CN114949190B - Application of antigen presenting cells and CAR-T cell combination in anti-tumor - Google Patents

Application of antigen presenting cells and CAR-T cell combination in anti-tumor Download PDF

Info

Publication number
CN114949190B
CN114949190B CN202210475661.4A CN202210475661A CN114949190B CN 114949190 B CN114949190 B CN 114949190B CN 202210475661 A CN202210475661 A CN 202210475661A CN 114949190 B CN114949190 B CN 114949190B
Authority
CN
China
Prior art keywords
ser
gly
leu
thr
val
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210475661.4A
Other languages
Chinese (zh)
Other versions
CN114949190A (en
Inventor
孙敏敏
郝瑞栋
李彦涛
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou Yimufeng Biotechnology Co ltd
Original Assignee
Suzhou Yimufeng Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Suzhou Yimufeng Biotechnology Co ltd filed Critical Suzhou Yimufeng Biotechnology Co ltd
Priority to CN202210475661.4A priority Critical patent/CN114949190B/en
Priority to CN202410324943.3A priority patent/CN118178626A/en
Publication of CN114949190A publication Critical patent/CN114949190A/en
Application granted granted Critical
Publication of CN114949190B publication Critical patent/CN114949190B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/195—Chemokines, e.g. RANTES
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/208—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0635—B lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Toxicology (AREA)
  • Mycology (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The invention relates to the technical field of biological medicines, in particular to an application of antigen presenting cells and CAR-T cells in combination in resisting tumors. More particularly, the invention relates to the use of an antigen presenting cell expressing a tumor antigen and a CAR-T cell whose chimeric antigen receptor extracellular domain targets recognition of the tumor antigen in the combined preparation of a medicament for the treatment of tumors.

Description

Application of antigen presenting cells and CAR-T cell combination in anti-tumor
Technical Field
The invention relates to the technical field of biological medicines, in particular to an application of antigen presenting cells and CAR-T cells in combination in resisting tumors.
Background
The development of immunotherapy in recent years has led to a profound change in the field of tumor treatment, in particular immune checkpoint therapies typified by inhibitors of the PD1/PDL1 pathway and adoptive cell therapies typified by CAR-T. Adoptive cell therapies include TIL, NK, TCR-T, CAR-T and the like. Wherein CD 19-targeted CAR-T therapy achieves excellent clinical efficacy in B-cell tumors, and two CAR-T products were FDA approved for treatment of B-cell leukemia or lymphoma in 2017.
Although CAR-T has made a major breakthrough in hematological neoplasms, its killing effect still needs to be further improved. In addition, the development of the CAR-T in the field of solid tumors is limited, one important reason is that the CAR-T cells can not be effectively and continuously amplified after entering the body, so that the continuous killing effect on tumor cells can not be maintained, and the treatment effect of the CAR-T on tumors is poor and the subsequent high recurrence rate is caused.
Disclosure of Invention
A first object of the present invention is to provide an antigen presenting cell expressing a tumor antigen and a CAR-T cell whose chimeric antigen receptor extracellular domain targets and recognizes the tumor antigen for use in the combined preparation of a medicament for treating tumor.
It is a second object of the present invention to provide an antigen presenting cell that expresses a tumor antigen and over-expresses a cytokine that facilitates activation of CAR-T cells.
A third object of the present invention is to provide a cell pharmaceutical preparation comprising a) or b):
a) Antigen presenting cells and CAR-T cells, individually packaged or mixed together;
Wherein the antigen presenting cell and the CAR-T cell are as defined above;
b) Antigen presenting cells as described above.
The antigen presenting cells and the CAR-T cells are combined, so that the proliferation of the CAR-T cells can be obviously promoted, the effect of killing tumors by the CAR-T cells can be enhanced, and the infiltration level of the CAR-T cells in solid tumors can be increased.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic representation of an adenovirus-based expression vector expression cassette;
FIG. 2 is a phenotypic assay after 1, 3, 5 days of DC induced maturation
FIG. 3 shows the detection of expression of CLDN18.2 on DC;
FIG. 4 is a detection of the positive rate of CAR molecules after construction of clDN18.2 CAR-T;
FIG. 5 is a graph showing detection of killing of CLDN18.2 CAR-T cells against different cell lines;
FIG. 6 is a graph of DC-182, DC-182.IL12, DC-182.CXCL9, DC-182.IL12.CXCL9 promoting proliferation of CLDN18.2 CAR-T in vitro;
FIG. 7 is a ratio of DC-182, DC-182.IL12, DC-182.CXCL9, DC-182.IL12.CXCL9 to increase the central memory phenotype of CLDN18.2 CAR-T in vitro;
FIG. 8 is a graph of the ability of DC-182, DC-182.IL12, DC-182.CXCL9, DC-182.IL12.CXCL9 to increase the killing of CLDN18.2 CAR-T by target cells NUGC4 in vitro;
FIG. 9 shows that DC-182, DC-182.IL12, DC-182.CXCL9, DC-182.IL12.CXCL9 increase levels of cytokine (IFN-. Gamma., TNF-. Alpha., IL 2) release when CLDN18.2 CAR-T is co-incubated with target cell NUGC4 in vitro;
FIG. 10 is a DC-182.IL12.CXCL9 promoting CAR-T cell tumor clearance;
FIG. 11 shows the proliferation of CAR-T in mouse peripheral blood;
FIG. 12 is immunofluorescent staining of CAR-T in mouse tumor tissue.
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Unless otherwise defined, all terms (including technical and scientific terms) used to describe the invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from the group consisting of "and/or", "and/or", it should be understood that, in the present application, the technical solutions include technical solutions that all use "logical and" connection, and also include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical schemes of all "logical or" connections), also include any and all combinations of A, B, C, D, i.e., the combinations of any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical schemes of all "logical and" connections).
The terms "comprising," "including," and "comprising," as used herein, are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.
The recitation of numerical ranges by endpoints of the present invention includes all numbers and fractions subsumed within that range, as well as the recited endpoint.
