CN114133457B

CN114133457B - Bispecific Chimeric Antigen Receptor (CAR) targeting ROR1 and CD33 and application thereof

Info

Publication number: CN114133457B
Application number: CN202111487581.2A
Authority: CN
Inventors: 张克礼; 雷林均
Original assignee: Henan Yuanchuang Life Stem Cell Bank Technology Co ltd; Zhengzhou Bain Biotechnology Co ltd; Zhengzhou Revo Gene Industry Co ltd
Current assignee: Henan Yuanchuang Life Stem Cell Bank Technology Co ltd; Zhengzhou Bain Biotechnology Co ltd; Zhengzhou Yuanchuang Gene Technology Co ltd
Priority date: 2021-12-08
Filing date: 2021-12-08
Publication date: 2022-08-19
Anticipated expiration: 2041-12-08
Also published as: CN114133457A

Abstract

The invention provides a bispecific Chimeric Antigen Receptor (CAR) targeting ROR1 and CD33 and application thereof, and the CAR has the following structure: ROR1 scFv-CD33 scFv-H-TM-C-CD3 ζ, wherein "-" is a linker peptide or a peptide bond; h is a hinge region; TM is a transmembrane domain; c is a costimulatory signal molecule; CD3 ζ is an intracellular signaling sequence. The chimeric antigen receptor can inhibit the growth of tumors in vivo and in vitro, prolong the life cycle, promote the secretion of cell factors and play a role in synergy anti-tumor.

Description

Bispecific Chimeric Antigen Receptor (CAR) targeting ROR1 and CD33 and application thereof

The technical field is as follows:

the invention belongs to the field of tumor immunotherapy, and particularly provides a bispecific Chimeric Antigen Receptor (CAR) targeting ROR1 and CD33 and application thereof in preparation of an antitumor drug.

Background art:

at present, cancer becomes one of the diseases with the highest fatality rate in the global scope, but the existing means such as surgical resection, chemoradiotherapy and the like have good treatment effect on early-stage tumors, are difficult to treat patients in middle and late stages, can cause serious adverse reaction of the patients, influence the life quality of the patients and increase the burden of the patients and families. In recent years, with the development of tumor immunotherapy, Chimeric Antigen Receptor T Cell (CART) therapy has been highly successful in anticancer. This therapy was originally proposed in 1989 by Gross and his colleagues, who fused the antigen binding region scFv of an antibody to the intracellular portion of the CD 3-zeta chain or fcsri γ to form a chimeric antigen receptor. The basic structure of the CAR includes a tumor-associated antigen (TAA) binding region (usually the scFV fragment derived from the antigen binding region of a monoclonal antibody), an extracellular Hinge region (hind area), a Transmembrane region (Transmembrane region), and an intracellular Immunoreceptor tyrosine-activated motif (ITAM). Then, in order to improve the anti-tumor capability of CART, researchers add co-stimulation signal molecules on the basis of the research of Gross et al, so that the killing capability of CART on tumor cells is greatly enhanced, and the study succeeds in the aspect of blood tumor treatment, Yesbarta (KTE-C10) of Kymiriah (CLT-019 and Kite pharmacy (purchased by Gilidard corporation)) of Nowa is approved by the US FDA to be listed in turn in 2017, the first CAR-T therapy Alkirschner injection (Rekaitt) in China is also approved to be listed in 2019, and the research enthusiasm for chimeric antigen receptor-related anti-tumor drugs is raised.

Early CART cells mostly use CD19 as a therapeutic target, but CD19 is subsequently found to be expressed not only in tumor cells but also in normal cells in relatively high expression level, so that the CART cells are misjudged to attack the normal cells and induce toxic reaction. To this end, researchers have begun to try to find new therapeutic targets and attempt to improve recognition specificity by constructing bispecific CART cells to reduce the appearance of off-target phenomena and to target two anti-tumor sites simultaneously.

