CN110885790B

CN110885790B - Targeting MMSA-1 chimeric antigen receptor modified T lymphocyte and preparation method and application thereof

Info

Publication number: CN110885790B
Application number: CN201911044528.8A
Authority: CN
Inventors: 周芙玲; 童西琴; 魏永长; 金艳霞; 丁路; 邵亮; 江宏强; 灿灿; 商豫凤; 吴八路
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2019-10-30
Filing date: 2019-10-30
Publication date: 2021-07-20
Anticipated expiration: 2039-10-30
Also published as: CN110885790A

Abstract

The invention provides a targeting MMSA-1 chimeric antigen receptor modified T lymphocyte and a preparation method and application thereof. The invention constructs a specific CAR-T cell line targeting MMSA-1, verifies the anti-myeloma activity in vitro and in vivo, and finds a new target for CAR-T immunotherapy of multiple myeloma. A specific MMSA-1 single-chain antibody (scFv) is obtained by humanizing and transforming a wild-type MMSA-1 antibody, the obtained MMSA-1 antibody gene fragment is modified and loaded into a plasmid, and MMSA-1 chimeric receptor T cells are prepared by lentivirus transfection. And detecting the expression of the CAR on the surface of the T cell, amplifying and transfecting the successful CAR-T cell in vitro, and finally carrying out in vitro and in vivo tests to detect the anti-myeloma effect of the CAR-T cell.

Description

Targeting MMSA-1 chimeric antigen receptor modified T lymphocyte and preparation method and application thereof

Technical Field

The invention belongs to the field of tumor immunotherapy, and particularly relates to a target MMSA-1 chimeric antigen receptor modified T lymphocyte and a preparation method and application thereof.

Background

Multiple Myeloma (MM) is a malignant clonal plasmacytosis, and Myeloma Bone Disease (MBD) is one of its major clinical manifestations, with bone destruction characterized by unbalanced bone remodeling. In the last decade, although clinical research aims at a new therapeutic drug, namely proteasome inhibitor bortezomib, and hematopoietic stem cell transplantation, and the like, the disease remission rate and the quality of life are higher than those of chemotherapy measures, the bone destruction and bone marrow cell residual focus are still the main problems facing the treatment of multiple myeloma. Therefore, it is important to improve the clinical efficacy of MM patients and to focus on finding precise therapeutic approaches that target MM cells in the bone marrow microenvironment.

The Science journal lists tumor immunotherapy as the first ten scientific breakthroughs in 2013, and one of the major developments was immune cell therapy using chimeric antigen receptor (chimeric antigen receptor) T cells. Unlike traditional adoptive immunotherapy, CAR-T cells are one type of T cells that are modified by genetic engineering methods to carry receptors that recognize tumor antigen-specific. The technical advantages include: 1) extremely low rejection risk; 2) can be widely used for various tumors; 3) the glycolipid non-protein antigen can also be utilized, so that the range of tumor antigens is expanded; 4) the action process is not limited by Main Histocompatibility Complex (MHC); 5) the CAR-T cell has an immunological memory function, can survive in vivo for a long time, and has the capability of completely eliminating tumor cells. The advent of CAR-T cells has advanced myeloma cell immunotherapy to a new stage, a very effective strategy for relapsed/refractory myeloma patients, both for transitional treatment of patient allogeneic stem cell transplantation and as a rescue strategy for disease relapse after transplantation. For CAR-T cell therapy against multiple myeloma, finding and evaluating specific myeloma cell targets is crucial.