Concentration values are referred to in this invention, the meaning of which includes fluctuations within a certain range. For example, it may fluctuate within a corresponding accuracy range. For example, 2%, may allow fluctuations within + -0.1%. For values that are larger or do not require finer control, it is also permissible for the meaning to include larger fluctuations. For example, 100mM, fluctuations in the range of.+ -. 1%,.+ -. 2%,.+ -. 5%, etc. can be tolerated. Molecular weight is referred to, allowing its meaning to include fluctuations of + -10%.
In the present invention, the terms "plurality", and the like refer to, unless otherwise specified, 2 or more in number.
In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
In the present invention, "preferred", "better", "preferred" are merely embodiments or examples which are better described, and it should be understood that they do not limit the scope of the present invention. In the present invention, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Unless otherwise indicated to the contrary by the intent and/or technical aspects of the present application, all references to which this application pertains are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in the present application, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. In the case of the cited documents, examples and preferred modes of the cited relevant technical features are also incorporated into the present application by reference, but are not limited to being able to implement the present application. It should be understood that when a reference is made to the description of the application in conflict with the description, the application is modified in light of or adaptive to the description of the application.
The invention relates to an antigen presenting cell and application of a CAR-T cell in combined preparation of a medicament for treating tumors, wherein the antigen presenting cell expresses a tumor antigen, and the chimeric antigen receptor extracellular domain of the CAR-T cell targets and recognizes the tumor antigen.
The invention combines antigen presenting cells and CAR-T cells to treat tumors, can obviously increase the infiltration level of the CAR-T cells in the tumors, promote the proliferation of the CAR-T cells and enhance the effect of killing the tumors by the CAR-T cells.
In some embodiments, the tumor antigen is over-expressed from a foreign gene in the antigen presenting cell.
In the present invention, "overexpression of exogenous gene" refers to a process in which a target gene is artificially introduced into a cell and expressed in a large amount. The usual method for introducing the target gene into the cell is to construct the target gene into a tool carrier, and the target gene is introduced into the cell to realize a large number of transcription and translation of the target gene, so that the overexpression of a gene product is realized; the target gene may be introduced into the cell by electrotransport or liposome transduction. The tool vectors generally include prokaryotic expression vectors, normal eukaryotic expression vectors, lentiviral vectors, adeno-associated viral vectors, adenoviral vectors, retroviral vectors, and the like.
"Overexpression" includes "overexpression of an exogenous gene" and "overexpression of an endogenous gene", which means that the cell itself can express the target gene, and the expression level can be increased by such means as CRISPR-dCAS9 transcriptional activation.
In some embodiments, the drug does not comprise an agent for nucleic acid transfection.
The term "tumor antigen" is a novel antigen that is highly expressed by tumor cells or that is present only in certain tumor cells but not in normal cells, and is usually produced by genetic variation (e.g., gene point mutation, gene deletion, gene translocation, gene fusion) of tumor cells, and the like, and is also called a tumor neo-antigen (neo-antigen).
CAR-T cells, i.e., T cells expressing chimeric antigen receptors, may be of the subclasses well known in the art, such as one or more of helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, MAIT cells, NKT cells, and γδ T cells.
In the present invention, tumors include solid tumors and hematological tumors. "hematological neoplasms" include mainly various leukemias, multiple myelomas, and malignant lymphomas. "solid tumor" includes: bone, bone connection, muscle, lung, trachea, heart, spleen, artery, vein, capillary, mouth, pharynx, esophagus, stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gall bladder, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, external pudendum, scrotum, testis, vas deferens, penis, eye, ear, nose, tongue, skin, brain stem, medulla oblongata, spinal cord, cerebrospinal fluid, nerve, thyroid, parathyroid gland, adrenal gland, pituitary gland, pineal gland, islet, thymus, gonad, sublingual gland, tumor generated by lesions in any of the parotid glands.
Tumor antigens described in the present invention are well known to those skilled in the art and include tumor specific antigens (tumor SPECIFIC ANTIGEN, TSA) and tumor associated antigens (tumor-associatedantigen, TAA), such as any one or more selected from the group consisting of: alpha-fetoprotein (AFP), alpha-actin-4, A3, antigen specific for the A33 antibody, ART-4, B7, ba 733, BAGE, brE3 antigen, BMCA, CA125, CAMEL, CAP-1, carbonic anhydrase IX、CASP-8/m、CCL19、CCL21、CD1、CD1a、CD2、CD3、CD4、CD5、CD8、CD11A、CD14、CD15、CD16、CD18、CD19、CD20、CD21、CD23、CD25、CD29、CD30、CD32b、CD37、CD40、CD40L、CD44、CD45、CD46、CD52、CD54、CD55、CD59、CD64、CD66a-e、CD67、CD70、CD70L、CD74、CD79a、CD79b、CD80、CD83、CD95、CD126、CD132、CD133、CD138、CD147、CD154、CDC27、CDK-4/m、CDKN2A、CLDN family protein, CTLA4, CXCR7, CXCL12, HIF-1 alpha, colon specific antigen p (CSAp), CEA (CEACAM-5), CEACAM-6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, ep-CAM, fibroblast activation protein alpha (FAP), fibroblast Growth Factor (FGF), flt-1, flt-3, folate receptor, G250 antigen, GAGE, GD2, gp100, GRO-beta, HLA-DR, HM1.24 Human Chorionic Gonadotrophin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1)、HSP70-2M、HST-2、Ia、IGF-1R、IFN-γ、IFN-α、IFN-β、IFN-λ、IL-4R、IL-6R、IL-13R、IL-15R、IL-17R、IL-18R、IL-2、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-23、IL-25、 insulin-like growth factor 1 (IGF-1), KC4 antigen, KS-1 antigen, KS1-4, le-Y, LDR/FUT, macrophage migration inhibitory factor (MIF)、MAGE、MAGE-3、MART1、MART-2、NY-ESO-1、TRAG-3、mCRP、MCP-1、MIP-1α、MIP-1β、MIF、MUC1、MUC2、MUC3、MUC4、MUC5ac、MUC13、MUC16、MUM-1/2、MUM-3、NCA66、NCA95、NCA90、 Mesothelin (MSLN), pancreatic mucin, PD1 receptor, prostate Stem Cell Antigen (PSCA), placental growth factor, p53, PLAGL2, prostaacid phosphatase, trophoblast cell surface antigen 2 (TROP 2), PSA, PRAME, PSMA, plGF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, S100, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor, TNF-alpha, tn antigen, thomson-Fredory antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A antigen, complement factors C3, C3a, C3B, C5a, C5, angiogenesis markers, bc1-2, bc1-6, kras, oncogene markers, and oncogene products. More preferred solid tumor specific antigens are selected from the group consisting of CLDN family proteins, ep-CAM, FAP, PSCA, TROP2 and MSLN. The CLDN family protein may be selected from CLDN1、CLDN2、CLDN3、CLDN4、CLDN5、CLDN6、CLDN7、CLDN8、CLDN9、CLDN10、CLDN11、CLDN12、CLDN15、CLDN16、CLDN18(CLDN18.1 or CLDN 18.2), CLDN20, CLDN23.
