CN117384968B - Chimeric Antigen Receptor (CAR) and use thereof for treating leukemia - Google Patents
Chimeric Antigen Receptor (CAR) and use thereof for treating leukemia Download PDFInfo
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Abstract
The invention relates to the technical field of chimeric antigen receptors, and provides a Chimeric Antigen Receptor (CAR) and application thereof for treating leukemia, wherein the chimeric antigen receptor provided by the invention can specifically recognize CD19 antigen, has good targeting property, CAR-T cells containing the chimeric antigen receptor show good killing ability in vitro, and experiments in vivo show good antigen targeting property and leukemia cell killing activity, so that the CAR-T cells prepared by the invention can be used for targeted treatment of leukemia.
Description
Technical Field
The invention relates to the technical field of chimeric antigen receptors, in particular to a Chimeric Antigen Receptor (CAR) and application thereof in treating leukemia.
Background
Currently, the field of cell therapy has become a popular direction for future human medical development due to its high precision and individuality. Cell therapy refers to a therapeutic method in which autologous, allogeneic or xenogeneic (non-human) cells of a human are used, and are infused back (or implanted) into the human body after an in vitro procedure. Among them, chimeric antigen receptor T cell immunotherapy (CAR-T cell therapy) is an adoptive cellular immunotherapy which has been developed in recent years, and is mainly performed by combining the specificity of chimeric antigen receptor with the immune action of T cells and then killing malignant tumor cells through specific recognition. The traditional field of CAR-T cell therapy is mainly to treat hematological malignant tumors, and has good clinical effect.
CD19 molecules are receptors on the surface of B lymphocytes that exert specific signal transduction, are present at various stages of B cell maturation, and are present and stably and continuously expressed at the B progenitor stage. CD19 is expressed as a cell surface differentiation antigen specific for B cell lines on the surface of pre-B cells and mature B cells only, but not in hematopoietic stem cells, plasma cells and other normal tissue cells. In addition, CD19 molecules are relatively exposed on the membrane, are easily accessible, have no significant internalization or shedding after binding to monoclonal antibodies, and do not undergo antigen modulation due to binding to antibodies. In addition, recent studies on how oncogenes MYC gene promote the development and progression of B-lymphocyte malignancies have found that CD19 is absolutely necessary for stabilizing MYC protein, and when MYC gene is stable and at a higher level, MYC gene promotes cancer progression, and patients with high MYC protein levels are more likely to die from lymphomas. CD19 may be the switch on the Myc gene pathway and controlling the on-off switch may represent a powerful tool for the treatment of lymphocytic tumors. Based on the above, CD19 is an important target for the treatment of CD19 positive acute B-lymphoblastic leukemia.
Disclosure of Invention
Aiming at the technical problems existing in the prior art, the invention provides a Chimeric Antigen Receptor (CAR) and application thereof in treating leukemia. The chimeric antigen receptor provided by the invention can specifically recognize CD19, has good targeting property, and can be used for targeted treatment of leukemia.
It is an object of the present invention to provide a CD 19-targeting CAR-T expression vector, characterized in that the expression vector comprises a CD 19-targeting CAR gene sequence;
further, the CD19 targeting CAR consists of, in order, a CD8a signal peptide, a CD19 targeting single chain antibody, a CD8a hinge region, a CD8a transmembrane domain, a CD28 protein costimulatory domain, and a CD3 zeta cell signaling domain;
further, the amino acid sequence of the CD19 targeting CAR-T is shown as SEQ ID NO.1, and the coded nucleotide sequence is shown as SEQ ID NO. 2;
further, the amino acid sequence of the CD8 alpha signal peptide sequence is shown as SEQ ID NO.3, and the coded nucleotide sequence is shown as SEQ ID NO. 4;
further, the amino acid sequence of the CD19 targeting single-chain antibody is shown as SEQ ID NO.5, and the coded nucleotide sequence is shown as SEQ ID NO. 6;
further, the amino acid sequence of the CD8a hinge region is shown as SEQ ID NO.7, and the encoding nucleotide sequence is shown as SEQ ID NO. 8;
further, the amino acid sequence of the CD8a transmembrane domain is shown as SEQ ID NO.9, and the coded nucleotide sequence is shown as SEQ ID NO. 10;
further, the amino acid sequence of the CD28 protein costimulatory domain is shown as SEQ ID NO.11, and the coded nucleotide sequence is shown as SEQ ID NO. 12;
further, the amino acid sequence of the CD3 zeta cell signal transduction domain is shown as SEQ ID NO.13, and the coded nucleotide sequence is shown as SEQ ID NO. 14;
another object of the present invention is to provide a method for constructing a CD 19-targeting CAR-T expression vector, wherein a CD 19-targeting chimeric antigen receptor gene is used as a target gene, and a recombinant lentiviral packaging system is used to construct the CD 19-targeting CAR-T expression vector.
