CN110606893B - Method for treating tumor by chimeric antigen receptor T cell targeting CD19 and CD20 double antigens - Google Patents
Method for treating tumor by chimeric antigen receptor T cell targeting CD19 and CD20 double antigens Download PDFInfo
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Abstract
The invention discloses a method for treating tumors by using chimeric antigen receptor T cells targeting CD19 and CD20 double antigens. The invention provides a chimeric antigen receptor, which comprises an extracellular recognition domain and an intracellular signal transduction domain, wherein the extracellular recognition domain sequentially comprises a signal peptide, a double antigen recognition region, a hinge region and a transmembrane region; the dual antigen recognition region comprises a CD20 single chain antibody (scfv) and a CD19 single chain antibody, wherein the CD19 single chain antibody is proximal to the transmembrane region. The invention has the advantages that DCAR-013-T cells can recognize any antigen of CD19 and CD20 and kill tumor cells; in B cell derived blood tumor and lymphoma patients, CD19 and CD20 are both negative tumor cells are few, and the double-target CAR aiming at the CD19 and the CD20 shows good in-vitro killing and clinical treatment effects, so that the superiority of the double-target design is reflected.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for treating tumors by using chimeric antigen receptor T cells of targeted CD19 and CD20 double antigens.
Background
The Chimeric Antigen Receptor (CAR) technology is to use a genetic engineering method to endow T cells with the capability of recognizing tumor cell surface antigens, and specifically recognize and kill tumor cells without the restriction of HLA (human leukocyte antigen). The structure of the CAR consists of an extracellular recognition domain and an intracellular signaling domain: the extracellular recognition domain can specifically recognize tumor surface specific protein (antigen), and the intracellular signal transduction structural domain is used for starting immune response of T lymphocytes after recognizing the tumor cell (target cell) surface protein, playing a role in cytotoxicity and killing the target cell.
The extracellular recognition domain of CAR consists of Signal Peptide (SP), the extracellular region of single chain antibody domain (scFv) that recognizes antigen, linker, hinge, and transmembrane regions (TM); the intracellular signaling domain consists of a costimulatory molecule and a CD3 ζ (CD 3 z) intracellular segment. The no costimulatory molecule is a first generation CAR, containing 1 costimulatory molecule (CD 28, 4-1BB, OX40, ICOS, etc.) as a second generation CAR, and 2 costimulatory molecules as a third generation CAR.
Disclosure of Invention
An object of the present invention is to provide a chimeric antigen receptor.
The chimeric antigen receptor provided by the invention comprises an extracellular recognition domain and an intracellular signal transduction structural domain, wherein the extracellular recognition domain sequentially comprises a signal peptide, a double-antigen recognition region, a hinge region and a transmembrane region;
the double antigen recognition region comprises a CD20 single chain antibody (scfv) and a CD19 single chain antibody, wherein the CD19 single chain antibody is adjacent to the transmembrane region.
In the chimeric antigen receptor, the CD20 single-chain antibody comprises a light chain variable region of the CD20 single-chain antibody and a heavy chain variable region of the CD20 single-chain antibody;
the CD19 single-chain antibody comprises a light chain variable region of the CD19 single-chain antibody and a heavy chain variable region of the CD19 single-chain antibody;
wherein, the four positional sequence relations among the light chain variable region of the CD20 single-chain antibody, the heavy chain variable region of the CD20 single-chain antibody, the light chain variable region of the CD19 single-chain antibody and the heavy chain variable region of the CD19 single-chain antibody are (1) or (2):
(1) The variable region of the light chain of the CD20 single-chain antibody, the variable region of the heavy chain of the CD19 single-chain antibody, and the variable region of the light chain of the CD19 single-chain antibody;
(2) The heavy chain variable region of the CD20 single-chain antibody, the light chain variable region of the CD19 single-chain antibody and the heavy chain variable region of the CD19 single-chain antibody.
In the chimeric antigen receptor, the heavy chain variable region of the CD20 single-chain antibody and the heavy chain variable region of the CD19 single-chain antibody are linked via a linking region a; or, the light chain variable region of the CD20 single-chain antibody and the light chain variable region of the CD19 single-chain antibody are connected through the connecting region A;
and/or, the light chain variable region of the CD20 single-chain antibody and the heavy chain variable region of the CD20 single-chain antibody are connected through a connecting region B;
and/or the light chain variable region of the CD19 single-chain antibody and the heavy chain variable region of the CD19 single-chain antibody are connected through a connecting region C.
In the chimeric antigen receptor, the variable region of the CD20 single-chain antibody light chain is a, b or c as follows:
a) Which consists of amino acid residues shown in 22 th to 127 th positions of a sequence 2 in a sequence table;
b) A peptide chain which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence defined by a) and has the same function;
c) A peptide chain having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the amino acid sequence defined in a) and having the same function;
the variable region of the CD20 single-chain antibody heavy chain is d or e or f as follows:
d) Which consists of amino acid residues shown in 133 th-252 th sites of a sequence 2 in a sequence table;
e) Peptide chains with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequences defined by the d);
f) A peptide chain having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the amino acid sequence defined in d) and having the same function;
the variable region of the CD19 single-chain antibody light chain is g or h or i as follows:
g) Which consists of amino acid residues shown in 403 th to 509 th in a sequence 2 in a sequence table;
h) Peptide chains with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequences defined in g);
i) A peptide chain having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the amino acid sequence defined in g) and having the same function;
the CD19 single-chain antibody heavy chain variable region is g or k or l as follows:
g) It consists of amino acid residues shown in 268-387 of a sequence 2 in a sequence table;
k) Peptide chains with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequences defined in g);
l) a peptide chain having a homology of 99% or more, 95% or more, 90% or more, 85% or more or 80% or more with the amino acid sequence defined in g) and having the same function;
and/or the presence of a gas in the gas,
the amino acid sequence of the connecting region A is 253 th to 267 th positions of the sequence 2
The amino acid sequence of the connecting region B is 128 th to 132 th positions of the sequence 2
The amino acid sequence of the connecting region C is 388-402 th of the sequence 2.