In 2005, a study reported that multiple myeloma antigen MMSA-1, which is specifically and highly expressed on MM cells with active proliferation, was screened by serological analysis using a recombinant cDNA expression library. Another study in 2006 found that human MMSA-1 is a membrane protein that is mainly localized on the cell membrane and regulates the proliferation and apoptosis of myeloma cells. One recent study induced HLA-a 0201 positive peripheral blood mononuclear cells to form mature Dendritic Cells (DCs) by stimulation with multiple cytokine combinations in vitro, and the results showed that DCs loaded with MMSA-1+ DKK1 combination polypeptides induced effector T cells with strong killing effect on HLA-a 0201 positive myeloma cells. As a marker molecule of MM cells, MMSA-1 meets the requirement of CAR-T treatment on antigen targeting, and is an ideal target. Therefore, the subject group is further to prepare autologous/allogeneic MMSA-1-specific CAR-T cells (MMSA-1 CAR-T) and to confirm their anti-myeloma activity in vitro and in vivo.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a targeting MMSA-1 chimeric antigen receptor modified T lymphocyte and a preparation method and application thereof. A specific CAR-T cell line targeting MMSA-1 is constructed, the anti-myeloma activity of the CAR-T cell line is verified in vitro and in vivo, and a new target is searched for CAR-T immunotherapy of multiple myeloma. A specific MMSA-1 single-chain antibody (scFv) is obtained by humanizing and transforming a wild-type MMSA-1 antibody, the obtained MMSA-1 antibody gene fragment is modified and loaded into a plasmid, and MMSA-1 chimeric receptor T cells are prepared by lentivirus transfection. And detecting the expression of the CAR on the surface of the T cell, amplifying and transfecting the successful CAR-T cell in vitro, and finally carrying out in vitro and in vivo tests to detect the anti-myeloma effect of the CAR-T cell.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a method of targeting a MMSA-1 chimeric antigen receptor modified T lymphocyte, comprising: the cell surface expresses a specific chimeric antigen receptor, can specifically recognize the MMSA-1 antigen on the surface of the myeloma cell and plays an anti-myeloma effect; the sequence of the MMSA-1CAR fragment is shown as SEQ ID NO. 1; the front end of the polypeptide is a CD8 die-penetrating signal peptide, and the sequence of the polypeptide is shown in SEQ ID NO. 2; the adjacent MMSA-1scFv single-chain antibody has the sequence shown in SEQ ID NO. 3; the rear end of the probe is sequentially provided with a CD8 transmembrane region, an intracellular 4-1BB costimulatory signal region and a CD3zeta TCR activation region, and the sequence of the probe is shown as SEQ ID NO. 4.

In a second aspect, the present invention provides a method for preparing the above-mentioned target MMSA-1 chimeric antigen receptor modified T lymphocyte, which comprises the following steps:

1) preparation of MMSA-1CAR plasmid:

after the wild type antibody is subjected to humanized transformation, MMSA-1scFv is obtained, an MluI enzyme cutting site and a CD8 membrane penetrating signal peptide are inserted before a fragment, and a BamHI enzyme cutting site is inserted after the fragment; carrying out double enzyme digestion on the synthesized pUC57-Amp plasmid containing MluI + CD8a-MMSA-1scFv + BamHI, carrying out agarose gel electrophoresis to identify the enzyme digestion effect, and recovering gel to obtain a modified gene fragment; meanwhile, carrying out MluI and BamHI double enzyme digestion on the lentiviral backbone plasmid pHR containing the CD8 transmembrane region, the 4-1BB costimulation signal region and the CD3zeta TCR activation region, and carrying out agarose gel electrophoresis identification to recover long fragments; connecting the modified MMSA-1scFv fragment to a lentiviral backbone plasmid pHR, extracting the plasmid, and obtaining a pHR-MMSA-1CAR plasmid after determining that the sequencing is correct, wherein the sequencing sequence of the pHR-MMSA-1CAR plasmid is shown as SEQ ID NO. 5;

2) preparation of MMSA-1 chimeric antigen receptor lentivirus:

dropwise adding the transfection reagent and the plasmids into serum-free and antibiotic-free RPMI1640, dropwise adding the final transfection reagent and plasmid mixed solution into a T175 culture bottle with the convergence of 293T cells being 60-70% for culture, and collecting 293T cell supernatant after 72h of transfection; collecting the supernatant of the 293T cells in a centrifuge tube to enable the fallen 293T cells to be centrifuged to the bottom of the tube, filtering the centrifuged supernatant by using a 0.4 mu M filter membrane, transferring the filtered supernatant into an ultracentrifuge tube, centrifuging the filtered supernatant for 2 hours at vacuum 25000rpm, discarding the supernatant, and re-suspending the concentrated lentivirus in lmL fresh culture medium;

3) construction and in vitro expansion of CAR-T cells:

50mL of fresh blood was taken and passed through the lymph nodeSubjecting the cell separation solution to density gradient centrifugation to separate mononuclear cells; the mononuclear cells are arranged in a 1-2X 10⁶Resuspend in CTSTMAIM VTM SFM medium/mL; 5% ICS, 50ng/mL of CD3 monoclonal antibody and 50ng/mL of CD28 monoclonal antibody are added simultaneously to activate T lymphocytes, and the cells are cultured for 48h at 37 ℃ with 5% CO 2;

after 2 days of culture, cells were harvested and resuspended to 1X10⁶Adding the concentrated lentivirus according to the MOI of 5, simultaneously adding IL-2 and polybrene with final concentration of 200U/mL, uniformly mixing, culturing at 37 ℃ for 6-8 hours by 5% CO2, and centrifuging at 300g for 5min to obtain fresh CTSTM AIM VTM SFM culture medium containing 200U/mL IL-2;

adding fresh CTSTM AIM VTM SFM medium containing 200U/mL IL-2 every 2-3 days to maintain the cell density at 1 × 10⁶about/mL, and performing amplification culture for 10-12 days.