It is another object of the present invention to provide a CAR-T cell obtained by transducing T lymphocytes with the above CAR-T expression vector.
One of the other objects of the invention is the use of the above-described CAR-T expression vector or CAR-T cells for the preparation of a medicament for the treatment of leukemia.
Further, the tumor is a CD19 overexpressing leukemia.
Further, the tumors include acute lymphocytic and acute myeloid leukemia.
The invention has the following advantages: the chimeric antigen receptor provided by the invention can specifically recognize CD19 antigen, has good targeting property, and CAR-T cells containing the chimeric antigen receptor show good killing capacity in vitro, and also show good antigen targeting property and leukemia cell killing activity in vivo experiments, so that the CAR-T cells prepared by the invention can be used for targeted treatment of leukemia.
Drawings
FIG. 1. Specific killing effect of CAR-T cells on leukemia cells;
FIG. 2. Survival effect of CAR-T cells on leukemia mouse model;
FIG. 3 effect of CAR-T cells on CD19+ cell number in leukemia mice.
Detailed Description
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art.
Example 1 design of CAR expression sequences and construction of lentiviruses
1.1 Design of CAR expression sequences
A CD19 targeting CAR sequentially comprises a CD8 alpha signal peptide, a CD19 targeting single-chain antibody, a CD8a hinge region, a CD8a transmembrane domain, a CD28 protein costimulatory domain and a CD3 zeta cell signaling domain, wherein the amino acid sequence is shown as SEQ ID NO.1, and the encoding nucleotide sequence is shown as SEQ ID NO. 2.
Wherein the amino acid sequence of the CD8 alpha signal peptide sequence is shown as SEQ ID NO.3, and the coded nucleotide sequence is shown as SEQ ID NO. 4; the amino acid sequence of the CD19 targeting single-chain antibody is shown as SEQ ID NO.5, and the encoding nucleotide sequence is shown as SEQ ID NO. 6; the amino acid sequence of the CD8a hinge region is shown as SEQ ID NO.7, and the coded nucleotide sequence is shown as SEQ ID NO. 8; the amino acid sequence of the CD8a transmembrane domain is shown as SEQ ID NO.9, and the coded nucleotide sequence is shown as SEQ ID NO. 10; the amino acid sequence of the CD28 protein costimulatory domain is shown as SEQ ID NO.11, and the coded nucleotide sequence is shown as SEQ ID NO. 12; the amino acid sequence of the CD3 zeta cell signal transduction domain is shown as SEQ ID NO.13, and the encoding nucleotide sequence is shown as SEQ ID NO. 14.
1.2 Construction of lentiviruses
Inserting the designed CAR gene sequence between EcoRI and BamH1 restriction enzyme cutting sites in a slow virus skeleton plasmid pCDH-EF1-MCS (purchased from Addgene), performing double restriction enzyme cutting on the restriction enzyme cutting sites at two ends of the designed gene sequence by using restriction enzyme, performing SDS-PAGE electrophoresis, selecting a fragment region with the same size as the designed enzyme cutting segment for glue recovery, and obtaining positive cloning plasmids with correct sequences through sequencing identification; mixing positive cloning plasmid with DH5 alpha competent escherichia coli, adding an LB liquid culture medium without antibiotics for culture, preparing an original bacterial liquid, inoculating the original bacterial liquid into 100mL of an Amp-resistant LB culture medium, and obtaining the bacterial liquid for plasmid extraction the next day by shaking table 200rpm at 37 ℃ for later use, and detecting the purity and concentration of the plasmid.
