CN106432460A - Tumor antigen protein and tumor vaccine - Google Patents
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Abstract
The invention discloses a tumor antigen protein and a tumor vaccine. The tumor antigen protein comprises a human tumor cell membrane MUC1-Xex peptide fragment, and an amino acid sequence of the MUC1-Xex peptide fragment is as shown in SEQ ID No: 1. The tumor vaccine contains the tumor antigen protein. The tumor antigen protein selects MUC1-Xex peptide fragment with an MUC1 target point as an antigen, thus having very good safety; more preferentially, an adjuvant immune stimulating protein is directly coupled with the MUC1-Xex peptide fragment by means of gene fusion recombinant expression, so that the immunogenicity is improved. The tumor vaccine selects a clinical commonly used aluminum adjuvant as an auxiliary material, so that the action time of the vaccine is prolonged. Finally, animal experiments prove that after being intramuscularly injected, the obtained tumor vaccine has good safety and effectiveness. The tumor vaccine has a good treatment effect on malignant tumors, especially for lung cancer, melanoma and colon cancer.
Description
Technical field
The present invention relates to the immunotherapy techniques field of tumor, particularly a kind of tumor antigen protein and tumor associated therewith
Vaccine.
Background technology
At present, the annual new cancer patient of China there are about 2,500,000, and because of cancer, the number of death is about 1,400,000.Future
Air, water pollution, environmental disruption may improve sickness rate and the case fatality rate of cancer further.The research of cancer treatment drugs is
International study hotspot now.In addition to operation, chemotherapy, radiotherapy, biological immune treatment quickly grows in recent years for treatment of cancer,
Cell therapy, Antybody therapy and the vaccine therapy gesture stood like the legs of a tripod of formation, have greatly enclose cancer and annihilate may.
Melanoma is a kind of cancer from melanocyte, occurs mainly in skin, also occur in once in a while oral cavity,
Small intestinal and eyes.Women the most often occurs in leg, and male mainly appears on back.In countries such as America and Europes, melanoma is the most normal
One of 10 big cancer species seen, every year European neopathy just have 100,000 many cases, dead patient has 20,000 many cases.90% trouble
Person, in early stage, can pass through excision.Patients with terminal poor prognosis, poor to chemotherapy, radiotherapy side effect, survival rate only has 17% within 5 years.
By 2011, purine precursors analog dacarbazine, cisplatin and vindesine etc. were used for the chemotherapy of patients with terminal, but do not find and prolong
The evidence of long life cycle.The patient of BRAF mutation, inhibitor Wei Luofeini, Da Lafeini, Sibutramine Hydrochloride replace Buddhist nun, mek inhibitor
Cobimetinib plays a role, but the continuing advances of disease also fail to solve.Auxiliary treatment such as gamma interferon is permissible
By 5 years life span extension 3-5%, but the side effect such as fatigue, liver enzyme rising, anorexia, headache, depression occur.Interleukin-2 is permissible
Disease is made to have alleviated, but large side effects.These years recently, FDA have approved 7 kinds of single therapies and 3 kinds of combined therapies, including exempting from
Epidemic disease treatment and lytic viruses treatment etc..Wherein studying the most intensive is CTLA-4 antibody, PD-1 Antybody therapy melanoma.
CTLA-4 antibody and PD-1 antibody are treated alone or in combination and are achieved in some melanoma patients (about 10%, 30%, 61%)
Preferably therapeutic effect, but general effect is to be improved, and large side effects treated, 38% patient of therapeutic alliance because
Treatment is exited in side effect.The side effect of CTLA-4 antibody includes diarrhoea/colitis, dermatitis, hepatitis and endocrinopathy；PD-1 resists
The side effect of body includes dermatitis, thyroid disease and pneumonopathy etc..
In terms of disease, occupying national malignant tumor morbidity primary is pulmonary carcinoma, and male lung cancer M ＆ M all accounts for
First of all malignant tumor, women sickness rate accounts for second, and mortality rate accounts for second.Pulmonary carcinoma is that M ＆ M increases
Long the fastest, one of malignant tumor maximum to population health and life threat.
At present, colorectal cancer has become as one of 3 kinds of modal cancers, and circumscribed colon intestinal cancer can be controlled by operation
Treat, and shift patient and only have small part can obtain multi-mode treatment.About 50% limitation colorectal cancer patients are controlled in operation
Transfer occurs after treatment and recurs.The prognosis of patient depends on tumor grade being interacted by host's tumor to be affected, and shows as
The T cell infiltration of focus.
Vaccine therapy cancer applications are convenient, effect is significant, deeply welcome by extensive patients.Inoculated tumour vaccine is immunization therapy
One of method, its principle is to produce specific cellular immunity and body using tumor cell or tumor antigen material induction body
Liquid immunoreation, that is, activate the immune system of patient itself, carrys out the anti-cancer ability of enhancing body, and then the growth of prevention tumor,
Diffusion and recurrence, are finally reached and control the purpose even removing tumor.There is curative effect height, high specificity, untoward reaction little etc. excellent
Point.A kind of method is exactly to inject the tumor associated antigen of purification or restructuring together with adjuvant, induces humoral immunization and cell
Immunity.Tested differentiation antigen Melan-A/MART-1, gp100 and tyrosinase；Cancer antigen MAGE-A3, NY-ESO-1,
Mutant antigen and virus antigen etc., but clinical effectiveness is unsatisfactory.Research shows, has a kind of molecule MUC1 and relation between tumor close
Cut, have become as the target spot of immunotherapy of tumors.
