CN111363009B

CN111363009B - Rectal cancer target antigen, CTL cell stimulated and cultured by rectal cancer target antigen and application of CTL cell

Info

Publication number: CN111363009B
Application number: CN202010191068.8A
Authority: CN
Inventors: 焦顺昌; 张嵘; 王海燕
Original assignee: Beijing Dingcheng Taiyuan Biotechnology Co ltd
Current assignee: Beijing Dingcheng Taiyuan Biotechnology Co ltd
Priority date: 2020-03-18
Filing date: 2020-03-18
Publication date: 2020-10-30
Anticipated expiration: 2040-03-18
Also published as: CN111363009A

Abstract

The invention provides a rectal cancer target antigen, a CTL cell cultured by the rectal cancer target antigen in a stimulation way and application thereof, belonging to the technical field of tumor treatment. The amino acid sequence of the rectal cancer target antigen is shown as SEQ ID No. 8; the rectal cancer target antigen is an effective tumor antigen obtained by prediction analysis and screening based on the detection result of the whole exon of the tumor tissue of a patient, and CTL cells obtained by culturing the rectal cancer target antigen have a specific killing effect on tumors; the rectal cancer target antigen provided by the invention can be applied to treatment of rectal cancer.

Description

Rectal cancer target antigen, CTL cell stimulated and cultured by rectal cancer target antigen and application of CTL cell

Technical Field

The invention belongs to the technical field of tumor treatment, and particularly relates to a rectal cancer target antigen, a CTL cell cultured by the rectal cancer target antigen in a stimulation manner, and an application of the CTL cell.

Background

Rectal cancer refers to cancer from the dentate line to the rectosigmoid junction, and is one of the most common malignancies of the digestive tract. Rectal cancer is low in location and is easily diagnosed by digital rectal examination and sigmoidoscope.

Surgical treatment and targeted drug therapy are adopted for treating rectal cancer; the operation treatment is in the core position in the treatment of the rectal cancer, but the rectal cancer is deeply positioned in the pelvic cavity, the anatomical relationship is complex, the operation is not easy to be thorough, and the postoperative recurrence rate is high. The targeted medicine mainly comprises bevacizumab and cetuximab, and inhibits the proliferation and metastasis of cancer cells mainly by combining with vascular endothelial growth factor and epidermal growth factor receptor.

With the popularization of precise treatment, the individuation and the variability of tumors cannot be met by conventional chemotherapy, radiotherapy and targeting, so that the search for the individualized tumor neoantigen and the culture and identification of CTL (cytotoxic T lymphocyte) of the individualized tumor neoantigen are effective methods for thoroughly eliminating the tumors.

Disclosure of Invention

In view of the above, the present invention aims to provide a rectal cancer target antigen, a CTL cell cultured by stimulation of the rectal cancer target antigen, and applications thereof; the rectal cancer target antigen provided by the invention is an effective tumor antigen obtained by prediction analysis and screening based on the detection result of the whole exon of the tumor tissue of a patient, and CTL cells obtained by culturing the rectal cancer target antigen have a specific killing effect on tumors; the target antigen specificity of the rectal cancer provided by the invention aims at the rectal cancer of the same HLA type, and personalized treatment is realized.

The invention provides a rectal cancer target antigen, and the amino acid sequence of the rectal cancer target antigen is shown as SEQ ID No. 8.

The invention provides application of the rectal cancer target antigen in preparing a medicine for treating rectal cancer.

The invention provides application of the rectal cancer target antigen in preparing CTL cells for treating rectal cancer.

The invention provides a CTL cell, wherein the CTL cell stimulates the cultured CTL cell for the rectal cancer target antigen.

The invention provides a preparation method of the CTL cell, which comprises the following steps:

1) adjusting the concentration of CTL cells to (2-3). times.10⁵cells/mL；

2) Mixing the CTL cells with the adjusted cell concentration, IL-2 and the rectal cancer target antigen to obtain a culture system;

3) and culturing the culture system to obtain the CTL cells cultured by the rectal cancer target antigen stimulation.

Preferably, the final concentration of the IL-2 in the culture system is 150-250U/mL.

Preferably, the final concentration of the rectal cancer target antigen in the culture system is 40-60 ng/mL.

Preferably, the temperature of the culture in the step 3) is 36-38 ℃, and the time of the culture is 22-26 h.

Preferably, the CTL cells described in step 1) are obtained by co-culturing PBMC cells and APC cells.