In some embodiments, the antigen presenting cell is a natural cell, such as a professional antigen presenting cell, including a dendritic cell, a macrophage, or a B cell. In some embodiments, the antigen presenting cell does not include an artificial antigen presenting cell (aAPC).
In some embodiments, the antigen presenting cell overexpresses a cytokine that facilitates activation of the CAR-T cell. The cytokine may be one or more of interleukin, IFN, TNF, CSF, chemokine, and growth factor. "activating CAR-T cells" is understood to mean inducing activation of T cells and/or promoting proliferation of T cells and/or promoting infiltration of T cells; in some embodiments, the cytokine assists the antigen presenting cell in activating CAR-T cells; in other embodiments, the cytokine directly activates the CAR-T cell. The interleukin may be selected from one or more of IL1 to IL 36. In some embodiments, the interleukin comprises IL12. The chemokines may be selected from one or more of CXCL 1-19, CCL 1-28, XCL1, XCL2, CX3CL 1. In some embodiments, the chemokine comprises CXCL9. In some embodiments the antigen presenting cells overexpress IL12 and CXCL9.
When antigen presenting cells express multiple proteins, such as a tumor antigen and one or more cytokines, they can be concatenated with a self-cleavable short peptide such as a 2A peptide (e.g., T2A, P2A, E2A, F A) for expression, or separately transferred into the antigen presenting cells.
In some embodiments, the extracellular domain is a sc-Fv, fab, scFab, scFab, scIgG or V H H fragment.
As used herein, a "Chimeric Antigen Receptor (CAR)" refers to a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain derived from a polypeptide other than the polypeptide from which the extracellular domain was derived, and at least one intracellular domain. "Chimeric Antigen Receptor (CAR)" is sometimes referred to as "chimeric receptor", "T-body" or "Chimeric Immune Receptor (CIR)". An "extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide that can bind to a particular antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to function in a cell as a domain that transmits a signal to cause activation or inhibition of a biological process. In some embodiments, the chimeric antigen receptor comprises a hinge region, a transmembrane region, and an intracellular signaling region;
As used herein, a "region" or "domain" comprised in the chimeric antigen receptor refers to a region in a polypeptide that can be folded into a specific structure independently of the other regions. These "regions" or "domains" may be sequences of murine or other animal origin, preferably human origin.
In some embodiments, the hinge region is the hinge region of CD8 or CD 28;
In some embodiments, the transmembrane domain is selected from one of the α, β or ζ chain 、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、KIRDS2、OX40、CD2、CD27、LFA-1(CD11a、CD18)、ICOS(CD278)、4-1BB(CD137)、GITR、CD40、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、IL2Rβ、IL2Rγ、IL7Rα、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D and NKG2C of a T cell receptor.
In some embodiments, the intracellular signaling region comprises a CD3 zeta signaling domain.
In some embodiments, the intracellular signaling region further comprises one or more of the following proteins or intracellular signaling regions thereof: CD28, 4-1BB, OX40, ICOS, CD27, CD40-MyD88, DAP12, DAP10, and 2B4.
In some embodiments, the tumor antigen is CLDN18.2 and the amino acid sequence of the extracellular domain is selected from the group consisting of SEQ ID NOs: 8 to 10.
In some embodiments, the amino acid sequence of the chimeric antigen receptor is selected from the group consisting of SEQ ID NOs: 5 to 7.
In some embodiments, the tumor antigen is CLDN18.2 and the antigen presenting cells express the amino acid sequence of SEQ ID NO:1 to 4.
SEQ ID NO: variants of any one of claims 1 to 10, which can correspond to SEQ ID NO: 1-10, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Variants may have deletions, additions or substitutions of one or more amino acids; substitutions are generally conservative, and substitutions are generally considered to be conservative substitutions in aliphatic amino acids Ala, val, leu and Ile with each other, exchange of hydroxyl residues Ser and Thr, exchange of acidic residues Asp and Glu, exchange of amide residues Asn and Gln, exchange of basic residues Lys and Arg, and exchange of aromatic residues Phe, tyr. Those skilled in The art know that, in general, single amino acid substitutions in The non-essential region of a polypeptide do not substantially alter biological activity (see, e.g., watson et al (1987) Molecular Biology of The Gene, the Benjamin/CummingsPub.Co., page 224, (4 th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.
According to yet another aspect of the invention, there is also provided an antigen presenting cell that expresses a tumor antigen and over-expresses a cytokine that facilitates activation of a CAR-T cell;
wherein the tumor antigen is as defined above;
The cytokines are as defined above;
the antigen presenting cells are as defined above.
The invention also relates to a cell pharmaceutical preparation comprising a) or b):
a) Antigen presenting cells and CAR-T cells, individually packaged or mixed together;
Wherein the antigen presenting cell and the CAR-T cell are as defined above;
b) Antigen presenting cells as described above.
