CN110606893A - 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 PDF

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CN110606893A
CN110606893A CN201810618266.0A CN201810618266A CN110606893A CN 110606893 A CN110606893 A CN 110606893A CN 201810618266 A CN201810618266 A CN 201810618266A CN 110606893 A CN110606893 A CN 110606893A
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CN110606893B (en
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邓宏魁
高君双
周士新
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Peking University
Beihao Stem Cell and Regenerative Medicine Research Institute Co Ltd
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Beihao Stem Cell and Regenerative Medicine Research Institute Co Ltd
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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 provided by the invention, which 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 close 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 hematological tumor and lymphoma patients, CD19 and CD20 are both negative tumor cells are few, and the double-target CAR aiming at CD19 and CD20 shows good in vitro killing and clinical treatment effects, so that the superiority of the double-target design is reflected.

Description

Method for treating tumor by chimeric antigen receptor T cell targeting CD19 and CD20 double antigens
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 genetic engineering method to endow T cells with the ability of recognizing tumor cell surface antigen 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 the intracellular segment of CD3 ζ (CD3 z). The no co-stimulatory molecule is a first generation CAR, contains 1 co-stimulatory molecule (CD28, 4-1BB, OX40, ICOS, etc.) as a second generation CAR, and contains 2 co-stimulatory 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 close to the transmembrane region.
In the chimeric antigen receptor, the CD20 single-chain antibody comprises the light chain variable region of the CD20 single-chain antibody and the heavy chain variable region of the CD20 single-chain antibody;
the CD19 single-chain antibody comprises a light chain variable region of a CD19 single-chain antibody and a heavy chain variable region of a CD19 single-chain antibody;
wherein, the positional sequence relationship 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 is (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 CD20 single-chain antibody, the variable region of the heavy chain of the CD19 single-chain antibody, 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 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 by 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 linked through the linking 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 linked by a linking 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 linked by a linking region.
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 the 133 nd-252 th site of the sequence 2 in the 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 the 403-509 th sequence of the sequence 2 in the 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 variable region of the CD19 single-chain antibody heavy chain is g or k or l as follows:
g) which 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 the 253-267 position of the sequence 2
The amino acid sequence of the connecting region B is the 128 th-132 th position of the sequence 2
The amino acid sequence of the connecting region C is the 388-402 th position 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 part of 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 signal 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 (CD3z) intracellular segment;
the intracellular signaling domain specifically includes intracellular costimulatory molecule 41-BB and signaling domain CD3 ζ;
the non-costimulatory molecule is a first generation CAR, contains 1 costimulatory molecule (CD28, 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 4-1 BB.
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) a peptide chain which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence limited by 1) and has the same function;
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 which expresses the chimeric antigen receptor and is obtained by transfecting the recombinant vector into the T cell by the lentivirus.
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 nucleic acid molecule or the recombinant vector, the expression cassette, the recombinant bacterium and the recombinant virus are applied to preparing T cells with increased IFN-gamma and/or IL-2 secretion under the induction of tumor cells.
The tumor is a tumor which is positive for both CD19 and CD 20;
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 cells are tumor cells which are positive for both CD19 and CD 20;
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 is then returned to cell therapy preparations 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. It is characterized by the outside scFv of CD20 and the inside scFv of CD19 (the side near the cell membrane). DCAR-013-T cells can recognize two antigens, CD19 or CD20, and either CD19 or CD20 of tumor cells can be recognized by the DCAR-013-T cells and killed directly.
A single CD19-CAR or CD20-CAR can only recognize and kill a single antigen of tumor cells, e.g., CD19-CAR can kill CD19 positive tumor cells, possibly leaving a few CD19 negative CD20 positive tumor cells that can rapidly proliferate, causing tumor recurrence or poor therapeutic efficacy.
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 hematological tumor and lymphoma patients, CD19 and CD20 are both negative tumor cells are few, and the double-target CAR aiming at CD19 and CD20 shows good in vitro killing and clinical treatment effects, so that the superiority of the double-target design is reflected.
From the spatial structures of CD19 and CD20, CD19 is a single transmembrane protein, an 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, and aiming at the structure, a long-short combined double-target CAR is designed, CD20-scFV recognizes a short CD20 extracellular region at the outer side, and CD19-scFV recognizes a 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 CD20 and CD19 is characterized by the fact that the Linker region (Linker) is flanked by 20-scFV and 19-scFV heavy chain variable regions, respectively, or both light chain variable regions, i.e., arranged as 20-VL-VH and 19-VH-VL, or 20-VH-VL and 19-VL-VH; the arrangement of the heavy chain variable region or the light chain variable region with 20 and 19 at two sides of the connecting region (Linker) is characterized in that the heavy chain variable region (or the light chain variable region) of 20 and the light chain variable region (or the heavy chain variable region) of 19 are prevented from forming a new single chain antibody structure, and the mismatched structure can cause non-specific recognition and killing of autologous cells.