The invention provides application of the CTL cell in preparing a medicine for treating rectal cancer.

The invention has the beneficial effects that: the rectal cancer target antigen provided by the invention is an effective tumor antigen obtained by prediction analysis and screening based on the sequencing result of the whole exon of the tumor tissue of a patient, and has a specific killing effect on tumors by using CTL cells obtained by stimulating and culturing the rectal cancer target antigen; after culturing the CTL cells, the cell phenotype was analyzed by flow analysis, and the result showed NKT (CD 56)⁺CD3⁺) The ratio was 22.67%, CD8⁺The proportion of T cells was 80.47%.

Drawings

FIG. 1 shows the screening results of target antigens for rectal cancer;

FIG. 2 is a graph showing the comparison of the killing efficiency of CTLs cultured with target antigen for rectal cancer to that of antigen polypeptide-loaded target cells and HLA-only target cells;

FIG. 3 shows the results of CTL flow analysis of cell phenotype in culture stimulated by rectal cancer target antigen.

Detailed Description

The invention provides a rectal cancer target antigen, the amino acid sequence of which is shown as SEQ ID No.8 and specifically comprises the following components: FIFPETVFM are provided.

In the present invention, the rectal cancer target antigen is preferably an effective tumor antigen obtained by sequencing the tumor tissue of a patient through a whole exon, analyzing the sequencing result and screening. The exon sequencing method is not particularly limited in the invention, and a conventional exon sequencing method in the field can be adopted. According to the invention, after the exon sequencing result is obtained, the exon sequencing result is preferably analyzed by adopting a tumor neoantigen analysis technology, in the invention, the analysis is preferably carried out by adopting Immunoming tumor individualized neoantigen analysis prediction software, and the copyright of the software has the registration number of 2019SR 0130720.

The invention provides application of the rectal cancer target antigen in preparing a medicine for treating rectal cancer. In the present invention, the rectal cancer target antigen is used for treating rectal cancer by stimulating the culture of CTL cells.

The invention also provides application of the rectal cancer target antigen in preparing CTL cells for treating rectal cancer. In the present invention, the rectal cancer target antigen is used for stimulating and culturing CTL cells, so that the obtained CTL cells have specific killing effect on the cells loaded with the rectal cancer target antigen.

The invention also provides a preparation method of the CTL cell, which comprises the following steps: 1) adjusting the concentration of CTL cells to (2-3). times.10⁵cells/mL; 2) mixing the CTL cells with the adjusted cell concentration, IL-2 and the rectal cancer target antigen to obtain a culture system; 3) and culturing the culture system to obtain the CTL cells cultured by the rectal cancer target antigen stimulation.

In the present invention, the CTL cells are preferably obtained by co-culturing PBMC cells with APC cells. In the present invention, the culturing step of the APC cell is preferably as follows: after the PBMC cells are subjected to resuscitation culture, the cells are obtained by resuspension culture with a rectal cancer target antigen solution. In the present invention, the culture medium for the resuscitation culture is preferably 1640+ 10% (v/v) FBS; the recovery culture time is preferably 22-26 h, and more preferably 24 h; the temperature of the recovery culture is preferably 37 ℃, and the environmental carbon dioxide concentration of the recovery culture is preferably 5%; the concentration of PBMC cells before resuscitation culture is preferably 1 × 10⁶cells/mL; the concentration of PBMC cells before the resuscitation culture is preferably adjusted by dilution with resuscitation medium. According to the invention, after the recovery culture, cells are preferably collected by centrifugation, the rotation speed of the centrifugation is preferably 1400-1600 rpm, more preferably 1500rpm, and the time of the centrifugation is preferably 4-6 min, more preferably 5 min.

In the present invention, the rectal cancer target antigen solution preferably comprises the following components: 1640+ 10% FBS + 150-250U/mL IL-2+ 1500-1700U/mL GM-CSF + 40-60 ng/mL rectal cancer target antigen, more preferably 1640+ 10% FBS +200U/mL IL-2+1600U/mL GM-CSF +50ng/mL rectal cancer target antigen. In the invention, the IL-2 is a cell growth factor, which can enable T cells to survive for a long time and stimulate the T cells to enter a human cell division cycle; the GM-CSF stimulates the colony formation of neutrophils and macrophages in vitro; the source of the IL-2 and GM-CSF in the present invention is not particularly limited, and IL-2 and GM-CSF conventionally used in the art may be used.