In some embodiments, the cytopharmaceutical formulation further comprises a pharmaceutically acceptable adjuvant. As used herein, a "pharmaceutically acceptable carrier" includes any material that, when combined with an active ingredient, allows the ingredient to remain biologically active and non-reactive with the subject's immune system. Examples include, but are not limited to, standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, and any of various types of wetting agents. Exemplary diluents for aerosol or parenteral administration are Phosphate Buffered Saline (PBS) or physiological (0.9%) saline. Compositions comprising such carriers are formulated by well known conventional methods (see, e.g., remington's Pharmaceutical Sciences, 18 th edition, a. Gennaro, mack Publishing co., easton, PA,1990; and Remington, THESCIENCE AND PRACTICE of Pharmacy, 21 st edition, mack Publishing, 2005).
In some embodiments, the cytopharmaceutical formulation is frozen or cryopreserved.
In some embodiments, the cytopharmaceutical formulation may be contained in a cell delivery vehicle (e.g., syringe, etc.).
In some embodiments, the cytopharmaceutical formulation does not comprise an agent for nucleic acid transfection.
In some embodiments, the cytopharmaceutical formulation comprises at least 10 5, or at least 10 6, or at least 10 7, or at least 10 8 CAR-T cells. In some embodiments, the cytopharmaceutical formulation comprises at least 10 5, or at least 10 6, or at least 10 7, or at least 10 8 antigen presenting cells.
According to yet another aspect of the present invention, there is also a method of treating a tumor in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a cytopharmaceutical formulation as described above.
The term "effective amount" refers to the amount that achieves treatment, prevention, alleviation and/or relief of a disease or condition of the present invention in a subject.
It will be appreciated that contemplated methods of treatment will also include administration of other immunotherapeutic entities, particularly preferably immunotherapeutic entities, including viral cancer vaccines (e.g., adenovirus vectors encoding cancer-specific antigens), bacterial cancer vaccines (e.g., non-pyrogenic escherichia coli expressing one or more cancer-specific antigens), yeast cancer vaccines, N-803 (also known as ALT-803, altor biosciences), and antibodies (e.g., binding to tumor-associated antigens or patient-specific tumor neoantigens), stem cell grafts (e.g., allogeneic or autologous), and tumor-targeting cytokines (e.g., NHS-IL12, IL-12 coupled to tumor-targeting antibodies or fragments thereof).
A "patient" is a mammal, including but not limited to humans, monkeys, pigs and other farm animals, sports animals, pets, primates, horses, dogs, cats, pandas, rodents (including mice, rats, guinea pigs), and the like.
In some embodiments, the CAR-T cells are administered simultaneously with the antigen presenting cells, preferably in a mixture. In some embodiments, the CAR-T cell is administered separately from the antigen presenting cell, e.g., the CAR-T cell is administered prior to the antigen presenting cell, or the CAR-T cell is administered prior to the antigen presenting cell; the interval may be1 to 50 days, for example 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40 days. The time interval is calculated from the last administration of CAR-T cells to the first administration of antigen presenting cells; or calculated as last administration of antigen presenting cells to first administration of CAR-T cells. Alternatively, the two may be administered. The number of administrations of CAR-T cells/antigen presenting cells is independently selected from 1,2, 3, 4, 5, 6 or more times.
In some embodiments, the ratio of the number of CAR-T cells to the antigen presenting cells is 50:1、45:1、40:1、35:1、30:1、25:1、20:1、15:1、10:1、5:1、3:1、2:1、1:1、1:2、1:3、1:5、1:10、1:15、1:20、1:25、1:30、1:35、1:40、1:45、1:50.
Embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to in the guidelines given in the present invention, and may be according to the experimental manuals or conventional conditions in the art, and may be referred to other experimental methods known in the art, or according to the conditions suggested by the manufacturer.
In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.
EXAMPLE 1 construction of DC cells expressing CLDN18.2
To construct DC cells that overexpress CLDN18.2, we first constructed an adenovirus-based expression vector whose coding sequences are shown in the following table, and see schematic figure 1. The adenovirus vector was synthesized and prepared by CRO corporation. See table 1 for specific sequences.
TABLE 1
DC cell preparation: peripheral blood PBMC were isolated by Ficoll lymphocyte separation, CD14+ monocytes were sorted by magnetic beads, GM-CSF (800 IU/ml) was added using DC-specific medium, IL-6 (500 IU/ml) was induced to differentiate into immature DC cells, immature DC cells were induced to mature by addition of IL-1beta (10 ng/ml), IL-6 (10 ng/ml), TNF-a (10 ng/ml), PGE2 (10 ng/ml), and CD80+CD86+CD83+ mature DC cell induction ratio was examined by FACS. The phenotypic assay results of DCs are shown in fig. 2, demonstrating that DC cells have successfully differentiated and induced maturation.
DC cell transduction: the expression of CLDN18.2 on DCs was examined 3 days after transduction by adding each adenovirus or empty adenovirus vector control H225 described above to mature DC cells, and the results were shown in fig. 3, where CLDN18.2 was successfully expressed on DC cells. Further, we examined secretion of IL12 or CXCL9 in the supernatant of DC cells after transduction of each vector, and the results are shown in Table 2.
TABLE 2 detection of secreted cytokines by DC cells
Virus (virus) Structure of the IL12 concentration (pg/ml) CXCL9 concentration (pg/ml)
Mock DC control ADV 501.02 3986.11
DC-182 CLDN18.2 477.57 3937.48
DC-182.IL12 CLDN18.2-IL12 28450.52 4860.22
DC-182.CXCL9 CLDN18.2-CXCL9 803.21 7672 (Detection line)
DC-182.IL12.CXCL9 CLDN18.2-IL12-CXCL9 30775.87 11985 (Detection upper line)
EXAMPLE 2CLDN18.2 CAR-T cell construction
Plasmid construction of cldn18.2 CAR:
The gene fragments of different module parts of the CAR are synthesized by CRO company according to the conventional gene synthesis technology after primer synthesis, and then cloned and constructed by using the conventional molecular cloning technology. The relevant sequences are shown in table 3. If not specifically stated, CLDN18.2 CAR-T cells shown in the examples below are all CAR1.
