CN112048021A - A chimeric antigen receptor targeting ROR2, expression gene, expression vector, T cell and application thereof - Google Patents

Abstract

The present invention provides a chimeric antigen receptor targeting ROR2, an expression gene, an expression vector, T cells and applications thereof. Anti-ROR2 chimeric antigen receptor with co-stimulator domain and intracellular signaling domain, and prepared CAR-T cells targeting ROR2, which have been verified in cell and animal experiments to have a significant therapeutic effect on ROR2-expressing tumors .

Description

A kind of chimeric antigen receptor targeting ROR2, expression gene, expression vector, T cell and application thereof

technical field

The invention relates to the field of biotechnology, in particular to a chimeric antigen receptor targeting ROR2, an expression gene, an expression vector, a T cell and applications thereof.

Background technique

Chimeric antigen receptor T cell (CAR-T) therapy is an emerging method for the treatment of malignant tumors, which has a very high cure rate in the treatment of some types of leukemia and lymphoma. In 2017, the U.S. FDA has approved 2 CAR-T cell therapies for the market. However, the current mature CAR-T cell therapies mainly target the B cell surface marker CD19, so the types of tumors that can be treated with this therapy are also greatly restricted. At present, many studies are developing new targets suitable for CAR-T cell therapy, in order to apply CAR-T cell therapy to a wider range of tumor types.

Receptor tyrosine kinase-like orphan receptor-2 (ROR2) is an important development-related regulatory protein, which is highly expressed in various tissues in early embryonic development. It plays an important role in proliferation and differentiation. After the second trimester, the expression level of ROR2 gradually decreased, except in some osteoblasts and uterine cells, ROR2 was basically not expressed in healthy adult tissues.

Current research shows that ROR2 is highly expressed in many tumor tissues, including osteosarcoma, melanoma, colorectal cancer, gastric cancer, breast cancer and other cancers, and cancer cells with higher ROR2 expression levels are more malignant and more likely to occur Metastasis, recurrence, the worse the prognosis. Therefore, many scholars believe that ROR2 is a tumor-related gene that can serve as an ideal drug target. However, there is currently no CAR-T cell therapy targeting ROR2 in the treatment of malignant tumors.

SUMMARY OF THE INVENTION

The main purpose of the present invention is to provide a chimeric antigen receptor targeting ROR2, aiming to construct a CAR-T cell therapy for tumors with high expression of ROR2, and its effectiveness and safety in the treatment of malignant tumors to evaluate.

In order to achieve the above object, the first aspect of the present invention provides an anti-ROR2 chimeric antigen receptor, including an anti-ROR2 single-chain antibody, and the anti-ROR2 single-chain antibody includes at least one of the following:

(1) a light chain variable region with amino acid sequences shown in CSSSSSVSYMHWYQ, IYDTSKLAS and CQQWSSNPPTFGAG, and a heavy chain variable region with amino acid sequences shown in YTITSYLMHWV, LEWIGYINPYN DGTKYNEKFKDKAT and CARSDVYYGVRFAYWGQG;

(2) a light chain variable region with amino acid sequences shown in CSSSSSVTYTYWYQ, IYDTSNLAS and CQQWSSYPFTFGSG, and a heavy chain variable region with amino acid sequences shown in YTFTSYLMHWV, LEWIGYINPYNDGTKYNEKFKDKAT and CARSDVYYGVRFAYWGQG;

or an amino acid sequence with at least one conservative amino acid substitution compared to (1) or (2);

Optionally, the anti-ROR2 single-chain antibody includes at least one of the following:

(a) the light chain variable region having the amino acid sequence shown in SEQ ID NO.1 and the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.2;

(b) a light chain variable region having the amino acid sequence shown in SEQ ID NO.3 and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO.4;

Or an amino acid sequence having at least one conservative amino acid substitution compared to (a) or (b).

In one embodiment, the chimeric antigen receptor comprises a linked signal peptide sequence, an anti-ROR2 single-chain antibody region, a transmembrane region, a costimulatory factor domain, and an intramembrane signal domain; wherein

The anti-ROR2 single-chain antibody region comprises an amino acid linking peptide linking the amino acid sequence of the anti-ROR2 single-chain antibody heavy chain variable region and the anti-ROR2 single-chain antibody light chain variable region;

Optionally, the transmembrane region includes at least one of the following:

CD4 transmembrane domain, CD8 transmembrane domain, CD28 transmembrane domain;

Optionally, the costimulator domain includes at least one of the following:

4-1BB costimulator domain, CD28 costimulator domain, ICOS costimulator domain, OX40 costimulator domain, CD27 costimulator domain;

Optionally, the intramembrane signaling domain includes at least one of the following:

CD3ζ chain intracellular signaling domain, Fc receptor FcεRIγ chain.

Optionally, the chimeric antigen receptor also includes a hinge region, and the hinge region includes at least one of the following:

CD8 hinge region, CD28 hinge region, IgG hinge region.

Optionally, the chimeric antigen receptor also includes regulatory elements or cytokines; wherein,

The regulatory element comprises an inducible Caspase9 suicide regulatory module;

The cytokine includes at least one of the following:

IL-7, CCL19, IL-15.

The second aspect of the present invention provides an expression gene of an anti-ROR2 chimeric antigen receptor, the gene encoding the anti-ROR2 chimeric antigen receptor described in the first aspect.

In one embodiment, the nucleotide sequence of the expression gene of the anti-ROR2 chimeric antigen receptor is selected from the group consisting of SEQ ID NO. Compared with the nucleotide sequence shown in .8, a sequence with more than 90% homology, optionally a sequence with more than 95% homology, preferably a sequence with more than 98% homology, more preferably a sequence with 99% homology % sequence homology above.

The third aspect of the present invention provides an expression vector, the expression vector is inserted with the expression gene described in the second aspect, or the expression vector can cause the host cell to express the expression of the first aspect after the host cell is transfected. The anti-ROR2 chimeric antigen receptor.

The fourth aspect of the present invention provides a T cell expressing an anti-ROR2 chimeric antigen receptor, wherein the T cell can express the anti-ROR2 chimeric antigen receptor described in the first aspect.

The fifth aspect of the present invention provides a pharmaceutical composition, the active ingredient of the pharmaceutical composition comprises the T cells expressing the anti-ROR2 chimeric antigen receptor according to the fourth aspect.

