CN111848822B

CN111848822B - CD19 and CD30 double-target chimeric antigen receptor and application thereof

Info

Publication number: CN111848822B
Application number: CN202010763253.XA
Authority: CN
Inventors: 汤朝阳; 秦乐; 吴迪; 邓殷健; 吴海鹏; 王翠花; 冯世忠; 冯嘉昆; 其他发明人请求不公开姓名
Original assignee: Guangdong Zhaotai Invivo Biomedicine Co ltd
Current assignee: Guangdong Zhaotai Invivo Biomedicine Co ltd
Priority date: 2020-07-31
Filing date: 2020-07-31
Publication date: 2022-02-15
Anticipated expiration: 2040-07-31
Also published as: CN111848822A

Abstract

The invention provides a CD19 and CD30 dual-target chimeric antigen receptor and application thereof, wherein the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain and a signal transduction domain; the antigen binding domains include anti-CD 19 single chain antibodies and anti-CD 30 single chain antibodies. The anti-CD 19 and CD30 double-target chimeric antigen receptor has targeting activity on CD19 positive and/or CD30 positive cells, and T cells expressing the anti-CD 19 and CD30 double-target chimeric antigen receptor have killing effects on tumor cells with low or no expression of CD19 antigen and tumor cells with low or no expression of CD30 antigen, so that the immune escape phenomenon is avoided, and the possibility of disease relapse is reduced.

Description

CD19 and CD30 double-target chimeric antigen receptor and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a CD19 and CD30 double-target chimeric antigen receptor and application thereof.

Background

A Chimeric Antigen Receptor (CAR) consists of a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane region, and an intracellular signaling region. Generally, CAR molecules comprise an antibody single chain fragment variable (scFv) with specific binding for a Tumor Associated Antigen (TAA), which is coupled to the cytoplasmic domain of a signaling molecule via a hinge and a transmembrane region.

In recent years, with the development of tumor immunology theory and clinical technology, Chimeric antigen receptor T-cell immunotherapy (CAR-T) has become one of the most promising tumor immunotherapy. At present, CAR-T cell therapy has been widely applied to the treatment of B cell malignant tumors, and CAR-T cells targeting CD19 are precursors for the treatment of B cell malignant tumors by CAR-T therapy, and provide an effective scheme for the treatment of B cell malignant tumors. However, medical diagnosis shows that tumor cells in some blood tumors do not express CD19 molecules, but express CD30 molecules, and the CAR-T cells only targeting CD19 molecules have poor treatment effect, and the tumor recurrence phenomenon appears in some patients after a period of time.

Therefore, there is a need to develop chimeric antigen receptors that target both CD19 and CD30 molecules, improving the targeting and clearance of CAR-T cells to tumor cells.

Disclosure of Invention

Aiming at the defects and practical requirements of the prior art, the invention provides the CD19 and CD30 double-target chimeric antigen receptor and the application thereof, wherein the chimeric antigen receptor can simultaneously target CD19 and CD30 molecules and has wide prospects in the aspect of treating hematological malignant diseases.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain, and a signaling domain;

the antigen binding domains include anti-CD 19 single chain antibodies and anti-CD 30 single chain antibodies.

Compared with a single-target chimeric antigen receptor, the anti-CD 19 and anti-CD 30 double-target chimeric antigen receptor has stronger targeting activity on CD19 positive and/or CD30 positive cells, has efficient targeting effect on tumor cells with little or no expression of CD19 antigen and tumor cells with little or no expression of CD30 antigen, and is favorable for avoiding the immune escape phenomenon.

Preferably, the anti-CD 19 single chain antibody comprises the amino acid sequence shown in SEQ ID NO. 1;

SEQ ID NO:1：

DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPPRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSAYTFGQGTKLEIKSGGGGQVQLVESGGGVVQPGRSLRLSCAASGFTFSRHGMHWVRQAPGKGLEWVAVIWYDGSNQYYVDSVKGRFTISRDNSKNTLDLQMNSLRVEDTAVYYCARRSITWYGGFDIWGQGTMVTVSSAQTTAPSVYPLAP。

preferably, the anti-CD 30 single chain antibody comprises an amino acid sequence shown in SEQ ID NO. 2;

SEQ ID NO:2：

QIQLVQSGAEVKKPGASVKVSCKASGYTFTDYYITWVRQAPGQGLEWMGWIYPGSGNTKYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCANYGNYWFAYWGQGTLVTVSSGSTSGSGKPGSSEGSTKGDIVMTQSPDSLAVSLGERATINCKASQSVDFDGDSYMNWYQQKPGQPPKLLIYAASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPWTFGQGTKVEIK。

preferably, the anti-CD 19 single chain antibody and the anti-CD 30 single chain antibody are linked by a linking peptide.

