CN109796536B

CN109796536B - Preparation method of CTL (cytotoxic T lymphocyte) targeting multiple epitopes of glioblastoma

Info

Publication number: CN109796536B
Application number: CN201910133847.XA
Authority: CN
Inventors: 李晨蔚; 邓婷婷
Original assignee: Shanghai Sunstem Biotechnology Co ltd
Current assignee: Shanghai Sunstem Biotechnology Co ltd
Priority date: 2019-02-22
Filing date: 2019-02-22
Publication date: 2021-09-17
Anticipated expiration: 2039-02-22
Also published as: CN109796536A

Abstract

The invention relates to the field of biotechnology development and application research. Specifically, the invention provides a preparation method of Cytotoxic T Lymphocytes (CTL) targeting multiple glioblastoma epitope. In the method, 3 glioma-associated antigen (GAA) epitope sequences are combined in series to synthesize a new artificial coding antigen sequence, the new artificial coding antigen sequence is cloned to a lentivirus expression vector, a virus is packaged and is transfected into dendritic cells, and the new artificial coding antigen sequence and the sorted CD3 are combined⁺T cells are co-cultured to stimulate proliferation and differentiation of activated antigen-specific T cells, so that the prepared CTL has specificity of targeting 3 GAA antigens. The GAA antigen-specific CTL prepared by the invention has the advantages of strong target specificity, no side effect, difficulty in causing immune escape and the like, and has great application prospect in the treatment of GAA-expressing glioblastoma.

Description

Preparation method of CTL (cytotoxic T lymphocyte) targeting multiple epitopes of glioblastoma

Technical Field

The invention relates to the field of biotechnology development and application research, in particular to a preparation method of cytotoxic T lymphocytes targeting multiple epitopes of glioblastoma multiforme.

Background

Brain glioma is the most common intracranial tumor, the incidence rate accounts for about 40% -50% of all intracranial tumors, wherein, Glioblastoma (GBM) is the most common glioma with the highest malignancy, the 5-year survival rate is only 5.1%, the most effective treatment scheme of GBM is the comprehensive treatment mainly comprising operation, radiotherapy and chemotherapy, the combined application of the chemotherapeutic drug temozolomide and radiotherapy only improves the median survival time by 3 months compared with the median survival time of the simple radiotherapy, and the median survival time is still less than 15 months after active treatment.

With the continuous improvement of genetic engineering and cell engineering techniques, the clinical research of the treatment of GBM by immunotherapy has achieved encouraging results, and has become the fourth treatment means of the comprehensive treatment of tumors besides surgery, radiotherapy and chemotherapy. The GBM immunotherapy mode includes active immunotherapy, such as Dendritic Cell (DC) vaccine, immune checkpoint inhibitor, and passive immunotherapy, including adoptive immune cell therapy, antibody drug therapy, and cytokine therapy. Adoptive immune cell T cell therapy is better suited to overcome the blood-brain barrier and the large number of immunosuppressive substances creating these problems in GBM treatment regimens than active immunotherapy, as adoptive therapy allows in vitro modification of these glioma-associated antigen (GAA) -specific Cytotoxic T Lymphocytes (CTLs) to enhance or confer new functions, such as the alteration of CTL cell trafficking by expressing cytokine receptors (CXCR3) or integrin receptors, and thus specific adoptive CTL therapy may be a promising approach in the currently existing GBM immunotherapy strategies.

Specific CTLs are derived in two ways: obtaining fresh tumor tissue of a patient through operation, separating Tumor Infiltrating Lymphocytes (TILs) in the fresh tumor tissue, obtaining a monoclonal through limited dilution, and then performing expanded culture to obtain the tumor infiltrating lymphocytes; can also induce the lymphocytes in the peripheral blood, lymph nodes or spleen of the patient to generate CTLs. Because the TIL preparation technology is difficult, the culture time is long, and the like, and cannot meet the requirements of future clinical treatment, Peripheral Blood Mononuclear Cells (PBMCs) of patients are often selected and collected, and the generation of specific T cells is induced in vitro by autologous or allogeneic antigens. The autologous or heterologous antigen used can be DC-loaded antigen, tumor cells or apoptosis products, synthetic HLA-restricted polypeptides.

However, most of the existing adoptive CTL therapies for brain glioma are not only insufficient to effectively kill tumor cells, but also induce tumor cells to generate immune escape, and the effect is still unsatisfactory.

Therefore, there is an urgent need in the art to develop a CTL therapy which is less likely to cause immune escape and can produce a stronger anti-tumor effect.

Disclosure of Invention

It is an object of the present invention to provide a CTL therapy which is less likely to cause immune escape and can produce a stronger antitumor effect.

