CN112300288A - Chimeric antigen receptor CAR of CIK cell and application thereof - Google Patents

Abstract

The application discloses a chimeric antigen receptor CAR of a CIK cell and application thereof. The CAR comprises: an extracellular antigen-binding domain that binds to a specific target antigen, a hinge and transmembrane domain, and an intracellular signaling domain; the intracellular signaling domain comprises a costimulatory signaling domain containing intracellular segments of: DAP12 and CD 226. Experiments demonstrated that DAP12 or DAP10 were used as hinge and transmembrane domains of CARs andpart of costimulatory signaling domain, which forms costimulatory signaling domain together with DNAM-1(CD226) or 2B4, etc., uses these costimulatory signaling molecules and CD3 zeta to activate immune cells, especially CD3⁺CD56⁺The related signal conduction path in the CIK inhibits and kills malignant tumor cells, so that the cytokine storm can be remarkably reduced, and the safety of treatment products is improved.

Description

Chimeric antigen receptor CAR of CIK cell and application thereof

Technical Field

The invention relates to the field of biological medicines, in particular to the technical field of immune cell therapy of malignant tumors, and particularly relates to a chimeric antigen receptor CAR and application thereof.

Background

Chimeric Antigen Receptors (CAR) generally consist of an extracellular domain, a transmembrane domain and an intracellular domain, and are capable of specifically recognizing tumor-associated antigens on the surface of target cells and promoting effector cells such as T cells, NK cells, NKT cells and the like where the Chimeric Antigen Receptors are located to activate, proliferate and kill the target cells through a receptor-activation signal mechanism.

The structural design of CAR molecules has been perfected with many generations of modifications. The first generation CARs contained a single chain variable fragment (scFv) that recognized a tumor cell surface antigen, a hinge and transmembrane structure, and an intracellular domain of CD3 ζ or FcR γ that activated T cells, but severely affected their clinical efficacy due to their short survival time in vivo. Second generation CARs with an addition of a costimulatory molecule, such as CD28, 4-1BB (CD137), OX-40(CD134), etc., in the intracellular domain have had great success in hematologic malignancies. A CAR comprising CD28 is capable of producing more IL-2; a CAR comprising 4-1BB can then enhance the viability of the cell. In order to obtain better proliferation capacity, cytokine secretion capacity and tumor lysis activity of CAR-T cells in vivo, researchers add a costimulatory molecule to the intracellular domain of the three generations of CAR. Fourth generation CARs control cytokine storms or enhance CAR-T cell function by adding a "suicide gene" or "cytokine gene". The two Anti-CD19 CAR-T currently on the market are second generation CAR. For the killing ability of tumor cells, the second generation CAR-T has been fully met, but with the risk of causing cytokine storm and nervous system toxicity.

CIK (cytokine-induced killer) cells, i.e. killer cells induced by various cytokinesThe human peripheral blood mononuclear cells are cultured together with a plurality of cytokines in vitro for a period of time to obtain a heterogeneous population of cells, which are also called NK cell-like T lymphocytes and have the advantages of strong anti-tumor activity of the T lymphocytes and non-MHC restricted tumor killing of the NK cells. CD3, CD56 double positive cells are the main effector cells, and also have CCR7^-、CD45RA⁺、CD62L^low、CD11a⁺、CD27⁺、CD28^-、NKG2D⁺、NKp30^low、NKp44^-、NKp46^-、NKG2A^-、CD94^-And the like. CIK cells for clinical application are cell subsets, including CD3⁺、CD56⁺CIK and CD3⁺And CD8⁺CTL (cytotoxic T lymphocyte), CD3 (Takara Shuzo)⁺And CD4⁺Th cells (helper T cells) of (1). CD3⁺CD56⁺The CIK of (2) recognizes the corresponding ligand on malignant tumor mainly through NKG2D, and plays a non-MHC restriction killing function after cell-cell contact. The CIK cells are widely used for treating malignant tumors and solid tumors of the blood system, and achieve certain clinical curative effect. The property that CIK cells do not substantially elicit graft versus host response (GVHD) also makes them good CAR vector cells.

At present, CAR-CIK is constructed mainly based on CD28 as a costimulatory molecule. However, the CAR-T cells taking CD28-CD3 zeta as an intracellular segment can generate more adverse reactions. In 2017 Juno, the company abandoned its CD28 zeta CAR and turned to a 4-1BB zeta CAR. Even the approved Yescata from Kite (also with CD28 as co-stimulatory molecule), has developed severe clinical neurotoxicity in clinical trials and is of widespread concern. How to fully develop CD3 in CIK cell subset⁺、CD56⁺How to control CD3 in the CIK cell subset⁺CD8⁺The potential cytokine storm and nervous system toxicity caused by CTL requires further research.

DAP12, DNAX-related protein 12, is a transmembrane molecule, and is connected with NKG2D-S molecule in the form of homodimer, the intracellular region of the molecule contains an Immunoreceptor Tyrosine Activation Motif (ITAM), and tyrosine in the motif can be recognized by other PTKs (such as Syk or ZAPT0) with SH2 domain and cell molecules after being phosphorylated under the action of Protein Tyrosine Kinase (PTK), so as to start and activate corresponding signal transduction pathways.

DNAM-1, namely CD226, also called PTA1 or TliSA1, transmembrane protein of about 65KDa is distributed on the surfaces of NK cells, platelets and monocytes such as dendritic cells, macrophages and T cells, is an important NK cell activation receptor, can stimulate cytokine secretion of the NK cells through PI-3K, SLP-76 and other ways, and can also promote CD4⁺And CD8⁺Co-stimulation of T cells and promotion of activation of CD8 by a non-classical antigen presenting cell pathway⁺A cell.

At present, CAR of CAR-CIK is mostly based on traditional scFv-CD8 alpha (TM) -CD28-CD3 zeta or scFv-CD8 alpha (TM) -4-1BB-CD3 zeta, and co-stimulatory molecules such as OX-40 and CD27 are increased to evolve. These CARs have achieved some effect in T cells, but the suitability for CIK is unknown. Andrea et al found CAR-modified CD3 of scFv-Fc-CD28-CD3 ζ -OX40⁺CD56⁺Cells, although able to secrete more interferon, result in more CD56⁺Cell Activation Induces Cell Death (AICD), thereby reducing its anti-tumor function in vivo.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a chimeric antigen receptor CAR of CIK cells and application thereof. The chimeric antigen receptor CAR has the effect of remarkably reducing the storm of the cell factor and is higher in safety.