TABLE 3 Table 3
Numbering device Structure of the Sequence(s)
CAR1 CLDN18.2 Binder1-CD28 Hinge-CD28TM-CD28 co-sim-CD3zeta SEQ ID NO：5
CAR2 CLDN18.2Binder2-CD28 Hinge-CD28TM-CD28 co-sim-CD3zeta SEQ ID NO：6
CAR3 CLDN18.2Binder3-CD28 Hinge-CD28TM-CD28 co-sim-CD3zeta SEQ ID NO：7
The amino acid sequences of CLDN18.2 Binder1, binder2 and Binder3 are shown in SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: shown at 10.
Viral packaging of cldn18.2 CAR
The extracted middle-extracted plasmid is packaged by slow virus, and the specific steps are as follows:
1) The state of 293T cells (including cell morphology, amplification status and passage number less than 15 generation) needs to be confirmed before the experiment;
2) 293T cells were plated on the first day (T75 flask was inoculated with approximately 6-7X 10 6 cells, medium volume 25ml, T175 flask was inoculated with approximately 15-18X 10 6 cells, medium volume 40 ml);
3) Before virus is packaged the next day, the confluence of 293T cells is required to be confirmed to be more than 70% -80% and less than 95%, the equal volume liquid exchange is carried out, the culture is continued after the liquid exchange, and the mixed liquid of Tube A and Tube B is waited for being prepared;
4) Tube A and Tube B (T175 and T75 are different in volume, as described below) are respectively configured, and inverted or uniformly mixed by low-speed oscillation;
T175 system:
Tube A：Opti-MEM(4.6ml)+Lipo3000(129μl)
tube B: opti-MEM (4.6 ml) +P3000 (111 μl) +helper plasmid PZ201/PZ202/PZ203 (41.4 μg,1:1:1 mass ratio) +CAR molecule plasmid (13.8 μg);
T75 system:
Tube A：Opti-MEM(2ml)+Lipo3000(55μl)
Tube B: opti-MEM (2 ml) +P3000 (46 μl) +helper plasmid PZ201/PZ202/PZ203 (18 μg,1:1:1 mass ratio) +CAR molecule plasmid (6 μg).
5) Adding Tube A into Tube B, shaking and mixing, and incubating for 15 minutes at room temperature;
6) Turning over the culture flask with the liquid changed in the step 3, enabling the culture medium to be positioned on the other surface of the culture flask, adding the Tube A+B mixture, slightly shaking and uniformly mixing, and slowly turning over the culture flask back to the front surface;
7) Placing the mixture into an incubator for culturing for 2 days, collecting supernatant, centrifuging for 10min at 500g, filtering the supernatant with a 0.45 μm filter to a 50ml centrifuge tube, wrapping a sealing film, centrifuging for 2h at 10000g at 4 ℃, and observing precipitation;
8) In a biosafety cabinet, discarding the supernatant, reversely buckling the centrifuge tube on surgical gauze, taking new gauze to gently wipe the inner wall of the tube after 3min, and taking care not to touch the sediment;
9) After the pellet was dissolved in 200. Mu. l X-vivo medium, 2. Mu.l was assayed for titer, the remainder labeled, LV virus ID number, preparation lot, preparation person and split-fill volume were designated, and stored at-80 ℃.
Construction of CLDN18.2 CAR-T cells
T cells were first isolated from healthy human PBMCs and activated using CD3/CD28 antibodies. T cells were infected with the lentivirus described above 24h after activation. After 24h the medium was replaced with complete medium (X-VIVO+5% FBS+IL2) to wash out residual lentiviruses. The positive rate of the CAR molecules was detected 2 days after cell expansion and used for functional experiments 5 days later. As shown in fig. 4, the CAR-T cell positive rate was 46% and the construction was successful.
Cldn18.2 CAR-T cell killing target cell ability validation
We further validated the ability of CLDN18.2CAR-T to kill target cells using LDH methods. NUGC4 cells were CLDN18.2 positive target cells and 293T cells were CLDN18.2 negative control cells. As shown in fig. 5, the results showed that CLDN18.2CAR-T was able to kill target cell NUGC4 efficiently and dose dependently, but not negative control cell 293T. It was demonstrated that CLDN18.2CAR-T cells were able to specifically kill CLDN18.2 positive target cells.
EXAMPLE 3 CLDN18.2 expressing DCs promote proliferation of CLDN18.2 CAR-T cells in vitro
To verify whether CLDN18.2 expressing DCs were able to promote CAR-T cell proliferation in vitro, we co-incubated 18.2 expressing DCs and CLDN18.2 CAR-T at different ratios (1:10, 1:20, 1:30) while co-incubating (1:10) CLDN18.2 CAR-T with non-transduced DC cells as controls and examined CAR-T cell proliferation after 5 days of co-incubation. The results are shown in FIG. 6A, where DC-182 significantly promotes the proliferative capacity of CAR-T relative to control DC cells. Detection of CAR positive rate results showed that stimulation of DC-182 could increase CAR positive rate (fig. 6B). We further studied the proliferative effect of increasing IL12 and/or CXCL9 on CAR-T, DC-182.cxcl9, DC-182.il12.cxcl9. As shown in FIGS. 6C and 6D, DC-182.IL12, DC-182.CXCL9, and DC-182.IL12.CXCL9 all promoted proliferation (6C) and increased positive rate (6D) of CAR-T cells.
Example 4 CLDN18.2 expressing DCs increased CLDN18.2 CAR-T cell central memory phenotype levels in vitro
We further verified whether CLDN18.2 expressing DCs could promote CAR-T cell proliferation in vitro, and we examined CCR7 and CD45RA expression on CAR-T cells five days after co-incubating 18.2 expressing DCs with CLDN18.2 CAR-T. As a result, as shown in FIG. 7, DC-182, DC-182.IL12, DC-182.CXCL9, DC-182.IL12.CXCL9 were able to increase the proportion of central memory phenotype T cells (Tcm). Wherein the proportion of Tcm is highest for DC-182.il12 group CAR-T cells.