In a first aspect of the invention, there is provided an epitope peptide combination comprising a Z0 sequence, wherein the Z0 sequence comprises a sequence that expresses two or more glioma-associated antigens;

wherein the two or more glioma-associated antigens are linked in series via a linking sequence.

In another preferred embodiment, the glioma-associated antigen is selected from the group consisting of: EGFR-VIII, CD133, CMV-PP65, IL13R alpha 2 or met-ex 14.

In another preferred embodiment, the glioma-associated antigens are EGFR-VIII, CD133 and CMV-PP65, respectively.

In another preferred embodiment, the linker sequence is a sequence selected from the group consisting of:

(a) a sequence shown as SEQ ID NO. 4;

(b) an amino acid sequence formed by substituting, deleting or adding one or more amino acid residues on the basis of the amino acid sequence shown in SEQ ID NO. 4.

In another preferred embodiment, the gene sequence encoding the junction sequence is selected from the group consisting of:

(a) 1 as shown in SEQ ID NO;

(b) the encoded sequence is an amino acid sequence formed by substituting, deleting or adding one or more amino acid residues on the basis of the amino acid sequence shown in SEQ ID NO. 4.

In another preferred embodiment, the Z0 sequence has the structure shown in formula I:

n terminal 'P1-L1-P2-L2-P3C terminal' (I)

Wherein, the elements P1, P2 and P3 are respectively the amino acid sequences of 3 glioma-associated antigens, and the sequence of the three can be interchanged; and, element L1 and element L2 are said linking sequences;

wherein the glioma-associated antigen is selected from the group consisting of: EGFR-VIII, CD133, CMV-PP65, IL13R alpha 2, or a combination thereof.

In another preferred embodiment, the 3 glioma-associated antigens are EGFR-VIII, CD133 and CMV-PP65, respectively.

In another preferred embodiment, the element Z0 has a structure represented by formula II:

n terminal 'P1-L1-P2C terminal' (II)

Wherein, elements P1 and P2 are amino acid sequences of 2 glioma-associated antigens respectively; and, element L1 is said linker sequence;

In another preferred embodiment, the 2 glioma-associated antigens are 2 glioma-associated antigens selected from the group consisting of EGFR-VIII, CD133 and CMV-PP 65.

In another preferred embodiment, the glioma-associated antigens EGFR-VIII, CD133 and CMV-PP65 may be naturally occurring or artificially synthesized.

In another preferred embodiment, the glioma-associated antigens EGFR-VIII, CD133 and CMV-PP65 may be wild, mutated, or spliced from different fragments of the wild-type amino acid sequence, or repeated and spliced from the same fragment of the wild-type amino acid sequence.

In another preferred embodiment, the splicing comprises adding the connecting sequence of the present invention to two sequences.

In another preferred example, the N-terminal of the Z0 sequence can also comprise an engineered sequence.

In another preferred embodiment, the engineering sequence can be designed according to different experimental means.

In another preferred embodiment, the glioma-associated antigen wherein the gene encoding EGFR-VIII comprises the sequence shown in SEQ ID NO. 5.

In another preferred embodiment, the amino acid sequence of EGFR-VIII in the glioma-associated antigen comprises the sequence shown in SEQ ID NO. 6.

In another preferred embodiment, the gene encoding CD133 of the glioma-associated antigen comprises the sequence shown in SEQ ID NO. 7.

In another preferred embodiment, the amino acid sequence of CD133 in the glioma-associated antigen comprises the sequence shown in SEQ ID NO. 8.

In another preferred embodiment, the gene encoding CMV-PP65 in the glioma-associated antigen comprises the sequence shown in SEQ ID NO. 9.

In another preferred embodiment, the amino acid sequence of CMV-PP65 in the glioma-associated antigen comprises the sequence shown in SEQ ID NO. 10.

In a second aspect of the invention, there is provided an isolated polynucleotide encoding a combination of epitope peptides according to the first aspect of the invention.

In a third aspect of the invention, there is provided a vector comprising a polynucleotide according to the second aspect of the invention.

In another preferred embodiment, the vector is a lentiviral vector.

In another preferred embodiment, the lentiviral vector is obtained by inserting the polynucleotide according to the second aspect of the present invention into an empty lentiviral vector.

In another preferred embodiment, the empty lentiviral vector is selected from the group consisting of: pLVX, ShRNA.

In another preferred embodiment, the empty lentiviral vector is pLVX.

In a fourth aspect of the invention, there is provided a lentivirus obtained by packaging the vector of the third aspect of the invention with a packaging vector.

In another preferred embodiment, the packaging carrier comprises: pLP1, pLP2, and pLP/VSVG.