In one aspect, the invention provides a chimeric antigen receptor CAR of a CIK cell, comprising: an extracellular antigen-binding domain (also referred to as an antigen-binding domain or antigen-specific binding domain, including scFv) that binds to a specific target antigen, a hinge and transmembrane domain, and an intracellular signaling domain;

the intracellular signaling domain comprises a costimulatory signaling domain (or costimulatory domain) containing the intracellular segment of the following protein: DAP12 and CD 226; the number of the intracellular sections can be specifically 1-3;

preferably, the intracellular segment sequence of DAP12 includes the amino acid sequence at position 314-365 of SEQ ID No. 1; more preferably, the intracellular segment sequence of DAP12 is encoded by the nucleotide sequence 940-1095 of SEQ ID No. 6; the intracellular segments of DAP12 may be 2-3 and in series;

preferably, the intracellular segment sequence of CD226 comprises the amino acid sequence of SEQ ID No.1 at position 366-431; more preferably, the intracellular segment sequence of CD226 is encoded by the nucleotide sequence 1096-1293 of SEQ ID No. 6; the intracellular segments of the CD226 can be 2-3 and connected in series.

In the chimeric antigen receptor CAR described above, the intracellular segment of DAP12 was replaced with the intracellular segment of DAP 10; preferably, the intracellular segment sequence of DAP10 includes the amino acid sequence at position 312-335 of SEQ ID No. 3; more preferably, the intracellular segment sequence of DAP10 is encoded by the nucleotide sequence at positions 934-1005 of SEQ ID No. 8; the intracellular segments of DAP10 may be 2-3 and in series;

and/or, the intracellular segment of CD226 can be replaced with the intracellular segment of 2B4, preferably, the intracellular segment sequence of 2B4 comprises the sequence after the intracellular segment knockout of 2B4 comprises the third ITSM (immune receptor tyrosine switching motif), more preferably, the intracellular segment sequence of 2B4 comprises the 356-and-475-th amino acid sequence of SEQ ID No.4 or the 356-and-419-th amino acid sequence of SEQ ID No.4, more preferably, the intracellular segment sequence of 2B4 is encoded by the 1066-and-1425-th nucleotide sequence of SEQ ID No.9 or the 1066-and-1257-th nucleotide sequence of SEQ ID No. 9; the intracellular segments of 2B4 can be 2-3 and connected in series.

In the CAR described above, optionally, the co-stimulatory signaling domain may further comprise any one or a combination of at least two of CD, 4-1BB, TLR, CD, PD-1, ICOS, LFA-1, CD, LIGHT, NKG2, B-H, CDS, ICAM-1, GITR, BAFFR, HVEM, SLAMF, NKp, CD160, CD α, CD β, IL2 γ, IL7 α, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, CD11, LFA-1, ITGAM, CD11, ITGAX, CD11, ITGB, CD-1, ITGB, TNFR, TRANCEL/KL, CD M, SLAF, CD160, ACA, TAAM, TAMG, TARG, CD, TARG, TAMP, TAMPE, CD, TARG, TAM, TARG.

In one embodiment, the CAR comprises an extracellular antigen-binding domain that specifically binds to a target polypeptide expressed on a tumor cell, e.g., a target antigen, the binding domain may comprise any protein, polypeptide, oligopeptide, or peptide possessing the ability to specifically recognize and bind to a biomolecule (e.g., a cell surface receptor or tumor protein, lipid, polysaccharide, or other cell surface target molecule or component thereof), the binding domain comprising any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner of the biomolecule of interest;

further, the extracellular binding domain comprises an antibody or antigen binding fragment thereof;

an antibody refers to a binding agent that is a polypeptide comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds an epitope of a target antigen, such as a peptide, lipid, polysaccharide, or nucleic acid containing an antigenic determinant, such as a nucleic acid recognized by an immune cell. Antibodies include antigen binding fragments, for example, camelid igs (camelid antibodies or VHH fragments thereof), Ig NAR, Fab fragments, Fab ' fragments, f (ab) ' 2 fragments, f (ab) ' 3 fragments, Fv, single chain Fv antibodies ("scFv"), bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disulfide stabilized Fv proteins ("dsFv"), and single domain antibodies (sdAb, nanobodies), or other antibody fragments thereof. In a preferred embodiment, the binding domain is an scFv.

The intracellular signaling domain transduces information effective CAR binding to the target antigen into the interior of an immune effector cell to elicit effector cell functions, e.g., activation, cytokine production, proliferation, and cytotoxic activity, including release of cytotoxic factors to the CAR-bound target cell or other cellular responses elicited using antigen binding with the extracellular CAR domain.

In one embodiment, the extracellular antigen-binding domain of the CAR is followed by one or more "spacer domains," which refer to regions that move the antigen-binding domain away from the surface of an effector cell to achieve proper cell/cell contact, antigen binding, and activation. The spacer domain may be derived from natural, synthetic, semi-synthetic or recombinant sources. In certain embodiments, the spacer domain is part of an immunoglobulin, including but not limited to one or more heavy chain constant regions, such as CH2 and CH 3. The spacer domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In one embodiment, the spacer domain comprises CH2 and CH3 of IgG1, IgG4, or IgD.

In some embodiments, the CAR includes a linker residue between the domains. A "variable region linker sequence" is an amino acid sequence that links the heavy chain variable region to the light chain variable region and provides a spacer function compatible with the interaction of the two sub-binding domains, such that the resulting polypeptide retains the specific binding affinity of the same target molecule as an antibody comprising the same light chain variable region and heavy chain variable region. In particular embodiments, the linker isolates one or more of a heavy or light chain variable domain, a hinge domain, a transmembrane domain, a costimulatory domain, and/or a primary signaling domain. In particular embodiments, the CAR comprises one, two, three, four, or five or more linkers. In particular embodiments, the linker is from about 1 amino acid to about 25 amino acids, from about 5 amino acids to about 20 amino acids, or from about 10 amino acids to about 20 amino acids, or any intermediate length amino acid. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acids in length.

In the above CAR, the intracellular signaling domain further comprises a primary signaling domain comprising any one or a combination of at least two of FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD22, CD79a, CD79b and CD66d, preferably, CD3 ζ, more preferably, the amino acid sequence at position 432 and 543 of SEQ ID No.1, more preferably, encoded by the nucleotide sequence at position 1294 and 1629 of SEQ ID No. 6.

