GD2-targeted CAR-T cells and their preparation and application

Abstract

The present invention provides a GD2-targeting CAR-T cell and its preparation and application. Specifically, the present invention provides a GD2-targeting CAR construct, which comprises the scFv fragment of the humanized GD2 antibody 3F8, the human Fc fragment, the ICOS transmembrane region, the ICOS and 4-1BB intracellular regions (co-stimulatory signals), and CD3ζ. The present invention also provides a CAR-T cell based on the above CAR, its preparation method and application. The CAR-T cell of the present invention can significantly inhibit the proliferation of tumor cells in a subcutaneous model of neuroblastoma, has a significant in vivo anti-tumor effect, and can be used for the targeted treatment of neuroblastoma.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a GD2-targeted CAR-T cell and its preparation and application. Background Art

[0002] Neuroblastoma is the most common solid tumor in children. 90% of the tumors occur in children under 10 years old, and the median age of diagnosis is 18 months. Neuroblastoma is a kind of neuroendocrine tumor that occurs during the development of the sympathetic nervous system. The clinical manifestations of the disease vary greatly. Tumors in infants and young children will have spontaneous regression and do not require treatment; while in slightly older patients, there is a high probability of tumor metastasis. Although the survival rate of some patients with low- and medium-risk neuroblastoma has been improved at present, for those high-risk patients (patients over 18 years old, determined according to the degree of tumor metastasis and genetic factors), the treatment effect is still not very satisfactory. At present, for this part of patients, the most effective treatment plan is to introduce anti-GD2 monoclonal antibody in the treatment.

[0003] Disialoganglioside GD2 is highly expressed in a variety of pediatric and adult tumors, including neuroblastoma, glioblastoma, melanoma, etc. GD2 is usually expressed during embryonic development, and its expression level in postnatal tissues is relatively low, and it is mainly limited to bone progenitor cells, the brain, peripheral nerves, and skin melanocytes, etc. In view of the above characteristics of GD2, a variety of GD2-targeted treatment strategies have been developed, including specific antibodies, drug conjugates, and chimeric antigen receptor-modified T cell therapies, etc.

[0004] Although the introduction of anti-GD2 monoclonal antibody can improve the survival rate of patients, about 50% of the patients will eventually relapse. These patients need more effective and more targeted therapies, such as chimeric antigen receptor-modified T cell (CAR-T) therapy. In recent years, CAR-T therapy has shown broad prospects in the treatment of hematological malignancies and also shown certain promise in the research of neuroblastoma. Malvina Prapa et al. linked the scFv fragment of a murine GD2 antibody to 4-1BB to construct a second-generation CAR structure to study the anti-tumor activity of GD2-CAR-T against neuroblastoma, and achieved certain curative effects, proving the feasibility of using GD2 as a target to treat neuroblastoma. However, the CAR-T cells constructed using the above CAR structure have low cytotoxicity, and the cytotoxic ability is not ideal at a low effector-to-target ratio in vitro. Sarah A. Richman et al. linked the scFv fragment of the murine GD2 antibody 14G2a to 4-1BB and H performed mutations, improved the affinity of the scFv fragment, and at the same time increased V L and V HThe length of the Linker in between to improve the stability of the scFv fragment. Based on this, they constructed three CAR structures respectively and studied the effects of different CAR structures on the anti-tumor activity of CAR-T cells. Their research proved that improving the affinity of the scFv fragment could effectively enhance the anti-tumor activity of CAR-T cells, but it was accompanied by very serious "on-target, off-tumor tissue" side effects. While increasing the length of the Linker between V L and V H could enable CAR-T cells to effectively home to the tumor site, but it could not improve the anti-tumor activity of CAR-T cells. It was speculated that this might be due to the too rapid exhaustion rate of CAR-T cells and the inability to continuously inhibit the proliferation of tumor cells. Therefore, it was necessary to design a CAR structure with high cytotoxicity, low side effects, and at the same time capable of continuously and efficiently exerting anti-tumor activity.

[0005] The CAR structure usually consists of three parts: an extracellular antigen-binding site, a transmembrane domain (TM domain), and an intracellular signal transduction structure. The main function of the TM domain is to anchor the CAR structure to the cell membrane, and common ones are molecules such as CD8 and CD28 that regulate T cell functions. The intracellular signal transduction structure is mainly composed of one or two co-stimulatory signals connected to CD3ζ. Commonly used co-stimulatory signals include CD28, 4-1BB, OX40, ICOS, etc. In 2018, Carl June first reported in the article the combined use of ICOS and 4-1BB in the CAR structure to construct a third-generation CAR containing two co-stimulatory molecules. They found that compared with the CAR structure with CD28, after introducing the intracellular region and transmembrane region of ICOS, it could promote the differentiation of CD4 + T cells into the Th1 / Th17 phenotype, and thus improve the anti-tumor activity of CD8 + T cells. At the same time, it was found that introducing the 4-1BB molecule into the CAR structure could effectively extend the survival time of CD8 + T cells.

[0006] Currently, there are still many challenges in the treatment of solid tumors with CAR-T therapy, including: lack of ideal treatment targets, homing obstacles, and poor persistence of CAR-T cells caused by the immunosuppressive microenvironment, etc. Therefore, there is still a need in this field to develop new CAR-T cells and treatment methods for solid tumors, especially neuroblastoma. Summary of the Invention

[0007] The object of the present invention is to provide a CAR-T cell targeting GD2 and its preparation and application.

[0008] In the first aspect of the present invention, a chimeric antigen receptor (CAR) is provided, and the antigen-binding domain of the chimeric antigen receptor comprises a heavy chain variable region and a light chain variable region.

[0009] The heavy chain variable region comprises the following complementarity-determining regions (CDRs):

[0010] CDR1 shown in SEQ ID NO:12,

[0011] CDR2 shown in SEQ ID NO:13, and

[0012] CDR3 shown in SEQ ID NO:14;

[0013] And the light chain variable region comprises the following complementarity-determining regions (CDRs):

[0014] CDR1’ shown in SEQ ID NO:15,

[0015] CDR2’ shown in SEQ ID NO:16, and

[0016] CDR3’ shown in SEQ ID NO:17.

[0017] In another preferred example, the antigen-binding domain comprises the antibody heavy chain variable region shown in SEQ ID NO:1 and the antibody light chain variable region shown in SEQ ID NO:2.

[0018] In another preferred example, the antibody heavy chain variable region and the antibody light chain variable region are connected by a linker peptide.

[0019] In another preferred example, the structure of the antigen-binding domain is shown in Formula I or II as follows:

[0020] V L -V H (I); V H -V L (II)

[0021] Wherein, V H is the antibody heavy chain variable region; V L is the antibody light chain variable region; "-" is a linker peptide or a peptide bond.

[0022] In another preferred example, the structure of the antigen-binding domain is shown in Formula II.

[0023] In another preferred example, the amino acid sequence of V H is as shown in SEQ ID NO:1, and the amino acid sequence of V L is as shown in SEQ ID NO:2.

[0024] In another preferred embodiment, the linker peptide is a sequence shown by 1-4 consecutive SEQ ID NO:4 (GGGGS), preferably 2-4, more preferably 3.

[0025] In another preferred embodiment, the antigen-binding domain binds to GD2, preferably human GD2.

[0026] In another preferred embodiment, the heavy chain variable region and the light chain variable region of the antigen-binding domain are derived from a humanized antibody.

[0027] In another preferred embodiment, the structure of the chimeric antigen receptor is shown by the following formula III:

[0028] L-scFv-H-TM-C-CD3ζ (III)

[0029] Wherein,

[0030] L is absent or a signal peptide sequence;

[0031] scFv is an scFv targeting GD2;

[0032] H is a hinge region;

[0033] TM is a transmembrane domain;

[0034] C is a co-stimulatory signal molecule;

[0035] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0036] In another preferred embodiment, the scFv targeting GD2 includes the heavy chain variable region of the antibody shown by SEQ ID NO:1, and the light chain variable region of the antibody shown by SEQ ID NO:2.