EXAMPLE 5 expression of CLDN18.2 DCs in vitro to enhance the ability of CLDN18.2 CAR-T cells to kill target cells and cytokine secretion
To verify the difference in the ability of CAR-T cells to kill target cells after in vitro stimulation of CLDN18.2 expressing DCs we first said that CAR-T cells were co-incubated with DC-182, DC-182.il12, DC-182.cxcl9, DC-182.il12.cxcl9 or Mock DC cells, respectively, for five days, after which each CAR-T cell was co-incubated with target cell NUGC4 (effective target ratio 1:3,1:1, 3:1) for killing ability. Results as shown in figure 8, the ability of DC-182, DC-182.il12, DC-182.cxcl9, DC-182.il12.cxcl9 stimulated CAR-T cells to kill NUGC4 was significantly higher than Mock DC or unstimulated CAR-T cells. We further analyzed the secretion levels of cytokine IFN- γ after co-incubation of stimulated CAR-T cells with NUGC4, and the results showed that modified DC stimulated CAR-T cells were both activated and released IFN- γ by NUGC4, with specific release levels ordered as DC-182.il12.cxcl9> DC-182.cxcl9> DC-182.il12> DC-182 (fig. 9). This result demonstrates that CXCL9 and IL12 are able to increase the level of DC-activated CAR-T.
EXAMPLE 6 expression of CLDN18.2 DCs promote tumor clearance of CLDN18.2 CAR-T cells in vivo
To further verify whether CLDN18.2 overexpressing DC cells could clear tumors in vivo in synergy with CAR-T, we performed NUGC4-luc tumor model based efficacy experiments.
NUGC4-luc tumor model construction: human gastric cancer cell NUGC-4-luc was cultured in RPMI1640 medium with 10% FBS in a 37℃incubator containing 5% CO 2. Cells were inoculated subcutaneously into mice prior to ten successive passages of culture. Mice were anesthetized with 3-4% isoflurane prior to inoculation. Approximately 5X 10 6 NUGC-4-Luc cells were resuspended in PBS and, after homogenization with an equal volume of Matrigel, inoculated into NCG mice by subcutaneous injection at a volume of 200. Mu.L. Tumors were randomly grouped according to tumor size and body weight when they grew to an average of about 91mm 3 or so. The day of grouping was defined as day 0. Sub-potent doses of CAR-T cells were injected on days 0 and 23 post-grouping. In Day 49, the tumor size reached 500-1500 mm 3 and the trend of rapid growth was seen, and mice of each group were given MOCK DC or DC-182.il12.cxcl9, respectively, to demonstrate whether the transduced DC cells had an adjuvant effect on CAR-T efficacy. As shown in fig. 10, none of the sub-doses of CAR-T inhibited tumor cell growth. Following DC administration, tumors in mice with DC-182.il12.cxcl9 all showed a rapid decrease in tumor volume after undergoing a short growth. In contrast, the tumors of mice given MOCK DC cells continued to grow and could not be controlled. The above results demonstrate that DC-182.IL12.CXCL9 has a significant enhancement effect on the killing of tumors by CAR-T.
EXAMPLE 7 CLDN18.2 expressing DCs promote CLDN18.2 CAR-T cell proliferation and tumor infiltration in vivo
To further explore the mechanism of action of DC-182.il12.cxcl9 in enhancing CAR-T function, we examined the level of CAR-T in mouse peripheral blood and the level of infiltration of CAR-T in tumors. First, the levels of CAR-T were measured using FCM before and 14 days after DC administration, and the results are shown in fig. 11, in which each group of mice CAR-T was at a lower level (0 to 0.5) in peripheral blood before DC administration, whereas the levels of DC-182.il12.cxcl9 were significantly increased 14 days after DC administration, while the MOCK DC group was not significantly changed. The above results demonstrate that DC-182.IL12.CXCL9 is effective in enhancing proliferation of CAR-T in vivo. We further collected tumor tissue at the end of the experiment and examined CAR-T infiltration in tumors by immunofluorescent staining with hCD 45. The results are shown in figure 12, treatment with DC-182.il12.cxcl9 significantly increased the level of CAR-T cell infiltration in tumors.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. The scope of the invention is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted in accordance with the contents of the claims.
