CN106367406A - UDP glycosyl transferase immunogen polypeptide and applications thereof - Google Patents
UDP glycosyl transferase immunogen polypeptide and applications thereof Download PDFInfo
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- CN106367406A CN106367406A CN201610741929.9A CN201610741929A CN106367406A CN 106367406 A CN106367406 A CN 106367406A CN 201610741929 A CN201610741929 A CN 201610741929A CN 106367406 A CN106367406 A CN 106367406A
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- polypeptide
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- glycosyl transferase
- immunogen
- tumor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
Abstract
The invention discloses UDP glycosyl transferase immunogen polypeptide, belongs to the field of anti-cancer vaccines, and in particular relates to optimized polypeptide of UDP glycosyl transferase immunogen, which can be taken as anti-cancer vaccines, used for enhancing T-cell immune response. An amino acid sequence is a brand-new sequence, is applied to the preparation of medicines for treating tumor, especially, the preparation of medicines for enhancing the T-cell immune response, as for tumor immunity cells, and the preparation of the medicines for CAR-T cell immunotherapy. The UDP glycosyl transferase immunogen polypeptide has the beneficial effects the brand-new sequence is adopted, and the UDP glycosyl transferase immunogen polypeptide can be applied to the immunogen in the tumor immunity cell technology, and has the functions of (1) promoting the T cell proliferation, (2) promoting the CD8+ immune response, (3) realizing the effective specific binding with MHC on the surfaces of the T cells, (4) promoting the binding between the T cells and the tumor cells, and (5) inhibiting the tumor growth in the bodies of the pancreatic cancer tumor-bearing mice. The UDP glycosyl transferase immunogen polypeptide is applied to the cell therapy including CAR-T as the target molecule of the cell therapy.
Description
Technical field
The present invention is relevant anti-cancer vaccine field.In particular it relates to the udp glycosyl transfer as anti-cancer vaccine
Enzyme immunogenic optimization polypeptide, strengthens t cellullar immunologic response.
Background technology
The sugared of catalytic activation connects glycosyl transferase (glycoprotein glucosyltransferase) in vivo
It is connected to different acceptor molecules, such as in albumen, nucleic acid, oligosaccharide, fat and small molecule, glycosylated product has work(a lot of biology
Energy.Glucosyltransferase (udp- glycosyl transferase) is the enzyme (glu- enzyme) only shifting glucosyl group in enzyme reaction, Fructus Vitis viniferae
FscMⅠ is the enzyme shifting together with the glycosidic bond of glucose during transfer.Udp- glycosyl transferase is glycoprotein, glycolipid
One of biosynthetic key enzyme of middle sugar chain.The cycle of the expression of udp- glycosyl transferase and cell is closely relevant. divides in cell
In the change stage, the gene of many glycosyl transferases is expression, occurs in that a series of saccharide heteroplasmons as differentiation antigen for this；One
Denier is reached maturity, and occurs in that other Glycoforms in cell surface.As transfructosylase, in mature cell, activity is very
Height, will produce canceration, occur in that the differentiation antigen of early stage simultaneously.Poly n- second phthalein Lactose in the bonded sugar chain of n- glucosides
The appearance of amine chain is regarded as the important symbol of tumor, and this kind of sugar chain can reduce the adhesion between cell and substrate, be conducive to cancer
The further invasion of cell.Therefore, the improper expression of udp- glycosyl transferase activity and the disease such as tumor, immune system
Occur, development has substantial connection, its inhibitor can be used for the new drug development of the diseases such as antitumor, anti-immunity system.
There is the antibody of the clinical trial testing antagonism udp- glycosyl transferase of tumor at present.This antibody can not only kill
Dead b lymphocytic tumors, simultaneously also can health b cell, thus weakening immune system.Research worker wants to design more
Effectively Therapeutic Method, can optionally kill tumor cell, retain the b cell of health simultaneously, produce safer, effectively face
Bed therapeutic effect.
Tumor cell immunization therapy is a kind of tumor treatment model that is emerging, having significant curative effect, is that one kind itself is exempted from
The novel method for the treatment of of epidemic disease anticancer.It is with biotechnology and biological preparation, the immunocyte gathering from the patient to be carried out
The method feeding back in patient body after In vitro culture and amplification, to excite, enhancing body autoimmune function, thus reaching treatment
The purpose of tumor.Tumor cell immunotherapy is the fourth-largest oncotherapy technology after operation, radiation and chemotherapy.