In a third aspect, the invention provides a use of the above-mentioned targeting MMSA-1 chimeric antigen receptor modified T lymphocyte for treating multiple myeloma.

The invention detects the specific killing effect of MMSA-1CAR-T cells on MM cell strains (RPMI-8226) and primary myeloma cells, and finds that the specific killing efficiency is increased along with the increase of an effective target ratio (CAR-T cell number/tumor cell number), and the specific killing effect is not generated on K562 cells which do not express MMSA-1 antigen. The intracellular flow cytometry detection shows that the synthesis amount of the intracellular granzyme B, the perforin and the IL-2 is increased to a certain degree after the MMSA-1CAR-T cells are stimulated by tumor cells. The secretion condition of the cytokine in the supernatant of the tumor cell killed by the MMSA-1CAR-T cell is detected by an ELISA kit, and a large amount of IL-2 and INF-gamma cytokines are found, so that a good killing effect is prompted. An immune deficiency mouse is inoculated subcutaneously through an RPMI-8226 cell line to construct a mouse MM tumor model, the anti-myeloma effect of the MMSA-1CAR-T cell in vivo is detected, and the result shows that the MMSA-1CAR-T cell can obviously reduce the tumor load and obviously prolong the survival period of the mouse.

The invention has the following advantages and beneficial effects:

1. the MMSA-1 antigen targeted by the MMSA-1CAR-T cell is a new antigen of multiple myeloma cell surface specificity, is expressed on the cell surface, and the HLA-A0201 restrictive MMSA-1 polypeptide screened by a bioinformatics method shows better anti-tumor effect in vitro and in vivo, has less toxic and side effects compared with the traditional chemotherapy and treatment and has stronger targeting property.

2. MMSA-1CAR-T cells can kill MM cells specifically in vitro.

3. The MMSA-1CAR-T cell can obviously reduce tumor load in vivo, obviously prolong the survival time of mice, and has clinical application potential.

Drawings

FIG. 1 flow cytometry was used to detect the affinity of MMSA-1 single chain antibodies to RPMI-8226 cells;

FIG. 2 flow cytometry is used to detect infection efficiency and cell proliferation curves;

FIG. 3 flow cytometry detects specific killing effect of MMSA-1CAR-T cells in vitro;

FIG. 4 flow cytometry and ELISA method for detecting cytokine secretion when MMSA-1CAR-T cells kill tumor cells;

FIG. 5A MM mouse model of cell line was constructed to explore the antimyeloma effect of MMSA-1CAR-T in vivo.

FIG. 6MMSA-1 CAR plasmid design and pattern diagrams.

Detailed Description

The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.

Firstly, cell strain: the RPMI-8226 cell line and the K562 cell line are obtained from ATCC (Manassas, VA), and the human primary myeloma cells are separated from bone marrow samples of patients with multiple myeloma in Hematology of China and south Hospital.

II, reagent: anti-his labeled antibody [ ifluor647]Lymphocyte separation (Tianjin level), CTSTM AIM VTM SFM medium (GIBCO, Cat. No. A3021002), ICS (GIBCO, A2596101), CD3 monoclonal antibody (Ebioscience), CD28 monoclonal antibody (Ebioscience), IL-2 (Quangang), polybrene (Sigma), Alexa Fluor647Affinipure Goat Anti-Mouse IgG, F (ab')2fragment specific (jackson immunoresearch), ZDHHC9 PolyclonalAntibody(Invitrogen)，FITC anti-human CD3(Biolegend)，Human IL-2ELISA MAX^TM Deluxe(Biolegend)，Human IFN-γELISA MAX^TM Deluxe(Biolegend)。

Example 1 construction and affinity flow assay of MMSA-1scFv

Preparation of MMSA-1CAR

After the wild type antibody is subjected to humanized transformation, MMSA-1scFv is obtained, an MluI enzyme cutting site and a CD8 transmembrane signal peptide are inserted before a fragment, a BamHI enzyme cutting site is inserted after the fragment, and the fragment is handed to a gene company (Jinweizhi) for synthesis. The pUC57-Amp plasmid containing MluI + CD8a-MMSA-1scFv + BamHI, which is synthesized by gene company, is subjected to double enzyme digestion by MluI (NEB) and BamHI (NEB), the enzyme digestion effect is identified by agarose gel electrophoresis, and the modified gene fragment is obtained by gel recovery. Meanwhile, the slow virus skeleton plasmid pHR which contains the CD8 transmembrane region, the 4-1BB costimulatory signal region and the CD3ZetaTCR activation region and is already existed in a laboratory is subjected to MluI and BamHI double enzyme digestion, and the gel is used for recovering long fragments after agarose gel electrophoresis identification. And connecting the modified MMSA-1scFv fragment to a lentiviral backbone plasmid pHR, extracting the plasmid, and obtaining the pHR-MMSA-1CAR plasmid after determining that the sequencing is correct.

