CN105177031A - Chimeric antigen receptor-modified T cells and uses thereof - Google Patents

Abstract

The present invention provides chimeric antigen receptor-modified T cells and uses thereof, wherein the chimeric antigen receptor contains a signal peptide, an extracellular binding region, an optional hinge region, a transmembrane region and an intracellular signal region, the signal peptide, the extracellular binding region, the optional hinge region, the transmembrane region and the intracellular signal region are sequentially connected, and the nucleotide sequence of the extracellular binding region is represented by SEQ ID No:4 or SEQ ID No:5.

Description

Chimeric antigen receptor modified T cells and uses thereof

·Technical field

The present invention relates to the fields of immunology and molecular biology, and more specifically relates to T cells modified by chimeric antigen receptors and uses thereof.

·Background technique

Most patients with B-cell malignancies, including Chronic Lymphocytic Leukemia (CLL), have a poor cure rate and prognosis with chemotherapy and targeted therapy. One approach to treating these patients is to genetically modify T cells to target antigens expressed on tumor cells through expression of Chimeric Antigen Receptor (CAR). CARs are antigen receptors designed to recognize cell surface antigens in a human leukocyte antigen-independent manner. Attempts to treat these types of patients with CAR-expressing genetically modified T cells have met with some success (Molecular Therapy, 2010, 18:4, 666-668; Blood, 2008, 112:2261-2271).

With the continuous development of Chimeric Antigen Receptor-T cell (CAR-T) technology, CAR-T can be divided into three generations.

The first generation of CAR-T cells consists of an extracellular binding region—single-chain fragment variable (scF _V ), a transmembrane region (transmembrane region, TM) and an intracellular signaling region—the immunoreceptor tyrosine activation motif. (immunoreceptortyrosine-based activationmotif, ITAM), wherein each part of the chimeric antigen receptor is connected in the following form: scFv-TM-CD3ζ. This kind of CAR-T cells can stimulate anti-tumor cytotoxic effects, but the secretion of cytokines is relatively small, and cannot stimulate long-lasting anti-tumor effects in vivo [ZhangT.et.al. ):11029-11036.].

The second-generation CAR-T cells developed subsequently added the intracellular signaling region of CD28 or CD137 (also known as 4-1BB), in which the parts of the chimeric antigen receptor were connected in the following form: scFv-TM-CD28-ITAM or scFv -TM-CD137-ITAM. The B7/CD28 or 4-1BBL/CD137 co-stimulation in the intracellular signal area can cause the continuous proliferation of T cells, and can increase the level of cytokines such as IL-2 and IFN-γ secreted by T cells, and at the same time increase the level of CAR-T in In vivo survival cycle and anti-tumor effect [DottiG.et.al.CD28costimulationimprovesexpansionandpersistenceofchimericantigenreceptormodifiedTcellsinlymphomapatients.JClinInvest,2011,121(5):1822-1826.].

In the third-generation CAR-T cells developed in recent years, the parts of the chimeric antigen receptor are connected in the following form: scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, which further improves the CAR -T survival cycle in vivo and its anti-tumor effect [Carpenito C., et al.

Although CAR-T cells have attractive prospects in tumor immunotherapy, some potential risks also need to be considered. For example, due to the low expression of specific antigens recognized by CAR in some normal tissues, CAR-T cells may damage normal tissues expressing corresponding antigens. In addition, excessive co-stimulatory signals in CAR will reduce the threshold required for effector cell activation, so that genetically modified T cells may also be activated under low-level or no antigen-triggered conditions, resulting in the release of a large number of cytokines so that A so-called "cytokine storm" can be triggered. This signal leakage (signalleakage) can lead to off-target cytotoxicity, resulting in non-specific tissue damage.

Compositions and methods for the treatment of human cancer are currently disclosed in Patent Application No. 201180067173.X, which involves the administration of genetically modified T cells to express a CAR, wherein the CAR includes an antigen binding region (i.e., an extracellular binding region), Transmembrane region, co-stimulatory signaling region and CD3ζ signaling region. However, the antigen-binding region scFv on the surface of the CAR-T cells is non-humanized (derived from mice), which makes the CAR-T cells themselves immunogenic.

