CN114907487A

CN114907487A - Novel chimeric antigen receptor and immune cell comprising same

Info

Publication number: CN114907487A
Application number: CN202111422360.7A
Authority: CN
Inventors: 王尧河; 周文平; 苗晋鑫
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2021-11-26
Filing date: 2021-11-26
Publication date: 2022-08-16

Abstract

The invention relates to a fusion protein capable of specifically binding to a tumor specific marker and an immune cell capable of expressing the fusion protein, such as a T cell or an NK cell. The fusion protein of the invention may comprise a Chimeric Antigen Receptor (CAR), interleukin 12(IL12), and an oxygen-dependent degradation region (ODD). The CAR of the invention comprises an ectodomain, a transmembrane domain and an endodomain, said ectodomain being capable of specifically binding to a tumor-specific antigen and activating T or NK cells via the transmembrane domain and endodomain. The present invention provides CAR-T cells or NK cells that target tumor-specific markers, utilizing CAR-T cells or NK cells to specifically kill tumor cells, such as myeloma or leukemia. The CAR-T or NK cell can be used as a therapeutic drug for tumor diseases, and provides a new method for preventing and treating tumors.

Description

Novel chimeric antigen receptor and immune cell comprising same

Technical Field

The invention relates to the field of biomedicine, in particular to a novel chimeric antigen receptor, an immune cell containing the same, and preparation and application thereof.

Background

Diffuse Large B Cell Lymphoma (DLBCL) is the most common type of malignant lymphoma, accounting for approximately 30% -40% of its incidence. DLBCL has better response to traditional chemotherapy and radiotherapy, and the addition of the monoclonal CD20 antibody rituximab further improves the curative effect and prognosis, so that more than half of patients can survive or be cured for a long time. At this stage, the therapeutic challenge is the refractory patients to relapse early, their life span is short, and there is no effective treatment. There is a need to find effective therapeutic methods and means for refractory DLBCL.

CAR-T is globally known as Chimeric Antigen Receptor T-Cell, and Chimeric Antigen Receptor T-Cell-based immunotherapy is the hot spot in current research. CAR-T treatment is based on the separation and culture of tumor patient autoimmune T cells, the use of transgenic technology will include specific recognition of tumor cell surface antigen molecules, such as specific single chain antibody scFv (single chain antibody fragment) and T cell costimulatory molecules of polycistronic protein expression on the T cell surface, will be genetically modified T cells into the patient in vivo, the returned CAR-T cells in vivo specific recognition and killing tumor cells.

In 2011, professor Carl H June published a second generation CAR-T treatment based on 4-1BB co-stimulation signals, targeting CD19, with an indication of chronic myelogenous leukemia (CLL) and successful cure of Emily 4 year old girls with CLL. CD19 CAR-T is applied to various hematopathy such as ALL, CLL, DLBCL and the like, wherein the remission rate of most of hematopathy ALL and CLL can reach 90%.

Compared with the remission rate of other hematologic diseases of up to 90%, the cure rate of the CD19 CAR-T on the DLBCL which is difficult to treat does not exceed 50%, and the immune microenvironment of the DLBCL limits the curative effect of the CD19 CAR-T to a certain extent, so that a suitable method for locally improving the DLBLC tumor immune microenvironment is urgently needed to be found.

Interleukin 12(Interleukin-12, IL-12), an originally discovered heterodimeric complex in EBV-transformed B cells covalently linked by p35 and p40 subunits. IL-12 is a multifunctional cytokine, discovered by Hsieh et al in 1993, naive CD4 ⁺ Cells induce the expression of transcription factor STAT1 under stimulation of interferon-gamma (IFN-gamma) released by natural killer cells (NK), and then STAT1 transcribes the expression of T-beta, which promotes the conversion of naive CD4+ cells into CD4 ⁺ TH1 effector cells, while expression of T-beta induces the expression of Interleukin-12receptor beta 2-subbunit (IL-12R beta 2) in CD4 ⁺ Th1 cell membrane surface, when IL-12 binds to IL-12R on CD4+ Th1 cell membrane surface, promoting CD4 ⁺ Th1 cells autocrine IFN-gamma and perform effector cell functions with other inflammatory factors, therefore IL-12 plays a bridge connecting role in innate immunity and adaptive cellular immunity. IL-12 immunoregulatory function is not limited to innate and adaptive immune bridge, IL-12 promotes CD4 ⁺ TH1 cell autocrine IFN-gamma, also can induce the release of other second and third proinflammatory cytokines, the release of these proinflammatory cytokines has cytotoxic effect on tumor cells, and the release of these proinflammatory cytokines also changes the local immune environment of tumor, prolongs CD8 to a certain extent ⁺ T cells survive, and the tumor killing effect is promoted. In general from an immune cell perspective, IL-12 contributes to the promotion of NK, NK-Tcells, CD4 ⁺ 、CD8 ⁺ T cells increase their survival rate and enhance their effector functions, especially their induced secretion of IFN-gamma, also have anti-tumor effects. From the viewpoint of tumor microenvironment, secretion of IFN-gamma can reduce the secretion of Vascular Endothelial Growth Factor (VEGF) by injured tumor cells, thereby inhibiting tumor metastasis. NK, NK-Tcells, CD4 stimulated by IL-12 ⁺ 、 CD8 ⁺ T cells produce chemokinesIP-10 and MIG, IP-10 and MIG inhibit the expression of endothelial Matrix Metalloproteinase (MMP), reduce the alteration of extracellular matrix (ECM) remodeling, and thus achieve the effect of reducing angiogenesis and tumor invasion.

Disclosure of Invention

The inventors of the present invention found that the combination of CD19 CAR-T with IL-12 for the treatment of DLBCL improves the remission rate of DLBCL from CD19 CAR-T. Meanwhile, IL-12 brings certain cytotoxicity while bringing the effect of killing the immunity, especially to the cytotoxic damage of the liver, so that the antitumor clinical application of IL-12 is greatly limited, and therefore, a solution for reducing the hepatotoxicity of IL-12 is urgently needed.

To overcome the toxic side effects of IL-12 and CAR cells when used in combination, the present invention co-expresses a fusion protein (IL-12ODD) with the CAR on the surface of an immune cell. The anaerobic glycolytic properties of tumor cells make them more adaptive to the anaerobic microenvironment local to the tumor. The CAR or the CAR immune cell designed by the invention ensures that secretory IL-12(IL-12ODD) expressed by oxygen pressure regulation can only play a role in the local anaerobic microenvironment of tumor secretion, while the oxygen pressure regulation ODD can start ubiquitination degradation pathway in the aerobic environment of normal tissues to degrade IL-12, and the IL-12ODD can only play an anti-tumor role in the high expression of IL-12 in the anaerobic microenvironment of tumor, thereby reducing the concentration of IL-12 in the normal tissues of peripheral blood and the toxicity of liver and lung caused by the IL-12 concentration.

In addition, the invention discovers that a short connecting peptide (linker) which is based on a single-chain antibody scFv and is connected with 15-20 amino acids of the heavy chain variable region and the light chain variable region of the antibody can improve the combined treatment effect of CAR immune cells and IL 12. Specifically, the invention applies a novel connecting peptide Linker218 and a traditional connecting peptide (G) ₄ S)3 compared with the Linker218, the single-chain antibody scFv can show a more flexible spatial structure without reducing the affinity, and the stability of the proteolytic enzyme system is increased.

The inventor co-expresses CD19 CAR-T containing a novel connecting peptide Linker218 and IL-12 on the surface of a T cell through self-cleavage peptide connection, after the CD19 CAR-T cell co-expressing IL-12 is transfused back, the CD19 CAR-T cell can home to lymph nodes to express IL-12, and the IL-12 can induce autoimmune cells to secrete IFN-gamma so as to improve DLBLC tumor immune microenvironment and inhibit DLBCL progression and metastasis, meanwhile, the survival period of the CD19 CAR-T cell is increased, the killing effect of the CD19 CAR-T cell is prolonged, and the curative effect of CD 19-T immunotherapy on difficult-to-treat DLBCL is improved.