In the chimeric antigen receptor, the signal peptide is a signal peptide of CD8A or a signal peptide of CSF1R, CSF2RA or CD 4;
the hinge region is all or partial peptide fragments of IgG1, igG4, igD and/or CD 8;
the transmembrane region is selected from the transmembrane structures of one or more of CD28, CD3 ζ, CD4, CD8, and CD 2;
the intracellular signaling domain comprises an intracellular co-stimulatory molecule and a signaling domain;
the intracellular co-stimulatory molecule is selected from one or more of CD28, 4-1BB, ICOS and OX 40;
the signaling domain is selected from one or more of CD3 ζ, CD5, CD28 and CD 124;
and/or, the intracellular signaling domain comprises a costimulatory molecule and a CD3 ζ (CD 3 z) intracellular segment;
the intracellular signaling domain specifically comprises intracellular costimulatory molecule 41-BB and signaling domain CD3 ζ;
the non-costimulatory molecule is a first generation CAR, contains 1 costimulatory molecule (CD 28, 4-1BB, OX40, ICOS, etc.) as a second generation CAR, contains 2 costimulatory molecules as a third generation CAR; the present invention employs intracellular costimulatory molecule 4-1BB.
And/or, the chimeric antigen receptor, in turn, consists of a signal peptide, a dual antigen recognition region, a hinge region, a transmembrane region, and an intracellular signal domain;
the chimeric antigen receptor is composed of a CD8a signal peptide, a double antigen recognition region, a Hinge region (Hinge), a transmembrane region (TM) and an intracellular signal domain in sequence;
wherein, the double antigen recognition region is composed of the light chain variable region of the CD20 single-chain antibody, the connection region B, the heavy chain variable region of the CD20 single-chain antibody, the connection region A, the heavy chain variable region of the CD19 single-chain antibody, the connection region C and the light chain variable region of the CD19 single-chain antibody in sequence;
or, the double antigen recognition region is composed of the heavy chain variable region of the CD20 single-chain antibody, the connection region B, the light chain variable region of the CD20 single-chain antibody, the connection region A, the light chain variable region of the CD19 single-chain antibody, the connection region C and the heavy chain variable region of the CD19 single-chain antibody in sequence.
In the chimeric antigen receptor, the chimeric antigen receptor is 1) or 2) or 3) as follows:
1) Which consists of amino acid residues shown in a sequence 2 in a sequence table;
2) Peptide chains with the same function and obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequences defined in 1);
3) A peptide chain having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the amino acid sequence defined in 1) and having the same function.
Nucleic acid molecules encoding the chimeric antigen receptors described above are also within the scope of the invention;
or, a recombinant vector, expression cassette, recombinant bacterium, recombinant virus or cell comprising said nucleic acid molecule.
The recombinant vector is a lentiviral vector which is used for introducing the nucleic acid molecule into a lentiviral vector to obtain the chimeric antigen receptor;
the recombinant virus is obtained by introducing the recombinant vector into a host cell and packaging;
or, the cell is an immune cell,
or, the immune cell is specifically a T cell or NK cell;
and/or, the cell is a T cell transfected by the lentivirus into the recombinant vector to obtain the T cell expressing the chimeric antigen receptor.
It is another object of the invention to provide a product.
The product provided by the invention comprises the chimeric antigen receptor, the nucleic acid molecule or the recombinant vector, the expression cassette, the recombinant bacterium, the recombinant virus or the cell.
The application of the chimeric antigen receptor or the nucleic acid molecule or the recombinant vector, the expression cassette, the recombinant bacterium, the recombinant virus or the cell in the preparation of products for preventing and/or treating tumors is also within the protection scope of the invention;
or the application of the chimeric antigen receptor or the nucleic acid molecule or the recombinant vector, the expression cassette, the recombinant bacterium, the recombinant virus or the cell in preparing a product for killing tumor cells is also within the protection scope of the invention;
or the application of the nucleic acid molecule or the recombinant vector, the expression cassette, the recombinant bacterium and the recombinant virus in preparing the T cell with increased IFN-gamma and/or IL-2 secretion induced by the tumor cell is also within the protection scope of the invention.
Wherein the tumor is a tumor that is both CD19 and CD20 positive;
and/or, the tumor is derived from any one or a plurality of combinations of B lymphocyte hematological tumors, leukemia, lymphoma, non-Hodgkin lymphoma, myeloma and the like;
and/or, the lymphoma is a large B-cell lymphoma;
and/or, the tumor cell is a tumor cell that is both CD19 and CD20 positive;
and/or the product is a medicament or a kit or an autologous transfusion preparation.
The T cells are ex vivo cells, which are autologous cells of the patient.
The invention connects scFv recognizing 2 tumor antigens CD19 and CD20 in series, the double recognition CAR (Dual-CAR) is subjected to lentivirus infection on autologous T lymphocytes of tumor patients, is subjected to in vitro amplification, and then is returned to a cell therapy preparation of the tumor patients. Specific chimeric antigen receptors for recognizing surface molecules CD19 and CD20 tumor cells are transfected into autologous T cells through a chronic viral vector, and the modified T cells are named as DCAR-013-T cells. The modified DCAR-013-T cells can specifically recognize and kill cells with the surface molecules of CD19 or CD 20. The scFv is characterized by the outer side of CD20 and the inner side (the side close to the cell membrane) of CD 19. DCAR-013-T cells recognize both antigens CD19 or CD20, and either CD19 or CD20 antigen of tumor cells can be recognized and directly killed by the DCAR-013-T cells.
A single CD19-CAR or CD20-CAR can only recognize and kill a single antigen of tumor cells, e.g., a CD19-CAR can kill CD 19-positive tumor cells, possibly leaving a few CD 19-negative CD 20-positive tumor cells that can proliferate rapidly, causing tumor recurrence or poor treatment.
The invention has the advantages that DCAR-013-T cells can recognize any antigen of CD19 and CD20 and kill tumor cells; in B cell derived blood tumor and lymphoma patients, CD19 and CD20 are both negative tumor cells are few, and the dual-target CAR aiming at the CD19 and the CD20 shows good in-vitro killing and clinical treatment effects, so that the superiority of the dual-target design is reflected.
From the spatial structures of CD19 and CD20, CD19 is a single transmembrane protein, the extracellular region is long and contains 270 amino acids, CD20 is a 4-time transmembrane protein, 2 extracellular regions are short and respectively contain 6 and 47 amino acids, aiming at the structure, a long-short combined double-target CAR is designed, CD20-scFV recognizes the short CD20 extracellular region at the outer side, and CD19-scFV recognizes the long CD19 extracellular region at the inner side.
