CN110734931A - humanized scFv chimeric antigen receptor T cells targeting CD19, and preparation method and application thereof - Google Patents
humanized scFv chimeric antigen receptor T cells targeting CD19, and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering and cytobiology, and particularly relates to humanized scFv chimeric antigen receptor T cells targeting CD19, a preparation method and application thereof, wherein a lentivirus expression vector containing a gene structure of a humanized scFv chimeric antigen receptor (anti-hCD 19 CAR) targeting CD19 is constructed, the anti-hCD19CAR expression vector is transduced to the T cells in a lentivirus mediated mode, the anti-hCD19CAR modified T cells can specifically kill CD19 positive tumor cells, the generation of human anti-mouse antibodies caused by murine CD19 genes can be effectively avoided, the immunogenicity of murine CAR can be reduced, the survival time of the CAR-T cells can be improved, the high-specificity is combined with human CD19 protein, the treatment effect of CAR-T is enhanced, and the safety and the effectiveness of treatment of the CD-T are improved.
Description
Technical Field
The invention belongs to the technical field of genetic engineering and cell biology, and particularly relates to humanized scFv chimeric antigen receptor (anti-hCD 19 CAR) T cells targeting CD19, and a preparation method and application thereof.
Background
In view of the poor curative effect, i.e. low Complete response rate (CR) or high recurrence rate, of the traditional therapies such as chemotherapy, radiotherapy and hematopoietic stem cell transplantation on hematological malignancies, for example, in Acute Lymphoblastic Leukemia (ALL), although the CR rate after chemotherapy can reach 80% -90%, the recurrence rate of about 70% and the Disease free survival rate (DFS) of about 30% in 3 years, researchers still need to search for better therapeutic methods.
With the development of tumor immunology theory and technology, cellular immunotherapy has advanced greatly in recent years, and is listed as the first ten scientific breakthroughs in 2013 by the Science journal. The target immunotherapy of tumor represented by T cells modified by Chimeric Antigen Receptor (CAR) is particularly outstanding in achievement, shows good targeting, killing and durability in vitro and clinical tests, and shows huge application potential and development prospect.
CAR-T cell therapy expresses a Single chain antibody fragment (scFv) for recognizing tumor-associated specific antigen and a fusion protein of a T cell activation sequence to the surface of a T cell by an exogenous gene transduction technology, so that the scFv is coupled with an activation proliferation signal domain in the T cell through a transmembrane region, is greatly amplified after being back-transfused into a patient body, and can show a strong anti-tumor effect in an antigen-dependent and non-MHC-restricted mode.
CD19 is transmembrane glycoproteins belonging to Ig superfamily members, expressed on B lineage cells (including pre-B cells, immature B cells, mature B cells and activated B cells) and follicular dendritic cells, and is an important membrane antigen involved in B lymphocyte differentiation, activation, proliferation and antibody production.most B cell malignant tumor cells express CD19 molecules, and hematopoietic stem cells and non-hematopoietic cells do not express CD19, so CD19 is the best marker for diagnosing B lymphocyte lineage tumors and identifying B lymphocytes, and is the most potential target for treating B lineage tumors.
In 8 months 2017, the U.S. FDA officially approved the first CAR-T drug kymeriah (CTL 019) for clinical treatment of relapsed or refractory (r/r) childhood and young adult B-cell acute lymphoblastic leukemia, which is a human historical milestone event in the same year 10 months the U.S. FDA approved the 2 nd CAR-T drug yescata (KTE-C10) for treatment of certain types of adult large B-cell lymphoma patients who either failed to respond to other therapies or relapsed after receiving at least 2 treatment regimens, including diffuse large B-cell lymphoma, transformed follicular lymphoma, primary mediastinal B-cell lymphoma.
The current data show a Complete Remission (CR) rate of 70-90% in patients with refractory relapse B-ALL to anti-CD19 CAR-T therapy. The regression of hematologic malignant cells is closely related to the level of proliferation of CAR-T cells and their survival time in the blood. CAR-T cell depletion in ALL patients was accompanied by recovery of normal B cells, and some patients developed a relapse of CD19+ leukemia. The CD19 antigen receptor used in CAR-T technology at present adopts murine gene, such as murine monoclonal antibody FMC63, but such murine gene fragment has possibility of causing the generation of Human anti-mouse antibody (HAMA) in the process of treating Human diseases. There are studies showing that the immunogenicity of murine CAR sequences may lead to the inability of CAR-T cells to activate and persist.
Disclosure of Invention
In response to the problems of the prior art, the present invention provides humanized scFv chimeric antigen receptor (anti-hCD 19 CAR) T cells targeting CD 19.
The invention also provides a preparation method of humanized scFv chimeric antigen receptor (anti-hCD 19 CAR) T cells targeting CD 19.
The invention also provides application of the humanized scFv chimeric antigen receptor (anti-hCD 19 CAR) T cell targeting CD 19.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the invention provides lentivirus expression vectors, which are characterized by comprising a humanized scFv chimeric antigen receptor (anti-hCD 19 CAR) gene structure targeting CD19, and the nucleotide sequences of the humanized scFv chimeric antigen receptor (anti-hCD 19 CAR) gene structure are SEQ ID NO.1, SEQ ID NO. 2 or SEQ ID NO. 3.
Further , the chimeric antigen receptor gene structure comprises in serial order a CD8 transmembrane signal peptide, anti-hCD19scFv, CD8 transmembrane region, 4-1BB costimulatory signal region, and CD3Zeta TCR activation region.