In summary, there is a strong demand in the field for CAR-T cells that overcome the above defects.

·Invention content

A first aspect of the present invention provides a nucleic acid molecule encoding a chimeric antigen receptor expressed on the surface of T cells, said chimeric antigen receptor comprising a signal peptide connected in sequence, an extracellular binding region, an optional hinge region (hinger region) ), a transmembrane region and an intracellular signal region (signalregion), wherein the nucleotide sequence of the extracellular binding region is shown in SEQ ID No: 4 or SEQ ID No: 5.

The second aspect of the present invention provides a vector comprising the nucleic acid molecule of the chimeric antigen receptor expressed on the surface of T cells according to the first aspect of the present invention.

The third aspect of the present invention provides a virus comprising the vector of the second aspect of the present invention, which includes the packaged infectious virus, and also includes the packaged virus to be packaged as an essential component of the infectious virus.

The fourth aspect of the present invention provides a genetically modified T cell comprising the nucleic acid sequence of the first aspect of the present invention or the vector of the second aspect of the present invention or the virus of the third aspect of the present invention.

A fifth aspect of the present invention provides a medicament comprising the T cell of the fourth aspect of the present invention.

The sixth aspect of the present invention provides the use of the T cell of the fourth aspect of the present invention for preparing a drug for treating tumors.

The antigen-binding region scFv on the surface of the CAR-T cells of the present invention is humanized, so its immunogenicity is greatly reduced.

·Description of drawings

The above and other aspects, features and advantages of the present invention will become more apparent from the following detailed description with reference to the accompanying drawings.

Figure 1 shows the detection results of T cells expressing chimeric antigen receptor modification.

Figure 2 shows the results of labeling CAR-T19 cells with the carbocyanine dye Did.

Figure 3 shows the results of labeling Daudi cells with the carbocyanine dye Did.

Figure 4 shows the results of detecting the killing effect of CAR-T19 cells on Daudi cells by flow cytometry.

Figure 5 shows the comparison results of the killing effect of T cells and CAR-T19 cells on the target cell Daudi.

Figure 6 shows the comparative results of the killing effect of CAR-T19 containing different scFv regions on the target cell Daudi.

Figure 7 shows the results of in vitro detection of the killing effect of different CAR-T19 on cells from patients with B lymphocytic leukemia.

·Detailed ways

As mentioned above, the first aspect of the present invention provides nucleic acid molecules encoding chimeric antigen receptors expressed on the surface of T cells, said chimeric antigen receptors comprising sequentially linked signal peptides, extracellular binding regions, optional hinge region, transmembrane region and intracellular signal region, wherein the nucleotide sequence of the extracellular binding region is shown in SEQ ID No:4 or SEQ ID No:5.

The term "nucleic acid" of the present invention may be in the form of DNA or in the form of RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The present invention also relates to variants of said nucleic acid, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention. Variants of the nucleic acid may be naturally occurring allelic variants or non-naturally occurring variants. These nucleic acid variants include substitution variants, deletion variants and insertion variants. As is well known to those of ordinary skill in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides without substantially altering its coding the function of the polypeptide.

In one embodiment, the signal peptide is a GMCSF signal peptide, the nucleotide sequence of which is shown in SEQ ID No: 1

In one embodiment, the signal peptide is a CD8 signal peptide, the nucleotide sequence of which is shown in SEQ ID No:2.

In one embodiment, the hinge region can be selected from the hinge regions of proteins such as CD8 or CD28. The CD8 or CD28 is a natural marker on the surface of T cells.

In a specific embodiment, the hinge region is a CD8 hinge region (CD8-hinge), and its nucleotide sequence is as shown in SEQ ID No:6.

In a specific embodiment, the hinge region is a CD28 hinge region (CD28-hinge), the nucleotide sequence of which is shown in SEQ ID No:7.

