CN105906705A - CD19 immunogen polypeptide and application thereof - Google Patents
CD19 immunogen polypeptide and application thereof Download PDFInfo
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- CN105906705A CN105906705A CN201610438413.7A CN201610438413A CN105906705A CN 105906705 A CN105906705 A CN 105906705A CN 201610438413 A CN201610438413 A CN 201610438413A CN 105906705 A CN105906705 A CN 105906705A
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Abstract
The invention belongs to the field of anticancer vaccines, discloses a CD19 immunogen polypeptide, particularly relates to an optimization polypeptide for the CD19 immunogen of an anticancer vaccine for enhancing T cell immunologic response and particularly relates to application of preparing medicine for tumor immune cells and T cell immune response enhancement and application of preparing medicine for CAR-T cell immune therapy, wherein an amino acid sequence is adopted as a brand new sequence and applied to preparing medicine for treating tumors. The CD19 immunogen polypeptide has the advantages that the brand new sequence is adopted, the CD19 immunogen polypeptide can be used for the immunogen for the tumor immune cell technology, T cells can be promoted, CD8+ immune response can be promoted, effective specific binding with MHC on the surfaces of the T cells can be achieved, binding of the T cells and tumor cells can be promoted, and tumor growth can be restrained inside tumor-bearing mice. The CD19 immunogen polypeptide is adopted as a target molecule of the cell therapy and applied to CAR-T and other cell therapies.
Description
Technical field
The present invention is relevant anti-cancer vaccine field.In particular it relates to be used as the CD19 of anti-cancer vaccine
Immunogenic optimization polypeptide, strengthens T cell immune response.
Background technology
B cell lymphoma is that the entity tumor that B cell occurs typically occurs in children and adult, is that one is attacked
Hit the cancer types of B cell in immune system.Including Hodgkin lymphoma and NHL.Its parting
Numerous, classical Hodgkin lymphoma and Nodular lymphocyte are principal mode Hodgkin lymphoma, are presently considered to be
Originate from the tumour of B cell.Diffusivity large B cell lymphoid tumor, follicular lymphoma, mucosa-associated lymphoid group
Knit lymthoma (MALT), small lymphocyte lymthoma/chronic lymphocytic leukemia, lymphoma mantle cell (MCL)
5 kinds of B cell NHLs are most commonly seen, account for the 3/4 of NHL.B cell lymphoma
Prognosis and treatment depend on lymphadenomatous particular type and stage and step.Current treatment means with chemotherapy are
Main, but chemotherapy is very big to actual bodily harm, later rehabilitation passive protective physical fitness also can become very poor.
Researcher finds that B cell lymphoma cell surface receptor CD19 plays key effect.CD19 is one
Plant B cell specific transferring film glycoprotein, be expressed in B system cell (not including mature plasme cell) and follicular dendritic
On shape cell, CD19 is a kind of important signal transduction molecule, the growth activation of regulation bone-marrow-derived lymphocyte and work
Change, regulation bone-marrow-derived lymphocyte antigen receptor or other surface receptors signal threshold value in play an important role, be with
Bone-marrow-derived lymphocyte breaks up, activate, breed and antibody produces relevant important membrane antigen, is diagnosis bone-marrow-derived lymphocyte
It is the preferably mark of tumour, such as chronic lymphocytic leukemia (CLL) and ALL (ALL).
There is the antibody of the clinical trial testing antagonism CD19 acceptor of tumour at present.This antibody can not only kill
Dead bone-marrow-derived lymphocyte system tumour, B cell that the most also can be healthy, thus weaken immune system.Researcher wishes
Prestige can design more effective methods for the treatment of, can optionally kill tumour cell, retains the B of health simultaneously
Cell, produces clinical therapeutic efficacy safer, effective.
Tumour cell immunization therapy be a kind of emerging, there is the tumor treatment model of significant curative effect, be a kind of from
The novel method for the treatment of of body immunity anticancer.It uses biotechnology and biologic product to gathering from the patient
The method that immunocyte feeds back in patient body after carrying out in vitro culture and amplification, excites, and strengthens body self
Immunologic function, thus reach to treat the purpose of tumour.Tumour cell immunotherapy is continue operation, radiation and chemotherapy
The fourth-largest oncotherapy technology afterwards.