The arrangement of the heavy chain variable regions (VH) and light chain variable regions (VL) of the two single chain antibody domains of CD20 and CD19 is characterized in that the Linker is flanked by the heavy chain variable regions of 20-scFV and 19-scFV, respectively, or both light chain variable regions, i.e., arranged according to 20-VL-VH and 19-VH-VL, or 20-VH-VL and 19-VL-VH; the heavy chain variable region or light chain variable region with 20 and 19 on two sides of the connecting region (Linker) is characterized in that the arrangement avoids the formation of a new single chain antibody structure by the heavy chain variable region (or light chain variable region) of 20 and the light chain variable region (or heavy chain variable region) of 19, and the mismatched structure may cause non-specific recognition and killing of autologous cells.
The lentivirus infected DCAR-013-T cells used in the invention had no effect on the patient's CD4 and CD8-T cell phenotype and slightly increased the percentage of memory T cells (CD 45RO + and CD62L +) (8%); can kill CD19+ or CD20+ or double positive tumor cells in vitro, but has no killing effect on CD19 negative and CD20 negative cells. The Complete Remission (CR) rate of 18 patients with relapsing refractory large B cell lymphoma in a clinical hospital is 38.9% (7/18), the Partial Remission (PR) rate is 44.44% (8/18), the total effective (CR + PR) rate reaches 83.33%, and the good clinical treatment effect of the double-target DCAR-013-T cells is shown.
Drawings
FIG. 1 is a schematic view of the molecular composition of DCAR-013.
FIG. 2 is a flow cytometric analysis of the phenotypes CD4 and CD8 of uninfected T cells and DCAR-013-T cells on day 3 of lentivirus infection.
FIG. 3 is a flow cytometric analysis of the phenotypes of uninfected T cells and DCAR-013-T cells on day 3 of viral infection, CD62L and CD45RO.
FIG. 4 is a phenotypic characterization of patient uninfected lentiviral T cells.
FIG. 5 shows the positive rate of human T cells against DCAR-013 lentivirus infection.
FIG. 6 shows the DCAR-013-T cell killing experiment in vitro.
FIG. 7 is a DCAR-013 factor release experiment.
FIG. 8 is a schematic diagram of the effect of DCAR-013 in recognizing and killing lymphoma cells.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of chimeric antigen receptor DCAR-013
The molecular composition of the chimeric antigen receptor DCAR-013 is schematically shown in FIG. 1, and sequentially comprises a CD8a signal peptide, a double antigen recognition region, a Hinge region (Hinge), a transmembrane region (TM) and an intracellular signal domain;
wherein, the double antigen recognition region consists of a light chain variable region of the CD20 single-chain antibody, a connection region B, a heavy chain variable region of the CD20 single-chain antibody, a connection region A, a heavy chain variable region of the CD19 single-chain antibody, a connection region C and a light chain variable region of the CD19 single-chain antibody in sequence;
or the double antigen recognition region consists of a heavy chain variable region of the CD20 single-chain antibody, a connecting region B, a light chain variable region of the CD20 single-chain antibody, a connecting region A, a light chain variable region of the CD19 single-chain antibody, a connecting region C and a heavy chain variable region of the CD19 single-chain antibody in sequence.
The amino acid sequence of the chimeric antigen receptor DCAR-013 is sequence 2, and the nucleotide sequence of the nucleic acid molecule encoding the chimeric antigen receptor DCAR-013 is sequence 1.
1. CD8a signal peptide
The amino acid sequence of the CD8a signal peptide is the 1 st-21 st amino acid of the sequence 2 in the sequence table;
2. double antigen recognition region
The double antigen recognition region consists of a light chain variable region (VL) of the anti-CD20scFv, a connecting region ethinker, a heavy chain variable region (VH) of the anti-CD20scFv, a connecting region methylinker, a heavy chain variable region (VH) of the anti-CD19scFv, a connecting region propylnker and a light chain variable region (VL) of the anti-CD19scFv in sequence;
or the double antigen recognition region consists of a heavy chain variable region (VH) of the anti-CD20scFv, a connecting region ethylinker, a light chain variable region (VL) of the anti-CD20scFv, a connecting region methylinker, a light chain variable region (VL) of the anti-CD19scFv, a connecting region propylkliner and a heavy chain variable region (VH) of the anti-CD19scFv in sequence.
The above-mentioned linker region A, linker region B and linker region C are different.
The amino acid sequence of the light chain variable region (VL) of the anti-CD20scFv is 22 th to 127 th in the sequence 2 in the sequence table, and the nucleotide sequence of the nucleic acid molecule for coding the anti-CD20scFv light chain variable region is 64 th to 381 th in the sequence 1;
the amino acid sequence of the connecting region ethylinker is 128 th to 132 th in a sequence 2 in a sequence table, and the nucleotide sequence of the nucleic acid molecule for coding the connecting region ethylinker is 382 th to 396 th in the sequence 1;
the amino acid sequence of the heavy chain variable region (VH) of the anti-CD20scFv is 133 th-252 th in the sequence 2 in the sequence table, and the nucleotide sequence of the nucleic acid molecule for coding the heavy chain variable region of the anti-CD20scFv is 397 th-756 th in the sequence 1;
the amino acid sequence of the linker of the connecting region A is 253 rd to 267 th in a sequence 2 in a sequence table, and the nucleotide sequence of the nucleic acid molecule for coding the connecting region A is 757 th to 801 th in the sequence 1.
The amino acid sequence of the heavy chain variable region (VH) of the anti-CD19scFv is 268 th to 387 th sites of a sequence 2 in a sequence table, and the nucleotide sequence of the nucleic acid molecule for coding the anti-CD19scFv heavy chain variable region is 802 th to 1161 th sites of the sequence 1;
the amino acid sequence of the connecting region linker is 388 th to 402 th of the sequence 2 in the sequence table, and the nucleotide sequence of the nucleic acid molecule for coding the connecting region linker is 1162 th to 1206 th of the sequence 1;
the amino acid sequence of the light chain variable region (VL) of the anti-CD19scFv is 403 th to 509 th in the sequence 2 in the sequence table, and the nucleotide sequence of the nucleic acid molecule for encoding the anti-CD19scFv light chain variable region is 1207 th to 1527 th in the sequence 1;
3. hinge region (Hinge)
The Hinge region (Hinge) adopts a Hinge region (Hinge) of Human CD8A, and the amino acid sequence of the Hinge region (Hinge) is from 510 th to 554 th in the sequence 2. The nucleic acid sequence corresponding to the Hinge region (Hinge) is 1528-1662 of SEQ ID No. 1.