Preferably, the connecting peptide comprises an amino acid sequence shown as SEQ ID NO. 3;

SEQ ID NO:3：GGGGSGGGGSGGGGSGGGGS。

preferably, the transmembrane domain comprises CD28 and/or CD8 a.

Preferably, the signalling domain comprises any one of CD3 ζ, 4-1BB, CD28, TLR1, TLR2, CD27, OX40 or DAP10, or a combination of at least two.

Preferably, the chimeric antigen receptor further comprises a signal peptide.

Preferably, the signal peptide comprises a GM-CSF signal peptide.

Preferably, the GM-CSF signal peptide comprises the amino acid sequence shown in SEQ ID NO. 4;

SEQ ID NO:4：MLLLVTSLLLCELPHPAFLL。

preferably, the chimeric antigen receptor consists of a GM-CSF signal peptide, an anti-CD 19 single chain antibody, a linker peptide, an anti-CD 30 single chain antibody, CD28 and CD3 zeta concatemeric.

Preferably, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 5;

SEQ ID NO:5：

MLLLVTSLLLCELPHPAFLLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPPRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSAYTFGQGTKLEIKSGGGGQVQLVESGGGVVQPGRSLRLSCAASGFTFSRHGMHWVRQAPGKGLEWVAVIWYDGSNQYYVDSVKGRFTISRDNSKNTLDLQMNSLRVEDTAVYYCARRSITWYGGFDIWGQGTMVTVSSAQTTAPSVYPLAPGGGGSGGGGSGGGGSGGGGSQIQLVQSGAEVKKPGASVKVSCKASGYTFTDYYITWVRQAPGQGLEWMGWIYPGSGNTKYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCANYGNYWFAYWGQGTLVTVSSGSTSGSGKPGSSEGSTKGDIVMTQSPDSLAVSLGERATINCKASQSVDFDGDSYMNWYQQKPGQPPKLLIYAASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPWTFGQGTKVEIKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

in a second aspect, the present invention provides a coding gene encoding the chimeric antigen receptor of the first aspect.

Preferably, the coding gene comprises an anti-CD 19 single chain antibody coding sequence and an anti-CD 30 single chain antibody coding sequence.

Preferably, the coding gene further comprises a linker peptide coding sequence.

Preferably, the coding gene further comprises a coding sequence for GM-CSF signal peptide, a coding sequence for CD28, and a coding sequence for CD3 zeta.

Preferably, the anti-CD 19 single chain antibody coding sequence comprises the nucleic acid sequence shown in SEQ ID NO. 6;

SEQ ID NO:6：

gacatccagatgacccagagccccagcaccctgagcgccagcgtgggcgaccgcgtgaccatcacctgccgcgccagccagagcatcagcagctggctggcctggtaccagcagaagcccggcaaggcccccaagctgctgatctacaaggccagcagcctggagagcggcgtgcccccccgcttcagcggcagcggcagcggcaccgagttcaccctgaccatcagcagcctgcagcccgacgacttcgccacctactactgccagcagtacaacagcgcctacaccttcggccagggcaccaagctggagatcaagtccggtggcggtggccaggtgcagctggtggagagcggcggcggcgtggtgcagcccggccgcagcctgcgcctgagctgcgccgccagcggcttcaccttcagccgccacggcatgcactgggtgcgccaggcccccggcaagggcctggagtgggtggccgtgatctggtacgacggcagcaaccagtactacgtggacagcgtgaagggccgcttcaccatcagccgcgacaacagcaagaacaccctggacctgcagatgaacagcctgcgcgtggaggacaccgccgtgtactactgcgcccgccgcagcatcacctggtacggcggcttcgacatctggggccagggcaccatggtgaccgtgagcagcgcccagaccaccgcccccagcgtgtaccccctggccccc。

preferably, the anti-CD 30 single chain antibody coding sequence comprises the nucleic acid sequence shown in SEQ ID NO. 7;