Udp glycosyl transferase can be used as the specific immunogens of tumor vaccine.However, udp glycosyl transferase molecular weight
Larger it is more difficult to obtain the high udp glycosyl transferase of purity.So, not only can bring to the specific t cell immunity in later stage tired
Difficulty, and the autoimmune of patient can be led to.Therefore, the invention provides a kind of high specificity, purity is many compared with high small molecule
Peptide is as immunogen.
Content of the invention
Goal of the invention
The present invention provides a kind of udp glycosyl transferase immunogen polypeptide, can be used for the immunogen of tumor cell immunity, strengthens
T cellullar immunologic response, has the characteristics that molecular weight is little, specific immunogenic is strong.
Technical scheme
Technical program of the present invention lies in providing a kind of udp glycosyl transferase immunogen polypeptide, sequence is
Vgiifntenvvvekscesnsissyiraksraleflpmdqakrrrsd (seq id no:1).This aminoacid sequence is complete
New sequence, the application in preparation is for tumor.Especially it is used for tumor vaccine cells in preparation, strengthen t cell
Application in immunne response medicine, and it is used for the application of car-t cellular immunotherapy in preparation.
Beneficial effect
The udp glycosyl transferase immunogen polypeptide of the present invention, for brand-new sequence, can be used in tumor vaccine cells technology
Immunogen.Have the beneficial effects that (1) promotes t cell proliferation, (2) promote cd8+ immunne response, and (3) can be with t cell surface
Mhc effectively specifically binds, and (4) promote the combination of t cell and tumor cell, and (5), in cancer of pancreas tumor-bearing mice body, can press down
The growth of tumor processed.As the target molecules of cell therapy, it is applied to the cell therapys such as car-t.
Specific embodiment
Polypeptide is by Shanghai raw work gill synthesis.
Embodiment 1
Detect the internal vigor of udp glycosyl transferase immunogen polypeptide with tumor model.
6-8 week old c57bl/6 mice, mice is randomly divided into 4 groups, male and female half and half, every group 10.(1) blank group；(2) many
Peptide low dose group；(3) polypeptide middle dose group；(4) polypeptide high dose group.Set up pancreatic tumour model, in inoculated tumour cell
The 3rd, 5,7 days afterwards, carry out immunity respectively.Scheme is: blank group adds the solvent of same volume, and experimental group polypeptide sets 3 agent
Amount: 5,10,20mg/kg, multi-point injection around tumor.After 21 days, observe mouse survival quantity, calculate survival rate.Result shows
Show, polypeptide can protect mice effectively, improve the survival rate of tumor-bearing mice, the survival rate of 20mg/kg reaches 47.4%.
Embodiment 2
Application competitive receptor-ligand affine method test polypeptide and the binding ability of mhc:
Binding ability various with hla for polypeptide is judged by the affine method of competitive receptor-ligand.Fixed concentration is 50 μ
The polypeptide of the polypeptide of labelling 125i of mol/l and variable concentrations (1-50 μm of ol/l) adds reaction system (phosphate buffer
And mhc, described mhc test kit is purchased from upper ingression Ke bio tech ltd, has hla-a1, a2, a3, a11 in test kit
And a24), reaction system forms two kinds of complex, i.e. candidate polypeptide mhc complex and 125i labeling polypeptide complex.Treat
Survey the combination of polypeptide and the competition of 125i labeling polypeptide and mhc.With ultra-filtration (product type microcon 30, amicon company)
Separate free polypeptide and polypeptide mhc complex, then measure the 125i exit dose of polypeptide mhc complex, by this exit dose with do not have
The exit dose of the polypeptide mhc complex sample of candidate polypeptide (not plus) of competitive suppression compares, and seeks candidate polypeptide in suppression
Make concentration when mhc is combined for 50% labeling polypeptide, i.e. ic50.Thus obtain, polypeptide is to hla-a1, a2, a3, a11 and a24
Ic50 value is respectively 7.43,4.32,8.51,2.67 and 0.32 μm of ol/l, all meets the standard (≤10 μm) of positive polypeptides.Cause
This, polypeptide is the immunogen polypeptide with effective binding energy power.