4. Transmembrane region (TM)
The transmembrane region (TM) is the transmembrane region of Human CD8a, and the amino acid sequence thereof is 555-578 th position of the sequence 2.
The transmembrane region corresponds to the nucleic acid sequence 1663-1734 of SEQ ID NO. 1.
5. Intracellular signaling domains
The intracellular signaling domain consists of, in turn, intracellular costimulatory molecule 41-BB and signaling domain CD3 ζ (CD 3 z).
The amino acid sequence of the intracellular costimulatory molecule 41-BB is 579-620 of the sequence 2; the nucleotide sequence is the 1735 th-1860 th of the sequence 1.
The amino acid sequence of the signal domain CD3 zeta (CD 3 z) is 621 th to 732 th sites of the sequence 2, and the nucleotide sequence is 1861 th to 2196 th sites of the sequence 1.
2. Preparation of Lentiviral vector expressing chimeric antigen receptor DCAR-013
Lentiviral vector pRRL-sin. Cppt. Pgk-gfp. Wpre is described in the following documents: zhao Y, stepto H, schneider CK. Development of the First World Health Organization viral Vector Standard: heated the Production Control and Standardization of Lentivirus-Based Gene Therapy products, human Gene Therapy methods.2017;28 (4):205-214..
The self-inactivating lentiviral vector (self-inactivating lentivirus vector) was obtained by replacing the DNA molecule between the BamH I and BstXI double cleavage sites of the lentiviral vector pRRL-SIN. CPPT. PGK-GFP. WPRE with the Elongation factor-1 alpha (EF 1A) promoter, and was named as Lenti-EF1A-GFP.
The lentiviral vector DCAR-013 for expressing the chimeric antigen receptor DCAR-013 is a vector obtained by replacing a nucleic acid molecule encoding the chimeric antigen receptor DCAR-013 shown in a sequence 1 in a sequence table with a nucleic acid sequence encoding GFP between XbaI and SalI cleavage sites of an inactivated lentiviral vector Lenti-EF1A-GFP, and the expression of the chimeric antigen receptor DCAR-013 is driven by an extended growth factor-1 alpha promoter.
3. Packaging of lentiviruses expressing chimeric antigen receptor DCAR-013
1. Lentiviral packaging
Packaging lentiviral particles comprising a protein encoding DCAR-013 in HEK293T cells using a three vector system;
HEK293T in DMEM medium containing 10% FBS;
reagents include the culture medium DMEM (ThermoFisher), fetal bovine serum FBS (Hyclone), pancreatin (Gibco), dulbecco's PBS (ThermoFisher), PEI (1 mg/mL, polyplus, opti-MEM (Gibco)) and 10cm cell culture dish (Corning);
packaging lentivirus particles containing the protein encoding DCAR-013 the protocol for preparation was as follows:
1) HEK293T cells are inoculated in a cell culture dish with the diameter of 10cm, and the cell confluency reaches 70-80% after 24 hours.
2) Preparation of a pre-transfection mixture: preparing a transfection mixed solution (a slow virus vector DCAR-013 expressing a chimeric antigen receptor DCAR-013, a plasmid psPAX2 encoding viral nucleocapsid proteins Gag/Pol and Rev, and a plasmid pMD2.G encoding viral envelope proteins are mixed according to a certain ratio) in a 15mL sterile centrifuge tube;
3) Transfection: adding 1mL of transfection mixed solution into each 10cm culture dish, and slowly and uniformly adding the transfection mixed solution; lightly shaking the whole body at 37 deg.C, and culturing in CO2 incubator at constant temperature.
4) Liquid changing: fresh DMEM was replaced 2 hours before transfection; 8mL of fresh DMEM pre-heated to 37 ℃ was added to each 10cm dish. And (3) changing the liquid after 6 hours of transfection, collecting virus supernatant after 48 hours to 72 hours of transfection, filtering by a 0.22 mu m filter, collecting filtrate to obtain the lentivirus particles expressing the DCAR-013 protein, or subpackaging and storing in a-80 ℃ refrigerator, or purifying the lentivirus.
2. Lentiviral purification
1) Pretreatment of centrifugation vessels
2) Soaking and washing 40ml of inner sleeve and metal outer tube used for centrifugation with 75% alcohol for 2 hours, pouring out the alcohol, rinsing with sterile water for three times, and irradiating with ultraviolet rays overnight in a biological safety cabinet;
3) Mixing the filtrates of the virus supernatants collected at different transfection time points obtained in the step 1, placing the mixture in a 50ml centrifuge tube, centrifuging the mixture at 2000rpm for 10min at room temperature, and centrifuging the mixture to remove cell debris;
4) Sucking the centrifuged supernatant by a 20ml syringe, filtering by a 0.22 mu m filter and collecting into a new 50ml centrifuge tube;
5) Transferring the filtered virus supernatant into a 40mL inner sleeve, transferring into a metal sleeve after balance, screwing a bottle cap, placing into a Beckman centrifuge, and centrifuging at 28000rpm and 4 ℃ for 150min;
6) Taking out the centrifuge tube, pouring out the supernatant in the inner sleeve, completely sucking the liquid at the tube opening by using absorbent paper as much as possible, adding 200 mu l of D-PBS, placing the inner sleeve in a clean 50mL centrifuge tube, and standing overnight at 4 ℃;
7) Subpackaging the virus overnight at 4 ℃ into a clean EP tube, taking a part of the virus to detect the virus titer, and storing the rest of the virus liquid in a refrigerator at-80 ℃ to obtain the lentivirus expressing the chimeric antigen receptor DCAR-013 (hereinafter referred to as DCAR-013 lentivirus).