The inventor of the invention constructs a third generation lentivirus plasmid expressing CD19 specific antigens 4-1BB containing a novel connecting peptide Linker218 and CD28 as intracellular activation signals in vitro, utilizes 293T to package lentiviruses with proper titer in vitro, and expresses CD19 specific antigens and intracellular activation signals in CD3 separated from peripheral blood ⁺ T cells and modified T cells kill a DLBLC system which is difficult to treat in vitro, and the combined effect of CD19 CAR-T, IL12 and IL12ODD is compared by methods of calculating a growth curve, calculating killing efficiency, in vivo and in vitro and the like.

In one aspect, the invention provides 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 further comprises a fusion protein of interleukin 12(IL12) and an oxygen-dependent degradation region (ODD) linked to and co-expressed with the intracellular domain, optionally, the intracellular domain is linked to the fusion protein through a self-cleaving protein (e.g., selected from the group consisting of T2A, P2A, E2A, F2A, or a combination thereof).

In one embodiment of the invention, the transmembrane domain of the invention is derived from a transmembrane domain selected from one or more of the group consisting of the α, β or zeta chain of the T cell receptor, CD3 ∈, CD4, CD5, CD8, CD8 α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD80, CD86, CD134, CD137, CD152, CD154 and ICOS.

In one embodiment of the invention, the intracellular signaling domain of the invention comprises a costimulatory signaling domain and is derived from: CD2, CD3 ζ, CD3 γ, CD3 δ, CD3 ε, CD4, CD5, CD7, CD22, CD27, CD28, CD30, CD40, CD66d, CD79a, CD79B, CD83, CD134, CD137, ICOS, CD154, 4-1BB, and one or more of OX40, LFA-1, LIGHT, NKG2C, and B7-H3.

In one embodiment of the invention, the CAR of the invention further comprises a hinge domain, e.g. derived from CD8a, between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

In one embodiment of the invention for the CAR of the invention wherein the extracellular antigen-binding domain is a molecule, e.g. an antibody (e.g. one or more of CD19, CD20, CD22, CD123, CD30, CD33, CD38, CD44, CD138, CD276, EGFR, BCMA, HER1, HER2, HER3, EpCAM, mesothelin (mesothelin), fap (fibroblast activation protein), Glypican-3, CEA, PSMA, IL13R α 2, CD171, GD2, CEACAM6, CLDN4, CLDN18.2, nk 2, LAMC2, CA9, CST1, EPPK1 and ANO 1), such as an antibody (Ig r, Fab fragment, Fab 'fragment, f Ig ab)' ₂ Fragment, F (ab)' ₃ Fragment, Fv, scFv, bis-scFv, (scFv) ₂ Minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins, and single domain antibodies (sdabs, nanobodies), bispecific antibodies, or trispecific antibodies).

For those commonly used in tumor cell surface antigens, the following is exemplarily explained.

CD19 is a surface protein expressed on B lymphocytes and follicular dendritic cells, belongs to a member of the immunoglobulin (Ig) superfamily, is located on the short arm of chromosome 16 (16p11.2), encodes a type I transmembrane glycoprotein of 556 amino acids, and has a molecular weight of 95 KD. CD19 is expressed only in normal and malignant B cells, and hardly in other tissues; second, CD19 is not lost during B cell malignant transformation, and refractory/relapsing cases remain effective; furthermore, CD19 is not expressed in hematopoietic stem cells and pro-B cells, and B cells can be efficiently replenished after treatment is stopped. Therefore, various leukemia treatment strategies targeting CD19 have better clinical effects in recent years, including bispecific antibodies of CD3 and CD19, CAR-T targeting CD19 and the like.

The CD20 antigen is a B cell differentiation antigen expressed only on the surface of pre-B cells and mature B cells, and is expressed in more than 95% of B cell lymphomas, but not in hematopoietic stem cells, plasma cells and other normal tissues. Several mabs targeting CD20 (e.g., Rituxan) have been used clinically successfully for the treatment of various lymphomas.

CD38 is a single-chain transmembrane glycoprotein of 45kDa, and the whole structure is divided into a short cytoplasmic tail at the N terminal, a single transmembrane domain and a long extracellular region at the C terminal. In adults, CD38 is found on most natural killer cells, T cells, B cells, monocytes/macrophages. There is also some expression on platelets and red blood cells. Certain mabs targeting CD38 have been approved for the treatment of multiple myeloma.

BCMA (B cell maturation antigen) is a type III transmembrane protein consisting of 185 amino acid residues. It belongs to a member of the TNF receptor family and binds to its ligand B cell activator BAFF or proliferation-inducing ligand APRIL to stimulate B cell proliferation. BCMA is normally expressed in mature B cells and plasma cells, has wide expression in Multiple Myeloma (MM), and is a very ideal immunotherapy target point of multiple myeloma.

The HER2 gene is located on human chromosome 17q21 and encodes a 185kD molecular weight transmembrane protein which has tyrosine kinase activity, normally exists in an inactive form, is involved in regulating normal cell differentiation, is normally expressed only in infancy and is expressed at low levels in a few tissues in adults. SA 2000; 97: 3444-3449 Slamon et al, Science 1987; 235: 177-182).

CEA (carcinoembryonic antigen) is a glycoprotein produced by large intestine cancer tissues, and can be used as an antigen to cause immune response of patients. It is widely present in digestive system cancer of endoembryonic leaf origin, and also in digestive vessel tissue of normal embryo, and may be present in trace amount in normal human serum. The carcinoembryonic antigen is a broad-spectrum tumor marker, can reflect the existence of various tumors to people, and is a better tumor marker for curative effect judgment, disease development, monitoring and prognosis estimation of colorectal cancer, breast cancer and lung cancer.

PSMA is 110kDa type II transmembrane protein, the gene is positioned on the short arm 11q of the chromosome and is expressed in normal prostate epithelial cells and prostate tumor cells, wherein the expression level in the tumor cells is up-regulated, and the PSMA is considered to be involved in the regulation and control of cell migration in the prostate carcinogenesis process. PSMA expression in prostate cancer is positively correlated with tumor grade and the presence or absence of hormone resistance, and strong expression of PSMA means higher recurrence rates. PSMA is now considered to be one of the references for prostate cancer.

More specifically, in the CAR of the present invention, wherein said IL12 has the amino acid sequence of SEQ ID No. 5; the ODD has the nucleotide sequence of SEQ ID NO: 7, preferably the CAR comprises the amino acid sequence of SEQ ID NO:1 and SEQ ID NO:3, or a pharmaceutically acceptable salt thereof.

The enhanced CAR of the invention optionally further comprises a tag sequence (e.g., Poly-His, Hemagglutenin, c-Myc, GST, Flag-tag, etc.) or IgG1-Fc protein sequence.

Yet another aspect of the invention provides a nucleotide sequence encoding a CAR, preferably the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO. 2 or comprises a nucleotide sequence consisting of SEQ ID NO. 2-10-4 in that order.

A third aspect of the invention provides an isolated CAR-T cell or CAR-NK cell or monocyte macrophage, wherein said cell is capable of expressing a CAR or comprises a polynucleotide of the invention.

In a fourth aspect, the present invention provides a vector comprising a polynucleotide as described above.

In a specific embodiment, the vector of the invention is an expression vector, such as a viral vector, preferably a retroviral vector, such as a lentiviral vector, preferably selected from the group consisting of human immunodeficiency virus 1(HIV-1), human immunodeficiency virus 2(HIV-2), visna-mei virus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), Equine Infectious Anaemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), Simian Immunodeficiency Virus (SIV), adenovirus and poxvirus.

A fifth aspect of the invention provides a pharmaceutical composition comprising a chimeric antigen receptor as provided by the invention of the claims, or comprising a CAR-T cell or a CAR-NK cell or a monocyte macrophage as provided by the invention, and optionally, a pharmaceutically acceptable carrier.

