Novel chimeric antigen receptor and immune cell containing the same

Abstract

The present invention is a novel chimeric antigen receptor and an immune cell comprising the same, and relates to a fusion protein that can specifically bind to a tumor-specific marker and an immune cell that can express the fusion protein, such as a T cell or a NK cell. The fusion protein of the present invention may include a chimeric antigen receptor (CAR), interleukin 12 (IL12) and an oxygen-dependent degradation region (ODD). The CAR of the present invention includes an extracellular domain, a transmembrane domain and an intracellular domain, and the extracellular domain can specifically bind to a tumor-specific antigen and activate T or NK cells through the transmembrane domain and the intracellular domain. The CAR-T cells or NK cells provided by the present invention target tumor-specific markers and use CAR-T cells or NK cells to specifically kill tumor cells, such as myeloma or leukemia. The CAR-T or NK cells of the present invention can be used as therapeutic drugs for tumor diseases, providing a new method for the prevention and treatment of tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a novel chimeric antigen receptor, immune cells containing the same, and preparation and application thereof. Background Art

[0002] Diffuse large B cell lymphoma (DLBCL) is the most common type of malignant lymphoma, accounting for about 30%-40% of its incidence. DLBCL responds well to traditional chemotherapy and radiotherapy. The addition of monoclonal CD20 antibody rituximab has further improved the efficacy and prognosis, allowing more than half of the patients to achieve long-term survival or cure. At this stage, the challenge of treatment is refractory patients with early relapse, who have a short survival period and no effective treatment. It is urgent to find effective treatments and methods for refractory DLBCL.

[0003] CAR-T stands for Chimeric Antigen Receptor T-Cell. Immunotherapy based on chimeric antigen receptor T cells is currently a hot topic of research. CAR-T therapy is based on isolating and culturing the autoimmune T cells of tumor patients, using transgenic technology to express molecules that specifically recognize tumor cell surface antigens, such as specific single-chain antibody scFv (single-chain antibody fragment) and polycistronic proteins of T cell co-stimulatory molecules on the surface of T cells, and then injecting the genetically modified T cells back into the patient's body. The injected CAR-T cells specifically recognize and kill tumor cells in the body.

[0004] In 2011, Professor Carl H June published a case of second-generation CAR-T therapy based on 4-1BB co-stimulatory signal and targeting CD19, with the indication of chronic myeloid leukemia (CLL), and successfully cured Emily, a 4-year-old girl suffering from CLL. Subsequently, CD19 CAR-T was applied to a variety of blood diseases such as ALL, CLL, DLBCL, and the remission rate of most blood diseases ALL and CLL can reach 90%.

[0005] Compared with the remission rate of up to 90% for other blood diseases, the cure rate of CD19 CAR-T for refractory DLBCL does not exceed 50%. The reason is that the immune microenvironment of DLBCL limits the efficacy of CD19 CAR-T to a certain extent. Therefore, there is an urgent need to find a suitable method to locally improve the immune microenvironment of DLBLC tumors.

[0006] Interleukin-12 (IL-12) was first discovered in EBV-transformed B cells as a heterodimer complex consisting of p35 and p40 subunits covalently linked. IL-12 is a cytokine with multiple functions. Hsieh et al. found in 1993 that primitive naive CD4 + Under the stimulation of interferon-γ (IFN-γ) released by natural killer cells (NK), cells are induced to express the transcription factor STAT1, which then transcribes the expression of T-bet, which in turn promotes the transformation of naive CD4+ cells into CD4 + TH1 effector cells, while T-bet expression induces interleukin-12 receptor β2-subunit (IL-12Rβ2) expression on CD4 + Th1 cell membrane surface, at this time IL-12 binds to the IL-12R on the surface of CD4+Th1 cell membrane, prompting CD4 + Th1 cells secrete IFN-γ and perform effector cell functions together with other inflammatory factors. Therefore, IL-12 plays a bridging role between innate immunity and adaptive cellular immunity. The immunomodulatory function of IL-12 is not limited to the bridging role between innate immunity and adaptive immunity. IL-12 also plays a role in promoting CD4 + While TH1 cells secrete IFN-γ, they can also induce the release of other secondary and tertiary pro-inflammatory cytokines, which also have a cytotoxic effect on tumor cells. The release of these pro-inflammatory factors also changes the local immune environment of the tumor and prolongs the CD8 + T cell survival promotes tumor killing effect. From the perspective of immune cells, IL-12 helps promote NK, NK-T cells, CD4 + 、CD8 + T cells increase their survival rate and enhance cell effector function, especially the IFN-γ induced by them, which also plays an anti-tumor role. From the perspective of tumor microenvironment, the secretion of IFN-γ can reduce the secretion of vascular endothelial growth factor (VEGF) by tumor cells, thereby inhibiting tumor metastasis. At the same time, NK, NK-T cells, and CD4 + 、CD8 + T cells can produce chemokines IP-10 and MIG, which inhibit the expression of matrix metalloproteinases (MMPs) in endothelial cells and reduce changes in extracellular matrix (ECM) remodeling, thereby reducing angiogenesis and tumor invasion. Summary of the invention

[0007] The inventors of the present invention have found that the combined use of CD19 CAR-T and IL-12 to treat DLBCL can improve the remission rate of CD19CAR-T for DLBCL. At the same time, while IL-12 brings about immune killing effects, it also brings about certain cytotoxicity, especially cytotoxic damage to the liver, which also greatly limits the clinical application of IL-12 in anti-tumor treatment. Therefore, it is also urgent to find a solution to reduce the hepatotoxicity of IL-12.

[0008] In order to overcome the toxic effects of IL-12 and CAR cells when used in combination, the present invention co-expresses the fusion protein (IL-12ODD) with CAR on the surface of immune cells. The anaerobic glycolysis characteristics of tumor cells enable them to be more adaptable to the local anaerobic microenvironment of the tumor. The CAR or CAR immune cells designed by the present invention allow the secretory IL-12 (IL-12ODD) expressed by oxygen pressure regulation to be secreted and exert its effect only in the local anaerobic microenvironment of the tumor, while in the aerobic environment of normal tissue, oxygen pressure regulation of ODD will activate the ubiquitination degradation pathway to degrade IL-12. The application of IL-12ODD allows IL-12 to exert its anti-tumor effect only when it is highly expressed in the anaerobic microenvironment of the tumor, thereby reducing the concentration of IL-12 in normal peripheral blood tissues and the liver and lung toxicity caused by it.

[0009] In addition, the present invention found that a 15-20 amino acid linker peptide (linker) based on the single-chain antibody scFv connecting the heavy chain variable region and the light chain variable region of the antibody can improve the therapeutic effect of the combination of CAR immune cells and IL12. Specifically, the present invention uses a new type of connecting peptide Linker218. Compared with the traditional connecting peptide (G4S)3, Linker218 can make the single-chain antibody scFv present a more flexible spatial structure without reducing affinity, and increase the stability to the proteolytic enzyme system.

[0010] The inventors of the present invention co-expressed CD19 CAR-T containing a novel connecting peptide Linker218 and IL-12 on the surface of T cells through a self-cleaving peptide connection. After the CD19 CAR-T cells co-expressing IL-12 were reinfused, the IL-12 expressed by the CD19 CAR-T cells after homing to the lymph nodes can induce autoimmune cells to secrete IFN-γ, thereby improving the DLBLC tumor immune microenvironment and inhibiting the progression and metastasis of DLBCL. At the same time, it increases the survival of CD19 CAR-T cells, prolongs the killing effect of CD19 CAR-T cells, and improves the efficacy of CD19 CAR-T immunotherapy for refractory DLBCL.

[0011] The inventors of the present invention constructed a third-generation lentiviral plasmid in vitro expressing a CD19-specific antigen 4-1BB containing a novel linker peptide Linker218 and CD28 as an intracellular activation signal, packaged a lentivirus of appropriate titer using 293T in vitro, and expressed the CD19-specific antigen and intracellular activation signal in CD3 isolated from peripheral blood. + T cells, modified T cells killed refractory DLBLC lines in vitro, and the effects of CD19 CAR-T combined with IL12 and IL12ODD were compared by calculating growth curves, calculating killing efficiency, and in vivo and in vitro methods.

[0012] On the one hand, the present invention provides a chimeric antigen receptor (CAR), which comprises (1) an extracellular antigen binding domain; (2) a transmembrane domain; and (3) an intracellular signaling domain, characterized in that the CAR also comprises a fusion protein of interleukin 12 (IL12) and an oxygen-dependent degradation domain (ODD) connected to and co-expressed with the intracellular domain, and optionally, the intracellular domain and the fusion protein are connected via a self-cleaving protein (e.g., selected from the group consisting of T2A, P2A, E2A, F2A, or a combination thereof).

[0013] In one embodiment of the present invention, the transmembrane domain of the present invention is derived from one or more transmembrane domains selected from the group consisting of α, β or ζ chain of the T cell receptor, CD3ε, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD80, CD86, CD134, CD137, CD152, CD154 and ICOS.

[0014] In one embodiment of the present invention, the intracellular signal transduction domain of the present invention comprises a co-stimulatory signaling domain and is from: CD2, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD7, CD22, CD27, CD28, CD30, CD40, CD66d, CD79a, CD79b, CD83, CD134, CD137, ICOS, CD154, 4-1BB and OX40, LFA-1, LIGHT, NKG2C and B7-H3, one or more.

[0015] In one embodiment of the present invention, the CAR of the present invention further comprises a hinge domain located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain, for example, derived from CD8α.

[0016] In one embodiment of the present invention, for the CAR of the present invention, the extracellular antigen binding domain is a molecule that can bind to a tumor cell surface antigen (e.g., CD19, CD20, CD22, CD123, CD30, CD33, CD38, CD44, CD138, CD276, EGFR, BCMA, HER1, HER2, HER3, EpCAM, mesothelin, FAP (Fibroblast activation protein), Glypican-3, CEA, PSMA, IL13Rα2, CD171, GD2, CEACAM6, CLDN4, CLDN18.2, NKGD2, LAMC2, CA9, CST1, EPPK1, and ANO1), such as an antibody (Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, scFv, double-scFv, (scFv)2, miniantibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins, and single-domain antibodies (sdAb, nanobodies), bispecific antibodies, or trispecific antibodies).

[0017] With respect to those commonly used among tumor cell surface antigens, they are exemplarily explained as follows.

[0018] CD19 is a surface protein expressed on B lymphocytes and follicular dendritic cells. It is a member of the immunoglobulin (Ig) superfamily and is located on the short arm of chromosome 16 (16p11.2). It encodes a type I transmembrane glycoprotein of 556 amino acids and has a molecular weight of 95KD. CD19 is only expressed in normal and malignant B cells and is almost not expressed in other tissues. Secondly, CD19 is not lost during the malignant transformation of B cells, and refractory / relapsed cases are still effective. Furthermore, CD19 is not expressed in hematopoietic stem cells and pro-B cells. After treatment is stopped, B cells can be effectively supplemented. Therefore, various leukemia treatment strategies targeting CD19 have achieved good clinical results in recent years, including bispecific antibodies of CD3 and CD19 and CAR-T targeting CD19.