In one embodiment, the transmembrane region can be selected from transmembrane regions of proteins such as CD8 or CD28.

In a specific embodiment, the transmembrane region is CD8 transmembrane region (CD8-TM), the nucleotide sequence of which is shown in SEQ ID No:8.

In a specific embodiment, the transmembrane region is CD28 transmembrane region (CD28-TM), the nucleotide sequence of which is shown in SEQ ID No:9.

The "extracellular binding region" comprises scFv (anti-CD19 scFv) that specifically recognizes human CD19 epitope.

The term "scFv" as used herein refers to an antibody fragment which is a recombinant protein comprising a heavy chain variable region (variable region of heavy chain, VH) and a light chain variable region (variable region of light chain, VL) connected by a linker, A linker brings these two domains into association to ultimately form the antigen binding site. The size of scFv is generally 1/6 of a whole antibody. A scFv is preferably an amino acid sequence encoded by one chain of nucleotides. The scFv used in the present invention can be further modified by conventional techniques known in the art alone or in combination, such as amino acid deletion, insertion, substitution, addition, and/or recombination and/or other modification methods. Methods for introducing such modifications into the DNA sequence of an antibody based on its amino acid sequence are well known to those skilled in the art (see, eg, Sambrook Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.). Said modifications are preferably carried out at the nucleic acid level. The aforementioned scFv may also include derivatives thereof.

The term "specific recognition" as used herein means that the bispecific antibody of the present invention does not cross-react or substantially does not cross-react with any polypeptide other than the target antigen. The degree of its specificity can be judged by immunological techniques, including but not limited to western blotting, immunoaffinity chromatography, flow cytometry and the like.

In one embodiment, the intracellular signaling region may be selected from intracellular signaling regions of CD3ζ, FcεRIγ, CD28, CD137, CD134 proteins, and combinations thereof. The CD3 molecule is composed of five subunits, among which the CD3ζ subunit (also known as CD3zeta, referred to as Z) contains three ITAM motifs, which are important signal conversion regions in the TCR-CD3 complex. CD3δZ is a mutated CD3ζ sequence without an ITAM motif, which is generally used as a construction component of a negative control in the embodiments of the present invention. FcεRIγ is mainly distributed on the surface of mast cells and basophils, and it contains an ITAM motif, which is similar to CD3ζ in structure, distribution and function. In addition, as mentioned above, CD28, CD137, and CD134 are co-stimulatory signal molecules. After binding with their respective ligands, the co-stimulatory effect produced by their intracellular signal segments causes the continuous proliferation of T cells and can increase the secretion of IL- 2 and IFN-γ and other cytokines, while improving the survival cycle and anti-tumor effect of CAR-T cells in vivo.

There are various combinations of intracellular signal regions used in the present invention, including signal regions or combinations thereof selected from the following:

CD28 signal region (CD28-signal), the nucleotide sequence of which is shown in SEQ ID No: 10;

CD137 signal region (CD137-signal), its nucleotide sequence is shown in SEQIDNo:11; And

CD3ζ signal region (CD3ζ-signal), its nucleotide sequence is shown in SEQ ID No: 12.

In a specific embodiment, the chimeric antigen receptor protein expressed on the surface of T cells of the present invention is selected from the group consisting of a signal peptide, an extracellular binding region, an optional hinge region, a transmembrane region and a cellular Chimeric Antigen Receptor Proteins in the Inner Signaling Domain:

GMCSF-scFv-S1-CD8-hinge-CD8-TM-CD137-signal-CD3ζ-signal, its nucleic acid sequence is shown in SEQIDNo:13;

GMCSF-scFv-S2-CD8-hinge-CD8-TM-CD137-signal-CD3ζ-signal, its nucleic acid sequence is shown in SEQIDNo:14;

GMCSF-scFv-S1-CD28-TM-CD28-signal-CD3ζ-signal, its nucleic acid sequence is shown in SEQIDNo:15;

GMCSF-scFv-S2-CD28-TM-CD28-signal-CD3ζ-signal, its nucleic acid sequence is shown in SEQIDNo:16;