1.3 lentivirus preparation
293T cells are inoculated to a 6cm culture dish 24h in advance, so that the cell fusion rate can reach 80% during transfection. The following day, 293T cells were observed, and when the cell state was confirmed to be good and the degree of fusion was 80% -90%, fresh medium was changed, and the recombinant lentiviral plasmid was mixed with packaging plasmid (psPAX 2 and pMD 2. G) using Lipofectamine 3000 kit (Cat#L 3000008) produced by Thermofiser, and then added dropwise to 293T cell culture dish after incubation according to the instructions, and the dish was gently shaken and thoroughly mixed. The dishes were placed at 37℃in 5% CO 2 Culturing in an incubator for 6-8 h, and then performing liquid exchange treatment. After continuous culture for 72 hours, collecting culture medium supernatant containing virus in the culture dish, filtering with a 0.45 μm filter membrane, transferring the filtrate into a sterile centrifuge tube, centrifuging at ultra-high speed, concentrating to obtain slow virus, packaging in virus tube, and storing at-80deg.C.
Example 2 preparation of CAR-T cells
2.1 Preparation of CAR-T cells
T cells were obtained from human peripheral blood using a T cell purification kit (Meitian and gentle, procedures were performed according to the product instructions) and then, after that, the amount of T cells was 4X 10 6 The T cells are added with 40 mu L of activated magnetic beads (Meitian gentle) and mixed uniformly, and IL-2 to 200IU/mL is added for activation.
Counting activated T cells, adjusting cell density with T cell culture medium to 5×10 5 The wells were placed in 24-well plates, 5mL of virus supernatant was added, and the plates were placed in an incubator for infection culture. And (3) supplementing 500 mu L of fresh culture medium for 24h of virus infection, collecting T cells after virus transduction after 48h of virus infection, centrifuging at 1500rpm for 5min, discarding supernatant, adding fresh culture medium to resuspension the cell suspension, and transferring to a culture flask for expansion culture to obtain CART-CD19 cells.
Example 3 functional detection of CAR-T cells
3.1 Cytotoxicity assay of CAR-T cells for killing human tumor cells
Respectively 1×10 6 CD19 positive B lymphoblastic leukemia cells Nalm-6 and Raji and CD19 non-expressing chronic myeloblastic leukemia cell K562 were added to the cell culture dish, and 1×10 cells were added to each group after they had been attached 4 Individual T cells or CAR-T cells. After 5 days of culture, the killing rate of the cells was analyzed using LDH detection kit instructions. The results are shown in FIG. 1: the CAR-T cell disclosed by the invention basically has no killing effect on chronic granulocytic leukemia cells K562 which do not express CD19, but has obvious killing effect on target cells Nalm-6 and Raji.
3.2 Therapeutic effect of CAR-T cells on advanced stage CD19 positive acute B-lymphoblastic leukemia
1X 10 by tail vein transplantation in immunodeficient NSG mice 6 CD19 + CD34 + CD38 - Leukemia cells were used to construct a mouse tumor model, and model mice were aliquoted into two groups (10 mice per group) and then returned intravenously 1X 10 4 The treatment status of mice was assessed after reinfusion and monitored continuously for one month.
The results are shown in fig. 2 and 3: compared with T cells, the CAR-T cells can obviously improve the survival rate of a model mouse, and compared with the proportion of CD19+ cells in the mouse before treatment, the CAR-T cells can obviously lower, so that the CAR-T cells can exert an obvious killing activity on CD19 positive B lymphoblastic leukemia cells in the bone marrow of an acute B lymphoblastic leukemia mouse positive with CD19 in the progressive stage.