[0019] The CD20 antigen is a B cell differentiation antigen that is only expressed on the surface of pre-B cells and mature B cells. It is expressed in more than 95% of B cell lymphomas, but not in hematopoietic stem cells, plasma cells and other normal tissues. Several monoclonal antibodies targeting CD20 (such as Rituxan) have been successfully used in the clinic to treat a variety of lymphomas.

[0020] CD38 is a 45kDa single-chain transmembrane glycoprotein with an overall structure of a short cytoplasmic tail at the N-terminus, a single transmembrane domain, and a long extracellular region at the C-terminus. In adults, CD38 is expressed on most natural killer cells, T cells, B cells, and monocytes / macrophages. It is also expressed to a certain extent on platelets and red blood cells. Certain monoclonal antibodies targeting CD38 have been approved for the treatment of multiple myeloma.

[0021] BCMA (B cell maturation antigen) is a type III transmembrane protein composed of 185 amino acid residues. It belongs to the TNF receptor family and can stimulate B cell proliferation by binding to its ligand B cell activating factor BAFF or proliferation inducing ligand APRIL. BCMA is normally expressed in mature B cells and plasma cells, and is also widely expressed in multiple myeloma (MM), making it an ideal immunotherapy target for multiple myeloma.

[0022] The HER2 gene is located on human chromosome 17q21 and encodes a transmembrane protein with a molecular weight of 185 kD. The protein has tyrosine kinase activity and exists in an inactive form under normal conditions. It participates in regulating normal cell differentiation and is usually only expressed in infancy. In adults, it is only expressed at low levels in a few tissues (SA 2000; 97: 3444-3449. Slamon et al., Science 1987; 235: 177-182).

[0023] CEA (carcinoembryonic antigen) is a glycoprotein produced by colorectal cancer tissue. As an antigen, it can induce an immune response in patients. It is widely present in digestive system cancers of endoderm origin, in the digestive tract tissues of normal embryos, and in trace amounts in normal human serum. Carcinoembryonic antigen is a broad-spectrum tumor marker that can reflect the presence of a variety of tumors. It is a good tumor marker for judging the efficacy of colorectal cancer, breast cancer, and lung cancer, as well as for disease progression, monitoring, and prognosis estimation.

[0024] PSMA is a 110kDa type II transmembrane protein. Its gene is located on the short arm of chromosome 11q. It is expressed in normal prostate epithelium and prostate tumor cells. The expression level in tumor cells is upregulated. Some studies believe that PSMA is involved in the regulation of cell migration during prostate cancer. In prostate cancer, the expression of PSMA is positively correlated with the grade of the tumor and the presence of hormone resistance. Strong expression of PSMA means a higher recurrence rate. PSMA is now considered one of the references for prostate cancer.

[0025] More specifically, in the CAR of the present invention, the IL12 has the amino acid sequence of SEQ ID NO: 5; the ODD has the amino acid sequence of SEQ ID NO: 7. Preferably, the CAR comprises the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3.

[0026] The improved CAR of the present invention optionally further comprises a tag sequence (eg, Poly-His, Hemagglutinin, c-Myc, GST, Flag-tag, etc.) or an IgG1-Fc protein sequence.

[0027] Another aspect of the present invention also provides nucleotides encoding CAR, preferably, the nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO: 2 or comprises a nucleotide sequence consisting of SEQ ID NO: 2-SEQ ID NO: 10-SEQ ID NO: 4 in sequence.

[0028] The third aspect of the present invention provides an isolated CAR-T cell or CAR-NK cell or mononuclear macrophage, wherein the cell is capable of expressing CAR or comprises a polynucleotide of the present invention.

[0029] The fourth aspect of the present invention provides a vector comprising the above-mentioned polynucleotide.

[0030] In a specific embodiment, the vector of the present invention is an expression vector, such as a viral vector, preferably a retroviral vector, such as a lentiviral vector, preferably selected from human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), Vesna-Medivirus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), simian immunodeficiency virus (SIV), adenovirus and poxvirus.

[0031] The fifth aspect of the present invention provides a pharmaceutical composition comprising the chimeric antigen receptor provided by the present invention, or the CAR-T cells or CAR-NK cells or mononuclear macrophages provided by the present invention, and optionally, a pharmaceutically acceptable carrier.

[0032] The fifth aspect of the present invention provides a method for preparing the CAR-T cells or CAR-NK cells or mononuclear macrophages provided by the present invention, which comprises introducing the vector provided by the present invention into T lymphocytes or natural killer cells or mononuclear macrophages.

[0033] The fifth aspect of the present invention provides a use. Specifically, the use refers to the use of the CAR provided by the present invention, or the CAR-T cell or CAR-NK cell provided by the present invention in the preparation of a drug for treating and / or preventing cancer, illustratively, the cancer is myeloma and leukemia.

[0034] The present invention also provides a method for preparing the CAR-T cell or CAR-NK cell or mononuclear macrophage of the present invention, or preparing the immune effector cell of the present invention, which comprises introducing the vector of the present invention into the cell.

[0035] Exemplarily, the method for preparing CAR-T cells of the present invention comprises the following steps:

[0036] (1) synthesizing and amplifying the CAR-self-cleaving protein-IL12-ODD protein gene, and cloning the protein gene into a lentiviral expression vector;

[0037] (2) using the lentiviral packaging plasmid and the lentiviral expression vector plasmid obtained in step (1) to infect 293T cells to package and prepare lentivirus; and

[0038] (3) Using the lentivirus obtained in step (2) to infect T cells to obtain CAR-T cells.

[0039] The present invention also provides the use of the following materials in preparing drugs for treating and / or preventing cancer:

[0040] (a) the CAR of the present invention; or

[0041] (b) The CAR-T cells or CAR-NK cells described in the present invention.

[0042] Exemplarily, the cancer is a tumor and related diseases that highly express tumor-specific markers, such as myeloma and leukemia.

[0043] The present invention also provides a method for treating and / or preventing cancer, comprising administering an effective amount of (a) the CAR described in the present invention; or (b) the CAR-T cells or CAR-NK cells or monocytes or macrophages described in the present invention to a subject.

[0044] For the fusion protein, CAR, etc. described in the present invention, the present invention also considers its variants, such as its identity sequence or humanized sequence, etc. Exemplarily, the identity sequence refers to an identity of about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1 ... or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0045] For the polynucleotides of the present invention, the present invention also contemplates degenerate sequences or complementary sequences thereof. Exemplarily, the degenerate sequence has a homology of about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0046] The CAR provided by the present invention can specifically bind to tumor-specific antigen tumor-specific markers and activate the T cells through the transmembrane domain and the co-stimulatory signal transduction region. The CAR-T cells can express fusion proteins with tumor-specific markers as target antigens, so they can specifically kill tumor cells and are used to treat tumor diseases, such as tumors with high expression of tumor-specific markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagram of the pEF plasmid structure inserted into the CD19CARIL12ODD construct.

[0048] Figure 2 ELISA was used to detect the IL12 secretion level of CAR19IL12ODD under normal oxygen (17%) and hypoxic (1%) conditions.

[0049] Figure 3 ,in: Figure 3 A: LDH was used to detect the in vitro killing efficiency of CAR19 on diffuse large B-cell lymphoma cell line Ly3; Figure 3 B: In vivo killing curve of CAR19 against diffuse large B-cell lymphoma cell line Ly3.

[0050] Figure 4 ,in: Figure 4 A: ELISA was used to detect the concentration of IL12 in peripheral blood after CAR19 killed the diffuse large B-cell lymphoma cell line Ly3 in vivo; Figure 4 B: Loss detection of the survival rate of CAR-T cells in peripheral blood after CAR19 killed the diffuse large B-cell lymphoma cell line Ly3 in vivo. DETAILED DESCRIPTION

[0051] The CART or NK cells of the present invention can target tumor-specific markers (such as CD19) and can be used to treat cancers associated with CD19, for example.

[0052] The high affinity of the CAR of the present invention enables CAR-T or NK cells to a) recognize tumor target cells with high, medium and low tumor marker surface expression, with low off-target reactivity, b) be activated against the tumor target cells, and c) kill the tumor target cells. Therefore, the CAR-T of the present invention can be used to treat a variety of cancers, such as lymphoma, multiple myeloma tumor cells and acute myeloid leukemia and B-NHL, such as follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma and chronic lymphocytic leukemia, etc.

[0053] In an in vitro co-culture system, the CAR-T or NK cells of the present invention can have a significant therapeutic effect or inhibitory effect on diffuse large B-cell lymphoma (DLBCL).

[0054] Animal experiments have confirmed that the CAR-T or NK cells of the present invention can significantly kill diffuse large B-cell lymphoma (DLBCL) and promote the infiltration of tumors.

[0055] At the same time, the experiments also confirmed the in vitro killing effect of the fusion protein CD19-PE24 containing the antibody of the present invention on RPMI 8226 cells.

[0056] Chimeric Antigen Receptor:

[0057] CAR comprises an extracellular extracellular domain derived from an antibody and an intracellular domain comprising a signal transduction module derived from a T cell signal transduction protein. In one embodiment, the extracellular domain may include a heavy chain variable region from an immunoglobulin, or a variable region comprising a heavy chain and a light chain, such as being constructed as a single chain variable fragment (scFv), preferably a single domain antibody (sdAb) with only a heavy chain variable region. sdAb is connected to a hinge region, which provides flexibility and transduces signals to an intracellular signal transduction domain via a transmembrane domain. The transmembrane domain is preferably derived from CD8α. In the first generation of CAR (the term "generation" is for an intracellular signal transduction domain domain), the intracellular signal transduction domain is composed of the ζ chain of the TCR complex. The second generation CAR is designed to contain a single costimulatory domain derived from CD28 or 4-1BB. The third generation CAR includes two costimulatory domains, such as 4-1BB-CD3ζ. The present invention preferably relates to a second or third generation CAR.

[0058] The present invention exemplarily provides a genetically engineered receptor for redirecting the cytotoxicity of immune effector cells to cancer cells.These genetically engineered receptors are referred to herein as chimeric antigen receptors (CAR).CAR is based on the antibody specificity of targeted antigens (eg, CD19) combined with the intracellular domain of activated T cell receptors or NK cell receptors, with a chimeric protein molecule of specific anti-CD19 cellular immune activity.In this article, the term "chimeric" refers to different proteins or DNA compositions from different sources.