The sixth aspect of the present invention provides the anti-ROR2 chimeric antigen receptor according to the first aspect, the expression gene of the anti-ROR2 chimeric antigen receptor according to the second aspect, the expression vector according to the third aspect, and the third aspect. Use of the T cells expressing the anti-ROR2 chimeric antigen receptor described in the fourth aspect in the preparation of a medicament for treating tumors.

The chimeric antigen receptor targeting ROR2 in the technical solution of the present invention is based on the ROR2 antibody structure independently developed by our laboratory, and the antibody has high specificity and high affinity.

The technical solution of the present invention is to construct an anti-ROR2 chimeric antigen receptor comprising a linked signal peptide sequence, an anti-ROR2 single-chain antibody region, a transmembrane region, a costimulatory factor domain and an intracellular signal domain, and prepare a targeting ROR2 The CAR-T cells have a significant therapeutic effect on tumors with high ROR2 expression in cell and animal experiments.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention, and for those of ordinary skill in the art, other drawings can also be obtained according to the structures shown in these drawings without creative efforts.

1 is a schematic structural diagram of an embodiment of an anti-ROR2 chimeric antigen receptor of the present invention;

Figure 2 is a graph showing the relative quantitative results of the expression level of ROR2 in peripheral blood mononuclear cells of healthy human beings;

Figure 3 is a graph showing the expression level of ROR2 protein in leukemia cells and healthy human peripheral blood mononuclear cells;

Figure 4 is a graph showing the expression level of ROR2 protein in bone marrow samples of leukemia patients and peripheral blood mononuclear cells of healthy people;

Figure 5 is a graph showing the expression level of ROR2 protein in a mouse model of lymphoma;

6 is a graph showing the results of the expression of CAR molecules in the CART cells constructed in an embodiment of the present invention;

FIG. 7 is a graph showing the result of binding ability between CART cells and target cells constructed in an embodiment of the present invention;

8-11 are graphs showing the results of in vitro killing ability of CART cells constructed in an embodiment of the present invention on different cell lines and clinical bone marrow samples;

Fig. 12 is a graph showing the experimental results of the CART cell treatment of leukemia model mice constructed in an embodiment of the present invention;

Figure 13 is a graph showing the experimental results of the CART cells constructed in an embodiment of the present invention for treating lymphoma model mice.

The realization, functional characteristics and advantages of the present invention will be further described with reference to the accompanying drawings in conjunction with the embodiments.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

The term "chimeric antigen receptor (CAR)" is the core component of CAR-T, conferring the ability of T cells to recognize tumor antigens in an MHC-independent manner. The basic design of CAR includes a tumor associated antigen (TAA) binding region (usually derived from the scFv segment of the antigen binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region and an intracellular signal Area.

The term "antibody" is an immunoglobulin molecule capable of binding to a specific antigen. It consists of two light chains with lighter molecular weight and two heavy chains with heavier molecular weight. The heavy chain (H chain) and the light chain (L chain) are connected by disulfide bonds to form a tetrapeptide chain molecule. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly, which is called the variable region (V region), and the carboxyl-terminal (C-terminal) is relatively stable and has little change, which is called the constant region (C region). The V regions of the L and H chains are called VL and VH, respectively.

In the variable region, the amino acid composition and sequence of certain regions have a higher degree of variation, which is called hypervariable region (Hypervariable region, HVR). Complementarity-determining region (CDR). There are three CDR regions on both the heavy chain variable region and the light chain variable region.

The term "single-chain antibody (scFv)" is formed by linking the variable region of the heavy chain and the variable region of the light chain of an antibody through a flexible short peptide (linker) composed of 10-25 amino acids, which is the smallest form of recombinant antibody. The small molecular size of single-chain antibodies brings the advantages of strong intratumoral penetration, rapid degradation in blood, and small negative feedback in the human body.

In some embodiments, the first aspect of the present invention provides an anti-ROR2 chimeric antigen receptor, including an anti-ROR2 single-chain antibody, the anti-ROR2 single-chain antibody has CDR sequences CSSSSSVSYMHWYQ, IYDTSKLAS and CQQWSSNPPTFGAG, respectively The light chain variable region of the indicated amino acid sequence, and the heavy chain variable region having the amino acid sequences indicated by the CDR sequences YTITSYLMHWV, LEWIGYI NPYNDGTKYNEKFKDKAT and CARSDVYYGVRFAYWGQG, respectively.

In other embodiments, the anti-ROR2 single-chain antibody has a light chain variable region whose CDR sequences are CSSSSSVTYTYWYQ, IYDTSNLAS, and CQQWSSYPFTFGSG, respectively, and a light chain variable region whose CDR sequences are YTFTSYLMHWV, LEWIGYINP YNDGTKYNEKFKDKAT, and CARSDVYYGVRFAYWGQG, respectively The heavy chain variable region of the amino acid sequence.

In other embodiments, the anti-ROR2 single-chain antibody can also be an amino acid sequence with at least one conservative amino acid substitution with the above-mentioned light chain variable region and heavy chain variable region. It is understood that the at least one conservative amino acid substitution may be a conservative amino acid substitution of the CDR sequences of the light chain variable region and the heavy chain variable region, or may be in addition to the CDR sequences of the light chain variable region and the heavy chain variable region. Conservative amino acid substitutions of the sequence. At least one conservative amino acid substitution includes one, two, three, four, five, etc. The number of conservative amino acid substitutions is not limited, as long as the function of the anti-ROR2 single-chain antibody after the conservative amino acid substitution remains unchanged. within the protection scope of the invention scheme.

In some embodiments, the anti-ROR2 single chain antibody has a light chain variable region of the amino acid sequence shown in SEQ ID NO.1 and a heavy chain variable region of the amino acid sequence shown in SEQ ID NO.2. In other embodiments, the anti-ROR2 single chain antibody has the light chain variable region of the amino acid sequence shown in SEQ ID NO.3 and the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.4.