Embodiment 3
The proliferation function of t lymphocyte: aseptic take mouse spleen, 1640 culture medium clean 3 times, 5ml piston grind,
200 mesh sieve net filtrations, make single cell suspension, are centrifuged (1000r/min, 5min), abandon supernatant, tris-nh4Cl cracks red thin
Born of the same parents, ice-water bath stands 3-5min, is centrifuged (1000r/min, 5min), abandons supernatant, with aseptic cold pbs washed cell twice.Finally
Add rpmi RPMI-1640 (5ml) suspension cell of 10% calf serum, cell counting, adjustment cell concentration is 5 × 106
Individual/ml, cultivates in 96 well culture plates.
Experiment set blank control group, model group (purchase of canavaline a, sigma company), each dosage of polypeptide (5,10,
20mg/ml) group.After each group is separately added into spleen lymphocyte suspension 100 μ l/ hole, blank control group adds RPMI-1640 100
μ l, model group adds cona (final concentration of 5 μ g/ml), and each dosage group of polypeptide adds on the basis of the polypeptide adding variable concentrations
Cona (final concentration of 5 μ g/ml).37 DEG C of cell culture incubator static gas wave refrigerator 48h, culture adds 20 μ l mtt in every hole after terminating, and continues
Continuous culture 4h, finally discards all solution in every hole, and every hole adds 100 μ ldmso, concussion, detects od value at 570nm with microplate reader,
Every hole set 5 parallel.
Experimental result shows, compares with model group, and polypeptide can promote the propagation (p < 0.01) of mouse spleen lymphocyte,
Dosage is that the scope of 5～20mg/ml assumes good dose-dependence, and the rate of increase is respectively 59.98,87.85 and
96.12%.
Embodiment 4
The immunogenicity determining of udp glycosyl transferase immunogen polypeptide: immunogen polypeptide is measured using Flow Cytometry
Impact to cd8+t cell.6-8w age c57bl/6 mice, male and female half and half, mice is randomly divided into 4 groups, every group 10.(1) blank
Group；(2) immunogen polypeptide low dose group；(3) immunogen polypeptide middle dose group；(4) polypeptide high dose group.Test the 0th, 7,
14 days, carry out immunity respectively.5,10 scheme is: blank group adds the solvent of same volume, and experimental group polypeptide sets 3 dosage:,
20mg/kg, multi-point injection near mouse back lymph node.After 30 days, eyeball takes blood 0.8ml, and anticoagulant heparin, after blood is centrifuged
Take hemocyte layer, crack erythrocyte with erythrocyte cracked liquid, wash away the erythrocyte of cracking, then use fluorescently-labeled monoclonal anti
After cd8+-fitc (purchased from Beijing Hua Taixin bio-medical technology company limited) incubation, fixation, carry out Flow Cytometry survey
Fixed, evaluate the impact to cd8+t cell for the immunogen polypeptide.
The effect to cd8+t lymphocyte for table 1 polypeptide
* p < 0.05, * * p < 0.01 is compared with blank group.
Experimental result is shown in Table 1, compares with blank group, and polypeptide can promote mice cd8+t lymphocyte, dosage be 5～
The scope of 20mg/kg assumes good dose-dependence, with the rate of increase highest of 20mg/kg, is 107.82%.
Embodiment 5
The t Cell binding assay of udp glycosyl transferase immunogen polypeptide: t cell and pancreas are evaluated using rosette
The binding ability of adenocarcinoma cell sw1990.Aseptic take Thymus of Guinea Pigs, 1640 culture medium clean 3 times, 5ml piston grind, 200
Mesh sieve net filtration, makes single cell suspension, is centrifuged 1000 revs/min, 10 minutes, abandons supernatant, adjusts cell concentration for 3 with hank ' s liquid
×106/ml.Cultivate in 96 well culture plates.