In the invention, the cells collected by centrifugation are resuspended in the rectal cancer target antigen solution for resuspension culture. In the present invention, the cell density after resuspension is preferably 1X 10⁶cells/mL, wherein the time for heavy suspension culture is preferably 22-26 h, and more preferably 24 h; the temperature of the resuspension culture is preferably 37 ℃, and the ambient carbon dioxide concentration of the resuscitation culture is preferably 5%. The present invention obtains APC cells after the resuspension culture.

In the present invention, CTL cells are obtained by co-culturing the APC cells and PBMC cells obtained by the above-mentioned culture. In the invention, the number ratio of the APC cells to the PBMC cells is preferably 1 (90-110), and more preferably 1: 100. In the present invention, the co-cultivation time is preferably 20 to 22 days, and more preferably 21 days. In the invention, after the co-culture is carried out for 48 hours, the IL-2 is added into the co-culture system every day, so that the final concentration of the IL-2 is 400-600U/mL, and more preferably 500U/mL.

After obtaining the CTL cells, the concentration of the CTL cells is adjusted to (2-3) x 10⁵cells/mL, more preferably 2.5X 10⁵cells/mL。

In the present invention, the CTL cell, IL-2 and the target antigen for rectal cancer after adjusting the cell concentration are mixed to obtain a culture system. In the invention, the final concentration of the IL-2 in the culture system is preferably 150-250U/mL, and more preferably 200U/mL; the final concentration of the rectal cancer target antigen in a culture system is preferably 40-60 ng/mL, and more preferably 50 ng/mL.

In the present invention, the culture system is cultured to obtain the rectal cancer target antigen-stimulated cultured CTL cells. In the invention, the temperature of the culture is preferably 36-38 ℃, more preferably 37 ℃, and the time of the culture is preferably 22-26 h, more preferably 24 h. After the culturing, the rectal cancer target antigen is preferably collected to stimulate the cultured CTL cells; the collection is preferably performed by a centrifugation method, the rotation speed of the centrifugation is preferably 1000-1500 rpm, and the time of the centrifugation is preferably 5-10 min.

The invention provides application of the CTL cell in preparing a medicine for treating rectal cancer. In the invention, the CTL cell can kill tumor cells specifically, and the killing power of the tumor cells loaded with the rectal cancer target antigen is far greater than that of the tumor cells not loaded with the rectal cancer target antigen. The dosage form of the medicament is not particularly limited, and the cell medicament dosage form which is conventional in the field can be adopted.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

The sequencing result of the whole exon of the tumor tissue adopts Immunoming tumor individualized new antigen analysis prediction software to analyze the tumor new antigen, and the copyright of the software has the registration number of 2019SR 0130720. Obtaining 24 prediction antigens by ancient cooking vessel peptide source biological information technology limited company; the predicted 24 antigens were synthesized (synthesized by Nanjing Kinsley polypeptide Synthesis part).

TABLE 1 polypeptide sequences and corresponding numbering

1. Co-culture for APC preparation:

1) resuscitating cryopreserved PBMC (2.5X 10)⁷cells), dilute the frozen stock solution with 1640+ 10% FBS, centrifuge at 1500rpm for 5min, and centrifuge at 10mL of 164Resuspend 0+ 10% FBS, 1X 10⁶cells/mL，37℃、5％CO₂Resuscitating and culturing for 24 h;

2) respectively preparing antigen polypeptide solutions, namely 1640+ 10% FBS +200U/mL IL-2+1600U/mL GM-CSF, wherein the final concentration of each antigen polypeptide is 50ng/mL, and uniformly mixing for later use;

3) centrifuging at 1500rpm for 5min, collecting cells, resuspending the cells with polypeptide solution, and adjusting the density to 1 × 10⁶cells/mL；

4)37℃、5％CO₂After 24h of culture, the cells are APC, and the cells are lightly blown and uniformly mixed for later use.