MUC1 is a kind of heterodimer transmembrane glycoprotein, by nitrogen end subunit (MUC1-N) and carbon teminal subunit (MUC1-
C) form.MUC1 unconventionality expression in the tumor tissues of multiple epithelial origins, it is closely related with the generation development of tumor.Abroad
Peptide based on VNTR with MUC1 (number different continuously repeat sequence, variable number tandemrepeats)
Vaccine has carried out III clinical trial phase, shows cancer vaccine overall treatment effect inconspicuous, may be late period lung with enter group
Cancer patient has relation.
Content of the invention
Specificity for the antigenic peptides based on the VNTR of MUC1 in prior art is not good, and peptide vaccine therapeutic effect is poor
Problem, one aspect of the present invention provide a kind of tumor antigen peptide, on the other hand the tumor epidemic disease prepared by this tumor antigen peptide is provided
Seedling.
We think, VNTR is not directly anchored to cell surface at research, and cancerous cell combination is more loose, is conventional vaccine
One of the reason weak curative effect, one aspect of the present invention, is from MUC1 target spot human oncocyte's film MUC1-Xex peptide fragment, this target spot
It is that normal cell is not expressed or low expression, cancer cell and the high expression of precancer cell, there is good safety, obtain
A kind of tumor antigen protein for this shot design.
The tumor antigen protein that the present invention provides, including human oncocyte's film MUC1-Xex peptide fragment, described MUC1-Xex peptide fragment
Aminoacid sequence such as SEQ ID NO:Shown in 1 (referring to Fig. 2).
Further, we have discovered that, loose mixing between the vaccine component polypeptide in conventional vaccine and adjuvant
Make another reason that the immunoreation exciting is specifically not its effect difference.The present invention is also carried out to this respect improveing, and then
Go out the scheme being more highly preferred to：Above-mentioned tumor antigen protein, also includes the immunostimulation egg merging with described MUC1-Xex peptide fragment
In vain.Immune stimulator plays skeptophylaxis stimulation, is that one kind can produce antibody and/or activation T by stimulating immune system
The albumen of cell effect.Immune stimulator and fusion, the N-terminal of the two polypeptide chain and the C-terminal order of connection of MUC1-Xex peptide fragment
And be not limited, can be that the C-terminal of immune stimulator connects the N-terminal of MUC1-Xex peptide fragment or the C of MUC1-Xex peptide fragment
End connects the N-terminal of immune stimulator.
It is further preferable that described immune stimulator is MBP, GST or KLH.MBP is maltose-binding protein (maltose
Binding protein), the immunogenicity of recombinant subunit vaccine can be improved, as immunostimulant, improve immunity of organism
Response.GST be glutathione S transferring enzymes (glutathione S-transferase), GST be by polygenes encode, have many
Plant one group of isozyme of function, molecular weight is 23～29KDa, can induce the immunoreation for fusion protein composition.KLH is
Keyhole limpet hemocyanin (keyhole limpet hemocyanin), is a glycoprotein, and its sugared content is about molecular weight
4%, glycosyl is the important feature causing KLH antigen, and the glycosyl of KLH includes mannose, galactose, N-acetyl-glucosamine, acetyl
Galactosamine, fucose etc..Because KLH antigenicity is strong, it is also the antigen coupling protein more commonly used, primary structure contains about
3400 aminoacid.There is the immunogenic effect of enhancement antigen.
Preferably, in above-mentioned tumor antigen protein, the aminoacid sequence such as SEQ ID NO of described MBP:Shown in 2；Described
The aminoacid sequence of GST such as SEQ ID NO:Shown in 3.
Present invention also offers the encoding gene of above-mentioned anti-tumor protein.
The present invention also provides the biomaterial containing above-mentioned encoding gene, and described biomaterial is expression vector, Host Strains,
Or transgenic cell line.
Present invention also offers a kind of preparation method of above-mentioned tumor antigen protein, comprise the following steps：
(1) aminoacid sequence according to described tumor antigen protein, designs and expands the coding obtaining tumor antigen protein
Gene, is then attached to pMAL-p5X plasmid, converts bacillus coli DH 5 alpha, cultivates to logarithm in the LB culture medium containing AMP
Trophophase, is subsequently adding IPTG abduction delivering；Through the culture centrifugation of IPTG abduction delivering, obtain culture precipitation and cultivate
Thing supernatant；
(2) the culture precipitation resuspended rear ultrasonication of Tris buffer, centrifugation, supernatant precipitation carries out following behaviour respectively
Make：
The Amylose resin of supernatant upper 8 post bed Tris buffer balances after micro-pore-film filtration, is buffered with 12 post bed Tris
With the Tris buffer solution elution containing maltose after liquid washing, obtain target protein 1；
Precipitation adds the dissolving of 8M carbamide Tris solutions overnight, carries out dialysis Tris buffer and remove carbamide after centrifugation, filters
Go up afterwards 8 post bed Tris buffer balance Amylose resin, with after 12 post bed Tris buffer solution with the Tris containing maltose
Buffer solution elution, obtains target protein 2；
(3) culture supernatant is degerming through inner pressed membrane filtration, and gained filtrate concentrates through external-compression type filter membrane again；Concentrated solution warp
Cross 10K filter membrane to dialyse 2 times, after centrifugation, upper Amylose resin purification obtains target protein 3；
Wherein, step (2) and step (3) no sequencing, described protein 1, protein 2 and protein 3 are not
Tumor antigen protein with form.