[0059] The CAR of the present invention includes binding to, for example, an extracellular domain (also referred to as a binding domain or antigen binding domain) of CD19, a transmembrane domain, and an intracellular domain or an intracellular signaling domain. Exemplarily, the binding of the anti-CD19 antigen binding domain of CAR to CD19 on the surface of the target cell causes the aggregation of CAR and delivers activation stimulation to cells containing CAR. CAR can specifically redirect immune effector cells, thereby inducing proliferation, cytokine production, phagocytosis, or cell killing of target antigen-expressing cells.

[0060] In some embodiments of the present invention, CAR comprises the following domains: a humanized extracellular binding domain that specifically binds to a cancer cell-specific surface antigen; a transmembrane domain; one or more intracellular signaling domains. In some embodiments, CAR sequentially comprises an extracellular binding domain of a humanized CD19 antigen binding fragment; one or more spacer regions; a transmembrane domain; one or more intracellular signaling domains; and an IL12-ODD fusion protein connected to the signal transduction domain by a self-cleaving protein.

[0061] "Extracellular antigen binding domain" or "extracellular binding domain" are used interchangeably and provide the CAR with the ability to specifically bind to a target antigen of interest (e.g., CD19). The binding domain can be derived from natural, synthetic, semisynthetic, or recombinant sources. Preferred are sdAbs of recombinant origin.

[0062] "Specific binding" should be understood by those skilled in the art, who are well aware of various experimental methods or means that can be used to test binding and binding specificity. Methods for determining equilibrium association or equilibrium dissociation constants are known in the art. In many protein-protein interactions, some cross-reactions or background binding may occur, but this does not impair the "specificity" of the binding between CAR and epitope. For example, "specific binding" describes the binding of anti-CD19 antibodies or their antigen-binding fragments (also including their CARs) to CD19, and its binding affinity is higher than background binding.

[0063] "Antigen (Ag)" refers to a compound, composition or substance that can stimulate antibody production or T cell response in an animal. In some embodiments of the invention, the target antigen is an epitope of a CD19 polypeptide. "Epitope" refers to the region of an antigen that binds to a binding agent. An epitope can be formed by continuous amino acids, or by non-continuous amino acids that result in the tertiary structure of a protein.

[0064] "Single-chain Fv" or "scFv" antibody fragments comprise the V H Domain and V L domains, wherein these domains are present in a single polypeptide chain and in either orientation (e.g., V L -V H or V H -V L ). Typically, the scFv polypeptide also comprises V H Domain and V L The polypeptide linker between the domains enables the scFv to form the desired structure for antigen binding. In a preferred embodiment, the CAR of the present invention comprises an antigen-specific binding domain, which is an scFv and can be a mouse, human or humanized scFv. Single-chain antibodies can be cloned from the V region genes of hybridomas that are specific to the desired target. In a particular embodiment, the antigen-specific binding domain is a humanized scFv that binds to a human CD19 polypeptide.

[0065] Antibodies and Antibody Fragments:

[0066] CAR comprises an extracellular antigen binding domain, which comprises an antibody or antibody fragment that binds to a tumor-specific marker (e.g., CD19) polypeptide. Therefore, the antibodies or antibody fragments of the present invention include, but are not limited to, polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single-chain fragments (scFv), single variable fragments (ssFv), single domain antibodies (e.g., VHH fragments from nanobodies), Fab fragments, F(ab')2 fragments, fragments produced by Fab expression libraries, anti-idiotypic antibodies, and epitope binding fragments or any combination of the above, provided that they have similar binding properties of the CAR of the present invention, preferably comprising corresponding CDRs, or VHH fragments as described herein. H and V L Region. Miniantibodies and multivalent antibodies such as bivalent antibodies, trivalent antibodies, tetravalent antibodies and pentavalent antibodies can also be used in the method of the present invention. The immunoglobulin molecule of the present invention can be any class (i.e., IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecules. Therefore, as used herein, the term antibody also includes antibodies and antibody fragments comprised by the CAR of the present invention, which are produced by modifying complete antibodies or resynthesized using recombinant DNA methods.

[0067] As used herein, "antibody" generally refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or immunoglobulin gene fragments. When the term "antibody" is used, it may also be considered to refer to an "antibody fragment". Optionally, an antibody or antibody fragment may be chemically conjugated to or expressed as a fusion protein with other proteins or other proteins. In some embodiments, the antibody or antigen-binding fragment of the present invention is included in a multispecific antibody, such as a bispecific antibody. Such multispecific antibodies can be produced by known methods, such as cross-linking two or more antibodies, antigen-binding fragments (e.g., scFv) of the same or different types. Exemplary methods for preparing multispecific antibodies include those described in PCT Patent Publication No. WO2013 / 163427, which is incorporated herein by reference in its entirety.

[0068] The affinity of a binding domain polypeptide of the invention and an antibody or antibody fragment or CAR protein can be readily determined using conventional techniques, such as by competitive ELISA (enzyme-linked immunosorbent assay), or using a surface plasmon resonance apparatus such as Biacore.

[0069] Humanized antibodies comprising one or more CDRs of an antibody or antibody fragment of the invention or comprising one or more CDRs derived from said antibody or antibody fragment can be prepared using methods known in the art. For example, four steps can generally be used to humanize a monoclonal antibody: (1) determining the nucleotide and predicted amino acid sequences of the starting antibody light chain and heavy chain variable domains; (2) designing a humanized antibody, i.e., determining which antibody framework region to use in the humanization process; (3) carrying out humanization methods / techniques; and (4) transfection and expression of the humanized antibody. For example, see, U.S. Patent No. 6,180,370.

[0070] The term humanized antibody means that at least a portion of the framework region of an immunoglobulin and a portion of an optional CDR region or other regions involved in binding are derived from or adjusted to human immunoglobulin sequences. Humanized, chimeric or partially humanized forms of mouse monoclonal antibodies can be prepared, for example, by recombinant DNA technology. The CDR region of a non-human antibody is connected to a human constant region by recombinant DNA technology to produce a humanized form of mouse antibody (Queen et al., 1989; WO 90 / 07861). Alternatively, the monoclonal antibody used in the method of the present invention can be a human monoclonal antibody. Human antibodies can be obtained, for example, using phage display methods (WO 91 / 17271; WO 92 / 01047).

[0071] As used herein, humanized antibodies also refer to forms of non-human (e.g., murine, camel, llama, shark) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies, such as VHH), that contain minimal sequence derived from non-human immunoglobulins.

[0072] As used herein, a human or humanized antibody or antibody fragment refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and can be prepared using any technique known in the art for preparing antibodies. A human antibody or fragment thereof can be selected by competitive binding experiments or other means to determine that it has the same epitope binding specificity as a particular mouse antibody.

[0073] Further explanation of chimeric antigen receptor

[0074] In certain embodiments, the CAR of the present invention may include linker residues added between the various domains for the proper spacing and conformation of the molecule, such as a linker comprising an amino acid sequence that connects V H Domain and V LDomain and provide a spacer function compatible with the interaction of the two sub-binding domains, so that the resulting polypeptide maintains specific binding affinity for the target molecule. The CAR of the present invention may include one, two, three, four or five or more linkers. In a particular embodiment, the length of the linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any suitable length of amino acids.

[0075] Exemplary examples of linkers include glycine polymers; glycine-serine polymers; glycine- Alanine polymers; Alanine-serine polymers; other flexible linkers known in the art, such as Whitlow linkers. Glycine and glycine-serine polymers are relatively unstructured and can therefore serve as connections between domains or some of the domains of a fusion protein (e.g., a CAR described herein).

[0076] In a particular embodiment, the binding domain of the CAR is followed by one or more "spacers" or "spacer polypeptides", which are equivalent to linkers, which move the antigen binding domain away from the effector cell surface to enable proper contact, antigen binding and activation between cells. In certain embodiments, the spacer region is a part of an immunoglobulin, including but not limited to one or more heavy chain constant regions, such as CH2 and CH3. The spacer region may include a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region amino acid sequence. In one embodiment, the spacer region includes the CH2 and CH3 domains of IgG1 or IgG4.

[0077] In some embodiments, the binding domain of CAR may be followed by one or more "hinge domains", which move the antigen binding domain away from the effector cell surface so that cells can properly contact, antigen bind and activate. CAR may include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be of natural, synthetic, semi-synthetic or recombinant origin. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Exemplary hinge domains suitable for CAR described herein include hinge regions derived from the extracellular region of type 1 membrane proteins (e.g., CD8α, CD4, CD28, PD1, CD152 and CD7), which may be wild-type hinge regions from these molecules, or may be altered. In another embodiment, the hinge domain includes PD1, CD152 or CD8α hinge regions.

[0078] "Transmembrane domain" is a part of CAR that fuses the extracellular binding part and the intracellular signaling domain and anchors CAR to the plasma membrane of immune effector cells. The TM domain can be derived from natural, synthetic, semi-synthetic or recombinant sources. The TM domain can be derived from α, β or ζ chain of T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154 and PD1. In one embodiment, the CAR of the present invention comprises a TM domain derived from CD8α or CD28.

[0079] In a particular embodiment, the CAR of the present invention includes an intracellular signaling domain. "Intracellular signaling domain" refers to the information transduction involved in combining effective anti-CD19CAR with human CD19 polypeptides to the interior of immune effector cells to induce effector cell functions (e.g., activation, production of cytokines, proliferation and cytotoxic activity, the cytotoxic activity including the release of cytotoxic factors to the target cells bound by CAR) or other cell reactions triggered by antigen binding extracellular CAR domains. The term "effector function" refers to the specialized functions of immune effector cells. For example, the effector function of T cells can be cytolytic activity or help or activity including cytokine secretion. The term "intracellular signaling domain" refers to the part of the protein that transduces effector function signals and instructs cells to perform specialized functions.

[0080] The CAR of the present invention comprises one or more costimulatory signaling domains to enhance the efficacy, expansion and / or memory formation of T cells or NK cells expressing CAR receptors. As used herein, the term "costimulatory signaling domain" refers to the intracellular signaling domain of the CAR molecule, providing a second signal required for the effective activation and function of T lymphocytes or NK cells after binding to an antigen.

[0081] protein

[0082] "Protein", "polypeptide fragment" and "polypeptide" are used interchangeably unless otherwise indicated and are used according to the conventional meaning, i.e., as an amino acid sequence. Proteins are not limited to a particular length, for example, they may comprise a full-length protein sequence or a fragment of a full-length protein, and may include post-translational modifications of the polypeptide (e.g., glycosylation, acetylation, phosphorylation, etc.) and other modifications known in the art, including naturally occurring and non-naturally occurring ones.