[0037] In another preferred embodiment, the L is a signal peptide of a protein selected from the group consisting of: CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0038] In another preferred embodiment, the L is a signal peptide derived from macrophage colony-stimulating factor.

[0039] In another preferred embodiment, the amino acid sequence of L is as shown by SEQ ID NO:5.

[0040] In another preferred embodiment, the H is a hinge region of a protein selected from the group consisting of: CD8, CD28, CD137, or a combination thereof.

[0041] In another preferred embodiment, the H is an Fc fragment, preferably a human Fc fragment.

[0042] In another preferred embodiment, the amino acid sequence of H is as shown in SEQ ID NO: 6.

[0043] In another preferred embodiment, the TM is the transmembrane region of a protein selected from the group consisting of: ICOS, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, GD2, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0044] In another preferred embodiment, the TM is the transmembrane region derived from ICOS.

[0045] In another preferred embodiment, the sequence of TM is as shown in SEQ ID NO: 7.

[0046] In another preferred embodiment, the C is a co-stimulatory signal molecule of a protein selected from the group consisting of: ICOS, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

[0047] In another preferred embodiment, the C is a co-stimulatory signal molecule derived from ICOS and 4-1BB.

[0048] In another preferred embodiment, the amino acid sequence of the co-stimulatory signal molecule derived from ICOS is as shown in SEQ ID NO: 8.

[0049] In another preferred embodiment, the amino acid sequence of the co-stimulatory signal molecule derived from 4-1BB is as shown in SEQ ID NO: 9.

[0050] In another preferred embodiment, the amino acid sequence of CD3ζ is as shown in SEQ ID NO: 10.

[0051] In another preferred embodiment, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO: 3.

[0052] In a second aspect of the present invention, there is provided a nucleic acid molecule encoding the chimeric antigen receptor (CAR) described in the first aspect of the present invention.

[0053] In another preferred embodiment, the nucleic acid molecule is isolated.

[0054] In another preferred embodiment, the nucleic acid molecule is as shown in SEQ ID NO: 11.

[0055] In a third aspect of the present invention, there is provided a vector, and the vector contains the nucleic acid molecule described in the second aspect of the present invention.

[0056] In another preferred embodiment, the vector is selected from the group consisting of: DNA, RNA, plasmid, lentiviral vector, adenoviral vector, adeno-associated viral vector (AAV), retroviral vector, transposon, or a combination thereof.

[0057] In another preferred embodiment, the vector is selected from the group consisting of: plasmid, viral vector.

[0058] In another preferred embodiment, the vector is in the form of viral particles.

[0059] In another preferred embodiment, the vector is a lentiviral vector.

[0060] In a fourth aspect of the present invention, there is provided a host cell, and the host cell contains the vector described in the third aspect of the present invention, or the nucleic acid molecule described in the second aspect of the present invention is integrated into the chromosome exogenously, or expresses the CAR described in the first aspect of the present invention.

[0061] In another preferred embodiment, the host cell includes eukaryotic cells and prokaryotic cells.

[0062] In another preferred embodiment, the host cell includes Escherichia coli.

[0063] In a fifth aspect of the present invention, there is provided an engineered immune cell, and the immune cell expresses the CAR described in the first aspect of the present invention.

[0064] In another preferred embodiment, the cell is an isolated cell, and / or the cell is a genetically engineered cell.

[0065] In another preferred embodiment, the immune cell is from a human or a non-human mammal (such as a mouse).

[0066] In another preferred embodiment, the cell includes T cells, NK cells.

[0067] In another preferred embodiment, the cell is a CAR-T cell or a CAR-NK cell, preferably a CAR-T cell.

[0068] In another preferred embodiment, the CAR in the immune cell is co-expressed with a cell suicide element.

[0069] In a sixth aspect of the present invention, there is provided a preparation, and the preparation contains the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, or the immune cell described in the fifth aspect of the present invention, and a pharmaceutically acceptable carrier.

[0070] In another preferred embodiment, the preparation is a liquid preparation.

[0071] In another preferred embodiment, the dosage form of the preparation is an injection.

[0072] In another preferred embodiment, the concentration of the CAR-T cells in the preparation is 1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0073] In another preferred embodiment, the preparation further comprises a second anti-tumor active ingredient, preferably including a second antibody or a chemotherapeutic agent.

[0074] In another preferred embodiment, the chemotherapeutic agent is selected from the group consisting of docetaxel, carboplatin, or a combination thereof.

[0075] In a seventh aspect of the present invention, there is provided the use of the chimeric antigen receptor according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, the vector according to the third aspect of the present invention, or the immune cell according to the fifth aspect of the present invention, or the preparation according to the sixth aspect of the present invention, for the preparation of a drug or preparation for preventing and / or treating cancer or tumor.

[0076] In another preferred embodiment, the tumor is selected from the group consisting of hematological tumors, solid tumors, or a combination thereof.

[0077] In another preferred embodiment, the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0078] In another preferred embodiment, the solid tumor is selected from the group consisting of gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or a combination thereof.

[0079] In another preferred embodiment, the tumor is a GD2-positive tumor.

[0080] In another preferred embodiment, the GD2-positive tumor is selected from the group consisting of neuroblastoma, glioma, melanoma.

[0081] In an eighth aspect of the present invention, there is provided a kit for preparing the host cell described in the fourth aspect of the present invention, the kit comprising a container, and the nucleic acid molecule described in the second aspect of the present invention, or the vector described in the third aspect of the present invention located within the container.

[0082] In a ninth aspect of the present invention, there is provided a method for preparing engineered immune cells, the immune cells expressing the CAR described in the first aspect of the present invention, the method comprising the following steps:

[0083] (a) providing immune cells to be engineered; and

[0084] (b) transducing the nucleic acid molecule described in the second aspect of the present invention or the vector described in the third aspect of the present invention into the immune cells, thereby obtaining the engineered immune cells.

[0085] In another preferred example, the engineered immune cells are CAR-T cells or CAR-NK cells.

[0086] In another preferred example, the method further comprises the step of detecting the function and effectiveness of the obtained engineered immune cells.

[0087] In a tenth aspect of the present invention, there is provided a method for treating a disease, comprising administering an appropriate amount of the vector described in the third aspect of the present invention, the immune cells described in the fifth aspect of the present invention, or the preparation described in the sixth aspect of the present invention to a subject in need of treatment.

[0088] In another preferred example, the disease is cancer or tumor.

[0089] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 Shows the construction and specific verification of the GD2-CAR vector.

[0091] Figure 1 A shows the schematic diagram of the PG3-GD2-CAR structure. PG3-GD2-CAR includes the scFv fragment of the humanized GD2 antibody 3F8, the human Fc fragment, the ICOS transmembrane region, the ICOS and 4-1BB intracellular regions (co-stimulatory signals), and CD3ζ.

[0092] Figure 1 B shows the expression level of GD2 on the surface of the neuroblastoma cell line.

[0093] Figure 1C shows the expression level of CAR on the surface of PG3-GD2-CAR-Jurkat cells detected by flow cytometry.

[0094] Figure 1 D shows the expression of CD25 and CD69 on the surface of PG3-GD2-CR-Jurkat cells after co-incubation for 24 h at an effector-to-target ratio of 1:1.

[0095] Figure 2 Shows the construction and verification of PG3-GD2-CAR-T cells.

[0096] Figure 2 A shows the expression level of CAR molecule on the surface of PG3-GD2-CAR-T cells detected by flow cytometry.