Wherein, the biomaterial such as pMAL-p5X plasmid, bacillus coli DH 5 alpha all can be bought by commercial sources and obtain.
Tris buffer is conventionally prepared or is directly bought commercial goods, and the present invention does not have particular/special requirement to it.
Present invention also offers application in the vaccine of preparation treatment malignant tumor for the above-mentioned tumor antigen protein.
Preferably, in above-mentioned application, described malignant tumor is one or more of melanoma, pulmonary carcinoma and colon cancer.
A kind of another aspect of the present invention, there is provided tumor vaccine, its composition includes above-mentioned tumor antigen protein and hydrogen
Aluminum adjuvant., with respect to adjuvants such as Water-In-Oils, the pain stimulation to patient and cicatrix residual are more faint for aluminum hydroxide adjuvant,
Pretreatment operation before patient injection is relatively easy, contrast single tumor antigen protein injection, can greatly enhancement antigen
Immunogenicity.
Test shows, the above-mentioned tumor vaccine of our transformations is for malignant tumor particularly pulmonary carcinoma, melanoma, colon cancer
All show good therapeutic effect.
Preferably, in above-mentioned tumor vaccine, endotoxin content is less than 500Eu/mg, and tumor antigen protein concentration is 1mg/
ml.
Tumor antigen protein and tumor vaccine that the present invention provides, have the advantages that：
The present invention first select MUC1 target spot human oncocyte's film MUC1-Xex peptide fragment, this target spot be normal cell do not express or
The high expression of low expression, cancer cell and precancer cell, has good safety.Secondly the adjuvant immunity selecting stimulates egg
Direct in vain and MUC1-Xex passes through the recombinant expressed coupling of gene fusion, increased the immunogenicity of MUC1-Xex.Then, select to face
The conventional aluminium adjuvant of bed, as adjuvant, increases the action time of vaccine.The tumor vaccine finally obtaining passes through intramuscular injection, animal
Experiment shows there is good safety and effectiveness.The tumor vaccine of the present invention shows to malignant tumor, particularly pulmonary carcinoma,
Melanoma, the good therapeutic effect of colon cancer.
Brief description
Fig. 1 is the gene coded sequence (Blocked portion indicates NdeI, NcoI restriction enzyme site respectively) of MUC1-Xex albumen.
The amino acid coding of Fig. 2 MUC1-Xex albumen.
Fig. 3 is the recombinant expressed result electrophoretogram of BMP-MUC1-Xex albumen, wherein, 1：Protein scale, from top to bottom
It is 97.4；66.4；43.0；31.0；21.1；14.2KD；2：DH5 α-pMAL-p5X after IPTG induction；3：After IPTG induction
DH5α-pMAL-p5X-BMP-MUC1-Xex；4：DH5 α-pMAL-p5X before IPTG induction；5：DH5 α before IPTG induction-
pMAL-p5X-BMP-MUC1-Xex.MBP is 416 aminoacid, molecular weight 47.8KD；MUC1-Xex is 147 aminoacid,
MBP-MUC1-Xex fusion protein amounts to 563 aminoacid, and MBP-MUC1-Xex molecular weight is 62KD, the left finger of arrow.
Fig. 4 is the purification result electrophoretogram of BMP-MUC1-Xex albumen, wherein, 1：DH5 α-pMAL- before IPTG induction
p5X-BMP-MUC1-Xex；2,19：DH5 α-pMAL-p5X-BMP-MUC1-Xex after IPTG induction；3：Thalline；4：Culture medium；
5：Thalline ultrasonication precipitates；6：Thalline ultrasonication supernatant；7：Ultrasonication supernatant crosses the prick post liquid of Amylose resin；8,
9：The conjugated protein (from broken supernatant, E1 and E2) of 10mM maltose eluting Amylose resin, arrow right refers to；10：Albumen
Matter scale, is 97.4 from top to bottom；66.4；43.0；31.0；21.1；14.2KD)11：The bacteria culture media of ammonium sulfate precipitation；
12：The bacteria culture media prick post liquid of ammonium sulfate precipitation；13,14：The conjugated protein of 10mM maltose eluting Amylose resin
(from culture medium supernatant, E2 and E3)；15：The inclusion body of carbamide dissolving；16：Solubilization of inclusion bodies thing crosses wearing of Amylose resin
Post liquid；17,18：The conjugated protein (from solubilization of inclusion bodies thing, E1 and E2) of 10mM maltose eluting Amylose resin.
Fig. 5 is the inhibitory action design sketch to melanoma for the MCMVax vaccine, wherein ip+im saline control table
Show that muscle physiological saline injection adds abdominal cavity physiological saline joint matched group；Im saline control represents muscle physiological
Saline injection matched group；Ip saline control represents abdominal cavity physiological saline matched group；Ip PD-1Ab represents abdominal cavity
PD-1 antibody injects treatment group；Im MCMVax represents muscle MCMVax injection treatment group；Im MCMVax+ip PD-1Ab represents
Intramuscular injection MCMVax vaccine adds the antibody combined treatment group of lumbar injection PD-1.
Fig. 6 is the inhibitory action design sketch to pulmonary carcinoma for the MCMVax.
Fig. 7 is the therapeutic effect figure to pulmonary carcinoma for the MCMVax.