3. Lentiviral titer assay
Pancreatin digestion counting HEK293T cells at 2X 10 5 Perwell was seeded in 6-well plates. Incubated in a 37 ℃ 5% C02 incubator until the cells are 50% confluent. The DCAR-013 lentivirus venom obtained in 2 above was added to EP tubes in an amount of 0.5. Mu.l, 1. Mu.l, 5. Mu.l, 10. Mu.l and 20. Mu.l, polybrene (polybrene) was added to a final concentration of 8. Mu.g/mL, and fresh medium was added to a final volume of lmL. The HEK293T cell culture solution was discarded, and each of the virus solutions (including stock solution) at the above concentrations was added to a 6-well plate, and incubated in a 37 ℃ 5% C02 incubator. After 48 hours 293T was trypsinized, the medium was neutralized and centrifuged for 4 minutes at 350g, the supernatant was discarded, the cells were washed 2 times with Phosphate Buffered Saline (PBS), the cells were dissolved in 50. Mu.l PBS, 1. Mu.l of an antibody against rabbit IgG (H + L) F (ab') 2-APC (Jackson, inc.) was added, after incubation for 30 minutes at 4 ℃, centrifuged for 4 minutes at 2000rpm, the supernatant was discarded, the cells were washed with PBS, and then 350. Mu.l PBS was added to resuspend the cells, and the CAR positivity of 293T cells was examined by flow cytometry.
Virus titer (TU/mL) = CAR positive cell rate × total number of cells/volume of lentivirus fluid (mL), and to avoid errors, only wells with a positive rate of 1-30% were selected for calculation, and the titer was calculated by taking the mean value of each group.
The number of cells was 2X 10 5 The 293T cells of (1) were added with 1. Mu.l of lentivirus, the positive rate of the F (ab') 2 antibody detection by flow cytometry was 15%, and the titer of the DCAR-013 lentivirus-infected 293T cells was 0.15X 2X 10 5 /0.001mL＝3.0×10 7 TU/mL。
4. CAR-T cell obtention expressing chimeric antigen receptor DCAR-013
1. Diluting RetroNectin to working concentration, adding to six well plates, about 2mL per well, placing in a 5% CO2 incubator at 37 ℃, taking out after 2 hours, sucking RetroNectin in the wells, and recovering to a clean 50mL centrifuge tube;
2. placing the three obtained lentivirus venom expressing the chimeric antigen receptor DCAR-013 stored at-80 ℃ in an ice box, and slowly melting;
3. taking the activated T cells in culture at 5X 10 6 Plating into 6-well plates;
the activated T cells were prepared as follows: mixing the collected autologous peripheral blood of the patient with sterile PBS in equal volume, diluting the peripheral blood, meanwhile, adding the lymphocyte separation solution into a 50mL sterile centrifuge tube, carefully adding the diluted peripheral blood to the upper layer of the lymphocyte separation solution according to a ratio of 1; the lymphocyte separation liquid divides peripheral blood in the centrifugal tube into four layers, wherein the first layer is a plasma layer, the second layer is an annular milky white lymphocyte layer (a leukocyte layer), the third layer is a transparent separation liquid layer, and the fourth layer is a red blood cell layer. Gently sucking the leukocyte layer, washing with sterile PBS for 2 times, and centrifuging for 8 minutes respectively; pouring off the supernatant, resuspending the white blood cells in PBS, and counting to obtain prepared peripheral blood mononuclear lymphocytes (PBMC); the PBMC were then added to 6-well plates, 2ml of PBMC-containing medium was added to each well, and 5X 10 cells were added 6 Each well was filled with 80. Mu.l of Human T cell activating CD3/CD28 antibody (Gibco Dynabeads Human T-Activator CD3/CD28, 11131D), and the cells were cultured at 37 ℃ in an incubator and stimulated with the CD3/CD28 antibody for 24-48 hours to become activated T cells.
4. Adding polybrene (polybrene) to a final concentration of 8 μ g/mL, dropwise adding DCAR-013 lentivirus, shaking, centrifuging at 2000rpm for 60min at room temperature;
5. incubated in a 5% CO2 incubator at 37 ℃ for 24 hours;
6. infecting lentivirus for the second time, and repeating the operation step 4;
7. after culturing in a 37 ℃ 5% CO2 incubator for 24 hours, the cells were transferred to another vessel and cultured.
8. After 72 hours of the second infection with lentivirus, lentivirus-infected CAR-T cells (hereinafter referred to as DCAR-013-T) were obtained.
T cells not infected with lentivirus were used as controls, as above, except that DCAR-013 lentivirus was not added.
The lentiviral-infected CAR-T cells were collected, washed 1 time with PBS, adjusted to a volume of 50. Mu.l, and 2. Mu.l each of mouse anti-human CD4-APC and CD8-PE flow antibodies (BD Biosciences) was added, with uninfected T cells as a control. After incubation at 4 ℃ for 60 minutes in the absence of light, centrifugation was carried out at 2000rpm for 4 minutes, the supernatant was discarded, the cells were washed with PBS2 times, 350. Mu.l of PBS was added to resuspend the cells, and the positive rates of CD4 and CD8 cells were determined by flow cytometry, as shown in FIG. 2. FL4 is anti-CD4, FL2 is anti-CD8, and the positive rates of CD4 and CD8 of uninfected T cells are 36.7 percent and 59.1 percent respectively; the positive rates of the DCAR-013-T cells, CD4 and CD8, were 35.8% and 59.8%, respectively, and there was no significant difference between the two.
Molecular phenotype characterization of memory T cells: cells that are double positive for CD62L and CD45RO are memory T cells. Lentivirally infected CAR-T cells and uninfected patient T cells were collected, washed 1 time with PBS, adjusted to a volume of 50. Mu.l, and 3. Mu.l each of mouse anti-human CD45RO-APC and CD62L-PE (BD Biosciences) flow-through antibodies was added. After incubating for 60 minutes at 4 ℃ in a dark place, centrifuging for 4 minutes at 2000rpm, discarding the supernatant, washing the cells for 2 times by PBS, then adding 350 mu L of PBS to resuspend the cells, and detecting the positive rates of CD45RO and CD62L cells by a flow cytometer; results as shown in figure 3, the double positive cells are memory T cells, the percentage of CD62L, CD45RO double positive cells of CAR-T cells infected with lentivirus is 68.6%, while the double positive percentage of CD62L and CD45RO of T cells of uninfected patients is 60.4%, indicating that: the memory T cells (Tmem) in the DCAR-013-T cell group were slightly higher than those in the control group by 8.2%.