The amino acid sequence of the CD8 transmembrane signal peptide is shown as SEQ ID NO.4, the anti-hCD19scFv comprises or is selected from any amino acid sequence shown as SEQ ID NO. 5 or SEQ ID NO. 6 or SEQ ID NO. 7, wherein the scFv shown as (b), (c) and (a) have the same function, and the scFv amino acid sequences shown as (b), (c) and (a) have more than 90% of consistency, (b), (c) and (a) all consist of a light chain variable region, a light chain connecting region and a heavy chain connecting region, the amino acid sequence of the CD8 transmembrane region is shown as SEQ ID NO.8, the amino acid sequence of the 4-1BB co-stimulation signal region is shown as SEQ ID NO.9, and the amino acid sequence of the CD3 ZeTCR activation region is shown as SEQ ID NO. 10.
The invention also provides a preparation method of humanized scFv chimeric antigen receptor T cells which contain the lentivirus expression vector and target CD19, and the humanized scFv chimeric antigen receptor T cells are prepared by the following method:
(1) constructing a plasmid vector: after the anti-CD19 scFv fragment is subjected to humanized transformation, obtaining anti-hCD19scFv, inserting an Mlu I enzyme cutting site and a CD8 transmembrane signal peptide into the fragment, inserting a BamH I enzyme cutting site into the fragment, synthesizing a pUC57-Amp plasmid containing Mlu I + CD8a-hCD19scFv + BamH I, performing double enzyme cutting by Mlu I and BamH I, identifying the enzyme cutting effect by agarose gel electrophoresis, and recovering gel to obtain a modified gene fragment; meanwhile, carrying out double enzyme digestion on the lentiviral backbone plasmid pHR containing the CD8 transmembrane region, the 4-1BB costimulatory signal region and the CD3Zeta TCR activation region by Mlu I and BamH I, and recovering long fragments from gel after agarose gel electrophoresis identification; connecting the modified anti-hCD19scFv fragment to a lentiviral backbone plasmid pHR, extracting the plasmid, and obtaining a pHR-anti-hCD19CAR plasmid after determining that the sequencing is correct;
(2) and (3) preparing lentivirus: mixing the pHR-anti-hCD19CAR plasmid carrying the target gene, a pCMV vector and a pMD.2G vector, transfecting into 293FT cells, replacing with a DMEM complete culture medium containing 10% FBS and 1% glutamine for culture after 6-8 h after transfection, collecting a culture solution after 48h, centrifuging, retaining a supernatant, filtering the supernatant by using a 0.45 mu m filter, retaining a filtrate, wherein the filtrate is a recombinant lentivirus solution, and concentrating for later use;
(3) anti-hCD19CAR-T preparation: taking 50mL of fresh blood, separating mononuclear cells by density gradient centrifugation of lymphocyte separating medium, and performing 1-2 × 10 centrifugation on the mononuclear cells6/mL resuspended CTSTMAIM VTM5% ICS, 50ng/mL of CD3 monoclonal antibody and 50ng/mL of CD28 monoclonal antibody are added into an SFM culture medium at the same time to activate T lymphocytes, and the cells are cultured for 48 hours at 37 ℃ with 5% CO 2; after 2 days of culture, cells were harvested and resuspended to 1X106Adding the concentrated recombinant lentivirus solution according to MOI = 5, simultaneously adding IL-2 and polybrene with final concentration of 500U/mL, uniformly mixing, and 5% CO at 37 DEG C2Culturing for 6-8 hours, centrifuging at 300g for 5min, and changing the liquid into fresh CTSTMAIM VTMSFM medium; adding fresh CTS every 2-3 daysTMAIM VTMSFM medium, maintaining cell density at 1X106about/mL, and the amplification culture is carried out for 10-12 days, step , in step (3), the CTSTMAIM VTMSFM medium contained 500U/mL IL-2.
The invention also provides application of humanized scFv chimeric antigen receptor T cells targeting CD19 in preparation of medicines for preventing and/or treating and/or adjunctively treating malignant tumors.
The malignant tumor cells include CD19 positive B lymphocyte leukemia or B lymphocyte tumor.
some common terms used in the present invention are described below:
CAR: chimeric antigen receptors
scFv Single chain antibody fragment
anti-hCD19 CAR: humanized scFv chimeric antigen receptor targeting CD19
anti-mCD19CAR targeting murine CD19scFv chimeric antigen receptor
T lymphocytes
The invention provides a preparation method of humanized scFv chimeric antigen receptor (anti-hCD 19 CAR) T cells of targeting CD19 and specifically killing CD19 malignant B cell tumors, wherein the chimeric antigen receptor can be combined with human CD19 protein with high specificity, a humanized antibody fragment of the targeting CD19 integrated in the CAR is engineered and expressed in the T cells by utilizing a genetic engineering technology, and the obtained CAR-T cells can be used for treating hematological malignancies expressing CD 19.
The anti-hCD19CAR T cell provided by the invention can specifically kill CD19 positive tumor cells, in-vitro tests prove that the anti-hCD19CAR T killing effect is achieved, the killing efficiency is over 95% when the ratio of effector cells to target cells is 3:1, and cytokines such as IL-2 and IFN-gamma are generated in the killing process.
The anti-hCD19CAR T cell provided by the invention can effectively protect B cell lymphoma loaded mice, and after 60 days of observation, the survival rate of the mice reaches 75 percent and is obviously higher than that of a tumor loaded mouse group transplanted with the anti-mCD19CAR-T cell.