Specific embodiment
In order that those skilled in the art more fully understand the present invention program, with reference to the accompanying drawings and detailed description
The present invention is described in further detail.
In following examples, unless otherwise specified, biomaterial used is commercial goods.
Embodiment 1 prepares MCMVax vaccine
1st, MUC1-Xex gene design and synthesis
MUC1-Xex encoding gene uses DNA design software to design, and carries out codon optimization to encoding gene, 5 ' draws at it
Enter NdeI restriction enzyme site, 3 ' ends introduce NcoI restriction enzyme site, such as SEQ ID NO:Shown in 4, synthetic gene is cloned into pUC57
Carrier, ammonia benzyl resistance.
2nd, the enzyme action of gene and sequencing identification
Carry out double digestion with NdeI and NcoI restriction endonuclease after gene chemical synthesis, connect into the pMAL-p5X matter through same enzyme action
Grain, DNA ligase connects, and is transformed into competence DH5 α antibacterial.With sieving on the agar plate containing ampicillin (AMP)
Choosing, selects monoclonal LB-AMP culture medium (i.e. LB culture medium containing AMP) culture, extracts plasmid enzyme restriction identification, then send
Carry out sequencing identification to Beijing Tian Yihuiyuan bio tech ltd, gene order is shown in that (Blocked portion indicates Fig. 1 respectively
NdeI, NcoI restriction enzyme site), full length gene 456bp.
Correctly above-mentioned plasmid is connected with MBP encoding gene after double digestion for sequencing, constitutes new fusion gene plasmid,
It is named as pMAL-p5X-MBP-MUC1-Xex plasmid, containing MUC1-Xex encoding gene and MBP encoding gene.
3rd, MUC1-N tandem repeat locus is recombinant expressed
DH5 α antibacterial containing pMAL-p5X-MBP-MUC1-Xex plasmid is cultivated with LB-AMP, treats OD600nmReach 0.5
Add the IPTG of final concentration of 1mM, abduction delivering 12h.The DH5 α antibacterial that same abduction delivering carries pMAL-p5X empty plasmid makees
For comparison.The bacterium solution before and after induction is taken to carry out protein electrophorese, coomassie brilliant blue R250 dyes.After IPTG induction, containing pMAL-
The DH5 α bacterial expression of p5X-MBP-MUC1-Xex plasmid goes out the protein of about 62KD, consistent with calculated value, sees Fig. 3.MBP
It is 416 aminoacid, molecular weight 47.8KD；MUC1-Xex is 147 aminoacid, and MBP-MUC1-Xex fusion protein amounts to 563
Individual aminoacid, MBP is located at the aminoterminal of MUC1-Xex, and the MBP-MUC1-Xex fusion protein molecule amount of acquisition is 62KD, Fig. 3 arrow
Left finger)
4th, the purification of MBP-MUC1-Xex fusion protein
The centrifugation of DH5 α bacteria-induction culture containing pMAL-p5X-MBP-MUC1-Xex plasmid, takes precipitation Tris to delay
Rush liquid (20mM Tris-HCl, 0.2M NaCl, 1mM EDTA) to hang, ultrasonication 10min, supernatant is collected by centrifugation, with micro-
After the membrane filtration of hole upper 8 post bed Tris buffer balance Amylose resin, with after 12 post bed Tris buffer solution with containing
The Tris buffer solution elution of 10mM maltose, obtains target protein 1.Ultrasonication collected after centrifugation precipitates, and adds 8M carbamide
Tris solutions overnight dissolves, and carries out dialysis Tris buffer and remove carbamide after centrifugation, upper 8 post bed Tris buffer balances after filtration
Amylose resin, obtain purpose with after 12 post bed Tris buffer solution with the Tris buffer solution elution containing 10mM maltose
Protein 2.After centrifugation, through inner pressed filter membrane (002N type, ＜ 200K, Beijing Xu Bang company) filtration sterilization, filtrate is again for bacterial solution
Concentrate through external-compression type filter membrane (CLW-002 type polysulfone material, ＞ 3K, Beijing Xu Bang company).Concentrated solution is through 10K filter membrane dialysis 2
Secondary, after centrifugation, upper Amylose resin purification obtains target protein 3.With SDS-PAGE and R250 dyeing point upper after G250 detection
Analysis.
Protein purification detection electrophoresis result is shown in Fig. 4.MBP-MUC1-Xex expressing fusion protein in escherichia coli, molecular weight
For 62KD (the 2nd road).Purified fusion protein MBP-MUC1-Xex from bacteria breaking supernatant, with Amylose resin-bonded and
10mM maltose eluting, obtain is the protein (the 8th and the 9th road) of about 62KD.It is purified into molecular weight from bacteria culture media
It is about the protein of 62KD, show that MBP-MUC1-Xex occurs in that secreting, expressing (the 13rd and the 14th road).Purification from inclusion body
The protein molecular weight going out is slightly larger, about 64KD (the 17th and the 18th road).
5th, the preparation of MCMVax vaccine
Experiment removes MBP-MUC1-Xex fusion protein endotoxin using Q-sepharose prick post, and endotoxic content is low
In 500Eu/mg, regulation MBP-MUC1-Xex fusion protein concentration is 1mg/ml.And mix with aluminum hydroxide adjuvant equal-volume
To vaccine MCMVax, 4 DEG C save backup.MCMVax vaccine is transparent, odorless, liquid with no taste.PH when 25 ± 0.5 DEG C is
6.0～8.0.