2. Affinity flow assay

Purified single chain antibodies were affinity sequenced using flow cytometry for antibody screening. RPMI-8226 cells (positive cell line, ATCC) and K562 cells (negative cell line, ATCC) were cultured as described in the specification. Briefly, each well is about 3X 10⁵Individual cells were seeded in 96-well circular bottom plates and then incubated with a single primary antibody at 4 ℃ for 30 minutes. Wild-type scFv (starting from 3 μm to 0.152nm) and 16 humanized scFv were serially diluted three-fold. The cells were washed 3 times with PBS buffer, and then with anti-his-labeled antibody [ ifluor647](3. mu.g/ml) was incubated in the dark for 30 minutes. Again, cells were washed 3 times with PBS buffer and then analyzed for binding to EC50 using fascibur (bd bioscience) and flowjo software.

As shown in FIG. 1, the affinity of the 16 scFv for RPMI-8226 compared to WT scFv was the highest by flow assay, VL2-G4S-VH 4.

Example 2 preparation of MMSA-1CAR-T cells and detection of infection efficiency

MMSA-1CAR-T cell preparation

50mL of fresh blood was taken, and mononuclear cells were separated by density gradient centrifugation using a lymphocyte separation medium (tertiary amine). The mononuclear cells are arranged in a 1-2X 10⁶Perml was resuspended in CTSTM AIM VTM SFM medium (GIBCO, cat # A3021002). T lymphocytes were activated by the simultaneous addition of 5% ICS (GIBCO, A2596101), 50ng/mL of CD3 monoclonal antibody (Ebioscience) and 50ng/mL of CD28 monoclonal antibody (Ebioscience), and cultured at 37 ℃ for 48 hours in 5% CO 2.

After 2 days of culture, cells were harvested and resuspended to 1X10⁶The concentrated lentivirus was added to the medium at MOI of 5, followed by addition of IL-2 (spring harbor) and polybrene (Sigma) at final concentrations of 200U/mL, mixed, incubated at 37 ℃ with 5% CO2 for 6-8 hours, and centrifuged at 300g for 5min to obtain fresh CTSTMAIM VTM SFM medium (containing 200U/mL IL-2).

Adding fresh CTSTM AIM VTM SFM culture medium (containing 200U/mL IL-2) every 2-3 days, and maintaining the cell density at 1 × 10⁶about/mL, and performing amplification culture for 10-12 days.

2. Infection efficiency detection and cell proliferation detection

The CAR-T cells were tested for CAR positivity by flow cytometry using Alexa Fluor647Affinipure Goat Anti-Mouse IgG, F (ab')2fragment specific (jackson immunoresearch). Collecting MMSA-1CAR-T cells cultured for 2 days after virus infection, and culturing at 5 × 10⁵mu.L of antibody was added to each sample, incubated at 4 ℃ for 15min in the dark, centrifuged at 400g for 5min, washed 1 time with 1mL of PBS, and the pellet was resuspended in 2% paraformaldehyde and then examined by a machine, a fascicularir (BD bioscience).

As shown in FIG. 2A, the infection efficiency of the lentivirus infected T cells after 48h flow detection is over 50%, indicating that over 50% of MMSA-1CAR-T cells are successfully obtained.

The cells were counted every 2 to 3 days using a hemocytometer and cell proliferation curves were plotted.

As shown in figure 2B, MMSA-1CAR-T cells proliferated well in vitro.

Example 3 flow cytometry detection of specific killing effect of MMSA-1CAR-T cells in vitro

By flow-typeThe cell technology detects the MMSA-1 antigen expression of K562 cell line, RPMI-8226 cell line and primary tumor cell. Taking K562 cell line, RPMI-8226 cell line and primary tumor cell each 5X 10⁵mu.L of ZDHHC9 Polyclonal Antibody (Invitrogen) Antibody and 1uL of secondary Antibody were added to each sample, incubated at 4 ℃ in the dark for 15min, centrifuged at 400g for 5min, washed 1 time with 1mL of PBS, and the pellet was resuspended in 2% paraformaldehyde and then examined by an onboard facscalibur (BD bioscience).

As shown in FIG. 3A, K562 does not express MMSA-1 molecules on the surface, while RPMI-8226 cells and primary MM tumor cells express MMSA-1 molecules on the surface.