[0083] In various embodiments, the CAR polypeptide or protein of the present invention is included in a signal (or leader) sequence at the N-terminus of the protein that can guide protein transfer during or after translation. Polypeptides can be prepared using various well-known recombinant and / or synthetic techniques. The polypeptide of the present invention specifically includes CAR disclosed herein, or has one or more (e.g., 1-20, 1-10 or 1-5) amino acid deletions, additions and / or replacement sequences disclosed herein.

[0084] Nucleic Acids

[0085] As used herein, the term "polynucleotide" refers to mRNA, RNA, genomic RNA (gRNA), positive strand RNA (RNA (+)), negative strand RNA (RNA (-)), genomic DNA (gDNA), complementary DNA (cDNA) or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. Preferably, the polynucleotides of the present invention include polynucleotides or variants having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% sequence identity with any reference sequence described herein, typically wherein the variant retains at least one biological activity of the reference sequence.

[0086] For example, the encoding CD19 binding domain-CD8α hinge-CD8 TM The sequence of the polynucleotide of the -4-1BB-CD3ζ-P2A-IL12-ODD fusion protein is any DNA sequence that can encode the fusion protein, preferably, the sequence is a nucleotide sequence consisting of SEQ ID NO: 2, or SEQ ID NO: 2-SEQ ID NO: 10-SEQ ID NO: 4, or a complementary sequence thereof. On the other hand, the polynucleotide encoding the CD19 binding domain-CD8α hinge-CD8 TM The sequence of the polynucleotide of the -4-1BB-CD3ζ-P2A-IL12-ODD fusion protein may be a polynucleotide or its complementary sequence that hybridizes with the polynucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence consisting of SEQ ID NO: 2-SEQ ID NO: 10-SEQ ID NO: 4 in sequence under stringent conditions and encodes the fusion protein;

[0087] The "stringent conditions" described herein may be any of low stringency conditions, medium stringency conditions, and high stringency conditions, preferably high stringency conditions. For example, "low stringency conditions" may be 30°C, 5×SSC, 5×Denhardt solution, 0.5% SDS, and 52% formamide; "medium stringency conditions" may be 40°C, 5×SSC, 5×Denhardt solution, 0.5% SDS, and 52% formamide; "high stringency conditions" may be 50°C, 5×SSC, 5×Denhardt solution, 0.5% SDS, and 52% formamide. Those skilled in the art should understand that the higher the temperature, the more polynucleotides with high homology can be obtained. In addition, those skilled in the art can select the comprehensive result formed by multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration that affect the stringency of hybridization to achieve the corresponding stringency.

[0088] In addition, the hybridizable polynucleotide may exemplarily be a polynucleotide that, when calculated using the default parameters set by the same search software such as FASTA and BLAST using the system, has about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 100% or more, 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more, 109% or more, 110% or more, 111% or more, 112% or more, 113% or more, 114% or more, 115% or more, 116% or more, 117% or more, 118% or more, 119% or more, 121% or more, 122% or more, 123% or more, 124% or more, 125% or more or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more identity to a polynucleotide.

[0089] The identity of nucleotide sequences can be determined using the BLAST algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc. Natl. Acad. Sci. USA 90: 5873, 1993). Programs BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When using BLASTN to analyze base sequences, the parameters are score = 100 and wordlength = 12; when using BLASTX to analyze amino acid sequences, the parameters are score = 50 and wordlength = 3; when using BLAST and Gapped BLAST programs, the default parameter values ​​can be set by the system of each program.

[0090] Polynucleotides can be prepared, manipulated and / or expressed using any of the various mature techniques known and available in the art. In order to express the desired polypeptide or protein, the nucleotide sequence encoding the polypeptide can be inserted into a suitable vector. Examples of vectors are plasmids, autonomously replicating sequences and transposable elements. Other exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)), bacteriophages (e.g., lambda phages or M13 phages) and animal viruses. Examples of animal virus vectors include, but are not limited to, retroviruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses and papovaviruses (e.g., SV40). Examples of expression vectors are pClneo vectors (Promega) for expression in mammalian cells; Lenti4 / V5-DESTTM, pLenti6 / V5-DESTTM and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentiviral-mediated gene transfer and expression in mammalian cells. In a particular embodiment, the coding sequence of the chimeric protein disclosed herein can be connected to such expression vectors for expressing the chimeric protein in mammalian cells. The "control element" or "regulatory sequence" present in the expression vector is a non-translated region of the vector (e.g., replication origin, promoter, enhancer, translation initiation signal (SD sequence or Kozak sequence) intron, polyadenylation sequence, 5' and 3' non-translated region), which interacts with host cell proteins for transcription and translation. The strength and specificity of such elements or sequences can vary. Depending on the vector system and host used, any number of suitable transcription and translation elements or sequences can be used, including broadly expressed promoters and inducible promoters.

[0091] Carrier

[0092] In a particular embodiment, cells (e.g., immune effector cells, such as T cells) are transduced with a retroviral vector (e.g., a lentiviral vector) encoding a CAR. For example, immune effector cells are transduced with a vector encoding a CAR, the vector comprising a humanized anti-CD19 antibody or antigen binding fragment binding to a CD19 polypeptide, the humanized anti-CD19 antibody or antigen binding fragment having a transmembrane domain and an intracellular signaling domain, so that these transduced cells can induce a CAR-mediated cytotoxic response.

[0093] Retrovirus is a common tool for gene delivery. In a particular embodiment, retrovirus is used to deliver polynucleotides encoding chimeric antigen receptors (CAR) to cells. As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy, and then covalently integrates its genomic DNA into the host genome. Once the virus is integrated into the host genome, it is called a "provirus". The provirus serves as a template for RNA polymerase II and guides the expression of RNA molecules that encode structural proteins and enzymes required to produce new virus particles.

[0094] Exemplary retroviruses suitable for use in particular embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), Gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), murine stem cell virus (MSCV), and Rous sarcoma virus (RSV)) and lentiviruses.

[0095] As used herein, the term "lentivirus" refers to a group (or genus) that includes many retroviruses. Exemplary lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedivirus (VMV) virus; caprine arthritis-encephalitis virus;

[0096] (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). In one embodiment, HIV-based vector backbones (i.e., HIV cis-acting sequence elements) are preferred. In a particular embodiment, a lentivirus is used to deliver a polynucleotide comprising a CAR to a cell.

[0097] The term "vector" is used herein to refer to a nucleic acid molecule that is capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is usually connected to a carrier nucleic acid molecule, for example, inserted into a carrier nucleic acid molecule. The vector may include a sequence that directs autonomous replication in the cell, or may include a sequence sufficient to allow integration into the host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-defective retroviruses and lentiviruses.

[0098] It will be apparent to those skilled in the art that the term "viral vector" is broadly used to refer to nucleic acid molecules (e.g., transfer plasmids) or viral particles that mediate nucleic acid transfer, and nucleic acid molecules include viral-derived nucleic acid elements that typically facilitate transfer or integration of nucleic acid molecules into the cellular genome. Viral particles typically include various viral components and sometimes host cell components in addition to nucleic acids.

[0099] The term viral vector may refer to a virus or viral particle capable of transferring nucleic acid into a cell, or the nucleic acid itself being transferred. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses. The term "retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements or portions thereof that are primarily derived from retroviruses. The term "lentivirus" refers to a genus of the Retroviridae family that is capable of efficiently infecting non-periodic and post-mitotic cells; they can transfer significant amounts of genetic information into the DNA of host cells, so that they are one of the most efficient methods of gene delivery vectors.

[0100] Therefore, in a preferred embodiment, the present invention relates to a method for transfecting cells with an expression vector encoding CAR. For example, in some embodiments, the vector comprises additional sequences, such as sequences that promote CAR expression, such as promoters, enhancers, poly-A signals and / or one or more introns. In a preferred embodiment, the flank of the CAR coding sequence is a transposon sequence, so that there is a transposase to allow the coding sequence to be integrated into the genome of the transfected cell.

[0101] In some embodiments, the genetically transformed cells are further transfected with a transposase, which promotes the integration of the CAR coding sequence into the genome of the transfected cells. In some embodiments, the transposase is provided as a DNA expression vector. However, in a preferred embodiment, the transposase is provided as an expressible RNA or protein so that the transposase does not undergo long-term expression in transgenic cells. For example, in some embodiments, the transposase is provided as mRNA (e.g., mRNA comprising a cap and a poly-A tail). Any transposase system can be used according to embodiments of the present invention. However, in some embodiments, the transposase is a salmon-type Tel-like transposase (SB). In some embodiments, the transposase is an engineered enzyme with increased enzymatic activity. Some specific examples of transposases include, but are not limited to, SB 10, SB 11 or SB 100X transposases (see, e.g., Mates et al., 2009, Nat Genet.41 (6): 753-61 or US9228180, which are incorporated herein by reference). For example, the method may comprise electroporating cells with mRNA encoding SB10, SB11, or SB100X transposase.

[0102] Sequence variants:

[0103] The sequence variants of the claimed nucleic acid, protein, antibody, antibody fragment and / or CAR (such as those defined by percentage sequence identity) are also included in the scope of the present invention, which maintains similar binding properties of the present invention. These variants show alternative sequences, but maintain substantially the same binding properties such as target specificity, because the specific sequences provided are known to be functional analogs or functional analogs. Sequence identity refers to the percentage of identical nucleotides or amino acids when performing sequence alignment.

[0104] As used herein, "sequence identity" refers to the degree of sequence identity based on nucleotide-nucleotide or based on amino acid-amino acid on a comparison window. Therefore, the "percentage of sequence identity" can be calculated by: comparing two optimal alignment sequences on a comparison window, determining that there are identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) on the two sequences to produce the number of matching positions, the number of matching positions divided by the total number of positions in the comparison window (i.e., window size), and the result multiplied by 100 to obtain the percentage of sequence identity. Included are nucleotides or polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any reference sequence described herein, typically wherein the polypeptide variant retains at least one biological activity of the reference polypeptide.

[0105] Those of ordinary skill in the art will appreciate that, due to the degeneracy of the genetic code, there are many nucleotide sequences encoding polypeptides or proteins as described herein. Some of these polynucleotides have minimal homology or sequence identity to the nucleotide sequence of any natural gene. Nevertheless, the present invention specifically contemplates polynucleotides that vary due to differences in codon usage. Deletions, substitutions and other changes that fall within the sequence of the sequence identity are also included in the present invention.

[0106] Protein sequence modifications that can occur by replacement are also included in the scope of the present invention. Replacement as defined herein is a modification of the amino acid sequence of a protein, whereby one or more amino acids are replaced by the same number of (different) amino acids, thereby producing a protein containing an amino acid sequence different from the primary protein. Replacements can be made that preferably do not significantly change the function of the protein. As with additions, replacements can be natural or artificial. It is well known in the art that amino acid replacements can be made without significantly changing the function of a protein. This is especially true when the modification involves a "conservative" amino acid replacement of an amino acid with another amino acid of similar properties. This type of "conservative" amino acid can be a natural or synthetic amino acid that can be replaced but will not significantly affect the structure and function of the protein due to size, charge, polarity and conformation. Typically, many amino acids can be replaced by conservative amino acids without adversely affecting the function of the protein.