The inhibition to melanoma for embodiment 2 MCMVax
Take 6-8 week old male C 57 BL/6 J mouse 15, be divided into matched group, MNRVax treatment group and MCMVax treatment group three
Group, every group 5, in every mice right axil subcutaneous vaccination B16-Muc1 melanoma cell 2.8 × 106Individual, after 5 days, tumor can
See.
Matched group injects 100 μ L normal saline respectively in every mice hind leg muscle, injects weekly 2 times, injects 4 altogether
Secondary；
50 μ g MNRVax inject respectively in every mice hind leg muscle in MNRVax treatment group, inject 2 times weekly, note altogether
Penetrate 4 times；
50 μ g MCMVax inject respectively in every mice hind leg muscle in MCMVax treatment group, inject weekly 2 times, and altogether 4
Secondary.
It is found that with respect to control group mice, swell in 14,18,20 after MCMVax treatment group mouse inoculation melanoma day
Tumor is all substantially reduced, and is shown in Table 1.14 days tumors after MNRVax treatment group mouse inoculation melanoma are substantially reduced, and show
MCMVax is better than MNRVax to the therapeutic effect of melanoma.
The inhibitory action to melanoma for the table 1.MCMVax
Note：*, p<0.05 (contrast matched group).
Embodiment 3 MCMVax merges the inhibition to melanoma for the MNRVax
Take 6-8 week old male C 57 BL/6 J mouse 24, be divided into matched group, MNRVax treatment group, MCMVax treatment group,
Four groups of MNRVax and MCMVax therapeutic alliance group, every group 6, thin in every mice right axil subcutaneous vaccination B16-Muc1 melanoma
Born of the same parents' cell 2.4 × 106Individual, after 9 days, tumor is visible.
In mice hind leg muscle ejection preparation respectively, 3 times a week, 3 times altogether.Wherein, matched group per injection 200 μ L life
Reason saline；MNRVax treatment group per injection 50 μ g MNRVax；MCM Vax treatment group per injection 50 μ g MCMVax；Joint
Treatment group's per injection 50 μ g MNRVax adds 50 μ g MCMVax.
It is found that with respect to control group mice, after MCMVax treatment group mouse inoculation melanoma 11,12,13,14,
Tumor is all substantially reduced within 15 days, is shown in Table 2.MNRVax treatment group mouse tumor diminishes, but and matched group no significant difference, combine and control
11,16 days tumors after treatment group mouse inoculation melanoma are all substantially reduced.After mouse inoculation melanoma 11,12,14 days
MCMVax single therapy is better than MNRVax and MCMVax therapeutic alliance.After mouse inoculation melanoma 12,13,14,15 days
MCMVax single therapy is better than MNRVax single therapy.
The inhibitory action to melanoma for the table 2.MCMVax
Note：*:p<0.05；**:p<0.01 (contrast normal saline)；
#:p<0.05；##:p<0.01 (contrast MNRVax)；
p<0.05 (contrast MNRVax+MCMVax).
Embodiment 4 MCMVax merges the inhibition to melanoma for the PD-1 antibody
Take 36 6-8 week old male C 57 BL/6 J mouses, right side oxter injection 1.6 × 106(50 μ L) B16-hMUC1 black
Plain oncocyte, the 11st day tangible formed to tumor after, the volume of measurement mouse tumor.It is divided into 6 groups, every group 6.
Muscle physiological saline injection matched group (im saline control)：Every leg muscle injects 100 μ L physiology salts
Water；
Muscle MCMVax injection treatment group (im MCMVax)：Every leg muscle injects 50 μ g/100 μ L MCMVax epidemic diseases
Seedling；
Abdominal cavity physiological saline matched group (ip saline control)：Every lumbar injection 100 μ L normal saline；
Abdominal cavity PD-1 antibody injection treatment group (ip PD-1Ab)：Every lumbar injection 150 μ g/100 μ L PD-1 mice resists
Body；
Muscle physiological saline injection adds abdominal cavity physiological saline joint matched group (ip+im saline control)：Often
Leg muscle is injected 100 μ L normal saline and is added lumbar injection 100 μ L normal saline；
Intramuscular injection MCMVax vaccine adds the antibody combined treatment group of lumbar injection PD-1 (im MCMVax+ip PD-1Ab)：
Every leg muscle is injected 50 μ g/100 μ L MCMVax vaccines and is added lumbar injection PD-1 antibody.
Inject 3 times weekly, continuous injection 1 week；Measure 6 times weekly, continuous measurement 2 weeks.According to length × wide2/ 2 calculating tumors
Volume.
The 2nd, 3,4 days after result display medication, MCMVax vaccine is all better than injection PD-1 to the therapeutic effect of melanoma
The effect of antibody.Vaccine is not observed and synergism in antibody, may be too fast relevant with animal tumor growth, see Fig. 5.
Show that MCMVax can significantly inhibit melanic growth, and the synergism of antibody is inconspicuous.
The inhibition to pulmonary carcinoma for embodiment 5 MCMVax
Take 6-8 week old male C 57 BL/6 J mouse 10, be divided into saline control group (control) and MCMVax treatment
Organize two groups, every group 5.
Treatment group hind leg muscle injection MCMVax, 10 μ g/100 μ L every time, interval is 7 days, 4 times altogether；Matched group is noted
Penetrate normal saline, every time 100 μ L, interval is 7 days, 4 times altogether.
Hereafter in the right axil subcutaneous vaccination lung carcinoma cell 5 × 10 of every mice5Individual, after 6 days, matched group tumor is visible.
