CN110172480B

CN110172480B - Double-antigen epitope fusion gene recombinant lentiviral vector, antigen presenting cell and CTL cell as well as construction method and application thereof

Info

Publication number: CN110172480B
Application number: CN201910500866.1A
Authority: CN
Inventors: 焦顺昌; 张嵘; 周子珊
Original assignee: Beijing Dingcheng Taiyuan Biotechnology Co Ltd
Current assignee: Beijing Dingcheng Taiyuan Biotechnology Co Ltd
Priority date: 2019-06-11
Filing date: 2019-06-11
Publication date: 2020-01-07
Anticipated expiration: 2039-06-11
Also published as: CN110172480A

Abstract

The invention provides a double-antigen epitope fusion gene recombinant vector, an antigen presenting cell, a CTL cell, a construction method and application thereof, and belongs to the technical field of tumor drugs. The double-antigen epitope fusion gene recombinant lentiviral vector comprises a fusion gene formed by MAGE-A3 gene and KRAS mutant gene. And after the recombinant virus vector is packaged into a recombinant lentivirus, introducing the double-antigen encoding gene into the genome of the DC cell by taking the recombinant lentivirus as a mediating vector to obtain the antigen presenting cell for simultaneously presenting MAGE-A3 and KRAS mutant double antigens. And (3) inducing the T cells by using the antigen presenting cells by using a DC-CTL technology to obtain specific CTL cells targeting MAGE-A3 and KRAS mutation. Based on the fact that the CTL cell has stronger double-antigen killing effect, the CTL cell is used for preparing the medicine for treating cancer or tumor.

Description

Double-antigen epitope fusion gene recombinant lentiviral vector, antigen presenting cell and CTL cell as well as construction method and application thereof

Technical Field

The invention belongs to the technical field of tumor drugs, and particularly relates to a double-antigen epitope fusion gene recombinant vector, an antigen presenting cell, a CTL cell, a construction method and application thereof.

Background

MAGE-a3, an endogenous tumor antigen, is degraded into small polypeptides after intracellular production and is presented on the cell surface as a complex with MHC (major histocompatibility complex) molecules. MAGE-a3 protein is expressed in a variety of tumor types, including melanoma, as well as other solid tumors such as gastric, lung, esophageal, bladder, head and neck squamous cell carcinoma, and the like. For the treatment of the above diseases, chemotherapy, radiotherapy and the like can be used, but both of them cause damages to normal cells themselves.

The KRAS gene is one of the ras gene family, a gene associated with human tumors. KRAS is involved in intracellular signal transmission, and when KRAS gene is mutated, the gene is activated permanently and can not produce normal ras protein, so that signal transmission in cells is disturbed, and the cells proliferate uncontrollably and become cancerous. Effectively identify the antigen expressed by the KRAS gene after mutation, and has important significance for treating cancers.

The treatment means of tumors include surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy. The conventional tumor treatment means is surgical treatment. This treatment is an active treatment without spreading the tumor, but is too harmful to the patient. Radiotherapy and chemotherapy are therapeutic treatments for already spread tumors, but cause great harm to the body and mind of patients. Currently, there is an increasing interest in targeted drug therapy, which prevents the growth of cancer cells through interaction with specific molecular targets essential for carcinogenesis, tumor growth. The characteristics of the targeted drug determine that the targeted drug is particularly suitable for being used by patients with weak advanced stages.

Immunotherapy is the stimulation or mobilization of the immune system of the body, enhancing the anti-tumor immunity of the tumor microenvironment, and thus controlling and killing tumor cells. The tumor vaccine for the treatment at present mainly comprises tumor cell tumor vaccine, genetic engineering vaccine, anti-idiotype antibody tumor vaccine and tumor vaccine based on antigen presenting cells. DC-CTL technology, a method of immunotherapy, is the combined synergistic treatment of tumors using both DC and anti-tumor effector cells (T lymphocytes). Through antigen presentation by the DC, T lymphocytes are activated, and effector cells, Cytotoxic T Lymphocytes (CTLs), which are the most important and confer anti-tumor specificity to the body, are generated. CTL recognizes antigen peptide fragments presented on the surface of tumor cells and bound to Main Histologic Complex (MHC) -class I molecules, and kills tumor cells by a secretory killing (apoptosis of target cells caused by exocytic particle release of effector molecules such as perforin, granzyme, lymphotoxin, TNF-related protein, etc.) or non-secretory killing (apoptosis induced by binding FasL and TRAIL to corresponding receptor molecules on the surface of target cells to initiate signal transduction pathways). Although CTLs have specificity and relatively high-potency tumoricidal effects, MHC restriction exists, and it has been reported that 25% to 75% of tumor cells have different forms of Human Leukocyte Antigen (HLA) phenotypic changes, resulting in impaired immune recognition, one of the major causes of tumor immune escape; meanwhile, although the specific killing effect of CTL is relatively high, limited targeting can only kill a small part of tumor cells, and the defects seriously limit the curative effect of CTL-based tumor immunotherapy. Therefore, enhancing the target killing effect on tumor cells and improving the multi-target effect on tumor cells are the problems of the current immunotherapy research.

Disclosure of Invention

In view of the above, the present invention aims to provide a recombinant lentiviral vector, an antigen presenting cell and a CTL cell, which enhance the targeted killing effect on tumor cells and improve the multi-target effect on tumor cells, and methods for constructing and using the same.

The invention provides a double-antigen epitope fusion gene recombinant lentiviral vector, which comprises a fusion gene formed by MAGE-A3 gene and KRAS mutant gene; wherein there is no linking sequence between MAGE-A3 gene and KRAS mutant gene; the protein after the corresponding amino acid mutation in the KRAS mutant gene comprises one of KRAS-G12D, KRAS-G12C, KRAS-G12V, KRAS-G12A, KRAS-G12S, KRAS-G13D and KRAS-Q61H.

Preferably, the amino acid sequence of the KRAS-G12D is shown as SEQ ID No. 2;

the amino acid sequence of the KRAS-G12C is shown as SEQ ID No. 3;

the amino acid sequence of the KRAS-G12V is shown as SEQ ID No. 4;

the amino acid sequence of the KRAS-G12A is shown as SEQ ID No. 5;

the amino acid sequence of the KRAS-G12S is shown as SEQ ID No. 6;

the amino acid sequence of the KRAS-G13D is shown as SEQ ID No. 7;

the amino acid sequence of the KRAS-Q61H is shown as SEQ ID No. 8.

Preferably, the nucleotide sequence of the MAGE-A3 gene is shown in SEQ ID No. 1.