The DCAR-013-T cells infected with the lentivirus used in the invention had no effect on the phenotype of CD4 and CD8-T cells of the patients, and the percentage of memory T cells (CD45RO + and CD62L +) was slightly increased (8%); tumor cells that are either CD19+ or CD20+ or double positive can be killed specifically in vitro, while there is no killing effect on CD 19-negative and CD 20-negative cells. The Complete Remission (CR) rate of 18 patients with relapsing refractory large B cell lymphoma is 38.9 percent (7/18), the Partial Remission (PR) rate is 44.44 percent (8/18), the total effective (CR + PR) rate is 83.33 percent, 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 of uninfected T cells and lentivirus-infected day 3 DCAR-013-T cells, CD4 and CD 8.
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 CD45 RO.
FIG. 4 is a phenotypic characterization of patient uninfected lentiviral T cells.
FIG. 5 shows the positive rate of human T cells for DCAR-013 lentivirus infection.
FIG. 6 shows the DCAR-013-T cell killing experiment in vitro.
FIG. 7 shows DCAR-013 factor release experiments.
FIG. 8 is a schematic diagram showing 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 a CD20 single-chain antibody, a connection region B, a heavy chain variable region of a CD20 single-chain antibody, a connection region A, a heavy chain variable region of a CD19 single-chain antibody, a connection region C and a light chain variable region of a CD19 single-chain antibody in sequence;
or the double antigen recognition region consists of a heavy chain variable region of a CD20 single-chain antibody, a connecting region B, a light chain variable region of a CD20 single-chain antibody, a connecting region A, a light chain variable region of a CD19 single-chain antibody, a connecting region C and a heavy chain variable region of a 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 in 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 aforementioned 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 the 128 th-position 132 of the sequence 2 in the sequence table, and the nucleotide sequence of the nucleic acid molecule for coding the connecting region ethylinker is the 382 nd-position 396 of the sequence 1;
the amino acid sequence of the heavy chain variable region (VH) of the anti-CD20scFv is the 133 th-252 th site of 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 the 397 th-756 th site of the sequence 1;
the amino acid sequence of the linker of the connecting region A is the 253 rd-267 th position of the sequence 2 in the sequence table, and the nucleotide sequence of the nucleic acid molecule of the connecting region A is the 757 th-801 th position of the sequence 1.
The amino acid sequence of the heavy chain variable region (VH) of the anti-CD19scFv is 268-387 of 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-CD19scFv is 802-1161 of the sequence 1;
the amino acid sequence of the connecting region linker is the 388 th-402 th site of the sequence 2 in the sequence table, and the nucleotide sequence of the nucleic acid molecule for coding the connecting region linker is the 1162 th-1206 th site of the sequence 1;
the amino acid sequence of the light chain variable region (VL) of the anti-CD19scFv is the 403-509 th site of the sequence 2 in the sequence table, and the nucleotide sequence of the nucleic acid molecule for encoding the light chain variable region of the anti-CD19scFv is the 1207-1527 th site of the sequence 1;
3. hinge region (Hinge)
The Hinge region (Hinge) is the Hinge region (Hinge) of Human CD8A, and the amino acid sequence of the Hinge region (Hinge) is the 510 th-554 th position of 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 at position 555-578 of the sequence 2.
The nucleic acid sequence corresponding to the transmembrane region is 1663-1734 of SEQ ID NO.
5. Intracellular signaling domains
The intracellular signaling domain consists of, in turn, intracellular costimulatory molecule 41-BB and signaling domain CD3 ζ (CD3 z).
The amino acid sequence of the intracellular costimulatory molecule 41-BB is the 579-620 site of the sequence 2; the nucleotide sequence is the sequence 1 position 1735-1860.
The amino acid sequence of the signal domain CD3 zeta (CD3z) is 621 th-732 th site of the sequence 2, and the nucleotide sequence is 1861 th-2196 th site of the sequence 1.
Secondly, preparation of a lentivirus vector expressing a 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-EF 1A-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.
Thirdly, packaging lentivirus 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 was cultured 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 (1mg/mL, Polyplus), Opti-MEM (Gibco)) and 10cm cell culture dishes (Corning);
packaging lentivirus particles comprising 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 confluence reaches 70-80% after 24 h.