The invention has the following beneficial effects:
(1) the anti-hCD19CAR T cell provided by the invention can effectively avoid the generation of human anti-mouse antibodies caused by murine CD19 genes, reduce the immunogenicity of murine CAR, improve the survival time of CAR-T cells, combine with human CD19 protein with high specificity, enhance the therapeutic effect of CAR-T, and improve the safety and effectiveness of CAR-T therapy.
Drawings
FIG. 1 is a schematic diagram of the modification of anti-hCD19scFv fragment according to the present invention.
FIG. 2 shows the construction of pHR-anti-hCD19CAR plasmid according to the present invention.
FIG. 3 is a schematic diagram showing the flow assay results of lentivirus titer according to the present invention.
FIG. 4 flow cytometry described herein detects anti-hCD19CAR T cell CAR protein expression.
FIG. 5 shows the specific killing results of anti-hCD19CAR T cells of the present invention in vitro killing CD19 positive tumor cells at different target ratios.
FIG. 6 is a schematic diagram showing the results of the content of IL-2 and IFN-gamma secreted to the outside of cells when anti-hCD19CAR T cells kill CD19 positive tumor cells in vitro.
FIG. 7 is a schematic diagram of tumor-bearing mouse survival curves after the anti-hCD19CAR T cells of the present invention are transplanted into a human B-cell lymphoma transplanted tumor mouse model.
The present invention is further illustrated in and will not be limited to the details of the embodiments illustrated in the drawings and described in the following description, it is understood that the embodiments of the invention are not limited to the examples disclosed herein, but are intended to cover modifications within the scope of the invention as expressed in the claims.
Example 1: plasmid vector construction
after the anti-CD19 scFv fragment is subjected to humanized transformation, the anti-hCD19scFv is obtained, the Mlu I enzyme cutting site and the CD8 transmembrane signal peptide are inserted in the front of the fragment, the BamH I enzyme cutting site is inserted in the back of the fragment (shown in figure 1), and the fragment is handed to a gene company (Jinwei Zhi) for synthesis. A pUC57-Amp plasmid containing Mlu I + CD8a-hCD19scFv + BamH I is synthesized by a gene company, and is subjected to double enzyme digestion of Mlu I and BamH I, agarose gel electrophoresis is performed to identify the enzyme digestion effect, and gel is recovered to obtain a modified gene fragment, which is shown in figure 1. Meanwhile, the slow virus skeleton plasmid pHR (see patent CN 108753774A) which is already in a laboratory and contains a CD8 transmembrane region, a 4-1BB costimulatory signal region and a CD3Zeta TCR activation region is subjected to double enzyme digestion of Mlu I and BamH I, and the gel is recovered into a long fragment after agarose gel electrophoresis identification. The modified anti-hCD19scFv fragment was ligated into the lentiviral backbone plasmid pHR, and the pHR-anti-hCD19CAR plasmid was obtained after plasmid extraction to confirm correct sequencing (as shown in FIG. 2).
Example 2 lentivirus preparation and titer detection:
and (2) mixing the lentivirus expression vector carrying the target gene, a pCMV vector and a pMD.2G vector, transfecting into 293FT cells (purchased from ATCC), replacing for 6-8 h after transfection with a complete culture medium for culture, collecting a culture solution after 48h, centrifuging, reserving a supernatant, filtering the supernatant by using a 0.45 mu m filter, and reserving a filtrate, wherein the filtrate is a solution of the recombinant lentivirus. Lenti-XTM Concentrator (Takara, cat: 631231) instructions for lentivirus concentration.
Virus titer was determined by gradient dilution method using a 24-well plate with 1X10 plates per well5Diluting the concentrated virus 10 times for each K562 cell, adding 1 μ L, 3 μ L, 10 μ L, and 30 μ L virus respectively, and adding 5% CO at 37 deg.C2After 48 hours of culture, 200. mu.L of cell fluid was taken per well for flow assay. Each sample was Labeled with 10. mu.L of FITC-Labeled HumanCD19 (20-291) Protein antibody, virus-free K562 was used as a negative control, incubated for 15min at room temperature in the absence of light, centrifuged for 5min at 400g, washed 2 times with 1mL PBS, and the pellet was resuspended in 200. mu.L of 2% paraformaldehyde and then examined on the machine (Millipore guava easy Cyte HT). As a result, as shown in FIG. 3, the proportion of positive cells gradually increased with the increase in the amount of virus added. When the positive rate is less than or equal to 10%, the virus particle number is considered to be equal to the cell number, so the titer is 1.35 multiplied by 10 after the virus is concentrated8IU/mL。
Example 3 preparation of CAR-T cells and detection of CAR-Positive Rate
1.anti-hCD19CAR-T 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 106/mL resuspended CTSTMAIM VTMSFM 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 1X106PermL, the lentivirus of example 2 after concentration was added at MOI = 5, together with IL-2 (spring harbor) at a final concentration of 500U/mL) Mixing with 4ug/mL polybrene (Sigma), mixing, culturing at 37 deg.C with 5% CO2 for 6-8 hr, centrifuging at 300g for 5min, and changing the filtrate to fresh CTSTMAIM VTMSFM medium (containing 500U/mL IL-2).
Adding fresh CTS every 2-3 daysTMAIM VTMSFM medium (containing 500U/mL IL-2) maintained cell density at 1X106about/mL, and performing amplification culture for 10-12 days.