In other embodiments, the anti-ROR2 single-chain antibody can also be an amino acid sequence with at least one conservative amino acid substitution with the above-mentioned light chain variable region and heavy chain variable region. It can be understood that at least one conservative amino acid substitution can also be a conservative amino acid substitution of the CDR sequences of the light chain variable region and the heavy chain variable region, or it can be the difference between the light chain variable region and the heavy chain variable region except the CDR sequences. Conservative amino acid substitutions outside the sequence. At least one conservative amino acid substitution includes one, two, three, four, five, etc. The number of conservative amino acid substitutions is not limited, as long as the function of the anti-ROR2 single-chain antibody after the conservative amino acid substitution remains unchanged. within the protection scope of the invention scheme.

The chimeric antigen receptor targeting ROR2 in the technical solution of the present invention is based on the ROR2 antibody structures A12 and B16 independently developed by our laboratory, and the antibody has high specificity and high affinity. Specifically, the light chain variable region of the amino acid sequence shown in SEQ ID NO.1 and the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.2 are the light chain variable region and the heavy chain of the anti-ROR2 antibody A12, respectively. variable region. The light chain variable region of the amino acid sequence shown in SEQ ID NO.3 and the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.4 are the light chain variable region and the heavy chain variable region of the anti-ROR2 antibody B16, respectively.

In some embodiments, the chimeric antigen receptor comprises a linked signal peptide sequence, an anti-ROR2 single-chain antibody region, a transmembrane region, a costimulator domain, and an intracellular signaling domain; wherein the anti-ROR2 The single-chain antibody region includes an amino acid linking peptide linking the amino acid sequence of the anti-ROR2 single-chain antibody heavy chain variable region and the anti-ROR2 single-chain antibody light chain variable region.

Specifically, the various parts of the chimeric antigen receptor may or may not be sequentially connected, as long as the anti-ROR2 chimeric antigen receptor can achieve its function.

Specifically, the signal peptide sequence is located outside the T cell after expression. The function of the signal peptide is to guide different proteins to reach different parts of the cell, such as the endoplasmic reticulum, lysosome, and cell membrane. The function of the signal peptide is to allow the CAR molecule to be expressed in the On the cell membrane, usually the CD8 signal peptide.

Optionally, the amino acid linker peptide may be an 18 amino acid (G4S3) linker peptide, a 218 (Whitlow) linker peptide, and the like.

Specifically, the amino acid linking peptide connects the variable region of the heavy chain of the anti-ROR2 single-chain antibody and the variable region of the light chain of the single-chain antibody of the anti-ROR2 can be the variable region of the heavy chain of the anti-ROR2 single-chain antibody, and the variable region of the single-chain antibody of the anti-ROR2 The structure in which the variable region of the light chain of the chain antibody is followed may also be a structure in which the variable region of the light chain of the anti-ROR2 single-chain antibody precedes and the variable region of the heavy chain of the anti-ROR2 single-chain antibody follows.

Optionally, the transmembrane region may be a CD4 transmembrane region, a CD8 transmembrane region, a CD28 transmembrane region, and the like.

Optionally, the costimulator domain can be at least one of the following: 4-1BB costimulator domain, CD28 costimulator domain, ICOS costimulator domain, OX40 costimulator domain, CD27 costimulator domain Stimulator domain.

Among them, the costimulatory factor domain of the second-generation CAR molecular structure is one of them, and the costimulatory factor domain of the third-generation CAR molecular structure is at least two of them. CD28 and 4-1BB are the most commonly involved costimulatory factors in CAR-T research. Molecularly, CD28 may significantly enhance the ability of CAR-T cells to kill tumor cells, while 4-1BB can prolong the survival time of CAR-T cells.

Optionally, the intracellular signaling domain can be the intracellular signaling domain of CD3ζ chain, Fc receptor FcεRIγ chain, and the like.

In some embodiments, the chimeric antigen receptor further includes a hinge region, optionally, the hinge region may be a CD8 hinge region, a CD28 hinge region, an IgG hinge region, or the like. After the hinge region is expressed, it is located between the anti-ROR2 single-chain antibody and the cell membrane. This region provides more flexibility for the CAR molecule to bind to the target antigen. It can be understood that, in some embodiments, the chimeric antigen receptor may not include a hinge region, as long as the chimeric antigen receptor can fulfill its function.

In some embodiments, the chimeric antigen receptor further includes a regulatory element or cytokine; wherein the regulatory element includes an inducible Caspase9 suicide regulatory module; the cytokine can be IL-7, CCL19, IL-15 Wait.

Specifically, other regulatory elements, such as inducible Caspase9 suicide regulation modules, or cytokines such as IL-7, CCL19, IL-15, etc., are added on the basis of the second-generation CAR molecule or the third-generation CAR molecular structure to enhance the ability of T cells to tissue infiltration.

In one embodiment, the chimeric antigen receptor comprises a sequence of a CD8 signal peptide, an anti-ROR2 antibody scFv light chain variable region, an 18 amino acid linker peptide, an anti-ROR2 antibody scFv heavy chain variable region, and a CD8 hinge region. , CD8 transmembrane region, 4-1BB costimulatory factor domain and CD3ζ chain signaling domain, see FIG. 1 , which is a schematic structural diagram of an anti-ROR2 chimeric antigen receptor according to an embodiment of the present invention. The amino acid sequence of the anti-ROR2 chimeric antigen receptor is selected from SEQ ID NO.5 or SEQ ID NO.6, and compared with SEQ ID NO.5 or SEQ ID NO.6, it has at least one conservative amino acid substitution amino acid sequence.

The technical solution of the present invention is constructed by constructing a linked signal peptide sequence, an anti-ROR2 single-chain antibody region (including the anti-ROR2 antibody scFv light chain variable region in front of the heavy chain variable region, and the heavy chain variable region in front of the light chain) Variable region in the latter two cases), hinge region, transmembrane region, costimulatory factor domain and anti-ROR2 chimeric antigen receptor of intracellular signal domain, wherein anti-ROR2 antibody scFv light chain variable region, connecting peptide The scFv heavy chain variable region of the anti-ROR2 antibody constitutes an anti-ROR2 single-chain antibody, and the chimeric antigen receptor is used to prepare CAR-T cells targeting ROR2, and its effect on ROR2-expressing tumors has been verified in cell and animal experiments. treatment effect is remarkable.

The second aspect of the present invention also provides an expression gene of an anti-ROR2 chimeric antigen receptor, the gene encoding the anti-ROR2 chimeric antigen receptor described in the first aspect.