Continue in MCMVax treatment group mice hind leg muscle injection MCMVax, 10 μ g/100 μ L every time, now interval is 3
My god, 4 times altogether.Matched group injecting normal saline, 100 μ L every time, interval is 3 days, 4 times altogether.It is found that with respect to right
According to group mice, 10,13,15,17 after treatment group's mouse inoculation lung carcinoma cell day, tumor all pole is substantially reduced, and sees Fig. 6.Show
MCMVax can significantly inhibit the growth of pulmonary carcinoma.
The therapeutic effect to pulmonary carcinoma for embodiment 6 MCMVax
Take 6-8 week old male C 57 BL/6 J mouse 12, be divided into saline control group (control) and MCMVax treatment
Organize two groups, every group 6.In the right axil subcutaneous vaccination lung carcinoma cell 5 × 10 of every mice5Individual, after 6 days, matched group tumor is visible.?
MCMVax treatment group mice hind leg muscle injection MCMVax, 50 μ g/100 μ L every time, now interval is 3 days, 3 times altogether.Right
According to group injecting normal saline, 100 μ L every time, interval is 3 days, 3 times altogether.It is found that with respect to control group mice, treating
After group mouse inoculation lung carcinoma cell 13 days, tumor pole is substantially reduced；After inoculation lung carcinoma cell 15 days, tumor is substantially reduced,
See Fig. 7.Show that MCMVax has therapeutic effect to pulmonary carcinoma.
Embodiment 7 MCMVax merges the inhibition to colon cancer for the MNRVav
Take 6-8 week old female Balb/c mice 24, be divided into matched group, MNRVax treatment group, MCMVax treatment group,
Four groups of MNRVax and MCMVax therapeutic alliance group, every group 6.In every mice right axil subcutaneous vaccination CT26WT colon cancer cell 1
×106Individual, after 3 days, tumor is visible.
Matched group：Every mice in hind leg muscle injecting normal saline, 100 μ L every time, 2 times a week, 6 times altogether；
MNRVax treatment group：Every mice injects MNRVax, 50 μ g every time, 2 times a week, 6 times altogether in hind leg muscle；
MCMVax treatment group：Every mice injects MCMVax, 50 μ g every time, 2 times a week, 6 times altogether in hind leg muscle；
Therapeutic alliance group：Every mice is injected 50 μ g MNRVax in hind leg muscle every time and adds 50 μ g MCMVax, and weekly 2
Secondary, 6 times altogether.
It was found that with respect to control group mice, 17 days tumors after MCMVax treatment group mouse inoculation colon cancer cell
It is substantially reduced, 28 days tumor poles after inoculation colon cancer are substantially reduced.MNRVax treatment group mice and matched group indifference, joint
26,28 days tumors after treatment group's mouse inoculation colon cancer are all substantially reduced, and are shown in Table 3.Show that MCMVax can significantly inhibit
The growth of CT26WT colon cancer, and the synergism of MNRVax is inconspicuous.
The inhibitory action to colon cancer for the table 3.MCMVax
Note：*:p<0.05；**:p<0.01 (contrast normal saline).
Embodiment 8 MCMVax merges the inhibition of PD-1 antibodies on colon cancer
Take 6-8 week old female C57BL/6J mice 28, be divided into matched group, MCMVax treatment group, PD-1 Antybody therapy group,
Four groups of the antibody combined treatment group of MCMVax and PD-1, every group 7, in every mice right axil subcutaneous vaccination MC38 colon cancer cell 3
×106Individual, after 7 days, tumor is visible.
Matched group：Inject 100 μ L normal saline+intraperitoneal injection of saline in mice hind leg muscle；
MCMVax treatment group：Inject 50 μ g MCMVax+ intraperitoneal injection of saline in mice hind leg muscle；
PD-1 Antybody therapy group：Inject 100 μ L normal saline+abdominal cavity 150 μ g PD-1 antibody in mice hind leg muscle；
Therapeutic alliance group：Inject 50 μ g MCMVax+ abdominal cavity 150 μ g PD-1 antibody in mice hind leg muscle.
Above each group, injection 2 times weekly, 3 times altogether.
It is found that with respect to control group mice, 28,31 after MCMVax treatment group mouse inoculation colon cancer day tumor shows
Write and reduce；14,31 days tumors after PD-1 Antybody therapy group mouse inoculation colon cancer are substantially reduced, and the 28th celestial pole is substantially reduced；
14,16,21 days tumors after therapeutic alliance group mouse inoculation colon cancer are substantially reduced, the 28th, 31 celestial poles be substantially reduced, be shown in Table 4.
Show that MCMVax can significantly inhibit the growth of MC38 colon cancer, and the synergism of antibody is inconspicuous.
Table 4.MCMVax merges the inhibitory action of PD-1 antibodies on colon cancer
Note：*:p<0.05；**:p<0.01 (contrast normal saline).
MNRVax vaccine clinical application process
First course for the treatment of 1 year, 1-4 pin is spaced one week, 5-15 pin interval January.Cancer early stage patient uses 1 year；Cancer
Disease mid-term patient increases by a course for the treatment of, and 16-27 pin is spaced January, and cancer early stage patient uses 2 years.Bilateral upper arm muscle injection.
The explanation of above example is only intended to help understand the core concept of the present invention.It should be pointed out that for this technology
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improvement can also be carried out to the present invention
And modification, these improve and modify and also fall in the protection domain of the claims in the present invention.