In a fifth aspect of the invention, there is provided a dendritic cell obtained by transfection of a lentivirus according to the fourth aspect of the invention.

In another preferred embodiment, the dendritic cells are isolated from peripheral blood mononuclear cells PBMCs.

In another preferred embodiment, the surface of the dendritic cell has a surface molecular marker selected from the group consisting of: CD80, CD83, HLA-DR, CD86, or a combination thereof.

In a sixth aspect of the invention there is provided a cytotoxic T lymphocyte having the specificity of a glioma-associated antigen as defined in the first aspect of the invention.

In another preferred embodiment, the cytotoxic T lymphocytes are isolated from peripheral blood mononuclear cells PBMCs.

In another preferred embodiment, the cytotoxic T lymphocyte is CD3⁺T lymphocytes.

In another preferred embodiment, the cytotoxic T lymphocyte is obtained by sensitizing dendritic cells according to the fifth aspect of the present invention.

In another preferred embodiment, the sensitization reaction refers to that the dendritic cell according to the fifth aspect of the present invention is activated at a rate of 1: (5-50), preferably 1: (8-30), and the more preferable ratio is 1: 10.

In another preferred embodiment, the obtained cytotoxic T lymphocyte of the sixth aspect of the present invention has a killing rate of glioma-associated antigen of not less than 40%, preferably not less than 50%, more preferably not less than 60%.

In a seventh aspect of the invention, there is provided a use of a dendritic cell according to the fifth aspect of the invention for the preparation of a cytotoxic T lymphocyte according to the sixth aspect of the invention.

In an eighth aspect of the present invention, there is provided a method for preparing cytotoxic T lymphocytes according to the sixth aspect of the present invention, comprising the steps of:

(I) providing a dendritic cell according to the fifth aspect of the present invention;

(II) sensitizing the isolated T lymphocytes with dendritic cells according to the fifth aspect of the invention to obtain cytotoxic T lymphocytes according to the sixth aspect of the invention.

In another preferred example, the method further comprises the steps of:

(i) providing a polynucleotide according to the second aspect of the invention;

(ii) inserting a polynucleotide sequence according to the second aspect of the invention into an empty lentiviral vector to obtain a lentiviral vector according to the third aspect of the invention;

(iii) packaging the obtained lentiviral vector according to the third aspect of the invention to obtain the lentivirus according to the fourth aspect of the invention;

(iv) transfecting the isolated mature dendritic cells with the obtained lentivirus according to the fourth aspect of the invention to obtain the dendritic cells according to the fifth aspect of the invention.

In another preferred example, in the step (iv), the method further comprises the steps of: isolation and maturation induction of dendritic cells.

In another preferred example, the medium used in the maturation induction of the dendritic cells is 1640.

In another preferred embodiment, the medium used in the induction of maturation of dendritic cells comprises a component selected from the group consisting of: IL-4, GM-CSF, autologous plasma.

In another preferred embodiment, the plasma is autologous plasma, wherein the autologous plasma is plasma from the same blood sample as the dendritic cells and cytotoxic T lymphocytes.

In another preferred embodiment, the concentration of the plasma in the culture medium used in the maturation induction of the dendritic cells is 0.5 to 10%, preferably 0.8 to 8%, more preferably 1 to 5%, and still more preferably 2%.

In another preferred embodiment, the concentration of IL-4 in the medium used in the induction of maturation of dendritic cells is 50-200ng/mL, preferably 60-150ng/mL, more preferably 80-120ng/mL, more preferably 100 ng/mL.

In another preferred embodiment, the culture medium used in the induction of maturation of dendritic cells has a concentration of GM-CSF of 50-200ng/mL, preferably 60-150ng/mL, more preferably 80-120ng/mL, more preferably 100 ng/mL.

In another preferred embodiment, in the step (iv), after the isolated mature dendritic cells are transfected by the obtained lentivirus according to the fourth aspect of the present invention, the method further comprises the steps of: dendritic cell differentiation and maturation promoter is added into dendritic cell culture solution.

In another preferred embodiment, the dendritic cell differentiation and maturation promoting agent is selected from the group consisting of: IFN-gamma, LPS, or a combination thereof.

In another preferred embodiment, the IFN- γ concentration is 10-100ng/mL, preferably 30-80ng/mL, more preferably 40-60ng/mL, more preferably 50 ng/mL.

In another preferred embodiment, the concentration of LPS is 10-100EU/mL, preferably 30-80EU/mL, more preferably 40-60EU/mL, more preferably 50 EU/mL.

In another preferred example, in step (II), the method further comprises the steps of: isolation of T lymphocytes.

In another preferred embodiment, the culture medium used in the co-culture is KBM 581.