[0107] In general, the nonpolar amino acids Gly, Ala, Val, Ile and Leu; the nonpolar aromatic amino acids Phe, Trp and Tyr; the neutral polar amino acids Ser, Thr, Cys, Gln, Asn and Met; the positively charged amino acids Lys, Arg and His; the negatively charged amino acids Asp and Glu represent conservative amino acid groups. This list is not exhaustive. For example, it is well known that Ala, Gly, Ser and sometimes Cys can be substituted for each other even if they belong to different groups.

[0108] Substitution variants remove at least one amino acid residue in the antibody molecule and insert a different residue in its place. For the occurrence of substitution mutagenesis, the most interesting position includes the hypervariable region, but FR changes are also considered. If such substitution leads to changes in biological activity, then a larger amount of changes can be introduced and the product screened.

[0109] Compositions and preparations

[0110] The composition of the present invention may include one or more polypeptides, polynucleotides, vectors comprising the polynucleotides, genetically modified immune effector cells, etc. as contemplated herein. Compositions include, but are not limited to, pharmaceutical compositions. "Pharmaceutical composition" refers to a composition formulated in a pharmaceutically acceptable or physiologically acceptable solution, which is administered to cells or animals alone or in combination with one or more other treatment modalities. It should also be understood that, if necessary, the composition of the present invention may also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or other various pharmaceutically active agents. There is virtually no restriction on other components that may also be included in the composition, provided that the additional components do not adversely affect the ability of the composition to deliver the intended therapy.

[0111] The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, and are commensurate with a reasonable benefit / risk ratio.

[0112] As used herein, "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant or emulsifier that has been approved by the U.S. Food and Drug Administration or the China Food and Drug Administration for use in humans or livestock. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats and oils, paraffins, silicones, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer; and any other compatible substances used in pharmaceutical formulations.

[0113] In a particular embodiment, the composition of the present invention comprises a certain amount of the immune effector cells expressing CAR of the present invention. As used herein, the term "amount" refers to an "effective amount" of genetically modified therapeutic cells (eg, T cells) that achieve a beneficial or desired preventive or therapeutic outcome (including clinical outcome).

[0114] "Prophylactically effective amount" refers to the amount of genetically modified therapeutic cells that is effective to achieve the desired prophylactic result. Usually, but not necessarily, the prophylactic effective amount is less than the therapeutic effective amount because the prophylactic dose is used in the subject before the disease or in the early stages of the disease. The term prevention does not necessarily mean to completely prohibit or prevent a particular medical condition. The term prevention also refers to reducing the risk of a certain medical condition occurring or the symptoms worsening.

[0115] A "therapeutically effective amount" of genetically modified therapeutic cells can vary depending on various factors, such as the disease state, age, sex, and weight of the individual, and the ability of stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutic beneficial effects outweigh any toxic or deleterious effects of the virus or transduced therapeutic cells. The term "therapeutically effective amount" includes an amount that is effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the exact amount of the composition of the invention to be administered can be determined by a physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). It can be generally specified that a pharmaceutical composition comprising T cells described herein can be administered in an amount of 10 2 Up to 10 10 cells / kg body weight, preferably 10 5 Up to 10 6 The amount of cells will depend on the end use of the composition and the cell type contained therein. For the purposes provided herein, the cells are typically in a volume of 1 L or less, and may be 500 mL or less, or even 250 mL or 100 mL or less. Thus, the desired cell density is typically greater than 10 6 cells / ml, usually greater than 10 7 cells / ml, usually 10 8 Clinically relevant numbers of immune cells can be distributed into multiple infusions that cumulatively equal or exceed 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 In some embodiments of the invention, lower numbers of cells may be administered, particularly because all infused cells will be redirected to a specific target antigen. The CAR-expressing cell composition may be administered multiple times at doses within these ranges. For the patient receiving treatment, the cells may be allogeneic, syngeneic, allogeneic, or autologous.

[0116] Typically, compositions comprising cells activated and expanded as described herein can be used to treat and prevent diseases occurring in immunocompromised individuals. In particular, compositions comprising CAR-modified T cells of the present invention are used to treat B cell malignancies. The CAR-modified T cells of the present invention can be administered alone, or as a pharmaceutical composition with a carrier, a diluent, an excipient and / or in combination with other components or other cytokines or cell populations. In a particular embodiment, the pharmaceutical composition of the present invention comprises a certain amount of genetically modified T cells, and one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

[0117] The pharmaceutical composition of the present invention comprising an immune effector cell group (eg, T cell) expressing CAR may include: buffer, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptide or amino acid (eg, glycine); antioxidant; chelating agent (eg, EDTA) or glutathione; adjuvant, such as aluminum hydroxide; and preservative. The composition of the present invention is preferably formulated for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.

[0118] Liquid pharmaceutical compositions, whether in the form of solutions, suspensions or other similar forms, may include one or more of the following: sterile diluents (e.g., water for injection), saline solutions (preferably physiological saline, Ringer's solution, isotonic sodium chloride), fixed oils (e.g., synthetic monoglycerides or diglycerides that can be used as solvents or suspension media), polyethylene glycols, glycerol, propylene glycol or other solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and buffers, such as acetates, citrates or phosphates, and agents for regulating osmotic pressure, such as sodium chloride or glucose. Parenteral preparations may be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0119] In a particular embodiment, the composition of the present invention includes a single effective amount of immune effector cells expressing CAR, or a combination thereof with one or more therapeutic agents. Therefore, the immune effector cell composition expressing CAR can be applied alone or in combination with other known cancer treatments, such as radiotherapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The composition can also be applied in combination with antibiotics. This area can accept such therapeutic agents as standard treatments for specific disease states (such as specific cancers) as described herein. Exemplary therapeutic agents considered include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies or other active and auxiliary agents.

[0120] Treatment

[0121] The genetically modified immune effector cells of the present invention provide improved methods for adoptive immunotherapy for treating cancers including, but not limited to, acute myeloid leukemia, chronic myeloid leukemia, nasopharyngeal carcinoma, glioma, colon cancer, gastric cancer, prostate cancer, renal cell carcinoma, cervical cancer and ovarian cancer, lung cancer (SCLC and NSCLC), gastric cancer, gallbladder cancer, and cancer-related paraneoplastic syndromes of cachexia, fatigue, asthenia, cachexia and hypercalcemia.

[0122] In another aspect of the present invention, a method for treating a disease using a CAR and CAR-T or NK according to the present invention as described herein is provided, wherein the method comprises administering to the patient a therapeutically effective amount of a CAR or CAR-T or NK as described herein, illustratively, the disease is selected from: acute myeloid leukemia, chronic myeloid leukemia, nasopharyngeal carcinoma, glioma, colon cancer, gastric cancer, prostate cancer, renal cell carcinoma, cervical cancer and ovarian cancer, lung cancer (SCLC and NSCLC), gastric cancer, gallbladder cancer, and cancer-related cachexia, fatigue, asthenia, cachexia and paraneoplastic syndromes of hypercalcemia.

[0123] As used herein, the terms "individual" and "subject" are generally used interchangeably and refer to any animal that exhibits symptoms of a disease, disorder, or condition that can be treated with gene therapy vectors, cell-based therapeutic agents, and methods disclosed elsewhere herein. In a preferred embodiment, the subject includes any animal that exhibits symptoms of a disease, disorder, or condition of the hematopoietic system that can be treated with gene therapy vectors, cell-based therapeutic agents, and methods disclosed elsewhere herein. Typical subjects include experimental animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, and livestock or pets (e.g., cats or dogs). Non-human primates are included, preferably human patients are included.

[0124] As used herein, "treatment" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even the smallest reduction in one or more measurable markers of the disease or condition being treated. Treatment may optionally involve alleviation or amelioration of the symptoms of the disease or condition, or delay in the progression of the disease or condition. "Treatment" does not necessarily mean complete eradication or cure of the disease or condition or its associated symptoms.

[0125] As used herein, "prevention" refers to a method of preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0126] In one embodiment, a method of treating cancer in a subject in need thereof comprises administering an effective amount, e.g., a therapeutically effective amount, of a composition comprising genetically modified immune effector cells of the invention. The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although appropriate dosages can be determined by clinical trials.

[0127] The administration of the composition of the present invention can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, blood transfusion, implantation or transplantation. In a preferred embodiment, the composition is administered parenterally. The phrase "parenteral administration" used herein refers to the mode of administration other than enteral and topical administration, usually by injection, including but not limited to intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the composition of the present invention is administered to a subject by direct injection into a tumor, lymph node or site of infection.

[0128] The English names in this article are not case-sensitive; CD19 CAR-T refers to CAR-T cells that can express the CD19-specific binding domain; CD8 TM Represents the transmembrane domain.

[0129] Relative to NK cells (including CAR-NK), the present invention provides a more detailed description of T cells (including CAR-T cells), but in general, these descriptions of T cells are also applicable to NK cells. Here, the description of "T cells" or their synonyms in the description of T cells is replaced with "NK cells" or their synonyms. This is necessary from the perspective of narrative simplicity. If it is judged that such a replacement is inappropriate in some cases based on the prior art, the replacement is not performed.

[0130] The terms and expressions which have been employed herein are used as terms of description rather than limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed.

[0131] It should be understood, therefore, that although the present invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be adopted by those skilled in the art and that such modifications and variations are considered to be within the scope of the present invention as defined by the appended claims.

[0132] In order to more clearly illustrate the present invention, it is now described in detail in conjunction with the following examples, but these examples are only exemplary descriptions of the present invention and cannot be interpreted as limiting the present application.

[0133] Example 1: Preparation of lentiviral vector

[0134] 1) Gene synthesis of lentiviral expression plasmid; 2) Gene synthesis of Kozal-CD8a signal-CD19 SCFV-CD8aTM-4-1BB-CD3zeta fusion gene sequence (its amino acid sequence is shown in SEQ ID NO: 1, its DNA sequence is shown in SEQ ID NO: 2, and its structural schematic diagram is shown in Figure 1 ); 3) Gene synthesis nucleotide sequence, the nucleotide sequence is: self-cleavage protein P2A gene (its amino acid sequence is shown in SEQ ID NO: 9, its DNA sequence is shown in SEQ ID NO: 10) -IL12ODD fusion gene sequence (its amino acid sequence is shown in SEQ ID NO: 3, its DNA sequence is shown in SEQ ID NO: 4); 4) The above gene synthesis fragments are inserted into the lentiviral expression plasmid using the multi-fragment homologous recombination method, and the recombinant vector is transformed into the Escherichia coli strain Stbl3, and the target recombinant plasmid is obtained after kanamycin screening and monoclonal sequencing; then the host bacteria containing the target recombinant plasmid are expanded and cultured, and an endotoxin-free plasmid is obtained using an endotoxin removal kit, that is, a plasmid vector containing the CD19CARIL12ODD gene fragment, and its structure is shown in Figure 1 ; 5) At the same time, the lentiviral packaging auxiliary plasmids pMDLgpRRE, pRRE, and pCMV-VSV-G were transformed into Escherichia coli strain Stbl3, selected with ampicillin, and sterile endotoxin-free plasmids were obtained using an endotoxin removal kit.