In the above CAR, the hinge and transmembrane domain comprises any one or at least two of the topological domains and transmembrane domains of CD8 a, CD28, 4-1BB, DAP12, DAP10, preferably, the topological domains and transmembrane domains of DAP12, or DAP 10;

more preferably, the topological domain and transmembrane domain sequences of DAP12 comprise the amino acid sequence at amino acid positions 280 and 313 of SEQ ID No.1, more preferably, are encoded by the nucleotide sequence at nucleotide positions 838 and 939 of SEQ ID No.6,

more preferably, the topological domain and transmembrane domain sequences of DAP10 comprise the amino acid sequences 262-311 of SEQ ID No.3, and more preferably are encoded by the nucleotide sequences 784-933 of SEQ ID No. 8.

The hinge and transmembrane domain may also include other domains derived from natural, synthetic, semi-synthetic or recombinant sources, and in one embodiment the hinge and transmembrane domain may also include or be derived from one or more transmembrane regions of the following group: the α or β chain of the T cell receptor, CD3 δ, CD3 ∈, CD3 γ, CD3 ζ, CD4, CD5, CD8 α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD-1.

In the above CAR, the specific target antigen against which the extracellular antigen-binding domain is directed comprises: any one or at least two of CD19, CD20, CD22, CD30, CD33, CD123, CD138, CD171, BCMA, ROR1, WT-1, Pr3, erB-B2, MUC1, LeY, PSCA, EGP-40, HER-2, TAG-72, CD44v7/8, MAGE-A1, GD3, CAIX, NY-ESO-1, GD2, EGFRIII, FAP, alpha Folate Receptor (alpha-Folate Receptor), IL-13R-a2, EGP-2, Mesothelin (Mesothelin), KDR, VEGF-R2, PSMA, CEA, glypican-3 (GPC3), IL-13; preferably, CD19, more preferably, the sequence of said extracellular antigen-binding domain comprises the amino acid sequence from position 28 to 269 of SEQ ID No.1, more preferably, encoded by the nucleotide sequence from position 82 to 807 of SEQ ID No. 6.

In the above CAR, at the N-terminus of the extracellular antigen-binding domain, further comprising a signal peptide, preferably the signal peptide is of DAP12, DAP10, CD8 α, or CD28, more preferably the signal peptide of DAP12 comprises amino acid sequence 1-27 of SEQ ID No.1, more preferably encoded by nucleotide sequence 1-81 of SEQ ID No.6, more preferably the signal peptide of DAP10 comprises amino acid sequence 1-19 of SEQ ID No.3, more preferably encoded by nucleotide sequence 1-57 of SEQ ID No.8, more preferably the signal peptide of CD8 α comprises amino acid sequence 1-21 of SEQ ID No.2, more preferably encoded by nucleotide sequence 1-63 of SEQ ID No. 7;

and/or, the CAR further comprises a protein Tag sequence, the protein Tag comprises any one or at least two of MyC, His, GST, HA, Flag, MBP, Avi Tag, SUMO, c-MyC Tag; preferably, c-Myc tag; the protein tag sequence comprises amino acid sequence 270-279 of SEQ ID No.1, more preferably, the protein tag sequence is encoded by sequence 808-837 of SEQ ID No.6, and the protein tag sequence can be used for detecting CAR transfection efficiency; preferably, the protein tag sequence is located between the extracellular antigen-binding domain and the hinge and transmembrane domains;

and/or, further comprises, following the amino acid sequence of said intracellular signaling domain, the amino acid sequence of: self-cleaving proteins (P2A, T2A, F2A, and/or IRES), and cytokines or suicide genes; in one embodiment, the self-cleaving protein may also be ligated after the peptide fragment of the cytokine or "suicide gene" prior to the CAR structure;

preferably, the amino acid sequence of the CAR comprises: the sequence of SEQ ID No.1 excluding position 270-279, the sequence of SEQ ID No.1, SEQ ID No.3, SEQ ID No.4 excluding position 420-475, the sequence of SEQ ID No.4, SEQ ID No.5 excluding position 400-455, or SEQ ID No. 5.

In another aspect, the invention also provides a nucleic acid molecule encoding a CAR as described in any preceding claim, which nucleic acid molecule is DNA or RNA,

preferably, the sequence of the nucleic acid molecule comprises: the sequence of SEQ ID No.6 excluding position 808-minus 837, the sequence of SEQ ID No.6, SEQ ID No.8, SEQ ID No.9 excluding position 1258-minus 1425, the sequence of SEQ ID No.9, SEQ ID No.10 excluding position 1198-minus 1365, and SEQ ID No. 10.

In some embodiments, any of the above described CARs can also be constructed using a nucleic acid molecule that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid molecule;

in some embodiments, any of the above CARs can also be constructed using different nucleic acid sequences that encode the same amino acid, using the degeneracy of the code.

In another aspect, the present invention also provides a recombinant vector comprising the nucleic acid molecule, wherein the recombinant vector is a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a simian viral vector, a vaccinia viral vector, a Sendai viral vector, an EB viral vector, a herpes simplex viral vector, a sleeping beauty transposon system plasmid, preferably a lentiviral vector,

in a specific embodiment, the lentiviral vector is a lentiviral expression vector pELPS-19-BB-z in which a CD19BB zeta fragment downstream of the EF-1 alpha promoter is replaced by any one of the nucleic acid molecules;

the nucleotide sequence of the CD19BB zeta segment is shown in SEQ ID No.7, and the amino acid sequence of the encoded CRA is shown in SEQ ID No. 2.

In another aspect, the present invention also provides a recombinant cell or a recombinant bacterium, wherein the recombinant cell or the recombinant bacterium comprises any one of the above CARs, or comprises any one of the above nucleic acid molecules, or comprises the recombinant vector;

preferably, the recombinant cell is an immune cell, preferably, a T cell, more preferably, a CIK cell, more preferably, CD3⁺、CD56⁺The CIK cell of (1);

on the other handThe invention also provides the application of any one of the CAR, the nucleic acid molecule and the recombinant vector in preparing immune cells with reduced cytokine storm; preferably, the cytokine is IL-6; preferably, the immune cell is a T cell, more preferably, a CIK cell, more preferably, CD3⁺CD56⁺The CIK cell of (1).