The invention provides a construction method of an antigen presenting cell for simultaneously presenting MAGE-A3 and KRAS mutant double antigens, which comprises the following steps:

A. mixing the recombinant lentiviral vector, the lentiviral packaging plasmid, a culture medium and a transfection reagent, and standing to obtain a transfection solution;

B. dropwise adding the transfection liquid into a culture medium containing T cells, culturing for 6-7 h, replacing a DEME culture medium containing 10% FBS by volume concentration, continuously culturing, and collecting a supernatant;

C. and (3) concentrating the supernatant, and infecting DC cells by using the obtained virus concentrated solution to obtain the antigen presenting cells which simultaneously present MAGE-A3 and KRAS mutant double antigens.

Preferably, the virus concentrate has a multiplicity of infection of DC cells of 5: 1.

The antigen presenting cell constructed by the construction method provided by the invention contains a fusion gene formed by MAGE-A3 gene and KRAS mutant gene in the genome of the antigen presenting cell.

The invention provides a construction method of specific CTL cells targeting MAGE-A3 and KRAS mutation, which comprises the following steps:

(1) separating and screening to obtain T cells expressing CD8 or CD3 on the surface;

(2) mixing the antigen presenting cells or the antigen presenting cells constructed by the construction method with the T cells obtained in the step (1) to obtain a culture system for co-culture;

(3) and after the co-culture is carried out for 2 days, adding IL-2 into the co-culture solution every day, continuing the co-culture, carrying out half-amount solution change when the culture solution turns yellow, and obtaining the harvested cells as the specific CTL when the co-culture is continued for 21-22 days.

Preferably, the ratio of the antigen presenting cells to the T cells is 1: 100-500; the concentration of the T cells is 1X 10⁶Per mL; the concentration of the antigen presenting cells is 1 × 10⁶/mL。

The invention provides specific CTL cells targeting MAGE-A3 and KRAS mutation.

The invention also provides application of the specific CTL cell in preparation of a medicine for treating cancer or tumor.

The double-antigen epitope fusion gene recombinant lentiviral vector provided by the invention comprises a fusion gene formed by MAGE-A3 gene and KRAS mutant gene. The invention takes a lentivirus plasmid as a basic expression vector, inserts the fusion gene into the lentivirus plasmid to obtain a recombinant vector which simultaneously expresses MAGE-A3 and KRAS mutant protein, and prepares for the subsequent construction of CTL with dual antigen specificity.

The invention provides an antigen presenting cell for simultaneously presenting MAGE-A3 and KRAS mutant double antigens and a construction method thereof, the invention takes lentivirus as a mediation carrier, a fusion gene formed by MAGE-A3 gene and KRAS mutant gene is introduced into the genome of the antigen presenting cell to obtain a stable genetic expression system, and the constructed antigen presenting cell can simultaneously present MAGE-A3 and KRAS mutant double antigens, thereby increasing the variety of the identified antigens and having wider application range.

The invention provides the specific CTL cell targeting MAGE-A3 and KRAS mutation and a construction method thereof, and the prepared CTL cell has specificity to MAGE-A3 and KRAS mutation antigen and can have the function of targeted killing of tumor cells with MAGE-A3 antigen and/or KRAS mutation antigen. Experiments prove that: CTL targeting both MAGE-A3 and KRAS mutations killed tumor cells more strongly than CTL targeting MAGE-A3 alone, but did not kill tumor cells that were positive for MAGE-A3 and KRAS mutations. Based on the killing effect of the specific CTL cells, the CTL cells provide a new method for immunotherapy of tumors or cancers.

Drawings

FIG. 1 shows the PCR detection of fusion genes of different MAGE-A3-KRAS mutations;

FIG. 2 is a diagram showing the cell phenotype of flow-assay CTL targeting MAGE-A3-G12D;

FIG. 3 is a diagram showing the cell phenotype of flow-assay CTL targeting MAGE-A3-G12C;

FIG. 4 is a diagram showing the cell phenotype of flow-assay CTL targeting MAGE-A3-G12V;

FIG. 5 is a diagram showing the cell phenotype of flow-assay CTL targeting MAGE-A3-G12A;

FIG. 6 is a diagram showing the cell phenotype of flow-assay CTL targeting MAGE-A3-G12S;

FIG. 7 is a diagram showing the cell phenotype of flow-tested CTLs targeting MAGE-A3-G13D;

FIG. 8 is a graph showing the cell phenotype of flow-assay CTLs targeting MAGE-A3-G13D;

FIG. 9 shows the killing effect of CTL against 293T-KRAS mutant target cells (MAGE-A3 negative + KRAS mutation);

FIG. 10 shows the killing effect of CTL on H358-KRAS mutant (MAGE-A3 positive + KRAS-mutant) target cells;

FIG. 11 shows the killing effect of CTL against H358(MAGE-A3 positive) target cells.

Detailed Description

In the invention, the amino acid sequence of KRAS-G12D is preferably shown as SEQ ID No. 2; the amino acid sequence of the KRAS-G12C is preferably shown as SEQ ID No. 3; the amino acid sequence of the KRAS-G12V is preferably shown as SEQ ID No. 4; the amino acid sequence of the KRAS-G12A is preferably shown as SEQ ID No. 5; the amino acid sequence of the KRAS-G12S is preferably shown as SEQ ID No. 6; the amino acid sequence of the KRAS-G13D is preferably shown as SEQ ID No. 7; the amino acid sequence of the KRAS-Q61H is preferably shown as SEQ ID No. 8. The nucleotide sequence of the MAGE-A3 is preferably shown as SEQID No. 1.

In the invention, the combination scheme of the fusion protein corresponding to the fusion gene comprises MAGE-A3-KRAS-G12D (SEQ ID No.9), MAGE-A3-KRAS-G12C (SEQ ID No.10), MAGE-A3-KRAS-G12V (SEQ ID No.11), MAGE-A3-KRAS-G12A (SEQ ID No.12), MAGE-A3-KRAS-G12S (SEQ ID No.13), MAGE-A3-KRAS-G13D (SEQ ID No.14) and MAGE-A3-KRAS-G61H (SEQ ID No. 15). The amino acid sequences of KRAS-G12D-MAGE-A3, KRAS-G13D-MAGE-A3, KRAS-G12V-MAGE-A3, KRAS-G12C-MAGE-A3, KRAS-G12S-MAGE-A3, KRAS-G12A-MAGE-A3 and KRAS-Q61H-MAGE-A3 are formed by combining SEQ ID No. 2-SEQ ID No.8 with SEQ ID No. 1. The nucleotide sequence of the fusion gene is a nucleotide sequence for coding amino acids shown in SEQ ID No. 9-SEQ ID No.15, and the nucleotide sequence comprises degenerate codons with different frequencies appearing in the usage bias of synonymous codons of different species. For illustration, a specific method for constructing a recombinant vector using the fusion gene is specifically described using one of the coding sequences in the fusion protein, but this should not be construed as limiting the scope of the present invention, for example, the nucleotide sequences encoding the amino acids shown in SEQ ID Nos. 9 to 15 are shown in SEQ ID Nos. 16 to 22.