[0135] Example 2: Preparation of CAR19IL12ODD Lentivirus

[0136] 1) Inoculate 5x10 62) 293T cells were placed in a 10 cm culture dish containing 10 ml DMEM + 10% FBS medium; 2) 24 hours later, 10 ml of fresh DMEM + 10% FBS medium was replaced, and the viral plasmids (CAR19IL12ODD: 25 μg, pMDLgpRRE: 7.5 ug, pRRE: 6 ug, pCMV-VSV-G: 6 ug) were mixed with 250 ul serum-free DMEM to prepare a plasmid suspension; 45 ul 1 ug / ml of transfection reagent (Linear Polyethylenimine (PEI) was mixed with 250ul serum-free DMEM to prepare PEI suspension; after standing at room temperature for 5 minutes, 250ul plasmid suspension was mixed with PEI suspension, and after 30 minutes, it was added to 293T cells and gently mixed; 3) after 8 hours, 20ml fresh DMEM+10% FBS culture medium was replaced; 4) after 60 hours, the culture medium supernatant was collected, centrifuged to remove cell debris, and the supernatant was filtered with a 0.45μm filter; then Amicon Ultra-15 CentrifugalFilter Unit with Ultracel-30 membrane (Merckmillipore, catalog number UFC903096) was used for centrifugation at 4°C and 1500g for 30 minutes to concentrate the virus supernatant 10 times to obtain the virus solution, and the virus was titered by gradient dilution method, and the virus was stored at -80°C after aliquoting.

[0137] Example 3: Preparation and functional verification of CAR19IL12ODD CAR-T cells

[0138] 1) Isolation of peripheral blood lymphocytes: collect peripheral blood, add peripheral blood lymphocyte separation solution equal to the blood sample, centrifuge at 800g for 20min, collect buffy coat lymphocytes, and wash twice with PBS; 2) Isolation of CD3 T cells from peripheral blood lymphocytes: resuspend 1X10 7 Lymphocytes were cultured in 5 ml of X-VIVO medium containing 5% FBS. TM 15 Cultured in 6-well plates, CD3 and CD8 antibodies were used to detect the proportion of CD3 T cells in peripheral lymphocytes. Human T-Expander CD3 / CD28 (Gibco, Catalog No. 11141D) was used to separate and stimulate CD3 T cells; 3) 2×10 5 CD3T cells and 5X10 5 IU virus was mixed in a 24-well plate and centrifuged at 30°C and 1500g for 90 min. After 4 h, 1 ml of X-VIVO containing 5% FBS was added. TM 15 ml of culture medium, gently pipette to mix, return to the incubator for culture, and replace with 1 ml of fresh 5% FBS X-VIVO medium after 24 hoursTM 15 culture medium;

[0139] 4) After 48 hours, adjust the T cell concentration to 5×10 5 / ml, and all the culture medium was replaced with fresh medium; 5) After 72 hours, the T cell concentration was adjusted to 5×10 5 / ml, WB verified the expression of CAR19IL12ODD protein; flow cytometry was used to detect and confirm the expression of CAR in CAR-T cells; CAR19IL12ODD CAR-T cells were cultured under normal oxygen concentration and hypoxic concentration, and ELESA was used to detect the secretion concentration of IL12 in the cell culture supernatant ( Figure 2 ), the results showed that CAR19IL12ODD detected low-dose IL12 secretion expression under normal oxygen concentration conditions (17%), while under anaerobic conditions (1%), IL12 secretion as high as 26pg / ml was secreted.

[0140] The above results show that the CAR19IL12ODD of the present invention can be expressed in CD3T cells, and the ODD domain can effectively control the secretion expression of IL12.

[0141] Example 4: LDH experiment

[0142] Detection of the in vitro killing effect of CAR19IL12ODD CAR-T cells on the DLBCL cell line Ly3 (CytoTox Non-Radioactive Cytotoxicity Assay, Promega, Catalog No. G1780): 1) Take CAR19IL12ODD CAR-T effector cells and target cells Ly3, wash twice with PBS, and count with trypan blue staining; 2) Add 1x10 41) Add 50ul of target cells in 5% FBS 1640 medium; 2) Set up gradient effector-target ratio experimental groups: add corresponding numbers of effector cells according to the effector-target ratio of 10:1, 5:1, 2.5:1, 1.25:1, 0.6:1, 0.3:1, and 0.15:1, and the volume of effector cells added to each well is 50ul; 3) Set up the following control groups: blank group (100ul 5% FBS 1640 medium), effector cell spontaneous group (50ul effector cells + 50ul 5% FBS 1640 medium), and target cell spontaneous group (50ul target cells + 50ul 5% FBS 1640 medium), target cell maximum release group (50ul target cells + 50ul 5) After 4 hours, take out the plate and centrifuge at 250g for 5 minutes (1 hour before this step, add 10ul 10x lysis buffer to the target cell maximum release group); 6) Take 50ul of the supernatant to a new flat-bottom 96-well plate and add 50ul Cytoto x Substrate, foil coated, react at room temperature for 30 minutes; 7) Add 50ul stop solution to each well to terminate the reaction, and measure the absorbance at 492nm with a microplate reader; 8) Calculate the killing efficiency, killing efficiency = (experimental group - effector cell spontaneous group - target cell spontaneous group) / (target cell maximum release group - target cell spontaneous group) X 100; The experimental results are as follows Figure 3 A. The CAR-T of the present invention can have a significant therapeutic effect or inhibitory effect on diffuse large B-cell lymphoma (DLBCL).

[0143] Example 5: Animal experiment to detect the in vivo killing effect of CAR19IL12ODD CAR-T cells on DLBCL cell line Ly3 and functional verification

[0144] Severely immunodeficient Syrian hamsters aged 4-6 weeks were selected and 1×10 7 / Ly3 diffuse large B-cell lymphoma tumor cells. Observe and measure the tumor size. After 2-4 weeks, the tumor grows to 250mm 3 The Syrian hamsters were randomly divided into groups of 5 and intraperitoneally injected with CAR-T cells cultured in vitro for 7-8 days at a dose of 1×10 7 / . Peripheral blood was collected 3, 6, 9, and 12 days after the reinfusion of CAR19IL12ODD CAR-T cells, that is, 17, 20, 23, and 26 days after tumor inoculation to detect IL12 concentration and CAR-T cell flow cytometry analysis showed that ( Figure 4 A and Figure 4B), IL12 significantly increased the survival time of CAR-T cells in vivo; continue to observe and measure the tumor size. When the tumor in the control group grows to the ethical limit, the experimental animals are killed according to ethical requirements, and the tumor tissue is taken for immunohistochemical staining. The following formula is used to calculate the change in tumor size and draw a tumor growth curve: tumor size = (1 / 2) * long diameter * short diameter squared; from Figure 3 B It can be seen that the CAR-T prepared in the examples of the present application has a significant inhibitory effect on human diffuse large B-cell lymphoma (DLBCL); the immunohistochemistry results (not shown) show that IL12 significantly promotes the tumor infiltration of CAR-T, and the expression of IL12 in CAR-T cells plays a role in breaking the tumor immune microenvironment of DLBLC. The above results show that the CAR19IL12ODD CAR-T cells prepared in the examples of the present application have a significant therapeutic effect or inhibitory effect on human diffuse large B-cell lymphoma (DLBCL) by breaking the immune microenvironment.