A fifth aspect of the invention provides a method of making a CAR-T cell or CAR-NK cell or monocyte macrophage as provided by the invention comprising introducing a vector as provided by the invention into a T lymphocyte or natural killer cell or monocyte macrophage.

A fifth aspect of the invention provides a use. In particular, the use refers to the use of a CAR provided herein, or a CAR-T cell or CAR-NK cell provided herein, in the manufacture of a medicament for the treatment and/or prevention of cancer, illustratively myeloma and leukemia.

The invention also provides a method of making a CAR-T cell or CAR-NK cell or monocyte macrophage of the invention, or an immune effector cell of the invention, comprising introducing a vector of the invention into the cell.

Illustratively, the method of making the CAR-T cells of the invention comprises the steps of:

(1) synthesizing and amplifying a CAR-self-cleaving protein-IL 12-ODD protein gene, cloning the protein gene onto a lentiviral expression vector;

(2) infecting 293T cells by using a lentivirus packaging plasmid and the lentivirus expression vector plasmid obtained in the step (1), packaging and preparing lentivirus; and

(3) and (3) infecting T cells by using the lentivirus obtained in the step (2) to obtain CAR-T cells.

The invention also provides the application of the following materials in preparing a medicament for treating and/or preventing cancer:

(a) a CAR of the present invention; or

(b) The CAR-T cell or CAR-NK cell of the invention.

Illustratively, the cancer is a tumor that is highly expressing a tumor-specific marker and related diseases, such as myeloma and leukemia.

The invention also provides a method of treating and/or preventing cancer, comprising administering to a subject in need thereof an effective amount of (a) a CAR of the invention; or (b) a CAR-T cell or CAR-NK cell or monocyte macrophage of the invention is administered to a subject.

For the fusion proteins, CARs, etc. described herein, variants thereof, e.g., their identity sequences or humanized sequences, etc., are also contemplated by the present invention. Illustratively, the identical sequence refers to 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, 99.2% or more, 99.3% or more, 99.4% or more, the same as the original sequence or a reference sequence, Or 99.9% or more.

Degenerate or complementary sequences are also contemplated by the present invention for the polynucleotides of the present invention. Illustratively, 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, with the original or reference sequence, Or 99.9% or more.

The CAR provided by the invention is capable of specifically binding to a tumor specific antigen, a tumor specific marker, and activating the T cell via a transmembrane domain and a costimulatory signaling region. The CAR-T cell can express a fusion protein with tumor specific markers as target antigens, so that the CAR-T cell can specifically kill tumor cells and is used for treating tumor diseases, such as tumor with high expression of tumor specific markers.

Drawings

FIG. 1 is a schematic diagram of the pEF plasmid structure inserted into the CD19CARIL12ODD construct.

Figure 2 is an ELISA assay of CAR19IL12ODD secreting IL12 levels under normal (17%) and low (1%) oxygen conditions, respectively.

FIG. 3, wherein: FIG. 3A LDH assay CAR19 killing efficiency in vitro on the diffuse large B-cell lymphoma cell line Ly 3; FIG. 3B in vivo killing curves of CAR19 on the diffuse large B-cell lymphoma cell line Ly 3.

FIG. 4, wherein: FIG. 4A ELISA detection of IL12 concentration in peripheral blood after in vivo killing of CAR19 on the diffuse large B-cell lymphoma cell line Ly 3; FIG. 4B loss assay CAR19 ratio of CAR-T cell survival in peripheral blood after in vivo killing of the diffuse large B-cell lymphoma cell line Ly 3.

Detailed Description

The CART or NK cells of the invention can target tumor-specific markers (e.g., CD19) and can, in turn, be used to treat, for example, CD 19-associated cancers.

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

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

Animal experiments prove that the CAR-T or NK cell can remarkably kill Diffuse Large B Cell Lymphoma (DLBCL) and promote infiltration of tumors.

Meanwhile, the in vitro killing effect of the fusion protein CD19-PE24 containing the antibody on RPMI 8226 cells is also proved through experiments.

Chimeric antigen receptor:

the CAR comprises an extracellular domain derived from an antibody and an intracellular domain comprising a signaling module derived from a T cell signaling protein. In one embodiment, the ectodomain may comprise a heavy chain variable region from an immunoglobulin, or comprise variable regions of both heavy and light chains, e.g. constructed as a single chain variable fragment (scFv), preferably a single domain antibody (sdAb) with only the heavy chain variable region. The sdabs are connected to a hinge region that provides flexibility and transduces signals to the intracellular signaling domain through the transmembrane domain. The transmembrane domain is preferably derived from CD8 α. In the first generation of CARs (the term "generation" is for the intracellular signaling domain), the intracellular signaling domain consists of the zeta chain of the TCR complex. Second generation CARs were designed to contain a single co-stimulatory domain derived from CD28 or 4-1 BB. Third generation CARs include two co-stimulatory domains, e.g., 4-1BB-CD3 ζ. The invention preferably relates to a second or third generation CAR.

The present invention illustratively provides genetically engineered receptors that redirect cytotoxicity of immune effector cells to cancer cells. These genetically engineered receptors are referred to herein as Chimeric Antigen Receptors (CARs). CARs are chimeric protein molecules with specific anti-e.g., CD19 cellular immune activity based on the specificity of an antibody targeting an antigen (e.g., CD19) in combination with the intracellular domain of an activated T cell receptor or NK cell receptor. In this context, the term "chimeric" refers to a composition of different proteins or DNAs of different origin.

The CARs of the invention include an extracellular domain (also referred to as a binding domain or antigen binding domain), a transmembrane domain, and an intracellular domain or intracellular signaling domain that binds, for example, CD 19. Illustratively, binding of the anti-CD 19 antigen-binding domain of the CAR to CD19 on the surface of the target cell results in aggregation of the CAR and delivery of an activation stimulus to the CAR-containing cell. CARs are capable of specifically redirecting immune effector cells, thereby triggering proliferation, cytokine production, phagocytosis, or cell killing of target antigen expressing cells.

In some embodiments of the invention, the 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, the CAR comprises, sequentially, the 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 linked to a signaling domain via a self-cleaving protein.

An "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., CD 19). The binding domain may be derived from natural, synthetic, semi-synthetic or recombinant sources. Preferred are sdabs of recombinant origin.

"specific binding" shall be understood by those skilled in the art as one will appreciate that various experimental methods or means that can be used to test binding and binding specificity are clearly known to those skilled in the art. Methods for determining equilibrium association or equilibrium dissociation constants are known in the art. In many protein-protein interactions, some cross-reactivity or background binding may occur, but this does not impair the "specificity" of binding between the CAR and the epitope. For example, "specific binding" describes the binding of an anti-CD 19 antibody or antigen-binding fragment thereof (also including their CARs) to CD19 with a binding affinity higher than background binding.

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

"Single chain Fv" or "scFv" antibody fragments comprise the V of an antibody _H Field and V _L Domains, wherein the 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 further comprises V _H Field and V _L Polypeptide linker between domains enabling scFv formationTo a desired structure for antigen binding. In preferred embodiments, the CAR of the invention comprises an antigen-specific binding domain that is an scFv and may be a murine, human or humanized scFv. Single chain antibodies can be cloned from the V region genes of hybridomas specific for the desired target. In particular embodiments, the antigen-specific binding domain is a humanized scFv that binds a human CD19 polypeptide.

Antibodies and antibody fragments:

the CAR comprises an extracellular antigen-binding domain comprising an antibody or antibody fragment that binds a tumor-specific marker (e.g., CD19) polypeptide. Thus, the antibodies or antibody fragments of the 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 a Fab expression library, anti-idiotype antibodies and epitope-binding fragments or a combination of any of the above, provided that they have similar binding properties of a CAR according to the invention, preferably comprising the corresponding CDR as described herein, or V _H And V _L And (4) a zone. Miniantibodies and multivalent antibodies such as diabodies, triabodies, tetrabodies and pentavalent antibodies may also be used in the methods of the invention. The immunoglobulin molecules of the present invention may be of any class (i.e., IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecules. Thus, as used herein, the term antibody also includes antibodies and antibody fragments encompassed by the CARs of the invention, which are produced by modifying an intact antibody or are synthesized de novo using recombinant DNA methods.