Example 2 application of DCAR-013-T cells
FIG. 8 is a schematic representation of the effect of DCAR-013 in recognizing and killing lymphoma cells; the upper part is lymphoma cells, and the lower part is DCAR-013-T cells.
1. Phenotypic characterization of T cells infected and not infected with chimeric antigen receptor DCAR-013
1. CAR-T cells expressing chimeric antigen receptor DCAR-013
Activated T cells were counted at 5X 10 6 Perwell into 6-well plates, the DCAR-013 lentivirus obtained from 3 of example 1, stored at-80 ℃ was placed in an ice box and allowed to thaw slowly; adding polybrene (polybrene) to a final concentration of 8 μ g/mL in a 6-well plate, slowly adding virus solution, shaking, and stirring to 200%Centrifuging at 0rpm at room temperature for 60min, and incubating in a 5% CO2 incubator at 37 deg.C for 24 hours; and then infecting the virus for the second time, repeating the operation, changing fresh culture solution after 12 hours, and detecting the positive rate of the CAR-T by flow cytometry 48-72 hours after infecting the virus for the second time.
2. Detection
The results of flow analysis of the patient autologous T cells after activation in example 1 (antibody is mouse anti-human CD3, BD Biosciences), the results are shown in fig. 4, and it can be seen that the phenotype of flow analysis of the patient autologous T cells after activation with CD3 and CD28 antibodies on day 3 of in vitro culture: the positive rate of CD3 is 98.5%; the positive rates for CD4 and CD8 were 36.7% and 59.1%, respectively.
After 5 days from the patient infected with lentivirus DCAR-013-T, the antibody was analyzed by flow cytometry as rabbit anti-mouse IgG (H + L) F (ab') 2 (Jackson) based on the principle that 20-scFV and 19-scFV encoded by DCAR-013 were of mouse origin, fab fragment was species-specific, and the patient autologous T cells did not have this structure, so that the expression of DCAR-013 could be detected with the Fab fragment antibody; as shown in FIG. 5, the upper part of the graph is a negative Control (Control), and the lower part is the DCAR-013-T positive rate at day 7 of viral infection detected by the anti-mouse Fab fragment antibody, minus the Control, and is about 23.9%.
2. DCAR-013-T cell in vitro killing experiment
Killing effect of DCAR-013-T cells on tumor cells expressing CD19 and CD20 only, and K562 being CD19 negative and CD20 negative hematological tumor cells (human chronic myelogenous leukemia cells; K562: no. 3111C0001CCC000039, cell center of institute of basic medicine of Chinese academy of medicine)
As shown in FIG. 6, when the effective target ratio (E: T) is 2.5 and 5, DCAR-013-T cells kill CD 19-positive or CD 20-positive tumor cells but not negative K562 cells, CD19 and CD20 and single-target CAR also kill target cells, and single CAR19-T and CAR20-T have higher killing rate on target cells and no killing effect on CD19-CD 20-K562 cells than uninfected T cells (Normal T); DCAR-013-T has a higher killing rate on target cells than CAR19-T and CAR20-T alone, up to 80% or more.
3. DCAR-013-T cell secretion cytokine gamma-interferon (IFN-gamma) and interleukin 2 (IL-2) experiment induced by target cell
After the T cells are stimulated and activated by the target cells, cytokines such as IFN-gamma and IL-2 are secreted, and the secretion amount of the cytokines can indirectly reflect the reactivity of the DCAR-013-T or 19CAR and 20CAR-T cells to the tumor target cells. ELISA detection kit for detection (Shenzhen Dake is company, dayou IFN-gamma and IL-2). Target cell: raji cells are lymphoma cells that are both CD19 and CD20 positive, and K562 cells are hematological tumor cells that are CD19 and CD20 negative. Normal T cells, 19CAR, 20CAR and DCAR-013-T effector cells were mixed with the target cells at the specified ratios, incubated for 6-12 hours, centrifuged at 1000rpm, and the supernatant from the isolated cells was collected in approximately 50. Mu.l aliquots into sterile EP tubes and stored at-80 ℃ until use. The gamma-interferon (IFN-gamma) and the interleukin 2 (IL-2) are detected by an ELISA method according to the method of Dake as a Dayou kit, and the method firstly uses the standards of the IFN-gamma and the IL-2 and the opposite standard curve. Adding standard and sample (standard preparation 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.3pg/ml, 15.6 pg/ml); adding samples into a 96-well plate, and incubating for 1 hour at 37 ℃; wash the plate 3 times with 200 μ l wash solution per well; add diluted 50. Mu.l streptavidin-HRP to each well for 30 minutes at room temperature; wash the plate 3 times with 200 μ l wash solution per well; add 50. Mu.l TMB to each well for 20 minutes at room temperature; adding a stop solution to stop the reaction; and reading an absorbance (OD) value by using an enzyme-labeling instrument, reading a 96-pore plate by using the enzyme-labeling instrument, measuring an OD value of 450nm, and calculating the contents of IFN-gamma and IL-2 in the sample by using the OD value according to a standard curve.
The results of the detection are shown in FIG. 7, which indicates that: 19CAR and DCAR-013-T are co-incubated with Raji cells, the amount of secreted IFN-gamma reaches more than 4000pg/mL, the content of IL-2 is more than 1000pg/mL, and the contents are far higher than those of other groups; 19CAR and DCAR-013-T were incubated with K562 cells and little IFN-. Gamma.and IL-2 were secreted. Shows that DCAR-013-T can recognize target cells and greatly improve the secretion of IFN-gamma and IL-2, and has almost no secretion of IFN-gamma and IL-2 for CD19 negative and CD20 negative K562.
3. Curative effect analysis of DCAR-013-T cell clinical treatment on large B cell lymphoma
A total of 18 patients with large B-cell lymphoma (from the Jiefu 301 hospital with informed consent) were all of the refractory relapsing type (treated by other methods with no significant effect), and 19 treatments of DCAR-013-T were completed.