In the present invention, the method for constructing the recombinant lentiviral vector preferably comprises the following steps:

artificially synthesizing a fusion gene;

adding enzyme cutting sites to two ends of the fused gene synthesized in the step I;

cloning the fusion gene connected with the enzyme cutting site obtained in the step II into a lentivirus vector to obtain a recombinant lentivirus vector.

In the invention, in order to verify that the recombinant lentiviral vector is correctly introduced with the fusion gene, the recombinant lentiviral vector is transformed into a competent cell, a monoclonal is picked for sequencing, a clone with a correct sequencing result is selected for subsequent experiments. In the present invention, the lentiviral vector comprises pCDH. The multiple cloning site of the fusion gene inserted into the lentiviral vector is BamH I/NheI.

B. adding the culture medium into the T cells, then dropwise adding the transfection solution, culturing for 6-7 h, replacing a DEME culture medium containing 10% FBS by volume concentration, continuously culturing, and collecting a supernatant;

The recombinant lentiviral vector, the lentiviral packaging plasmid, the culture medium and the transfection reagent are mixed and kept stand to obtain the transfection solution.

In the invention, a recombinant lentiviral vector, a lentiviral packaging plasmid and a culture medium are mixed in advance to form a lentiviral system, and the culture medium and a transfection reagent are mixed to form a transfection reagent system. The volume ratio of the recombinant lentiviral vector to the lentiviral packaging plasmid to the culture medium is 1:1: 125. The volume ratio of the culture medium to the transfection reagent is 125: 18. The mixing ratio of the lentivirus system and the transfection reagent is 127: 143. The medium is preferably 1640 (FBS-free) medium. The transfection reagent is preferably lipofectamine 2000. The standing is preferably carried out at room temperature for 25 min.

In the present invention, the T cell is preferably a 293T cell. The temperature of the culture is preferably 37 ℃, and the culture is preferably performed at 5% CO₂In an incubator. The volume of the transfection solution added is preferably 1ml per 10ml of the culture medium. The culture medium is replaced and the supernatant is preferably collected after 48 hours of further culture. The method of collecting the supernatant is preferably centrifugation. The centrifugal force of the centrifugation is preferably 4000g, the time of the centrifugation is preferably 5min, and the temperature of the centrifugation is preferably 4 ℃.

After obtaining the supernatant, the invention concentrates the supernatant, and infects DC cells with the obtained virus concentrated solution to obtain the antigen presenting cells which simultaneously present MAGE-A3 and KRAS mutation double antigens.

The method of concentration in the present invention is not particularly limited, and a concentration method known in the art may be used. In the present invention, the virus concentrate preferably has a multiplicity of infection of DC cells of 5: 1. The source of the DC cells is not particularly limited in the present invention, and those known in the art can be used.

The antigen presenting cell constructed by the construction method provided by the invention contains a fusion gene formed by MAGE-A3 and KRAS mutant genes in the genome of the antigen presenting cell.

In the present invention, PCR identification was performed using specific primers in order to verify that the constructed antigen-presenting cells have the above-described fusion gene introduced therein. Primers for identifying fusion genes: f: ATGCCTCTTGAGCAGAGGAG (SEQ ID No. 23); r12: GGATCCCTGG ATGGTTAAAGCGCTCTTTCC CACT (SEQ ID No. 24); r13: CAGCTGGATGGTTAAAGCGCTCTT (SEQ ID No. 25); r61: GTACTGGTCC CTCATTGCACTGTAC (SEQ ID No. 26); f and R12 are used as primers to identify MAGE-A3+ KRAS-G12N mutation, namely, the scheme of mutating MAGE-A3+ KRAS-G12 into D, C, V, S, A; identifying MAGE-A3+ KRAS-G13D mutation scheme by taking F and R13 as primers; and F and R61 are used as primers to identify the mutation scheme of MAGE-A3+ KRAS-Q61H.

(3) and after the co-culture is carried out for 2 days, adding IL-2 into the co-culture solution every day, continuing the co-culture, carrying out half-amount solution change when the culture medium turns yellow, and obtaining the cells, namely the specific CTL when the co-culture lasts for 21-22 days.

T cells expressing CD8 or CD3 on the surface are obtained by separation and screening. The separation and screening method preferably adopts an immunomagnetic bead method for separation.

After the T cells are obtained, the antigen presenting cells or the antigen presenting cells constructed by the construction method are mixed with the T cells obtained in the step (1), and the obtained culture system is co-cultured.

In the present invention, the concentration of the T cells is preferably 1X 10⁶Per mL; the concentration of the T cells is preferably adjusted with X-VIVO medium. The ratio of the antigen presenting cells to the T cells is preferably 1: 100-500, and more preferably 1: 200. The concentration of the antigen-presenting cells is preferably 1X 10⁶/mL。

In the present invention, the co-cultivation conditions are preferably as follows: 5% CO₂，37℃。

And after the co-culture is continued for 2 days, adding IL-2 into the co-culture solution every day, continuing the co-culture, performing half-amount liquid change when the culture medium turns yellow, and when the co-culture is continued for 21-22 days, obtaining the harvested cells, namely the specific CTL.

In the present invention, the final concentration of IL-2 is 50 to 1000IU/mL, and more preferably 50 to 60 IU/mL. The continued co-cultivation is preferably continued for 3 d. The new culture solution is X-VIVO culture medium containing 50IU/mL IL-2 during half-volume liquid change. Supplementing culture medium according to cell growth condition to maintain cell density at 0.5-1 × 10⁶/mL。

In the present invention, in order to verify that the cultured cells are CTL cells, the collected CTL cells are subjected to flow typing.

The invention provides specific CTL cells targeting MAGE-A3 and KRAS mutation.

In the present invention, the drug is preferably an injection solution. The concentration of specific CTL cells in the injection is preferably 1-3 multiplied by 10⁷and/mL. The injection dosage of the injection is preferably 50-200. The cancer or tumor preferably includes all tumors that are positive for KRAS mutations.

The double antigen epitope fusion gene recombinant vector, antigen presenting cell and CTL cell provided by the present invention and their construction method will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Construction of fusion Gene expression vector for MAGE-A3 and KRAS mutations

1) Artificially synthesizing the coding sequence of the fusion protein, and cloning the coding sequence to a lentiviral plasmid expression vector;

the fusion protein scheme comprises MAGE-A3+ KRAS-G12D, MAGE-A3-KRAS-G12C, MAGE-A3-KRAS-G12V, MAGE-A3-KRAS-G12A, MAGE-A3-KRAS-G12S, MAGE-A3-KRAS-G13D, MAGE-A3-KRAS-G61H;

2) and transforming the obtained recombinant vector into competent cells, selecting a monoclonal antibody, sequencing, extracting the plasmid by using a plasmid extraction kit after the sequencing result is correctly compared, and performing subsequent experiments by using an operation method according to the manufacturer's instructions.