SEQUENCE LISTING
<110>Cai Jiong
<120>Tumor antigen protein and tumor vaccine
<130> P20160123
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 147
<212> PRT
<213>Artificial sequence
<400> 1
Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala
1 5 10 15
Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Leu Ser
20 25 30
Thr Gly Val Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln
35 40 45
Phe Asn Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu
50 55 60
Gln Arg Asp Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly
65 70 75 80
Phe Leu Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val
85 90 95
Gln Leu Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Met
100 105 110
Glu Thr Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn
115 120 125
Leu Thr Ile Ser Asp Val Ser Val Ser Gly Val Pro Phe Pro Phe Ser
130 135 140
Ala Gln Ser
145
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<212> PRT
<213>Artificial sequence
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Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr
1 5 10 15
Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys
20 25 30
Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu
35 40 45
Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu
50 55 60
His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly
65 70 75 80
Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr
85 90 95
Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln
100 105 110
Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys
115 120 125
Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn
130 135 140
Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala
145 150 155 160
Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn
165 170 175
Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly
180 185 190
Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
195 200 205
Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu
210 215 220
Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu
225 230 235 240
Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp
245 250 255
Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val
260 265 270
Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu
275 280 285
Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu
290 295 300
Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn
305 310 315 320
Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu
325 330 335
Leu Val Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys
340 345 350
Gly Glu Ile Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala
355 360 365
Val Arg Thr Ala Val Ile Asn Ala Ala Ser Gly Arg Gln Thr Val Asp
370 375 380
Glu Ala Leu Lys Asp Ala Gln Thr Asn Ser Ser Ser Asn Asn Asn Asn
385 390 395 400
Asn Asn Asn Asn Asn Asn Leu Gly Ile Glu Gly Arg Ile Ser His Met
405 410 415
Ser Met Gly Gly Arg Asp Ile Val Asp Gly Ser Glu Phe Pro Ala Gly
420 425 430
Asn
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20 25 30
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35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Glu Val Leu
210 215 220
Phe Gln Gly Pro Leu Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly
225 230 235 240
Arg Ile Val Thr Asp
245
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<212> DNA
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<400> 4
catatgtctg gccatgcatc tagcactcca ggtggtgaaa aagaaacttc tgcaactcaa 60
cgttcctctg ttccaagcag cactgagaaa aacgcgctgt ctaccggcgt gtctttcttc 120
tttctgtctt tccacattag caacctgcag ttcaactcct ccctggaaga cccgagcacc 180
gactattatc aggaactgca acgcgatatc agcgaaatgt tcctgcagat ctataaacag 240
ggcggtttcc tgggcctgtc taacatcaaa ttccgtccgg gctccgtcgt tgtacagctg 300
actctggcgt ttcgtgaagg taccatcaac gtgcacgata tggagactca gttcaaccaa 360
tataaaactg aggcagctag ccgttataac ctgaccattt ctgatgtgag cgtttccggc 420
gtgccattcc ctttttccgc ccagtcctaa ccatgg 456
Claims (10)
1. a kind of tumor antigen protein is it is characterised in that include human oncocyte's film MUC1-Xex peptide fragment, described MUC1-Xex peptide fragment
Aminoacid sequence such as SEQ ID NO:Shown in 1.
2. tumor antigen protein according to claim 1 is it is characterised in that also include merging with described MUC1-Xex peptide fragment
Immune stimulator；Described immune stimulator is preferably MBP, GST or KLH.
3. tumor antigen protein according to claim 2 is it is characterised in that the aminoacid sequence such as SEQ ID of described MBP
NO:Shown in 2；The aminoacid sequence of described GST such as SEQ ID NO:Shown in 3.
4. the encoding gene of the tumor antigen protein described in claim 1 or 2.
5. the biomaterial containing the encoding gene described in claim 4, described biomaterial is expression vector, Host Strains, or
Transgenic cell line.
6. the preparation method of the tumor antigen protein described in claim 1 or 2 is it is characterised in that comprise the following steps：
(1) aminoacid sequence according to described tumor antigen protein, designs and expands the encoding gene obtaining tumor antigen protein,
It is then attached to pMAL-p5X plasmid, convert bacillus coli DH 5 alpha, cultivate to logarithmic growth in the LB culture medium containing AMP
Phase, it is subsequently adding IPTG abduction delivering；Through the culture centrifugation of IPTG abduction delivering, obtain on culture precipitation and culture
Clearly；
(2) the culture precipitation resuspended rear ultrasonication of Tris buffer, centrifugation, supernatant precipitation carries out following operation respectively：
The Amylose resin of supernatant upper 8 post bed Tris buffer balances after micro-pore-film filtration, is washed with 12 post bed Tris buffer
With the Tris buffer solution elution containing maltose after washing, obtain target protein 1；
Precipitation adds the dissolving of 8M carbamide Tris solutions overnight, carries out dialysis Tris buffer and removes carbamide, upper 8 after filtration after centrifugation
The Amylose resin of post bed Tris buffer balance, with being buffered with the Tris containing maltose after 12 post bed Tris buffer solution
Liquid eluting, obtains target protein 2；
(3) culture supernatant is degerming through inner pressed membrane filtration, and gained filtrate concentrates through external-compression type filter membrane again；Concentrated solution passes through
10K filter membrane is dialysed 2 times, and after centrifugation, upper Amylose resin purification obtains target protein 3；
Wherein, step (2) and step (3) no sequencing, described protein 1, protein 2 and protein 3 are not similar shape
The tumor antigen protein of formula.