Under the guidance of this theory, a large number of novel anti-tumor targets have been developed, and among them, receptor tyrosine kinase like orphan receptor 1 (ROR 1) is one of the most spotlighted novel targets. ROR1) is a transmembrane protein belonging to the Receptor Tyrosine Kinase (RTK) family, Tyrosine Kinase (TK) being a group of tumor-specific antigens. ROR1 was originally found in neuroblastoma strains and was named as a neurotrophin tyrosine kinase receptor-related protein, and was defined as an orphan receptor because its natural ligand was not found. ROR1 is involved in processes such as intercellular signaling, intracellular signaling, etc., regulating cell proliferation, differentiation, metastasis and survival, ROR1 is highly expressed in various tumors such as leukemia, prostate cancer, ovarian cancer, testicular cancer, adrenal cancer, breast cancer, lung cancer, malignant melanoma, pancreatic cancer, bladder cancer, colon cancer, etc., but is slightly expressed in normal postpartum tissues (Hojjar-Farsangi M, Jeddi-Tehrani M, Daneshmanesh AH, et al. Spontaneous Immunity aging the Receptor type Kinase ROR1 in Patents with Chronic Lymphocytic leukemia LeucoS. Ples one.2015,10(11): e 0142310.). Due to this property, ROR1 is an ideal anti-tumor target and diagnostic tumor marker. Researchers have developed various antibodies or nucleic acid inhibitors against ROR1 for treating malignant tumors, but few CART cells related to ROR1 have been reported, such as CA2973964a1, WO2020160050a1, US20180265593a1, CN107557337A, WO2017127664a1, etc., which disclose ROR1 antibodies or chimeric antigen receptors, respectively. Bispecific CART studies involving targeting ROR1 are more rare, as CN109503716A discloses a bispecific chimeric antigen receptor consisting of a signal peptide, two specific antigen binding fragments, an extramembranous spacer, a transmembrane region, and an intracellular costimulatory signal region, and the description mentions that the first antigen can be selected from ROR1 and the first antigen is CD38, but it actually provides BCMA & CD38 chimeric antigen receptor, and bispecific CART comprising ROR1 target has not been studied.

Therefore, the invention provides a novel bispecific CART for solving the technical problems and lays a technical foundation for further developing related antitumor drugs and therapies, wherein the problem is that the CART cell which can fully exert antitumor activity based on ROR1 target is still lacked, particularly how to develop a bispecific CART cell comprising ROR1 target and how to select a second antigen capable of generating synergistic effect so as to improve the targeting property, effectiveness and safety of tumor therapy.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a bispecific chimeric antigen receptor comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain targets an antigen of interest such as ROR1, BCMA, c-Met, B7-H6 and is scFv, preferably ROR1 scFv; the first antigen binding domain targets target antigens such as CD19, CD20, CD33, CD7, CD3 and the like, and is scFv, preferably CD33 scFv.

While various target targets for chimeric antigen receptors have been reported in the prior art, including but not limited to ROR1, BCMA, c-Met, B7-H6, D19, CD20, CD33, CD7, CD3, CD123, PD-1, etc., there are no unified standards and practices and even there are completely opposite teachings and experimental results for how to select target targets for chimeric antigen receptors with dual specific resistance. Therefore, the combination of ROR1 and CD33 is screened by the inventor through a large amount of experiments, and compared with other bispecific antigen combinations, the combination can effectively recognize and kill leukemia cells.

In certain embodiments, the chimeric antigen receptor comprises the structure: ROR1 scFv-CD33 scFv-H-TM-C-CD3 ζ, wherein "-" is a linker peptide or peptide bond; h is a hinge region; TM is a transmembrane domain; c is a costimulatory signal molecule; CD3 ζ is an intracellular signaling sequence; ROR1 scFv included the heavy chain CDR regions shown in SEQ ID NOS: 1-3 and the light chain CDR regions shown in SEQ ID NOS: 4-6; the CD33 scFv included the heavy chain CDR regions shown in SEQ ID NOS: 7-9 and the light chain CDR regions shown in SEQ ID NOS: 10-12.

In certain embodiments, the ROR1 scFv comprises a heavy chain variable region as set forth in SEQ ID NO:13 or having greater than 85% homology thereto and a light chain variable region as set forth in SEQ ID NO:14 or having greater than 85% homology thereto.