SEQ ID NO:7：

cagatccagctggtgcagagcggcgccgaggtgaagaagcccggcgccagcgtgaaggtgagctgcaaggccagcggctacaccttcaccgactactacatcacctgggtgagacaggcccccggccagggcctggagtggatgggctggatctaccccggcagcggcaacaccaagtacaacgagaagttcaagggcagagtgaccatgaccagagacaccagcatcagcaccgcctacatggagctgagcagactgagaagcgacgacaccgccgtgtactactgcgccaactacggcaactactggttcgcctactggggccagggcaccctggtgaccgtgagcagcggcagcaccagcggcagcggcaagcccggcagcagcgagggcagcaccaagggcgacatcgtgatgacccagagccccgacagcctggccgtgagcctgggcgagagagccaccatcaactgcaaggccagccagagcgtggacttcgacggcgacagctacatgaactggtaccagcagaagcccggccagccccccaagctgctgatctacgccgccagcaacctggagagcggcgtgcccgacagattcagcggcagcggcagcggcaccgacttcaccctgaccatcagcagcctgcaggccgaggacgtggccgtgtactactgccagcagagcaacgaggacccctggaccttcggccagggcaccaaggtggagatcaag。

preferably, the linker peptide coding sequence comprises the nucleic acid sequence set forth in SEQ ID NO 8;

SEQ ID NO:8：

ggtggaggcggcagtggcggaggtgggagcggagggggcggttccggtggcgggggatct。

preferably, the GM-CSF signal peptide coding sequence comprises the nucleic acid sequence shown in SEQ ID NO. 9;

SEQ ID NO:9：

atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctg。

preferably, the encoding gene of the chimeric antigen receptor comprises a nucleic acid sequence shown as SEQ ID NO. 10;

SEQ ID NO:10：

atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctggacatccagatgacccagagccccagcaccctgagcgccagcgtgggcgaccgcgtgaccatcacctgccgcgccagccagagcatcagcagctggctggcctggtaccagcagaagcccggcaaggcccccaagctgctgatctacaaggccagcagcctggagagcggcgtgcccccccgcttcagcggcagcggcagcggcaccgagttcaccctgaccatcagcagcctgcagcccgacgacttcgccacctactactgccagcagtacaacagcgcctacaccttcggccagggcaccaagctggagatcaagtccggtggcggtggccaggtgcagctggtggagagcggcggcggcgtggtgcagcccggccgcagcctgcgcctgagctgcgccgccagcggcttcaccttcagccgccacggcatgcactgggtgcgccaggcccccggcaagggcctggagtgggtggccgtgatctggtacgacggcagcaaccagtactacgtggacagcgtgaagggccgcttcaccatcagccgcgacaacagcaagaacaccctggacctgcagatgaacagcctgcgcgtggaggacaccgccgtgtactactgcgcccgccgcagcatcacctggtacggcggcttcgacatctggggccagggcaccatggtgaccgtgagcagcgcccagaccaccgcccccagcgtgtaccccctggcccccggtggaggcggcagtggcggaggtgggagcggagggggcggttccggtggcgggggatctcagatccagctggtgcagagcggcgccgaggtgaagaagcccggcgccagcgtgaaggtgagctgcaaggccagcggctacaccttcaccgactactacatcacctgggtgagacaggcccccggccagggcctggagtggatgggctggatctaccccggcagcggcaacaccaagtacaacgagaagttcaagggcagagtgaccatgaccagagacaccagcatcagcaccgcctacatggagctgagcagactgagaagcgacgacaccgccgtgtactactgcgccaactacggcaactactggttcgcctactggggccagggcaccctggtgaccgtgagcagcggcagcaccagcggcagcggcaagcccggcagcagcgagggcagcaccaagggcgacatcgtgatgacccagagccccgacagcctggccgtgagcctgggcgagagagccaccatcaactgcaaggccagccagagcgtggacttcgacggcgacagctacatgaactggtaccagcagaagcccggccagccccccaagctgctgatctacgccgccagcaacctggagagcggcgtgcccgacagattcagcggcagcggcagcggcaccgacttcaccctgaccatcagcagcctgcaggccgaggacgtggccgtgtactactgccagcagagcaacgaggacccctggaccttcggccagggcaccaaggtggagatcaagattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc。

in a third aspect, the present invention provides an expression vector, wherein the expression vector is a lentiviral vector comprising the coding gene of the second aspect.

In a fourth aspect, the present invention provides a recombinant lentivirus which is a mammalian cell transfected with an expression vector and a helper plasmid as described in the third aspect.

In a fifth aspect, the invention provides a CAR-T cell expressing the chimeric antigen receptor of the first aspect.