CAR-T cell CAR-Positive Rate detection
The CAR-T cells were tested for CAR-positivity by flow cytometry using FITC-Labeled HumanCD19 (20-291) Protein. Collecting anti-hCD19CAR-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 1 h in the dark, centrifuged at 400g for 5min, washed 3 times with 1mL of PBS, and the pellet was resuspended in 200. mu.L of 2% paraformaldehyde and examined on the machine (Millipore guava easy Cyte HT).
The results are shown in FIG. 4, and the CAR positive rate in anti-hCD19CAR T cells reaches 60.8%.
Example 4CAR-T cell functional validation
Detection of killing efficiency of anti-hCD19CAR-T cells in vitro
The K562 cells and the K562-CD19 cells are lymphoma cell strains, the surface of the K562 cells does not express CD19 molecules, and the surface of the K562-CD19 cells expresses CD19 molecules (see patent CN 107365798A). Two cells were seeded 1:1 in 96-well plates at 1X10 per well5T cells, anti-hCD19CAR-T cells and anti-mCD19CAR-T cells, which were cultured for 9 days, were inoculated in E: T =1:1, 3:1, 6:1, respectively, 3 duplicate wells per group. 5% CO at 37 ℃2After 24 hours of incubation, 100. mu.L of supernatant was aspirated from each well and stored at-80 ℃ for further use. The remaining cells were added to each well 1. mu.L of PE anti-human CD19 (Biolegend) and 1. mu.L of FITTAinti-human CD3 (Biolegend), incubated at room temperature for 15min in the absence of light, centrifuged at 400g for 5min, washed 2 times with 1mL of PBS, and the pellet resuspended in 200. mu.L of 2% paraformaldehyde and then examined on the machine (Millipore guava easy Cyte HT). As shown in FIG. 5, the anti-hCD19CAR-T cells kill K562-CD19 cells specifically, the average specific killing rate is 61%, 97% and 99% when the effective target ratio is 1:1, 3:1 and 6:1, and the killing rate is increased along with the increase of the effective target ratio. anti-hCDThe killing efficiency of the 19CAR-T cells is higher than that of the anti-mCD19CAR-T cell group. Two sets of paired t-tests P<0.05, with significant differences.
2. ELISA detection of cytokine concentration in killer supernatant
The concentrations of IL-2 and IFN- γ in the samples of E: T =3:1 killing supernatants were determined by ELISA, using a HumanIL-2 ELISA MAX Deluxe (Biolegend, 431804) or a Human IFN- γ ELISA MAX Deluxe (Biolegend, 430104) protocol according to the kit instructions, and 3 duplicate wells were made for all standards and samples. The specific operation method comprises the following steps:
a) all reagents were equilibrated for 1 h at room temperature before use of the kit.
b) antibody coating, before days of detection, antibody is diluted according to the instruction, 100 mu l/well is added to a 96-well enzyme label plate, and the plate is placed at 4 ℃ overnight.
c) The following day the coating solution was discarded, washed 4 times with PBST, and 200. mu.l of blocking solution was added to each well and blocked for 1 h at room temperature.
d) 500pg/ml → 7.81 pg/ml standard preparation according to the kit instructions, prepare 7 Eppendorf tubes, label 500pg/ml, 250pg/ml, 125 pg/ml, 62.5 pg/ml, 31.3 pg/ml, 15.6 pg/ml, 7.81 pg/ml, prepare 500pg/ml to 1ml, add 500. mu.L of sample dilution to the remaining 6 tubes, add 500. mu.L of standard to the labeled 250pg/ml tube, mix well, take 500. mu.L out similarly, add tubes, and push the same until sample tubes.
e) Sample dilution: the sample was diluted 10-fold with 40. mu.l sample plus 360. mu.l sample diluent.
f) Sample adding: the blocking solution was discarded, and washed 4 times with PBST, and blank wells (sample diluent), standard wells, and sample wells to be tested were set, respectively. Accurately adding 100 mu l of sample on an enzyme-labeled coated plate, and incubating for 2 h at room temperature.
g) Adding a detection antibody: samples were discarded, washed 4 times with PBST, and 100. mu.l of accurately detected antibody was incubated at room temperature for 1 h.
h) Adding an enzyme: the detection antibody was discarded, washed 4 times with PBST, 100. mu.l of enzyme-labeled reagent was added to each well, and incubated at room temperature for 30 min.
i) Color development: discarding the enzyme-labeled reagent, washing with PBST for 5 times, adding 100 μ l of color-developing agent into each well, shaking gently, mixing, and developing at room temperature in dark place for 30 min.
j) And (4) terminating: the reaction was stopped by adding 100. mu.l of stop solution to each well (blue color immediately turned yellow).
k) And (3) determination: the absorbance (OD value) of each well was measured sequentially at a wavelength of 450nm with the blank well being zeroed.
As shown in FIG. 6, after the anti-hCD19CAR-T cells and the anti-mCD19CAR-T cells are respectively cultured in a mixed manner with target cells, a large amount of IL-2 and IFN-gamma cytokines can be secreted to kill the cells, and the cytokine content of the anti-hCD19CAR-T group is higher than that of the anti-mCD19CAR-T cell group, so that the difference between the anti-hCD19CAR-T cell group and the T cell group is significant.