In certain embodiments, the CD33 scFv comprises the heavy chain variable region as set forth in SEQ ID NO. 15 or more% homology thereto and the light chain variable region as set forth in SEQ ID NO. 16 or more% homology thereto.

In certain embodiments, the co-stimulatory signaling molecule is selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40 or CD3, preferably CD28 and 4-1BB, more preferably 4-1 BB. The presence of costimulatory factors is considered essential in the chimeric antigen receptor structure, and the lack of costimulatory factors in the first generation CART makes CART cells difficult to be activated efficiently; also, improper selection of co-stimulatory factors may lead to serious adverse effects, and malignant adverse effects leading to patient death have also been reported. As such, the invention selects relatively mature and mild-activity 4-1BB as a co-stimulatory factor, and constructs a relevant chimeric antigen receptor.

In certain embodiments, the amino acid sequence of the chimeric antigen receptor is as set forth in SEQ ID NO 17 or has greater than 85% homology thereto.

Further, a nucleic acid molecule is provided, which codes the bispecific chimeric antigen receptor, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 18 or has more than 85% homology with the nucleic acid molecule.

Further, an immune cell is provided, said immune cell expressing said bispecific chimeric antigen receptor.

In some embodiments, the immune cells are T cells or NK cells, both of which are immunocompetent cells in vivo that exert an anti-tumor effect, and can exert significant anti-tumor activity after introduction of the chimeric antigen receptor structure provided by the present invention.

Further, provides an application of the bispecific chimeric antigen receptor or the nucleic acid molecule or the immune cell in preparing tumor drugs. Such tumors include, but are not limited to: ewing's sarcoma, neuroblastoma, neuroendocrine tumor, glioblastoma, melanoma, skin cancer, pancreatic cancer, lung cancer, cholangiocarcinoma, breast cancer, medullary thyroid cancer, ovarian cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, cervical cancer, endometrial cancer, esophageal cancer, stomach cancer, head and neck cancer, glioma, lymphoma, leukemia, myeloma, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, hodgkin's lymphoma, non-hodgkin's lymphoma, and bladder cancer, among others.

Advantageous effects

The invention provides a novel targeted ROR1 and CD33 bispecific chimeric antigen receptor, which can be simultaneously combined with two tumor targets of ROR1 and CD33 with high specificity, thereby obviously improving the tumor targeting property and preventing off-target effect; experiments show that the target combination of ROR1 and CD33 can obviously provide killing effect on leukemia cells and lymphoma cells relative to other anti-tumor targets; the ROR1 and CD33 targeted bispecific CART cells can promote the secretion of cytokines, and the expression levels of IFN-gamma and TNF-alpha are obviously improved, so that the therapy can mediate the cytokines to exert the synergistic tumor inhibition effect besides activating the immune function of T cells; the ROR1 and CD33 targeted bispecific CART cell can obviously inhibit the growth of tumors in vivo, improve the survival cycle of experimental animals and play a role in resisting tumors in vivo.

Drawings

FIG. 1: survival rate of K562 cells

FIG. 2: raji cell survival rate;

FIG. 3: IFN- γ expression level;

FIG. 4: TNF-alpha expression level;

FIG. 5 is a schematic view of: survival cycle of mouse Raji tumor model.

Detailed Description

Example 1: CART cell preparation

1.1 chimeric antigen receptor design

The bispecific chimeric antigen receptor provided by the invention has the following structure: ROR1 scFv-X scFv-H-TM-C-CD3 ζ wherein "-" is a linker peptide or peptide bond; h is a hinge region; TM is a transmembrane domain; c is a costimulatory signal molecule; CD3 ζ is an intracellular signaling sequence; the X scFv is selected from scfvs targeting CD19, CD20, CD33, CD7, or CD 3. The amino acid sequence and nucleotide sequence of each scFv were prepared and stored by the inventors in early experiments.