CD19 can be as the specific immunogens of tumor vaccine.But, CD19, by the albumen of 95KDa, divides
Son amount is bigger, it is more difficult to obtain the CD19 that purity is high.So, the Specific T cell immunity in later stage can not only be given
Bring difficulty, and the autoimmunity of patient can be caused.Therefore, the invention provides a kind of high specificity, pure
Spend higher micromolecule polypeptide as immunogene.
Summary of the invention
Goal of the invention
The present invention provides a kind of CD19 immunogene polypeptide, can be used for the immunogene of tumour cell immunity, strengthens T
Cellullar immunologic response, has the advantages that molecular weight is little, specific immunogenic is strong.
Technical scheme
Technical program of the present invention lies in providing a kind of CD19 immunogene polypeptide, sequence is
DNAVLQCLKGTSDGPTQQLTWSRESPLK FLKLSLGLPGLGIHMRPLASW
(SEQ ID NO:1).This amino acid sequence is brand-new sequence, in preparation in tumor
Application.Especially in preparation for tumor vaccine cells, strengthen the application in T cell immune response medicine,
And the application of CAR-T cellular immunotherapy it is used in preparation.
Beneficial effect
The CD19 immunogene polypeptide of the present invention, for brand-new sequence, can be used in tumor vaccine cells technology
Immunogene.Having the beneficial effects that (1) promotes T cell, (2) promote CD8+ immune response, (3) energy and T
The MHC of cell surface is the most specific binding, and (4) promote the combination of T cell and tumour cell, (5)
In tumor-bearing mice body, the growth of tumour can be suppressed.As the target molecules of cell therapy, it is applied to CAR-T
Etc. cell therapy.
Detailed description of the invention
Polypeptide is by Shanghai raw work gill synthesis.
Embodiment 1
The proliferation function of T lymphocyte: aseptic take mouse spleen, 1640 culture mediums clean 3 times, and 5ml injects
Device core grinds, and 200 eye mesh screens filter, and make single cell suspension, and centrifugal (1000r/min, 5min) abandons
Clearly, Tris-NH4CL cracks red blood cell, and ice-water bath stands 3-5min, centrifugal (1000r/min, 5min), abandons
Supernatant, with aseptic cold PBS washed cell twice.The RPMI 1640 being eventually adding 10% calf serum cultivates
Liquid (5ml) suspension cell, cell count, adjusting cell concentration is 5 × 106Individual/ml, in 96 well culture plates
Middle cultivation.
Experiment set blank group, model group (concanavalin A, sigma company buy), each dosage of polypeptide (3,
6,12mg/ml) group.After each group is separately added into spleen lymphocyte suspension 100 μ l/ hole, blank group adds
RPMI-1640 100 μ l, model group adds ConA (final concentration of 5 μ g/ml), and polypeptide each dosage group is adding
ConA (final concentration of 5 μ g/ml) is added on the basis of the polypeptide of variable concentrations.37 DEG C of cell culture incubator static gas wave refrigerator
48h, cultivates every hole after terminating and adds 20 μ l MTT, continue to cultivate 4h, finally discard all solution in every hole, often
Hole adds 100 μ lDMSO, concussion, with ELIASA detection 570nm at OD value, every hole set 5 parallel.
Table 1 polypeptide proliferation function to T lymphocyte
* P < 0.05, * * P < 0.01 is compared with model group.
Experimental result is shown in Table 1, compares with model group, and many Toplink promote the propagation of mouse spleen lymphocyte,
Dosage be 3～12mg/ml scope present good dose-dependence, with the rate of increase of 20mg/ml
Height, is 153.6%.
Embodiment 2
The immunogenicity determining of CD19 immunogene polypeptide: use Flow Cytometry to measure immunogene polypeptide pair
The impact of CD8+T cell.6-8w C57BL/6 in age mouse, mouse is randomly divided into 4 groups, often group 10.(1)
Blank group；(2) immunogene polypeptide low dose group；(3) dosage group in immunogene polypeptide；(4) polypeptide high dose
Group.At the 0th, 7,14 days of test, carry out immunity respectively.Scheme is: blank group adds same volume
1,2,4mg/Kg solvent, experimental group polypeptide sets 3 dosage:, multiple spot note near mouse back lymph node
Penetrate.After 30 days, eyeball takes blood 0.8ml, anticoagulant heparin, takes haemocyte layer, split with red blood cell after centrifugal for blood
Solve liquid and crack red blood cell, wash away the red blood cell of cracking, then (purchase with the anti-CD8+-FITC of fluorescently-labeled monoclonal
From Beijing Hua Taixin bio-medical technology Co., Ltd) hatch, fixing after, carry out Flow Cytometry mensuration,
Evaluate the impact on CD8+T cell of the immunogene polypeptide.
