CN110922492B - Fusion peptide, CTP-mediated DC vaccine for inducing CML cellular immune response and preparation method thereof - Google Patents
Fusion peptide, CTP-mediated DC vaccine for inducing CML cellular immune response and preparation method thereof Download PDFInfo
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/25—Tumour necrosing factors [TNF]
Abstract
The invention discloses a fusion peptide, wherein the amino acid sequence of the fusion peptide is shown as SEQ ID No. 1 or 2. Also disclosed is an isolated polynucleotide encoding the aforementioned fusion peptide. Also disclosed is a DC vaccine for CTP-mediated induction of a CML cellular immune response, obtained by co-incubating the aforementioned fusion peptide with mature dendritic cells. Also discloses a preparation method of the vaccine, which comprises the following steps: 1) synthesizing the fusion peptide sequence; 2) culturing mature dendritic cells; 3) and co-incubating and washing the fusion peptide and the mature dendritic cells to obtain the DC vaccine. After a mouse is immunized by the dendritic cell vaccine, specific cellular immune response in the mouse is stimulated, cytotoxic T lymphocytes are induced and generated, and a slow granular cell line BaF3-P210 is killed, so that the feasibility of CTP mediated formation of DC vaccine and DC vaccine induced immune efficacy is proved.
Description
Technical Field
The invention relates to the technical field of tumor therapeutics and immunology, in particular to a fusion peptide, a CTP-mediated DC vaccine for inducing CML cellular immune response and a preparation method thereof.
Background
Chronic Myelogenous Leukemia (CML) is a group of malignant proliferative tumors derived from bone marrow hematopoietic stem cells. The main pathogenic mechanism is that the gene is derived from abl gene on chromosome 9 and bcr gene on chromosome 22, and the gene is disrupted, translocated and fused to form bcr-abl fusion gene. The gene can code and generate BCR-ABL fusion protein with strong tyrosine kinase activity, thereby promoting the occurrence and development of diseases. Currently, the clinical application of tyrosine kinase inhibitors such as imatinib, dasatinib and nilotinib, developed for the tyrosine kinase activity of BCR-ABL fusion proteins, has led to a milestone progression for chronic myelogenous therapy, but fails to clear the malignant clones of CML, leading to relapse and development of drug resistance.
Adoptive Cellular Immunotherapy (ACI), such as Tumor Infiltrating Lymphocytes (TIL), Lymphokine Activated Killer cells (LAK), Natural Killer cells (Natural Killer, NK), and Dendritic Cells (DC), has been applied to clinical applications in sequence, and has good therapeutic effects and fewer adverse reactions.
anti-CML immunotherapy has received much attention because of its ability to produce specific clearance of chronic myeloid leukemia cells. BCR-ABL fusion proteins encoded by BCR-ABL fusion genes are CML-specific cytogenetic markers. The research reports that the antigenicity of the BCR-ABL fusion site is limited in the BCR-ABL fusion site and is presented to the cell surface by MHC class molecules, and the amino acid sequence does not exist in normal cells, so the BCR-ABL fusion site is a very ideal cellular immunotherapy target. This is the basis for immunotherapy of chronic myeloid leukemia. It has been shown that BCR-ABL antigen-specific Cytotoxic T lymphocytes (C)TL) response plays a critical role in anti-CML immunity, presenting exogenous antigen to CD8 by loading DCs (dendritic cells) with BCR-ABL antigen+T lymphocyte, inducing to form specific CTL response has become a new CML treatment strategy, but the treatment effect is not significant because the presenting efficiency of exogenous antigen through DC is not high.
Dendritic cells are the antigen presenting cells with the most powerful functions at present, and a cross antigen presenting path also exists in cells. It can stimulate the body to generate specific cellular immune response after cross-presenting exogenous tumor antigen. The efficiency of cross antigen presentation directly determines the immune killing effect. However, the existing antigen loading systems can not fully stimulate the potential of cross antigen presentation of DC vaccines, which is a great problem which puzzles immunologists and clinical tumor workers for a long time and also is a bottleneck problem of improving the anti-CML immunotherapy efficiency.
The theory of cross antigen presentation considers that exogenous antigen forms endocytosis body after being taken by DC through endocytosis dependent path, exogenous antigen must enter cytoplasm from endocytosis body 'leakage' to be regarded as endogenous antigen by DC, then processing is carried out, and the exogenous antigen enters MHC-I molecule cross presentation path. In other words, to activate the cross-presentation potential of DCs, exogenous antigens must be "internalized" by cytoplasmic localization. Currently, various novel antigen loading tools such as cell penetrating peptide, liposome, nanoparticle and the like have been developed based on the cross antigen presentation theory, but because the above approaches still enter the DC through endocytosis to deliver the antigen, the leakage rate of the antigen leaking from the pinocytosis into the cytoplasm is low, so that the development of the DC cross antigen presentation potential is limited, and the CTL response cannot be activated strongly. Therefore, if the exogenous antigen can directly penetrate the DC envelope and be localized in the cytoplasm by the "endocytosis-independent pathway", the problem of "blocking cross presentation due to low endogenous efficiency of the exogenous antigen" can be effectively solved.