Example 2

And (3) slow virus packaging:

1) the day before transfection, 4X 10 cells were previously transfected⁶293T (Canada) cells (10mL DMEM + 10% FBS) on 10cm dishes;

2) taking a 15mL centrifuge tube marked as A, adding the following reagents and mixing uniformly:

lentiviral plasmid 12. mu.g

Lentiviral packaging plasmid 6. mu.g + 6. mu.g

1640 culture Medium 500. mu.L

3) Taking a 15mL centrifuge tube as B, respectively adding 500 μ L1640 (no FBS) culture medium and 72 μ L lipofectamine 2000, mixing, and standing at room temperature for 5 min;

4) transferring the liquid in tube A into tube B, mixing, standing at room temperature for 25min,

5)293T cells are taken out from the incubator, the culture solution is discarded, and 10mL 1640 (without FBS) culture solution is added;

6) 1mL of the mixture was added dropwise to 293T cell culture medium, gently mixed, and incubated at 37 ℃ with 5% CO₂Culturing for 6h in an incubator;

7) changing normal culture solution DEME + 10% FBS for culture

8) After 48h, the virus solution was harvested, the culture medium was transferred to a 50mL centrifuge tube at 4000g, centrifuged for 5min, the supernatant virus solution was collected at 4 ℃, filtered through a 0.45 μm filter, and then stored at 4 ℃.

Lentiviral concentration

1) According to the volume ratio of the lentivirus supernatant to the concentrated solution (purchased from syngeneic genes) in the ratio of 5:1, mixing, and standing at 4 deg.C overnight.

2) Centrifuging at 4000g for 30min at 4 deg.C to concentrate lentivirus;

3) discard the supernatant and suck all the liquid with a pipette

4) Each tube was resuspended in 150. mu.L PBS, mixed well, aliquoted, and 10. mu.L was retained for titer determination.

Example 3

1. Isolation of DC and T cells:

1) collecting blood singly, and separating Peripheral Blood Mononuclear Cells (PBMC);

2) PBMC were adjusted to a concentration of 1X 10 with 1640 medium containing 10% FBS⁶cells/mL in Petri dishes at 37 ℃ 5% CO₂Culturing in an incubator, standing overnight; collecting suspension cells, and marking the suspension cells as T cells for later use;

3) and (3) blowing and beating the cells attached to the bottom of the culture dish by using a 1640 culture medium containing 10% FBS, and collecting adherent mononuclear cells to obtain the DC cells for later use.

2. Lentivirus infected DC

DCs isolated from the lentivirus prepared in example 2 were infected with lentivirus, the lentivirus having an MOI of 5: 1; 12-well plates, 1mL in total volume, were added 1. mu.L Polybrene per well to obtain antigen-presenting cells (i-DC) with double antigens of MAGE-A3+ KRAS-G12D.

3. Identifying double-antigen presenting cells:

extracting RNA of DC infected by lentivirus, carrying out reverse transcription, and carrying out PCR identification by using a specific primer of a fusion gene, wherein the fusion gene identification primer: primers for identifying fusion genes: f: ATGCCTCTTG AGCAGAGGAG (SEQ ID No. 23); r12: GGATCCCTGGATGGTTAAAG CGCTCTTTCC CACT (SEQ ID No. 24); r13: CAGCTGGATGGTTAAAGCGC TCTT (SEQ ID No. 25); r61: GTACTGGTCC CTCATTGCACTGTAC (SEQ ID No. 26); f and R12 are used as primers to identify MAGE-A3+ KRAS-G12N mutation, namely, the scheme of mutating MAGE-A3+ KRAS-G12 into D, C, V, S, A; identifying MAGE-A3+ KRAS-G13D mutation scheme by taking F and R13 as primers; f and R61 are used as primers to identify MAGE-A3+ KRAS-Q61H mutation.

And (3) amplification procedure: (1) 5min at 95 ℃; (2) 1min at 95 ℃, 1min at 62 ℃, 2min at 72 ℃ and 25 cycles; (3) 5min at 72 ℃.

The results are shown in FIG. 1, where 1: Marker; 2, positive control; 3: negative control; 4: MAGE-A3-G12D; 5: MAGE-A3-G12C; 6: MAGE-A3-G12V; 7: MAGE-A3-G12A; 8: MAGE-A3-G12S; 9: MAGE-A3-G13D; 10: MAGE-A3-Q61H. As can be seen from the amplified electrophoretic bands of FIG. 1, the desired band was identified, indicating that the fusion gene of MAGE-A3 and KRAS mutations was expressed.

Example 4

1. Isolation of T cells:

1) centrifuging the T + B cell sap obtained in the step 2) in the example 3 for 10min at 300g, completely removing supernatant, collecting precipitated cells, reselecting with PBS, sieving the cells with a 30-micron cell sieve, and counting after sieving;

2) the cells were resuspended in isolation buffer (PBS, pH 7.2, 0.5% BSA +2mM EDTA) every 10 th⁷cells were resuspended in 80. mu.L of isolation buffer;

3) every 10 th⁷cells, 20. mu.L of CD8/CD3 labeled magnetic beads were added;

4) after uniformly mixing, incubating for 15min at the temperature of 2-8 ℃;

5) every 10 th⁷Adding 1-2 mL of separation buffer into cells, centrifuging for 10min under the condition of 300g, and completely removing supernatant;

6) will 10⁸Resuspending cells within cells with 500 μ L of separation buffer to obtain cell suspension;

7) rinsing the separation column with a separation buffer; MS: 500 μ L, LS: 3 mL;

8) transferring the cell suspension to a separation column;

9) collecting unbound cells, washing with separation buffer for 3 times, and adding separation buffer when liquid completely flows out; MS: 3X 500. mu.L;

10) removing the column from the separator and placing it on a suitable separation tube;

11) adding a proper amount of separation buffer solution, immediately pushing out the cells, and collecting the T cells for later use.

Example 5

DC-induced CTL cells targeting MAGE-A3 and KRAS mutations

1) Adjusting CD3+ T cells or CD8+ T cells to 1X 10 with X-VIVO medium⁶2mL of the suspension was inoculated into a 6-well plate,the i-DC prepared in example 3 was prepared as follows: 200 portions into the wells for co-cultivation, now labeled Day 0;

2) culturing for 48h, adding IL-2 every Day from Day3 according to the volume of the culture medium until the final concentration of IL-2 in the culture solution is 50 IU/mL;

4) after Day 5, when the culture medium turns yellow, half-volume liquid change is carried out by using an X-VIVO culture medium containing 50IU/mL IL-2; supplementing culture medium according to cell growth condition, and maintaining cell density at 0.5-1 × 10⁶/mL；

5) And (5) harvesting the cells by Day 21, namely obtaining the specific CTL cells.