In one embodiment, the nucleotide sequence of the expression gene of the anti-ROR2 chimeric antigen receptor is selected from the group consisting of SEQ ID NO. Compared with the nucleotide sequence shown in .8, a sequence with more than 90% homology, optionally a sequence with more than 95% homology, preferably a sequence with more than 98% homology, more preferably a sequence with 99% homology % sequence homology above.

The third aspect of the present invention provides an expression vector, the expression vector is inserted with the expression gene described in the second aspect, or the expression vector can cause the host cell to express the expression of the first aspect after the host cell is transfected. The anti-ROR2 chimeric antigen receptor.

In one embodiment, the expression vector is a lentiviral expression vector.

Lentiviral vector refers to a viral vector derived from human immunodeficiency virus-1 (HIV-1). The lentiviral vector contains the genetic information required for packaging, transfection, and stable integration, and is the main component of the lentiviral vector system. part. Lentiviral expression vectors carrying foreign genes are packaged into infectious virus particles with the assistance of lentiviral packaging plasmids and cell lines. By infecting cells or living tissues, the exogenous genes can be stored in cells or living tissues. Express.

The fourth aspect of the present invention provides a T cell expressing an anti-ROR2 chimeric antigen receptor, wherein the T cell can express the anti-ROR2 chimeric antigen receptor described in the first aspect.

Specifically, the preparation method of the T cells expressing the anti-ROR2 chimeric antigen receptor can be by lentivirus infection of T cells to introduce the expression gene of the anti-ROR2 chimeric antigen receptor, or CRISPR gene editing technology can be used to modify the T cells. The cells were edited to knock in the expression gene of the anti-ROR2 chimeric antigen receptor at the TCR locus.

Specifically, CRISPR technology is used to edit T cells, and the anti-ROR2 CAR molecular sequence is knocked in at the TCR locus, so that not only the CAR sequence is expressed, but also the interaction of the donor's TCR-MHC molecule is destroyed, thereby reducing the graft. Against the risk of host disease, the source of T cells used to prepare CAR-T cells is not limited to the patient itself, and the scope of application can be expanded.

The fifth aspect of the present invention provides a pharmaceutical composition, the active ingredient of the pharmaceutical composition comprises the T cells expressing the anti-ROR2 chimeric antigen receptor according to the fourth aspect.

The sixth aspect of the present invention provides the anti-ROR2 chimeric antigen receptor according to the first aspect, the expression gene of the anti-ROR2 chimeric antigen receptor according to the second aspect, the expression vector according to the third aspect, and the third aspect. Use of the T cells expressing the anti-ROR2 chimeric antigen receptor described in the fourth aspect in the preparation of a medicament for treating tumors.

In one embodiment, the tumor is acute myeloid leukemia or lymphoma.

The embodiments of the present invention will be described in detail below in conjunction with specific examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

Example 1 Expression and distribution of ROR2 in blood cancer

The expression of ROR2 in malignant tumors determines the effectiveness of ROR2-targeting CAR-T cell therapy. Since CAR-T cell therapy has outstanding therapeutic effect in hematological diseases, we first detected ROR2 in hematological cancer and leukemia. To further study the efficacy of CAR-T cell therapy targeting ROR2 on leukemia.

First, the expression of ROR2 mRNA in leukemia cell lines and healthy human peripheral blood was detected. We selected 3 acute lymphoblastic leukemia (ALL) cell lines (REH, RS4; 11, CCRF), 1 chronic myeloid leukemia (CML) cell line K562, and 1 acute myeloid leukemia (AML) cell line THP -1, and magnetic bead sorting (Miltenyi Biotec, 130-097-054) of peripheral blood mononuclear cells (PBMC) from healthy volunteers to obtain T cells, B cells and monocytes, respectively, and then RNA extraction was performed on the samples using an RNA extraction kit (Kang Weishi, CW0560), and cDNA was obtained by reverse transcription of the extracted RNA using a reverse transcription kit (Promega, A2791). Using the obtained cDNA as a template, primers were designed to amplify the ROR2 gene, and β-actin was used as an internal reference to perform gel electrophoresis on the PCR results. The PCR amplification primer sequences of the ROR2 gene used were: upstream primer: GAATATGGTTCACGACTGCG, downstream primer: CAGTTCCATGCGTACGTTTT.

The experimental results are shown in Figure 2. There is weak ROR2 mRNA expression in monocytes and T cells, but no ROR2 expression in B cells, while CML cell line K562 and AML cell line THP-1 have higher levels of ROR2 mRNA Express.

The expression of ROR2 was further verified at the protein level. The Santa Cruz anti-human ROR2 antibody (sc-374174) was used for immunohybridization, GAPDH was used as an internal control, and the wild-type CML cell line K562 was used as a positive control to use CRISPR. -K562 cells in which ROR2 expression was knocked out by Cas9 technology (marked as: K562-R2KO) were used as a negative control. The results showed that T cells, B cells and monocytes of healthy people did not express ROR2 at the protein level. The acute lymphoblastic leukemia human T-ALL cell line Jurkat has higher levels of ROR2 protein expression, as shown in Figure 3.

At the same time, immunoprecipitation-immunoblotting experiments were performed on clinical bone marrow samples of various leukemia people to detect the expression of ROR2. 3 cases of acute myeloid leukemia human bone marrow samples (marked as: AML-1, AML-2, AML-3) and 1 case of chronic myeloid leukemia human bone marrow samples (marked as: CML) were obtained from cooperative hospitals, B cell malignant transformation 1 case of human bone marrow samples caused by acute lymphoblastic leukemia (marked as: B-ALL), wild-type human embryonic kidney cells HEK293T (marked as: 293T) cells were used as positive controls to knock out ROR2 expression using CRISPR-Cas9 technology. HEK293T cells (marked as: 293T-ROR2KO) were used as negative control, immunoprecipitated with ROR2 antibody (marked as: A12) developed by our laboratory, and commercialized ROR2 antibody (Santa Cruz, sc-374174) (marked as: ROR2) was subjected to immunoblotting, and the results are shown in Figure 4. Compared with PBMCs from two healthy donors, ROR2 was up-regulated in AML, B-ALL, T-ALL and CML samples.