Two cells were mixed as 1: 1 in 96-well plates, 1X10 per well⁵The following components are added according to E: t is 1: 1. 3: 1. 5: 1 MMSA-1CAR-T cells and T cells cultured for 9 days were inoculated separately, 3 duplicate wells per group. Incubated at 37 ℃ for 24 hours with 5% CO 2. The remaining cells were added to each well 1. mu.L of ZDHHC9 Polyclonal Antibody (Invitrogen), 1. mu.L of secondary Antibody, and 1. mu.L of FITC anti-human CD3(Biolegend), incubated at room temperature in the dark for 15min, centrifuged at 400g for 5min, washed 2 times with 1mL of PBS, and the pellet was resuspended in 2% paraformaldehyde and then examined in a machine facscalibur (BD bioscience).

As shown in FIGS. 3B-C, MMSA-1CAR-T cells specifically kill MMSA-1 antigen-positive cells, and the efficiency of specific killing increases with increasing effective-to-target ratio (P.ltoreq.0.001). But has no specific killing effect on K562 cells which do not express MMSA-1 antigen.

Example 4 detection of cytokine secretion in tumor cells killed by MMSA-1CAR-T cells by flow cytometry and ELISA method

T5h stimulated by PMA, ION and BFA was used as a positive control for intracellular flow detection of cytokines, MMSA-1CAR-T was used as a negative control with BFA added for 5h, and intracellular cytokine release after MMSA-1CAR-T stimulation by RPMI-8226 tumor cell line and BFA was detected. According to the kit instruction, the final precipitate is resuspended by membrane-breaking buffer and then is detected by a facscalibur (BD bioscience).

As shown in FIG. 4A, there was some increase in the synthesis of granzyme B, perforin and IL-2 following stimulation of MMSA-1CAR-T cells by tumor cells.

Determination of E by ELISA: t is 3: 1 killing IL-2 and IFN-gamma concentration in supernatant samples, Human IL-2 ELISA MAX according to kit instructions^TM Deluxe(Biolegend)、Human IFN-γELISA MAX^TMDeluxe (biolegend) protocol, 3 duplicate wells were made for all standards and samples.

As shown in FIG. 4B, the supernatant of the tumor cell killing MMSA-1CAR-T cells had a large amount of IL-2 and INF-gamma cytokines, suggesting that the killing effect was good.

In vivo experiments to verify the antimyeloma effect of MMSA-1CAR-T cells [ example 5 ]

8-12 week NSG mice, from the university of Nanjing institute of model animals, 25-30g, 5 x10⁶RPMI-8226 cells were injected subcutaneously to establish a mouse MM tumor model.

The T cell group is control group, MMSA-1CAR-T cell group is treatment group, each of the control group and the treatment group comprises 8 mice, and the infusion amount of T cells and CAR-T cells is 2 x10⁷. Tumor burden was measured twice weekly after CAR-T cell treatment using IVIS spectrometer (Perkinelmer, boston, ma). The IVIS spectrometer use parameter method comprises the following steps: luc-labeled cells were tumor-bearing in mice, and when mice were injected with the substrate fluorescein (luciferin), the substrate concentration: 150mg/kg, luciferase can catalyze luciferin to be oxidized into oxyuciferin, bioluminescence can be emitted in the oxidation process, and the imaging time of the mouse is generally controlled to be 3-5min, so that statistical data can be obtained. The cytokine concentration in the mouse serum was determined as described in example 4, following the ELISA kit protocol.

As shown in FIG. 5A, MMSA-1CAR-T cells were able to significantly reduce tumor burden and maintained for a period of at least 34 days with significant differences between groups 2 (P ≦ 0.001).

As shown in FIG. 5B, MMSA-1CAR-T cells significantly improved survival of mice and maintained for a period of at least 60 days, with significant differences between groups 2 (P.ltoreq.0.001).

As shown in FIG. 5C, there was a large amount of cytokine secretion in the mouse serum after the MMSA-1CAR-T cells were injected, and there was a difference between the time points at which the secretion amount and the maximum secretion amount of different cytokines appeared.