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 "antibody fragments". Optionally, the antibody or antibody fragment may be chemically conjugated to or expressed as a fusion protein with other proteins or with other proteins. In some embodiments, the antibodies or antigen-binding fragments of the invention are comprised on a multispecific antibody, e.g., a bispecific antibody. Such multispecific antibodies may be produced by known methods, e.g. cross-linking two or more antibodies, antigen-binding fragments (e.g. scFv) of the same or different types. Exemplary methods of making multispecific antibodies include those described in PCT patent publication No. WO2013/163427, which is incorporated herein by reference in its entirety.

The affinity of the binding domain polypeptides and antibodies or antibody fragments or CAR proteins of the invention can be readily determined using conventional techniques, for example by competitive ELISA (enzyme-linked immunosorbent assay), or using surface plasmon resonance devices (e.g. Biacore).

Humanized antibodies comprising one or more CDRs of or derived from an antibody or antibody fragment of the invention can be prepared using methods known in the art. For example, monoclonal antibodies can generally be humanized using four steps: (1) determining the nucleotide and predicted amino acid sequences of the starting antibody light and heavy chain variable domains; (2) designing a humanized antibody, i.e., determining which antibody framework regions are used in the humanization process; (3) (ii) developing humanization methods/techniques; and (4) transfection and expression of humanized antibodies. See, for example, U.S. patent No. 6,180,370.

The term humanized antibody means that at least a portion of the framework regions and optionally a portion of the CDR regions or other regions involved in binding of an immunoglobulin are derived from or adapted to human immunoglobulin sequences. Humanized, chimeric or partially humanized forms of mouse monoclonal antibodies can be prepared, for example, by recombinant DNA techniques. Humanized forms of mouse antibodies can be generated by joining CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques (Queen et al, 1989; WO 90/07861). Alternatively, the monoclonal antibody used in the methods of the invention may be a human monoclonal antibody. Human antibodies can be obtained, for example, using phage display methods (WO 91/17271; WO 92/01047).

As used herein, humanized antibodies also refer to forms of non-human (e.g., murine, camel, llama, shark) antibodies that are specific for a minimal sequence containing sequences derived from non-human immunoglobulinsChimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab ', F (ab') ₂ Or other antigen-binding subsequences of antibodies, such as VHH.

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 may 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 otherwise to determine that it has the same epitope binding specificity as a particular mouse antibody.

Further description of chimeric antigen receptors

In certain embodiments, the CARs of the invention may comprise linker residues between the domains added for proper spacing and conformation of the molecule, e.g., a linker comprising an amino acid sequence that links V _H Domain and V _L Domains and provide spacer functions compatible with the interaction of the two sub-binding domains such that the resulting polypeptide retains specific binding affinity for the target molecule. The CAR of the invention may comprise one, two, three, four, or five or more linkers. In particular embodiments, 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.

Illustrative examples of linkers include glycine polymers; glycine-serine polymers; glycine-alanine polymer; alanine-serine polymers; other flexible joints are known in the art, such as a wheatstone joint. Glycine and glycine-serine polymers are relatively unstructured and thus can serve as a link between domains of a fusion protein or some of the domains (e.g., a CAR as described herein).

In particular embodiments, the binding domain of the CAR is followed by one or more "spacers" or "spacer polypeptides", corresponding to linkers, that move the antigen binding domain away from the surface of the effector cell to enable proper cell-to-cell contact, antigen binding and activation. In certain embodiments, the spacer region is part of an immunoglobulin, including but not limited to one or more heavy chain constant regions, such as CH2 and CH 3. The spacer region may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In one embodiment, the spacer region comprises the CH2 and CH3 domains of IgG1 or IgG 4.

In some embodiments, the binding domain of the CAR may be followed by one or more "hinge domains" that distance the antigen binding domain from the surface of the effector cell to enable proper cell-to-cell contact, antigen binding and activation. The CAR may comprise 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 comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Exemplary hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins (e.g., CD8a, 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 comprises a PD1, CD152, or CD8a hinge region.

The "transmembrane domain" is a portion of the CAR that fuses the extracellular binding moiety and the intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell. The TM domain may be derived from natural, synthetic, semi-synthetic or recombinant sources. The TM domain may be derived from the α, β or ζ chain of a T cell receptor, CD3 ∈, CD3 ζ, CD4, CD5, CD8 α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD 1. In one embodiment, the CAR of the invention comprises a TM domain derived from CD8a or CD 28.

In particular embodiments, the CAR of the invention comprises an intracellular signaling domain. By "intracellular signaling domain" is meant information involved in the binding of an effective anti-CD 19CAR to a human CD19 polypeptide that is transduced into the interior of an immune effector cell to elicit effector cell functions (e.g., activation, cytokine production, proliferation, and cytotoxic activity, including release of cytotoxic factors to target cells to which the CAR binds) or other cellular responses elicited by antigen-binding extracellular CAR domains. The term "effector function" refers to a specialized function of an immune effector cell. For example, the effector function of a T cell may be cytolytic activity or help or activity including cytokine secretion. The term "intracellular signaling domain" refers to the portion of a protein that transduces effector function signals and directs a cell to perform a specialized function.

The CARs of the invention comprise one or more co-stimulatory signaling domains to enhance the efficacy, expansion, and/or memory formation of T cells or NK cells expressing the CAR receptor. As used herein, the term "co-stimulatory signaling domain" refers to the intracellular signaling domain of the CAR molecule, providing the secondary signal required for effective activation and function of T lymphocytes or NK cells upon binding to an antigen.

Protein

"protein", "polypeptide fragment" and "polypeptide" are used interchangeably unless indicated to the contrary, and are used according to conventional meanings, i.e., as amino acid sequences. Proteins are not limited to a particular length, e.g., they may comprise full-length protein sequences or fragments of full-length proteins, and may include post-translational modifications of the polypeptide (e.g., glycosylation, acetylation, phosphorylation, etc.) as well as other modifications known in the art, including both naturally occurring and non-naturally occurring.

In various embodiments, the CAR polypeptides or proteins of the invention comprise a signal (or leader) sequence at the N-terminus of the protein that can direct protein transfer upon or after translation. Polypeptides may be prepared using a variety of well-known recombinant and/or synthetic techniques. The polypeptides of the invention specifically include the CARs of the disclosure, or sequences having one or more (e.g., 1-20, 1-10, or 1-5) amino acid deletions, additions, and/or substitutions to the CARs disclosed herein.

Nucleic acid

The term "polynucleotide" as used herein refers to mRNA, RNA, genomic RNA (grna), positive strand RNA (+), negative strand RNA (-), genomic DNA (gdna)), complementary DNA (cdna), or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. Preferably, a polynucleotide of the invention includes a polynucleotide or variant 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 to any of the reference sequences described herein, typically wherein the variant retains at least one biological activity of the reference sequence.

Illustratively, the molecules of the invention encode the CD19 binding domain-CD 8 alpha hinge-CD8 ^TM The sequence of the polynucleotide of the 4-1BB-CD3 ζ -P2A-IL12-ODD fusion protein is any DNA sequence capable of encoding the fusion protein, preferably, the sequence is SEQ ID NO: 2. or the nucleotide sequence consisting of SEQ ID NO 2-10-4 or the complementary sequence thereof. In another aspect, the invention features a polypeptide encoding the CD19 binding domain-CD 8 α hinge-CD8 ^TM The sequence of the polynucleotide of the-4-1 BB-CD3 ζ -P2A-IL12-ODD fusion protein may be a sequence that hybridizes under stringent conditions with the full complement of the sequence set forth in SEQ ID NO:2 or the nucleotide sequence consisting of SEQ ID NO. 2-10-4 in sequence, and encoding the polynucleotide of the fusion protein or the complementary sequence thereof;

the "stringent conditions" as used herein may be any of low stringency conditions, medium stringency conditions or high stringency conditions, and preferably high stringency conditions. Illustratively, "low stringency conditions" can be conditions of 30 ℃, 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 52% formamide; "moderate stringent conditions" can be 40 degrees C, 5x SSC, 5x Denhardt liquid, 0.5% SDS, 52% formamide conditions; the "high stringency conditions" may be 50 ℃ in 5 XSSC, 5 XDenhardt's solution, 0.5% SDS, 52% formamide. It will be appreciated by those skilled in the art that higher temperatures will result in polynucleotides with higher homology. In addition, one skilled in the art can select the result of combining multiple factors, such as temperature, probe concentration, probe length, ionic strength, time, salt concentration, etc., that affect the stringency of hybridization to achieve the corresponding stringency.