1.2 viral vectors transfection of T cells

In the invention, a plurality of bispecific chimeric antigen receptor structures are constructed so as to verify the combined effect of the ROR1 target and other anti-tumor targets such as CD19, CD20, CD33, CD7 or CD3, and the combined effects are respectively marked as ROR1-CD19, ROR1-CD20, ROR1-CD33, ROR1-CD7 and ROR1-CD3 for convenient differentiation and subsequent detection. The chimeric antigen receptor gene fragment is introduced into psb1576 vector, the vector is introduced into competent cells, plasmid is extracted by using a plasmid extraction kit (purchased from Axygen company), whether the target gene sequence is correct or not is identified by sequencing, and positive clone with correct sequencing is selected for subsequent experiments. The positive cloning plasmid was transferred to GeneChem Biotechnology, Shanghai Proyka corporation (GeneChem Biotechnology) to synthesize a lentiviral vector.

200mL of peripheral blood were collected from healthy volunteers and informed consent was given. Peripheral Blood Mononuclear Cells (PBMC) were collected by Ficoll gradient centrifugation and cultured in 1640 medium containing CD3mAb, IL-2(100U/mL) and 10% FBS. After the T cells are adjusted to a logarithmic growth phase, lentivirus infection is carried out, and the lentivirus titer reaches 2 x 10 ⁸ TU/mL or more. Resuspending the cells in 1640 medium and adjusting the cell density to 1X 10 ⁶ cells/mL, adding lentivirus into 24-well culture plate according to MOI of 10, mixing, placing at 37 deg.C and 5% CO ₂ And (5) culturing in an incubator. After 48h of culture, the medium was replaced with fresh 1640 medium, half of the medium was replaced every other day, and the total culture was carried out for about 96h while maintaining IL-2100U/mL. And (3) detecting the transfection rate of the CAR modified T cells by using a flow cytometer, wherein the positive rate is more than 95%.

Example 2 in vitro tumor cell killing Effect of CART cells

In order to verify the tumor killing effect of the CART cells, K562 (chronic myelogenous leukemia cell line) and Raji (lymphoma cell line) are selected as research objects in the invention.

2.1 tumor cell culture

Removing and recovering K562 cells and Raji cells from liquid nitrogen at 1X 10 ⁶ one/mL inoculated in RPMI 1640 complete medium (containing 10% FBS), 37 ℃, 5% CO ₂ And (5) performing sterile culture in a cell culture box. Counting and passaging for 1 time every 48h, observing the cell state, and performing subsequent experiments when the cell survival rate reaches more than 80%.

2.2CART cell Co-culture

Separately harvesting CART cells and the above tumor cells, washing with sterile PBS 3 times, mixing CART cells and tumor cells at a ratio of 2:1, and adjusting cell density to 1 × 10 ⁶ Inoculating to 96-well plate, CO-culturing CART cells and tumor cells at 37 deg.C under 5% CO ₂ Co-culturing in an incubator for 48 h. Sterile PBS solution was used as negative control.

2.3 detection of cell killing efficiency by MTT method

After 2 days of co-culture of the CART cells and the tumor cells, centrifuging to obtain supernatant, adding 20 mu L of MTT into each hole for treatment for 4 hours, then removing the MTT, adding 150 mu L of DMSO, and shaking in a shaking table for 10 min; meanwhile, blank comparison is set: i.e.the same procedure as the test, except that no cells were added. And (3) adjusting zero with blank holes at the wavelength of 490nm, measuring the light absorption value of each hole on an enzyme-labeling instrument, and recording the OD value detected by the enzyme-labeling instrument. Calculating the relative survival rate (cell viability) of the cells: cell viability (%) (OD of experimental group-blank group OD)/(OD of control group-blank group OD) × 100%.

As shown in fig. 1, the bispecific CART cells provided in the present invention can strongly kill tumor cells, and specifically, in K562 cells, ROR1 target seems to be more effective in combination with CD7 or CD33 target, and both the bispecific CART cells targeting ROR1 and CD7 and those targeting ROR1 and CD33 can achieve about 30% cell survival rate, but more CD19 and CD20 targets are reported, while the bispecific CART cells combined with ROR1 do not achieve satisfactory tumor cell killing effect, and the tumor cell survival rate is significantly higher than that of the combination of CD33 and CD 7.