[0145] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Sequence Listing <110> Zhengzhou University <120> Novel chimeric antigen receptor and immune cell containing the same <130> 21SG1F7645 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 522 <212> PRT <213> Artificial sequence <220> <223> CAR amino acid sequence <400> 1 Ala Thr Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu 1 5 10 15 Leu Leu His Ala Ala Arg Pro Ala Ala Ala Asp Ile Gln Met Thr Gln 20 25 30 Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser 35 40 45 Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln 50 55 60 Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu 65 70 75 80 His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 85 90 95 Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr 100 105 110 Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr 115 120 125 Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser 130 135 140 Gly Glu Gly Ser Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro 145 150 155 160 Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser 165 170 175 Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro 180 185 190 Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr 195 200 205 Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn 210 215 220 Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp 225 230 235 240 Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr 245 250 255 Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala 260 265 270 Ala Ala Arg Ser Phe Ser His Phe Val Pro Val Phe Leu Pro Ala Lys 275 280 285 Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 290 295 300 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 305 310 315 320 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 325 330 335 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 340 345 350 Val Ile Thr Leu Tyr Cys Asn His Arg Asn His Met Gly Gly Thr Ser 355 360 365 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 370 375 380 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 385 390 395 400 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg 405 410 415 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 420 425 430 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 435 440 445 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 450 455 460 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 465 470 475 480 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 485 490 495 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 500 505 510 Ala Leu His Met Gln Ala Leu Pro Pro Arg 515 520 <210> 2 <211> 1566 <212> DNA <213> Artificial sequence <220> <223> CAR nucleotide sequence <400> 2 gccaccatgg ccttaccagt gaccgccttg ctcctgccgc tggccttgct gctccacgcc 60 gccaggccgg cggccgctga catccagatg acacagacta catcctccct gtctgcctct 120 ctgggagaca gagtcaccat cagttgcagg gcaagtcagg acattagtaa atatttaaat 180 tggtatcagc agaaaccaga tggaactgtt aaactcctga tctaccatac atcaagatta 240 cactcaggag tcccatcaag gttcagtggc agtgggtctg gaacagatta ttctctcacc 300 attagcaacc tggagcaaga agatattgcc acttactttt gccaacaggg taatacgctt 360 ccgtacacgt tcggaggggg gactaagttg gaaataacag gctccacctc tggatccggc 420 aagcccggat ctggcgaggg atccaccaag ggcgaggtga aactgcagga gtcaggacct 480 ggcctggtgg cgccctcaca gagcctgtcc gtcacatgca ctgtctcagg ggtctcatta 540 cccgactatg gtgtaagctg gattcgccag cctccacgaa agggtctgga gtggctggga 600 gtaatatggg gtagtgaaac cacatactat aattcagctc tcaatccag actgaccac 660 atcaggaca actccaagag ccaagtttc ttaaaaatga acagtctgca aactgatgac 720 acagccattt actactgtgc siacattat tactacggtg gtagctatgc tatggactac 780 tggggtcaag gaacctcagt caccgtctc tcagcggccg cagatcttt cagccactc 840 gtgccggtct tcctgccagc gaagcccacc accgaccg cgccgcgacc accacaccg 900 ggcccacca tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg 960 gggggcgcag tgcacacgag ggggctggac tcgcctgtg attackactacat ctgggcgccc 1020 ttggccggga cttgtggggt ccttctcctg tcacctt tcacctta ctgcaccac 1080 aggaaccata tgggaggac tgtaaacgg ggcagaaaga aactcctgta tatattcaa 1140 spaccatta tgagaccagt acaactact cagaggaag atggctgtag ctgccgattt 1200 ccagagaag agagaggg agagactg agatygagt tcagcaggc cgcagacgcc 1260 cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 1320 gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga 1380 aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 1440 tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1500 cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 1560 cctcgc 1566 <210> 3 <211> 841 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of IL12ODD fusion protein <400> 3 Met Gly His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu 1 5 10 15 Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val 20 25 30 Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu 35 40 45 Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln 50 55 60 Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys 65 70 75 80 Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val 85 90 95 Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp 100 105 110 Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe 115 120 125 Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp 130 135 140 Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg 145 150 155 160 Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser 165 170 175 Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu 180 185 190 Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile 195 200 205 Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr 210 215 220 Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn 225 230 235 240 Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp 245 250 255 Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr 260 265 270 Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg 275 280 285 Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala 290 295 300 Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser 305 310 315 320 Glu Trp Ala Ser Val Pro Cys Ser Val Pro Gly Val Gly Val Pro Gly 325 330 335 Val Gly Ala Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe 340 345 350 Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met 355 360 365 Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu 370 375 380 Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu 385 390 395 400 Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser 405 410 415 Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys 420 425 430 Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu 435 440 445 Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met 450 455 460 Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile 465 470 475 480 Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln 485 490 495 Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu 500 505 510 Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg 515 520 525 Val Met Ser Tyr Leu Asn Ala Ser Val Asp Gln Gln Thr Glu Cys Val 530 535 540 Leu Lys Pro Val Glu Ser Ser Asp Met Lys Met Thr Gln Leu Phe Thr 545 550 555 560 Lys Val Glu Ser Glu Asp Thr Ser Ser Leu Phe Asp Lys Leu Lys Lys 565 570 575 Glu Pro Asp Ala Leu Thr Leu Leu Ala Pro Ala Ala Gly Asp Thr Ile 580 585 590 Ile Ser Leu Asp Phe Gly Ser Asn Asp Thr Glu Thr Asp Asp Gln Gln 595 600 605 Leu Glu Glu Val Pro Leu Tyr Asn Asp Val Met Leu Pro Ser Pro Asn 610 615 620 Glu Lys Leu Gln Asn Ile Asn Leu Ala Met Ser Pro Leu Pro Thr Ala 625 630 635 640 Glu Thr Pro Lys Pro Leu Arg Ser Ser Ala Asp Pro Ala Leu Asn Gln 645 650 655 Glu Val Ala Leu Lys Leu Glu Pro Asn Pro Glu Ser Leu Glu Leu Ser 660 665 670 Phe Thr Met Pro Gln Ile Gln Asp Gln Thr Pro Ser Pro Ser Asp Gly 675 680 685 Ser Thr Arg Gln Ser Ser Pro Glu Pro Asn Ser Pro Ser Glu Tyr Cys 690 695 700 Phe Tyr Val Asp Ser Asp Met Val Asn Glu Phe Lys Leu Glu Leu Val 705 710 715 720 Glu Lys Leu Phe Ala Glu Asp Thr Glu Ala Lys Asn Pro Phe Ser Thr 725 730 735 Gln Asp Thr Asp Leu Asp Leu Glu Met Leu Ala Pro Tyr Ile Pro Met 740 745 750 Asp Asp Asp Phe Gln Leu Arg Ser Phe Asp Gln Leu Ser Pro Leu Glu 755 760 765 Ser Ser Ser Ala Ser Pro Glu Ser Ala Ser Pro Gln Ser Thr Val Thr 770 775 780 Val Phe Gln Gln Thr Gln Ile Gln Glu Pro Thr Ala Asn Ala Thr Thr 785 790 795 800 Thr Thr Ala Thr Thr Asp Glu Leu Lys Thr Val Thr Lys Asp Arg Met 805 810 815 Glu Asp Ile Lys Ile Leu Ile Ala Ser Pro Ser Pro Thr His Ile His 820 825 830 Lys Glu Thr Thr Ser Ala Thr Ser Ser 835 840 <210> 4 <211> 2523 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of IL12ODD fusion protein <400> 4 atgggtcacc agcagttggt catctcttgg ttttccctgg tttttctggc atctcccctc 60 gtggccatat gggaactgaa gaaagatgtt tatgtcgtag aattggattg gtatccggat 120 gcccctggag aaatggtggt cctcacctgt gacacccctg aagaagatgg tatcacctgg 180 accttggacc agagcagtga ggtcttaggc tctggcaaaa ccctgaccat ccaagtcaaa 240 gagtttggag atgctggcca gtacacctgt cacaaaggag gcgaggttct aagccattcg 300 ctcctgctgc ttcacaaaaa ggaagatgga atttggtcca ctgatatttt aaaggaccag 360 aaagaaccca aaaataagac ctttctaaga tgcgaggcca agaattattc tggacgtttc 420 acctgctggt ggctgacgac aatcagtact gatttgacat tcagtgtcaa aagcagcaga 480 ggctcttctg acccccaagg ggtgacgtgc ggagctgcta cactctctgc agagagagtc 540 agaggggaca acaaggagta tgagtactca gtggagtgcc aggaggacag tgcctgccca 600 gctgctgagg agagtctgcc cattgaggtc atggtggatg ccgttcacaa gctcaagtat 660 gaaactaca ccagcagctt cttcatcagg gacatcatca aacctgaccc acccaagaac 720 ttgcagctga agccattaaa gaattctcgg caggtggagg tcagctggga gtaccctgac 780 acctggagta ctccacattc ctacttctcc ctgacattct gcgttcaggt ccagggcaag 840 agcaagagag aaaagaaaga tagagtcttc acggacaaga cctcagccac ggtcatctgc 900 cgcaaaaatg ccagcattag cgtgcgggcc caggaccgct actatagctc atcttggagc 960 gaatgggcat ctgtgccctg cagtgttcct ggagtagggg tacctggggt gggcgccaga 1020 aacctccccg tggccactcc agacccagga atgttcccat gccttcacca ctcccaaaac 1080 ctgctgaggg ccgtcagcaa catgctccag aaggccagac aaactctaga attttaccct 1140 tgcacttctg aagagattga tcatgaagat atcacaaaag ataaaaccag cacagtggag 1200 gcctgtttac cattggaatt aaccaagaat gagagttgcc taaattccag agagacctct 1260 ttcatacta atgggagttg cctggcctcc agaaagacct cttttatgat ggccctgtgc 1320 1380 aagctgctga tggatcctaa gaggcagatc ttctagatc aaaacatgct ggcagttatt 1440 gatgagctga tgcaggccct gatttcaac agtgagactg tgccacaaa atcctccctt 1500 gagaaccgg atttttata aactaaaatc aagctctgca tactctca tgctttcaga 1560 attcgggcag tgactattga tagatgatg agctatctga atgcttccgt cgaccaacaa 1620 acagaatgtg tccttaaacc ggttgaatct tcagatatga aaatgactca gctattcacc 1680 aaagttgaat cagaagatac aagtagccctc tttgacaac ttagaagga acctgatgct 1740 ttaactttgc tggccccagc cgctggagac acaatcatat ctttagatt tggcagcac 1800 gacagaaa ctgatgacca gcaacttgag gaagtaccat tatataatga tgtaatgctc 1860 ccctcacccca acgaaaatt acgaatata aatttggcaa tgtctccatt acccaccgct 1920 gaaacgccaa agccactcg aagtagtgct gaccctgcac tcaatcaaga agttgcatta 1980 aaattagaac caaatccaga gtcactggaa ctttcttta ccatgcccca gattcaggat 2040 cagacaccta gtccttccga tggaagcact agacaagtt cacctgagcc taatagtccc 2100 agtgaatt gtttttatgt ggatagtgat atggtcaatg aattcaagtt ggaattggta gaaaaacttt ttgctgaaga cacagaagca aagaacccat tttctactca ggacacagat ttagacttgg agatgttagc tccctatatc ccaatggatg atgacttcca gttacgttcc ttcgatcagt tgtcaccatt agaaagcagt tccgcaagcc ctgaaagcgc aagtcctcaa agcacagtta cagtattcca gcagactcaa attack ctactgctaa tgccaccact accactgcca accctgatga attack gtgacaaaag accgtatgga agacattaa atttgattg catctccatc tcctacccac attack aaactactag tgccacatca tca 2523 <210> 5 <211> 536 <212> PRT <213> The snowstorm <220> <223> IL12 active ingredient <400> 5 Met Gly His Gln Gln Leu Val Ile Ser Trp Download, Listen and View free Met Gly His Gln Gln MP3, Video and Lyrics 1 5 10 15 Ala Ser Pro Leu Val Free Mp3 Download Ala Ser Pro Leu Val Free Mp3 Music Download 20 25 30 Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu 35 40 45 Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln 50 55 60 Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys 65 70 75 80 Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val 85 90 95 Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp 100 105 110 Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe 115 120 125 Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp 130 135 140 Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg 145 150 155 160 Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser 165 170 175 Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu 180 185 190 Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile 195 200 205 Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr 210 215 220 Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn 225 230 235 240 Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp 245 250 255 Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr 260 265 270 Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg 275 280 285 Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala 290 295 300 Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser 305 310 315 320 Glu Trp Ala Ser Val Pro Cys Ser Val Pro Gly Val Gly Val Pro Gly 325 330 335 Val Gly Ala Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe 340 345 350 Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met 355 360 365 Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu 370 375 380 Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu 385 390 395 400 Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser 405 410 415 Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys 420 425 430 Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu 435 440 445 Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met 450 455 460 Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile 465 470 475 480 Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln 485 490 495 Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu 500 505 510 Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg 515 520 525 Val Met Ser Tyr Leu Asn Ala Ser 530 535 <210> 6 <211> 1608 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of IL12 protein <400> 6 atgggtcacc agcagttggt catctcttgg ttttccctgg tttttctggc atctcccctc 60 gtggccatat gggaactgaa gaaagatgtt tatgtcgtag aattggattg gtatccggat 120 gcccctggag aaatggtggt cctcacctgt gacacccctg aagaagatgg tatcacctgg 180 accttggacc agagcagtga ggtcttaggc tctggcaaaa ccctgaccat ccaagtcaaa 240 gagtttggag atgctggcca gtacacctgt cacaaaggag gcgaggttct aagccattcg 300 ctcctgctgc ttcacaaaaa ggaagatgga atttggtcca ctgatatttt aaaggaccag 360 aaagaaccca aaaataagac ctttctaaga tgcgaggcca agaattattc tggacgtttc 420 acctgctggt ggctgacgac aatcagtact gatttgacat tcagtgtcaa aagcagcaga 480 ggctcttctg acccccaagg ggtgacgtgc ggagctgcta cactctctgc agagagagtc 540 agggggaca acaaggagta tgagtactca gtggagtgcc aggaggacag tgcctgccca gctgctgagg agagtctgcc cattgaggtc atggtggatg ccgttcacaa gctcaagtat 660 720. gaaaactaca ccagcagctt cttcatcagg gacatcatca aacctgaccc acccaagaac ttgcagctga agccattaaa gaattctcgg caggtggagg tcagctggga gtaccctgac acctggagta ctccacattc ctacttctcc ctgacattct gcgttcaggt ccagggcaag 840 agcaagagag aaaagaaga taggtcttc acggacaaga cctcagccac ggtcatctgc cgcaaaaatg cgcattag cgtgcgggcc cgcgct actatagctc atcttggagc gaatgggcat ctgtgccctg cagtgttcct ggagtagggg tacctggggt gggcgccaga 1020 aacctccccg tggccactcc agacccagga atgttcccat gccttcacca ctcccaaaac 1080 ctgctgaggg ccgtcagcaa catgctccag aaggccagac aaactctaga attttaccct tgcacttctg aagagattga tcatgaagat atcacaaaag ataaaaccag cacagtggag gcctgtttac cattggaatt aaccaagaat gagagttgcc taaattccag agagacctct 1260 ttcataacta atgggagttg cctggcctcc agaaagacct cttttatgat ggccctgtgc 1320 cttagtagta tttatgaaga cttgaagatg taccaggtgg agttcaagac catgaatgca 1380 aagctgctga tggatcctaa gaggcagatc tttctagatc aaaacatgct ggcagttatt 1440 gatgagctga tgcaggccct gaatttcaac agtgagactg tgccacaaaa atcctccctt 1500 gaagaaccgg atttttataa aactaaaatc aagctctgca tacttcttca tgctttcaga 1560 attcgggcag tgactattga tagagtgatg agctatctga atgcttcc 1608 <210> 7 <211> 305 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of ODD protein <400> 7 Val Asp Gln Gln Thr Glu Cys Val Leu Lys Pro Val Glu Ser Ser Asp 1 5 10 15 Met Lys Met Thr Gln Leu Phe Thr Lys Val Glu Ser Glu Asp Thr Ser 20 25 30 Ser Leu Phe Asp Lys Leu Lys Lys Glu Pro Asp Ala Leu Thr Leu Leu 35 40 45 Ala Pro Ala Ala Gly Asp Thr Ile Ile Ser Leu Asp Phe Gly Ser Asn 50 55 60 Asp Thr Glu Thr Asp Asp Gln Gln Leu Glu Glu Val Pro Leu Tyr Asn 65 70 75 80 Asp Val Met Leu Pro Ser Pro Asn Glu Lys Leu Gln Asn Ile Asn Leu 85 90 95 Ala Met Ser Pro Leu Pro Thr Ala Glu Thr Pro Lys Pro Leu Arg Ser 100 105 110 Ser Ala Asp Pro Ala Leu Asn Gln Glu Val Ala Leu Lys Leu Glu Pro 115 120 125 Asn Pro Glu Ser Leu Glu Leu Ser Phe Thr Met Pro Gln Ile Gln Asp 130 135 140 Gln Thr Pro Ser Pro Ser Asp Gly Ser Thr Arg Gln Ser Ser Pro Glu 145 150 155 160 Pro Asn Ser Pro Ser Glu Tyr Cys Phe Tyr Val Asp Ser Asp Met Val 165 170 175 Asn Glu Phe Lys Leu Glu Leu Val Glu Lys Leu Phe Ala Glu Asp Thr 180 185 190 Glu Ala Lys Asn Pro Phe Ser Thr Gln Asp Thr Asp Leu Asp Leu Glu 195 200 205 Met Leu Ala Pro Tyr Ile Pro Met Asp Asp Asp Phe Gln Leu Arg Ser 210 215 220 Phe Asp Gln Leu Ser Pro Leu Glu Ser Ser Ser Ala Ser Pro Glu Ser 225 230 235 240 Ala Ser Pro Gln Ser Thr Val Thr Val Phe Gln Gln Thr Gln Ile Gln 245 250 255 Glu Pro Thr Ala Asn Ala Thr Thr Thr Thr Ala Thr Thr Asp Glu Leu 260 265 270 Lys Thr Val Thr Lys Asp Arg Met Glu Asp Ile Lys Ile Leu Ile Ala 275 280 285 Ser Pro Ser Pro Thr His Ile His Lys Glu Thr Thr Ser Ala Thr Ser 290 295 300 Ser 305 <210> 8 <211> 915 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of ODD protein <400> 8 gtcgaccaac aaacagaatg tgtccttaaa ccggttgaat cttcagatat gaaaatgact 60 cagctattca ccaaagttga atcagaagat acaagtagcc tctttgacaa acttaagaag 120 gaacctgatg ctttaactt gctggcccca gccgctggag acacaatcat atctttagat 180 tttggcagca acgacacaga aactgatgac cagcaacttg aggaagtacc attatatat 240 gatgtaatgc tcccctcacc caacgaaaaa tcagaata taaatttggc aatgtctcca 300 ttacccaccg ctgaaacgcc aaagccactt cgaagtagtg ctgaccctgc actcaatcaa 360 gaagttgcat taaaattaga accaatcca gagtcactgg aactttctt taccatgccc 420 cagattcagg atcagacacc tagtccttcc gatggaagca ctagacaaag ttcacctgag 480 cctaatagtc ccagtgaata ttgttttt gtggatagtg atatggtca tgaattcaag 540 ttggaatttgg tagaaaact tttgctgaa gandacagaag caagaaccc atttctctact 600 caggacacag atttagactt ggagatgtta gctccctata tcccaatgga tgatgacttc 660 cagttacgtt ccttcgatca gttgtcacca ttagaaagca gttccgcaag ccctgaaagc 720 gcaagtcctc aaagcacagt tacagtattc cagcagactc aaatacaaga acctactgct 780 aatgccacca ctaccactgc caccactgat gattaaaaa cagtgacaaa agaccgtag 840 gaagacatta aaatattgat tgcatctcca tctcctaccc acatacataa agaaactact 900 agtgccacat catca 915 <210> 9 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of self-cleaving protein P2A <400> 9 Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 1 5 10 15 Pro Gly Pro <210> 10 <211> 57 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of self-cleaving protein P2A <400> 10 gccacgaact tctctctgtt aaagcaagca ggagatgttg aagaaaaccc cgggcct 57