2) Preparation of a pre-transfection mixture: preparing a transfection mixture (a mixture of a lentiviral vector DCAR-013 expressing 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 in 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; gently shaking the whole body, the whole body and the whole body, and culturing at 37 ℃ in a constant-temperature incubator containing 5% CO 2.
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 for 2 hours by using 75% alcohol, pouring out the alcohol, rinsing the inner sleeve and the metal outer tube for three times by using sterile water, and irradiating the inner sleeve and the metal outer tube with ultraviolet rays overnight in a biological safety cabinet;
3) mixing the filtrates obtained in step 1 and collecting virus supernatants, placing in a 50ml centrifuge tube, centrifuging at 2000rpm at room temperature for 10min, and centrifuging 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 150 min;
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 determination
Pancreatin digestion count HEK293T cells at 2X 105Perwell was inoculated into 6-well plates. The cells were incubated in a 37 ℃ 5% C02 incubator until 50% fusion. 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 cell culture medium of HEK293T 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 5% C02 incubator at 37 ℃. After 48H, 293T was trypsinized, the medium was neutralized, centrifuged at 350g for 4 minutes, the supernatant was discarded, the cells were washed 2 times with Phosphate Buffered Saline (PBS), the cells were dissolved in 50. mu.l of PBS, 1. mu.l of an antibody against rabbit anti-mouse IgG (H + L) F (ab')2-APC (Jackson Co., Ltd.) was added, after incubation at 4 ℃ for 30 minutes, centrifuged at 2000rpm for 4 minutes, the supernatant was discarded, the cells were washed with PBS, and then 350. mu.l of PBS was added to resuspend the cells, and the CAR-positive rate of 293T cells was examined by flow cytometry.
And (3) the virus titer (TU/mL) is CAR positive cell rate multiplied by total cell number/slow virus fluid volume (mL), in order to avoid errors, only holes with positive rate of 1-30% are selected for calculation, and the average value of each group is taken to calculate the titer.
The number of cells was 2X 105The 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 DCAR-013 lentivirus infected 293T cells was 0.15X 2X 105/0.001mL=3.0×107TU/mL。
Fourthly, obtaining CAR-T cells expressing chimeric antigen receptor DCAR-013
1. Diluting retroNectin to a working concentration, adding about 2mL of the retroNectin into a six-hole plate, placing the six-hole plate into a 5% CO2 incubator at 37 ℃, taking out the six-hole plate after 2 hours, sucking the retroNectin in holes, and recycling the retroNectin into 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 106Plating into 6-well plates;
the activated T cells were prepared as follows: mixing collected autologous peripheral blood of a patient with sterile PBS in equal volume, diluting the peripheral blood, meanwhile, adding lymphocyte separation liquid into a 50mL sterile centrifuge tube, carefully adding the diluted peripheral blood into the upper layer of the lymphocyte separation liquid according to the proportion of 1:1, 400g, and centrifuging for 20 minutes at room temperature; 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; decanting the supernatant, resuspending the leukocytes in PBS, and counting to obtain prepared peripheral blood mononuclear lymphocytes (PBMC); PBMC was added to 6-well plates, 2ml of PBMC-containing medium was added to each well, and 5X 10 cells were added6Each well was filled with 80. mu.l of Human T cell activating CD3/CD28 antibody (Gibco Dynabeads Human T-Activator CD3/CD28, 11131D), and cultured in an incubator at 37 ℃ with the CD3/CD28 antibody stimulating the cells for 24-48 hours to obtain 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. culturing in 5% CO2 incubator at 37 deg.C for 24 hr;
6. infecting lentivirus for the second time, and repeating the operation step 4;
7. after culturing in a 5% CO2 incubator at 37 ℃ for 24 hours, the cells were transferred to another vessel for further culturing.
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 lentivirus-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, and the results are shown in FIG. 2. FL4 is anti-CD4, FL2 is anti-CD8, and the positive rates of uninfected T cells CD4 and CD8 are 36.7% and 59.1% respectively; the positive rates of DCAR-013-T cell CD4 and CD8 were 35.8% and 59.8%, respectively, and there was no significant difference between the two.
Molecular phenotypic characterization of memory T cells: the cells with double positive CD62L and CD45RO are memory T cells. The lentivirus-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 rate 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) of DCAR-013-T cell group were slightly higher than those of the control group, and the difference between the two was 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.