As shown in fig. 2, among Raji cells, CART cells also exhibited strong tumor killing ability, and the survival rate of Raji cells was significantly reduced when co-cultured with CART cells. Unlike K562 cells, bispecific CART cells targeting ROR1 and CD19 showed strong killing ability, and CART cells with similar activity still included those targeting ROR1 and CD7 and those targeting ROR1 and CD33, both of which could bring the survival rate of tumor cells to below 40%. In contrast, the ROR1 target combined with CD20 and CD3 had relatively weak tumor killing ability.

Example 3CART promotes cytokine release

The CART cell and Raji cell provided by the invention are co-cultured, and the specific method is as in section 2.2. After 48h of co-culture, cell supernatants were collected and assayed for IFN-. gamma.and TNF-. alpha.content using an ELISA kit (purchased from Solarenbio).

As shown in fig. 3, the expression level of IFN- γ was greatly increased after co-culture of CART cells and Raji cells, in which the bispecific CART targeting ROR1 and CD33 had the strongest ability to promote IFN- γ secretion, while the bispecific CART targeting ROR1 and CD7 and ROR1 and CD20 had the next highest ability, and the bispecific CART targeting ROR1 and CD19 and the bispecific CART targeting ROR1 and CD3 had relatively weak ability to induce IFN- γ secretion.

Similar situation appears in the TNF-alpha expression regulation, as shown in fig. 4, CART can increase the TNF-alpha expression level in cell supernatant, dual-specificity CART targeting ROR1 and CD33 still shows strong cytokine induction ability, and the TNF-alpha expression level reaches the highest after the CART cell treatment; and other bispecific CART has similar capacity of promoting TNF-alpha expression and can also obviously promote TNF-alpha secretion compared with a control group.

Example 4 Targeted CART screening and identification

On the basis of the experiment, the bispecific chimeric antigen receptor which has stronger tumor cell killing capacity and cytokine secretion promoting capacity and is targeted to ROR1 and CD33 is selected as a research object for subsequent research. The chimeric antigen receptor has the following structure: ROR1 scFv-CD33 scFv-H-TM-C-CD3 ζ wherein "-" is a linker peptide or peptide bond; h is a hinge region; TM is a transmembrane domain; c is a costimulatory signal molecule; CD3 ζ is an intracellular signaling sequence. The ROR1 scFv and the CD33 scFv are target antigen-specific binding antibody fragments screened and obtained in early studies by the inventors, wherein the ROR1 scFv comprises heavy chain CDR regions shown as SEQ ID NO. 1-3 and light chain CDR regions shown as SEQ ID NO. 4-6; the CD33 scFv included the heavy chain CDR regions shown in SEQ ID NOS: 7-9 and the light chain CDR regions shown in SEQ ID NOS: 10-12.

As further identified, the ROR1 scFv included the heavy chain variable region shown in SEQ ID NO. 13 and the light chain variable region shown in SEQ ID NO. 14; the CD33 scFv comprises the heavy chain variable region as shown in SEQ ID NO. 15 and the light chain variable region as shown in SEQ ID NO. 16.

In the chimeric antigen receptor structure, the costimulatory factor is an essential part, the structure domain can effectively transmit intracellular and extracellular signals and mediate and maintain the activation state of T cells, and 4-1BB which is used in the prior research is selected as the costimulatory factor. In addition, the conventional elements of the chimeric antigen receptor such as the CD8 hinge region, the CD28 transmembrane region and the CD3 zeta intracellular signal region which are already reported are selected and combined together to form the structure of the chimeric antigen receptor, and the amino acid sequence of the structure is shown as SEQ ID NO. 17. Subsequently, through gene sequence analysis and codon optimization treatment, the nucleotide sequence of the chimeric antigen receptor disclosed by the invention is preferably selected, and is shown as SEQ ID NO. 18.