Claims

1. A chimeric antigen receptor (CAR), comprising (1) an extracellular antigen binding domain; (2) a transmembrane domain; and (3) an intracellular signaling domain, wherein: The CAR also comprises a fusion protein of interleukin 12 (IL12) and an oxygen-dependent degradation domain (ODD) connected to and co-expressed with the intracellular domain, wherein the sequence of the IL12 is the amino acid sequence of SEQ ID NO: 5; and the sequence of the ODD is the amino acid sequence of SEQ ID NO:

7.

2. The CAR according to claim 1, wherein the intracellular domain is connected to the fusion protein via a self-cleaving protein.

3. The CAR according to claim 2, wherein the self-cleaving protein is selected from the group consisting of T2A, P2A, E2A, F2A, or a combination thereof.

4. The CAR of claim 1, wherein the transmembrane domain is derived from one or more transmembrane domains selected from the group consisting of α, β or ζ chain of a T cell receptor, CD3ε, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD80, CD86, CD134, CD137, CD152, CD154 and ICOS.

5. The CAR according to claim 1, wherein the intracellular signal transduction domain comprises a co-stimulatory signaling domain and is from: CD2, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD7, CD22, CD27, CD28, CD30, CD40, CD66d, CD79a, CD79b, CD83, CD134, CD137, ICOS, CD154, 4-1BB and OX40, LFA-1, LIGHT, NKG2C and B7-H3.

6. The CAR according to any one of claims 1-5, further comprising a hinge domain located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.

7. The CAR of claim 6, wherein the hinge domain is derived from CD8α.

8. The CAR according to claim 1, wherein the extracellular antigen binding domain is a molecule that can bind to a tumor cell surface antigen.

9. The CAR according to claim 8, wherein the tumor cell surface antigen that can bind is CD19.

10. The CAR according to claim 8, wherein the molecule capable of binding to a tumor cell surface antigen is an antibody selected from the group consisting of: Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, scFv, bi-scFv, (scFv)2, mini antibody, double-chain antibody, three-chain antibody, four-chain antibody, disulfide bond-stabilized Fv protein, and single domain antibody, bispecific antibody or trispecific antibody.

11. The CAR according to claim 1, wherein the CAR comprises the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO:

3.

12. The CAR according to claim 1, further comprising a tag sequence or an IgG1-Fc protein sequence.

13. The CAR according to claim 12, wherein the tag sequence is Poly-His, Hemagglutinin, c-Myc, GST or Flag-tag.

14. Nucleotides encoding the CAR of any one of claims 1-13.

15. The nucleotide according to claim 14, wherein the nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO:2, or comprises the nucleotide sequence of SEQ ID NO:2-SEQ ID NO:10-SEQ ID NO:4 connected in sequence.

16. An isolated CAR-T cell or CAR-NK cell or mononuclear macrophage, characterized in that: The CAR-T cell or CAR-NK cell or mononuclear macrophage is capable of expressing the CAR described in any one of claims 1-13; the CAR-T cell or CAR-NK cell or mononuclear macrophage comprises the nucleotide described in claim 14 or 15.

17. A vector comprising the nucleotide according to claim 14 or 15.

18. The vector according to claim 17, wherein the vector is an expression vector.

19. The vector according to claim 18, wherein the vector is a viral vector.

20. The vector according to claim 18, which is selected from a retroviral vector or an oncolytic virus.

21. The vector of claim 18, which is selected from the group consisting of a lentiviral vector, human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), Vesna-Medivirus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), simian immunodeficiency virus (SIV), adenovirus, and poxvirus.

22. A pharmaceutical composition comprising the chimeric antigen receptor of any one of claims 1 to 13, comprising the cell of claim 16, and optionally, a pharmaceutically acceptable carrier.

23. A method for preparing the cell of claim 16, comprising introducing the vector of any one of claims 17 to 21 into T lymphocytes, natural killer cells or mononuclear macrophages.

24. Use of the CAR according to any one of claims 1-13 in the preparation of a medicament for treating and / or preventing diffuse large B-cell lymphoma.

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