3. anti-hCD19CAR-T cell in vivo killing experiment
The effect of anti-hCD19CAR-T of the present invention on the survival of mice was examined using a human B-cell lymphoma transplantation tumor model.
1) Construction of mouse model of human B cell lymphoma
SPF-grade NCG immunodeficient mice 6-8 weeks, 1x10 tail vein injection5A transplanted tumor model was constructed from Raji cells/300. mu.l of physiological saline.
2) anti-hCD19CAR-T cell transplantation assay
4 days after tumor inoculation, experimental mice were randomized into 3 groups of 8 mice each: infection of empty plasmid T cell group (Mock group), anti-hCD19CAR-T cell group and anti-mCD19CAR-T cell group, tail vein injection of corresponding T cells 3X 107500 μ l of physiological saline. And observing the survival number of the mice, recording the survival period of the mice and calculating the survival rate. The observation was continued for 60 days.
As shown in FIG. 7, after 60 days of continuous observation and recording, the tumor-bearing mice transplanted with anti-hCD19CAR-T cells still have 75% survival rate, which is significantly higher than that of the tumor-bearing mice group transplanted with anti-mCD19CAR-T cells (50% survival rate), while the Mock group died completely at 24 days. The anti-hCD19CAR-T cells provided by the invention have obvious effect of prolonging the survival time of disease model mice.
As shown in FIG. 7, after 60 days of continuous observation and recording, the tumor-bearing mice transplanted with anti-hCD19CAR-T cells still have 75% survival rate, which is significantly higher than that of the tumor-bearing mice group transplanted with anti-mCD19CAR-T cells (50% survival rate), while the Mock group died completely at 24 days. The anti-hCD19CAR-T cells provided by the invention have obvious effect of prolonging the survival time of disease model mice.
The examples prove that the anti-hCD19CAR-T cell provided by the invention has the advantages of inhibiting tumor cell proliferation and specifically killing CD19 positive tumor cells, is expected to avoid human anti-mouse antibody reaction caused by murine CD19CAR-T, and can be used for treating CD19 positive B lymphocyte leukemia or B lymphocyte tumor.
Comparative example 1
The patent (CN 104788573A) discloses a chimeric antigen receptor hCD19scFv-CD8A-CD28-CD 3zeta gene structure, the prepared CAR-T cells kill CD19 positive tumor cells specifically, and the IL-2 content (about 1250 pg/ml) in the co-culture supernatant of the CAR-T cells and target cells is lower than that of the invention.
Comparative example 2
The patent (CN 107880128A) discloses anti-CD19 fully human antibodies or antibody fragments, wherein the CAR gene structure comprises a CSF2RA signal peptide sequence, a CD19scFv, a C-myc detection tag, a hinge region, a CD8 transmembrane structure, a CD28 functional signaling domain, a 4-1BB functional signaling domain and a CD3Zeta functional signaling domain which are connected in sequence, the prepared CAR-T cells are cultured with CD19 positive target cells, and the IL-2 content (about 500 pg/ml) and the IFN-gamma content (about 1250 pg/ml) in culture supernatant are lower than those of the invention.
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.
<110> Qilu cell therapy engineering Co., Ltd, Shandong province
<120> humanized scFv chimeric antigen receptor T cells targeting CD19, and preparation method and application thereof
<160>10
<210>1
<211>1554
<212>DNA
<213>Artificial Sequence
<220>
<223>
<400>1
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggagcaga aaaaaaccgc tatcgcgatc gcagttgcac tggctggttt cgctaccgtt 120
gcgcaggccg atattcaaat gacccagagc ccgtcgtccc tgtccgcaag cgtcggtgat 180
cgtgtgacca ttacctgccg cgcaagccaa gacatctcca aatatctgaa ctggtaccag 240
caaaaaccgg gtaaagcggt gaaactgctg atttatcata cctcgcgtct gcacagcggc 300
gttccgtctc gctttagtgg ctccggttca ggcaccgatt tcaccctgac gatcagctct 360
ctgcagccgg aagactttgc cacgtattac tgtcaacagg gtaacacgct gccgtacacc 420
ttcggtcagg gcaccaaact ggaaatcaaa ggtggcggtg gctcgggtgg cggtggcagc 480
ggtggcggtg gccaagtcca actgcaagaa tcgggtccgg gcctggtgaa accgtctgaa 540
acgctgtccc tgacctgtac cgtgtcgggt gttagcctgc cggattatgg tgtgagctgg 600
attcgtcagc cgccgggtaa aggtctggaa tggattggcg ttatctgggg ttctgaaacc 660
acgtattaca acagtgccct gaaatcccgc gtcaccatct caaaagatac gtcgaaaaat 720
caagtgtccc tgaaactgag ctctgttacc gcggccgaca cggcagtcta ttactgcgct 780
aaacattatt actacggtgg tagttatgcg atggattact ggggtcaggg cacgatggtt 840
acggtctcct cccaccatca ccatcaccat ggatccacca cgacgccagc gccgcgacca 900
ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc tgcgcccaga ggcgtgccgg 960
ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgcctgtga tatctacatc 1020
tgggcgccct tggccgggac ttgtggggtc cttctcctgt cactggttat caccctttac 1080
tgctccctaa aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 1140
ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa 1200
