of the body of the mice intersection hypodermic layer, the tumor piece is inoculated 2 times repeatedly, forms animal model for tumour; Mice is divided into three groups at random, six every group; The tumor that goes down to posterity is cut into cubic tumor piece of the same size under magnifier, about 8mm3 size, and it is subcutaneous to be inoculated in the right front axil back of the body of mice intersection respectively with the trocar.
Each group all connects from the tumor piece and begins to carry out the immunity first time the last week, and the immunity back was carried out the immunity second time on the 21st day for the first time.Wherein, the each back leg intramuscular injection of every mice of matched group PBS adds oil phase ISA720200ul, the each back leg intramuscular injection of every mice of experimental group G17CRM197 bacterin preparation according to the invention 200ul.The tumor of weighing piece weight was calculated tumour inhibiting rate, detected antibody titer simultaneously with mice execution in the 40th day.
The result: tumor growth was obvious after the tumor piece was inoculated the 5th day, measured and respectively organized the tumor area, and size is close, there was no significant difference (P>0.05), show that the homogeneity of tumor growth is better, inoculate the 7th day beginning experimental group tumor area from the tumor piece and dwindle than matched group.Particularly in one week of back of immunity for the second time, experimental group tumor area obviously begins less than matched group, and difference has significance (P < 0.05); Experimental group tumor area is littler than matched group in the time of the 40th day.
Weight change according to tumor is calculated, and the tumour inhibiting rate of therapy vaccine preparation according to the invention reaches about 30% respectively for the tumour inhibiting rate of two kinds of tumors.The tumor weight situation of change is as shown in the table.
Table 4 in-vivo tumour weight change table
| Divide into groups | Gastric cancer group mouse tumor weight | Colorectal cancer group mouse tumor weight |
| Matched group antibody group | 1098.7±123.4mg758.5±65.6mg | 968.3±137.1mg699.1±58.6mg |
This shows that therapy vaccine preparation according to the invention has tangible tumor inhibition effect.
Sequence table
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Claims (6)
1. therapy vaccine preparation comprises a kind ofly itself having immunogenic immunogenic substances, have the active vaccine for man adjuvant of immunostimulant, be suitable for the oil-phase medium of water in oil emulsion and as the buffer of water;
Described immunogenic substances be a kind of have stimulate the active polypeptide immunogen of tumor growth or this polypeptide immunogen through the spacer peptide formed by 7 aminoacid cross-linking products by means of bi-functional cross-linking agent and carrier protein;
Described polypeptide immunogen aminoacid sequence is: pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu; Described spacer peptide aminoacid sequence is: Ser-Ser-Pro-Pro-Pro-Pro-Cys; Described bi-functional cross-linking agent is: ε-dimaleoyl imino caproic acid N-hydroxy-succinamide ester;
Described carrier protein is a diphtheria toxin muton CRM 197;
Described vaccine for man adjuvant is the oligodeoxynucleotide that contains at least one non-methylated CpG dinucleotide; This oligodeoxynucleotide is a thio-modification, and its length is 20-30 nucleotide; It is one of following that the nucleotides sequence of said oligodeoxynucleotide is classified as:
5’-TCGTATCGTATCGTATCGTG-3’
5’-TCGTTCGTTCGTTCGTTCGTT-3’
5’-TCGATTCGATTCGATTCGTA-3’
5’-TCGATCGATCGATCGATCGATCGTA-3’
5’-TCGTATCGTATCGTATCGTATCGTG-3’
5’-TCGATTCGATTCGATTCGATTCGTA-3’；
The described oil-phase medium that is suitable for water in oil emulsion is ISA720;
Described buffer is phosphate buffer, Tris salt buffer or acetate buffer;
Described therapy vaccine preparation is a water in oil emulsion, and every vaccinating agent contains and has immunocompetent immunogen 10 μ g-1mg; Contain oligodeoxynucleotide adjuvant 10 μ g-10mg; Containing oil-phase medium ISA720 is 0.05-1ml.
2. therapy vaccine preparation according to claim 1 is characterized in that, it is one of following that the nucleotides sequence of said oligodeoxynucleotide is classified as:
5’-TCGTATCGTATCGTATCGTG-3’
5’-TCGTTCGTTCGTTCGTTCGTT-3’
5’-TCGATTCGATTCGATTCGTA-3’。
3. therapy vaccine preparation according to claim 1 and 2 is characterized in that every vaccinating agent contains and has immunocompetent immunogen 50 μ g-0.5mg.
4. therapy vaccine preparation according to claim 1 and 2 is characterized in that every vaccinating agent contains oligodeoxynucleotide adjuvant 25 μ g-5mg.
5. therapy vaccine preparation according to claim 1 and 2 is characterized in that it is 0.1-0.5ml that every vaccinating agent contains oil-phase medium ISA720.
6. the application of the described therapy vaccine preparation of claim 1 in preparation anti-alimentary tract tumor medicine is suitable for intramuscular injection or subcutaneous injection.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5023077A (en) * | 1989-01-24 | 1991-06-11 | Aphton Corporation | Immunogenic compositions and methods for the treatment and prevention of gastric and duodenal ulcer disease |
| CN1271733A (en) * | 2000-04-04 | 2000-11-01 | 中国预防医学科学院病毒学研究所 | CpG oligonucleotide with specific immunostimulation activity to human immune cell |
| WO2004004687A2 (en) * | 2002-07-03 | 2004-01-15 | Aphton Corporation | Liposomal vaccine |
| CN101050236A (en) * | 2006-04-06 | 2007-10-10 | 海南天源康泽医药科技有限公司 | Immunogen by using mutant CRM197 of diphtheria toxin as carrier, preparation method, and application |
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2008
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5023077A (en) * | 1989-01-24 | 1991-06-11 | Aphton Corporation | Immunogenic compositions and methods for the treatment and prevention of gastric and duodenal ulcer disease |
| CN1271733A (en) * | 2000-04-04 | 2000-11-01 | 中国预防医学科学院病毒学研究所 | CpG oligonucleotide with specific immunostimulation activity to human immune cell |
| WO2004004687A2 (en) * | 2002-07-03 | 2004-01-15 | Aphton Corporation | Liposomal vaccine |
| CN101050236A (en) * | 2006-04-06 | 2007-10-10 | 海南天源康泽医药科技有限公司 | Immunogen by using mutant CRM197 of diphtheria toxin as carrier, preparation method, and application |
Non-Patent Citations (1)
| Title |
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| Fearon K. et al.A minimal human immunostimulatory CpG motif that potently induces IFN-gamma and IFN-alpha production.《European Journal of Immunology》.2003,第33卷2114-2122. * |
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