Cytoplasmic Transduction Peptide (CTP) is a newly reported transduction peptide that can carry a peptide fragment to penetrate the cell membrane and localize to the cytoplasm exclusively, and functions in transduction in an "endocytosis-independent" mechanism. Our previous experiments demonstrated that CTP showed high transduction activity and cytoplasmic localization bias in mouse promyelocytes, pre-B lymphocytes and various human blood system progenitors, which were difficult to transfect by various conventional methods. It has no obvious toxic side effect on cell because it has no influence on nucleus.
Disclosure of Invention
The invention aims to provide a fusion peptide, a CTP-mediated DC vaccine for inducing CML cellular immune response and a preparation method thereof.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a fusion peptide, the amino acid sequence of the fusion peptide is shown as SEQ ID No. 1 or 2.
An isolated polynucleotide encoding the fusion peptide described above.
A DC vaccine for inducing CML cell immune response mediated by CTP, which is obtained by co-incubating the fusion peptide with mature dendritic cells.
The preparation method of the DC vaccine comprises the following steps:
1) synthesizing the fusion peptide sequence of claim 1;
2) culturing mature dendritic cells;
3) and co-incubating and washing the fusion peptide and the mature dendritic cells to obtain the DC vaccine.
The specific method for culturing the mature dendritic cells in the step 2) comprises the following steps: extracting mouse bone marrow cells, lysing erythrocytes, culturing in 1640 medium containing granulocyte-macrophage colony stimulating factor, interleukin-4, and 10% fetal calf serum for 7 days, and adding tumor necrosis factor-alpha to stimulate dendritic cells to mature on the eighth day.
The invention has the beneficial effects that: designing an antigen peptide segment which is fused with CTP and is derived from a BCR-ABL fusion site, wherein the antigen is derived from 9 amino acid sequences of a BCR-ABL oncoprotein fusion region in the cytoplasm of the chronic granulocytic cell and can be presented to the surface of the chronic granulocytic leukemia cell through MHC class molecules. The synthesized fusion peptide segment directly penetrates through DC cell membrane and is positioned in cytoplasm under the mediation of CTP, and specific CTL response of CML is induced by activating 'MHC-I class molecule cross presentation pathway of exogenous antigen' so as to utilize efficient cytoplasm positioning potential of Cytoplasm Transduction Peptide (CTP). Experiments prove that after a mouse is immunized by the dendritic cell vaccine, specific cellular immune response in the mouse is stimulated, cytotoxic T lymphocytes are induced and generated, a slow granular cell line BaF3-P210 is killed, and feasibility of CTP mediated formation of DC vaccine and DC vaccine induced immune efficiency is proved. The DC vaccine design strategy in the invention also has important guidance and reference significance for immunotherapy of leukemia caused by various fusion genes.
Drawings
FIG. 1 is a diagram of immunofluorescence detection of the location of CTP fusion peptide fragments in DC cytoplasm.
FIG. 2 is a laser confocal microscope image of the sublocalization fluorescence of CTP fusion peptide segment DC cytoplasmic organelle.
FIG. 3 shows the results of ELISA assays for IL-2 levels in co-culture supernatants.
FIG. 4 shows the results of flow cytometry to detect the expression of CD69, CD137 and CD107a molecules on T lymphocytes after in vivo stimulation by DC vaccine.
FIG. 5 shows the results of the assay for lactate dehydrogenase release for determining the lysis efficiency of cells.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1 preparation of CTP-mediated lentine-specific dendritic cell vaccine
Designing and synthesizing 2 new fusion peptide segments and peptide segments of the two peptide segments added with HA labels as experimental groups (simultaneously setting 5 control groups) according to the peptide segment sequence of CTP and the sequence derived from BCR-ABL oncoprotein fusion site; for the requirement of subsequent positioning experiment, HA tag protein is added into the existing sequence, and the name and specific amino acid sequence of the peptide fragment are shown in Table 1.
TABLE 1
Dendritic cells loaded with peptide fragments and obtained after co-culture of the peptide fragments of SEQ ID Nos. 1-9 in the table 1 and mature dendritic cells are respectively named as: DC/CTP-GF, DC/CTP-SS, DC/CTP-HA-GF, DC/CTP-HA-SS, DC/GF, DC/SS, DC/HA-GF, DC/HA-SS, DC/CTP.