In another aspect, the invention further provides the use of any one of the CAR, the nucleic acid molecule, the recombinant vector, or the recombinant cell or recombinant bacterium described above in the preparation of a product for treating tumor.

The invention has the following beneficial effects:

experiments prove that DAP12 or DAP10, as a hinge and transmembrane domain and a partial costimulatory signaling domain of CAR, and DNAM-1(CD226) or 2B4 and the like form a costimulatory signaling domain, and the costimulatory signaling molecules and CD3 zeta are utilized to activate immune cells, particularly CD3⁺CD56⁺The related signal conduction path in the CIK inhibits and kills malignant tumor cells, so that the cytokine storm can be remarkably reduced, and the safety of treatment products is improved.

Drawings

Figure 1 is a lentiviral vector expressing a novel CAR wherein the target antigen for the scFv is CD 19.

FIG. 2 is a lentiviral vector expressing a conventional CAR, where the target antigen for the scFv is CD 19.

FIG. 3 shows the inhibition of killing of target cells by two CAR-CIK 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 packaging and purification of lentiviruses

The CAR in lentiviral vector pels-19-BB-z (whose sequence is described in patent 2017108531905) shown in fig. 2, which contained a conventional CAR structure (designated CAR-CK, with the structure SP-scFv-tag-CD8 α -4-1BB-CD3 ζ, i.e., CD19BB ζ located downstream of EF1 α promoter), was replaced with 7 different novel CAR structures (CAR-1 to CAR-7) shown in table 1, respectively, to obtain 7 different recombinant lentiviral vectors, wherein fig. 1 shows a recombinant lentiviral vector containing CAR-7 (with the structure SP-scFv-tag-DAP12-DNAM-1(CD226) -CD3 ζ, i.e., CD19-12D- ζ located downstream of EF1 α promoter).

The eight different recombinant lentiviral vectors are respectively packaged and purified according to the following methods to obtain eight different lentiviruses:

1. taking a 10cm culture dish as an example, HEK293T cells are inoculated in a DMEM medium containing 10% fetal bovine serum; when the virus grows to 70-80% of fusion degree, starting to package the virus;

2. removing the original culture medium by suction, adding 50% volume of fresh culture medium, and continuing incubation;

3. as pELPS (expressing a novel CAR or a traditional CAR): psPAX 2: PMD2.0G is 8: 6: 2(4:3:1), transfecting the cells of step 2 by adopting a calcium transfer method;

4. after 12h, changing the liquid;

5. after 36h, collecting cell suspension, filtering by a 0.44um filter membrane, and centrifuging at 50000g for 2 hours;

6. and collecting the precipitate to obtain the slow virus expressing the CAR structure.

TABLE 1 compositions of different CAR

Sequences of the segments in tables 2 and 1

In tables 1 and 2, SP represents a signal peptide sequence, TM represents a transmembrane domain, tag represents a tag sequence, -represents none, + represents inclusion, G1 represents costimulatory signaling domain 1, G2 represents costimulatory signaling domain 2, X represents a primary signaling domain, and No. represents SEQ ID no.

Example 2 obtaining of CAR-CIK cells

First, obtaining CIK cells

1. Collecting mononuclear cells from peripheral blood, umbilical cord blood and bone marrow by density gradient centrifugation;

2. by 2X 10⁶The cells are planted in X-VIVO 15 culture medium containing 1000U/ml IFN-gamma;

3. after 24h, adding OKT-3 and IL-2 to make the concentrations reach 50-100ng/ml and 1000U/ml respectively;

4. half changing the solution every 2-3 days later, and adding X-VIVO 15 culture medium containing 300-;

5. and 3-7 days, after the CIK cell subset reaches the fold increase period, starting cell transfection.

II, respectively transfecting CIK cell subsets by using CAR gene-loaded lentiviruses

1. Changing culture medium, and adjusting cell density to 0.1-1 × 10⁶/ml；

2. Eight different lentiviruses obtained in example 1 were added at MOI of 5-10, respectively, and the supernatants were resuspended;

3. 1000g, room temperature, centrifuging for 60 min;

4. and continuously culturing for 24h, and changing the liquid.

III, CAR-CIK culture and detection

Half-changing the liquid every 2-3 days later; by day 21, cell suspensions were collected to obtain 7 different novel CAR-CIK cells: CAR-1-CIK, CAR-2-CIK, CAR-3-CIK, CAR-4-CIK, CAR-5-CIK, CAR-6-CIK, and CAR-7-CIK, and 1 traditional CAR-CIK cell; FITC-CD3, PE-CD56, PerCP-Cy5.5-c-Myc antibodies incubated CAR-7-CIK and CAR-CK-CIK, and expression of CAR structures in different subsets of CIK cells was flow-assayed.

As a result: the transfection efficiency of lentiviruses expressing the novel CAR structure is not significantly different from that of conventional CAR structure, p >0.05, and the results of the novel CAR-CIK represented by CAR-7-CIK are shown in Table 3.

TABLE 3 results of CAR expression in various subsets of cells in different CAR-CIK cells

Cell subpopulation cell types	CAR-7-CIK (New pattern)	CAR-CK-CIK (tradition)
			CD3+CD56+	25.6％±16.3％	27.5％±17.6％
CD3-CD56+	0	0
			CD3+CD56-	42.1％±13.9％	46.6％±15.7％

Example 3 killing of Raji cell lines by different CAR-CIK cells

Respectively culturing the eight CAR-CIK cells obtained in the example 2 and target cell-Raji cells (human lymphoma cells) in a mixing manner for 24h according to the effective target ratio of 1:1, 1:5 and 1:25 (effective target ratio of E: T), setting the CIK cells which are not transfected by lentivirus as a control group, and detecting the inhibiting and killing capacity of the CAR-CIK on the target cells by adopting a luciferase quantitative killing efficiency evaluation method; wherein, the results of CAR-7-CIK are shown in FIG. 3; when the E: T is 1:1 and 1:5, the novel CAR-CIK represented by CAR-7-CIK has no obvious difference in killing of target cells compared with the traditional CAR-CIK, and the results of CAR-1-CIK, CAR-2-CIK, CAR-3-CIK, CAR-4-CIK, CAR-5-CIK and CAR-6-CIK are not obviously different from the results of CAR-7-CIK.