In addition, the polynucleotide hybridizable to the polynucleotide shown in SEQ ID NO:2 or a polynucleotide having a nucleotide sequence consisting of SEQ ID NO 2-SEQ ID NO 10-SEQ ID NO 4 in this order, 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.

The identity of nucleotide sequences can be determined using the algorithm rules BLAST of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc. Natl. Acad. Sci. USA 90:5873,1993). The programs BLASTN, BLASTX based on the rules of the BLAST algorithm have been developed (AltschulSF, et: J Mol Biol 215:403,1990). When BLASTN is used to analyze a nucleotide sequence, the parameters are, for example, score (100), Wordlength (12); when BLASTX is used to analyze an amino acid sequence, the parameters are set to score 50 and Wordlength 3; when BLAST and Gapped BLAST programs are used, default parameter values can be set for the system using each program.

Polynucleotides may be prepared, manipulated and/or expressed using any of a variety of well-established techniques known and available in the art. To express the desired polypeptide or protein, the nucleotide sequence encoding the polypeptide may be inserted into a suitable vector. Examples of vectors are plasmids, autonomously replicating sequences and transposable elements. Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes (e.g., Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), bacteriophages (e.g., lambda phage or M13 phage), and animal viruses. Examples of animal viral vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papovaviruses (e.g., SV 40). Examples of expression vectors are the pClneo vector (Promega) for expression in mammalian cells; lenti4/V5-DESTTM, pLenti6/V5-DESTTM and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In particular embodiments, the coding sequence of the chimeric proteins disclosed herein can be ligated into such expression vectors for expression of the chimeric proteins in mammalian cells. "control elements" or "regulatory sequences" present in an expression vector are the untranslated regions of the vector (e.g., origins of replication, promoters, enhancers, translational initiation signal (SD sequence or Kozak sequence) introns, polyadenylation sequences, 5 'and 3' untranslated regions) that interact with host cell proteins for transcription and translation. The strength and specificity of such elements or sequences may vary. Depending on the vector system and host used, any number of suitable transcription and translation elements or sequences may be used, including broadly expressing promoters and inducible promoters.

Carrier

In particular embodiments, a cell (e.g., an immune effector cell, such as a T cell) is 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 comprising a humanized anti-CD 19 antibody or antigen-binding fragment that binds a CD19 polypeptide, the humanized anti-CD 19 antibody or antigen-binding fragment having a transmembrane domain and an intracellular signaling domain, such that these transduced cells can elicit a CAR-mediated cytotoxic response.

Retroviruses are a common tool for gene delivery. In particular embodiments, the retrovirus is used to deliver a polynucleotide encoding a Chimeric Antigen Receptor (CAR) to a cell. As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into linear double-stranded DNA copies, followed by covalent integration of 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 directs the expression of RNA molecules encoding structural proteins and enzymes required for the production of new viral particles.

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), murine 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.

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

(CAEV); equine Infectious Anemia Virus (EIAV); feline Immunodeficiency Virus (FIV); bovine Immunodeficiency Virus (BIV); and Simian Immunodeficiency Virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred. In particular embodiments, the lentivirus is used to deliver a polynucleotide comprising the CAR to a cell.

The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is typically linked to, e.g., inserted into, a vector nucleic acid molecule. The vector may comprise sequences that direct autonomous replication in the cell, or may comprise sequences sufficient to allow integration into the DNA of the host cell. 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.

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

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

Thus, in a preferred embodiment, the invention relates to a method of transfecting a cell with an expression vector encoding a CAR. For example, in some embodiments, the vector comprises additional sequences, such as sequences that facilitate expression of the CAR, e.g., a promoter, an enhancer, a poly-a signal, and/or one or more introns. In a preferred embodiment, the CAR coding sequence is flanked by transposon sequences such that a transposase is present to allow integration of the coding sequence into the genome of the transfected cell.

In some embodiments, the genetically transformed cell is further transfected with a transposase that promotes integration of the CAR-encoding sequence into the genome of the transfected cell. In some embodiments, the transposase is provided as a DNA expression vector. However, in preferred embodiments, the transposase is provided as an expressible RNA or protein such that long term expression of the transposase does not occur in the transgenic cell. 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 may be used in accordance with 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 can include electroporating cells having mRNA encoding SB 10, SB 11, or SB 100X transposase.

Sequence variants:

also included within the scope of the invention are sequence variants (e.g., those defined by percent sequence identity) of the claimed nucleic acids, proteins, antibodies, antibody fragments, and/or CARs that maintain similar binding properties of the invention. These variants show alternative sequences but retain essentially the same binding properties such as target specificity, since the particular sequence provided is known to be a functional analog or functional analog. Sequence identity refers to the percentage of identical nucleotides or amino acids when aligned.

As used herein, the recitation of "sequence identity" refers to the degree to which nucleotide-based or amino acid-based sequences are identical over a comparison window. Thus, the "percentage of sequence identity" can be calculated by: comparing the two optimally aligned sequences over a comparison window, determining the number of positions at which the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, gin, Cys, and Met) is present on the two sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield 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.

One of ordinary skill in the art will appreciate that due to the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide or protein as described herein. Some of these polynucleotides have minimal homology or sequence identity to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Deletions, substitutions and other changes in the sequence that fall within the described sequence identity are also encompassed by the invention.

Modifications of the protein sequence which may occur by substitution are also included within the scope of the present invention. Substitutions, as defined herein, are modifications made to 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 a different amino acid sequence than the primary protein. Substitutions may be made which preferably do not significantly alter the function of the protein. As with the addition, the replacement may be natural or artificial. It is well known in the art that amino acid substitutions can be made without significantly altering the function of the protein. This is particularly true when the modification involves a "conservative" amino acid substitution of one amino acid for another with similar properties. Such "conservative" amino acids may be natural or synthetic amino acids that may be substituted for size, charge, polarity, and conformation without significantly affecting the structure and function of the protein. In general, many amino acids can be substituted for conservative amino acids without adversely affecting the function of the protein.

In general, the nonpolar amino acids Gly, Ala, Val, Ile and Leu; the non-polar aromatic amino acids Phe, Trp and Tyr; 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 a conserved group of amino acids. 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.

Substitutional variants remove at least one amino acid residue from the antibody molecule and insert a different residue in its place. For the generation of substitutional mutagenesis, the positions of most interest include the hypervariable regions, but FR alterations are also contemplated. If such substitutions result in a change in biological activity, a greater number of changes can be introduced and the product screened.

Compositions and formulations

The compositions of the invention may comprise one or more polypeptides, polynucleotides, vectors comprising the polynucleotides, genetically modified immune effector cells, and the like, 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 for administration to a cell or animal, either alone or in combination with one or more other therapeutic modalities. It is also understood that the compositions 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 various other pharmaceutically active agents, if desired. There is virtually no limitation on the 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.

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, commensurate with a reasonable benefit/risk ratio.

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, flavoring agent, 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 chinese 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 carboxymethyl cellulose, ethyl cellulose and cellulose acetate; gum tragacanth; malt; gelatin; talc; cocoa butter, wax, animal and vegetable oil, paraffin, organic silicon, bentonite, silicic acid and 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; buffering agents 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.