The number of treatment sessions before group entry was 6-18, and 5 patients had been treated with autologous stem cell transplantation before treatment, CART was divided into three dose groups (see table 1), the first dose group: 4 patients, 2 (small-load patients) received CCR for 8-10 months, 2 (large-load patients) PD; a second dose group of 6 humans, 2 (low-load patients) receive a CCR of 6-7 months, 3 (high-load 2, super-load 1) receive PR,1 (super-load patients) PD; third dose group 3 patients achieved CR (wherein 1 heavy-loaded patient achieved PR by the second dose group, two CART treatments achieved CR), 2 patients with light load achieved CCR 6-7 months, 5 patients with heavy load achieved CCR 1-2 months for continued PR,1 patient with heavy load achieved PD.
Table 1 shows the therapeutic effect of DCAR-013-T cells on the clinical treatment of large B cell lymphoma
Note: CCR or CR was complete remission, PR was partial remission, PD was disease progression, SD was disease stabilization.
The above results indicate that the total remission (CR) rate of the patients with 18 accepted relapsing large B-cell lymphomas which are difficult to treat is 38.9% (7/18), the Partial Remission (PR) rate is 44.44% (8/18) and the total effective (CR + PR) rate is 83.33% by DCAR-013-T cell treatment.
Sequence listing
<110> Beihao stem cell and regenerative medicine research institute Co., ltd
<120> a method for treating tumor by chimeric antigen receptor T cell targeting CD19 and CD20 double antigens
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<170> PatentIn version 3.5
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gatgtctggg gcgcagggac cacggtcacc gtgagcgaag ctgccgcaaa agaggcagct 780
gccaaggaag cagccgctaa agaggtgaaa ctgcaggagt caggacctgg cctggtggcg 840
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gggggcgcag tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc 1680
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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Ile Val Leu Ser Gln Ser Pro Ala Ile Leu
20 25 30
Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser
35 40 45
Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro
50 55 60
Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser
85 90 95
Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ile
100 105 110
Ser Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly
115 120 125
Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Val
130 135 140
Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr
145 150 155 160
Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly
165 170 175
Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr
180 185 190
Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
195 200 205
Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
210 215 220
Val Tyr Tyr Cys Ala Arg Ala Gln Leu Arg Pro Asn Tyr Trp Tyr Phe
225 230 235 240
Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Glu Ala Ala Ala
245 250 255
Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Val Lys Leu Gln
260 265 270
Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr
275 280 285
Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile
290 295 300
Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly
305 310 315 320
Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile
325 330 335
Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu
340 345 350
Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr
355 360 365
Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
370 375 380
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
385 390 395 400
Gly Ser Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser
405 410 415
Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser
420 425 430
Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu
435 440 445
Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe
450 455 460
Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu
465 470 475 480
Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu
485 490 495
Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Thr Thr Thr
500 505 510
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
515 520 525
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
530 535 540
His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
545 550 555 560
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
565 570 575
Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
580 585 590
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
595 600 605
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
610 615 620
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu
625 630 635 640
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
645 650 655
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
660 665 670
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
675 680 685
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
690 695 700
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
705 710 715 720
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
725 730
Claims (20)
1. A chimeric antigen receptor, which consists of a signal peptide, a double antigen recognition region, a hinge region, a transmembrane region and an intracellular signal structural domain in sequence;
the double antigen recognition region is composed of a CD20 single-chain antibody and a CD19 single-chain antibody, and the CD19 single-chain antibody is close to the transmembrane region; the CD20 single-chain antibody consists of a light chain variable region of the CD20 single-chain antibody and a heavy chain variable region of the CD20 single-chain antibody; the CD19 single-chain antibody consists of a light chain variable region of the CD19 single-chain antibody and a heavy chain variable region of the CD19 single-chain antibody; wherein, the light chain variable region of the CD20 single-chain antibody, the heavy chain variable region of the CD20 single-chain antibody, the light chain variable region of the CD19 single-chain antibody and the heavy chain variable region of the CD19 single-chain antibody are in a positional sequence relationship among the light chain variable region of the CD20 single-chain antibody, the heavy chain variable region of the CD19 single-chain antibody and the light chain variable region of the CD19 single-chain antibody;
the signal peptide is a signal peptide of CD8A, CSF1R, CSF2RA or CD 4;
the hinge region adopts a hinge region of Human CD8A, and the amino acid sequence of the hinge region is from 510 th to 554 th in the sequence 2;
the transmembrane region is a transmembrane region of Human CD8a, and the amino acid sequence of the transmembrane region is 555-578 th site of a sequence 2;
the intracellular signaling domain consists of, in order, an intracellular costimulatory molecule 41-BB and the signaling domain CD3 ζ (CD 3 z);
the amino acid sequence of the intracellular costimulatory molecule 41-BB is 579-620 of the sequence 2;
the amino acid sequence of the signal domain CD3 zeta (CD 3 z) is 621 th to 732 th in the sequence 2;
the heavy chain variable region of the CD20 single-chain antibody and the heavy chain variable region of the CD19 single-chain antibody are connected through a connecting region A;
the light chain variable region of the CD20 single-chain antibody and the heavy chain variable region of the CD20 single-chain antibody are connected through a connecting region B;
the light chain variable region of the CD19 single-chain antibody and the heavy chain variable region of the CD19 single-chain antibody are connected through a connecting region C;
the CD20 single-chain antibody light chain variable region consists of amino acid residues shown in 22 th to 127 th sites of a sequence 2 in a sequence table;
the heavy chain variable region of the CD20 single-chain antibody consists of amino acid residues shown in 133 th-252 th sites of a sequence 2 in a sequence table;
the CD19 single-chain antibody light chain variable region consists of amino acid residues shown in 403 th to 509 th of a sequence 2 in a sequence table;
the CD19 single-chain antibody heavy chain variable region consists of amino acid residues shown in 268-387 of a sequence 2 in a sequence table;
the amino acid sequence of the connecting region A is 253 th to 267 th positions of the sequence 2;
the amino acid sequence of the connecting region B is 128 th to 132 th of the sequence 2;
the amino acid sequence of the connecting region C is 388-402 th of the sequence 2.
2. The chimeric antigen receptor according to claim 1, wherein:
the chimeric antigen receptor consists of amino acid residues shown in a sequence 2 in a sequence table.
3. A nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 1-2.
4. A recombinant vector comprising the nucleic acid molecule of claim 3.
5. The recombinant vector according to claim 4, characterized in that: the recombinant vector is a lentiviral vector obtained by introducing the nucleic acid molecule of claim 3 into a lentiviral vector to express the chimeric antigen receptor of any one of claims 1-2.