[0097] Figure 2 B shows the expression of CAR protein in PG3-GD2-CAR-T cells detected by Western blot. Among them, cell lysates of PG3-GD2-CAR-T cells (lane 1) and T cells (lane 2) were separated by SDS-PAGE under reducing conditions. Mouse anti-CD3ζ antibody was used as the primary antibody, and goat anti-mouse horseradish peroxidase (HRP) antibody was used as the secondary antibody. Endogenous CD3ζ expression was detected at 35 KD in both T cells and CAR-T cells. However, exogenous CD3ζ expression was detected at 75 KD only in CAR-T cells.

[0098] Figure 3 Shows the in vitro functional verification of PG3-GD2-CAR-T cells.

[0099] Figure 3 A shows the growth inhibitory effect of PG3-GD2-CAR-T cells on neuroblastoma cells in vitro detected by xCELLigence real-time cell analyzer (RTCA). The two types of cells were co-incubated for 48 hours at the effector-to-target ratio shown in the figure, and the adherent growth index of the target cells was detected every 15 min.

[0100] Figure 3 B shows the release levels of Granzyme-B, IFN-γ, IL-2 and TNF-α in the supernatant after co-incubating PG3-GD2-CAR-T cells with three types of target cells for 48 h at the effector-to-target ratio shown in the figure.

[0101] Figure 4 Shows the in vivo functional verification of PG3-GD2-CAR-T cells.

[0102] Figure 4 A shows a schematic diagram of a neuroblastoma xenograft model.

[0103] Figure 4 Panel B shows the growth curve of tumor volume in mice and the ratio of tumor weight to body weight at euthanasia. Compared with T cells, PG3-GD2-CAR-T cells were able to substantially inhibit tumor growth (*p = 0.0103, **p = 0.0049 by t-test, mean ± SEM).

[0104] Figure 4 Panel C shows the contents of red blood cells, white blood cells, hemoglobin, and platelets in the peripheral blood of mice at euthanasia. There were no significant differences between the T cell group and the PG3-GD2-CAR-T group of mice (by t-test, mean ± SEM).

[0105] Figure 4 Panel D shows the comparison of tumor sizes between the two groups of mice in the CAR-T group and the T cell group.

[0106] Figure 5 The immunohistochemical results are shown.

[0107] Figure 5 Panel A shows the HE staining results of mouse tumors, indicating obvious pathological changes in the histological structure of tumors in the PG3-GD2-CAR-T group of mice, while there were no obvious changes in the control T cell group. Among them, the two tumors on the left represent the PG3-GD2-CAR-T group, and the two randomly selected tumors on the right represent the T cell group.

[0108] Figure 5 Panel B shows the IHC staining results of mouse tumors. The results show that there were more T cell infiltrations in the PG3-GD2-CAR-T experimental group. Among them, the two tumors on the left represent the PG3-GD2-CAR-T group, and the two randomly selected tumors on the right represent the T cell group.

[0109] Figure 5 Panel C shows the results of TUNEL analysis. The results show that compared with the T cell group, more apoptotic cells could be detected in the PG3-GD2-CAR-T experimental group. Detailed implementation methods

[0110] After extensive and in-depth research, the present inventor unexpectedly discovered for the first time a third-generation CAR vector targeting GD2. The inventor connected the scFv fragment of the GD2 humanized antibody to the transmembrane region and intracellular region of ICOS through the human Fc fragment. The intracellular region of ICOS was the first co-stimulatory molecule, and then the intracellular region of 4-1BB was connected as the second co-stimulatory molecule of the CAR structure, and then connected to CD3ζ to construct PG3-GD2-CAR. The results of in vitro functional experiments showed that when PG3-GD2-CAR-T cells were co-incubated with neuroblastoma cells, the tumor cell growth could be effectively inhibited at the effector-to-target ratios of 1:2, 1:1, and 4:1, respectively. At the same time, compared with the control T cells, a significant increase in the release of cytokines (including IFN-γ, TNF-α, Granzyme-B, and IL-2) was detected in the supernatant after the co-incubation of CAR-T cells and tumor cells. The results of animal experiments also showed that PG3-GD2-CAR-T cells could significantly inhibit the proliferation of tumor cells in the subcutaneous model of neuroblastoma and had a significant in vivo anti-tumor effect. Therefore, the PG3-GD2-CAR-T cells constructed in the present invention can be used for the targeted treatment of neuroblastoma.

[0111] Term

[0112] To facilitate a better understanding of the present disclosure, certain terms are first defined. As used in this application, unless otherwise clearly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0113] The term "about" may refer to a value or a component within an acceptable error range of a specific value or a component determined by a person of ordinary skill in the art, which will depend in part on how the value or the component is measured or determined.

[0114] The term "administer" refers to physically introducing the product of the present invention into a subject using any of a variety of methods and delivery systems known to those of skill in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0115] The term "antibody" (Ab) shall include, but not be limited to, immunoglobulins that specifically bind to an antigen and contain at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each H chain contains a heavy-chain variable region (abbreviated herein as VH) and a heavy-chain constant region. The heavy-chain constant region contains three constant domains, CH1, CH2, and CH3. Each L chain contains a light-chain variable region (abbreviated herein as VL) and a light-chain constant region. The light-chain constant region contains one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0116] It should be understood that in this article, amino acid names are identified by a single English letter in the international common usage, and the corresponding three-letter abbreviations of amino acid names are: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), Val (V).

[0117] GD2

[0118] Disialoganglioside GD2 (abbreviated as GD2) belongs to ganglioside sphingolipids and is highly expressed in various pediatric and adult tumors, including neuroblastoma, glioblastoma, melanoma, etc. GD2 is usually expressed during embryonic development, with a relatively low expression level in postnatal tissues and is mainly limited to bone progenitor cells, the brain, peripheral nerves, and skin melanocytes, etc. Given the above characteristics of GD2, a variety of GD2-targeted therapeutic strategies have been developed, including specific antibodies, drug conjugates, and chimeric antigen receptor-modified T cell therapies, etc.

[0119] Chimeric antigen receptor (CAR)

[0120] The design of CARs has gone through the following process: The first-generation CARs only had one intracellular signaling component, CD3ζ or FcγRI molecule. Since there was only one activation domain intracellularly, it could only induce short-term T cell proliferation and less cytokine secretion, and could not provide long-term T cell proliferation signals and sustained in vivo anti-tumor effects. Therefore, it did not achieve good clinical efficacy. The second-generation CARs introduced a co-stimulatory molecule, such as CD28, 4-1BB, OX40, ICOS, on the basis of the original structure, and had greatly improved functions compared with the first-generation CARs, further enhancing the persistence of CAR-T cells and their killing ability against tumor cells. Based on the second-generation CARs, some new immune co-stimulatory molecules, such as CD27, CD134, were tandemly arranged to develop into the third-generation and fourth-generation CARs.

[0121] The chimeric antigen receptor (CAR) of the present invention is a third-generation CAR, including an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen-binding domain). The intracellular domain includes a co-stimulatory signaling region and a ζ-chain portion. The co-stimulatory signaling region refers to a part of the intracellular domain including a co-stimulatory molecule. The co-stimulatory molecule is a cell surface molecule required for the effective response of lymphocytes to antigens, rather than an antigen receptor or their ligands.

[0122] Between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR, a linker can be incorporated. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that serves to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The linker can include 0 - 300 amino acids, preferably 2 to 100 amino acids, and most preferably 3 to 50 amino acids.

[0123] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen-binding domain targeting GD2. When the CAR of the present invention is expressed in T cells, it can recognize antigens based on antigen-binding specificity. When it binds to its associated antigen, it affects tumor cells, causing the tumor cells not to grow, being induced to die, or being affected in other ways, and resulting in the reduction or elimination of the tumor burden of the patient. The antigen-binding domain is preferably fused with the intracellular domain from one or more of the co-stimulatory molecule and the ζ-chain.