In particular embodiments, the compositions of the invention comprise an amount of an immune effector cell of the invention that expresses a CAR. As used herein, the term "amount" refers to an "effective amount" of genetically modified therapeutic cells (e.g., T cells) to achieve a beneficial or desired prophylactic or therapeutic result, including a clinical result.

By "prophylactically effective amount" is meant an amount of genetically modified therapeutic cells effective to achieve the desired prophylactic result. Typically, but not necessarily, the prophylactically effective amount is less than the therapeutically effective amount because the prophylactic dose is used in the subject prior to or at an early stage of the disease. The term preventing does not necessarily mean completely prohibiting or preventing a particular medical condition. The term preventing also refers to reducing the risk of the occurrence of a medical condition or worsening of a symptom.

The "therapeutically effective amount" of the genetically modified therapeutic cells may vary depending on various factors, such as the disease state, age, sex, and weight of the individual, and the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also an amount such that the therapeutically beneficial effect outweighs any toxic or detrimental effects of the virus or transduced therapeutic cells. The term "therapeutically effective amount" includes an amount effective to "treat" a subject (e.g., a patient). When indicating a therapeutic amount, the precise 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, extent of infection or metastasis, and patient (subject) condition. It may be generally stated that a pharmaceutical composition comprising T cells as described herein may be in the range of 10 ² To 10 ¹⁰ Individual cells/kg body weight, preferably 10 ⁵ To 10 ⁶ Agent for per cell/kg body weightAmounts (including all integer values within these ranges) are administered. The number of cells will depend on the end use of the composition and the cell type contained therein. For the uses provided herein, the cells are typically 1L or less in volume and may be 500mL or less, even 250mL or 100mL or less. Thus, the desired cell density is typically greater than 10 ⁶ Individual cell/ml, usually greater than 10 ⁷ Individual cell/ml, usually 10 ⁸ Individual cells/ml or higher. Clinically relevant numbers of immune cells can be apportioned 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 And (4) cells. In some embodiments of the invention, a lower number of cells may be administered, particularly because all infused cells will be redirected to a particular target antigen. The CAR-expressing cell composition can be administered multiple times at doses within these ranges. For patients receiving treatment, the cells may be allogeneic, syngeneic, allogeneic or autologous.

In general, compositions comprising cells activated and expanded as described herein are useful for treating and preventing diseases that occur in immunocompromised individuals. In particular, compositions comprising the CAR-modified T cells of the invention are useful for treating B cell malignancies. The CAR-modified T cells of the invention can be administered alone, or as a pharmaceutical composition in combination with a carrier, diluent, excipient, and/or with other components or other cytokines or groups of cells. In a particular embodiment, the pharmaceutical composition of the invention comprises an amount of genetically modified T cells, and one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

A pharmaceutical composition of the invention comprising a population of immune effector cells (e.g., T cells) that express a CAR can comprise: buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids (e.g., glycine); an antioxidant; chelating agents (e.g., EDTA) or glutathione; adjuvants, such as aluminum hydroxide; and a preservative. The compositions of the present invention are preferably formulated for parenteral administration, for example intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.

Liquid pharmaceutical compositions, whether in solution, suspension, or other similar form, 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 mono-or diglycerides which may be used as a solvent or suspending medium), polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; 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 the osmotic pressure, such as sodium chloride or glucose. The parenteral formulations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. The injectable pharmaceutical composition is preferably sterile.

In particular embodiments, the compositions of the invention comprise an effective amount of an immune effector cell expressing a CAR alone, or in combination with one or more therapeutic agents. Thus, the immune effector cell composition expressing the CAR can be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, photodynamic therapy, and the like. The compositions may also be administered in combination with an antibiotic. Such therapeutic agents are accepted in the art as standard treatments for particular disease states (e.g., particular cancers) as described herein. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies or other active and ancillary agents.

Method of treatment

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

In another aspect of the invention there is provided a CAR and CAR-T or NK according to the invention as described herein for use in the treatment of a disease, wherein the method comprises administering to the patient a therapeutically effective amount of a CAR or CAR-T or NK as described herein, exemplarily the disease is selected from: acute myelogenous leukemia, chronic myelogenous 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-associated cachexia, fatigue, weakness, cachexia and hypercalcemia of secondary tumor syndrome.

As used herein, the terms "individual" and "subject" are generally used interchangeably and refer to any animal exhibiting symptoms of a disease, disorder, or condition that can be treated with the gene therapy vectors, cell-based therapeutics, and methods disclosed elsewhere herein. In preferred embodiments, the subject includes any animal exhibiting symptoms of a disease, disorder or condition of the hematopoietic system that can be treated with the gene therapy vectors, cell-based therapeutics and methods disclosed elsewhere herein. Typical subjects include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, and livestock or pets (e.g., cats or dogs). Including non-human primates, and preferably including human patients.

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 minimal reduction in one or more measurable markers of the disease or condition being treated. Treatment may optionally involve alleviation or amelioration of symptoms of the disease or disorder, or delay in progression of the disease or disorder. "treating" does not necessarily mean completely eradicating or curing the disease or disorder or symptoms associated therewith.

As used herein, "preventing" refers to a method of preventing, inhibiting, or reducing the likelihood of occurrence or recurrence of a disease or disorder. It also refers to delaying the onset or recurrence of a disease or disorder, or delaying the onset or recurrence of symptoms of a disease or disorder. As used herein, "preventing" and similar words also include reducing the strength, impact, symptoms, and/or burden of a disease or disorder prior to its onset or recurrence.

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 a genetically modified immune effector cell of the invention. The number and frequency of administration will be determined by factors such as the condition of the patient and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

Administration of the compositions of the invention may 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" as used herein refers to modes of administration other than enteral and topical administration, typically by injection, including, but not limited to, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcontracting, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the compositions of the invention are administered to a subject by direct injection into a tumor, lymph node or site of infection.

English names appearing herein are case-insensitive; CD19 CAR-T represents a CAR-T cell capable of expressing a CD19 specific binding domain; CD8 ^TM Representing the transmembrane domain.

The invention has been described in more detail for T cells (including CAR-T cells) relative to NK cells (including CAR-NK), but in general these descriptions for T cells apply to NK cells as well. Herein, the description of the occurrence of "T cells" or synonyms thereof in the context of a description of T cells is incorporated herein by the replacement of "NK cells" or synonyms thereof. This is necessary from the standpoint of brevity of description. If it is determined according to the prior art that such a replacement is not appropriate in some cases, the replacement is not performed.

The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding 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 claimed.

Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

For a more clear illustration of the invention, reference is now made in detail to the following examples, which are intended to be purely exemplary of the invention and are not to be interpreted as limiting the application.

Example 1: preparation of Lentiviral vectors

1) Gene synthesis of lentivirus expression plasmid; 2) gene synthesis Kozal-CD8a signal-CD19 SCFV-CD8aTM-4-1BB-CD3zeta fusion gene sequence (the amino acid sequence is shown as SEQ ID NO:1, the DNA sequence is shown as SEQ ID NO:2, and the structure schematic diagram is shown as FIG. 1); 3) a gene-synthesized nucleotide sequence, the nucleotide sequence being: the self-splicing protein P2A gene (the amino acid sequence is shown as SEQ ID NO:9, the DNA sequence is shown as SEQ ID NO: 10) -IL12ODD fusion gene sequence (the amino acid sequence is shown as SEQ ID NO:3, and the DNA sequence is shown as SEQ ID NO: 4); 4) inserting the gene synthetic fragments into a lentivirus expression plasmid by using a multi-fragment homologous recombination method, transforming a recombinant vector into an escherichia coli strain Stbl3, and obtaining a target recombinant plasmid through kanamycin screening and monoclonal sequencing; then expanding and culturing host bacteria containing the target recombinant plasmid, and obtaining sterile endotoxin-free plasmid by using an endotoxin removal kit, namely a plasmid vector containing a CD19CARIL12ODD gene segment, wherein the structure of the plasmid vector is shown in figure 1; 5) meanwhile, the lentiviral vector packaging auxiliary plasmids pMDLgpRRE, pRRE and pCMV-VSV-G are respectively transformed into an escherichia coli strain Stbl3, ampicillin screening is carried out, and a sterile endotoxin-free plasmid is obtained by using an endotoxin removal kit.