In the invention, the T cells expressing anti-CD 19 and CD30 double-target chimeric antigen receptors have high-efficiency targeting activity and killing efficacy on CD19 positive and/or CD30 positive cells, and have killing effects on tumor cells with little or no expression of CD19 antigen and tumor cells with little or no expression of CD30 antigen, thereby being beneficial to avoiding the immune escape phenomenon and reducing the possibility of disease relapse.

Preferably, the CAR-T cell has integrated into its genome the gene encoding the second aspect.

Preferably, the CAR-T cell comprises the expression vector of the third aspect and/or the recombinant lentivirus of the fourth aspect.

In a sixth aspect, the present invention provides a method of producing a CAR-T cell according to the fifth aspect, the method comprising the step of introducing into a T cell a gene encoding the chimeric antigen receptor according to the first aspect.

In a seventh aspect, the present invention provides a chimeric antigen receptor of the first aspect, a coding gene of the second aspect, an expression vector of the third aspect, a recombinant lentivirus of the fourth aspect, or a CAR-T cell of the fifth aspect, for use in the preparation of a medicament for the treatment of a disease.

Preferably, the disease comprises a hematological tumor.

Preferably, the disease comprises a CD19 positive and/or CD30 positive disease.

Compared with the prior art, the invention has the following beneficial effects:

(1) compared with a single-target chimeric antigen receptor, the anti-CD 19 and anti-CD 30 double-target chimeric antigen receptor has stronger targeting activity on CD19 positive and/or CD30 positive cells, has efficient targeting effect on tumor cells with little or no expression of CD19 antigen and tumor cells with little or no expression of CD30 antigen, and is favorable for avoiding the immune escape phenomenon;

(2) the T cell expressing the anti-CD 19 and CD30 double-target chimeric antigen receptor has high-efficiency targeting activity and killing effect on CD19 positive cells and/or CD30 positive cells, has killing effect on tumor cells with small or non-expressed CD19 antigen expression quantity and tumor cells with small or non-expressed CD30 antigen expression quantity, is favorable for avoiding immune escape phenomenon, and reduces the possibility of disease relapse.

Drawings

FIG. 1 shows the killing efficiency of WT, 1928zT and 19.30.28.zT on tumor cells K562-CD19-GL at different E: T ratios;

FIG. 2 shows the killing efficiency of WT, 3028zT and 19.30.28.zT on tumor cells K562-CD30-GL at different E: T ratios.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1 construction of CAR molecular vectors

In the embodiment, firstly, a coding gene (SEQ ID NO:10) of anti-CD 19 and anti-CD 30 double-target chimeric antigen receptor is synthesized by genes, and a restriction enzyme Pme1 restriction site and a protection base thereof, and a restriction enzyme Spe1 restriction enzyme site and a protection base thereof are respectively added at the C end and the N end of the coding gene;

the coding gene is subjected to double enzyme digestion by restriction enzymes Pme1 and Spe1, an enzyme digestion product containing a sticky end is obtained by agar gel electrophoresis recovery, the enzyme digestion product is connected into a linearized pWPXld-eGFP plasmid (containing the sticky end) which is also subjected to double enzyme digestion by Pme1 and Spe1, and the connection reaction is carried out in the presence of T4 DNA polymerase (Invitrogen company) to obtain the lentiviral vector containing the coding gene of the CAR targeting double targets of CD19 and CD 30.

In this example, the antigen binding domains of CAR (anti CD19scFv-CD28-CD3 zeta) and CAR (anti CD30 scFv-CD28-CD3 zeta) against CD19scFv and CD30 scFv were constructed simultaneously, and corresponding lentiviral vectors were constructed.

Example 2 Lentiviral packaging

In order to introduce CAR molecules into T cells, recombinant lentiviruses were prepared using 293T cells, and lentivirus packaging was performed when 293T cells were plated at 80-90% on 100mm plates:

2h before virus packaging, the culture medium is replaced by DMEM containing 1% fetal calf serum, and the addition amount is 6mL/100mm culture dish;

preparing a plasmid mixed solution as shown in table 1, wherein the pWPXld-expression plasmid comprises a lentiviral vector containing a coding gene of a CAR targeting CD19 and CD30 double targets, a lentiviral vector containing a coding gene of a CAR targeting CD19 single target, and a lentiviral vector containing a coding gene of a CAR targeting CD30 single target, and the pWPXld-eGFP plasmid is an empty vector containing no CAR coding gene;

TABLE 1

Adding 36 μ g PEI into another 500 μ L opti-MEM medium, mixing, and standing at room temperature for 5 min;

mixing the plasmid mixed solution shown in the table 1 with PEI, blowing, beating and uniformly mixing, and standing at room temperature for 25-30 min;

dropwise adding the mixed solution to 293T cells cultured in a 100mm culture dish;

after culturing for 6h, changing the culture medium into DMEM containing 1% fetal calf serum, and adding the DMEM into a culture dish with the volume of 7mL/100 mm;

collecting virus supernatant 24h, 48h and 72h after packaging, and simultaneously supplementing a culture medium to 293T cells, wherein the addition amount is 7mL/100mm culture dish;

centrifugation at 1000g for 10min, filtration through a 0.45 μm filter, to obtain recombinant lentivirus expressing CAR or a blank control eGFP lentivirus, and storage at 4 ℃ for future use.