ggaggatgtg aactgagagt gaagttcagc aggagcgcag acgcccccgc gtacaagcag 1260
ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 1320
gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1380
gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 1440
atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 1500
gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg cggc 1554
<210>2
<210>2
<211>1554
<212>DNA
<213>Artificial Sequence
<220>
<223>
<400>2
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggagcaga aaaaaaccgc tatcgcgatc gcagttgcac tggctggttt cgctaccgtt 120
gcgcaggccg atattcaaat gacccagagc ccgtcgtccc tgtccgcaag cgtcggtgat 180
cgtgtgacca ttacctgccg cgcaagccaa gacatctcca aatatctgaa ctggtaccag 240
caaaaaccgg gtaaagcggt gaaactgctg atttatcata cctcgcgtct gcacagcggc 300
gttccgtctc gctttagtgg ctccggttca ggcaccgatt tcaccctgac gatcagctct 360
ctgcagccgg aagactttgc cacgtattac tgtcaacagg gtaacacgct gccgtacacc 420
ttcggtcagg gcaccaaact ggaaatcaaa ggtggcggtg gctcgggtgg cggtggcagc 480
ggtggcggtg gccaagtcca actgcaagaa tcgggtccgg gcctggtcaa accgtcagaa 540
acgctgtcag ttacctgtac cgtctcgggt gtgagcctgc cggattatgg tgtcagctgg 600
attcgtcagc cgccgggtaa aggtctggaa tggctgggcg ttatctgggg ttctgaaacc 660
acgtattaca acagtgccct gaaatcccgc ctgaccattt caaaagatac gtcgaaaaat 720
caagtgtccc tgaaaatgag ctctctgacc gcggccgaca cggcagttta ttactgcgct 780
aaacattatt actacggcgg ctcttatgct atggattatt ggggtcaggg cacgatggtt 840
acggtttctt cccaccatca ccatcaccat ggatccacca cgacgccagc gccgcgacca 900
ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc tgcgcccaga ggcgtgccgg 960
ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgcctgtga tatctacatc 1020
tgggcgccct tggccgggac ttgtggggtc cttctcctgt cactggttat caccctttac 1080
tgctccctaa aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 1140
ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa 1200
ggaggatgtg aactgagagt gaagttcagc aggagcgcag acgcccccgc gtacaagcag 1260
ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 1320
gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1380
gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacagtgagattggg 1440
atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 1500
gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg cggc 1554
<210>3
<211>1555
<212>DNA
<213>Artificial Sequence
<220>
<223>
<400>3
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggagcaga aaaaaaccgc tatcgcgatc gcagttgcac tggctggttt cgctaccgtt 120
gcgcaggccg acattcagat gacccagagc ccgtcctccc tgagtgcgag cgttggcgac 180
cgtgtgacca ttacctgccg tgcttcccaa gacatttcca aatatctgaa ctggtaccag 240
caaaaaccgg gtaaagcggt gaaactgctg atttatcata cctcgcgtct gcacagcggc 300
gttccgtctc gctttagtgg ctccggttca ggcaccgatt tcaccctgac gatcagctct 360
ctgcagccgg aagactttgc cacgtacttc tgtcaacagg gtaacacgct gccgtacacc 420
ttcggtcagg gcaccaaact ggaaatcaaa ggtggcggtg gctcgggtgg cggtggcagc 480
ggtggcggtg gccaagtcca actgcaagaa tcgggtccgg gcctggtgaa accgtctgaa 540
acgctgtccc tgacctgtac cgtgtcgggt gttagcctgc cggattatgg tgtgagctgg 600
attcgtcagc cgccgggtaa aggtctggaa tggattggcg ttatctgggg ttctgaaacc 660
acgtattaca acagtgccct gaaatcccgc gtcaccatct caaaagatac gtcgaaaaat 720
caagtgtccc tgaaactgag ctctgttacc gcggccgaca cggcagtcta ttactgcgct 780
aaacattatt actacggtgg tagttatgcg atggattact ggggtcaggg cacgatggtt 840
acggtctcct cccaccatca ccatcaccat ggatccacca cgacgccagc gccgcgacca 900
ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc tgcgcccaga ggcgtgccgg 960
ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgcctgtga tatctacatc 1020
tgggcgccct tggccgggac ttgtggggtc cttctcctgt cactggttat caccctttac 1080
tgctccctaa aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 1140
ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa 1200
ggaggatgtg aactgagagt gaagttcagc aggagcgcag acgcccccgc gtacaagcag 1260
ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 1320
gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1380
gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 1440
atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 1500
gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg cggcb 1555
<210>4
<211>23
<212>PRT
<213>Artificial Sequence
<220>
<223>
<400>4
MALPVTALLL PLALLLHAAR PEQ 23
<210>5
<211>267
<212>PRT
<213>Artificial Sequence
<220>
<223>
<400>5
KKTAIAIAVA LAGFATVAQA DIQMTQSPSS LSASVGDRVT ITCRASQDIS KYLNWYQQKP 60
GKAVKLLIYH TSRLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ GNTLPYTFGQ 120
GTKLEIKGGG GSGGGGSGGG GQVQLQESGP GLVKPSETLS LTCTVSGVSL PDYGVSWIRQ 180
PPGKGLEWIG VIWGSETTYY NSALKSRVTI SKDTSKNQVS LKLSSVTAAD TAVYYCAKHY 240
YYGGSYAMDY WGQGTMVTVS SHHHHHH 267
<210>6
<211>267
<212>PRT
<213>Artificial Sequence
<220>
<223>
<400>6
KKTAIAIAVA LAGFATVAQA DIQMTQSPSS LSASVGDRVT ITCRASQDIS KYLNWYQQKP 60
GKAVKLLIYH TSRLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYFCQQ GNTLPYTFGQ 120
GTKLEIKGGG GSGGGGSGGG GQVQLQESGP GLVKPSETLS LTCTVSGVSL PDYGVSWIRQ 180
PPGKGLEWIG VIWGSETTYY NSALKSRVTI SKDTSKNQVS LKLSSVTAAD TAVYYCAKHY 240