Example 5CART inhibits tumor growth in vivo

The Raji cells were recovered and cultured in the same manner as in section 2.1. 40B-NDG nude mice of 6-8 weeks old are prepared and normally bred for 3 days to adapt to experimental environment. After qualified quarantine, each nude mouse is injected intravenously at 1 × 10 ⁶ And Raji cells. After 3 days of tumor formation, the tumor volume was randomly divided into two groups of 20 tumor volumes, namely ROR1-CD33 group and control group, wherein ROR1-CD33 group was injected into tail vein at 1X 10 times after 3 days of tumor formation ⁶ Bispecific CART cells targeting ROR1 and CD33, control group was injected with equal volume of saline. Nude mice survival was monitored and recorded daily.

As shown in figure 5, the bispecific CART cell targeting ROR1 and CD33 provided by the invention can inhibit the growth of tumor in vivo, remarkably improve the life cycle of experimental animals, and the nude mice which are not treated die at 43 days, while the nude mice treated by the bispecific CART cell targeting ROR1 and CD33 have the maximum life cycle of 68 days, which indicates that the CART can kill tumor cells at the cell level in vitro, can also play a strong tumor inhibition role in the in vivo environment, prolong the life cycle of animals, inhibit the occurrence and development of tumor, and provide a basis for further developing novel antitumor drugs.

While this invention has been particularly shown and described with references to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

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610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

Met Gln Ala Leu Pro Pro Arg

755

<210> 18

<211> 2277

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gatattgtgc tgtttcagag cccgaccagc ctggcggtgg gccagcgccc gaccattagc 60