2. Co-cultivation

1) PBMC were collected by centrifugation, resuspended in 1640+ 10% FBS, and count adjusted to 1X 10⁶cells/mL；

2) According to PBMC: adding APC in the proportion of 100:1, and mixing uniformly, and recording as Day 0;

3)37℃、5％CO₂after culturing for 48h, supplementing IL-2 according to the total volume every day, and ensuring that the final concentration of IL-2 is 200U/mL;

4) when the cells are cultured to Day 21, collecting the cells to be CTL;

3. screening of tumor neoantigens:

1) CTL cells obtained by the above culture were collected, resuspended in 1640+ 10% FBS, and the count was adjusted to 2X 10⁶cells/mL；

2)10mL CTL cells +10mL 1640+ 10% FBS cells were adjusted to 1X 10⁶cells/mL, then adding 8 μ L of 500U/μ L IL-2, the final concentration of IL-2 is 200U/mL, and then dividing into 96-well plates (flat bottom) with 200 μ L/well;

5) adding different antigens, namely the No. 1-24 antigens, into each hole, wherein the volume is 5 mu L, and the concentration is 2 mu g/mL of polypeptide; setting positive control OKT3167 ng/mL, medium control and CTL;

6)37℃5％CO₂after 24h of culture, centrifugation is carried out at 1500rpm for 10min, and 170 mu L of supernatant is transferred to a new 96-well plate;

7) in order to avoid sucking cells, the new 96-well plate in the step 6) is centrifuged at 1500rpm for 10min, and 110 μ L of supernatant is transferred to the new 96-well plate;

8) and (3) detecting the release amount of IFN-gamma in the cell supernatant by using an R & D ELISA kit so as to screen the antigen.

The screening results are shown in FIG. 1, and IFN-gamma release level exceeds that of the control, and the IFN-gamma release level is considered to be effective new antigen, wherein the polypeptide No.8 is effective new colorectal cancer specific antigen obtained by screening.

4. Construction of target cells

1) HLA-B1501 gene synthesis (trusted Jinweizhi Biotechnology Co., Ltd.) was carried out by artificial synthesis, and the synthesized gene was constructed between Nhe I and Bam HI enzyme cleavage sites on the pCDH expression vector.

HLA-B1501(SEQ ID No.25)：

atgcgggtcacggcgccccgaaccgtcctcctgctgctctcgggagccctggccctgaccgagacctgggccggctcccactccatgaggtatttctacaccgccatgtcccggcccggccgcggggagccccgcttcatcgcagtgggctacgtggacgacacccagttcgtgaggttcgacagcgacgccgcgagtccgaggatggcgccccgggcgccatggatagagcaggaggggccggagtattgggaccgggagacacagatctccaagaccaacacacagacttaccgagagagcctgcggaacctgcgcggctactacaaccagagcgaggccgggtctcacaccctccagaggatgtacggctgcgacgtggggccggacgggcgcctcctccgcgggcatgaccagtccgcctacgacggcaaggattacatcgccctgaacgaggacctgagctcctggaccgcggcggacacggcggctcagatcacccagcgcaagtgggaggcggcccgtgaggcggagcagtggagagcctacctggagggcctgtgcgtggagtggctccgcagatacctggagaacgggaaggagacgctgcagcgcgcggaccccccaaagacacatgtgacccaccaccccatctctgaccatgaggccaccctgaggtgctgggccctgggcttctaccctgcggagatcacactgacctggcagcgggatggcgaggaccaaactcaggacaccgagcttgtggagaccagaccagcaggagatagaaccttccagaagtgggcagctgtggtggtgccttctggagaagagcagagatacacatgccatgtacagcatgaggggctgccgaagcccctcaccctgagatgggagccatcttcccagtccaccatccccatcgtgggcattgttgctggcctggctgtcctagcagttgtggtcatcggagctgtggtcgctactgtgatgtgtaggaggaagagctcaggtggaaaaggagggagctactctcaggctgcgtccagcgacagtgcccagggctctgatgtgtctctcacagcttga。

Transforming the obtained recombinant plasmid into competent cells, selecting a monoclonal antibody for sequencing, selecting the clone with the correct sequencing result, and carrying out subsequent experiments;

2) the plasmid extraction was carried out with Tiangen plasmid extraction kit (purchased from Tiangen Biochemical technology, Beijing) Ltd.) according to the manufacturer's instructions.

3) And (3) slow virus packaging: recovering 293T cells, and packaging the cells for lentiviruses after two generations; cell transfection: (T175 flask) cell density 2X 10⁷One/bottle; a15 mL centrifuge tube (labeled A) was taken, and packaged plasmids 4K (17. mu.g), 6K (34. mu.g) and the desired plasmid (51. mu.g) were added to a Buffer containing 1mL jetPRIME, and gently mixed. Slowly dripping jetPRIME (marked as B) into the centrifuge tube A, adding while shaking the centrifuge tube A at a constant speed to obtain solution C, and standing for 10 min; old medium was decanted from T175, 18mL DMEM (without antibiotics and serum) was added to C, added to a T175 flask, and placed at 37 ℃ in 5% CO₂Culturing in an incubator. After 4-6 h of transfection, the medium containing the transfection complex was aspirated off and replaced with fresh medium preheated at 37 ℃. Cultured for 48h and 72h and collected.