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tagccgcgtt ctaacgacaa tatgtacaag cctaattgtg tagcatctgg cttactgaag 480

cagaccctat catctctctc gtaaactgcc gtcagagtcg gtttggttgg acgaaccttc 540

tgagtttctg gtaacgccgt cccgcacccg gaaatggtca gcgaaccaat cagcagggtc 600

atcgctagcc agatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt 660

gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc 720

gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg 780

ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac 840

ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg aatggtgcac 900

tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc 960

cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac 1020

cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg 1080

aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta 1140

gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 1200

aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 1260

ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 1320

ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 1380

agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 1440

tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 1500

tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 1560

ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 1620

gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 1680

acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 1740

tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 1800

gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 1860

actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 1920

aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 1980

cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 2040

tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 2100

cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 2160

tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 2220

ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 2280

ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 2340

cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 2400

aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtctttct 2460

agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 2520

tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 2580

ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 2640

cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 2700

atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 2760

ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 2820

tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 2880

gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 2940

gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 3000

cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 3060

gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 3120

tcattaatgc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca 3180

gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc 3240

ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata 3300

gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg 3360

ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag 3420

ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagcttgga 3480

cacaagacag gcttgcgaga tatgtttgag aataccactt tatcccgcgt cagggagagg 3540

cagtgcgtaa aaagacgcgg actcatgtga aatactggtt tttagtgcgc cagatctcta 3600

taatctcgcg caacctattt tcccctcgaa cactttttaa gccgtagata aacaggctgg 3660

gacacttcac atgagcgaaa aatacatcgt cacctgggac atgttgcaga tccatgcacg 3720

taaactcgca agccgactga tgccttctga acaatggaaa ggcattattg ccgtaagccg 3780

tggcggtctg taccgggtgc gttactggcg cgtgaactgg gtattcgtca tgtcgatacc 3840

gtttgtattt ccagctacga tcacgacaac cagcgcgagc ttaaagtgct gaaacgcgca 3900

gaaggcgatg gcgaaggctt catcgttatt gatgacctgg tggataccgg tggtactgcg 3960

gttgcgattc gtgaaatgta tccaaaagcg cactttgtca ccatcttcgc aaaaccggct 4020

ggtcgtccgc tggttgatga ctatgttgtt gatatcccgc aagatacctg gattgaacag 4080

ccgtgggata tgggcgtcgt attcgtcccg ccaatctccg gtcgctaatc ttttcaacgc 4140

ctggcactgc cgggcgttgt tctttttaac ttcaggcggg ttacaatagt ttccagtaag 4200

tattctggag gctgcatcca tgacacaggc aaacctgagc gaaaccctgt tcaaaccccg 4260

ctttaaacat cctgaaacct cgacgctagt ccgccgcttt aatcacggcg cacaaccgcc 4320

tgtgcagtcg gcccttgatg gtaaaaccat ccctcactgg tatcgcatga ttaaccgtct 4380

gatgtggatc tggcgcggca ttgacccacg cgaaatcctc gacgtccagg cacgtattgt 4440

gatgagcgat gccgaacgta ccgacgatga tttatacgat acggtgattg gctaccgtgg 4500

cggcaactgg atttatgagt gggccccgga tctttgtgaa ggaaccttac ttctgtggtg 4560

tgacataatt ggacaaacta cctacagaga tttaaagctc taaggtaaat ataaaatttt 4620

taagtgtata atgtgttaaa ctactgattc taattgtttg tgtattttag attccaacct 4680

atggaactga tgaatgggag cagtggtgga atgcctttaa tgaggaaaac ctgttttgct 4740

cagaagaaat gccatctagt gatgatgagg ctactgctga ctctcaacat tctactcctc 4800

caaaaaagaa gagaaaggta gaagacccca aggactttcc ttcagaattg ctaagttttt 4860

tgagtcatgc tgtgtttagt aatagaactc ttgcttgctt tgctatttac accacaaagg 4920

aaaaagctgc actgctatac aagaaaatta tggaaaaata ttctgtaacc tttataagta 4980

ggcataacag ttataatcat aacatactgt tttttcttac tccacacagg catagagtgt 5040