cgccgcgcga gcccgagcag cacctattgg cagtatatgc atccgccgac caaccagcag 120

aaaccgggcc agaaaccgcg cctgctgatt tatggcacca gccgcgaact gatttattat 180

aacctggaaa gcggcgtgcc gctgaccatt agcagcctgc agccgagcgg caccgaattt 240

accctgaaca ttcatccggt ggaagaagat gatgcggcga cctattattg cagcggcgcg 300

gaaggcgaat gcattacctg cgtgaccaaa acctttaacc gcagcctgga atgcggcggc 360

ggcggcagcg gcggcggcgg cagccaggtg cagctgaccg aaagcggccc gggcctggat 420

gtggcgccga gccagagccc gagcattacc tgcaccgtga gcacccaggc ggataaagaa 480

attaccagcg tgcattggtg cccgcagccg ccgggcaaat ggctgatgtg gctgggcgtg 540

ctgagcggca actatccgag cgcgctgctg agcattagca aagataacca tcagggcagc 600

aaaagccagg attttctgaa aatgaccagc ctgcagctga ccgatgatac cgcgatgtat 660

tattgcaacg gccagaaacg cgtgccggaa aacctgccga aaaccgtgct gtttaaccat 720

caggattggt atctgaacgg cggcggcggc agcggcggcg gcggcagcgg cggcggcggc 780

agcggcggcg gcggcagcga tattcagacc caggaaagcc cgagcagcct gagcgcgatt 840

gtgggcgatg tggtgaccat ttgcaaaagc cgcagccaga ccagctggga aagctggttt 900

cagcagccgg gcaaagcgca gcagccgaaa acctggattt atcgcccggg ctgccgcgat 960

ggcgtgccga gccgctttag cggcagcccg agcggccagg attataccct gatgattagc 1020

agcctgcagc cggaagatgc ggcgacctat ctgagcaaca aatggggctg cctgaacccg 1080

tttggcggcg gcaccgatga tgaaccgaaa ggcggcggcg gcagcggcgg cggcggcagc 1140

caggtgcagt gggtgcagag cggcgatttt aaaccgggcg cgagcgtgac caaagtgagc 1200

gcgagcggct attttaccag ctgcaaagcg agcacctggg tggatcaggc gccgggcctg 1260

attgaatgga ttggcagcct gcgcagcgaa caggaaagca aaaaagcgac cctgacctgc 1320

ggcgatgcgg ataccagcga tagcaccgcg tatatggaac tgcgcgaact gcgcagcgat 1380

gatacctatt attgcgcgag cgtggataaa cgcatttgct ggatgtttgg ctattggtgg 1440

ggcacccaga ccaccgtgga agtgagcagc aaaaccacca ccccggcgcc gcgcccgccg 1500

accccggcgc cgaccattgc gagccagccg ctgagcctgc gcccggaagc gtgccgcccg 1560

gcggcgggcg gcgcggtgca tacccgcggc ctggattttg cgtgcgatac caccaccccg 1620

gcgccgcgcc cgccgacccc ggcgccgacc attgcgagcc agccgctgag cctgcgcccg 1680

gaagcgtgcc gcccggcggc gggcggcgcg gtgcataccc gcggcctgga ttttgcgtgc 1740

gatatttata tttgggcgcc gctggcgggc acctgcggcg tgctgctgct gagcctggtg 1800

attaccctgt attgcaaacg cggccgcaaa aaactgctgt atatttttaa acagccgttt 1860

atgcgcccgg tgcagaccac ccaggaagaa gatggctgca gctgccgctt tccggaagaa 1920

gaagaaggcg gctgcgaact gcgcgtgaaa tttagccgca gcgcggatgc gccggcgtat 1980

aaacagggcc agaaccagct gtataacgaa ctgaacctgg gccgccgcga agaatatgat 2040

gtgctggata aacgccgcgg ccgcgatccg gaaatgggcg gcaaaccgcg ccgcaaaaac 2100

ccgcaggaag gcctgtataa cgaactgcag aaagataaaa tggcggaagc gtatagcgaa 2160

attggcatga aaggcgaacg ccgccgcggc aaaggccatg atggcctgta tcagggcctg 2220

agcaccgcga ccaaagatac ctatgatgcg ctgcatatgc aggcgctgcc gccgcgc 2277

Claims

1. A bispecific chimeric antigen receptor targeting ROR1 and CD33, wherein said chimeric antigen receptor comprises the structure: ROR1 scFv-CD33 scFv-H-TM-C-CD3 ζ, wherein "-" is a linker peptide or peptide bond; h is a hinge region; TM is a transmembrane domain; c is a costimulatory signal molecule; CD3 ζ is an intracellular signaling sequence; the sequences of heavy chain CDR1-3 in ROR1 scFv are respectively shown as SEQ ID NO. 1-3, and the sequences of light chain CDR1-3 are respectively shown as SEQ ID NO. 4-6; the sequences of heavy chain CDR1-3 in the scFv of CD33 are respectively shown in SEQ ID NO. 7-9, and the sequences of light chain CDR1-3 are respectively shown in SEQ ID NO. 10-12.

2. The bispecific chimeric antigen receptor according to claim 1, wherein the ROR1 scFv comprises the heavy chain variable region as shown in SEQ ID NO. 13 and the light chain variable region as shown in SEQ ID NO. 14.

3. The bispecific chimeric antigen receptor of claim 1, wherein the CD33 scFv comprises the heavy chain variable region as shown in SEQ ID NO. 15 and the light chain variable region as shown in SEQ ID NO. 16.

4. The bispecific chimeric antigen receptor according to claim 1, wherein the costimulatory signal molecule is selected from the group consisting of CD28 or 4-1 BB.

5. The bispecific chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the chimeric antigen receptor is represented by SEQ ID NO 17.

6. A nucleic acid molecule encoding the bispecific chimeric antigen receptor of any one of claims 1 to 4.

7. The nucleic acid molecule of claim 6, wherein the nucleotide sequence of said nucleic acid molecule is set forth in SEQ ID NO. 18.

8. An immune cell expressing the bispecific chimeric antigen receptor of any one of claims 1 to 4.

9. The immune cell of claim 8, wherein the immune cell is a T cell.

10. Use of the bispecific chimeric antigen receptor of any one of claims 1 to 4 or the nucleic acid molecule of any one of claims 6 to 7 or the immune cell of any one of claims 8 to 9 for the preparation of a medicament for the treatment of a tumor, said tumor being a leukemia or a lymphoma.