The above-described embodiments are provided to illustrate the gist of the present invention, but are not intended to limit the scope of the present invention. It will be understood by those skilled in the art that various modifications and equivalent substitutions may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A CD 19-targeting CAR-T expression vector, wherein the expression vector comprises a CD 19-targeting CAR gene sequence; the CD19 targeting CAR consists of a CD8 alpha signal peptide, a CD19 targeting single-chain antibody, a CD8a hinge region, a CD8a transmembrane domain, a CD28 protein costimulatory domain and a CD3 zeta cell signaling domain in sequence; the amino acid sequence of the CD19 targeting CAR-T is shown as SEQ ID NO.1, and the encoding nucleotide sequence is shown as SEQ ID NO. 2.
2. The CD19 targeted CAR-T expression vector of claim 1, wherein the amino acid sequence of the CD8a signal peptide sequence is shown in SEQ ID No.3 and the nucleotide sequence encoded thereby is shown in SEQ ID No. 4.
3. The CD19 targeted CAR-T expression vector of claim 1, wherein the CD19 targeted single chain antibody has an amino acid sequence shown in SEQ ID No.5 and a nucleotide sequence encoded by the same is shown in SEQ ID No. 6.
4. The CD19 targeted CAR-T expression vector of claim 1, wherein the CD8a hinge region has the amino acid sequence shown in SEQ ID No.7 and the nucleotide sequence encoded thereby is shown in SEQ ID No. 8.
5. The CD19 targeted CAR-T expression vector of claim 1, wherein the CD8a transmembrane domain has the amino acid sequence shown in SEQ ID No.9 and the nucleotide sequence encoded thereby is shown in SEQ ID No. 10.
6. The CD19 targeted CAR-T expression vector of claim 1, wherein the CD28 protein costimulatory domain has the amino acid sequence shown in SEQ ID No.11 and the nucleotide sequence encoded thereby is shown in SEQ ID No. 12.
7. The CD19 targeted CAR-T expression vector of claim 1, wherein the amino acid sequence of the CD3 zeta cell signaling domain is shown in SEQ ID No.13 and the nucleotide sequence encoded thereby is shown in SEQ ID No. 14.
8. The method for constructing a CD 19-targeted CAR-T expression vector according to any one of claims 1 to 7, wherein the CD 19-targeted CAR-T expression vector is constructed by using a recombinant lentiviral packaging system with the CD 19-targeted chimeric antigen receptor gene as a target gene.
9. A CAR-T cell obtained by transduction of T lymphocytes using the CAR-T expression vector of any one of claims 1-7.
10. Use of the CAR-T expression vector of any one of claims 1-7 or the CAR-T cell of claim 9 in the manufacture of a medicament for the treatment of leukemia, wherein the leukemia is acute B-lymphocyte leukemia.
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| CN104788573A (en) * | 2015-05-08 | 2015-07-22 | 中国医学科学院血液病医院（血液学研究所） | Chimeric antigen receptor hCD19scFv-CD8a-CD-28-CD3zata and application thereof |
| CN107428830A (en) * | 2015-01-05 | 2017-12-01 | 依奈特制药公司 | Monomer FC domains |
| WO2020108642A1 (en) * | 2018-11-30 | 2020-06-04 | Beijing Meikang Geno-Immune Biotechnology Co., Ltd. | Cd19-and cd30-based combined car-t immunotherapy |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107428830A (en) * | 2015-01-05 | 2017-12-01 | 依奈特制药公司 | Monomer FC domains |
| CN104788573A (en) * | 2015-05-08 | 2015-07-22 | 中国医学科学院血液病医院（血液学研究所） | Chimeric antigen receptor hCD19scFv-CD8a-CD-28-CD3zata and application thereof |
| WO2020108642A1 (en) * | 2018-11-30 | 2020-06-04 | Beijing Meikang Geno-Immune Biotechnology Co., Ltd. | Cd19-and cd30-based combined car-t immunotherapy |
Non-Patent Citations (1)
| Title |
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| A novel and efficient CD22 CAR-T therapy induced a robust antitumor effect in relapsed/refractory leukemia patients when combined with CD19 CAR-T treatment as a sequential therapy;Yu Zhang;Experimental Hematology & Oncology;第11卷(第15期);1-18 * |
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