Example 3T cell activation and lentivirus transfection

Separating Peripheral Blood Mononuclear Cells (PBMC) from whole blood by using a Ficoll density gradient centrifugation kit (GE company), removing red blood cells, and then separating T cells by using MACS Pan-T magnetic beads;

the separated T cells were diluted with a medium (AIM-V medium + 5% FBS + penicillin 100U/mL + streptomycin 0.1mg/mL) to a cell concentration of 2.5X 10⁶Per mL for standby;

activating T cells by using CD2/CD3/CD 28T cell activation and expansion kit (Meitian whirlwind company), namely mixing coated magnetic beads with T cells at a ratio of 1:2, and finally, the density of the T cells is 5 x 10⁶Per mL/cm²Mixing, and standing at 37 deg.C and 5% CO₂Culturing and stimulating for 48h in an incubator;

after 48h of T cell activation, beads were removed, 300g was centrifuged for 5min, supernatant was removed, the T cells were resuspended in fresh medium, CAR-expressing recombinant lentivirus or blank control eGFP lentivirus (MOI 10) was added, and 8. mu.g/mL polybrene and 300IU/mL IL-2 were added, placed at 37 ℃ and 5% CO₂Culturing in an incubator;

after 24h, centrifuging for 5min at 300g, removing supernatant, and resuspending T cells in fresh culture medium containing 300IU/mL IL-2 to obtain CAR-T cells;

maintenance of CAR-T cell density at 1X 10⁶About one/mL, half the liquid change is carried out every 2-3 days, and after two weeks, the number of CAR-T cells is expanded by 100 times.

CAR-T cells constructed in this example were 19.30.28.zT (expressing anti-CD 19 and CD30 dual-target CARs), 1928zT (expressing anti-CD 19 single-target CAR), 3028zT (expressing anti-CD 30 single-target CAR), while the WT control group (transfection blank control eGFP lentivirus) was set.

Example 4 expression of CAR molecules by T cells

Since the lentiviral vector expressing the CAR molecule carries the eGFP gene, the expression of the CAR molecule on the T cell can be indicated by eGFP, and the eGFP on the T cell is detected in this example by flow cytometry, acearocyte.

As a result, the expression efficiency of WT cells on CAR molecules was found to be 0.9%, 1928zT on CAR molecules was found to be 22.8%, 3028zT on CAR molecules was found to be 7.6%, and 19.30.28.zT on CAR molecules was found to be 10.4%.

Example 5 in vitro testing of the killing function of CAR-T cells against tumor cells K562-CD19-GL

WT prepared in example 3, 1928zT and 19.30.28.zT were compared to 1X 10 zT, respectively⁴Mixing tumor cells K562-CD19-GL at E: T ratio of 4:1, 2:1, 1:2, 1:4, 1:8, and 1:16, adding into 96-well plate with 3 multiple wells, centrifuging at 250g for 5min, standing at 37 deg.C and 5% CO₂Co-culturing for 18h in an incubator;

after 18h, adding 100. mu.L/well Luciferase substrate (1X) into a 96-well plate, suspending and mixing the cells evenly, immediately measuring RLU (relative light unit) by a multifunctional microplate reader for 1 second, and comparing the killing effect of WT, 1928zT and 19.30.28.zT on K562-CD19-GL in vitro by using a Luciferase (Luciferase) quantitative killing efficiency evaluation method, wherein the killing proportion calculation formula is as follows: 100% × (control well reading-experimental well reading)/control well reading (blank reading without cells negligible)

The results are shown in figure 1, the killing efficiency of 1928zT and 19.30.28.zT to K562-CD19-GL in vitro is obviously higher than that of WT, and under the condition that E: T is very small, namely the tumor target cells are far larger than effector T cells, 19.30.28.zT can also show stronger tumor killing activity.