Example 6

Flow typing identification of CTL cells

1) Collecting the prepared specific CTL cells, and centrifuging for 5min at 1000 rpm;

2)1×10⁶cells/tube, adding CD3, CD4, CD8 and CD56 antibody, and keeping away from light for 30min at room temperature;

3) washing twice with PBS, and centrifuging at 1000rpm for 5 min; resuspend with PBS, and test on the machine.

The detection results are shown in FIGS. 2-8. As can be seen from FIGS. 2 to 8, in the CTLs induced by different antigen combinations, the ratio of NK is not higher than 10%, the ratio of T cells is higher than 75%, and the ratio of CD8+ T cells is higher than 70%.

Example 7

LDH method based killing experiment of specific CTL cells

1) Collecting the target cells prepared in example 5, centrifuging at 1000rpm for 5min, and removing the supernatant;

2) washing with PBS, centrifuging at 1000rpm for 5min, and removing supernatant;

3) after resuspension in 1640 medium containing 2% FBS, the counts were adjusted to 8X 10⁴cells/mL, divided into 96-well plates (U-shaped bottom), 50 μ L/well for use;

4) collecting effector cells, and centrifuging at 1000rpm for 5 min;

5) washing with PBS, and centrifuging at 1000rpm for 5 min;

6) cells were resuspended in 1640 medium containing 2% FBS, counted, and dispensed into 96-well plates (U-bottom) at 50 μ L/well, setting the effective target ratio to 10: 1. 5: 1. 2.5: 1 and 1.25: 1;

7) at 37 ℃ 5% CO₂After 3.5h of co-incubation, LDH detection was performed.

Comparative example 1

Preparation method of CTL cell targeting MAGE-A3

(1) Artificially synthesizing a coding sequence of MAGE-A3;

(2) inserting the coding sequence of MAGE-A3 into a lentivirus plasmid to form a recombinant lentivirus plasmid;

(3) transforming the recombinant lentivirus plasmid into escherichia coli, culturing, sequencing a bacterial colony, and extracting the recombinant lentivirus plasmid from positive bacterial liquid after the sequence comparison is correct;

(4) constructing lentiviruses and DC cells from the extracted recombinant lentivirus plasmid according to the method of the embodiment 2-3;

(5) performing PCR amplification verification on the constructed DC cell to obtain a band with a target length, which indicates that the constructed DC cell can express MAGE-A3 protein;

(6) the DC cells were induced into T cells by the method of example 5 to obtain single antigen-induced CTLs.

The killing effect of the CTL cells obtained was examined based on the LDH method, which was the same as that of example 7.

Killing effect result of targeting double antigen specificity CTL

Using the CTL induced by the single antigen prepared in comparative example 1 as a control, and a killing experiment was performed simultaneously with the CTL induced by the DC expressing the double antigens, 293T-KARS mutation (293T (MAGE-A3-), H358 cell (MAGE-A3+) and H358-KRAS mutation (MAGE-A3+ KRAS-G12D) were selected as target cells for the target cells, respectively, and the killing efficiencies of the two CTLs were compared.

The results show that: DC-induced CTL with single antigen MAGE-A3 had killing effect only against H358 and H358KRAS mutant cells, but no killing effect against 293T-KRAS mutant cells (FIG. 9), because single antigen-induced CTL recognized only target cells with MAGE-A3 antigen, whereas 293T-KRAS mutant cells had only KRAS mutant antigen and no MAGE-A3 antigen, and thus had no killing effect (FIG. 3). The CTL induced by the DC expressing the double antigens has higher killing effect on three target cells (figures 9-11), and the killing effect on the H358-KRAS mutation of the target cells expressing the double antigens is obviously higher than that of the CTL induced by a single antigen (figure 10).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Beijing ancient cooking peptide source Biotechnology Ltd

<120> double-antigen epitope fusion gene recombinant lentiviral vector, antigen presenting cell and CTL cell as well as construction method and application thereof

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agccaatcct atgaggactc cagcaaccaa gaagaggagg ggccaagcac cttccctgac 300

ctggagtccg agttccaagc agcactcagt aggaaggtgg ccgagttggt tcattttctg 360

ctcctcaagt atcgagccag ggagccggtc acaaaggcag aaatgctggg gagtgtcgtc 420

ggaaattggc agtatttctt tcctgtgatc ttcagcaaag cttccagttc cttgcagctg 480

gtctttggca tcgagctgat ggaagtggac cccatcggcc acttgtacat ctttgccacc 540

tgcctgggcc tctcctacga tggcctgctg ggtgacaatc agatcatgcc caaggcaggc 600

ctcctgataa tcgtcctggc cataatcgca agagagggcg actgtgcccc tgaggagaaa 660

atctgggagg agctgagtgt gttagaggtg tttgagggga gggaagacag tatcttgggg 720

gatcccaaga agctgctcac ccaacatttc gtgcaggaaa actacctgga gtaccggcag 780

gtccccggca gtgatcctgc atgttatgaa ttcctgtggg gtccaagggc cctcgttgaa 840

accagctatg tgaaagtcct gcaccatatg gtaaagatca gtggaggacc tcacatttcc 900

tacccacccc tgcatgagtg ggttttgaga gagggggaag ag 942

<210> 2

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

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

1 5 10 15

Ala Leu Thr Ile Gln

20

<210> 3

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Cys Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln

20

<210> 4

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln

20

<210> 5

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Ala Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln

20

<210> 6

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Ser Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln

20

<210> 7

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Glu Tyr Lys Leu Val Val Val Gly Ala Gly Asp Val Gly Lys Ser Ala

1 5 10 15

Leu Thr Ile Gln Leu

20

<210> 8

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly His Glu Glu Tyr Ser Ala

1 5 10 15

Met Arg Asp Gln Tyr

20

<210> 9

<211> 335

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu

1 5 10 15

Glu Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala

20 25 30

Thr Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val

35 40 45

Thr Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser

50 55 60

Pro Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp

65 70 75 80

Ser Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser

85 90 95

Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys

100 105 110

Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu

115 120 125

Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln

130 135 140

Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu

145 150 155 160

Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr

165 170 175

Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp

180 185 190

Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile

195 200 205

Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu

210 215 220

Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly

225 230 235 240

Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu

245 250 255

Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu

260 265 270

Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His

275 280 285

His Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu

290 295 300

His Glu Trp Val Leu Arg Glu Gly Glu Glu Thr Glu Tyr Lys Leu Val

305 310 315 320

Val Val Gly Ala Asp Gly Val Gly Lys Ser Ala Leu Thr Ile Gln

325 330 335

<210> 10

<211> 335

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Met Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu

1 5 10 15

Glu Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala

20 25 30

Thr Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val

35 40 45

Thr Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser

50 55 60

Pro Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp

65 70 75 80

Ser Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser

85 90 95

Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys

100 105 110

Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu

115 120 125

Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln

130 135 140

Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu

145 150 155 160

Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr

165 170 175

Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp

180 185 190

Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile

195 200 205

Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu

210 215 220

Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly

225 230 235 240

Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu

245 250 255

Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu

260 265 270

Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His

275 280 285

His Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu

290 295 300

His Glu Trp Val Leu Arg Glu Gly Glu Glu Thr Glu Tyr Lys Leu Val

305 310 315 320

Val Val Gly Ala Cys Gly Val Gly Lys Ser Ala Leu Thr Ile Gln

325 330 335

<210> 11

<211> 335

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Met Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu

1 5 10 15

Glu Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala

20 25 30

Thr Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val

35 40 45

Thr Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser

50 55 60

Pro Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp

65 70 75 80

Ser Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser

85 90 95

Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys

100 105 110

Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu

115 120 125

Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln

130 135 140

Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu

145 150 155 160

Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr

165 170 175

Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp

180 185 190

Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile

195 200 205

Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu

210 215 220

Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly

225 230 235 240

Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu

245 250 255

Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu

260 265 270

Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His

275 280 285

His Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu

290 295 300

His Glu Trp Val Leu Arg Glu Gly Glu Glu Thr Glu Tyr Lys Leu Val

305 310 315 320

Val Val Gly Ala Val Gly Val Gly Lys Ser Ala Leu Thr Ile Gln

325 330 335

<210> 12

<211> 335

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Met Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu

1 5 10 15

Glu Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala

20 25 30

Thr Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val

35 40 45

Thr Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser

50 55 60

Pro Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp

65 70 75 80

Ser Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser

85 90 95

Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys

100 105 110

Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu

115 120 125

Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln

130 135 140

Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu

145 150 155 160

Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr

165 170 175

Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp

180 185 190

Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile

195 200 205

Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu

210 215 220

Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly

225 230 235 240

Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu

245 250 255

Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu

260 265 270

Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His

275 280 285

His Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu

290 295 300

His Glu Trp Val Leu Arg Glu Gly Glu Glu Thr Glu Tyr Lys Leu Val

305 310 315 320

Val Val Gly Ala Ala Gly Val Gly Lys Ser Ala Leu Thr Ile Gln

325 330 335

<210> 13

<211> 335

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Met Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu

1 5 10 15

Glu Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala

20 25 30

Thr Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val

35 40 45

Thr Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser

50 55 60

Pro Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp

65 70 75 80

Ser Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser

85 90 95

Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys

100 105 110

Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu

115 120 125

Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln

130 135 140

Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu

145 150 155 160

Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr

165 170 175

Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp

180 185 190

Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile

195 200 205

Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu

210 215 220

Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly

225 230 235 240

Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu

245 250 255

Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu

260 265 270

Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His

275 280 285

His Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu

290 295 300

His Glu Trp Val Leu Arg Glu Gly Glu Glu Thr Glu Tyr Lys Leu Val

305 310 315 320

Val Val Gly Ala Ser Gly Val Gly Lys Ser Ala Leu Thr Ile Gln

325 330 335

<210> 14

<211> 335

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Met Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu

1 5 10 15

Glu Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala

20 25 30

Thr Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val

35 40 45

Thr Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser

50 55 60

Pro Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp

65 70 75 80

Ser Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser

85 90 95

Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys

100 105 110

Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu

115 120 125

Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln

130 135 140

Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu

145 150 155 160

Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr

165 170 175

Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp

180 185 190

Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile

195 200 205

Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu

210 215 220

Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly

225 230 235 240

Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu

245 250 255

Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu

260 265 270

Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His

275 280 285

His Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu

290 295 300

His Glu Trp Val Leu Arg Glu Gly Glu Glu Glu Tyr Lys Leu Val Val

305 310 315 320

Val Gly Ala Gly Asp Val Gly Lys Ser Ala Leu Thr Ile Gln Leu

325 330 335

<210> 15

<211> 335

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Met Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu

1 5 10 15

Glu Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala

20 25 30

Thr Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val

35 40 45

Thr Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser

50 55 60

Pro Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp

65 70 75 80

Ser Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser

85 90 95

Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys

100 105 110

Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu

115 120 125

Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln

130 135 140

Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu

145 150 155 160

Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr

165 170 175

Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp

180 185 190

Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile

195 200 205

Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu

210 215 220

Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly

225 230 235 240

Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu

245 250 255

Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu

260 265 270

Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His

275 280 285

His Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu

290 295 300

His Glu Trp Val Leu Arg Glu Gly Glu Glu Cys Leu Leu Asp Ile Leu

305 310 315 320

Asp Thr Ala Gly His Glu Glu Tyr Ser Ala Met Arg Asp Gln Tyr

325 330 335

<210> 16

<211> 1005

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atgcctcttg agcagaggag tcagcactgc aagcctgaag aaggccttga ggcccgagga 60

gaggccctgg gcctggtggg tgcgcaggct cctgctactg aggagcagga ggctgcctcc 120

tcctcttcta ctctagttga agtcaccctg ggggaggtgc ctgctgccga gtcaccagat 180

cctccccaga gtcctcaggg agcctccagc ctccccacta ccatgaacta ccctctctgg 240

agccaatcct atgaggactc cagcaaccaa gaagaggagg ggccaagcac cttccctgac 300

ctggagtccg agttccaagc agcactcagt aggaaggtgg ccgagttggt tcattttctg 360

ctcctcaagt atcgagccag ggagccggtc acaaaggcag aaatgctggg gagtgtcgtc 420

ggaaattggc agtatttctt tcctgtgatc ttcagcaaag cttccagttc cttgcagctg 480

gtctttggca tcgagctgat ggaagtggac cccatcggcc acttgtacat ctttgccacc 540

tgcctgggcc tctcctacga tggcctgctg ggtgacaatc agatcatgcc caaggcaggc 600

ctcctgataa tcgtcctggc cataatcgca agagagggcg actgtgcccc tgaggagaaa 660

atctgggagg agctgagtgt gttagaggtg tttgagggga gggaagacag tatcttgggg 720

gatcccaaga agctgctcac ccaacatttc gtgcaggaaa actacctgga gtaccggcag 780

gtccccggca gtgatcctgc atgttatgaa ttcctgtggg gtccaagggc cctcgttgaa 840

accagctatg tgaaagtcct gcaccatatg gtaaagatca gtggaggacc tcacatttcc 900

tacccacccc tgcatgagtg ggttttgaga gagggggaag agaccgagta caagctggtg 960

gtggtgggag ccgacggagt gggaaagagc gctttaacca tccag 1005

<210> 17

<211> 1005

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atgcctcttg agcagaggag tcagcactgc aagcctgaag aaggccttga ggcccgagga 60