Collecting cell supernatant with E: T being 1:1 group, and detecting the release level of cytokines such as IFN-gamma, IL-6 and the like by using an ELISA method; the results for the novel CAR-CIK represented by CAR-7-CIK are shown in Table 4; the IFN-gamma of the novel CAR-CIK has no statistical difference with the IFN-gamma of the traditional CAR-CIK, and the secretion of the cytokine storm key factor IL-6 of the novel CAR-CIK is lower than that of the traditional CAR-CIK, so that the safety is higher.

The results of CAR-1-CIK, CAR-2-CIK, CAR-3-CIK, CAR-4-CIK, CAR-5-CIK, CAR-6-CIK were not significantly different from CAR-7-CIK.

TABLE 4 cytokine secretion profiles of two CAR-CIK cells

	IL-6*	γ-IFN
			CAR-7-CIK (New pattern)	243.1±74.6pg/ml	4046.9±594.2pg/ml
CAR-CK-CIK (tradition)	367.5±68.7pg/ml	3973.5±630.7pg/ml

In table 4 indicates significance of difference p < 0.05.

Those not described in detail in this specification are within the skill of the art. The above description is only an example of the present application and is not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

SEQUENCE LISTING

<110> Jinan Seal Biotechnology Ltd

<120> chimeric antigen receptor CAR of CIK cell and application thereof

<130> JH-CNP190627

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<170> PatentIn version 3.5

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<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 Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

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

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

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

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Glu Gln Lys Leu Ile Ser Glu Glu Asp