[0124] As used herein, both "antigen-binding domain" and "single-chain antibody fragment" refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments having antigen-binding activity. An Fv antibody contains the variable region of the heavy chain and the variable region of the light chain of an antibody, but no constant region, and is the smallest antibody fragment with all antigen-binding sites. Generally, an Fv antibody also contains a polypeptide linker between the VH and VL domains and can form the structure required for antigen binding. The antigen-binding domain is usually an scFv (single-chain variable fragment). The size of an scFv is generally 1 / 6 of that of a complete antibody. The single-chain antibody is preferably an amino acid sequence of a single chain encoded by a single nucleotide chain. As a preferred embodiment of the present invention, the scFv contains an antibody that specifically recognizes GD2, preferably a humanized single-chain antibody.

[0125] For the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0126] Specifically, in a preferred embodiment of the present invention, in order to improve the viability of CAR-T cells in the tumor microenvironment, a novel third-generation CAR vector targeting GD2 was constructed using ICOS and 4-1BB as co-stimulatory domains, which sequentially includes the single-chain antibody sequence of a humanized GD2 antibody, a human Fc fragment, the transmembrane region and intracellular region sequence of human ICOS, the intracellular region sequence of human 4-1BB, and the sequence of human CD3ζ.

[0127] Furthermore, CAR-T cells specifically targeting GD2 (PG3-GD2-CAR-T) were constructed by lentiviral transfection. Then, the killing activity of PG3-GD2-CAR-T cells against GD2-positive neuroblastoma cell lines was verified by in vitro killing experiments, and the in vivo anti-tumor activity of PG3-GD2-CAR-T cells against neuroblastoma cell lines was evaluated by mouse experiments. The results showed that when the PG3-GD2-CAR-T cells constructed in the present invention were co-incubated with neuroblastoma cells, at effector-to-target ratios of 1:2, 1:1, and 4:1, they had obvious inhibitory effects on neuroblastoma cells and could produce a specific cytokine profile (including IFN-γ, TNF-α, Granzyme-B, and IL-2); the results of animal experiments showed that the PG3-GD2-CAR-T cells constructed in the present invention could significantly inhibit the proliferation of tumor cells in mice and had significant in vivo anti-tumor effects. In summary, the PG3-GD2-CAR-T cells constructed in the present invention provide a new method or strategy for targeted treatment of neuroblastoma.

[0128] vector

[0129] Nucleic acid sequences encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by directly isolating it from cells and tissues containing the gene using standard techniques. Optionally, the gene of interest can be produced synthetically.

[0130] The present invention also provides a vector into which the expression cassette of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term and stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus because they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.

[0131] Briefly, the expression cassette or nucleic acid sequence of the present invention is generally operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration in eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initial sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0132] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0133] The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into a vector such as, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particular vectors of interest include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.

[0134] Further, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0135] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into the vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0136] Additional promoter elements, such as enhancers, can modulate the frequency of transcriptional initiation. Typically, these are located in the region 30 - 110 bp upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is maintained when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by up to 50 bp before activity begins to decline. Depending on the promoter, it has been shown that individual elements can act cooperatively or independently to initiate transcription.

[0137] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turn off the expression when the expression is not desired. Examples of inducible promoters include but are not limited to the metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0138] To assess the expression of the CAR polypeptide or a portion thereof, the expression vector introduced into the cell may also contain either or both of a selectable marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells sought to be transfected or infected with the viral vector. In other aspects, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.

[0139] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifest by some readily detectable property such as enzymatic activity. After the DNA has been introduced into the recipient cells, the expression of the reporter gene is assayed at an appropriate time. Suitable reporter genes can include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Typically, constructs having at least 5 flanking regions that show the highest levels of reporter gene expression are identified as promoters. Such promoter regions can be ligated to reporter genes and used to evaluate the ability of a reagent to modulate promoter-driven transcription.

[0140] Methods for introducing genes into cells and for expressing genes in cells are known in the art. In the context of expression vectors, the vector can be readily introduced into a host cell by any method in the art, e.g., mammalian, bacterial, yeast, or insect cells. For example, an expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0141] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0142] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0143] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0144] In the case of using non-viral delivery systems, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for introducing nucleic acids into host cells (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid can be associated with lipids. Nucleic acids associated with lipids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via a linking molecule associated with both the liposome and the oligonucleotide, entrapped within liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, included as a suspension in lipids, included in micelles or complexed with micelles, or otherwise associated with lipids. The lipids, lipid / DNA or lipid / expression vector associated with the composition are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as micelles or with a "collapsed" structure. They can also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which occur naturally in the cytoplasm as well as in such compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols and aldehydes.

[0145] In a preferred embodiment of the present invention, the vector is a lentiviral vector.

[0146] Formulation

[0147] The present invention provides a preparation comprising the CAR-T cells of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the concentration of the CAR-T cells in the formulation is 1×10 3 -1×10 8 cells / ml, more preferably 1×10 4 -1×10 7 cells / ml.

[0148] In one embodiment, the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The formulations of the present invention are preferably formulated for intravenous administration.

[0149] Therapeutic applications

[0150] The present invention includes therapeutic applications with cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of the present invention. The transduced T cells can target the marker GD2 of tumor cells, co-activate T cells, and induce a T cell immune response, thus significantly improving their killing efficiency against tumor cells.

[0151] Accordingly, the present invention also provides a method for stimulating a T cell-mediated immune response against a target cell population or tissue in a mammal, which comprises the step of administering the CAR-T cells of the present invention to the mammal.

[0152] In one embodiment, the present invention includes a type of cell therapy, in which autologous T cells (or allogeneic donors) of a patient are isolated, activated and genetically modified to generate CAR-T cells, which are then injected into the same patient. This approach has a very low probability of developing graft-versus-host disease, and the antigen is recognized by T cells in an MHC-unrestricted manner. In addition, one type of CAR-T can treat all cancers expressing the antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0153] In one embodiment, the CAR-T cells of the present invention can undergo robust in vivo T cell expansion and can last for a prolonged amount of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy procedure, where CAR-modified T cells induce an immune response specific to the antigen-binding domain in the CAR. For example, anti-GD2 CAR-T cells induce a specific immune response against GD2-positive cells.

[0154] Although the data disclosed herein specifically disclose a lentiviral vector comprising an anti-GD2 scFv, an Fc hinge and an ICOS transmembrane and intracellular region, a 4-1BB intracellular region and a CD3ζ signaling domain, the present invention should be construed to include any number of variations to each of the components of the construct.

[0155] Treatable cancers include tumors that are not vascularized or are substantially non-vascularized, as well as vascularized tumors. Cancers can include non-solid tumors (such as hematological tumors, e.g., leukemia and lymphoma) or can include solid tumors. Cancer types treatable with the CARs of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignancies, such as sarcoma, carcinoma, and melanoma. Also included are adult tumors / cancers and pediatric tumors / cancers.

[0156] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemia, including acute leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, granulomonocytic, monocytic, and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and myelodysplasia.

[0157] Solid tumors are abnormal masses of tissue that generally do not contain cysts or areas of fluid. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell types that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors such as sarcoma and carcinoma include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, and ovarian cancer.

[0158] In a preferred embodiment, treatable cancers are GD2-positive tumors, such as neuroblastoma, glioma, melanoma, etc.

[0159] The CAR-modified T cells of the invention can also be used as a vaccine type for ex vivo immunization and / or in vivo therapy of a mammal. Preferably, the mammal is a human.

[0160] For ex vivo immunization, at least one of the following occurs in vitro prior to administering the cells into a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding the CAR into the cells, and / or iii) cryopreserving the cells.

[0161] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing the CARs disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be human, and the CAR-modified cells can be autologous with respect to the recipient. Optionally, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.

[0162] In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

[0163] The present invention provides a method for treating a tumor, which comprises administering to a subject in need thereof a therapeutically effective amount of the CAR-modified T cells of the present invention.

[0164] The CAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention can comprise a population of target cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0165] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The quantity and frequency of administration will be determined by factors such as the condition of the patient, and the type and severity of the patient's disease - although appropriate doses can be determined by clinical trials.