GMCSF-scFv-S1-CD8-hinge-CD8-TM-CD28-signal-CD137-signal-CD3ζ-signal, its nucleic acid sequence is shown in SEQIDNo:17;

GMCSF-scFv-S2-CD8-hinge-CD8-TM-CD28-signal-CD137-signal-CD3ζ-signal, its nucleic acid sequence is shown in SEQIDNo:18;

As described above, the second aspect of the present invention provides a vector comprising the nucleic acid molecule of the chimeric antigen receptor expressed on the surface of T cells according to the first aspect of the present invention.

In a specific embodiment, the vector used in the present invention is a lentiviral plasmid vector pLenti-CMV-eGFP. The plasmid belongs to the third-generation self-inactivating lentiviral vector system. There are three plasmids in this system, namely the packaging plasmid psPAX2 encoding the protein Gag/Pol and the Rev protein; the envelope plasmid pVSVG encoding the VSV-G protein; and the empty vector pLenti - CMV-eGFP, which can be used for recombination to introduce the nucleic acid sequence of interest, that is, the nucleic acid sequence encoding CAR. The expression of enhanced green fluorescent protein (eGFP) was regulated by the elongation factor-1α (elongationfactor-1α, EF-1α) promoter in the empty vector pLenti-CMV-eGFP (itself a mock in subsequent experiments). The recombinant expression vector pLenti-CMV-eGFP containing the target nucleic acid sequence encoding CAR achieves co-expression of eGFP and CAR through ribosomal skipping sequence 2A (F2A for short) from food and mouth disease virus (food and mouth virus disease, FMDV).

In another embodiment, the vector used in the present invention is a retroviral vector pMSCV-Ubc-GFP, and the plasmid is used to overexpress the nucleic acid sequence of interest, that is, the nucleic acid sequence encoding CAR.

As mentioned above, the third aspect of the present invention provides a virus comprising the vector of the second aspect of the present invention, which includes the packaged virus with infectious ability, and also includes the packaged virus to be packaged as an essential component of the infectious virus. Virus.

In one embodiment, the virus is a lentivirus comprising the pLenti-CMV-eGFP-F2A-CAR recombinant vector of the second aspect of the present invention. In another embodiment, the virus is a retrovirus comprising the pMSCV-Ubc-GFP-CAR recombinant vector of the second aspect of the present invention. It should be understood that other viruses known in the art to transfect T cells and their corresponding plasmid vectors can also be used in the present invention.

As mentioned above, the fourth aspect of the present invention provides genetically modified T cells transformed with the nucleic acid sequence of the first aspect of the present invention or the vector of the second aspect of the present invention or the virus of the third aspect of the present invention.

Conventional nucleic acid transformation methods in the art, including non-viral and viral transformation methods, can be used in the present invention. Non-viral based transformation methods include electroporation and transposon methods. Recently, the nucleofector nucleofector developed by Amaxa can directly introduce exogenous genes into the nucleus to obtain efficient transformation of target genes. In addition, the transformation efficiency of transposon systems based on Sleeping Beauty system or PiggyBac transposon has been greatly improved compared with ordinary electroporation. The combined application of nucleofector transfection instrument and SB Sleeping Beauty transposon system has been Reported [DaviesJK., etal.CombiningCD19redirectionandalloanergizationtogeneratetumor-specifichumanTcellsforallogeneiccelltherapyofB-cellmalignancies.CancerRes,2010,70(10):OF1-10.], this method can not only have a higher transformation efficiency but also can achieve site-specific integration of the target gene.