Example 2: preparation of CAR19IL12ODD lentivirus

1) Inoculation 5X10 ⁶ In a 10cm dish containing 10ml of DMEM + 10% FBS medium; 2) after 24 hours, 10ml of fresh DMEM + 10% FBS medium was replaced, and the viral plasmid (CAR19IL12 ODD: 25 ug, pMDLgpRRE 7.5ug, pRRE 6ug, pCMV-VSV-G6 ug) and 250ul serum-free DMEM to prepare a plasmid suspension; 45ul of 1ug/ml transfection reagent (PEI) was mixed with 250ul of serum-free DMEM to prepare a PEI suspension; standing at room temperature for 5 minutes, mixing 250ul of plasmid suspension with PEI suspension, adding the mixture into 293T cells after 30 minutes, and gently mixing; 3) after 8 hours, 20ml of fresh DMEM + 10% FBS medium was replaced; 4) collecting culture medium supernatant after 60 hr, centrifuging to remove cell debris, and filtering the supernatant with 0.45 μm filter; then, the virus supernatant was concentrated 10 times by centrifugation at 1500g for 30 minutes at 4 ℃ using an Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-30 membrane (Merckmillipore, Cat. UFC903096) to obtain a virus solution, the virus was titrated by a gradient dilution method, and the virus was stored at-80 ℃ after being split-packed.

Example 3: preparation and functional validation of CAR19IL12ODD CAR-T cells

1) Separation of peripheral blood lymphocytes: collecting peripheral blood, adding peripheral blood lymphocyte separation solution with the same amount as that of the blood sample, centrifuging for 20min at 800g, collecting leucocyte layer lymphocytes, and washing twice with PBS; 2) isolation of CD3T cells in peripheral blood lymphocytes: resuspending 1X10 ⁷ Lymphocytes in 5ml of 5% FBS-containing X-VIVO ^TM 15 culture based on antibodies against CD3 and CD8 in 6-well plates to detect the proportion of CD3T cells in peripheral lymphocytes, using

Human T-Expander CD3/CD28(Gibco, cat # 11141D) isolated and stimulated CD3T cells; 3) 2X10 ⁵ CD3T thinCell and 5X10 ⁵ Mixing IU virus in 24-well plate, centrifuging at 30 deg.C and 1500g for 90min, after 4 hr, adding 1ml of X-VIVO containing 5% FBS ^TM 15 medium culture medium, gently pumping and mixing, putting back into the incubator for culture, and after 24 hours, replacing 1ml of fresh 5% FBS X-VIVO ^TM 15 culture medium;

4) after 48 hours, the T cell concentration was adjusted to 5X10 ⁵ Ml, change fresh medium completely; 5) after 72 hours, the T cell concentration was adjusted to 5X10 ⁵ WB validation CAR19IL12ODD protein expression; detecting and confirming CAR expression in CAR-T cells with flow cytometry; CAR19IL12ODD CAR-T cells were cultured under normoxic and hypoxic conditions, respectively, and IL12 secretion concentrations in cell culture supernatants were measured using ELESA (fig. 2), which showed that CAR19IL12ODD detected low dose IL12 secretion expression under normoxic conditions (17%) and secreted IL12 secretion up to 26pg/ml under anaerobic conditions (1%).

The above results show that the CAR19IL12 ODDs of the invention are all expressed in CD3T cells, and that the ODD domains are effective in controlling the secretory expression of IL 12.

Example 4: LDH experiment

Detection of the in vitro killing Effect of CAR19IL12ODD CAR-T cells on DLBCL cell line Ly3 (CytoTox)

Non-radioactive cytotoxin Assay, Promega, cat # G1780): 1) taking CAR19IL12ODD CAR-T effector cells and target cells Ly3, washing twice with PBS, and counting by trypan blue staining; 2) adding 1x10 to each hole in U-shaped 96-hole plate ⁴ In 1640 medium with 50ul 5% FBS; 3) setting a gradient effective target ratio experimental group: according to the effective target ratio of 10: 1. 5: 1. 2.5: 1. 1.25: 1. 0.6: 1. 0.3: 1. And 0.15: 1 adding a corresponding number of effector cells, the volume of effector cells added per well being 50 ul; 4) The following control groups were set: blank group (100ul 1640 medium of 5% FBS), effector cell spontaneous group (50ul 1640 medium of effector cells +50ul 1640 medium of 5% FBS), and target cell spontaneous group (50ul 1640 medium of target cells +50ul 1640 medium of 5% FBS), target cell maximum release group (50ul target cells)+50ul of 1640 medium with 5% FBS), 5% CO at 37 ℃ ₂ Culturing in an incubator; 5) after 4h, the plate was removed and centrifuged at 250g for 5min (1 h before this step, 10ul of 10 × lysate was added to the maximal release group of target cells); 6) 50ul of supernatant was transferred to a new flat bottom 96 well plate, 50ul of Cytoto x was added to each well

Coating a substrate and tin foil paper, and reacting for 30min at room temperature; 7) adding 50ul of stop solution into each well to terminate the reaction, and measuring the light absorption value at 492nm of an enzyme-labeling instrument; 8) calculating killing efficiency, which is (experimental group-effector cell spontaneous group-target cell spontaneous group)/(target cell maximum release group-target cell spontaneous group) X100; experimental results as shown in fig. 3A, CAR-T of the present invention can have significant therapeutic or inhibitory effect on Diffuse Large B Cell Lymphoma (DLBCL).

Example 5: animal experiment for detecting in vivo killing effect and function verification of CAR19IL12ODD CAR-T cells on DLBCL cell line Ly3

Selecting 4-6 week-old severe immunodeficiency Syria hamster, inoculating 1 × 10 subcutaneous flank of forelimb after one week of acclimatization ⁷ Ly3 only diffused large B cell lymphoma tumor cells. The tumor size was observed and measured, and after 2-4 weeks, the tumor grew to 250mm ³ In this case, Syrian hamsters were randomly divided into 5 groups, and CAR-T cells cultured in vitro for 7 to 8 days were intraperitoneally injected at an administration rate of 1 × 10 ⁷ A/only. IL12 concentrations were measured 3, 6, 9, 12 days after CAR19IL12ODD CAR-T cells reinfused, i.e. 17, 20, 23, 26 days after tumor inoculation and CAR-T cell flow assays showed that IL12 significantly increased CAR-T cell survival in vivo (fig. 4A and 4B); continuously observing and measuring the size of the tumor, when the tumor of the control group grows to the ethical limit, killing the experimental animal according to the ethical requirement, taking the tumor tissue for immunohistochemical staining, calculating the change of the tumor size by using the following formula, and drawing a tumor growth curve, wherein the tumor size is (1/2) the square of the long diameter and the short diameter; as can be seen from FIG. 3B, CAR-T prepared in the examples of this application has significant inhibitory effect on human diffuse large B-cell lymphoma (DLBCL); immunohistochemistry results (not shown) showed that IL12 significantly promoted tumor infiltration of CAR-T,expression of IL12 in CAR-T cells plays a role in breaking the tumor immune microenvironment of DLBLC. The above results indicate that the CAR19IL12ODD CAR-T cells prepared in the examples of the present application have significant therapeutic or inhibitory effect on human Diffuse Large B Cell Lymphoma (DLBCL) by breaking immune microenvironment.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents and the like included within the spirit and scope of the present invention.