[0166] When referring to an "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences in the condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein can be administered at a dose of 10 4 to 10 9 cells / kg body weight, preferably 10 5 to 10 6administered at a dose of cells / kg body weight (including all integer values within those ranges). The T cell compositions can also be administered multiple times at these doses. The cells can be administered using infusion techniques known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a particular patient can be readily determined by one of ordinary skill in the medical arts by monitoring the patient's signs of disease and thus adjusting the treatment.

[0167] The administration of the subject compositions can be effected in any convenient manner, including by spraying, injection, ingestion, infusion, implantation, or transplantation. The compositions described herein can be administered subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally to a patient. In one embodiment, the T cell compositions of the invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the invention are preferably administered by i.v. injection. The compositions of T cells can be directly injected into a tumor, lymph node, or site of infection.

[0168] In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination (e.g., before, concurrently, or after) with any number of relevant forms of treatment, including but not limited to treatment with the following agents: such agents as antiviral therapy, cidofovir, and interleukin-2, cytarabine (also known as ARA-C), or treatment of patients with MS with natalizumab or treatment of patients with psoriasis with efalizumab or other treatment of patients with PML. In further embodiments, the T cells of the invention can be used in combination with: chemotherapy, radiation, immunosuppressive agents, such as, cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the invention are administered to a patient in combination (e.g., before, concurrently, or after) with bone marrow transplantation, the use of chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide. For example, in one embodiment, a subject can undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered before or after surgery.

[0169] The doses of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be effected in accordance with practices accepted in the art. Generally, for each treatment or each course of treatment, 1×10 6 to 1×10 10Modified T cells of the present invention (e.g., CAR-T cells) are administered to a patient by, for example, intravenous infusion.

[0170] The main advantages of the present invention include:

[0171] (a) The CAR-T cells constructed according to the present invention can continuously and efficiently inhibit the growth of GD2 + neuroblastoma cells, and the inhibitory effect on GD2 high SH-SY5Y cells is particularly obvious.

[0172] (b) The CAR-T cells constructed according to the present invention have high cytotoxicity and good persistence, and can continuously and efficiently inhibit the tumor progression in a xenograft mouse model constructed with neuroblastoma tumor cells.

[0173] (c) The CAR-T cells constructed according to the present invention do not cause obvious changes in the blood picture of mouse peripheral blood and have good safety.

[0174] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0175] General materials and methods:

[0176] 1. Cell lines and cell culture

[0177] The human neuroblastoma cell lines SK-N-SH, SK-N-AS, and SH-SY5Y cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. SK-N-SH cells were cultured in MEM medium (Inviitrogen), supplemented with 10% fetal bovine serum (FBS; Gibco), 1% non-essential amino acids (NEAA; Gibco), and 1 mM Sodium Pyruvate (NaP; Gibco). SK-N-AS cells were cultured in DMEM medium (Inviitrogen), supplemented with 10% FBS and 1% NEAA. SH-SY5Y cells were cultured in MEM / F-12 (1:1) medium (Inviitrogen), supplemented with 10% FBS, 1% NEAA, and 1 mM NaP. Jurkat cells (ATCC) were cultured in RPMI-1640 medium (Inviitrogen), supplemented with 10% FBS. T cells extracted from healthy human peripheral blood were cultured in TexMacs TM GMP medium, supplemented with IL-7 and IL-15. HEK293 cells were cultured in DMEM medium, supplemented with 10% FBS. All cells were cultured in an incubator at 37°C with 5% CO2.

[0178] 2. Construction of PG3-GD2-CAR

[0179] The novel third-generation CAR vector targeting GD2 constructed in the present invention sequentially includes the single-chain antibody sequence of the humanized GD2 antibody, the human Fc fragment, the transmembrane region and intracellular region sequence of human ICOS, the intracellular region sequence of human 4-1BB, and the human CD3ζ sequence.

[0180] 3. Preparation and transduction of lentivirus

[0181] The preparation of lentivirus refers to the conventional method in the prior art.

[0182] CAR-T cells were prepared as follows: The peripheral blood of healthy humans was mixed and diluted equally with PBS, and then added slowly along the tube wall to Ficoll according to the ratio of Ficoll: diluted peripheral blood = 1:2 for density gradient centrifugation at 800×g for 30 min. The mononuclear cells in the middle foggy layer were aspirated, washed twice with PBS, counted, and placed in a 24-well plate at 5×10 6 / well, and 20 μl of CD3 / CD28 magnetic beads were added to each well for activation. After 48 h of activation, the cells were counted, and 5×10 5 cells were transferred to a 48-well plate, and the virus concentrate was added for transfection (MOI = 10). The total system was 200 μl. After 16 h of transfection, 1 ml of medium was added to each well for culture. The entire T cell culture process was carried out in TexMacs TMPerformed in GMP medium, adding IL-7 and IL-15.

[0183] 4. Construction of PG3-GD2-CAR-Jurkat cells

[0184] To prove the specificity of the PG3-GD2-CAR constructed in the present invention, PG3-GD2-CAR-Jurkat cells (overexpressing PG3-GD2-CAR on Jurkat cells) were constructed. The cultured Jurkat cells were counted, and 1×10 6 cells were transferred to a 48-well plate, and the PG3-GD2-CAR virus concentrate was added for transfection. After 14 h of transfection, the supernatant was removed by centrifugation, and fresh medium was added for culture. When the number of cells was sufficient, the cells were stained with APC-labeled FC antibody and sorted on a flow cytometer until the positive cell rate reached 100%. The sorted cells were expanded in culture.

[0185] 5. Flow cytometry and Western blotting

[0186] To detect the expression of GD2-CAR on the cell surface, T cells and Jurkat cells were stained with APC-fluorescently labeled human IgG FC antibody. To detect the expression level of GD2 antigen on the surface of target cells, three strains of target cells were stained with PE-labeled GD2 antibody. Specifically, the cells were co-incubated with the antibody at 37 °C for 20 min, then washed twice with PBS, resuspended in 300 μl PBS, and detected and analyzed on a flow cytometer.

[0187] The total protein of PG3-GD2-CAR-T cells was extracted, and then mouse-derived anti-CD3ζ antibody (BD Biosciences) was used as the primary antibody, and goat anti-mouse horseradish peroxidase (HRP) antibody (Solarbio, Beijing, China) was used as the secondary antibody. Western blotting detection was performed according to the conventional methods of the prior art.

[0188] 6. In vitro cytotoxicity and cytokine detection

[0189] The xCELLigence real-time cell analyzer (RTCA) of ACEA Biosciences was used to detect the in vitro anti-tumor activity of PG3-GD2-CAR-T cells against neuroblastoma. SK-N-SH, SK-N-AS and SH-SY5Y cells were collected, and the cell concentration was adjusted to 1×10 4 / 100 ul. Pipette 100 ul per well into the plate and place the plate into the instrument. Incubate for 24 h until the cells adhere, then add CAR-T cells. Resuspend the PG3-GD2-CAR-T cells in the medium of the target cells and count them. Add the corresponding target cells at effector-to-target ratios of 1:2, 1:1, and 4:1. Supplement the blank wells with medium to a final volume of 200 ul, and monitor the cell growth index every 15 min.

[0190] After co-incubating for 48 h, aspirate the supernatant. Detect the cytokine content in the supernatant using a Cytometric Bead Array (CBA; BD) kit.

[0191] 7. Xenograft model

[0192] To verify the anti-tumor effect of PG3-GD2-CAR-T cells in vivo, a subcutaneous tumor model in mice was established using the neuroblastoma cell line SK-N-AS. First, female B-NSG mice aged 6 - 8 weeks, 5 mice per group, were selected. On day 0, the mice were subcutaneously injected with SK-N-AS cells (1.5×10 7 / mouse) mixed with Matrigel (BD Biosciences) at a ratio of 1:1. On day 7, T cells and PG3-GD2-CAR-T cells (CAR positive rate 35%) were injected via the tail vein. Subsequently, the tumor volume of the mice was measured. When the tumor grew to 1000 mm 3 , the mice were euthanized. The length and width of the tumor were recorded, and the tumor volume was calculated according to the following formula (volume = length * width 2 / 2). All mouse experiments were carried out with the approval of the local institutional and committee responsible for animal experiments.