gaggccctgg gcctggtggg tgcgcaggct cctgctactg aggagcagga ggctgcctcc 120

tcctcttcta ctctagttga agtcaccctg ggggaggtgc ctgctgccga gtcaccagat 180

cctccccaga gtcctcaggg agcctccagc ctccccacta ccatgaacta ccctctctgg 240

agccaatcct atgaggactc cagcaaccaa gaagaggagg ggccaagcac cttccctgac 300

ctggagtccg agttccaagc agcactcagt aggaaggtgg ccgagttggt tcattttctg 360

ctcctcaagt atcgagccag ggagccggtc acaaaggcag aaatgctggg gagtgtcgtc 420

ggaaattggc agtatttctt tcctgtgatc ttcagcaaag cttccagttc cttgcagctg 480

gtctttggca tcgagctgat ggaagtggac cccatcggcc acttgtacat ctttgccacc 540

tgcctgggcc tctcctacga tggcctgctg ggtgacaatc agatcatgcc caaggcaggc 600

ctcctgataa tcgtcctggc cataatcgca agagagggcg actgtgcccc tgaggagaaa 660

atctgggagg agctgagtgt gttagaggtg tttgagggga gggaagacag tatcttgggg 720

gatcccaaga agctgctcac ccaacatttc gtgcaggaaa actacctgga gtaccggcag 780

gtccccggca gtgatcctgc atgttatgaa ttcctgtggg gtccaagggc cctcgttgaa 840

accagctatg tgaaagtcct gcaccatatg gtaaagatca gtggaggacc tcacatttcc 900

tacccacccc tgcatgagtg ggttttgaga gagggggaag agaccgagta caagctggtg 960

gtggtgggag cctgtggagt gggaaagagc gctttaacca tccag 1005

<210> 18

<211> 1005

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atgcctcttg agcagaggag tcagcactgc aagcctgaag aaggccttga ggcccgagga 60