ctgctattaa taactatgct caaaaattgt gtacctttag ctttttaatt tgtaaagggg 5100

ttaataagga atatttgatg tatagtgcct tgactagaga tcataatcag ccataccaca 5160

tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat 5220

aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa 5280

agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt 5340

ttgtccaaac tcatcaatgt atcttatcat gtctggatca actggataac tcaagctaac 5400

caaaatcatc ccaaacttcc caccccatac cctattacca ctgccaatta cctagtggtt 5460

tcatttactc taaacctgtg attcctctga attattttca ttttaaagaa attgtatttg 5520

ttaaatatgt actacaaact tagtagttgg aagggctaat tcactcccaa agaagacaag 5580

atatccttga tctgtggatc taccacacac aaggctactt ccctgattag cagaactaca 5640

caccagggcc aggggtcaga tatccactga cctttggatg gtgctacaag ctagtaccag 5700

ttgagccaga taaggtagaa gaggccaata aaggagagaa caccagcttg ttacaccctg 5760

tgagcctgca tgggatggat gacccggaga gagaagtgtt agagtggagg tttgacagcc 5820

gcctagcatt tcatcacgtg gcccgagagc tgcatccgga gtacttcaag aactgctgat 5880

atcgagcttg ctacaaggga ctttccgctg gggactttcc agggaggcgt ggcctgggcg 5940

ggactgggga gtggcgagcc ctcagatcct gcatataagc agctgctttt tgcctgtact 6000

gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 6060

ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 6120

tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 6180

agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca 6240

ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc 6300

caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta 6360

agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa 6420

aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc 6480

ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc 6540

ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg 6600

tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc 6660

aaaacaaaag taagaccacc gcacagcaag cggccggccg ctgatcttca gacctggagg 6720

aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga 6780

accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc 6840

agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc 6900

agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca 6960

gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg 7020

catcaagcag ctccaggcaa gaatcctggc tgtgaaagat acctaaagga tcaacagctc 7080

ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct 7140

agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac 7200

agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag 7260

caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg 7320

tttaacataa caaattggct gtggtatata aaattattca taatgatagt aggaggcttg 7380

gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat 7440

tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga 7500

atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatct 7560

cgacggtatc gccaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg 7620

gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa 7680

ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat 7740

ccagtttgga tcgataagct tgatatcgaa ttcctgcagc cccgataaaa taaaagattt 7800

tatttagtct ccagaaaaag gggggaatga aagaccccac ctgtaggttt ggcaagctag 7860

ctgcagtaac gccattttgc aaggcatgga aaaataccaa accaagaata gagaagttca 7920

gatcaagggc gggtacatga aaatagctaa cgttgggcca aacaggatat ctgcggtgag 7980

cagtttcggc cccggcccgg ggccaagaac agatggtcac cgcagtttcg gccccggccc 8040

gaggccaaga acagatggtc cccagatatg gcccaaccct cagcagtttc ttaagaccca 8100

tcagatgttt ccaggctccc ccaaggacct gaaatgaccc tgcgccttat ttgaattaac 8160

caatcagcct gcttctcgct tctgttcgcg cgcttctgct tcccgagctc tataaaagag 8220

ctcacaaccc ctcactcggc gcgccagtcc tccgacagac tgagtcgccc gggggggatc 8280

tggagctctc gagaattctc acgcgtatgg ccttaccagt gaccgccttg ctcctgccgc 8340

tggccttgct gctccacgcc gccaggccgg agcagaaaaa aaccgctatc gcgatcgcag 8400

ttgcactggc tggtttcgct accgttgcgc aggccgatat tcaaatgacc cagagcccgt 8460

cgtccctgtc cgcaagcgtc ggtgatcgtg tgaccattac ctgccgcgca agccaagaca 8520

tctccaaata tctgaactgg taccagcaaa aaccgggtaa agcggtgaaa ctgctgattt 8580

atcatacctc gcgtctgcac agcggcgttc cgtctcgctt tagtggctcc ggttcaggca 8640

ccgatttcac cctgacgatc agctctctgc agccggaaga ctttgccacg tattactgtc 8700

aacagggtaa cacgctgccg tacaccttcg gtcagggcac caaactggaa atcaaaggtg 8760

gcggtggctc gggtggcggt ggcagcggtg gcggtggcca agtccaactg caagaatcgg 8820

gtccgggcct ggtgaaaccg tctgaaacgc tgtccctgac ctgtaccgtg tcgggtgtta 8880

gcctgccgga ttatggtgtg agctggattc gtcagccgcc gggtaaaggt ctggaatgga 8940

ttggcgttat ctggggttct gaaaccacgt attacaacag tgccctgaaa tcccgcgtca 9000

ccatctcaaa agatacgtcg aaaaatcaag tgtccctgaa actgagctct gttaccgcgg 9060

ccgacacggc agtctattac tgcgctaaac attattacta cggtggtagt tatgcgatgg 9120

attactgggg tcagggcacg atggttacgg tctcctccca ccatcaccat caccatggat 9180

ccaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg tcgcagcccc 9240

tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac acgagggggc 9300

tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt ggggtccttc 9360

tcctgtcact ggttatcacc ctttactgct ccctaaaacg gggcagaaag aaactcctgt 9420

atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa gatggctgta 9480

gctgccgatt tccagaagaa gaagaaggag gatgtgaact gagagtgaag ttcagcagga 9540