The effect to CD8+T lymphocyte of table 2 polypeptide
* P < 0.05, * * P < 0.01 is compared with model group.
Experimental result is shown in Table 2, compares with blank group, and many Toplink promote mouse CD8+T lymphocyte, at dosage
Be 1～4mg/Kg scope present good dose-dependence, the rate of increase with 4mg/Kg is the highest, for
156.82%.
Embodiment 3
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 competitive affine method of receptor-ligand.Will be fixing dense
Degree is polypeptide and the polypeptide addition reaction of variable concentrations (1-50 μm ol/L) of the mark 125I of 50 μm ol/L
(phosphate buffer and MHC, described MHC kit is purchased from the upper ingression limited public affairs of Ke's biotechnology to system
, there are HLA-A1, A2, A3, A11 and A24 in department in kit), reaction system is formed two kinds of compounds,
I.e. candidate polypeptide MHC compound and 125I labeling polypeptide compound.Candidate polypeptide is competing with 125I labeling polypeptide
Strive the combination with MHC.Separate free many with hyperfiltration (product type Microcon 30, Amicon company)
Peptide and polypeptide MHC compound, then measure the 125I exit dose of polypeptide MHC compound, by this exit dose
Compare with the exit dose of the polypeptide MHC compound (not adding the sample of candidate polypeptide) not having Reverse transcriptase,
Ask candidate polypeptide at suppression 50% labeling polypeptide concentration when MHC is combined, i.e. IC50.Thus obtain, many
Peptide is respectively 8.67,6.87,3.22,6.53 to the IC50 value of HLA-A1, A2, A3, A11 and A24
With 0.22 μm ol/L, all meet the standard (≤10 μm ol/L) of positive polypeptides.Therefore, polypeptide is for having effectively
The immunogene polypeptide of binding ability.
Embodiment 4
The T cell binding tests of CD19 immunogene polypeptide: use rosette to evaluate T cell and people
Diffuse Large B-Cell Lymphoma (diffuse large B-cell lymphoma, DLBCL) activating B cell
The binding ability of (activated B-cell, ABC) type cell line OCI-Ly3.Aseptic take Thymus of Guinea Pigs, 1640
Culture medium cleans 3 times, and 5ml piston grinds, and 200 eye mesh screens filter, and make single cell suspension, centrifugal
1000 revs/min, 10 minutes, abandon supernatant, adjust cell concentration for 3 × 10 with Hank ' s liquid6/ml.In 96 holes
Culture plate is cultivated.
Experiment sets blank group, each dosage of polypeptide (3,6,12mg/ml) group.Each group is separately added into thymus gland and drenches
Behind bar cell suspension 100 μ l/ hole, blank group adds RPMI-1640 500 μ l, and polypeptide each dosage group is adding
The polypeptide of variable concentrations.37 DEG C of cell culture incubators, cultivate 48h, cultivate and add 100 μ l/ holes in every hole after terminating
(cell concentration is 1 × 10 to OCI-Ly3 cell6/ ml, containing 10% hyclone), mixing, 500 revs/min, centrifugal
5 minutes.Abandoning supernatant, add a small amount of glutaraldehyde, rock gently, make cell suspend, add violet stain, 400 show
Micro mirror is observed.The T lymphocyte of all more than 3 tumour cells of combination is garland positive cell.Count 100 T
Cell is formed the lymphocyte percentage of garland, is the Percentage bound of T cell and OCI-Ly3 cell.Every hole sets
5 parallel.
Experimental result is shown in Table 3, compares with blank group, and CD19 immunogene polypeptide can increase T cell with conspicuousness
With the Percentage bound (P < 0.01) of OCI-Ly3 cell, the Percentage bound of low middle high dose group is respectively 34.35,45.30
With 54.72%.Good dose-dependence is presented in the scope that dosage is 3～12mg/ml.