In one embodiment of the present invention, the method for transforming T lymphocytes with chimeric antigen receptor gene modification is based on viruses such as retroviruses or lentiviruses. The method has the advantages of high transformation efficiency, stable expression of exogenous genes, and can shorten the time for culturing T lymphocytes in vitro to reach the clinical level. On the surface of the transgenic T lymphocytes, the transformed nucleic acid is expressed through transcription and translation. In vitro cytotoxicity experiments on various cultured tumor cells prove that the transgenic T lymphocytes expressing chimeric antigen receptors on the surface of the present invention have highly specific tumor cell killing effect (also known as cytotoxicity). Therefore, the nucleic acid molecule encoding the chimeric antigen receptor, the plasmid comprising the nucleic acid, the virus comprising the plasmid, and the transgenic T lymphocytes transformed with the nucleic acid, plasmid or virus of the present invention can all be effectively used for immunotherapy of tumors.

As mentioned above, the fifth aspect of the present invention provides a medicament comprising the T cell of the fourth aspect of the present invention.

In one embodiment, the medicament further includes pharmaceutically acceptable diluents, excipients or carriers and the like.

As mentioned above, the sixth aspect of the present invention provides the use of the T cells of the fourth aspect of the present invention for preparing a drug for treating tumors.

The content of the present invention is illustrated below through specific examples. It should be understood that the specific examples are for illustrative purposes only, and do not mean that the content of the present invention is limited to the specific examples.

Embodiment 1: Preparation of the nucleotide sequence of the CAR molecule

The CAR molecule of the present invention comprises a signal peptide, an extracellular binding region, an optional hinge region, a transmembrane region and an intracellular signal region. The nucleotide sequence of the extracellular binding region is in the antigen binding region of the anti-CD19 chimeric antigen receptor (named here as anti-CD19scFv-S0, referred to as scFv-S0, which is derived from mice, see JImmunother .2009September; 32(7):689-702.) was obtained on the basis of the nucleotide sequence (SEQ ID No: 3) through humanized transformation. It should be understood that the principle of antibody humanization is to change the framework region (framework region, FM) to a human sequence to the greatest extent while ensuring the affinity of the antibody, so as to reduce the immunogenicity of the antibody. In this example, the antigen recognition region in SEQIDNo:3 was kept unchanged, and the rest of the sequences were changed accordingly, more than 40 kinds of humanized designs were carried out, and these sequences were synthesized by gene synthesis method, The other parts of the CAR molecule are cloned from the human cDNA library using PCR technology, and then bridged and connected to finally prepare the nucleotide sequence of the CAR molecule. These CAR molecules were transferred into T cells, and the T cells containing these CAR molecules containing these humanized sequences were compared with the T cells containing the CAR molecules containing scFv-S0 on the killing ability of target cells, and two final screenings were obtained. A humanized modified extracellular binding region sequence - the killing rate of CAR-T19 containing these two humanized modified sequences is slightly better than that of CAR-T19 containing scFv-S0 or has no difference ( See Figure 6 in Example 4), which are respectively called anti-CD19scFv-S1 (abbreviated as scFv-S1, whose nucleotide sequence is shown in SEQ ID No: 4) and anti-CD19scFv-S2 (abbreviated as scFv-S2 , whose nucleotide sequence is shown in SEQ ID No: 5). Taking SEQIDNo: 15 and SEQIDNo: 16 as examples, the steps for preparing the nucleotide sequence of the CAR molecule will be described in detail below.

First carry out primer design, the primer sequence used in the present embodiment is as follows:

1-1: 5'-ATGCTTCTCCTGGTGACAAG-3'

1-2: 5'-TGGGATCAGGAGGAATGCTG-3'

2-1: 5'-TACATCTGGGCGCCCTTGGCCGG-3'

2-2: 5'-GGAGCGATAGGCTGCGAAGTCGCG-3'

3-1: 5'-AGAGTGAAGTTCAGCAGGAGCG-3'

3-2: 5'-TTAGCGAGGGGGCAGGGCCT-3'

4-1: 5'-ATGCTTCTCCTGGTGACAAGCC-3'

4-2: 5'-TGAGGAGACGGTGACTGAGGTTCCTTGG-3'

5-1: 5'-GCGGCCGCAATTGAAGTTATGTA-3'

5-2: 5'-TTAGCGAGGGGGCAGGGCCTGC-3'

6-1: 5'-CTAGACTAGTATGCTTCTCCTGGTGACAAGCC-3'