Secondly, a dendritic cell culture method comprises the following steps: extracting mouse bone marrow cells, lysing erythrocytes, and culturing with 1640 medium (Gibco, USA) containing 10ng/L granulocyte-macrophage colony stimulating factor (R & D Systems, USA), 5ng/L interleukin-4 (IL-4, R & D Systems, USA) and 10% fetal bovine serum for 7 days; addition of 5ng/L tumor necrosis factor-alpha (i.e., TNF-alpha, R & D Systems, USA) at day eight stimulated dendritic cell maturation.
Thirdly, observation of cytoplasm location of synthesized peptide fragment cells
(1) After co-culturing the peptide fragments in table 1 with mature dendritic cells for 30 minutes, respectively, washing five times via Phosphate Buffered Saline (PBS);
(2) centrifuging, discarding most of the supernatant, resuspending the cell pellet with the remaining supernatant, and uniformly smearing the cells on a clean slide;
(3) methanol-20 ℃ fixation overnight;
(4) after the fixation is finished, washing methanol remained on the glass slide;
(5) permeabilization: permeabilization with 1% Triton-100 for 15min, followed by three washes with PBS;
(6) and (3) sealing: blocking with goat serum at 4 deg.C for 1 hr;
(7) incubating the primary antibody: diluting the HA-Tag monoclonal antibody from rabbit with goat serum at a ratio of 1: 1000, directly adding onto a glass slide, and incubating at 4 deg.C overnight;
(8) after overnight washing, 1% Triton-100 was used as 1: diluting Cy 3-labeled goat anti-rabbit monoclonal antibody at the ratio of 800, adding the diluted antibody to a slide in an amount of 200. mu.l per slide, and incubating for 1 hour in a 37 ℃ water bath cabinet;
(9) wash slides, 4', 6-diamidino-2-phenylindole (DAPI) as 1: diluting the solution with phosphate buffer salt solution according to the proportion of 1000, and dyeing the nucleus;
(10) washing the glass slide for three times by using phosphate buffer salt solution, drying, sealing the glass slide by using glycerol, and waiting for observation; the results are shown in FIG. 1, and the CTP fusion peptide segment has good positioning effect in cytoplasm.
Fourthly, performing synthetic peptide fragment organelle sub-localization observation:
(1) after co-culturing the peptide fragments in table 1 with mature dendritic cells for 30 minutes, respectively, washing five times with phosphate buffered saline;
(2) adding endoplasmic reticulum red fluorescent probe (Biyunyan, Shanghai), and performing specific dyeing steps according to manufacturer instructions;
(3) after finishing the dyeing of the endoplasmic reticulum red fluorescent probe according to the specification, resuspending the cells by phosphate buffer solution, uniformly coating the cells on a glass slide, fixing the cells by methanol after air drying, and then counterdyeing by the endoplasmic reticulum red fluorescent probe, wherein the specific operation steps are carried out according to the specification of a manufacturer;
the results are shown in FIG. 2: most of the peptide fragments are localized on the endoplasmic reticulum in the cytoplasm.
Fifth, antigen loading method
And co-incubating the synthesized CTP fusion peptide segment with mature dendritic cells at the final concentration of 10 mu mol/L, and washing the cells for three times by using phosphate buffer salt solution after the incubation is carried out overnight to obtain the dendritic cells loaded with the specific antigen, namely the DC vaccine loaded with the CTP fusion peptide segment.
Sixth, detection of cross antigen presentation efficiency
Combining the specific antigen loaded dendritic cells obtained in the fifth step with CD8+T lymphocyte sorting magnetic beads (kit from Meitian whirlwind, Germany) sorted primary CD8+T lymphocytes were co-cultured for 20 hours, after which the supernatant was collected and applied to ELISA (kit purchased from R)&D Systems, USA) the secretion amount of interleukin-2 (IL-2) in the supernatant was detected, and the specific operation was performed according to the kit manual. The results are shown in FIG. 3: the IL-2 secretion detected in the CTP fusion peptide segment experimental group is obviously higher, which indicates that the DC vaccine loaded by the CTP fusion peptide segment can more effectively initiate cross antigen presentation reaction.
Example 2 application of CTP fusion peptide fragment loaded DC vaccine
First, mouse immunization strategy
Will be 3X 106Incubating the mature dendritic cells and 10 mu mol/L CTP fusion peptide segments together overnight, washing, injecting into subcutaneous lymph nodes of the inguinal of the mouse, and recording as the first day; after 7 days, carrying out secondary immunization; seven more days later, the spleen and peripheral blood lymphocytes of the mice were collected.
Second, mouse spleen lymphocyte isolation
Collecting spleen of mouse, preparing single cell suspension in mortar, and filtering with filter screen; adding a mouse spleen lymphocyte separation solution (from the last ocean biologicals science and technology Limited liability company in Tianjin) and the prepared single cell suspension into a centrifuge tube according to the ratio of 1: 1, wherein the lower layer is the spleen lymphocyte separation solution, the upper layer is the single cell suspension, and the two layers are required to be clear in boundary and cannot be mixed; centrifuging at 2000r/min, sucking leukocyte layer, and washing to obtain spleen lymphocytes.