260 265 270

Leu Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

275 280 285

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala

290 295 300

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

305 310 315 320

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

325 330 335

Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile

340 345 350

Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp

355 360 365

Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

370 375 380

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

385 390 395 400

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

405 410 415

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

420 425 430

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

<210> 3

<211> 513

<212> PRT

<213> Artificial sequence

<400> 3

Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala

1 5 10 15

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

20 25 30

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

35 40 45

Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys

50 55 60

Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn

85 90 95

Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr

100 105 110

Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys

130 135 140

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

145 150 155 160

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

165 170 175

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile

180 185 190

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

195 200 205

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn

210 215 220

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

225 230 235 240

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

245 250 255

Val Thr Val Ser Ser Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala

260 265 270

Phe Tyr Pro Gly Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser

275 280 285

Leu Pro Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu

290 295 300

Leu Ile Val Gly Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro

305 310 315 320

Ala Gln Glu Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly Ile

325 330 335

Val Ile Phe Leu Asn Arg Arg Arg Arg Arg Glu Arg Arg Asp Leu Phe

340 345 350

Thr Glu Ser Trp Asp Thr Gln Lys Ala Pro Asn Asn Tyr Arg Ser Pro

355 360 365

Ile Ser Thr Ser Gln Pro Thr Asn Gln Ser Met Asp Asp Thr Arg Glu

370 375 380

Asp Ile Tyr Val Asn Tyr Pro Thr Phe Ser Arg Arg Pro Lys Thr Arg

385 390 395 400

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

405 410 415

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

420 425 430

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

435 440 445

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Arg

<210> 4

<211> 587

<212> PRT

<213> Artificial sequence

<400> 4

Met Gly Gly Leu Glu Pro Cys Ser Arg Leu Leu Leu Leu Pro Leu Leu

1 5 10 15

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

20 25 30

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

35 40 45

Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln

50 55 60

Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg

65 70 75 80

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

85 90 95

Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr

100 105 110

Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly

115 120 125

Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

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

145 150 155 160

Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val

165 170 175

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys

180 185 190

Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

195 200 205

Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys

210 215 220

Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala

225 230 235 240

Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

245 250 255

Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gln Ala Gln

260 265 270

Ser Asp Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile

275 280 285

Val Met Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr

290 295 300

Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala

305 310 315 320

Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu

325 330 335

Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro

340 345 350

Tyr Tyr Lys Trp Arg Arg Lys Arg Lys Glu Lys Gln Ser Glu Thr Ser

355 360 365

Pro Lys Glu Phe Leu Thr Ile Tyr Glu Asp Val Lys Asp Leu Lys Thr

370 375 380

Arg Arg Asn His Glu Gln Glu Gln Thr Phe Pro Gly Gly Gly Ser Thr

385 390 395 400

Ile Tyr Ser Met Ile Gln Ser Gln Ser Ser Ala Pro Thr Ser Gln Glu

405 410 415

Pro Ala Tyr Thr Leu Tyr Ser Leu Ile Gln Pro Ser Arg Lys Ser Gly

420 425 430

Ser Arg Lys Arg Asn His Ser Pro Ser Phe Asn Ser Thr Ile Tyr Glu

435 440 445

Val Ile Gly Lys Ser Gln Pro Lys Ala Gln Asn Pro Ala Arg Leu Ser

450 455 460

Arg Lys Glu Leu Glu Asn Phe Asp Val Tyr Ser Arg Val Lys Phe Ser

465 470 475 480

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

580 585

<210> 5

<211> 567

<212> PRT

<213> Artificial sequence

<400> 5

Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala

1 5 10 15

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

20 25 30

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

35 40 45

Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys

50 55 60

Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn

85 90 95

Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr

100 105 110

Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys

130 135 140

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

145 150 155 160

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

165 170 175

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile

180 185 190

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

195 200 205

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn

210 215 220

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

225 230 235 240

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

245 250 255

Val Thr Val Ser Ser Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala

260 265 270

Phe Tyr Pro Gly Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser

275 280 285

Leu Pro Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu

290 295 300

Leu Ile Val Gly Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro

305 310 315 320

Ala Gln Glu Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly Trp

325 330 335

Arg Arg Lys Arg Lys Glu Lys Gln Ser Glu Thr Ser Pro Lys Glu Phe

340 345 350

Leu Thr Ile Tyr Glu Asp Val Lys Asp Leu Lys Thr Arg Arg Asn His

355 360 365

Glu Gln Glu Gln Thr Phe Pro Gly Gly Gly Ser Thr Ile Tyr Ser Met

370 375 380

Ile Gln Ser Gln Ser Ser Ala Pro Thr Ser Gln Glu Pro Ala Tyr Thr

385 390 395 400

Leu Tyr Ser Leu Ile Gln Pro Ser Arg Lys Ser Gly Ser Arg Lys Arg

405 410 415

Asn His Ser Pro Ser Phe Asn Ser Thr Ile Tyr Glu Val Ile Gly Lys

420 425 430

Ser Gln Pro Lys Ala Gln Asn Pro Ala Arg Leu Ser Arg Lys Glu Leu

435 440 445

Glu Asn Phe Asp Val Tyr Ser Arg Val Lys Phe Ser Arg Ser Ala Asp

450 455 460

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

465 470 475 480

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

485 490 495

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

500 505 510

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

515 520 525

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

530 535 540

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

545 550 555 560

Met Gln Ala Leu Pro Pro Arg

565

<210> 6

<211> 1629

<212> DNA

<213> Artificial sequence

<400> 6

atggggggac ttgaaccctg cagcaggctc ctgctcctgc ctctcctgct ggctgtaagt 60

ggtctccgtc ctgtccaggc cgacatccag atgacacaga ctacatcctc cctgtctgcc 120

tctctgggag acagagtcac catcagttgc agggcaagtc aggacattag taaatattta 180

aattggtatc agcagaaacc agatggaact gttaaactcc tgatctacca tacatcaaga 240

ttacactcag gagtcccatc aaggttcagt ggcagtgggt ctggaacaga ttattctctc 300

accattagca acctggagca agaagatatt gccacttact tttgccaaca gggtaatacg 360

cttccgtaca cgttcggagg ggggaccaag ctggagatca caggtggcgg tggctcgggc 420

ggtggtgggt cgggtggcgg cggatctgag gtgaaactgc aggagtcagg acctggcctg 480

gtggcgccct cacagagcct gtccgtcaca tgcactgtct caggggtctc attacccgac 540

tatggtgtaa gctggattcg ccagcctcca cgaaagggtc tggagtggct gggagtaata 600

tggggtagtg aaaccacata ctataattca gctctcaaat ccagactgac catcatcaag 660

gacaactcca agagccaagt tttcttaaaa atgaacagtc tgcaaactga tgacacagcc 720

atttactact gtgccaaaca ttattactac ggtggtagct atgctatgga ctactggggc 780

caaggaacct cagtcaccgt ctcctcagaa caaaaactca tctcagaaga ggatctgcag 840

gcccagagcg attgcagttg ctctacggtg agcccgggcg tgctggcagg gatcgtgatg 900

ggagacctgg tgctgacagt gctcattgcc ctggccgtgt acttcctggg ccggctggtc 960

cctcgggggc gaggggctgc ggaggcagcg acccggaaac agcgtatcac tgagaccgag 1020

tcgccttatc aggagctcca gggtcagagg tcggatgtct acagcgacct caacacacag 1080

aggccgtatt acaaaattgt cattttcctt aacagaagga gaaggagaga gagaagagat 1140

ctatttacag agtcctggga tacacagaag gcacccaata actatagaag tcccatctct 1200

accagtcaac ctaccaatca atccatggat gatacaagag aggatattta tgtcaactat 1260

ccaaccttct ctcgcagacc aaagactaga gttagagtga agttcagcag gagcgcagac 1320

gcccccgcgt acaagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 1380

gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 1440

agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 1500

gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 1560

taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 1620

ccccctcgc 1629

<210> 7

<211> 1488

<212> DNA

<213> Artificial sequence

<400>7

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120

accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180

ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240

tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300

caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360

ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420

ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480

ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540

cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600

tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660

gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720

cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc 780

gtctcctcag aacaaaaact catctcagaa gaggatctga ccacgacgcc agcgccgcga 840

ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 900

cggccagcgg cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatatctac 960

atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 1020

tactgcaaac ggggcagaaa gaaactcctg tatatattca aacaaccatt tatgagacca 1080

gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 1140

ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg cccccgcgta caagcagggc 1200

cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1260

aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 1320

ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1380

aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 1440

accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgc 1488

<210> 8

<211> 1539

<212> DNA

<213> Artificial sequence

<400> 8

atgatccatc tgggtcacat cctcttcctg cttttgctcc cagtggctgc agctcaggac 60

atccagatga cacagactac atcctccctg tctgcctctc tgggagacag agtcaccatc 120

agttgcaggg caagtcagga cattagtaaa tatttaaatt ggtatcagca gaaaccagat 180

ggaactgtta aactcctgat ctaccataca tcaagattac actcaggagt cccatcaagg 240

ttcagtggca gtgggtctgg aacagattat tctctcacca ttagcaacct ggagcaagaa 300

gatattgcca cttacttttg ccaacagggt aatacgcttc cgtacacgtt cggagggggg 360

accaagctgg agatcacagg tggcggtggc tcgggcggtg gtgggtcggg tggcggcgga 420

tctgaggtga aactgcagga gtcaggacct ggcctggtgg cgccctcaca gagcctgtcc 480

gtcacatgca ctgtctcagg ggtctcatta cccgactatg gtgtaagctg gattcgccag 540

cctccacgaa agggtctgga gtggctggga gtaatatggg gtagtgaaac cacatactat 600

aattcagctc tcaaatccag actgaccatc atcaaggaca actccaagag ccaagttttc 660

ttaaaaatga acagtctgca aactgatgac acagccattt actactgtgc caaacattat 720

tactacggtg gtagctatgc tatggactac tggggccaag gaacctcagt caccgtctcc 780

tcaacgactc caggagagag atcatcactc cctgcctttt accctggcac ttcaggctct 840

tgttccggat gtgggtccct ctctctgccg ctcctggcag gcctcgtggc tgctgatgcg 900

gtggcatcgc tgctcatcgt gggggcggtg ttcctgtgcg cacgcccacg ccgcagcccc 960

gcccaagaag atggcaaagt ctacatcaac atgccaggca ggggcattgt cattttcctt 1020

aacagaagga gaaggagaga gagaagagat ctatttacag agtcctggga tacacagaag 1080

gcacccaata actatagaag tcccatctct accagtcaac ctaccaatca atccatggat 1140

gatacaagag aggatattta tgtcaactat ccaaccttct ctcgcagacc aaagactaga 1200

gttagagtga agttcagcag gagcgcagac gcccccgcgt acaagcaggg ccagaaccag 1260

ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 1320

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1380

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1440

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1500

acctacgacg cccttcacat gcaggccctg ccccctcgc 1539

<210> 9

<211> 1761

<212> DNA

<213> Artificial sequence

<400> 9

atggggggac ttgaaccctg cagcaggctc ctgctcctgc ctctcctgct ggctgtaagt 60

ggtctccgtc ctgtccaggc cgacatccag atgacacaga ctacatcctc cctgtctgcc 120

tctctgggag acagagtcac catcagttgc agggcaagtc aggacattag taaatattta 180

aattggtatc agcagaaacc agatggaact gttaaactcc tgatctacca tacatcaaga 240

ttacactcag gagtcccatc aaggttcagt ggcagtgggt ctggaacaga ttattctctc 300

accattagca acctggagca agaagatatt gccacttact tttgccaaca gggtaatacg 360

cttccgtaca cgttcggagg ggggaccaag ctggagatca caggtggcgg tggctcgggc 420

ggtggtgggt cgggtggcgg cggatctgag gtgaaactgc aggagtcagg acctggcctg 480

gtggcgccct cacagagcct gtccgtcaca tgcactgtct caggggtctc attacccgac 540

tatggtgtaa gctggattcg ccagcctcca cgaaagggtc tggagtggct gggagtaata 600

tggggtagtg aaaccacata ctataattca gctctcaaat ccagactgac catcatcaag 660

gacaactcca agagccaagt tttcttaaaa atgaacagtc tgcaaactga tgacacagcc 720

atttactact gtgccaaaca ttattactac ggtggtagct atgctatgga ctactggggc 780

caaggaacct cagtcaccgt ctcctcacag gcccagagcg attgcagttg ctctacggtg 840

agcccgggcg tgctggcagg gatcgtgatg ggagacctgg tgctgacagt gctcattgcc 900

ctggccgtgt acttcctggg ccggctggtc cctcgggggc gaggggctgc ggaggcagcg 960

acccggaaac agcgtatcac tgagaccgag tcgccttatc aggagctcca gggtcagagg 1020

tcggatgtct acagcgacct caacacacag aggccgtatt acaaatggag gagaaagagg 1080

aaggagaagc agtcagagac cagtcccaag gaatttttga caatttacga agatgtcaag 1140

gatctgaaaa ccaggagaaa tcacgagcag gagcagactt ttcctggagg ggggagcacc 1200

atctactcta tgatccagtc ccagtcttct gctcccacgt cacaagaacc tgcatataca 1260

ttatattcat taattcagcc ttccaggaag tctggatcca ggaagaggaa ccacagccct 1320

tccttcaata gcactatcta tgaagtgatt ggaaagagtc aacctaaagc ccagaaccct 1380

gctcgattga gccgcaaaga gctggagaac tttgatgttt attccagagt gaagttcagc 1440

aggagcgcag acgcccccgc gtacaagcag ggccagaacc agctctataa cgagctcaat 1500

ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1560

gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1620

aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1680

cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1740

atgcaggccc tgccccctcg c 1761

<210> 10

<211> 1701

<212> DNA

<213> Artificial sequence

<400> 10

atgatccatc tgggtcacat cctcttcctg cttttgctcc cagtggctgc agctcaggac 60

atccagatga cacagactac atcctccctg tctgcctctc tgggagacag agtcaccatc 120

agttgcaggg caagtcagga cattagtaaa tatttaaatt ggtatcagca gaaaccagat 180

ggaactgtta aactcctgat ctaccataca tcaagattac actcaggagt cccatcaagg 240

ttcagtggca gtgggtctgg aacagattat tctctcacca ttagcaacct ggagcaagaa 300

gatattgcca cttacttttg ccaacagggt aatacgcttc cgtacacgtt cggagggggg 360

accaagctgg agatcacagg tggcggtggc tcgggcggtg gtgggtcggg tggcggcgga 420

tctgaggtga aactgcagga gtcaggacct ggcctggtgg cgccctcaca gagcctgtcc 480

gtcacatgca ctgtctcagg ggtctcatta cccgactatg gtgtaagctg gattcgccag 540

cctccacgaa agggtctgga gtggctggga gtaatatggg gtagtgaaac cacatactat 600

aattcagctc tcaaatccag actgaccatc atcaaggaca actccaagag ccaagttttc 660

ttaaaaatga acagtctgca aactgatgac acagccattt actactgtgc caaacattat 720

tactacggtg gtagctatgc tatggactac tggggccaag gaacctcagt caccgtctcc 780

tcaacgactc caggagagag atcatcactc cctgcctttt accctggcac ttcaggctct 840

tgttccggat gtgggtccct ctctctgccg ctcctggcag gcctcgtggc tgctgatgcg 900

gtggcatcgc tgctcatcgt gggggcggtg ttcctgtgcg cacgcccacg ccgcagcccc 960

gcccaagaag atggcaaagt ctacatcaac atgccaggca ggggctggag gagaaagagg 1020

aaggagaagc agtcagagac cagtcccaag gaatttttga caatttacga agatgtcaag 1080

gatctgaaaa ccaggagaaa tcacgagcag gagcagactt ttcctggagg ggggagcacc 1140

atctactcta tgatccagtc ccagtcttct gctcccacgt cacaagaacc tgcatataca 1200

ttatattcat taattcagcc ttccaggaag tctggatcca ggaagaggaa ccacagccct 1260

tccttcaata gcactatcta tgaagtgatt ggaaagagtc aacctaaagc ccagaaccct 1320

gctcgattga gccgcaaaga gctggagaac tttgatgttt attccagagt gaagttcagc 1380

aggagcgcag acgcccccgc gtacaagcag ggccagaacc agctctataa cgagctcaat 1440

ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1500

gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1560

aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1620

cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1680

atgcaggccc tgccccctcg c 1701

Claims (10)

1. A chimeric antigen receptor CAR of CIK cells, characterized in that it comprises: an extracellular antigen-binding domain that binds to a specific target antigen, a hinge and transmembrane domain, and an intracellular signaling domain;

the intracellular signaling domain comprises a costimulatory signaling domain containing intracellular segments of: DAP12 and CD 226;

preferably, the intracellular segment sequence of DAP12 includes the amino acid sequence at position 314-365 of SEQ ID No. 1; more preferably, the intracellular segment sequence of DAP12 is encoded by the nucleotide sequence 940-1095 of SEQ ID No. 6;

preferably, the intracellular segment sequence of CD226 comprises the amino acid sequence of SEQ ID No.1 at position 366-431; more preferably, the intracellular segment sequence of CD226 is encoded by the nucleotide sequence 1096-1293 of SEQ ID No. 6.