Example 6 in vitro testing of the killing function of CAR-T cells against tumor cells K562-CD30-GL

WT prepared in example 3, 3028zT and 19.30.28.zT were compared with 1X 10, respectively⁴Mixing tumor cells K562-CD30-GL at E: T ratio of 4:1, 2:1, 1:2, 1:4, 1:8, and 1:16, adding into 96-well plate with 3 multiple wells, centrifuging at 250g for 5min, standing at 37 deg.C and 5% CO₂Co-culturing for 18h in an incubator;

after 18h, 100. mu.L/well Luciferase substrate (1X) was added to the 96-well plate, the cells were resuspended and mixed well, RLU (relative light unit) was immediately measured by a multifunctional microplate reader for 1 second, and the killing effect of WT, 3028zT and 19.30.28.zT on K562-CD30-GL was compared in vitro by the Luciferase (Luciferase) quantitative killing efficiency evaluation method, and the killing ratio was calculated as follows: 100% × (control well reading-experimental well reading)/control well reading (blank reading without cells negligible)

As shown in FIG. 2, the killing efficiency of 3028zT and 19.30.28.zT on K562-CD30-GL in vitro is significantly higher than that of WT, and in the case of small E: T, i.e. the tumor target cells are far larger than the effector T cells, 19.30.28.zT can also show stronger tumor killing activity.

In conclusion, the anti-CD 19 and CD30 double-target chimeric antigen receptor constructed by the invention has targeting activity on CD19 positive and/or CD30 positive cells, and T cells expressing the anti-CD 19 and CD30 double-target chimeric antigen receptor have killing effects on tumor cells with low or no expression of CD19 antigen and tumor cells with low or no expression of CD30 antigen, so that the immune escape phenomenon is avoided, and the possibility of disease relapse is reduced.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Guangdong Shoutai biomedical science and technology Co., Ltd

<120> CD19 and CD30 dual-target chimeric antigen receptor and application thereof

<130> 20200730

<160> 10

<170> PatentIn version 3.3

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<212> PRT

<213> Artificial sequence

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Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

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Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Glu Gly Ser Thr Lys Gly Asp Ile Val Met Thr Gln Ser Pro Asp