[0193] 8. Histological examination and TUNEL

[0194] Tumor tissues from 2 mice in the T cell group and 2 mice in the PG3-GD2-CAR-T group were randomly selected, embedded in paraffin, sectioned (3 μm thick), and subjected to immunohistochemical analysis. After dewaxing and rehydrating the paraffin sections, they were stained with hematoxylin-and-eosin to observe the pathological changes in the tumor tissues. After dewaxing and rehydrating the paraffin sections, they were immersed in 0.01 M citrate buffer and microwave repaired for 5 mins, then blocked with methanol solution containing 0.3% hydrogen peroxide. They were stained with CD3 antibody (diluted 1:100) and biotinylated secondary antibody, and finally, freshly prepared DAB chromogenic solution was added. The infiltration of T cells in the tumor tissues was observed. After dewaxing and rehydrating the paraffin sections, they were treated with proteinase K, quenched with 3% hydrogen peroxide solution, and then stained with a TUNEL kit. The apoptosis of tumor cells in the tissues was observed.

[0195] 9. Data analysis

[0196] Statistical analysis was performed using GraphPad Prism software (version 5.0). Paired t-tests were used to compare differences between groups. Brackets in the figures indicate the groups that were compared, and *, **, and *** indicate P < 0.05, < 0.01, and < 0.001, respectively.

[0197] Example 1 Construction and Specificity Verification of GD2-CAR Vector

[0198] In this example, a novel third-generation CAR structure targeting GD2 was constructed, which included a single-chain antibody sequence of a humanized GD2 antibody, a human Fc fragment, the transmembrane and intracellular regions of ICOS (the intracellular region of ICOS was the first co-stimulatory molecule), and then the intracellular region of 4-1BB was ligated as the second co-stimulatory molecule, and then connected to the signaling region of CD3ζ. Figure 1 A is a schematic diagram of the CAR structure. Subsequently, the PG3-GD2-CAR structure was subcloned into a lentiviral expression vector and named the PG3-GD2-CAR plasmid.

[0199] Three neuroblastoma cell lines, SK-N-SH, SK-N-AS, and SH-SY5Y, were selected as target cells for this experiment to detect the expression of GD2 on the cell surface.

[0200] The results were as Figure 1 shown in B. SK-N-SH cells were GD2-negative cells, SK-N-AS cells were weakly positive cells, and SH-SY5Y cells were strongly positive cells.

[0201] The constructed PG3-GD2-CAR vector was packaged into lentivirus. After concentration, the lentivirus was transfected into Jurkat cells to construct PG3-GD2-CAR-Jurkat cells. After virus transfection, the cells were sorted with anti-human Fc antibody and expanded in culture. The expression level of CAR on the surface of PG3-GD2-CAR-Jurkat cells was detected by flow cytometry.

[0202] The results were as Figure 1 shown in C. More than 90% of the cells were CAR + cells.

[0203] The PG3-GD2-CAR-Jurkat cells were separately co-cultured with SK-N-SH (GD2 neg ), SK-N-AS (GD2 low ), and SH-SY5Y (GD2 high) Co-culture the cells at an effector-to-target ratio of 1:1 for 24 h. Subsequently, detect the expression of CD25 and CD69 on the surface of PG3-GD2-CAR-Jurkat cells.

[0204] The results are as Figure 1 shown in low D. Compared with the control Jurkat cells, after co-culture with SK-N-AS (GD2 high ) and SH-SY5Y (GD2 neg ) cells, the expression of CD25 and CD69 on the surface of PG3-GD2-CAR-Jurkat cells increased significantly, while there was no significant change in the expression of CD25 and CD69 on the surface of CAR-Jurkat cells after co-culture with SK-N-SH (GD2

[0205]

[0206] The above results indicate that the PG3-GD2-CAR-Jurkat constructed in this example can be specifically activated by GD2-positive tumor cells, but not by GD2-negative tumor cells. Example 2 Construction of PG3-GD2-CAR-T cells

[0207] After activating PBMCs from healthy donors for 48 h, transfect them with PG3-GD2-CAR virus to construct PG3-GD2-CAR-T cells. After culturing CAR-T cells for 6 - 10 days, detect the expression level of CAR protein on the surface of T cells by anti-Fc antibody, and detect the expression level of CD3ζ in CAR-T cells by Western Blotting.

[0208] The detection results of the CAR protein expression level are as Figure 2 shown in

[0209] A. Approximately 40% (±10%) of T cells stably express CAR. Figure 2 The detection results of the CD3ζ expression level are as

[0210] shown in

[0211] Example 3 In vitro functional verification of PG3-GD2-CAR-T cells.To evaluate the in vitro biological functions of PG3-GD2-CAR-T cells, the in vitro killing of neuroblastoma by PG3-GD2-CAR-T cells was detected using an xCELLigence real-time cell analyzer (RTCA). SK-N-SH is a GD2-negative cell, and SK-N-AS and SH-SY5Y are GD2-positive tumor cells. T cells and PG3-GD2-CAR-T cells were used as effector cells, and the effector-to-target ratios were 1:2, 1:1, and 4:1, respectively, and they were co-incubated for 48 h. Meanwhile, the release of cytokines in the supernatant was detected.

[0212] The RTCA detection results are as Figure 3 shown in A. Compared with control T cells, PG3-GD2-CAR-T cells could significantly inhibit the growth of GD2 + neuroblastoma cells, and the inhibitory effect on SH-SY5Y cells with high expression of GD2 was particularly obvious. However, for GD2 - neuroblastoma cells, there was no obvious difference in the inhibitory effects between the T cell group and the PG3-GD2-CAR-T group.

[0213] The cytokine release is as Figure 3 shown in B. Compared with the T cell group, there was a significant increase in cytokine release in the supernatant of the PG3-GD2-CAR-T group, including Granzyme-B, TNF-α, IFN-γ, and IL-2.

[0214] Example 4 In Vivo Function Verification of PG3-GD2-CAR-T Cells

[0215] To further evaluate the in vivo anti-tumor effect of PG3-GD2-CAR-T, a mouse subcutaneous transplanted tumor model was constructed using SK-N-AS cells. Figure 4 A is a simple diagram of the animal experiment. On day 0, NSG mice were subcutaneously injected with SK-N-AS cells (1.5×10 7 / mouse), and on day 7, T cells and PG3-GD2-CAR-T cells (CAR positive rate 35%) were injected via the tail vein. Subsequently, the growth of tumors in the mice was detected. When the tumor grew to 1000 mm 3 in size, the mice were euthanized.

[0216] The results of the tumor growth experiment are as Figure 4 shown in B and 4D. In the T cell group, the tumor volume of the mice rapidly increased to 1000 mm around one month 3As described above, tumor growth in the mice of the PG3-GD2-CAR-T group was significantly inhibited, and the tumors in 3 mice completely disappeared. The results indicated that PG3-GD2-CAR-T cells could preferably home to the tumor tissue site and exert a continuous anti-tumor effect.

[0217] Peripheral blood of the mice was collected to detect red blood cells, white blood cells, hemoglobin and platelets. The results were as Figure 4 shown in Figure C. There were no significant differences between the mice of the PG3-GD2-CAR-T group and the control T cell group, indicating that PG3-GD2-CAR-T cells would not cause obvious changes in the blood picture in the peripheral blood of the mice and had good safety.

[0218] Finally, tumor tissues of 2 randomly selected mice in the T cell group and tumor tissues of 2 mice in the PG3-GD2-CAR-T group were selected for immunohistochemical analysis.