Sequence listing

<110> Zhengzhou university

<120> novel chimeric antigen receptor and immune cell comprising 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 tcaaatccag actgaccatc 660

atcaaggaca actccaagag ccaagttttc ttaaaaatga acagtctgca aactgatgac 720

acagccattt actactgtgc caaacattat tactacggtg gtagctatgc tatggactac 780

tggggtcaag gaacctcagt caccgtctcc tcagcggccg caagatcttt cagccacttc 840

gtgccggtct tcctgccagc gaagcccacc acgacgccag cgccgcgacc accaacaccg 900

gcgcccacca tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg 960

gggggcgcag tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc 1020

ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaaccac 1080

aggaaccata tgggaggaac tagtaaacgg ggcagaaaga aactcctgta tatattcaaa 1140

caaccattta tgagaccagt acaaactact caagaggaag atggctgtag ctgccgattt 1200

ccagaagaag aagaaggagg atgtgaactg agagtgaagt tcagcaggag 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> IL12ODD fusion protein nucleotide sequence

<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

gaaaactaca 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

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 atgcttccgt cgaccaacaa 1620

acagaatgtg tccttaaacc ggttgaatct tcagatatga aaatgactca gctattcacc 1680

aaagttgaat cagaagatac aagtagcctc tttgacaaac ttaagaagga acctgatgct 1740

ttaactttgc tggccccagc cgctggagac acaatcatat ctttagattt tggcagcaac 1800

gacacagaaa ctgatgacca gcaacttgag gaagtaccat tatataatga tgtaatgctc 1860

ccctcaccca acgaaaaatt acagaatata aatttggcaa tgtctccatt acccaccgct 1920

gaaacgccaa agccacttcg aagtagtgct gaccctgcac tcaatcaaga agttgcatta 1980

aaattagaac caaatccaga gtcactggaa ctttctttta ccatgcccca gattcaggat 2040

cagacaccta gtccttccga tggaagcact agacaaagtt cacctgagcc taatagtccc 2100

agtgaatatt gtttttatgt ggatagtgat atggtcaatg aattcaagtt ggaattggta 2160

gaaaaacttt ttgctgaaga cacagaagca aagaacccat tttctactca ggacacagat 2220

ttagacttgg agatgttagc tccctatatc ccaatggatg atgacttcca gttacgttcc 2280

ttcgatcagt tgtcaccatt agaaagcagt tccgcaagcc ctgaaagcgc aagtcctcaa 2340

agcacagtta cagtattcca gcagactcaa atacaagaac ctactgctaa tgccaccact 2400

accactgcca ccactgatga attaaaaaca gtgacaaaag accgtatgga agacattaaa 2460

atattgattg catctccatc tcctacccac atacataaag aaactactag tgccacatca 2520

tca 2523

<210> 5

<211> 536

<212> PRT

<213> Artificial sequence

<220>

<223> IL12 protein amino acid sequence

<400> 5

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

530 535

<210> 6

<211> 1608

<212> DNA

<213> Artificial sequence

<220>

<223> IL12 protein nucleotide sequence

<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

agaggggaca acaaggagta tgagtactca gtggagtgcc aggaggacag tgcctgccca 600

gctgctgagg agagtctgcc cattgaggtc atggtggatg ccgttcacaa gctcaagtat 660

gaaaactaca 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

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> ODD protein amino acid sequence

<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 ctttaacttt gctggcccca gccgctggag acacaatcat atctttagat 180

tttggcagca acgacacaga aactgatgac cagcaacttg aggaagtacc attatataat 240

gatgtaatgc tcccctcacc caacgaaaaa ttacagaata taaatttggc aatgtctcca 300

ttacccaccg ctgaaacgcc aaagccactt cgaagtagtg ctgaccctgc actcaatcaa 360

gaagttgcat taaaattaga accaaatcca gagtcactgg aactttcttt taccatgccc 420

cagattcagg atcagacacc tagtccttcc gatggaagca ctagacaaag ttcacctgag 480

cctaatagtc ccagtgaata ttgtttttat gtggatagtg atatggtcaa tgaattcaag 540

ttggaattgg tagaaaaact ttttgctgaa gacacagaag caaagaaccc attttctact 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 gaattaaaaa cagtgacaaa agaccgtatg 840

gaagacatta aaatattgat tgcatctcca tctcctaccc acatacataa agaaactact 900

agtgccacat catca 915

<210> 9

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> self-cleaving protein P2A amino acid sequence

<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> self-cleaving protein P2A nucleotide sequence

<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 further comprises a fusion protein of interleukin 12(IL12) and an oxygen-dependent degradation region (ODD) linked to and co-expressed with the intracellular domain, optionally, the intracellular domain is linked to the fusion protein through a self-cleaving protein (e.g., selected from the group consisting of T2A, P2A, E2A, F2A, or a combination thereof).

2. The CAR of claim 1, wherein the transmembrane domain is derived from a transmembrane domain selected from one or more of the group consisting of the alpha, beta or zeta chain of a T cell receptor, CD3 epsilon, CD4, CD5, CD8, CD8 alpha, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD80, CD86, CD134, CD137, CD152, CD154 and ICOS.

3. The CAR of claim 1, the intracellular signaling domain comprising a costimulatory signaling domain and being from: one or more of CD2, CD3 ζ, CD3 γ, CD3 δ, CD3 ε, 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.

4. The CAR of any one of claims 1-3, further comprising a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

5. The CAR of claim 4, wherein the hinge domain is derived from CD8 a.

6. As claimed in claim 1The CAR of (a), wherein the extracellular antigen-binding domain is a molecule, e.g., an antibody (Ig, Ig R, Fab fragment, Fab 'fragment, F' (F) fragment, CD 6757, CD123, CD30, CD33, CD38, CD44, CD138, CD276, EGFR, BCMA, HER1, HER2, HER3, EpCAM, mesothelin (mesothelin), FAP (fibroblast activation protein), Glypain-3, CEA, PSMA, IL13R alpha 2, CD171, GD2, CEACAM6, CLDN4, CLDN18.2, NKGD2, LAMC2, CA9, CST1, EPPK1, and NAANO 1), which binds to a tumor cell surface antigen (e.g., CD19, CD20, CD22, CD 3970, CD2, CD 387, CD3 ', and NAANO 1)' ₂ Fragment, F (ab)' ₃ Fragment, Fv, scFv, bis-scFv, (scFv) ₂ Minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein and single domain antibody (sdAb, nanobody), bispecific antibody or trispecific antibody).

7. The CAR of claim 1, wherein the IL12 has an 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 sequence of SEQ ID NO:1 and SEQ ID NO 3.

8. The fusion protein of claim 1, further comprising a tag sequence (e.g., Poly-His,

Hemagglutenin, c-Myc, GST, Flag-tag, etc.) or IgG1-Fc protein sequence.

9. A nucleotide sequence encoding the CAR of any one of claims 1-8, preferably the nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID No. 2, or comprises the nucleotide sequences set forth in SEQ ID No. 2-SEQ ID No. 10-SEQ ID No. 4 linked sequentially.

10. An isolated CAR-T cell or CAR-NK cell or monocyte macrophage, wherein said CAR-T cell or CAR-NK cell or monocyte macrophage is capable of expressing a CAR of any one of claims 1-8; the CAR-T cell or CAR-NK cell or monocyte macrophage comprising a polynucleotide of claim 9.

11. A vector comprising the polynucleotide of claim 9.

12. The vector of claim 11, wherein the vector is an expression vector, such as a viral vector, preferably a retroviral vector or an oncolytic virus, such as a lentiviral vector, preferably selected from the group consisting of human immunodeficiency virus 1(HIV-1), human immunodeficiency virus 2(HIV-2), visna-midie virus (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.

13. A pharmaceutical composition comprising the chimeric antigen receptor of any one of claims 1-8, comprising the cell of claim 10, and optionally, a pharmaceutically acceptable carrier.

14. A method for preparing the cell of claim 10, comprising introducing the vector of claim 11 or 12 into a T lymphocyte or a natural killer cell or a monocyte macrophage.

15. Use of a cell according to any one of claims 1-8 in the manufacture of a medicament for the treatment and/or prevention of cancer, illustratively myeloma and leukemia.