The impact that T cell is combined by table 3 polypeptide
* P < 0.05, * * P < 0.01 is compared with blank group.
Embodiment 5
The internal vigor of employment diffusivity large B cell lymphoid tumor Transplanted tumor model detection CD19 immunogene polypeptide.
6-8 SCID mice in age, mouse is randomly divided into 4 groups, male and female half and half, often group 10.(1) blank group；
(2) polypeptide low dose group；(3) dosage group in polypeptide；(4) polypeptide high dose group.Set up people's diffusivity big
B cell lymphoma Transplanted tumor model, the 3rd, 5,7 days after inoculated tumour cell, carry out immunity respectively.
1,2,4mg/Kg scheme is: blank group adds the solvent of same volume, and experimental group polypeptide sets 3 dosage:,
Multi-point injection around tumour.After 21 days, observe mouse survival quantity, calculate survival rate.Result shows,
Dosage 1,2, the polypeptide of 4mg/Kg can effectively protect mouse, improve the survival rate of tumor-bearing mice, survival rate
Reach 65.87,70.98 and 82.98%.
SEQUENCE
LISTING
<110>
Pu Luoda bio tech ltd, Suzhou
<120>
A kind of CD19 immunogene polypeptide and application thereof
<130>
<160>
1
<170>
PatentIn version 3.3
<210>
1
<211>
37
<212>
PRT
<213>
Artificial sequence
<400>
1
Asp Asn Ala
Val Leu Gln Lys Lys Lys Leu Ser Asp Gly Pro Thr Gln
1
5
10
15
Gln Leu Thr
Trp Ser Arg Glu Ser Pro Leu Lys Leu Leu Leu Ser Leu
20
25
30
Gly Leu Pro
Gly Leu
35
Claims (4)
1. a CD19 immunogene polypeptide, it is characterised in that: described peptide sequence is SEQ ID NO:1.
Polypeptide the most according to claim 1 is in the application in tumor.
Application the most according to claim 2, it is characterised in that: described polypeptide for tumor vaccine cells, strengthens the application in T cell immune response medicine in preparation.
Application the most according to claim 2, it is characterised in that: the application of CAR-T cellular immunotherapy it is used in preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610438413.7A CN105906705A (en) | 2016-06-19 | 2016-06-19 | CD19 immunogen polypeptide and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610438413.7A CN105906705A (en) | 2016-06-19 | 2016-06-19 | CD19 immunogen polypeptide and application thereof |
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| Publication Number | Publication Date |
|---|---|
| CN105906705A true CN105906705A (en) | 2016-08-31 |
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ID=56750490
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610438413.7A Pending CN105906705A (en) | 2016-06-19 | 2016-06-19 | CD19 immunogen polypeptide and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111544585A (en) * | 2019-02-11 | 2020-08-18 | 北京卡替医疗技术有限公司 | Adjuvant capable of boosting immune cells to expand in vivo |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101612126A (en) * | 2009-07-21 | 2009-12-30 | 浙江大学 | The preparation and the purposes of CD19 monoclonal antibody immunity liposome |
| CN101943704A (en) * | 2010-04-30 | 2011-01-12 | 北京大学人民医院 | Novel application of anti-CD19 antibody and anti-CD5 antibody |
-
2016
- 2016-06-19 CN CN201610438413.7A patent/CN105906705A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101612126A (en) * | 2009-07-21 | 2009-12-30 | 浙江大学 | The preparation and the purposes of CD19 monoclonal antibody immunity liposome |
| CN101943704A (en) * | 2010-04-30 | 2011-01-12 | 北京大学人民医院 | Novel application of anti-CD19 antibody and anti-CD5 antibody |
Non-Patent Citations (1)
| Title |
|---|
| 冯元怡等: "ConA刺激小鼠T淋巴细胞期事件研究", 《北京医科大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111544585A (en) * | 2019-02-11 | 2020-08-18 | 北京卡替医疗技术有限公司 | Adjuvant capable of boosting immune cells to expand in vivo |
| WO2020164465A1 (en) * | 2019-02-11 | 2020-08-20 | 北京卡替医疗技术有限公司 | Adjuvant capable of promoting expansion of immune cells in vivo |
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