gcgcagacgc ccccgcgtac aagcagggcc agaaccagct ctataacgag ctcaatctag 9600

gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct gagatggggg 9660

gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag aaagataaga 9720

tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc aaggggcacg 9780

atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc cttcacatgc 9840

aggccctgcc ccctcgcggc taagcggccg cgactctaga gtcgacctgc aggcatgcaa 9900

gcttgatatc aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac 9960

tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt 10020

gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt 10080

gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt 10140

gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg 10200

gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg 10260

ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc 10320

atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt 10380

ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc 10440

tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc 10500

cgcctccccg cat 10513

Claims

1. A modified T lymphocyte targeting an MMSA-1 chimeric antigen receptor, wherein: the cell surface expresses a specific chimeric antigen receptor, can specifically recognize the MMSA-1 antigen on the surface of the myeloma cell and plays an anti-myeloma effect; the sequence of the MMSA-1CAR fragment is shown as SEQ ID NO. 1; the front end of the polypeptide is a CD8 transmembrane signal peptide, and the sequence of the polypeptide is shown in SEQ ID NO. 2; the adjacent MMSA-1scFv single-chain antibody has the sequence shown in SEQ ID NO. 3; the rear end of the probe is sequentially provided with a CD8 transmembrane region, an intracellular 4-1BB costimulatory signal region and a CD3zeta TCR activation region, and the sequence of the probe is shown as SEQ ID NO. 4.

2. A method for preparing the MMSA-1 targeted chimeric antigen receptor modified T lymphocyte of claim 1, comprising:

1) preparation of MMSA-1CAR plasmid:

after the wild type antibody is subjected to humanized transformation, MMSA-1scFv is obtained, an Mlu I enzyme cutting site and a CD8 membrane penetrating signal peptide are inserted in front of a fragment, and a BamH I enzyme cutting site is inserted behind the fragment; carrying out double enzyme digestion on the synthesized pUC57-Amp plasmid containing Mlu I + CD8a-MMSA-1scFv + BamH I, carrying out agarose gel electrophoresis to identify the enzyme digestion effect, and recovering gel to obtain a modified gene fragment; meanwhile, carrying out double enzyme digestion on a lentivirus framework plasmid pHR containing a CD8 transmembrane region, a 4-1BB costimulatory signal region and a CD3zeta TCR activation region by Mlu I and BamH I, and recovering long fragments from gel after agarose gel electrophoresis identification; connecting the modified MMSA-1scFv fragment to a lentiviral backbone plasmid pHR, extracting the plasmid, and obtaining a pHR-MMSA-1CAR plasmid after determining that the sequencing is correct, wherein the sequencing sequence of the pHR-MMSA-1CAR plasmid is shown as SEQ ID NO. 5;

2) preparation of MMSA-1 chimeric antigen receptor lentivirus:

dropwise adding the transfection reagent and the plasmids into serum-free and antibiotic-free RPMI1640, dropwise adding the final transfection reagent and plasmid mixed solution into a T175 culture bottle with the convergence of 293T cells being 60-70% for culture, and collecting 293T cell supernatant after 72h of transfection; collecting the supernatant of the 293T cells in a centrifuge tube to enable the fallen 293T cells to be centrifuged to the bottom of the tube, filtering the centrifuged supernatant by using a 0.4 mu M filter membrane, transferring the filtered supernatant into an ultracentrifuge tube, centrifuging the filtered supernatant for 2 hours at vacuum 25000rpm, discarding the supernatant, and re-suspending the concentrated lentiviruses in l mL of fresh culture medium;

3) construction and in vitro expansion of CAR-T cells:

taking 50mL of fresh blood, and separating mononuclear cells by performing density gradient centrifugation on lymphocyte separation liquid; the mononuclear cells are arranged in a 1-2X 10⁶Resuspend in CTSTMAIM VTM SFM medium/mL; 5% ICS, 50ng/mL of CD3 monoclonal antibody and 50ng/mL of CD28 monoclonal antibody are added simultaneously to activate T lymphocytes, and the cells are cultured for 48h at 37 ℃ with 5% CO 2;

after 2 days of culture, cells were harvested and resuspended to 1X10⁶Adding the concentrated lentivirus according to MOI of 5, adding IL-2 and 4ug/mLpolybrene at final concentration, mixing, culturing at 37 deg.C with 5% CO2 for 6-8 hr, centrifuging at 300g for 5min, and changing into fresh CTSTMAIM VTM SFM culture medium containing 200U/mL IL-2;

adding fresh CTSTMAIM VTM SFM culture medium containing 200U/mLIL-2 every 2-3 days to maintain cell density at 110⁶about/mL, and performing amplification culture for 10-12 days.

3. Use of the MMSA-1 targeted chimeric antigen receptor-modified T lymphocyte of claim 1 in the preparation of a medicament for treating multiple myeloma.