Peripheral blood lymphocyte separation procedure as above, the lymphocyte separation solution used was mouse peripheral blood lymphocyte separation solution (purchased from tianjin tertiary ocean corporation).
The results show that: the leucocyte layer with a proper thickness is obtained, namely the number of the lymphocytes can meet the requirement of subsequent experiments.
Third, CTL cell amplification and magnetic bead sorting
The mitomycin C-treated target cells BaF3-P210 (collection of the present laboratory construct) were cultured at a ratio of 3: 1 as feeder cells, and RPMI 1640 complete T lymphocyte culture medium containing 50. mu. mol/L2-mercaptoethanol was added on day one. After 1 day, mouse interleukin-2 (IL-2) was added to a final concentration of 10. mu. mol/L. At the time of culture to day 3 and day 6, 1ml of the medium was removed, and fresh safe RPMI 1640 medium containing 10. mu. mol/L of IL-2 was added. After seven days of incubation, effector CTL cells were collected. Collecting effector CTL cells by using Dryowa American day and whirlwind CD8+The T lymphocyte magnetic bead sorting kit comprises the specific steps according to instructions.
Activation of T lymphocyte (CD69, CD137 expression detection)
The CTL cells obtained by sorting in the third step and target cells BaF3-P210 are incubated together according to the ratio of 3: 1, APC-labeled mouse anti-CD 69 monoclonal antibody (purchased from eBioscience, USA) is directly added, the cells are collected after 12h and washed, FITC-labeled mouse anti-CD 8 monoclonal antibody (purchased from eBioscience, USA) is labeled, and flow detection is carried out after washing.
CD137 expression was detected using an APC-labeled mouse anti-CD 137 monoclonal antibody (purchased from eBioscience, USA) in the same manner as for CD69, but after 24 h.
The results are shown in FIG. 4: the expression quantity of CD69 and CD137 on T lymphocytes obtained by the immunization of the CTP fusion peptide segment experimental group is obviously higher than that of the control group, which indicates that the T lymphocytes in the experimental group have better activity.
Degranulation effect of T lymphocyte
Co-incubating CTL cells obtained by sorting in the third step with target cells BaF3-P210 according to the ratio of 3: 1, directly adding mouse anti-CD 107a monoclonal antibody (purchased from eBioscience, USA) marked by APC (immunoglobulin antigen), collecting the cells after 24h, washing, marking the cells with mouse anti-CD 8 monoclonal antibody (purchased from eBioscience, USA) marked by FITC (immunoglobulin antigen), and carrying out flow detection after washing;
as a result: the higher amount of the T lymphocyte expression CD107a in the CTP fusion peptide segment experimental group shows that the degranulation effect of the T lymphocyte in the group is more obvious and enough cytotoxic components can be released.
Sixthly, cytotoxic action of induced CTL cells on target cells
Co-incubating the CTL cells sorted in step three with the target cells BaF3-P210 according to different effective target ratios in a 96-well plate, each well containing 1X 10 target cells4Three duplicate wells were set up in a final volume of 200. mu.l. After 4 hours of culture, the supernatant was collected and the killing effect of effector CTL cells on target cells was examined using lactate dehydrogenase release kit (Promega, USA).
The results are shown in FIG. 5: the cytolysis efficiency in the CTP fusion peptide segment experimental group is more obvious, which shows that the DC vaccine loaded by the CTP fusion peptide segment can induce CML specific cellular immune response, thereby killing the slow granulocyte.
Sequence listing
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<120> fusion peptide, CTP-mediated DC vaccine for inducing CML cellular immune response and preparation method thereof
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Claims (3)
1. A CTP-mediated DC vaccine that induces a CML cellular immune response, characterized by: the DC vaccine is a dendritic cell loaded with the fusion peptide obtained by co-incubating the fusion peptide shown as SEQ ID No:1 or 2 with a mature dendritic cell.
2. The method of preparing the DC vaccine of claim 1, comprising the steps of:
1) synthesizing a fusion peptide sequence shown as SEQ ID No. 1 or 2;
2) culturing mature dendritic cells;
3) and co-incubating and washing the fusion peptide and the mature dendritic cells to obtain the DC vaccine.
3. The method according to claim 2, wherein the step 2) of culturing the mature dendritic cells comprises the following steps: extracting mouse bone marrow cells, lysing erythrocytes, culturing in 1640 medium containing granulocyte-macrophage colony stimulating factor, interleukin-4, and 10% fetal calf serum for 7 days, and adding tumor necrosis factor-alpha to stimulate dendritic cells to mature on the eighth day.
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