In another preferred embodiment, the co-culture medium comprises a component selected from the group consisting of: IL-7, IL-15, plasma.

In another preferred embodiment, the concentration of the plasma in the culture medium used in the maturation induction of the dendritic cells is 1 to 50%, preferably 3 to 30%, more preferably 5 to 20%, and still more preferably 10%.

In another preferred embodiment, the concentration of IL-7 in the medium used in the induction of maturation of dendritic cells is 0.5-30ng/mL, preferably 1-20ng/mL, more preferably 5-15ng/mL, more preferably 10 ng/mL.

In another preferred embodiment, the concentration of IL-15 in the medium used in the induction of maturation of dendritic cells is 0.5-30ng/mL, preferably 1-20ng/mL, more preferably 5-15ng/mL, more preferably 10 ng/mL.

In another preferred embodiment, the obtained cytotoxic T lymphocyte of the sixth aspect of the present invention has a purity of 90%, preferably 92%, more preferably 95%.

In a ninth aspect of the invention, there is provided a use of the cytotoxic T lymphocyte of the sixth aspect of the invention for the preparation of a pharmaceutical composition or formulation for the treatment of cancer.

In another preferred embodiment, the cancer comprises brain glioma.

In another preferred example, the brain glioma comprises: low-grade brain glioma, high-grade brain glioma.

In another preferred embodiment, the high grade brain glioma comprises a brain glioblastoma.

In a tenth aspect of the present invention, there is provided a method of treating cancer, comprising the steps of: administering to a subject in need of treatment cytotoxic T lymphocytes according to the sixth aspect of the invention.

In another preferred embodiment, the cancer comprises brain glioma.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a schematic diagram of the construction of a lentiviral vector carrying the coding sequences for three GAA antigens.

FIG. 2 is a diagram showing the flow analysis of markers after lentivirus transfection of DC cells and induction of maturation.

FIG. 3 is a diagram showing the flow analysis of the amplification-cultured CTLs.

FIG. 4 shows the detection of tumor cell killing by antigen-specific CTL.

Detailed Description

The present inventors have made extensive and intensive studies and, as a result, have developed a method for producing CTLs specific to a plurality of tumor-associated antigens for the first time through a large number of screenings. The inventor firstly constructs a lentivirus expression vector containing 3 GAA antigen epitope sequences (EGFR-VIII, CD133, CMV-pp65) which are connected in series; packaging, concentrating and detecting the slow virus particles with low degree; then performing transfection and maturation induction on dendritic cells; finally, antigen-loaded mature DCs and isolated CD3⁺T lymphocytes were co-cultured to thereby sensitize CTLs. The CTL prepared by the method shows better killing effect and stronger specificity in mid-autumn cell killing experiments, and the killing rate can reach 60%. The present invention has been completed based on this finding.

Term(s) for

The glioma-associated antigen of the invention

The presence of glioma-associated antigens may make them targets for immunotherapy.

EGFR-VIII is an EGFR variant gene product that is expressed in 30-50% of GBM patients and not in normal tissues. CD133 is a glioma tumor stem cell marker, and glioma stem cells expressing CD133 have an unconstrained self-renewal capacity and a surprising capacity to form tumors in vitro. Most patients with malignant glioma are infected with Cytomegalovirus (CMV), which plays a positive role in the process of glioma formation, CMV-PP65 protein is an important envelope protein of CMV, the expression of which is clearly correlated with viral replication, and CMV-PP65 is expressed in more than 90% of GBM tissues, but not in the surrounding normal brain tissues.

In the present invention, genes encoding the above three antigens are introduced into DCs, then maturation is induced by the addition of maturation inducers, and specific CTLs are prepared by coculture with T lymphocytes, and then returned to the body to attack tumors.

However, the immune attack of a single antigen is not only insufficient to effectively kill tumor cells, but also induces the tumor cells to generate immune escape. The lentiviral vector employed in the present invention can simultaneously express epitope peptides of 3 GAAs (EGFR-VIII, CD133, CMV-pp65), and thus can produce CTLs producing specificities targeting 3 GAAs. Targeting multiple antigens in GBM may mediate the activation of more immune T cells, less immune escape and may result in stronger anti-tumor effects.

Specific CTL

DCs are widely distributed in various tissues and organs, are full-time antigen presenting cells with the strongest functions in vivo, have unique capacity of stimulating T cell proliferation, and are the only in vivo cells capable of activating

Antigen presenting cells of T cells. The DC loaded with the tumor antigen can directly stimulate the DC through the tumor antigen, but the method has complex manufacturing process and poor quality control and can generate immune escape; therefore, it is a preferred embodiment of the present invention to directly introduce a gene encoding a tumor-associated antigen into DC. Compared with reverse transcription and adenovirus vectors, the lentivirus vector can transfect cells in an active mitosis phase, slowly-dividing cells and cells in a terminal division phase, has the advantages of large capacity of transferred gene segments, long and stable expression time of target genes, difficulty in inducing immune response and the like, and is a vector widely used for laboratory research and clinical tests at present.