[0219] The results of HE staining were as Figure 5 shown in Figure A. Obvious pathological changes occurred in the histological structure of the tumor tissues of the mice in the PG3-GD2-CAR-T group, while there were no obvious changes in the control T cell group.

[0220] IHC staining was performed on CD3 molecules in the tumor tissues. The results were as Figure 5 shown in Figure B. There were more T cell infiltrations in the PG3-GD2-CAR-T experimental group.

[0221] Finally, the apoptosis of cells in the tumor tissues was detected by TUNEL analysis. The results were as Figure 5 shown in Figure C. Compared with the T cell group, more apoptotic cells could be detected in the PG3-GD2-CAR-T experimental group.

[0222] Discussion

[0223] For CAR-T cell therapy, the selection of targets is very important for the safety and feasibility of treatment. Given the high expression of GD2 in various pediatric and adult tumors including neuroblastoma and its low expression in normal tissues after birth, multiple research groups have developed various GD2-targeted treatment strategies including CAR-T therapy. The research experience of GD2-CAR-T has established the feasibility of using GD2 as a target for the treatment of neuroblastoma and has good curative effects. However, the curative effect of CAR-T in solid tumors is not as strong as that in hematological malignancies, and there are many challenges in designing CAR-T cells for solid tumors. These include the persistence and potency of T cells, the lack of tumor-specific targets, and the immunosuppressive tumor microenvironment, etc.

[0224] The present invention constructs a novel third-generation CAR structure targeting GD2, which connects the scFv fragment of a humanized GD2 antibody with the transmembrane region and intracellular region of ICOS, and simultaneously connects the intracellular region of 4-1BB as the second co-stimulatory molecule in the intracellular region of its CAR structure, and then connects it with CD3ζ. Constructing a third-generation CAR structure with ICOS and 4-1BB as its two co-stimulatory molecules in the CAR structure can effectively prolong the survival period of CAR-T cells in vivo and improve their anti-tumor efficacy.

[0225] The experimental results show that PG3-GD2-CAR-T cells exhibit strong anti-tumor activity both in vitro and in vivo against GD2 + neuroblastoma cells. First, the CAR structure was constructed on Jurkat cells to verify its specificity. After co-incubating PG3-GD2-CAR-Jurkat cells with a neuroblastoma cell line, the surface activation markers CD25 and CD69 increased with the increase in the GD2 expression level. This indicates that the PG3-GD2-CAR-Jurkat constructed in the present invention can be specifically activated by GD2-positive tumor cells, but not by GD2-negative tumor cells, and has good specificity. Subsequently, PG3-GD2-CAR-T cells (positive rate 40% ± 10%) were successfully constructed, and their growth inhibitory effect on a neuroblastoma cell line was detected by xECLLigence real-time cell analyzer (RTCA) in vitro. The results show that PG3-GD2-CAR-T cells can continuously and efficiently inhibit the growth of GD2 + neuroblastoma cells at a low effector-to-target ratio, and the inhibitory effect on GD2 high SH-SY5Y cells is particularly obvious. And for GD2 -For neuroblastoma cells, no obvious difference was observed between the T cell group and the PG3-GD2-CAR-T group. The same result was obtained from the detection of cytokines in the supernatant, indicating that the PG3-GD2-CAR-T cells constructed in the present invention have high specificity and cytotoxicity. In the mouse experiment, it was found that the PG3-GD2-CAR-T cells could also efficiently and continuously inhibit the growth of neuroblastoma in mice. Compared with the control T cell group, the tumor growth in the CAR-T group was significantly inhibited, there was more T cell infiltration in the tumor tissue, the tumor tissue structure was more severely damaged, and more tumor cells underwent apoptosis. This indicates that the PG3-GD2-CAR-T cells constructed in the present invention can accurately reach the tumor site and have high cytotoxicity. The tumors of three mice completely disappeared and did not relapse, indicating that the PG3-GD2-CAR-T cells constructed in the present invention have high persistence and can continuously and efficiently play an anti-tumor effect. There were no significant differences in red blood cells, white blood cells, hemoglobin, and platelets in the peripheral blood of mice between the PG3-GD2-CAR-T group and the control T cell group, indicating that the PG3-GD2-CAR-T cells of the present invention do not cause obvious changes in the blood picture of the peripheral blood of mice and have good safety.

[0226] In summary, the present invention constructs a novel third-generation CAR-T cell (containing ICOS and 41BB co-stimulatory signals) based on the GD2 humanized antibody sequence, and proves the anti-tumor effect on neuroblastoma tumor cells through a series of experiments. It not only confirms the specific cytotoxicity of PG3-GD2-CAR-T cells against GD2-positive neuroblastoma at a low effector-to-target ratio through in vitro experiments, but also proves through animal experiments that the PG3-GD2-CAR-T cells have high cytotoxicity and good persistence, and can continuously and efficiently inhibit the tumor progression in a xenograft mouse model constructed with neuroblastoma tumor cells. In short, the PG3-GD2-CAR-T cells constructed in the present invention provide a new method or strategy for the treatment of solid tumors such as neuroblastoma.

[0227] All the documents mentioned in the present invention are cited in this application as references, just as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0228] The sequences involved in the sequence listing of this application are as follows:

[0229]