SEQUENCE LISTING
<110> Su Yi Mufeng Biotech Co., ltd
<120> Antigen presenting cells and application of CAR-T cell combination in anti-tumor
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 261
<212> PRT
<213> artificial sequence
<220>
<223> DC-182
<400> 1
Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile
1 5 10 15
Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr
20 25 30
Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly
35 40 45
Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg
50 55 60
Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg
65 70 75 80
Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val
85 90 95
Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser
100 105 110
Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser
115 120 125
Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val
130 135 140
Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly
145 150 155 160
Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe
165 170 175
Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met
180 185 190
Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala
195 200 205
Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly
210 215 220
Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile
225 230 235 240
Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser
245 250 255
Lys His Asp Tyr Val
260
<210> 2
<211> 823
<212> PRT
<213> artificial sequence
<220>
<223> DC-182.IL12
<400> 2
Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile
1 5 10 15
Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr
20 25 30
Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly
35 40 45
Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg
50 55 60
Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg
65 70 75 80
Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val
85 90 95
Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser
100 105 110
Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser
115 120 125
Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val
130 135 140
Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly
145 150 155 160
Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe
165 170 175
Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met
180 185 190
Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala
195 200 205
Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly
210 215 220
Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile
225 230 235 240
Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser
245 250 255
Lys His Asp Tyr Val Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys
260 265 270
Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Cys His Gln Gln
275 280 285
Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu Ala Ser Pro Leu Val
290 295 300
Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp
305 310 315 320
Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro
325 330 335
Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu
340 345 350
Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala
355 360 365
Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu
370 375 380
Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu
385 390 395 400
Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala
405 410 415
Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser
420 425 430
Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro
435 440 445
Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg
450 455 460
Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser
465 470 475 480
Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp
485 490 495
Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile
500 505 510
Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro
515 520 525
Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr
530 535 540
Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val
545 550 555 560
Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys
565 570 575
Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg
580 585 590
Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val
595 600 605
Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
610 615 620
Gly Ser Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro
625 630 635 640
Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu
645 650 655
Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu
660 665 670
Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala
675 680 685
Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg
690 695 700
Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr
705 710 715 720
Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys
725 730 735
Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp
740 745 750
Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp
755 760 765
Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys
770 775 780
Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys
785 790 795 800
Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val
805 810 815
Met Ser Tyr Leu Asn Ala Ser
820
<210> 3
<211> 408
<212> PRT
<213> artificial sequence
<220>
<223> DC-182.CXCL9
<400> 3
Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile
1 5 10 15
Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr
20 25 30
Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly
35 40 45
Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg
50 55 60
Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg
65 70 75 80
Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val
85 90 95
Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser
100 105 110
Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser
115 120 125
Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val
130 135 140
Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly
145 150 155 160
Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe
165 170 175
Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met
180 185 190
Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala
195 200 205
Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly
210 215 220
Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile
225 230 235 240
Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser
245 250 255
Lys His Asp Tyr Val Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys
260 265 270
Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Lys Lys Ser Gly
275 280 285
Val Leu Phe Leu Leu Gly Ile Ile Leu Leu Val Leu Ile Gly Val Gln
290 295 300
Gly Thr Pro Val Val Arg Lys Gly Arg Cys Ser Cys Ile Ser Thr Asn
305 310 315 320
Gln Gly Thr Ile His Leu Gln Ser Leu Lys Asp Leu Lys Gln Phe Ala
325 330 335
Pro Ser Pro Ser Cys Glu Lys Ile Glu Ile Ile Ala Thr Leu Lys Asn
340 345 350
Gly Val Gln Thr Cys Leu Asn Pro Asp Ser Ala Asp Val Lys Glu Leu
355 360 365
Ile Lys Lys Trp Glu Lys Gln Val Ser Gln Lys Lys Lys Gln Lys Asn
370 375 380
Gly Lys Lys His Gln Lys Lys Lys Val Leu Lys Val Arg Lys Ser Gln
385 390 395 400
Arg Ser Arg Gln Lys Lys Thr Thr
405
<210> 4
<211> 969
<212> PRT
<213> artificial sequence
<220>
<223> DC-182.IL12.CXCL9
<400> 4
Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile
1 5 10 15
Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr
20 25 30
Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly
35 40 45
Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg
50 55 60
Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg
65 70 75 80
Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val
85 90 95
Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser
100 105 110
Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser
115 120 125
Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val
130 135 140
Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly
145 150 155 160
Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe
165 170 175
Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met
180 185 190
Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala
195 200 205
Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly
210 215 220
Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile
225 230 235 240
Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser
245 250 255
Lys His Asp Tyr Val Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys
260 265 270
Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Cys His Gln Gln
275 280 285
Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu Ala Ser Pro Leu Val
290 295 300
Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp
305 310 315 320
Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro
325 330 335
Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu
340 345 350
Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala
355 360 365
Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu
370 375 380
Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu
385 390 395 400
Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala
405 410 415
Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser
420 425 430
Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro
435 440 445
Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg
450 455 460
Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser
465 470 475 480
Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp
485 490 495
Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile
500 505 510
Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro
515 520 525
Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr
530 535 540
Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val
545 550 555 560
Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys
565 570 575
Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg
580 585 590
Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val
595 600 605
Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
610 615 620
Gly Ser Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro
625 630 635 640
Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu
645 650 655
Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu
660 665 670
Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala
675 680 685
Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg
690 695 700
Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr
705 710 715 720
Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys
725 730 735
Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp
740 745 750
Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp
755 760 765
Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys
770 775 780
Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys
785 790 795 800
Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val
805 810 815
Met Ser Tyr Leu Asn Ala Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu
820 825 830
Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Lys Lys Ser
835 840 845
Gly Val Leu Phe Leu Leu Gly Ile Ile Leu Leu Val Leu Ile Gly Val