For lymphoma samples, we detected the protein of ROR2 in 2 tumor samples (labeled as LY0001 and LY0002) in 2 tumor samples (labeled as LY0001 and LY0002) of established lymphoma patient-derived xenograft (PDX) mice by immunoprecipitation-immunoblotting method. Expression, with wild-type CML cell line K562 as a positive control, the results are shown in Figure 5, showing that there is expression of ROR2 in mouse tumor samples, but the expression level is weaker than that of lymphoma cell line Jeko-1.

The above experiments show that ROR2 is highly expressed in tumor cells, while low or almost no expression of ROR2 in normal cells, indicating that ROR2 can be used as a target for tumor therapy, and its safety is reliable.

Example 2 Preparation of CAR-T cells targeting ROR2

2.1 Lentiviral packaging of CAR molecules targeting ROR2

The DNA sequences of A12-CAR molecule (SEQ ID NO.7) and B16-CAR molecule (SEQ ID NO.8) were cloned into the multi-cloning site of lentiviral expression vector CD532A, and verified by double-enzyme digestion and electrophoresis with NehⅠ and EcoRI , and performed next-generation sequencing to identify positive clones. The positive clones were selected and transformed into Stbl3 E. coli competent amplification plasmids, and the plasmids were extracted with endotoxin removal kit (qiagen). The extracted CAR expression plasmid was co-transfected with the packaging plasmids psPAX and PMD2.G into 293T cells by PEI transfection method for lentiviral packaging.

The day before transfection, 293T cells were seeded in a 10 cm culture dish, and the confluence of the cells was about 50% after 24 h. The next day, the PEI method was used for transfection, the total amount of plasmid was 16 μg, and the ratio was CAR expression plasmid: psPAX:PMD2.G=4:3:1, and the amount (volume) of PEI was 3 times the total amount of plasmid, i.e. 48 μl. 8 hours after transfection, 8 ml of new medium was changed. After 48 hours, the virus supernatant was collected, and new 8ml medium was added to continue culturing for 24 hours. The virus supernatant was collected again and mixed with the previously collected virus, and the virus was concentrated with virus concentrate (Syngentech, 306011). The virus packaged per 10 cm dish can infect 1.5x10 ⁵ primary T cells after concentration, and the infection method is described below.

2.2 Virus-transfected T cells packaged with CAR molecules

Human T-Activator CD3/CD28(Invitrogen,11161D)磁珠对得到的T细胞进行激活，每1x10⁶个细胞添加25μl磁珠，激活3天后进行感染，助染试剂采用RetroNectin(TAKARA，T100A)。 ^The peripheral blood (about 5ml) of healthy donors (volunteers) was obtained, and PBMCs were separated by density gradient centrifugation (ficoll method). The obtained peripheral blood mononuclear cells were isolated from CD3+ cells using MojoSort ^™ Human CD3 TCell Isolation Kit, and positive cells were cultured in supplemented 10% FBS (Hyclone), 30U/ml IL-2 (PEPROTECH, AF-200-02) and 1% P/S double antibody in X-VIVO 15 (Lonza, 04-418Q) medium at a density of 1×10 ⁶ cells/ml. with Dynabeads

The obtained T cells were activated with Human T-Activator CD3/CD28 (Invitrogen, 11161D) magnetic beads, 25 μl of magnetic beads were added per 1×10 ⁶ cells, and the infection was carried out after 3 days of activation. RetroNectin (TAKARA, T100A) was used as an auxiliary staining reagent.

Untreated 6-well plates were coated the day before infection: 40 μl RetroNectin diluted in 2 ml PBS was added to plate wells overnight at 4°C. The next day, the supernatant was aspirated, the wells of the plate were blocked with PBS containing 2% BSA, washed once with 2 ml of PBS for 30 min at room temperature, and then the concentrated viruses and controls that packaged the A12-CAR molecule and B16-CAR molecule respectively were added. The CD532A empty virus was loaded with MOI of 5 (approximately corresponding to the virus produced in a 10cm dish before concentration), and PBS was added to make up to 2ml, centrifuged at 2000g for 2 hours at 32°C. After centrifugation, aspirate the supernatant, and carefully add 1ml PBS to wash once, then add activated T cells ^1.5x105 /well, and add T cell culture medium (add 10% FBS, 100U/ml IL-2 and 1% P /S double antibody X-VIVO 15) to 2ml, continue to culture at 37°C, 5% CO2 incubator, adjust the cell density to ^1x106 cells/ml every 1-2 days, and obtain ROR2-A12-CART cells respectively and ROR2-B16-CART cells.

Test Example 1 CAR expression of CAR-T cells targeting ROR2 and verification of the ability to bind target molecules

The expression of CAR molecules in T cells was detected by flow cytometry on the third day after infection. The method was as follows: ^2x105 cells were washed twice with PBS containing 2% FBS; cells were labeled with anti-mouse-Fab antibody (Jackson ImmunoResearch, 115-065-072) diluted 1:400, and incubated on ice for 30 min ; Wash once with PBS containing 2% FBS; incubate with 1:500 dilution of PE-labeled streptavidin R-Phycoerythrin Streptavidin (Jackson ImmunoResearch, 016-110-084) on ice for 15 min, with PBS containing 2% FBS Wash three times; test on the machine. The expression of CAR molecules is shown in Figure 6. The expression rates of A12-CAR and B16-CAR are about 60% and 50%, respectively, and the expression effect is relatively good.

Simultaneously, the edited T cells were incubated with biotinylated ROR2 protein Biotinylated Human ROR2/NTRKR2 Protein, His, Avitag ^TM (AcroBiosystems, RO2-H82E3), 2 μg/ml, incubated on ice for 30 min, and then PE-labeled chain Mycovidin (Jackson ImmunoResearch, 016-110-084) was combined with it (1:500 dilution, incubated on ice for 15 min) to detect the binding ability of ROR2-CAR-T to the target protein ROR2. The results are shown in Figure 7. ROR2-A12-CART and ROR2-B16-CAR-T cells can bind to ROR2 protein, and the binding rate is about 25% and 50%, respectively, and the binding ability to target protein is strong.