Experiment sets blank control group, each dosage of polypeptide (5,10,20mg/ml) group.Each group is separately added into thymic lymphocytes
Behind suspension 100 μ l/ hole, blank control group adds RPMI-1640 500 μ l, and each dosage group of polypeptide is adding the many of variable concentrations
Peptide.37 DEG C of cell culture incubators, cultivate 48h, and culture adds 100 μ l/ hole sw1990 cells in every hole after terminating (cell concentration is 1
×106/ ml, containing 10% hyclone), mix, 500 revs/min, be centrifuged 5 minutes.Abandon supernatant, plus a small amount of glutaraldehyde, gently shake
Dynamic, make cell suspension, plus violet stain, 400 power microscopes observations.The t lymphocyte of all more than 3 tumor cells of combination is flower
Ring positive cell.Form the lymphocyte percentage of garland, the as knot of t cell and sw1990 cell in 100 t cells of numeration
Conjunction rate.Every hole set 5 parallel.
Experimental result shows, compares with blank group, and udp glycosyl transferase immunogen polypeptide can increase t cell with significance
With the combination rate (p < 0.01) of sw1990 cell, the combination rate respectively 75.13,88.47 and 97.14% of low middle high dose group.
Assume good dose-dependence in the scope that dosage is 5～20mg/ml.
sequence listing
<110>Suzhou Pu Luoda bio tech ltd
<120>udp glycosyl transferase immunogen polypeptide and application thereof
<130>
<160> 1
<170> patentin version 3.3
<210> 1
<211> 46
<212> prt
<213>artificial sequence
<400> 1
val gly ile ile phe asn thr glu asn val val val glu lys ser cys
1 5 10 15
glu ser asn ser ile ser ser tyr ile arg ala lys ser arg ala leu
20 25 30
glu phe leu pro met asp gln ala lys arg arg arg ser asp
35 40 45
Claims (4)
1. a kind of udp glycosyl transferase immunogen polypeptide it is characterised in that: described peptide sequence be seq id no:1.
2. application in preparation is for tumor for the polypeptide according to claim 1.
3. according to claim 2 application it is characterised in that: described polypeptide preparation be used for tumor vaccine cells, increase
Application in strong t cellullar immunologic response medicine.
4. application according to claim 3 is it is characterised in that described polypeptide is used for car-t cellular immunotherapy in preparation
Medicine in application.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610741929.9A CN106367406A (en) | 2016-08-28 | 2016-08-28 | UDP glycosyl transferase immunogen polypeptide and applications thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610741929.9A CN106367406A (en) | 2016-08-28 | 2016-08-28 | UDP glycosyl transferase immunogen polypeptide and applications thereof |
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| Publication Number | Publication Date |
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| CN106367406A true CN106367406A (en) | 2017-02-01 |
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| CN201610741929.9A Pending CN106367406A (en) | 2016-08-28 | 2016-08-28 | UDP glycosyl transferase immunogen polypeptide and applications thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1345963A (en) * | 2000-09-29 | 2002-04-24 | 上海博德基因开发有限公司 | Novel polypeptide-UDP glycosyltransferase (UGT) and cobalamin conjugated protein 9.57 and polynucleotide for encoding said polypeptide |
| CN101198620A (en) * | 2005-06-17 | 2008-06-11 | 曼康公司 | Methods and compositions to elicit multivalent immune responses against dominant and subdominant epitopes, expressed on cancer cells and tumor stroma |
| CN105859865A (en) * | 2016-05-05 | 2016-08-17 | 南方医科大学 | Dual anti-tumor polypeptide based on Eps8-EGFR binding domain |
-
2016
- 2016-08-28 CN CN201610741929.9A patent/CN106367406A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1345963A (en) * | 2000-09-29 | 2002-04-24 | 上海博德基因开发有限公司 | Novel polypeptide-UDP glycosyltransferase (UGT) and cobalamin conjugated protein 9.57 and polynucleotide for encoding said polypeptide |
| CN101198620A (en) * | 2005-06-17 | 2008-06-11 | 曼康公司 | Methods and compositions to elicit multivalent immune responses against dominant and subdominant epitopes, expressed on cancer cells and tumor stroma |
| CN105859865A (en) * | 2016-05-05 | 2016-08-17 | 南方医科大学 | Dual anti-tumor polypeptide based on Eps8-EGFR binding domain |
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