6-2: 5'-CGACGCGTTTAGCGAGGGGGCAGGGCCTGC-3'

Using the human cDNA library as a template, using 1-1 and 1-2, 2-1 and 2-2, 3-1 and 3-2 as primers, the corresponding CAR molecular parts were cloned by PCR, respectively GMCSF, CD28-TM+CD28-signal (the two parts are connected) and CD3ζ-signal. The GMCSF+scFv fragment was obtained by bridging primers 4-1 and 4-2, and the CD28-TM+CD28-signal+CD3ζ-signal fragment was obtained by bridging primers 5-1 and 5-2, followed by bridging primers 6-1 and 6 -2 Obtain the nucleotide sequence of the complete CAR molecule, and the enzyme cutting sites are SpeI and MluI.

Embodiment 2: the construction of the viral vector comprising the nucleic acid sequence of CAR molecule

The nucleotide sequence of the CAR molecule prepared in Example 1 was double-digested with SpeI (Fermentas) and MluI (Fermentas), ligated with T4 ligase (Fermentas) and inserted into the SpeI-MluI site of the lentiviral pLenti-CMV-eGFP vector point, transformed into competent E.coli (DH5α), and after the sequence was correct, the plasmid was extracted and purified using Qrigene’s plasmid purification kit for subsequent lentiviral packaging experiments.

Example 3: Construction of CAR-T cells

In this example, the recombinant viral vector prepared in Example 2 was first packaged into a virus, and then the virus was infected into T cells to achieve chimeric antigen receptor modified T cells. The present invention employs transformation methods based on viruses such as retroviruses or lentiviruses. This method has the advantages of high transformation efficiency, stable expression of exogenous genes, and can shorten the time for culturing T cells in vitro to reach clinical-grade quantities. Specific steps are as follows:

Lentivirus packaging: Day 1: Inoculate 293T cells into a 10cm culture dish to ensure that the cell confluence is 80-90% during transfection on the second day; Day 2: Transfection, replace the medium of 293T 1 hour before transfection It is 5mL serum-free DMEM high-glucose medium; add 500μD DMEM to the EP tube, then add 4μgpR8.74, 400ngpVSVG, 4μgpLenti-CMV-eGFP-F2A-CAR and vortex for 8s to mix; add the plasmid (pR8.74+pLenti- CMV-eGFP-F2A-CAR) 3 times the volume of PEI (1 μg/μL, a total of 25.2 μL), vortexed for 20 seconds to mix, and left to stand at room temperature for 15 minutes; the mixed solution was added dropwise to 293T cells; transfected for 6 hours Afterwards, the medium was replaced with full medium containing 10% FBS. After 48 hours of transfection, collect the cell culture supernatant, centrifuge at 3000 rpm at 4°C for 15 minutes, take the supernatant, filter the virus liquid with a 0.45 μm PVDF filter membrane, aliquot into EP tubes, and store at -80°C until T cells are infected.

Recombinant lentivirus infection of T cells: CD8 ⁺ T lymphocytes isolated from the peripheral blood of healthy people were cultured and activated and then infected with the above recombinant lentivirus at MOI=5. T cells were infected with 2×10 ⁵ / mL density for passage. The expression of different chimeric antigen receptors was detected on the infected CD8 ⁺ T lymphocytes on the 7th day of culture. Since eGFP and CAR were co-expressed, the positive cells detected for eGFP were positive cells expressing chimeric antigen receptors (results shown in Figure 1 As shown, the left picture is the representative picture of the cells under the light microscope, the right picture is the fluorescence expression picture of eGFP, and the left picture and the right picture are the same field of view).