Test Example 2 In vitro killing ability verification of CAR-T cells targeting ROR2

KASUMI and THP-1 cells are representative AML cell lines in which ROR2 is negative in KASUMI and expressed at lower levels in THP-1 (see Figures 2, 3). We introduced ROR2 gene into KASUMI and THP-1 cells by lentivirus infection, and constructed ROR2-overexpressing KASUMI-ROR2 and THP-1-ROR2 cells. Using these four cells as the target cells killed by ROR2-CAR-T cells, the in vitro killing ability of ROR2-CAR-T cells was detected by lactate dehydrogenase (LDH) release assay.

The target cells were seeded in a round-bottomed 96-well plate at the number of 1×10 ⁴ cells/well, and the target cell natural release enzyme activity control well and the maximum release enzyme activity control well were set at the same time, and three duplicate wells were set in each group. Background blank control wells (adding medium only), and volume control wells (adding medium and lysate).

非放射性细胞毒性检测试剂盒(Promega，G1780)对杀伤后细胞释放的乳酸脱氢酶进行检测，通过下述计算公式计算细胞毒性百分比。According to the ratio of effector to target (E:T) of 10:1, 1x10 ⁵ effector cells/well (Vector-T, A12-CAR-T, B16-CAR-T) were added to the target cell well, and the cells were mixed with no The medium of phenol red indicator was supplemented to 100ul and placed in a 37°C, 5% CO2 incubator for 12h. Using CytoTox

The non-radioactive cytotoxicity detection kit (Promega, G1780) was used to detect the lactate dehydrogenase released by the cells after killing, and the cytotoxicity percentage was calculated by the following formula.

The calculation formula is:

The killing of KASUMI and KASUMI-ROR2 cells is shown in Figure 8. A12-CAR-T and B16-CAR-T did not kill ROR2-negative KASUMI cells, while A12-CAR-T inhibited ROR2-overexpressing KASUMI-ROR2 cells. The killing rate was about 20%, and the killing rate of B16-CAR-T against ROR2-overexpressing KASUMI-ROR2 cells was about 40%. It shows that the killing of target cells by ROR2-CAR-T is specific and effective.

The killing of THP-1 and THP-1-ROR2 cells is shown in Figure 9. The killing rates of A12-CAR-T and B16-CAR-T on THP-1 cells with weak ROR2 expression are about 20% and 40%; A12 - The killing rate of CAR-T and B16-CAR-T to THP-1-ROR2 cells overexpressing ROR2 is about 60% and 70%, indicating that the killing ability of ROR2-CAR-T cells to target cells is dependent on the target antigen. increased expression.

At the same time, we also verified the in vitro killing ability of bone marrow cells from clinical leukemia patients. Due to the high heterogeneity of cells in clinical samples, we standardized the killing results to make the killing rate of Vector-T cells 10%. The standardized killing rates of A12-CAR-T and B16-CAR-T on bone marrow cells of AML patients were approximately 40% and 60%, see Figure 10.

To clarify the safety of ROR2-CAR-T cells, bone marrow cells (Lonza, 2M-125C) donated by healthy donors were purchased and the effect of ROR2-CAR-T cells on bone marrow of healthy donors was detected by lactate dehydrogenase release assay. The killing of cells, the results are shown in Figure 11, the standardized killing rate of A12-CAR-T and B16-CAR-T on bone marrow cells of healthy donors is less than 10%.

Test Example 3 Treatment of leukemia model mice with CAR-T cells targeting ROR2

We used lentivirus infection to introduce GFP/Luciferase into THP-1 cells to enable bioluminescence to obtain THP-1-GFP/Luc cells, and on this basis, additionally introduced ROR2 gene to construct ROR2 overexpressing cells THP -1-ROR2OE-GFP/Luc. Thus, a leukemia mouse model was constructed. Establishment of leukemia model in NCG immunodeficient mice with THP-1-GFP/Luc cells and THP-1-ROR2OE-GFP/Luc cells: On day 0, 5x10 ⁵ THP-1-GFP/Luc were injected intraperitoneally cells or THP-1-ROR2OE-GFP/Luc cells, 9 THP-1-GFP/Luc cell tumor-bearing mice, and 12 THP-1-ROR2-GFP/Luc cell tumor-bearing mice. The bioluminescence of mice injected with luciferase substrate was detected by a small animal in vivo imaging system, and the groups were evenly grouped according to the fluorescence value. The THP-1-GFP/Luc cell tumor-bearing mice were divided into 3 groups: PBS group, A12-CAR-T cell treatment group, B16-CAR-T cell treatment group, 3 mice in each group; THP-1 -ROR2OE-GFP/Luc cell tumor-bearing mice were divided into 3 groups: PBS group, A12-CAR-T cell treatment group, B16-CAR-T cell treatment group, 4 mice in each group.

^On day 1, CAR-T cell therapy was performed by tail vein injection at 1.5x10 cells per mouse. On the second day, the fluorescence value of mice after injection of luciferase substrate was detected by in vivo imaging of small animals, and then detected twice a week. The detection results are shown in Figure 12.

As can be seen from Figure 12, compared with the control group, A12-CAR-T can significantly inhibit the growth of THP-1-GFP/Luc cells, and B16-CAR-T can also effectively inhibit the growth of THP-1-GFP/Luc cells. Compared with the control group, B16-CAR-T can significantly inhibit the growth of THP-1-ROR2OE-GFP/Luc cells, and A12-CAR-T can also effectively inhibit the growth of THP-1-ROR2OE-GFP/Luc cells. Growth of THP-1-ROR2OE-GFP/Luc cells, but the effect was weaker than that of B16-CAR-T.

Test Example 4 Treatment of lymphoma PDX model mice by CAR-T cells targeting ROR2

The establishment of lymphoma model mice adopts the patient-derived xenograft (PDX) mouse model. Immunoprecipitation-immunoblot detection showed ROR2 expression in both lymphoma PDX mouse tumor samples, see Figure 5. We selected one of them for the construction of the lymphoma PDX mouse model: the second generation PDX tumor samples were digested with Tumor Dissociation Kit (Miltenyi Biotec, 130-095-929), and the cells were subjected to erythrocyte analysis after obtaining a single cell suspension. Lysis and counting, by subcutaneous injection, tumor-bearing NCG mice, 5× ^{10 6} mice per mouse, 3 mice in each group.