Compared with other immune cell treatment methods, the DC-CTL technology based on gene modification DC in-vitro stimulation activation of T lymphocytes has the advantages that antigen genes can be continuously and efficiently expressed, expressed multiple antigen peptides can effectively induce CTL cells to generate specific immune response for a long time, and endogenous antigens integrated into DC genome expression can be more favorably activated to CD8 through a cytosol pathway⁺The T cell has the advantages of safety, effectiveness, strong targeting specificity and the like, and can show wide application prospect in the immunotherapy of tumors.

The method of the invention

The invention provides a preparation method of specific cytotoxic T lymphocytes targeting multiple GBM epitopes.

For the purpose of realizing the preparation of the specific cytotoxic T lymphocyte targeting multiple epitopes of GBM, the core steps comprise:

step 1) constructing a lentivirus expression vector, packaging lentiviruses and concentrating;

step 2) separating and inducing mature DCs;

step 3) mixing the DC matured in the step 2) with CD3⁺T cells are co-cultured to obtain specific Cytotoxic T Lymphocytes (CTL) aiming at 3 tandem GAA epitope sequences.

Preferably, in step 1), the third generation lentiviral vector system is transfected into 293FT (human embryonic kidney cell) cells by calcium phosphate precipitation transient transfection method to prepare lentiviral particles;

preferably, the DC induction culture medium in the step 2) adopts 1640 culture medium of autologous plasma (2-10%), IL-4 (25-100 ng/mL) and GM-CSF (50-100 ng/mL); after the DC is induced for 4-5 days, adding 10-20 MOI virus to infect the DC; after 5-6 days of induction, supplementing IFN-gamma (50-100 ng/mL) and LPS (50-100 EU/mL) to promote DC differentiation and maturation; and after 6-7 days of induction, collecting mature DCs.

Preferably, step 3) we used magnetic bead negative selection to separate CD3 from PBMC⁺The ratio of the mature DC cells to the T cells in co-culture is 1: 10-1: 30, and the culture medium is KBM581 containing IL7(5-20ng/ml) and IL15(5-20 ng/ml).

Preferably, the number of times of sensitizing the T cells by the DC is 2, and each time lasts for 4-7 days; after T cells are sensitized with DC, a preferable CTL medium is KBM581 containing IL7(5-20ng/mL), IL15(5-20ng/mL) and IL2 (500-1000U/mL).

The invention adopts Linker sequence (shown as SEQ ID NO: 1), connects antigen epitope sequences of 3 GAAs (EGFR-VIII, CD133, CMV-pp65) in series, adds start and stop codons at the head and tail ends of the series respectively, establishes new coding antigen sequence (shown as SEQ ID NO: 2), and finally clones the sequence into a lentivirus expression vector.

The main advantages of the invention include:

1) an antigen gene modified DC vaccine constructed based on a lentiviral vector and an activated antigen-specific CTL are effective treatment means without side effect and strong target specificity.

2) The antigen gene can be expressed continuously and efficiently, and the expressed multiple antigen peptide can effectively induce CTL cells to generate specific immune response for a long time.

3) Integration into genomic expression of DC cellsThe antigen of origin can activate CD8 through the intrinsic pathway⁺T cells.

4) Because 3 GAA epitope peptides can be simultaneously expressed and generated based on the lentiviral vector, 3 targeted GAA specific Cytotoxic T Lymphocytes (CTL) can be simultaneously prepared and generated, and the lentiviral vector has the advantages of broad spectrum, difficulty in causing immune escape and the like in the actual treatment of GAA tumors.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The materials and reagents used in the examples were all commercially available products unless otherwise specified.

Example 1: construction of Lentiviral expression vectors containing encoded antigen sequences

Construction of lentiviral expression vectors: a schematic diagram of construction of a lentivirus expression vector carrying an antigen coding sequence is shown in figure 1, firstly, an antigen epitope sequence of 3 GAAs (EGFR-VIII, CD133 and CMV-pp65) is connected in series (preferably the sequence of the serial combination is CMV-pp65-EGFR-VIII-CD 133) by adopting a Linker sequence (shown in SEQ ID NO: 1), and a start codon and a stop codon are respectively added at the head end and the tail end of the serial sequence to create a new artificial antigen coding sequence (shown in SEQ ID NO: 2; abbreviated as PEC) with the sequence size of 858 bp; then adding BshT1 and Mlu1 restriction enzyme site sequences into two ends of a new artificial antigen coding sequence and carrying out gene synthesis; finally, after double digestion of the synthesized antigen coding sequence fragment and a lentivirus expression empty vector (pLVX) by BshT1 and Mlu1 restriction enzymes, the antigen coding sequence is cloned into a lentivirus expression vector sequence under the action of T4 ligase to construct a lentivirus expression vector (pLVX-PEC) containing the antigen coding sequence.