7. application in the vaccine of preparation treatment malignant tumor for the tumor antigen protein described in claim 1 or 2.
8. application according to claim 7 is it is characterised in that described malignant tumor is melanoma, pulmonary carcinoma and colon cancer
One or more of.
9. a kind of tumor vaccine is it is characterised in that composition includes the tumor antigen protein described in claim 1 or 2 and hydroxide
Aluminium adjuvant.
10. tumor vaccine according to claim 9, it is characterised in that endotoxin in vaccine content is less than 500Eu/mg, swells
Tumor antigen protein concentration is 1mg/ml.
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| CN201610831406.3A CN106432460A (en) | 2016-09-19 | 2016-09-19 | Tumor antigen protein and tumor vaccine |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109081873A (en) * | 2018-08-10 | 2018-12-25 | 固安鼎泰海规生物科技有限公司 | A kind of antitumor recombination NMM fused antigen Plasmid DNA vaccines |
| CN110464840A (en) * | 2019-09-06 | 2019-11-19 | 北京微九九科技有限公司 | A kind of preparation method of tumor vaccine and the tumor vaccine prepared using this method |
| CN111298111A (en) * | 2020-04-08 | 2020-06-19 | 长春康悦生物科技有限公司 | Medicine for treating and/or preventing cancer and application |
| WO2021110120A1 (en) * | 2019-12-04 | 2021-06-10 | 华中师范大学 | Anti-tumor vaccine molecule, preparation method therefor and use thereof |
| CN113713092A (en) * | 2021-08-05 | 2021-11-30 | 齐鲁工业大学 | Application of chemokine receptor CCR6 in preparation of antitumor drugs |
| CN114316066A (en) * | 2021-11-11 | 2022-04-12 | 元本(珠海横琴)生物科技有限公司 | MNR2 protein purification method |
| WO2023077924A1 (en) * | 2021-11-04 | 2023-05-11 | 元本(珠海横琴)生物科技有限公司 | Vaccine against pancreatic cancer, and medical use thereof |
| WO2023077925A1 (en) * | 2021-11-04 | 2023-05-11 | 元本(珠海横琴)生物科技有限公司 | Drug for treating and/or preventing cancer and use thereof |
| CN116751801A (en) * | 2023-07-21 | 2023-09-15 | 中国人民解放军海军军医大学 | Method for preparing tumor-associated antigen NY-ESO-1 by taking pGEX6P1 as vector and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8129506B2 (en) * | 2003-10-24 | 2012-03-06 | Genzyme Corporation | Modulation of the interaction of MUC1 with MUC1 ligands |
-
2016
- 2016-09-19 CN CN201610831406.3A patent/CN106432460A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8129506B2 (en) * | 2003-10-24 | 2012-03-06 | Genzyme Corporation | Modulation of the interaction of MUC1 with MUC1 ligands |
Non-Patent Citations (3)
| Title |
|---|
| BOUILLEZ A等: "NP_002447.4", 《GENBANK》 * |
| OOSTERKAMP,H.M.等: "AAD10858.1", 《GENBANK》 * |
| 张立新等: "肿瘤MUC1/Y的克隆及其胞外段在大肠杆菌中的可溶性表达、纯化和鉴定", 《中国生物化学与分子生物学报》 * |
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|---|---|---|---|---|
| CN109081873A (en) * | 2018-08-10 | 2018-12-25 | 固安鼎泰海规生物科技有限公司 | A kind of antitumor recombination NMM fused antigen Plasmid DNA vaccines |
| CN109081873B (en) * | 2018-08-10 | 2022-04-22 | 固安鼎泰海规生物科技有限公司 | Anti-tumor recombinant NMM fusion antigen plasmid DNA vaccine |
| CN110464840A (en) * | 2019-09-06 | 2019-11-19 | 北京微九九科技有限公司 | A kind of preparation method of tumor vaccine and the tumor vaccine prepared using this method |
| WO2021110120A1 (en) * | 2019-12-04 | 2021-06-10 | 华中师范大学 | Anti-tumor vaccine molecule, preparation method therefor and use thereof |
| CN111298111A (en) * | 2020-04-08 | 2020-06-19 | 长春康悦生物科技有限公司 | Medicine for treating and/or preventing cancer and application |
| CN113713092A (en) * | 2021-08-05 | 2021-11-30 | 齐鲁工业大学 | Application of chemokine receptor CCR6 in preparation of antitumor drugs |
| WO2023077924A1 (en) * | 2021-11-04 | 2023-05-11 | 元本(珠海横琴)生物科技有限公司 | Vaccine against pancreatic cancer, and medical use thereof |
| WO2023077925A1 (en) * | 2021-11-04 | 2023-05-11 | 元本(珠海横琴)生物科技有限公司 | Drug for treating and/or preventing cancer and use thereof |
| CN114316066A (en) * | 2021-11-11 | 2022-04-12 | 元本(珠海横琴)生物科技有限公司 | MNR2 protein purification method |
| CN114316066B (en) * | 2021-11-11 | 2023-07-14 | 元本(珠海横琴)生物科技有限公司 | Purification method of MNR2 protein |
| CN116751801A (en) * | 2023-07-21 | 2023-09-15 | 中国人民解放军海军军医大学 | Method for preparing tumor-associated antigen NY-ESO-1 by taking pGEX6P1 as vector and application |
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