T细胞，A12-CAR-T细胞，B16-CAR-T细胞，5x10⁶个/只，治疗后每2～3天测量肿瘤大小，参见图13，结果显示A12-CAR-T和B16-CAR-T细胞治疗能够有效抑制肿瘤大小。The length and width of the tumor were measured with a vernier caliper, and the tumor size was estimated according to the formula that the tumor size was approximately equal to the length x width x width/2. The mice were treated with CAR-T cells when the tumor size was approximately equal to 500 mm ³ . Inject PBS 200 μl/a via tail vein, unedited

T cells, A12-CAR-T cells, B16-CAR-T cells, 5x10 ⁶ cells/cell, tumor size was measured every 2-3 days after treatment, see Figure 13, the results showed that A12-CAR-T and B16-CAR-T T cell therapy can effectively suppress tumor size.

This shows that CAR-T cells targeting ROR2 can inhibit the growth of cancer cells in leukemia mice in vivo, and can also inhibit the growth of tumor cells in lymphoma mice.

The above descriptions are only optional embodiments of the present invention, and are not intended to limit the scope of the present invention. Under the inventive concept of the present invention, any equivalent structural transformations made by using the contents of the description and drawings of the present invention, or direct/indirect Applications in other related technical fields are included in the scope of patent protection of the present invention.

SEQUENCE LISTING

<110> Shenzhen University

<120> A chimeric antigen receptor targeting ROR2, expression gene, expression vector, T cell and application thereof

<130> 2020-08-18

<160> 8

<170> PatentIn version 3.3

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

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

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

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Ile Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

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

1 5 10 15

Ser Val Arg Met Ser Cys Lys Ala Ala Gly Tyr Thr Ile Thr Ser Tyr

20 25 30

Leu Met His Trp Val Lys Gln Arg Pro Gly Gln Asp Leu Glu Trp Ile

35 40 45

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

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Asp Val Tyr Tyr Gly Val Arg Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser

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

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Thr Tyr Thr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

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

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Ser Val Arg Met Ser Cys Lys Ala Ala Gly Tyr Thr Phe Thr Ser Tyr

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Leu Met His Trp Val Lys Gln Arg Pro Gly Gln Asp Leu Glu Trp Ile

35 40 45

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

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Asp Val Tyr Tyr Gly Val Arg Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

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

20 25 30

Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser

35 40 45

Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro

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Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala

65 70 75 80

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

85 90 95

Ser Met Glu Ala Glu Asp Ala Ala Ile Tyr Tyr Cys Gln Gln Trp Ser

100 105 110

Ser Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ala Ser Val Arg Met Ser Cys Lys Ala Ala Gly Tyr Thr Ile Thr Ser

165 170 175

Tyr Leu Met His Trp Val Lys Gln Arg Pro Gly Gln Asp Leu Glu Trp

180 185 190

Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys

195 200 205

Phe Lys Asp Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala

210 215 220

Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr

225 230 235 240

Cys Ala Arg Ser Asp Val Tyr Tyr Gly Val Arg Phe Ala Tyr Trp Gly

245 250 255

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

260 265 270

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

275 280 285

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

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Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

305 310 315 320

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

325 330 335

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

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Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

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Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

370 375 380

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

385 390 395 400

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

405 410 415

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

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

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

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

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Met Gln Ala Leu Pro Pro Arg

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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

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

20 25 30

Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser

35 40 45

Ser Val Thr Tyr Thr Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro

50 55 60

Arg Leu Leu Ile Tyr Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val

65 70 75 80

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

85 90 95

Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Ala Ser Val Arg Met Ser Cys Lys Ala Ala Gly Tyr Thr Phe Thr Ser

165 170 175

Tyr Leu Met His Trp Val Lys Gln Arg Pro Gly Gln Asp Leu Glu Trp

180 185 190

Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys

195 200 205

Phe Lys Asp Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Val

210 215 220

Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr

225 230 235 240

Cys Ala Arg Ser Asp Val Tyr Tyr Gly Val Arg Phe Ala Tyr Trp Gly

245 250 255

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

260 265 270

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

275 280 285

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

290 295 300

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

305 310 315 320

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

325 330 335

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

340 345 350

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

355 360 365

Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

His Met Gln Ala Leu Pro Pro Arg

485

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<211> 1476

<212> DNA

<213> Artificial sequences

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gctagcatgg ccttaccagt gaccgccttg ctcctgccgc tggccttgct gctccacgcc 60