Example 2: lentiviral particle packaging, concentration and titer detection

1) Virus packaging and concentration: 293FT (human embryonic Kidney) cells from P10-P12 in a 10cm dish at 8X 10⁶The cell mass of (3) was plated. Plasmid transfection by calcium phosphate-DNA coprecipitation: adding the constructed pLVX-PEC lentiviral expression vector and packaging vectors pLP1, pLP2 and pLP/VSVG into 2.5M CaCl₂In solution with ddH₂O complement volume 500ul, shake, stand for 5 minutes, slowly add plasmid and CaCl on vortex shaker₂The mixed solution was added dropwise to an equal volume of 500ul of 2 × HBS solution at pH7.0 at a constant rate, the bubbling was completed five times, 1ml of the mixture was added to an adherent 293FT petri dish, and the incubator was placed. And after overnight, removing the old culture medium, adding 10-15 mL of fresh culture medium, continuing culturing, collecting culture medium supernatant containing the lentivirus particles after transfection for 48 hours, centrifuging the supernatant, and filtering the supernatant by using a 0.4um filter to remove cell debris. The collected virus-containing culture medium was ultracentrifuged (30000rpm, 4 ℃, 2h) with 20% sucrose as a cushion, and PBS was added to concentrate and purify the lentiviral particles, which were then packaged and stored at-80 ℃.

2) And (3) virus titer detection: logarithmic growth of HT1080 cells at 6X 10³The cell amount of each hole is paved on a 96-well plate, a DMEM culture medium containing 10% FBS is added into each hole, the serum-free DMEM culture medium is used for carrying out gradient dilution on the fluorescence expression virus in a 10-fold manner in the 96-well plate with the round bottom in the next day, each gradient is used for carrying out three multiple holes, 90ul of the culture medium is discarded in each hole in the 96-well plate paved with HT1080 cells, 90ul of the diluted virus liquid in the 96-well plate with the round bottom is correspondingly added, 100ul of the DMEM culture medium containing 10% FBS is replaced after 24 hours, the fluorescence number of the hole with the moderate fluorescence expression amount is counted after infection is carried out for 72 hours, the volume ratio of the fluorescence number to the virus liquid in the hole is the titer of the fluorescence expression virus, the titer of the fluorescence expression virus is made into a standard curve, and the cq value of the fluorescence expression virus and the target virus obtained by combining a QPCR method is combined, and the titer of the target virus can be calculated.

Example 3: transfection, maturation Induction of DCs and preparation of specific CTL

1) DC transfection and maturation induction: collecting peripheral blood of volunteer, separating autologous plasma by Ficoll-Hypaque (polysucrose-diatrizoate) density gradient separation method (inactivating at 56 deg.C for 30min, and preserving at 4 deg.CPresent) and peripheral blood mononuclear cells PBMC, prepared by treating the PBMC with serum-free RPMI-1640 at 5-7X 10⁷One cell mass was plated in a T75 flask, and after 1h, suspension cells were collected for isolation of CD3+ T lymphocytes, and adherent cells were replaced with a 1640 medium containing IL-4(100ng/mL), GM-CSF (100ng/mL) and autologous plasma (2%) to stimulate monocyte differentiation to DC. Immature DCs collected in suspension in 24-well plates were infected on day 5 with concentrated lentivirus particles and lentivirus volume required was calculated from pre-experimental optimal MOI values. 24 hours after transfection, the medium was replaced with fresh DC induction medium and IFN-. gamma.was added (50ng/mL) and LPS (50EU/mL) to promote differentiation and maturation of the DCs. On day 7, the maturation status of the DC was observed by microscope, and the expression levels of the DC surface molecular markers CD80, CD83 and HLA-DR were measured by flow cytometry, as shown in FIG. 2. The results show that the expression levels of CD80, CD83 and HLA-DR on the surface of the DC at 7 days after the DC is subjected to transfection and induction culture are obviously enhanced, which indicates that the cultured DC is mature and the antigen presenting capability is enhanced.