130 135 140

Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala

145 150 155 160

Ser Gln Ser Val Asp Phe Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln

165 170 175

Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn

180 185 190

Leu Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val

210 215 220

Tyr Tyr Cys Gln Gln Ser Asn Glu Asp Pro Trp Thr Phe Gly Gln Gly

225 230 235 240

Thr Lys Val Glu Ile Lys

245

<210> 3

<211> 20

<212> PRT

<213> Artificial sequence

<400> 3

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 4

<211> 20

<212> PRT

<213> Artificial sequence

<400> 4

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu

20

<210> 5

<211> 749

<212> PRT

<213> Artificial sequence

<400> 5

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser

20 25 30

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

35 40 45

Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

50 55 60

Lys Leu Leu Ile Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Pro

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn

100 105 110

Ser Ala Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Gly

115 120 125

Gly Gly Gly Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln

130 135 140

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

145 150 155 160

Ser Arg His Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

165 170 175

Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Gln Tyr Tyr Val

180 185 190

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

195 200 205

Thr Leu Asp Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val

210 215 220

Tyr Tyr Cys Ala Arg Arg Ser Ile Thr Trp Tyr Gly Gly Phe Asp Ile

225 230 235 240

Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Gln Thr Thr Ala

245 250 255

Pro Ser Val Tyr Pro Leu Ala Pro Gly Gly Gly Gly Ser Gly Gly Gly

260 265 270

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Gln Leu

275 280 285

Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val

290 295 300

Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr Ile Thr Trp

305 310 315 320

Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Tyr

325 330 335

Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe Lys Gly Arg Val

340 345 350

Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser

355 360 365

Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Asn Tyr Gly

370 375 380

Asn Tyr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

385 390 395 400

Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Ser Glu Gly Ser

405 410 415

Thr Lys Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val

420 425 430

Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val

435 440 445

Asp Phe Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly

450 455 460

Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly

465 470 475 480

Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

485 490 495

Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln

500 505 510

Gln Ser Asn Glu Asp Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu

515 520 525

Ile Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys

530 535 540

Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser

545 550 555 560

Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val

565 570 575

Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile

580 585 590

Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr

595 600 605

Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln

610 615 620

Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys

625 630 635 640

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

645 650 655

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

660 665 670

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

675 680 685

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

690 695 700

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

705 710 715 720

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

725 730 735

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

740 745

<210> 6

<211> 732

<212> DNA

<213> Artificial sequence

<400> 6

gacatccaga tgacccagag ccccagcacc ctgagcgcca gcgtgggcga ccgcgtgacc 60

atcacctgcc gcgccagcca gagcatcagc agctggctgg cctggtacca gcagaagccc 120

ggcaaggccc ccaagctgct gatctacaag gccagcagcc tggagagcgg cgtgcccccc 180

cgcttcagcg gcagcggcag cggcaccgag ttcaccctga ccatcagcag cctgcagccc 240

gacgacttcg ccacctacta ctgccagcag tacaacagcg cctacacctt cggccagggc 300

accaagctgg agatcaagtc cggtggcggt ggccaggtgc agctggtgga gagcggcggc 360

ggcgtggtgc agcccggccg cagcctgcgc ctgagctgcg ccgccagcgg cttcaccttc 420

agccgccacg gcatgcactg ggtgcgccag gcccccggca agggcctgga gtgggtggcc 480

gtgatctggt acgacggcag caaccagtac tacgtggaca gcgtgaaggg ccgcttcacc 540

atcagccgcg acaacagcaa gaacaccctg gacctgcaga tgaacagcct gcgcgtggag 600

gacaccgccg tgtactactg cgcccgccgc agcatcacct ggtacggcgg cttcgacatc 660

tggggccagg gcaccatggt gaccgtgagc agcgcccaga ccaccgcccc cagcgtgtac 720

cccctggccc cc 732

<210> 7

<211> 738

<212> DNA

<213> Artificial sequence

<400> 7

cagatccagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc gactactaca tcacctgggt gagacaggcc 120