2. The CAR of claim 1, wherein the intracellular segment of DAP12 is replaced with an intracellular segment of DAP 10; preferably, the intracellular segment sequence of DAP10 includes the amino acid sequence at position 312-335 of SEQ ID No. 3; more preferably, the intracellular segment sequence of DAP10 is encoded by the nucleotide sequence at positions 934-1005 of SEQ ID No. 8;

the CD226 intracellular segment is replaced with the 2B4 intracellular segment, preferably, the 2B4 intracellular segment sequence includes the 2B4 intracellular segment knockout sequence including the third ITSM sequence, more preferably, the 2B4 intracellular segment sequence includes the 356-475 amino acid sequence of SEQ ID No.4 or the 356-419 amino acid sequence of SEQ ID No.4, more preferably, the 2B4 intracellular segment sequence is encoded by the 1066-1425 nucleotide sequence of SEQ ID No.9 or the 1066-1257 nucleotide sequence of SEQ ID No. 9.

3. The CAR according to claim 1 or 2, characterized in that the intracellular signaling domain further comprises a primary signaling domain, preferably CD3 ζ, more preferably the amino acid sequence at amino acids 432 and 543 of SEQ ID No.1, more preferably encoded by the nucleotide sequence at nucleotide 1294 and 1629 of SEQ ID No. 6;

the hinge and transmembrane domains include any one or at least two of the topological domains and transmembrane domains of CD8 a, CD28, 4-1BB, DAP12, DAP10, preferably, the topological domains and transmembrane domains of DAP12, or DAP 10;

more preferably, the topological domain and transmembrane domain sequences of DAP12 comprise the amino acid sequence at amino acid positions 280 and 313 of SEQ ID No.1, more preferably, are encoded by the nucleotide sequence at nucleotide positions 838 and 939 of SEQ ID No.6,

more preferably, the topological domain and transmembrane domain sequences of DAP10 comprise the amino acid sequences 262-311 of SEQ ID No.3, and more preferably are encoded by the nucleotide sequences 784-933 of SEQ ID No. 8.

4. A CAR according to any one of claims 1 to 3 wherein the specific target antigen against which the extracellular antigen-binding domain is directed comprises: any one or at least two of CD19, CD20, CD22, CD30, CD33, CD123, CD138, CD171, BCMA, ROR1, WT-1, Pr3, erB-B2, MUC1, LeY, PSCA, EGP-40, HER-2, TAG-72, CD44v7/8, MAGE-A1, GD3, CAIX, NY-ESO-1, GD2, EGFRIII, FAP, alpha folate receptor, IL-13R-a2, EGP-2, mesothelin, KDR, VEGF-R2, PSMA, CEA, glypican-3, IL-13; preferably, CD19, more preferably, the sequence of said extracellular antigen-binding domain comprises the amino acid sequence from position 28 to 269 of SEQ ID No.1, more preferably, encoded by the nucleotide sequence from position 82 to 807 of SEQ ID No. 6.

5. A CAR according to any of claims 1 to 4, characterized in that at the N-terminus of the extracellular antigen-binding domain it further comprises a signal peptide, preferably the signal peptide is a signal peptide of DAP12, DAP10, CD8 a, or CD28, more preferably the signal peptide of DAP12 comprises the amino acid sequence from position 1 to 27 of SEQ ID No.1, more preferably encoded by the nucleotide sequence from position 1 to 81 of SEQ ID No.6, more preferably the signal peptide of DAP10 comprises the amino acid sequence from position 1 to 19 of SEQ ID No.3, more preferably encoded by the nucleotide sequence from position 1 to 57 of SEQ ID No.8, more preferably the signal peptide of CD8 a comprises the amino acid sequence from position 1 to 21 of SEQ ID No.2, more preferably encoded by the nucleotide sequence from position 1 to 63 of SEQ ID No. 7;

and/or, the CAR further comprises a protein Tag sequence, the protein Tag comprises any one or at least two of MyC, His, GST, HA, Flag, MBP, Avi Tag, SUMO, c-MyC Tag; preferably, c-Myc tag; preferably, the protein tag sequence is located between the extracellular antigen-binding domain and the hinge and transmembrane domains;

and/or, further comprising, after the coding sequence of the intracellular signaling domain, the coding sequence of a protein: a self-cleaving protein, and a cytokine or suicide gene;

preferably, the amino acid sequence of the CAR comprises: the sequence of SEQ ID No.1 excluding position 270-279, the sequence of SEQ ID No.1, SEQ ID No.3, SEQ ID No.4 excluding position 420-475, the sequence of SEQ ID No.4, SEQ ID No.5 excluding position 400-455, or SEQ ID No. 5.

6. A nucleic acid molecule encoding the CAR of any one of claims 1-5;

preferably, the sequence of the nucleic acid molecule comprises: the sequence of SEQ ID No.6 excluding position 808-837, the sequence of SEQ ID No.6, SEQ ID No.8, SEQ ID No.9 excluding position 1258-1425, the sequence of SEQ ID No.9, SEQ ID No.10 excluding position 1198-1365, or SEQ ID No. 10.

7. A recombinant vector comprising the nucleic acid molecule according to claim 6, wherein the recombinant vector is a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a simian viral vector, a vaccinia viral vector, a Sendai viral vector, an EB viral vector, a herpes simplex viral vector, a sleeping beauty transposon system, or a plasmid, preferably a lentiviral vector.

8. A recombinant cell or recombinant bacterium, characterized in that: the recombinant cell or the recombinant bacterium comprising the CAR of any one of claims 1-5, or comprising the nucleic acid molecule of claim 6, or comprising the recombinant vector of claim 7;

preferably, the recombinant cell is an immune cell, preferably, a T cell, more preferably, a CIK cell, more preferably, CD3⁺CD56⁺The CIK cell of (1).

9. Use of a CAR according to any one of claims 1 to 5, or comprising a nucleic acid molecule according to claim 6, or comprising a recombinant vector according to claim 7, for the preparation of an immune cell with reduced cytokine storm;

preferably, the cytokine is IL-6,

preferably, the immune cell is a T cell, more preferably, a CIK cell, more preferably, CD3⁺CD56⁺The CIK cell of (1).

10. Use of a CAR according to any one of claims 1 to 5, a nucleic acid molecule according to claim 6, a recombinant vector according to claim 7, or a recombinant cell or recombinant bacterium according to claim 8 for the manufacture of a product for the treatment of a tumour.

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