2) Preparation of antigen-specific CTLs: suspending cells collected after PBMC is spread in a bottle for 1h, and separating CD3 by adopting a magnetic bead negative selection method⁺T lymphocytes, counted and cryopreserved to liquid nitrogen, revived when DC induced maturation by day 7, antigen-loaded mature DCs and CD3⁺T cells were co-cultured at a ratio of 1:10 to sensitize T lymphocytes, and the medium was KBM581 containing IL7(10ng/mL), IL15(10ng/mL) and autologous plasma (10%), and co-cultured for 2 times for 4 days each; after completion of 2 rounds of sensitization, the cells were collected by flow analysis after supplementing every two days with an amplification medium (KBM 581 containing 500U/mL IL 2), and when the cells proliferated to a considerable amount on day 14.

As shown in FIG. 3, the cultured CD3 was amplified⁺The CTL cells have higher purity.

Example 4: tumor cell killing experiment of antigen-specific CTL

In order to verify the specific killing effect of antigen-specific CTL on GAA tumor cells, in this example, first, a U87 cell line (U87-Ag) stably expressing 3 GAAs (EGFR-VIII, CD133, CMV-pp65) was constructed as an experimental group, and an in vitro killing verification experiment of antigen-specific CTL was performed using wild-type U87 and intestinal cancer cell line HT29 as control groups. On the 14 th day of T lymphocyte specific amplification, CTL and tumor target cells are respectively added into a 96-well plate at the ratio of 5:1, 10:1 and 20:1 for co-incubation for 16 hours, then CCK8 kit reagent is added for incubation for 1 hour, OD values of all groups of cells are detected by an enzyme-labeling instrument, and finally the specific killing efficiency of the CTL on cell strains is calculated.

As shown in FIG. 4, the results showed that the prepared CTL targeting 3 GAA had a good killing effect and strong specificity on the glioma antigen-containing cells. This immunotherapeutic approach holds great promise for clinical treatment of GBM.

Compared with the method of singly using one or two antigens in 3 kinds of GAA as the therapeutic target spots and using 3 kinds of GAA as the target spots, the invention can effectively avoid the immune escape of the tumor simultaneously expressing three kinds of antigen molecules.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

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Gly Ser Thr Thr Glu Arg Lys Thr Pro Arg Val Thr Gly Gly Gly Ala