cccggccagg gcctggagtg gatgggctgg atctaccccg gcagcggcaa caccaagtac 180

aacgagaagt tcaagggcag agtgaccatg accagagaca ccagcatcag caccgcctac 240

atggagctga gcagactgag aagcgacgac accgccgtgt actactgcgc caactacggc 300

aactactggt tcgcctactg gggccagggc accctggtga ccgtgagcag cggcagcacc 360

agcggcagcg gcaagcccgg cagcagcgag ggcagcacca agggcgacat cgtgatgacc 420

cagagccccg acagcctggc cgtgagcctg ggcgagagag ccaccatcaa ctgcaaggcc 480

agccagagcg tggacttcga cggcgacagc tacatgaact ggtaccagca gaagcccggc 540

cagcccccca agctgctgat ctacgccgcc agcaacctgg agagcggcgt gcccgacaga 600

ttcagcggca gcggcagcgg caccgacttc accctgacca tcagcagcct gcaggccgag 660

gacgtggccg tgtactactg ccagcagagc aacgaggacc cctggacctt cggccagggc 720

accaaggtgg agatcaag 738

<210> 8

<211> 60

<212> DNA

<213> Artificial sequence

<400> 8

ggtggaggcg gcagtggcgg aggtgggagc ggagggggcg gttccggtgg cgggggatct 60

<210> 9

<211> 60

<212> DNA

<213> Artificial sequence

<400> 9

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

<210> 10

<211> 2247

<212> DNA

<213> Artificial sequence

<400> 10

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

gacatccaga tgacccagag ccccagcacc ctgagcgcca gcgtgggcga ccgcgtgacc 120

atcacctgcc gcgccagcca gagcatcagc agctggctgg cctggtacca gcagaagccc 180

ggcaaggccc ccaagctgct gatctacaag gccagcagcc tggagagcgg cgtgcccccc 240

cgcttcagcg gcagcggcag cggcaccgag ttcaccctga ccatcagcag cctgcagccc 300

gacgacttcg ccacctacta ctgccagcag tacaacagcg cctacacctt cggccagggc 360

accaagctgg agatcaagtc cggtggcggt ggccaggtgc agctggtgga gagcggcggc 420

ggcgtggtgc agcccggccg cagcctgcgc ctgagctgcg ccgccagcgg cttcaccttc 480

agccgccacg gcatgcactg ggtgcgccag gcccccggca agggcctgga gtgggtggcc 540

gtgatctggt acgacggcag caaccagtac tacgtggaca gcgtgaaggg ccgcttcacc 600

atcagccgcg acaacagcaa gaacaccctg gacctgcaga tgaacagcct gcgcgtggag 660

gacaccgccg tgtactactg cgcccgccgc agcatcacct ggtacggcgg cttcgacatc 720

tggggccagg gcaccatggt gaccgtgagc agcgcccaga ccaccgcccc cagcgtgtac 780

cccctggccc ccggtggagg cggcagtggc ggaggtggga gcggaggggg cggttccggt 840

ggcgggggat ctcagatcca gctggtgcag agcggcgccg aggtgaagaa gcccggcgcc 900

agcgtgaagg tgagctgcaa ggccagcggc tacaccttca ccgactacta catcacctgg 960

gtgagacagg cccccggcca gggcctggag tggatgggct ggatctaccc cggcagcggc 1020

aacaccaagt acaacgagaa gttcaagggc agagtgacca tgaccagaga caccagcatc 1080

agcaccgcct acatggagct gagcagactg agaagcgacg acaccgccgt gtactactgc 1140

gccaactacg gcaactactg gttcgcctac tggggccagg gcaccctggt gaccgtgagc 1200

agcggcagca ccagcggcag cggcaagccc ggcagcagcg agggcagcac caagggcgac 1260

atcgtgatga cccagagccc cgacagcctg gccgtgagcc tgggcgagag agccaccatc 1320

aactgcaagg ccagccagag cgtggacttc gacggcgaca gctacatgaa ctggtaccag 1380

cagaagcccg gccagccccc caagctgctg atctacgccg ccagcaacct ggagagcggc 1440

gtgcccgaca gattcagcgg cagcggcagc ggcaccgact tcaccctgac catcagcagc 1500

ctgcaggccg aggacgtggc cgtgtactac tgccagcaga gcaacgagga cccctggacc 1560

ttcggccagg gcaccaaggt ggagatcaag attgaagtta tgtatcctcc tccttaccta 1620

gacaatgaga agagcaatgg aaccattatc catgtgaaag ggaaacacct ttgtccaagt 1680

cccctatttc ccggaccttc taagcccttt tgggtgctgg tggtggttgg gggagtcctg 1740

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1800

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1860

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cagagtgaag 1920

ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1980

ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 2040

gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 2100

aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 2160

aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 2220

cttcacatgc aggccctgcc ccctcgc 2247

Claims

1. A chimeric antigen receptor, which is composed of a GM-CSF signal peptide, an anti-CD 19 single chain antibody, a linker peptide, an anti-CD 30 single chain antibody, CD28 and CD3 zeta in tandem;

the amino acid sequence of the anti-CD 19 single-chain antibody is shown in SEQ ID NO. 1;

the amino acid sequence of the anti-CD 30 single-chain antibody is shown in SEQ ID NO. 2;

the amino acid sequence of the connecting peptide is shown as SEQ ID NO. 3;

the amino acid sequence of the GM-CSF signal peptide is shown in SEQ ID NO. 4;

the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 5.

2. A coding gene encoding the chimeric antigen receptor of claim 1;

the coding gene comprises an anti-CD 19 single-chain antibody coding sequence and an anti-CD 30 single-chain antibody coding sequence;

the coding gene further comprises a connecting peptide coding sequence;

the coding gene also comprises a GM-CSF signal peptide coding sequence, a CD28 coding sequence and a CD3 zeta coding sequence;

the coding sequence of the anti-CD 19 single-chain antibody is shown in SEQ ID NO. 6;

the coding sequence of the anti-CD 30 single-chain antibody is shown in SEQ ID NO. 7;

the coding sequence of the connecting peptide is shown as SEQ ID NO. 8;

the coding sequence of the GM-CSF signal peptide is shown in SEQ ID NO. 9;

the nucleic acid sequence of the coding gene of the chimeric antigen receptor is shown as SEQ ID NO. 10.

3. An expression vector, wherein the expression vector is a lentiviral vector comprising the coding gene of claim 2.

4. A recombinant lentivirus prepared from a mammalian cell transfected with the expression vector of claim 3 and a helper plasmid.

5. A CAR-T cell expressing the chimeric antigen receptor of claim 1;

the CAR-T cell having the coding gene of claim 2 integrated into its genome.

6. The CAR-T cell of claim 5, wherein the CAR-T cell comprises the expression vector of claim 3 and/or the recombinant lentivirus of claim 4.

7. A method of producing a CAR-T cell according to claim 5 or 6, which comprises the step of introducing into a T cell a gene encoding the chimeric antigen receptor of claim 1.

8. Use of the chimeric antigen receptor of claim 1, the coding gene of claim 2, the expression vector of claim 3, the recombinant lentivirus of claim 4 or the CAR-T cell of claim 5 or 6 for the preparation of a medicament for the treatment of a disease;

the disease is a CD19 positive and/or CD30 positive tumor.

9. The use according to claim 8, wherein the disease is a hematological tumor.