YYGGSYAMDY WGQGTMVTVS SHHHHHH 267
<210>7
<211>267
<212>PRT
<213>Artificial Sequence
<220>
<223>
<400>7
KKTAIAIAVA LAGFATVAQA DIQMTQSPSS LSASVGDRVT ITCRASQDIS KYLNWYQQKP 60
GKAVKLLIYH TSRLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ GNTLPYTFGQ 120
GTKLEIKGGG GSGGGGSGGG GQVQLQESGP GLVKPSETLS VTCTVSGVSL PDYGVSWIRQ 180
PPGKGLEWLG VIWGSETTYY NSALKSRLTI SKDTSKNQVS LKMSSLTAAD TAVYYCAKHY 240
YYGGSYAMDY WGQGTMVTVS SHHHHHH 267
<210>8
<211>69
<212>PRT
<213>Artificial Sequence
<220>
<223>
<400>8
TTTPAPRPPT PAPTIASQPL SLRPEACRPA AGGAVHTRGL DFACDIYIWA PLAGTCGVLL 60
LSLVITLYC
69
<210>9
<211>43
<212>PRT
<213>Artificial Sequence
<220>
<223>
<400>9
SLKRGRKKLL YIFKQPFMRP VQTTQEEDGC SCRFPEEEEG GCE 43
<210>10
<211>114
<212>PRT
<213>Artificial Sequence
<220>
<223>
<400>10
LRVKFSRSAD APAYKQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP RRKNPQEGLY 60
NELQKDKMAE AYSEIGMKGE RRRGKGHDGL YQGLSTATKD TYDALHMQAL PPRG 114
Claims (7)
- lentivirus expression vectors, which is characterized in that the vectors comprise a humanized scFv chimeric antigen receptor (anti-hCD 19 CAR) gene structure targeting CD19, and the nucleotide sequence of the vectors is SEQ ID NO.1, SEQ ID NO. 2 or SEQ ID NO. 3.
- 2. The lentiviral expression vector of claim 1, wherein the chimeric antigen receptor gene construct comprises, in serial order, a CD8 transmembrane signal peptide, anti-hCD19scFv, a CD8 transmembrane region, a 4-1BB co-stimulatory signal region, and a CD3Zeta TCR activation region.
- 3. The lentiviral expression vector of claim 2, wherein the CD8 transmembrane signal peptide has the amino acid sequence shown in SEQ ID No.4, the anti-hCD19scFv comprises or is selected from any amino acid sequence selected from the group consisting of SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, the CD8 transmembrane region has the amino acid sequence shown in SEQ ID No.8, the 4-1BB co-stimulatory signal region has the amino acid sequence shown in SEQ ID No.9, and the CD3Zeta TCR activation region has the amino acid sequence shown in SEQ ID No. 10.
- 4, A method for preparing CD19 targeted humanized scFv chimeric antigen receptor T cells comprising the lentiviral expression vector of claim 1, wherein the method comprises the steps of:(1) constructing a plasmid vector: after the anti-CD19 scFv fragment is subjected to humanized transformation, obtaining anti-hCD19scFv, inserting an Mlu I enzyme cutting site and a CD8 transmembrane signal peptide into the fragment, inserting a BamH I enzyme cutting site into the fragment, synthesizing a pUC57-Amp plasmid containing Mlu I + CD8a-hCD19scFv + BamH I, performing double enzyme cutting by Mlu I and BamH I, identifying the enzyme cutting effect by agarose gel electrophoresis, and recovering gel to obtain a modified gene fragment; meanwhile, carrying out double enzyme digestion on the lentiviral backbone plasmid pHR containing the CD8 transmembrane region, the 4-1BB costimulatory signal region and the CD3Zeta TCR activation region by Mlu I and BamH I, and recovering long fragments from gel after agarose gel electrophoresis identification; connecting the modified anti-hCD19scFv fragment to a lentiviral backbone plasmid pHR, extracting the plasmid, and obtaining a pHR-anti-hCD19CAR plasmid after determining that the sequencing is correct;(2) and (3) preparing lentivirus: mixing the pHR-anti-hCD19CAR plasmid carrying the target gene, a pCMV vector and a pMD.2G vector, transfecting into 293FT cells, replacing with a DMEM complete culture medium containing 10% FBS and 1% glutamine for culture after 6-8 h after transfection, collecting a culture solution after 48h, centrifuging, retaining a supernatant, filtering the supernatant by using a 0.45 mu m filter, retaining a filtrate, wherein the filtrate is a recombinant lentivirus solution, and concentrating for later use;(3) anti-hCD19CAR-T preparation: 50mL of fresh blood was taken and passed through lymphocytesSubjecting the separation solution to density gradient centrifugation to separate mononuclear cells, wherein the single mononuclear cells are subjected to density gradient centrifugation according to the ratio of 1-2 × 106/mL resuspended CTSTMAIM VTM5% ICS, 50ng/mL of CD3 monoclonal antibody and 50ng/mL of CD28 monoclonal antibody are added into an SFM culture medium at the same time to activate T lymphocytes, and the cells are cultured for 48 hours at 37 ℃ with 5% CO 2; after 2 days of culture, cells were harvested and resuspended to 1X106Adding the concentrated recombinant lentivirus solution according to MOI = 5, simultaneously adding IL-2 and polybrene with final concentration of 500U/mL, uniformly mixing, and 5% CO at 37 DEG C2Culturing for 6-8 hours, centrifuging at 300g for 5min, and changing the liquid into fresh CTSTMAIM VTMSFM medium; adding fresh CTS every 2-3 daysTMAIM VTMSFM medium, maintaining cell density at 1X106about/mL, and performing amplification culture for 10-12 days.