gaggccctgg gcctggtggg tgcgcaggct cctgctactg aggagcagga ggctgcctcc 120

tcctcttcta ctctagttga agtcaccctg ggggaggtgc ctgctgccga gtcaccagat 180

cctccccaga gtcctcaggg agcctccagc ctccccacta ccatgaacta ccctctctgg 240

agccaatcct atgaggactc cagcaaccaa gaagaggagg ggccaagcac cttccctgac 300

ctggagtccg agttccaagc agcactcagt aggaaggtgg ccgagttggt tcattttctg 360

ctcctcaagt atcgagccag ggagccggtc acaaaggcag aaatgctggg gagtgtcgtc 420

ggaaattggc agtatttctt tcctgtgatc ttcagcaaag cttccagttc cttgcagctg 480

gtctttggca tcgagctgat ggaagtggac cccatcggcc acttgtacat ctttgccacc 540

tgcctgggcc tctcctacga tggcctgctg ggtgacaatc agatcatgcc caaggcaggc 600

ctcctgataa tcgtcctggc cataatcgca agagagggcg actgtgcccc tgaggagaaa 660

atctgggagg agctgagtgt gttagaggtg tttgagggga gggaagacag tatcttgggg 720

gatcccaaga agctgctcac ccaacatttc gtgcaggaaa actacctgga gtaccggcag 780

gtccccggca gtgatcctgc atgttatgaa ttcctgtggg gtccaagggc cctcgttgaa 840

accagctatg tgaaagtcct gcaccatatg gtaaagatca gtggaggacc tcacatttcc 900

tacccacccc tgcatgagtg ggttttgaga gagggggaag agaccgagta caagctggtg 960

gtggtgggag ccgtcggagt gggaaagagc gctttaacca tccag 1005

<210> 19

<211> 1003

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

atgcctcttg agcagaggag tcagcactgc aagcctgaag aaggccttga ggcccgagga 60

gaggccctgg gcctggtggg tgcgcaggct cctgctactg aggagcagga ggctgcctcc 120

tcctcttcta ctctagttga agtcaccctg ggggaggtgc ctgctgccga gtcaccagat 180

cctccccaga gtcctcaggg agcctccagc ctccccacta ccatgaacta ccctctctgg 240

agccaatcct atgaggactc cagcaaccaa gaagaggagg ggccaagcac cttccctgac 300

ctggagtccg agttccaagc agcactcagt aggaaggtgg ccgagttggt tcattttctg 360

ctcctcaagt atcgagccag ggagccggtc acaaaggcag aaatgctggg gagtgtcgtc 420

ggaaattggc agtatttctt tcctgtgatc ttcagcaaag cttccagttc cttgcagctg 480

gtctttggca tcgagctgat ggaagtggac cccatcggcc acttgtacat ctttgccacc 540

tgcctgggcc tctcctacga tggcctgctg ggtgacaatc agatcatgcc caaggcaggc 600

ctcctgataa tcgtcctggc cataatcgca agagagggcg actgtgcccc tgaggagaaa 660

atctgggagg agctgagtgt gttagaggtg tttgagggga gggaagacag tatcttgggg 720

gatcccaaga agctgctcac ccaacatttc gtgcaggaaa actacctgga gtaccggcag 780

gtccccggca gtgatcctgc atgttatgaa ttcctgtggg gtccaagggc cctcgttgaa 840

accagctatg tgaaagtcct gcaccatatg gtaaagatca gtggaggacc tcacatttcc 900

tacccacccc tgcatgagtg ggttttgaga gagggggaag accgagtaca agctggtggt 960

ggtgggagcc gccggagtgg gaaagagcgc tttaaccatc cag 1003

<210> 20

<211> 1005

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

atgcctcttg agcagaggag tcagcactgc aagcctgaag aaggccttga ggcccgagga 60

gaggccctgg gcctggtggg tgcgcaggct cctgctactg aggagcagga ggctgcctcc 120

tcctcttcta ctctagttga agtcaccctg ggggaggtgc ctgctgccga gtcaccagat 180

cctccccaga gtcctcaggg agcctccagc ctccccacta ccatgaacta ccctctctgg 240

agccaatcct atgaggactc cagcaaccaa gaagaggagg ggccaagcac cttccctgac 300

ctggagtccg agttccaagc agcactcagt aggaaggtgg ccgagttggt tcattttctg 360

ctcctcaagt atcgagccag ggagccggtc acaaaggcag aaatgctggg gagtgtcgtc 420

ggaaattggc agtatttctt tcctgtgatc ttcagcaaag cttccagttc cttgcagctg 480

gtctttggca tcgagctgat ggaagtggac cccatcggcc acttgtacat ctttgccacc 540

tgcctgggcc tctcctacga tggcctgctg ggtgacaatc agatcatgcc caaggcaggc 600

ctcctgataa tcgtcctggc cataatcgca agagagggcg actgtgcccc tgaggagaaa 660

atctgggagg agctgagtgt gttagaggtg tttgagggga gggaagacag tatcttgggg 720

gatcccaaga agctgctcac ccaacatttc gtgcaggaaa actacctgga gtaccggcag 780

gtccccggca gtgatcctgc atgttatgaa ttcctgtggg gtccaagggc cctcgttgaa 840

accagctatg tgaaagtcct gcaccatatg gtaaagatca gtggaggacc tcacatttcc 900

tacccacccc tgcatgagtg ggttttgaga gagggggaag agaccgagta caagctggtg 960

gtggtgggag ccagcggagt gggaaagagc gctttaacca tccag 1005

<210> 21

<211> 1005

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

atgcctcttg agcagaggag tcagcactgc aagcctgaag aaggccttga ggcccgagga 60

gaggccctgg gcctggtggg tgcgcaggct cctgctactg aggagcagga ggctgcctcc 120

tcctcttcta ctctagttga agtcaccctg ggggaggtgc ctgctgccga gtcaccagat 180

cctccccaga gtcctcaggg agcctccagc ctccccacta ccatgaacta ccctctctgg 240

agccaatcct atgaggactc cagcaaccaa gaagaggagg ggccaagcac cttccctgac 300

ctggagtccg agttccaagc agcactcagt aggaaggtgg ccgagttggt tcattttctg 360

ctcctcaagt atcgagccag ggagccggtc acaaaggcag aaatgctggg gagtgtcgtc 420

ggaaattggc agtatttctt tcctgtgatc ttcagcaaag cttccagttc cttgcagctg 480

gtctttggca tcgagctgat ggaagtggac cccatcggcc acttgtacat ctttgccacc 540

tgcctgggcc tctcctacga tggcctgctg ggtgacaatc agatcatgcc caaggcaggc 600

ctcctgataa tcgtcctggc cataatcgca agagagggcg actgtgcccc tgaggagaaa 660

atctgggagg agctgagtgt gttagaggtg tttgagggga gggaagacag tatcttgggg 720

gatcccaaga agctgctcac ccaacatttc gtgcaggaaa actacctgga gtaccggcag 780

gtccccggca gtgatcctgc atgttatgaa ttcctgtggg gtccaagggc cctcgttgaa 840

accagctatg tgaaagtcct gcaccatatg gtaaagatca gtggaggacc tcacatttcc 900

tacccacccc tgcatgagtg ggttttgaga gagggggaag aggagtacaa gctggtggtg 960

gtgggagccg gagacgtggg aaagagcgct ttaaccatcc agctg 1005

<210> 22

<211> 1005

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

atgcctcttg agcagaggag tcagcactgc aagcctgaag aaggccttga ggcccgagga 60

gaggccctgg gcctggtggg tgcgcaggct cctgctactg aggagcagga ggctgcctcc 120

tcctcttcta ctctagttga agtcaccctg ggggaggtgc ctgctgccga gtcaccagat 180

cctccccaga gtcctcaggg agcctccagc ctccccacta ccatgaacta ccctctctgg 240

agccaatcct atgaggactc cagcaaccaa gaagaggagg ggccaagcac cttccctgac 300

ctggagtccg agttccaagc agcactcagt aggaaggtgg ccgagttggt tcattttctg 360

ctcctcaagt atcgagccag ggagccggtc acaaaggcag aaatgctggg gagtgtcgtc 420

ggaaattggc agtatttctt tcctgtgatc ttcagcaaag cttccagttc cttgcagctg 480

gtctttggca tcgagctgat ggaagtggac cccatcggcc acttgtacat ctttgccacc 540

tgcctgggcc tctcctacga tggcctgctg ggtgacaatc agatcatgcc caaggcaggc 600

ctcctgataa tcgtcctggc cataatcgca agagagggcg actgtgcccc tgaggagaaa 660

atctgggagg agctgagtgt gttagaggtg tttgagggga gggaagacag tatcttgggg 720

gatcccaaga agctgctcac ccaacatttc gtgcaggaaa actacctgga gtaccggcag 780

gtccccggca gtgatcctgc atgttatgaa ttcctgtggg gtccaagggc cctcgttgaa 840

accagctatg tgaaagtcct gcaccatatg gtaaagatca gtggaggacc tcacatttcc 900

tacccacccc tgcatgagtg ggttttgaga gagggggaag agtgtctctt ggatattctc 960

gacacagcag gtcacgagga gtacagtgca atgagggacc agtac 1005

<210> 23

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

atgcctcttg agcagaggag 20

<210> 24

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ggatccctgg atggttaaag cgctctttcc cact 34

<210> 25

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

cagctggatg gttaaagcgc tctt 24

<210> 26

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

gtactggtcc ctcattgcac tgtac 25

Claims

1. The double-antigen epitope fusion gene recombinant lentiviral vector is characterized by comprising a fusion gene formed by MAGE-A3 gene and KRAS mutant gene; wherein there is no linking sequence between MAGE-A3 gene and KRAS mutant gene;

the protein after the corresponding amino acid mutation in the KRAS mutant gene comprises one of KRAS-G12D, KRAS-G12C, KRAS-G12V, KRAS-G12A, KRAS-G12S, KRAS-G13D and KRAS-Q61H.

2. The recombinant lentiviral vector of claim 1, wherein the KRAS-G12D has the amino acid sequence shown in SEQ ID No. 2;

the amino acid sequence of the KRAS-G12C is shown as SEQ ID No. 3;

the amino acid sequence of the KRAS-G12V is shown as SEQ ID No. 4;

the amino acid sequence of the KRAS-G12A is shown as SEQ ID No. 5;

the amino acid sequence of the KRAS-G12S is shown as SEQ ID No. 6;

the amino acid sequence of the KRAS-G13D is shown as SEQ ID No. 7;

the amino acid sequence of the KRAS-Q61H is shown as SEQ ID No. 8.

3. The recombinant lentiviral vector of claim 1 or 2, wherein the nucleotide sequence of the MAGE-a3 gene is represented by SEQ ID No. 1.

4. A method for constructing an antigen presenting cell for simultaneously presenting MAGE-A3 and KRAS mutant double antigens is characterized by comprising the following steps:

A. mixing the recombinant lentiviral vector of any one of claims 1 to 3, a lentiviral packaging plasmid, a culture medium and a transfection reagent, and standing to obtain a transfection solution;

5. The method of claim 4, wherein the virus concentrate has a multiplicity of infection of DC cells of 5: 1.

6. The antigen-presenting cell constructed according to the construction method of claim 4 or 5, wherein the genome of the antigen-presenting cell comprises a fusion gene comprising MAGE-A3 gene and KRAS mutant gene.

7. A method of constructing specific CTL cells targeting MAGE-a3 and KRAS mutations comprising the steps of:

(2) co-culturing the culture system obtained by mixing the antigen-presenting cell according to claim 6 or the antigen-presenting cell obtained by the construction method according to claim 4 or 5 with the T cell obtained in step (1);

8. The method of claim 7, wherein the ratio of the antigen-presenting cells to the T cells is 1:100 to 500; the concentration of the T cells is 1X 10⁶Per mL; the concentration of the antigen presenting cells is 1 × 10⁶/mL。

9. Specific CTL cells targeting MAGE-A3 and KRAS mutations obtained by the construction method of claim 7.

10. Use of specific CTL cells according to claim 9 in the preparation of a medicament for the treatment of cancer or tumor.