gccaggccgg atatcgttct cactcaaagt cctgctatca tgtcagcctc acctggagag 120

aaggtcacta tgacctgctc agccagtagc tcagtgtcat acatgcattg gtatcagcag 180

aagtctggca ctagccctaa gcgctggatc tacgacacta gcaaactggc cagtggtgtg 240

cctgcacggt tcagcgggag tggtagtggg actagctata gcctgaccat ttccagcatg 300

gaagccgagg acgctgctat ctactactgc cagcagtggt ctagtaaccc accaactttc 360

ggtgcaggga ccaaactcga actgaagaca ggtggcggtg gctcgggcgg tggtgggtcg 420

ggtggcggcg gatctgaggt gcaagtgcaa ctccaacaat ctggtcctga actggttaag 480

cctggtgcct ctgttagaat gtcttgcaaa gccgctggtt acactatcac atcatacctc 540

atgcactggg tcaagcagag acctgggcag gacctggagt ggatcggcta catcaaccct 600

tacaacgacg gcaccaagta caacgaaaag tttaaggata aagctactct gaccagcgat 660

aagtcatcct ccaccgccta catggagctc agctccctga cctcagagga cagtgcagtg 720

tactactgcg ccaggagcga tgtgtactac ggcgttagat tcgcttactg gggacaggga 780

accctggtta ccgtgagtac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 840

atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca 900

gtgcacacga gggggctgga cttcgcctgt gatatctaca tctgggcgcc cttggccggg 960

acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaaacg gggcagaaag 1020

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 1080

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gagagtgaag 1140

ttcagcagga gcgcagacgc ccccgcgtac aagcagggcc agaaccagct ctataacgag 1200

ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260

gagatgggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320

aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380

aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440

cttcacatgc aggccctgcc ccctcgctaa gaattc 1476

<210> 8

<211> 1476

<212> DNA

<213> Artificial sequences

<400> 8

agcatggcct taccagtgac cgccttgctc ctgccgctgg ccttgctgct ccacgccgcc 60

aggccggata tagttctgac acaaagtccc gctatcatga gtgcttcacc aggagagaag 120

gtgacaatga cctgcagcgc ctcatccagt gttacctaca catattggta tcaacagaaa 180

cccggcagct ctcctagact cctcatctat gatacttcta acctggcctc tggcgtccca 240

gtcaggttct ctggctcagg cagtgggact tcttacagtc tgaccatcag tagaatggag 300

gcagaggacg cagctacata ctactgccag cagtggtcta gctatccatt cacttttggc 360

tccggaacta aactcgaaat caagacaggt ggcggtggct cgggcggtgg tgggtcgggt 420

ggcggcggat ctgaggtgca actccaactg cagcaatctg gtcctgaact ggttaagcct 480

ggagcatcag tgcgcatgag ttgcaaggct gcaggatata ctttcactag ctatctgatg 540

cactgggtca aacagagacc tgggcaggat ctggaatgga tcgggtacat aaacccatat 600

aatgatggaa ccaagtacaa cgagaaattc aaggacaagg ccaccctcac ctccgacaag 660

agttcatcta ccgtgtatat ggagctgtca tccctgacct cagaggactc cgctgtgtat 720

tactgcgcaa ggagtgatgt ttactacggc gttcggttcg cctattgggg acaggggaca 780

ctcgtgaccg tctctgcaac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 840

atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca 900

gtgcacacga gggggctgga cttcgcctgt gatatctaca tctgggcgcc cttggccggg 960

acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaaacg gggcagaaag 1020

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 1080

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gagagtgaag 1140

ttcagcagga gcgcagacgc ccccgcgtac aagcagggcc agaaccagct ctataacgag 1200

ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260

gagatgggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320

aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380

aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440

cttcacatgc aggccctgcc ccctcgctaa gaattc 1476

Claims (10)

1. A chimeric antigen receptor against ROR2, comprising a single chain antibody against ROR2, said single chain antibody against ROR2 comprising at least one of:

(1) a light chain variable region having an amino acid sequence set forth in CSASSSVSYMHWYQ, IYDTSKLAS and CQQWSSNPPTFGAG, and a heavy chain variable region having an amino acid sequence set forth in YTITSYLMHWV, LEWIGYINPYNDGTKYNEKFKDKAT and CARSDVYYGVRFAYWGQG;

(2) a light chain variable region having an amino acid sequence set forth in CSASSSVTYTYWYQ, IYDTSNLAS and CQQWSSYPFTFGSG, and a heavy chain variable region having an amino acid sequence set forth in YTFTSYLMHWV, LEWIGYINPYNDGTKYNEKFKDKAT and CARSDVYYGVRFAYWGQG;

or an amino acid sequence having at least one conservative amino acid substitution as compared to (1) or (2);

optionally, the single chain antibody against ROR2 comprises at least one of:

(a) a light chain variable region having an amino acid sequence shown in SEQ ID No.1 and a heavy chain variable region having an amino acid sequence shown in SEQ ID No. 2;

(b) a light chain variable region having an amino acid sequence shown in SEQ ID No.3 and a heavy chain variable region having an amino acid sequence shown in SEQ ID No. 4;

or an amino acid sequence having at least one conservative amino acid substitution as compared to (a) or (b).

2. The chimeric antigen receptor against ROR2, according to claim 1, wherein the chimeric antigen receptor comprises linked signal peptide sequence, single-chain antibody region against ROR2, transmembrane region, costimulatory factor domain, and intracellular signal region; wherein

The anti-ROR 2 scFv region comprises an amino acid sequence of an amino acid linker peptide linking the scFv of the anti-ROR 2 scFv to the scFv of the anti-ROR 2 scFv;

optionally, the transmembrane region comprises at least one of:

a CD4 transmembrane region, a CD8 transmembrane region, a CD28 transmembrane region;

optionally, the co-stimulatory factor domain comprises at least one of:

4-1BB co-stimulatory factor domain, CD28 co-stimulatory factor domain, ICOS co-stimulatory factor domain, OX40 co-stimulatory factor domain, CD27 co-stimulatory factor domain;

optionally, the intracellular signaling domain comprises at least one of:

intracellular signal domain of CD3 zeta chain, Fc receptor FcRI gamma chain.

3. The chimeric antigen receptor of claim 2, which is resistant to ROR2, further comprising a hinge region, wherein the hinge region comprises at least one of:

CD8 hinge region, CD28 hinge region, IgG hinge region.

4. The chimeric antigen receptor against ROR2, according to claim 2 or 3, further comprising a regulatory element or cytokine; wherein,

the regulatory element comprises an inducible Caspase9 suicide regulatory module;

the cytokine includes at least one of:

IL-7，CCL19，IL-15。

5. an expressed gene of a chimeric antigen receptor against ROR2, wherein the gene encodes a chimeric antigen receptor against ROR2 as claimed in any one of claims 1 to 4.

6. The ROR 2-resistant chimeric antigen receptor expression gene as claimed in claim 5, wherein the ROR 2-resistant chimeric antigen receptor expression gene has a nucleotide sequence selected from the group consisting of a sequence shown in SEQ ID No.7 or SEQ ID No.8, and a sequence having homology of 90% or more, optionally a sequence having homology of 95% or more, preferably a sequence having homology of 98% or more, and more preferably a sequence having homology of 99% or more compared with the nucleotide sequence shown in SEQ ID No.7 or SEQ ID No. 8.

7. An expression vector having the expression gene of claim 5 or 6 inserted therein, or capable of causing a host cell to express the chimeric antigen receptor against ROR2 of any one of claims 1 to 4 after transfection of the host cell.

8. A T cell expressing a chimeric antigen receptor against ROR2, wherein the T cell is capable of expressing the chimeric antigen receptor against ROR2 as claimed in any one of claims 1 to 4.

9. A pharmaceutical composition comprising the T cell of claim 8 expressing the chimeric antigen receptor against ROR2 as an active ingredient.

10. Use of the anti-ROR 2 chimeric antigen receptor of any one of claims 1 to 4, the anti-ROR 2 chimeric antigen receptor expression gene of claim 5 or 6, the expression vector of claim 7, the anti-ROR 2 chimeric antigen receptor expressing T cell of claim 8 in the preparation of a medicament for treating a tumor.

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