- 5. The method according to claim 4, wherein, in the step (3), the CTS isTMAIM VTMSFM medium contained 500U/mL IL-2.
- Use of humanized scFv chimeric antigen receptor T-cells targeting CD19 according to any of claims 1-3 to for the preparation of a medicament for the prevention and/or treatment and/or adjuvant treatment of malignancies.
- 7. The use of claim 7, wherein said malignant cells comprise a CD19 positive B-lymphocyte leukemia or B-lymphoma.
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| CN111135307A (en) * | 2020-02-05 | 2020-05-12 | 中山大学附属第八医院（深圳福田） | Preparation method and application of CAR-T cell-based drug delivery system |
| CN111849917A (en) * | 2020-07-22 | 2020-10-30 | 武汉大学 | Fourth generation CAR-T cell for fusion expression of type I IL-10 receptor and preparation method and application thereof |
| CN112111014A (en) * | 2020-09-22 | 2020-12-22 | 廖文强 | Antioxidant chimeric antigen receptor and immune cell |
| CN112522203A (en) * | 2020-11-23 | 2021-03-19 | 中山大学附属第五医院 | Cell vesicle for expressing chimeric antigen receptor and preparation method and application thereof |
| CN112679618A (en) * | 2020-08-13 | 2021-04-20 | 上海市第一人民医院 | TRBC 1-targeted humanized chimeric antigen receptor, T cell and application |
| WO2021213235A1 (en) * | 2020-04-20 | 2021-10-28 | 浙江瑞加美生物科技有限公司 | Technique for preparing universal humanised car19-dnt cells and application therefor |
| CN114457037A (en) * | 2022-01-26 | 2022-05-10 | 徐州医科大学 | Construction method and application of C57BL/6J mouse-derived CD19 chimeric antigen receptor T cells |
| CN114752615A (en) * | 2022-04-21 | 2022-07-15 | 新乡医学院 | CD 19-targeted CAR-T cells overexpressing ATP5IF1 gene and application thereof |
| CN114921496A (en) * | 2022-04-29 | 2022-08-19 | 上海驯鹿生物技术有限公司 | Construction method and application of humanized immune system animal model with NK (Natural killer) cell and ADCC (advanced Charge coupled device) capabilities |
| CN115716879A (en) * | 2022-11-15 | 2023-02-28 | 北京瑜阳科技有限公司 | Chimeric Antigen Receptor (CAR) and anti-cancer application thereof |
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| CN111849917A (en) * | 2020-07-22 | 2020-10-30 | 武汉大学 | Fourth generation CAR-T cell for fusion expression of type I IL-10 receptor and preparation method and application thereof |
| CN112679618A (en) * | 2020-08-13 | 2021-04-20 | 上海市第一人民医院 | TRBC 1-targeted humanized chimeric antigen receptor, T cell and application |
| CN112111014A (en) * | 2020-09-22 | 2020-12-22 | 廖文强 | Antioxidant chimeric antigen receptor and immune cell |
| CN112522203B (en) * | 2020-11-23 | 2023-08-11 | 中山大学附属第五医院 | Cell vesicle for expressing chimeric antigen receptor, and preparation method and application thereof |
| CN112522203A (en) * | 2020-11-23 | 2021-03-19 | 中山大学附属第五医院 | Cell vesicle for expressing chimeric antigen receptor and preparation method and application thereof |
| CN114457037A (en) * | 2022-01-26 | 2022-05-10 | 徐州医科大学 | Construction method and application of C57BL/6J mouse-derived CD19 chimeric antigen receptor T cells |
| CN114457037B (en) * | 2022-01-26 | 2023-12-22 | 徐州医科大学 | Construction method and application of C57BL/6J mouse-derived CD19 chimeric antigen receptor T cells |
| CN114752615A (en) * | 2022-04-21 | 2022-07-15 | 新乡医学院 | CD 19-targeted CAR-T cells overexpressing ATP5IF1 gene and application thereof |
| CN114752615B (en) * | 2022-04-21 | 2023-09-22 | 新乡医学院 | CD 19-targeted CAR-T cell overexpressing ATP5IF1 gene and application thereof |
| CN114921496A (en) * | 2022-04-29 | 2022-08-19 | 上海驯鹿生物技术有限公司 | Construction method and application of humanized immune system animal model with NK (Natural killer) cell and ADCC (advanced Charge coupled device) capabilities |
| CN114921496B (en) * | 2022-04-29 | 2023-03-10 | 上海驯鹿生物技术有限公司 | Construction method and application of humanized immune system animal model with NK (natural killer) cell and ADCC (advanced cellular ADCC) capabilities |
| CN115716879A (en) * | 2022-11-15 | 2023-02-28 | 北京瑜阳科技有限公司 | Chimeric Antigen Receptor (CAR) and anti-cancer application thereof |
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