[0230] Sequence Listing <110> Boshengji Medical Technology (Suzhou) Co., Ltd. <120> GD2-targeted CAR-T cells and their preparation and application <130> P2020-0475 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 119 <212> PRT <213> Artificial Sequence <400> 1 Gln Val Gln Leu Val Glu Ser Gly Pro Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Ile Ser Cys Ala Val Ser Gly Phe Ser Val Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ala Gly Gly Ile Thr Asn Tyr Asn Ser Ala Phe Met 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ser Arg Gly Gly His Tyr Gly Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 2 <211> 107 <212> PRT <213> Artificial Sequence <400> 2 Glu Ile Val Met Thr Gln Thr Pro Ala Thr Leu Ser Val Ser Ala Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Arg Tyr Ser Gly Val Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Glu Phe Thr Phe Thr Ile Ser Ser Val Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Phe Gly 85 90 95 Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val 100 105 <210> 3 <211> 704 <212> PRT <213> Artificial Sequence <400> 3 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Val Glu Ser Gly Pro Gly 20 25 30 Val Val Gln Pro Gly Arg Ser Leu Arg Ile Ser Cys Ala Val Ser Gly 35 40 45 Phe Ser Val Thr Asn Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly 50 55 60 Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ala Gly Gly Ile Thr Asn 65 70 75 80 Tyr Asn Ser Ala Phe Met Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser 85 90 95 Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Met Tyr Tyr Cys Ala Ser Arg Gly Gly His Tyr Gly Tyr Ala Leu 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met 145 150 155 160 Thr Gln Thr Pro Ala Thr Leu Ser Val Ser Ala Gly Glu Arg Val Thr 165 170 175 Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp Val Thr Trp Tyr 180 185 190 Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ser Ala Ser 195 200 205 Asn Arg Tyr Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Tyr Gly 210 215 220 Thr Glu Phe Thr Phe Thr Ile Ser Ser Val Gln Ser Glu Asp Phe Ala 225 230 235 240 Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Phe Gly Gln Gly Thr Lys 245 250 255 Leu Glu Ile Lys Arg Thr Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 260 265 270 Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe 275 280 285 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 290 295 300 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 305 310 315 320 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 325 330 335 Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr 340 345 350 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 355 360 365 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 370 375 380 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 385 390 395 400 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 405 410 415 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 420 425 430 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 435 440 445 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 450 455 460 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 465 470 475 480 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Trp Leu Pro Ile 485 490 495 Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile 500 505 510 Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn 515 520 525 Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg 530 535 540 Leu Thr Asp Val Thr Leu Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 545 550 555 560 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 565 570 575 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 580 585 590 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 595 600 605 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 610 615 620 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 625 630 635 640 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 645 650 655 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 660 665 670 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 675 680 685 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 690 695 700 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <400> 4 Gly Gly Gly Gly Ser 1 5 <210> 5 <211> 22 <212> PRT <213> Artificial Sequence <400> 5 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro 20 <210> 6 <211> 229 <212> PRT <213> Artificial Sequence <400> 6 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 7 <211> 23 <212> PRT <213> Artificial Sequence <400> 7 Trp Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly 1 5 10 15 Cys Ile Leu Ile Cys Trp Leu 20 <210> 8 <211> 35 <212> PRT <213> Artificial Sequence <400> 8 Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr 1 5 10 15 Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp 20 25 30 Val Thr Leu 35 <210> 9 <211> 42 <212> PRT <213> Artificial Sequence <400> 9 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 10 <211> 112 <212> PRT <213> Artificial Sequence <400> 10 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 11 <211> 2115 <212> DNA <213> Artificial Sequence <400> 11 atgctgctgc tggtgacctc tctgctgctc tgcgaactgc ctcacccagc ctttctgctg 60 atcccccagg tgcagctggt ggaatcagga ccaggagtgg tgcagccagg aagatctctg 120 aggatctctt gcgcagtgtc cggcttcagc gtgaccaatt acggcgtgca ttgggtccgg 180 cagcctccag gaaaaggact cgagtggctc ggcgtgattt gggcaggagg catcaccaac 240 tacaacagcg ccttcatgag ccggctgacc atcagcaagg acaacagcaa gaacaccgtg 300 tacctgcaga tgaacagcct gcgcgcagag gacaccgcca tgtactattg cgccagcagg 360 ggaggacact acggctacgc cctggactat tggggacagg gaaccctcgt gacagtgtct 420 tctggcggag gaggaagcgg aggaggagga tctggaggag gcggcagcga gattgtgatg 480 acccagaccc cagccacact gtctgtgtct gccggagaac gcgtgaccat cacttgcaag 540 gcctctcaga gcgtgtctaa cgacgtgacc tggtaccagc agaagccagg acaggctcct 600 aggctgctga tctacagcgc cagcaacagg tacagcggag tgccagccag attcagcgga 660 tcaggctacg gcaccgagtt caccttcacc atcagcagcg tgcagagcga ggactttgcc 720 gtgtactttt gccagcagga ctacagcagc ttcggacagg gcaccaagct ggagatcaag 780 cggacagtgg agagcaaata cggccctcct tgccctcctt gtccagcccc agagtttgag 840 ggaggaccta gcgtgttcct gttccctccc aagcccaagg acaccctgat gatcagcagg 900 acccccgaag tgacttgcgt ggtggtggac gtgtctcagg aggaccccga ggtgcagttc 960 aattggtacg tggacggagt ggaagtgcac aacgccaaga ccaagcccag agaggagcag 1020 ttccagagca cctacagggt ggtgtccgtg ctgacagtgc tgcaccagga ttggctgaac 1080 ttccagagca cctacagggt ggtgtccgtg ctgacagtgc tgcaccagga ttggctgaac 1080 ggcaaggagt acaagtgcaa ggtgtccaac aagggcctgc ccagcagcat cgagaagacc 1140 ggcaaggagt acaagtgcaa ggtgtccaac aagggcctgc ccagcagcat cgagaagacc 1140 atcagcaagg ccaaaggcca gcctagagaa cctcaggtgt acaccctgcc cccttctcag 1200 atcagcaagg ccaaaggcca gcctagagaa cctcaggtgt acaccctgcc cccttctcag 1200 gaggagatga ccaagaacca ggtgtccctg acttgcctcg tgaagggctt ctaccccagc 1260 gaggagatga ccaagaacca ggtgtccctg acttgcctcg tgaagggctt ctaccccagc 1260 gatatcgccg tggagtggga atctaacggc cagccagaga acaactacaa gaccaccccc 1320 gatatcgccg tggagtggga atctaacggc cagccagaga acaactacaa gaccaccccc 1320 ccagtgctgg acagcgacgg cagcttcttc ctgtacagca ggctgaccgt ggacaaaagt 1380 ccagtgctgg acagcgacgg cagcttcttc ctgtacagca ggctgaccgt ggacaaaagt 1380 cgctggcagg agggcaacgt gttcagttgc agcgtgatgc acgaggccct gcacaaccac 1440 cgctggcagg agggcaacgt gttcagttgc agcgtgatgc acgaggccct gcacaaccac 1440 tacacccaga agagcctgag cctgagcctg ggcaagtggc tgcctattgg ttgcgcagct 1500 tacacccaga agagcctgag cctgagcctg ggcaagtggc tgcctattgg ttgcgcagct 1500 ttcgtcgtcg tctgcatcct gggttgcatc ctgatttgct ggctgaccaa gaagaagtac 1560 ttcgtcgtcg tctgcatcct gggttgcatc ctgatttgct ggctgaccaa gaagaagtac 1560 agctctagcg tgcacgaccc taacggcgag tacatgttca tgcgggccgt caacaccgcc 1620 agctctagcg tgcacgaccc taacggcgag tacatgttca tgcgggccgt caacaccgcc 1620 aaaaagagcc ggctgaccga cgtgacactg aagaggggcc ggaagaagct gctgtacatc 1680 aaaaagagcc ggctgaccga cgtgacactg aagaggggcc ggaagaagct gctgtacatc 1680 ttcaagcagc ccttcatgcg gccagtgcag acaacacagg aggaagacgg ctgcagttgc 1740 ttcaagcagc ccttcatgcg gccagtgcag acaacacagg aggaagacgg ctgcagttgc 1740 aggtttccag aggaggaaga gggcggttgc gagctgagag tgaagttcag caggagcgcc 1800 gacgctccag cctatcagca gggacagaac cagctgtaca acgagctgaa cctgggcagg 1860 agggaggaat acgacgtgct ggacaagcgg aggggaagag atccagagat gggcggcaag 1920 cctagaagga agaaccccca ggagggcctg tacaacgagc tgcagaagga caagatggcc 1980 gaggcttaca gcgagatcgg catgaagggc gagaggagaa gaggcaaagg ccacgacgga 2040 ctgtatcagg gcctgagcac agccaccaag gacacctacg acgctctgca catgcaggct 2100 ctgcctccta gatga 2115 <210> 12 <211> 7 <212> PRT <213> Artificial Sequence <400> 12 Gly Phe Ser Val Thr Asn Tyr 1 5 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <400> 13 Trp Ala Gly Gly Ile 1 5 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <400> 14 Arg Gly Gly His Tyr Gly Tyr Ala Leu Asp Tyr 1 5 10 <210> 15 <211> 11 <212> PRT <213> Artificial Sequence <400> 15 Lys Ala Ser Gln Ser Val Ser Asn Asp Val Thr 1 5 10 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <400> 16 Ser Ala Ser Asn Arg Tyr Ser 1 5 <210> 17 <211> 6 <212> PRT <213> Artificial Sequence <400> 17 Gln Gln Asp Tyr Ser Ser 1 5

Claims

1. An engineered immune cell, characterized in that, The engineered immune cells express a chimeric antigen receptor, and the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:3; And the engineered immune cells are CAR-T cells.

2. A preparation, characterized in that, The preparation contains the engineered immune cells described in claim 1 and a pharmaceutically acceptable carrier.

3. Use of the engineered immune cells according to claim 1 or the preparation according to claim 2, characterized in that, It is used for preparing a drug for treating GD2-positive neuroblastoma.

4. A method for preparing engineered immune cells, characterized in that, The immune cells express a CAR with an amino acid sequence as shown in SEQ ID NO:3, and the method includes the following steps: (a) Providing immune cells to be transformed; and (b) Transducing a nucleic acid molecule encoding a CAR with an amino acid sequence as shown in SEQ ID NO:3 or a vector containing the nucleic acid molecule into the immune cells, thereby obtaining the engineered immune cells.

Images