4) Lentivirus transfection of 3T3 cells: DMEM was used to prepare whole cell culture medium containing 7% FBS, designated DMEM. DMEM was used to adjust the 3T3 cell concentration to 5X 10⁵Perml, add 6 well plates, 1mL per well, incubate at 37 ℃ overnight. DMEM medium in 6-well plates was discarded, and 1mL of AIM-V-P mixed virus solution was added to each well. Transferring the culture medium into a T75 culture bottle after 3 days of culture, and adding puromycin for screening; after screening for 6 days, 3T3 cells which stably express HLA-B1501 are obtained and are marked as 3T 3-HLA;

5. killing experiment-LDH method

1) Collecting target cells 3T3 and 3T3-HLA, and centrifuging at 1000rpm for 5 min;

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

3) adding No.8 polypeptide solution, wherein the concentration of No.8 polypeptide is 50ng/mL, the volume is 3mL, carrying out effective new antigen-loading, and carrying out loading for 4h at 37 ℃;

4) centrifuging at 1000rpm for 5min, removing excess solution, and washing with PBS for 2 times;

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

6) collecting CTL cells, and centrifuging at 1000rpm for 5 min;

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

8) after counting the resuspended cells in 1640+ 2% FBS, they were dispensed into 96-well plates (U-bottom), 50 μ L/well, and the effective target ratio (CTL: 3T3-HLA or CTL: 3T 3-HLA-loaded polypeptide) is 40:1, 20:1, 10:1, 5:1, 2.5:1 and 1.25: 1; 5% CO at 37 ℃₂After the incubation for 20h, performing LDH detection, wherein the kit for LDH detection is cytotoxin LDH Assay

The manufacturer: dojindo has a product number CK 12.

As shown in FIG. 2, the antigen-stimulated CTL has higher killing efficiency on target cells loaded with antigen polypeptide than target cells loaded with HLA alone, indicating that the cultured CTL can recognize the antigen and has specific killing effect, and can be used as the first choice antigen for treating rectal cancer.

6. Flow-type typing

1) Collecting CTL, centrifuging at 1000rpm for 5 min;

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;

4) and (5) resuspending by PBS and detecting on a machine.

The screened potent neoantigens were cultured and analyzed for cell phenotype by flow cytometry, and the results are shown in FIG. 3, NKT (CD 56)⁺CD3⁺) The ratio was 22.67%, 80.47% was CD8⁺A T cell; NK can generate nonspecific killing, the low NK proportion indicates that the nonspecific killing is small, and the specific killing effect is obvious.

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

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ccggagtatt gggaccggga gacacagatc tccaagacca acacacagac ttaccgagag 300

agcctgcgga acctgcgcgg ctactacaac cagagcgagg ccgggtctca caccctccag 360

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Claims

1. A rectal cancer target antigen, wherein the amino acid sequence of the rectal cancer target antigen is shown as SEQ ID No. 8.

2. Use of the rectal cancer target antigen of claim 1 in the manufacture of a medicament for the treatment of rectal cancer.

3. A CTL cell, wherein said CTL cell is cultured in the presence of a rectal cancer target antigen according to claim 1.

4. The method for producing CTL cells according to claim 3, comprising the steps of:

1) adjusting CTL cell concentration to 2X 10⁵cells/mL～3×10⁵cells/mL；

2) Mixing the CTL cell, IL-2 and the rectal cancer target antigen as set forth in claim 1 after adjusting the cell concentration to obtain a culture system;

5. The method according to claim 4, wherein the final concentration of IL-2 in the culture system is 150 to 250U/mL.

6. The method of claim 4 or 5, wherein the final concentration of the target antigen for rectal cancer in the culture system is 40-60 ng/mL.

7. The method according to claim 4, wherein the temperature of the culture in step 3) is 36 to 38 ℃ and the time of the culture is 22 to 26 hours.

8. The method according to claim 4, wherein the CTL cells in step 1) are obtained by co-culturing the PBMC cells and the APC cells.

9. Use of the CTL cell according to claim 3 or the CTL cell obtained by the method according to any one of claims 4 to 8 for the preparation of a medicament for the treatment of rectal cancer.