Embodiment 4: Construction of in vitro killing experiment model

The target cells used in the in vitro cell model are Daudi cells. The Daudi cell line is derived from a patient with Burkitt's lymphoma (Burkitt's lymphoma). It is a typical B lymphoblastoid cell line and is widely used in basic and preclinical research on the mechanism of leukemia formation and treatment. Daudi cells highly express the B cell antigen CD19, which can be recognized and killed by CAR-T19 (CD19-targeting CAR-T) cells. Therefore, we used Daudi cells as target cells (T), CAR-T19 cells as effector cells (E), and T cells as control cells (C) to construct an in vitro killing cell model. This model simulates to a high degree the therapeutic effect of CAR-T19 cells or T cells on B lymphocytic leukemia in vivo.

1) Establish two models for monitoring cell killing effect

After T cells recognize target cells, they undergo apoptosis and eventually lyse. Among them, the late apoptosis process and the lysis process of cells overlap but are not exactly the same. Therefore, we established two in vitro models for detecting cell killing effects for apoptosis and lysis.

1-1) Detection of target cell apoptosis by flow cytometry

First, we used the carbocyanine dye Did to label CAR-T19 cells, and the Did-positive cells detected by flow cytometry reached 99.17%, and only 0.83% of the CAR-T19 cells were Did-negative (Figure 2).

In addition, under the same detection index, 99.94% of Daudi cells were Did negative (Fig. 3).

Therefore, we first labeled CAR-T19 cells with Did, and then mixed them with Daudi cells according to a certain ET ratio to kill them; then detected the apoptosis level of Did-negative cells by flow cytometry, and the results obtained could reflect the CAR-T19 The level of cell killing to Daudi cells. We used AnnexinV-FITC/PI to detect cell apoptosis. FITC/PI double-negative cells were normal cells, FITC-positive and PI-negative cells were early apoptotic cells, and FITC/PI double-positive cells were late apoptotic cells. According to the ET ratio of 1:2, the apoptosis of Did-negative cells was detected after 4 hours of killing, and the results are shown in Figure 4: early apoptosis (LR=lowright) was 4.32%, and late apoptosis (UR=upright) was 4.55%, with an overall kill rate of 8.87%.

1-2) Detection of target cell lysis by fluorescence method

Calcein-AM is a cell staining reagent that can be used to fluorescently label living cells. Because it has enhanced hydrophobicity based on Calcein, it can easily penetrate the membrane of living cells. When it enters the cytoplasm, it will be hydrolyzed into Calcein by esterase and stay in the cell, thus emitting strong green fluorescence. We first labeled target cells Daudi with Calcein-AM, and then used CAR-T19 cells to kill them. After the Daudi cells are killed and lysed by CAR-T19, Calcein will be released into the supernatant, and the fluorescence intensity of the supernatant (TESTRELEASE) will be detected. Fluorescence intensity (MAXIMUMRELEASE) of the maximum lysis of Daudi cells treated with TritonX-100). The percentage of cell lysis was calculated according to the following formula: [(TESTRELEASE-SPONTANEOUSRELEASE)/(MAXIMUMRELEASE-SPONTANEOUSRELEASE)]*100.

As shown in Table 1, in the case of ET ratio of 1:2, the lysis rate of DAUDI cells was 4.77% after killing for 4 hours. The results obtained were essentially similar to those of late apoptosis detected by flow cytometry.

Table 1 The lysis rate of DAUDI cells detected by CAR-T19 cells by fluorescence method

2) Comparison of the killing effect of T cells and CAR-T19 cells

After establishing the model of in vitro cell killing effect, we compared the constructed CAR-T19 cells (containing GMCSF–scFv-S1–CD28-TM–CD28-signal–CD3ζ-signal or GMCSF–scFv-S2–CD28-TM–CD28 -signal–CD3ζ-signal CAR-T19 cells, respectively referred to as CAR-T19(S1), CAR-T19(S2)), and the killing effect of T cells without modification on the target cell Daudi. The detection steps are the same as those described in 1), and the results are shown in Figure 5. Under the same conditions, the killing effect of CAR-T19 cells was increased by at least 20 times (setting the killing rate of T cells as 1, CAR-T19(S1) and CAR-T19 (S2) killing rates were 20.5, 22), respectively.