65 70 75 80

Met Ala Gly Ala Ser Thr Gly Gly Gly Gly Ser Pro Ala Ser Arg Ala

85 90 95

Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys

100 105 110

Gly Gly Gly Ser Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Gly Asn

115 120 125

Tyr Val Val Thr Asp His Gly Ser Cys Gly Gly Gly Ser Pro Ala Ser

130 135 140

Arg Ala Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly

145 150 155 160

Ser Cys Gly Gly Gly Ser Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys

165 170 175

Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys Gly Gly Gly Ser Pro

180 185 190

Ala Ser Arg Ala Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp

195 200 205

His Gly Ser Cys Gly Gly Gly Gly Ser Arg Leu Pro Ile Gln Asp Ile

210 215 220

Leu Ser Ala Phe Ser Val Tyr Val Asn Asn Thr Glu Ser Gly Gly Gly

225 230 235 240

Ser Ile Gly Tyr Phe Glu His Tyr Leu Gln Trp Ile Glu Phe Ser Ile

245 250 255

Ser Glu Lys Val Ala Gly Gly Gly Ser Ile Gly Lys Ala Thr Val Phe

260 265 270

Leu Leu Pro Ala Leu Ile Phe Ala Val Lys Leu Ala Lys

275 280 285

<210> 4

<211> 5

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 4

Gly Gly Gly Gly Ser

1 5

<210> 5

<211> 363

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 5

ccggcgagtc gggctctgga ggaaaagaaa ggtaattatg tggtgacaga tcacggctcg 60

tgcggtggtg gtagtccggc gagtcgggct ctggaggaaa agaaaggtaa ttatgtggtg 120

acagatcacg gctcgtgcgg tggtggtagt ccggcgagtc gggctctgga ggaaaagaaa 180

ggtaattatg tggtgacaga tcacggctcg tgcggtggtg gtagtccggc gagtcgggct 240

ctggaggaaa agaaaggtaa ttatgtggtg acagatcacg gctcgtgcgg tggtggtagt 300

ccggcgagtc gggctctgga ggaaaagaaa ggtaattatg tggtgacaga tcacggctcg 360

tgc 363

<210> 6

<211> 121

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 6

Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr

1 5 10 15

Asp His Gly Ser Cys Gly Gly Gly Ser Pro Ala Ser Arg Ala Leu Glu

20 25 30

Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys Gly Gly

35 40 45

Gly Ser Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Gly Asn Tyr Val

50 55 60

Val Thr Asp His Gly Ser Cys Gly Gly Gly Ser Pro Ala Ser Arg Ala

65 70 75 80

Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys

85 90 95

Gly Gly Gly Ser Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Gly Asn

100 105 110

Tyr Val Val Thr Asp His Gly Ser Cys

115 120

<210> 7

<211> 207

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 7

cgtcttccta ttcaggatat actctcagca ttctctgttt atgttaataa cactgaaagt 60

ggtggtggta gtataggata ttttgaacat tatctgcagt ggatcgagtt ctctatcagt 120

gagaaagtgg caggtggtgg tagtatagga aaagctactg tatttttact tccggctcta 180

atttttgcgg taaaactggc taagtga 207

<210> 8

<211> 68

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 8

Arg Leu Pro Ile Gln Asp Ile Leu Ser Ala Phe Ser Val Tyr Val Asn

1 5 10 15

Asn Thr Glu Ser Gly Gly Gly Ser Ile Gly Tyr Phe Glu His Tyr Leu

20 25 30

Gln Trp Ile Glu Phe Ser Ile Ser Glu Lys Val Ala Gly Gly Gly Ser

35 40 45

Ile Gly Lys Ala Thr Val Phe Leu Leu Pro Ala Leu Ile Phe Ala Val

50 55 60

Lys Leu Ala Lys

65

<210> 9

<211> 186

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 9

gccggcatcc tggcccgcaa cctggtgccc atggttgcta cggttcaggg tcagaatctg 60

aagtaccagg agttcttctg ggacgccaac gacatctacc gcatcttcgc cgaaggtggt 120

ggtagtacca ccgagcgcaa gacgccccgc gttaccggcg gcggcgccat ggcgggcgcc 180

tccact 186

<210> 10

<211> 62

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 10

Ala Gly Ile Leu Ala Arg Asn Leu Val Pro Met Val Ala Thr Val Gln

1 5 10 15

Gly Gln Asn Leu Lys Tyr Gln Glu Phe Phe Trp Asp Ala Asn Asp Ile

20 25 30

Tyr Arg Ile Phe Ala Glu Gly Gly Gly Ser Thr Thr Glu Arg Lys Thr

35 40 45

Pro Arg Val Thr Gly Gly Gly Ala Met Ala Gly Ala Ser Thr

50 55 60

Claims

1. An epitope fusion peptide, wherein said epitope fusion peptide comprises the sequence of Z0, and said sequence of Z0 comprises the sequences of three glioma-associated antigens;

wherein the three glioma-associated antigens are linked in series by a linking sequence, and the glioma-associated antigens are EGFR-VIII, CD133 and CMV-PP65, respectively, wherein the Z0 sequence has the following structure (I):

n-terminal ` CMV-PP65-EGFR-VIII-CD 133C-terminal ` (I);

wherein, the amino acid sequence of EGFR-VIII is shown as SEQ ID NO 6; the amino acid sequence of CD133 is shown in SEQ ID NO. 8; the amino acid sequence of CMV-PP65 is shown in SEQ ID NO: 10.

2. The epitope fusion peptide of claim 1, wherein said Z0 sequence further comprises the sequence of glioma-associated antigen IL13R α 2.

3. An isolated polynucleotide encoding the epitope fusion peptide of claim 1.

4. A vector comprising the polynucleotide of claim 3.

5. The vector of claim 4, wherein the vector is a lentiviral vector.

6. A lentivirus obtained by packaging the vector of claim 4 in a packaging vector.

7. A dendritic cell obtained by transfection of the lentivirus of claim 6.

8. A cytotoxic T lymphocyte cell, wherein said cytotoxic T lymphocyte cell has the specificity of the glioma-associated antigen of claim 1.

9. Use of a dendritic cell according to claim 7 for the preparation of a cytotoxic T lymphocyte according to claim 8.

10. A method for preparing the cytotoxic T lymphocyte of claim 8, comprising the steps of:

(I) providing a dendritic cell according to claim 7;

(II) sensitizing the isolated T lymphocytes with the dendritic cells of claim 7, thereby obtaining the cytotoxic T lymphocytes of claim 8.

11. The method of claim 10, wherein the method further comprises the steps of:

(i) providing a polynucleotide according to claim 3;

(ii) inserting said polynucleotide sequence into an empty lentiviral vector to obtain the lentiviral vector of claim 5;

(iii) packaging the obtained lentivirus vector to obtain the lentivirus of claim 6;

(iv) transfecting the isolated mature dendritic cells with the obtained lentivirus to obtain the dendritic cells of claim 7.

12. Use of the cytotoxic T lymphocyte of claim 8, for the preparation of a pharmaceutical composition or formulation for the treatment of cancer, wherein the cancer is brain glioma.