6. An expression cassette comprising the nucleic acid molecule of claim 3.
7. A recombinant bacterium comprising the nucleic acid molecule of claim 3.
8. A recombinant virus comprising the nucleic acid molecule of claim 3.
9. The recombinant virus of claim 8, wherein: the recombinant virus is obtained by introducing the recombinant vector of claim 4 or 5 into a host cell and packaging.
10. A cell comprising the nucleic acid molecule of claim 3.
11. The cell of claim 10, wherein: the cell is an immune cell.
12. The cell of claim 11, wherein: the immune cell is a T cell or an NK cell.
13. The cell of claim 12, wherein: the cell is a T cell which expresses the chimeric antigen receptor and is obtained by transfecting the recombinant vector of claim 4 or 5 into the T cell through a lentivirus.
14. A product comprising the chimeric antigen receptor of any one of claims 1-2, the nucleic acid molecule of claim 3, the recombinant vector of claim 4 or 5, the expression cassette of claim 6, the recombinant bacterium of claim 7, the recombinant virus of claim 8 or 9, or the cell of any one of claims 10-13;
the product is a medicament or a kit or an autologous transfusion preparation.
15. Use of the chimeric antigen receptor of any one of claims 1 to 2, the nucleic acid molecule of claim 3, the recombinant vector of claim 4 or 5, the expression cassette of claim 6, the recombinant bacterium of claim 7, the recombinant virus of claim 8 or 9, or the cell of any one of claims 10 to 13 for the preparation of a product for the prevention and/or treatment of a tumor; the tumor is a tumor which is positive for both CD19 and CD 20.
16. Use according to claim 15, characterized in that:
the tumor is derived from any one or a combination of a plurality of leukemia, lymphoma and myeloma.
17. Use according to claim 16, characterized in that: the lymphoma is large B-cell lymphoma.
18. Use according to claim 16, characterized in that: the lymphoma is non-Hodgkin lymphoma or Hodgkin lymphoma.
19. Use according to claim 15, characterized in that: the tumor is a hematological tumor of B lymphocyte.
20. Use of the chimeric antigen receptor of any one of claims 1 to 2, the nucleic acid molecule of claim 3, the recombinant vector of claim 4 or 5, the expression cassette of claim 6, the recombinant bacterium of claim 7, the recombinant virus of claim 8 or 9, or the cell of any one of claims 10 to 13 for the preparation of a product for killing tumor cells; the tumor cells are tumor cells with double positive CD19 and CD 20.
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| CN110981971B (en) * | 2019-12-25 | 2022-12-16 | 华夏源（上海）细胞基因工程股份有限公司 | Double-target chimeric antigen receptor targeting CD19 and CD20, expression vector and application thereof |
| CN113402612A (en) | 2020-03-17 | 2021-09-17 | 西比曼生物科技（香港）有限公司 | Combined chimeric antigen receptor targeting CD19 and CD20 and application thereof |
| CN113549155B (en) * | 2020-04-23 | 2023-03-31 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Chimeric antigen receptor for simultaneously targeting CD19 and CD20 and application thereof |
| WO2022007802A1 (en) * | 2020-07-07 | 2022-01-13 | 深圳市菲鹏生物治疗股份有限公司 | T lymphocyte and application thereof |
| CN111848822B (en) * | 2020-07-31 | 2022-02-15 | 广东昭泰体内生物医药科技有限公司 | CD19 and CD30 double-target chimeric antigen receptor and application thereof |
| CN112794916B (en) * | 2021-04-08 | 2021-08-10 | 正大天晴药业集团南京顺欣制药有限公司 | Trispecific antigen binding construct, construction method and application |
| CN113684184A (en) * | 2021-06-21 | 2021-11-23 | 北京大学 | Method for preparing chimeric antigen receptor NK (natural killer) cells of targeted CD19 from human pluripotent stem cells and application of method |
| CN114478803B (en) * | 2022-02-11 | 2023-09-26 | 北京大学深圳研究生院 | Construction and application of novel bispecific chimeric antigen receptor |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105384825A (en) * | 2015-08-11 | 2016-03-09 | 南京传奇生物科技有限公司 | Bispecific chimeric antigen receptor based on variable domains of heavy chain of heavy-chain antibody and application thereof |
| CN106755109A (en) * | 2016-12-05 | 2017-05-31 | 刘晓明 | A kind of slow virus carrier for expressing people's CD19 and CD20 Chimeric antigen receptor gene |
| CA3040343A1 (en) * | 2016-10-19 | 2018-04-26 | California Institute For Biomedical Research | Chimeric antigen receptor effector cell switches with humanized targeting moieties and/or optimized chimeric antigen receptor interacting domains and uses thereof |
-
2018
- 2018-06-15 CN CN201810618266.0A patent/CN110606893B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105384825A (en) * | 2015-08-11 | 2016-03-09 | 南京传奇生物科技有限公司 | Bispecific chimeric antigen receptor based on variable domains of heavy chain of heavy-chain antibody and application thereof |
| CA3040343A1 (en) * | 2016-10-19 | 2018-04-26 | California Institute For Biomedical Research | Chimeric antigen receptor effector cell switches with humanized targeting moieties and/or optimized chimeric antigen receptor interacting domains and uses thereof |
| CN106755109A (en) * | 2016-12-05 | 2017-05-31 | 刘晓明 | A kind of slow virus carrier for expressing people's CD19 and CD20 Chimeric antigen receptor gene |
Non-Patent Citations (2)
| Title |
|---|
| "T Cells Expressing CD19/CD20 Bispecific Chimeric Antigen Receptors Prevent Antigen Escape by Malignant B Cells";Eugenia Zah等;《Cancer Immunol Res》;20160408;第4卷(第6期);第499页左栏第2段、第500页左栏第2段、第500页左栏最后一段、第500页右栏倒数第2段、504页右栏第2段、506页右栏第5段和图1-3 * |
| "利用嵌合抗原受体的T细胞（CAR-T）治疗复发和难治B细胞淋巴瘤/白血病的转化医学研究";袁顺宗等;《中国实验血液学杂志》;20140813;第22卷(第4期);第1137-1141页 * |
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