Phenotypic characterization of T cells of the infected and uninfected chimeric antigen receptor DCAR-013
1. CAR-T cells expressing chimeric antigen receptor DCAR-013
Activated T cells were counted at 5X 106Perwell 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, centrifuging at 2000rpm at room temperature for 60min, and culturing in a 5% CO2 incubator at 37 deg.C for 24 hr; 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 of
The results of flow analysis of patient autologous T cells after activation in example 1 (antibody mouse anti-human CD3, BD Biosciences) are shown in fig. 4, and it can be seen that the phenotype of flow analysis of patient autologous T cells cultured in vitro on day 3 after activation with CD3 and CD28 antibodies: the positive rate of CD3 was 98.5%; the positive rates of 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%.
Second, DCAR-013-T cell killing experiment in vitro
Killing effect of DCAR-013-T cell on single expression CD19 and single expression CD20 tumor cell, and CD19 negative and CD20 negative blood tumor cell as K562 (human chronic myelogenous leukemia cell; K562: number 3111C0001CCC000039, cell center of basic medical research institute 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 killed CD 19-positive or CD 20-positive tumor cells but not negative K562 cells, CD19 and CD20 killed target cells with single-target CAR, and individual CAR19-T and CAR20-T killed target cells with higher killing rate than uninfected T cells (Normal T) but not K562 cells with CD19-CD 20-; compared with the single CAR19-T and CAR20-T, DCAR-013-T has a high killing rate on target cells, which can reach more than 80%.
Third, DCAR-013-T cell is induced by target cell to secrete cytokine gamma-interferon (IFN-gamma) and interleukin 2(IL-2) experiment
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 of the separated cells was collected in about 50. mu.l to 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 a method used by a Dake Dayou kit, and the method firstly uses IFN-gamma and IL-2 standard products and an 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-well plate by using the enzyme-labeling instrument, measuring an OD value of 450nm, referring to a standard curve by using the OD value, and calculating the contents of IFN-gamma and IL-2 in the sample.
The results are shown in FIG. 7, which shows 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 K562 which is negative for CD19 and CD 20.
Analysis of curative effect of DCAR-013-T cells on clinical treatment of large B cell lymphoma
A total of 18 patients with large B-cell lymphomas (from the hospital of the liberty military 301, 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 (light-loaded patients) received CCR for 8-10 months, 2 (heavy-loaded 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 observation of the curative 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 38.9% (7/18), Partial Remission (PR) rate of 44.44% (8/18), and total effective (CR + PR) rate of 83.33% in 18 patients with relapsing large B-cell lymphoma who were refractory to treatment with DCAR-013-T cells.
Sequence listing
<110> Beihao stem cell and regenerative medicine research institute Co., Ltd
<120> a method for treating tumor by using chimeric antigen receptor T cell targeting CD19 and CD20 double antigens
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 2196
<212> DNA
<213> Artificial sequence
<400> 1
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggacatcg tgctgtcgca gtctccagca atcctgtctg catctccagg ggagaaggtc 120
acaatgactt gcagggccag ctcaagtgta agttacatgc actggtacca gcagaagcca 180
ggatcctccc ccaaaccctg gatttatgcc acatccaacc tggcttctgg agtccctgct 240
cgcttcagtg gcagtgggtc tgggacctct tactctctca caatcagcag agtggaggct 300
gaagatgctg ccacttatta ctgccagcag tggattagta acccacccac gttcggtgct 360
gggaccaagc tggagctgaa gggaggtgga ggaagccagg ttcagctggt ccagtcaggg 420
gctgagctgg tgaagcctgg ggcctcagtg aagatgtcct gcaaggcttc tggctacaca 480
tttaccagtt acaatatgca ctgggtaaag cagacacctg gacagggcct ggaatggatt 540
ggagctattt atccaggaaa tggtgatact tcctacaatc agaagttcaa aggcaaggcc 600
acattgactg cagacaaatc ctccagcaca gcctacatgc agctcagcag cctgacatct 660
gaggactctg cggtctatta ctgtgcaaga gcgcaattac gacctaacta ctggtacttc 720
gatgtctggg gcgcagggac cacggtcacc gtgagcgaag ctgccgcaaa agaggcagct 780
gccaaggaag cagccgctaa agaggtgaaa ctgcaggagt caggacctgg cctggtggcg 840
ccctcacaga gcctgtccgt cacatgcact gtctcagggg tctcattacc cgactatggt 900
gtaagctgga ttcgccagcc tccacgaaag ggtctggagt ggctgggagt aatatggggt 960
agtgaaacca catactataa ttcagctctc aaatccagac tgaccatcat caaggacaac 1020
tccaagagcc aagttttctt aaaaatgaac agtctgcaaa ctgatgacac agccatttac 1080
tactgtgcca aacattatta ctacggtggt agctatgcta tggactactg gggccaagga 1140
acctcagtca ccgtctcctc aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 1200
ggatctgaca tccagatgac acagactaca tcctccctgt ctgcctctct gggagacaga 1260
gtcaccatca gttgcagggc aagtcaggac attagtaaat atttaaattg gtatcagcag 1320
aaaccagatg gaactgttaa actcctgatc taccatacat caagattaca ctcaggagtc 1380
ccatcaaggt tcagtggcag tgggtctgga acagattatt ctctcaccat tagcaacctg 1440
gagcaagaag atattgccac ttacttttgc caacagggta atacgcttcc gtacacgttc 1500
ggagggggga ccaagctgga gatcacaacc acgacgccag cgccgcgacc accaacaccg 1560
gcgcccacca tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg 1620
gggggcgcag tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc 1680
ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaaacgg 1740
ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 1800
caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 1860
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 1920
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 1980
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 2040
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 2100
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 2160
tacgacgccc ttcacatgca ggccctgccc cctcgc 2196
<210> 2
<211> 732
<212> PRT
<213> Artificial sequence
<400> 2
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 (10)

1. A chimeric antigen receptor comprising an extracellular recognition domain and an intracellular signaling domain, wherein the extracellular recognition domain comprises, in order, a signal peptide, a dual antigen recognition region, a hinge region, and a transmembrane region;
the double antigen recognition region comprises a CD20 single-chain antibody and a CD19 single-chain antibody, wherein the CD19 single-chain antibody is close to the transmembrane region.
2. The antigen receptor of claim 1, wherein:
the CD20 single-chain antibody comprises a light chain variable region of a CD20 single-chain antibody and a heavy chain variable region of a CD20 single-chain antibody;
the CD19 single-chain antibody comprises a light chain variable region of a CD19 single-chain antibody and a heavy chain variable region of a CD19 single-chain antibody;
wherein, the positional sequence relationship 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 is (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 CD20 single-chain antibody, the variable region of the heavy chain of the CD19 single-chain antibody, 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 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.
3. The antigen receptor of claim 2, wherein:
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; 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 linked through the linking 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 linked by a linking 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 linked by a linking region.
4. The chimeric antigen receptor according to claim 2 or 3, characterized in that:
the variable region of the CD20 single-chain antibody light chain is a or 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 the 133 nd-252 th site of the sequence 2 in the 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 the 403-509 th sequence of the sequence 2 in the 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 variable region of the CD19 single-chain antibody heavy chain is g or k or l as follows:
g) which 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 the 253-267 position of the sequence 2
The amino acid sequence of the connecting region B is the 128 th-132 th position of the sequence 2
The amino acid sequence of the connecting region C is the 388-402 th position of the sequence 2.
5. The chimeric antigen receptor according to any one of claims 1-4, wherein:
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 part of 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 signal 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 (CD3z) intracellular segment;
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;
and/or, 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) a peptide chain which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence limited by 1) and has the same function;
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.
6. A nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 1-5;
or, a recombinant vector, expression cassette, recombinant bacterium, recombinant virus or cell comprising said nucleic acid molecule.
7. The recombinant vector or cell of claim 6, wherein:
the recombinant vector is a lentiviral vector which is obtained by introducing the nucleic acid molecule into a lentiviral vector and expresses the chimeric antigen receptor of any one of claims 1-5;
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 which expresses the chimeric antigen receptor and is obtained by transfecting the recombinant vector into the T cell by the lentivirus.
8. A product comprising the chimeric antigen receptor of any one of claims 1-5, the nucleic acid molecule of claim 6, or the recombinant vector, expression cassette, recombinant bacterium, recombinant virus, or cell.
9. Use of the chimeric antigen receptor of any one of claims 1 to 5 or the nucleic acid molecule of claim 6 or the recombinant vector, expression cassette, recombinant bacterium, recombinant virus or cell for the preparation of a product for the prevention and/or treatment of a tumor;
or the chimeric antigen receptor of any one of claims 1 to 4 or the nucleic acid molecule of claim 6 or the recombinant vector, expression cassette, recombinant bacterium, recombinant virus or cell for use in the preparation of a product for killing tumor cells;
or the chimeric antigen receptor of any one of claims 1 to 4 or the nucleic acid molecule of claim 6 or the recombinant vector, expression cassette, recombinant bacterium or recombinant virus, for preparing a T cell with increased secretion of IFN- γ and/or IL-2 induced by a tumor cell.
10. The product according to claim 8 or the use according to claim 9, characterized in that:
the tumor is a tumor which is positive for both CD19 and CD 20;
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 cells are tumor cells which are positive for both CD19 and CD 20;
and/or the product is a medicament or a kit or an autologous transfusion preparation.
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