3) Comparison of the killing effects of CAR-T19 containing different scFv regions

In this example, two CAR-T19s of the present invention (the above-mentioned CAR-T19 (S1), CAR-T19 (S1), CAR- T19(S2)) and CAR-T19 (including GMCSF–scFv-S0–CD28-TM–CD28-signal–CD3ζ-signal (the nucleic acid The CAR-T19 whose sequence is shown in SEQ ID No: 19, referred to as CAR-T19 (S0)) was compared for its killing ability on target cells. The specific detection steps are the same as those described in 1), and the results are shown in Figure 6. The humanized modification of scFv did not affect the killing efficiency of CAR-T19 on target cells.

4) In vitro detection of the killing effect of different CAR-T19 on the cells of patients with B lymphocytic leukemia

In this embodiment, at first isolate the cell of B lymphocytic leukemia patient, mainly be divided into two steps: the first step, isolate PBMC (peripheral blood mononuclear cell) from patient's bone marrow (this step is a person skilled in the art known); the second step is to isolate B cells from PBMCs. The specific steps of the second step are as follows: Lymphocytes are mixed with sheep red blood cells (SRBC) treated with bromide diaminoisosulfide (AET for short), wherein all T lymphocytes can adsorb AET-SRBC to form a firm Stable and huge E-rosette, the percentage of the E-rosette is higher than the percentage of the E-rosette formed by T lymphocytes and normal untreated SRBC, and the formation is fast, not easy to fall off, and has good repeatability. Then use the lymphocyte stratification solution to separate, at this time AET-E-rosette is easy to sink at the bottom of the tube, and B lymphocytes can be directly taken from the interface of the stratification solution. In this example, the killing effect of different CAR-T19s of the present invention on B lymphocytic leukemia patient cells was tested in vitro. The results are shown in Figure 7: Compared with unmodified T cells, different CAR-T19s of the present invention (represented by different CAR molecules in the figure) have significantly enhanced killing ability on B lymphocytic leukemia patient cells.

Claims (10)

1. coding is expressed in the nucleic acid molecule of the Chimeric antigen receptor on T cell surface, described Chimeric antigen receptor comprise be linked in sequence signal peptide, the outer land of born of the same parents, cross-film district and intracellular signal district, the nucleotide sequence of the outer land of wherein said born of the same parents is as shown in SEQIDNo:4 or SEQIDNo:5.

2. the nucleic acid molecule of claim 1, wherein said Chimeric antigen receptor also comprises hinge area between land and cross-film district outside its born of the same parents.

3. the nucleic acid molecule of claim 2, the nucleotide sequence of wherein said signal peptide is as shown in SEQIDNo:1 or SEQIDNo:2, the nucleotide sequence of described hinge area is as shown in SEQIDNo:6 or SEQIDNo:7, the nucleotide sequence in described cross-film district is as shown in SEQIDNo:8 or SEQIDNo:9, described intracellular signal district is selected from the intracellular signal district of CD3 ζ, Fc ε RI γ, CD28, CD137, CD134 albumen, and combination, the nucleotide sequence in preferred described intracellular signal district is made up of SEQIDNo:10 and/or SEQIDNo:11 and/or SEQIDNo:12.

4. the nucleic acid molecule any one of claim 1-3, its sequence is as shown in SEQIDNo:13, SEQIDNo:14, SEQIDNo:15, SEQIDNo:16, SEQIDNo:17 or SEQIDNo:18.

5. carrier, it comprises the nucleic acid molecule any one of claim 1-4.

6. the carrier of claim 5, it is slow virus plasmid vector pLenti-CMV-eGFP or retroviral vector pMSCV-Ubc-GFP.

7. virus, it comprises the carrier of claim 5 or 6.

8.T cell, wherein transforms the nucleic acid molecule of having the right any one of requirement 1-4 or the carrier of claim 5 or 6 or the virus of claim 7.

9. comprise the medicine of the T cell of claim 8, preferred described medicine also comprises pharmaceutically useful thinner, vehicle or carrier.

10. the T cell of claim 8 is for the preparation of the purposes of medicine for the treatment of tumour.