850 855 860
Gln Gly Thr Pro Val Val Arg Lys Gly Arg Cys Ser Cys Ile Ser Thr
865 870 875 880
Asn Gln Gly Thr Ile His Leu Gln Ser Leu Lys Asp Leu Lys Gln Phe
885 890 895
Ala Pro Ser Pro Ser Cys Glu Lys Ile Glu Ile Ile Ala Thr Leu Lys
900 905 910
Asn Gly Val Gln Thr Cys Leu Asn Pro Asp Ser Ala Asp Val Lys Glu
915 920 925
Leu Ile Lys Lys Trp Glu Lys Gln Val Ser Gln Lys Lys Lys Gln Lys
930 935 940
Asn Gly Lys Lys His Gln Lys Lys Lys Val Leu Lys Val Arg Lys Ser
945 950 955 960
Gln Arg Ser Arg Gln Lys Lys Thr Thr
965
<210> 5
<211> 361
<212> PRT
<213> artificial sequence
<220>
<223> CAR1
<400> 5
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly
20 25 30
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
35 40 45
Ser Ile Phe Asn Ile Pro Val Met Gly Trp Tyr Arg Gln Ala Pro Gly
50 55 60
Lys Gln Arg Glu Leu Val Ala Gly Ile Ser Thr Gly Gly Thr Thr Asn
65 70 75 80
Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
85 90 95
Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Asn Val Leu Val Val Ser Gly Ile Gly Ser Thr
115 120 125
Leu Glu Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ile Glu
130 135 140
Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr
145 150 155 160
Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro
165 170 175
Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu
180 185 190
Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
195 200 205
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
210 215 220
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
225 230 235 240
Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser
245 250 255
Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu
260 265 270
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
275 280 285
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
290 295 300
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
305 310 315 320
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
325 330 335
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
340 345 350
Leu His Met Gln Ala Leu Pro Pro Arg
355 360
<210> 6
<211> 487
<212> PRT
<213> artificial sequence
<220>
<223> CAR2
<400> 6
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Gln Gln Pro Gly Ala Glu
20 25 30
Leu Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly
35 40 45
Tyr Thr Phe Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly
50 55 60
Gln Gly Leu Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr
65 70 75 80
Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys
85 90 95
Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp
100 105 110
Ser Ala Val Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp
115 120 125
Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr
145 150 155 160
Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met
165 170 175
Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn
180 185 190
Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu
195 200 205
Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr
210 215 220
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln
225 230 235 240
Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro
245 250 255
Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Ile Glu Val Met
260 265 270
Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile
275 280 285
His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro
290 295 300
Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys
305 310 315 320
Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser
325 330 335
Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg
340 345 350
Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg
355 360 365
Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp
370 375 380
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
385 390 395 400
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
405 410 415
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
420 425 430
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
435 440 445
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
450 455 460
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
465 470 475 480
Met Gln Ala Leu Pro Pro Arg
485
<210> 7
<211> 492
<212> PRT
<213> artificial sequence
<220>
<223> CAR3
<400> 7
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
20 25 30
Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln
35 40 45
Ser Leu Phe Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln
50 55 60
Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr
65 70 75 80
Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
85 90 95
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val
100 105 110
Tyr Tyr Cys Gln Asn Ala Tyr Ser Phe Pro Tyr Thr Phe Gly Gly Gly
115 120 125
Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
145 150 155 160
Ile Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly
165 170 175
Ser Ile Ser Ser Gly Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly
180 185 190
Lys Gly Leu Glu Trp Ile Gly Tyr Ile His Tyr Thr Gly Ser Thr Asn
195 200 205
Tyr Asn Pro Ala Leu Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser
210 215 220
Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
225 230 235 240
Ala Ile Tyr Tyr Cys Ala Arg Ile Tyr Asn Gly Asn Ser Phe Pro Tyr
245 250 255
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Thr Thr Thr Pro Ala
260 265 270
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
275 280 285
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
290 295 300
Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly
305 310 315 320
Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile
325 330 335
Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met
340 345 350
Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro
355 360 365
Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe
370 375 380
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
385 390 395 400
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
405 410 415
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
420 425 430
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
435 440 445
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
450 455 460
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
465 470 475 480
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 8
<211> 142
<212> PRT
<213> artificial sequence
<220>
<223> Binder1
<400> 8
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly
20 25 30
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
35 40 45
Ser Ile Phe Asn Ile Pro Val Met Gly Trp Tyr Arg Gln Ala Pro Gly
50 55 60
Lys Gln Arg Glu Leu Val Ala Gly Ile Ser Thr Gly Gly Thr Thr Asn
65 70 75 80
Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
85 90 95
Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Asn Val Leu Val Val Ser Gly Ile Gly Ser Thr
115 120 125
Leu Glu Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
130 135 140
<210> 9
<211> 268
<212> PRT
<213> artificial sequence
<220>
<223> Binder2
<400> 9
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Gln Gln Pro Gly Ala Glu
20 25 30
Leu Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly
35 40 45
Tyr Thr Phe Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly
50 55 60
Gln Gly Leu Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr
65 70 75 80
Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys
85 90 95
Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp
100 105 110
Ser Ala Val Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp
115 120 125
Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr
145 150 155 160
Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met
165 170 175
Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn
180 185 190
Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu
195 200 205
Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr
210 215 220
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln
225 230 235 240
Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro
245 250 255
Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
260 265
<210> 10
<211> 267
<212> PRT
<213> artificial sequence
<220>
<223> Binder3
<400> 10
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
20 25 30
Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln
35 40 45
Ser Leu Phe Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln
50 55 60
Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr
65 70 75 80
Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
85 90 95
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val
100 105 110
Tyr Tyr Cys Gln Asn Ala Tyr Ser Phe Pro Tyr Thr Phe Gly Gly Gly
115 120 125
Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
145 150 155 160
Ile Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly
165 170 175
Ser Ile Ser Ser Gly Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly
180 185 190
Lys Gly Leu Glu Trp Ile Gly Tyr Ile His Tyr Thr Gly Ser Thr Asn
195 200 205
Tyr Asn Pro Ala Leu Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser
210 215 220
Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
225 230 235 240
Ala Ile Tyr Tyr Cys Ala Arg Ile Tyr Asn Gly Asn Ser Phe Pro Tyr
245 250 255
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
260 265

Claims (9)

1. Use of an antigen presenting cell and a CAR-T cell in combination for the preparation of a medicament for the treatment of gastric cancer, wherein the antigen presenting cell is a dendritic cell and expresses a tumor antigen, the chimeric antigen receptor ectodomain of the CAR-T cell targets and recognizes the tumor antigen, and the amino acid sequence of the ectodomain is selected from the group consisting of SEQ ID NOs: 8-10, and wherein the antigen presenting cell overexpresses a cytokine that facilitates activation of the CAR-T cell, the cytokine comprising an interleukin comprising IL12 and a chemokine comprising CXCL9, and the tumor antigen is as set forth in SEQ ID NO: CLDN18.2 as shown in 1.
2. The use of claim 1, wherein the tumor antigen is over-expressed from a foreign gene in the antigen presenting cell.
3. The use of claim 1, wherein the chimeric antigen receptor comprises a hinge region, a transmembrane region, and an intracellular signaling region.
4. The use according to claim 3, wherein the hinge region and transmembrane region are selected from one of the alpha, beta or zeta chain 、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、KIRDS2、OX40、CD2、CD27、LFA-1、ICOS、4-1BB、GITR、CD40、BAFFR、HVEM、SLAMF7、NKp80、CD160、CD19、IL2Rβ、IL2Rγ、IL7Rα、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、DNAM1、SLAMF4、CD84、CD96、CEACAM1、CRTAM、Ly9、CD16、PSGL1、CD100、SLAMF6、SLAM、BLAME、SELPLG、LTBR、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D and NKG2C of a T cell receptor.
5. The use of claim 3, wherein the intracellular signaling region comprises a CD3 zeta signaling domain.
6. The use of claim 5, wherein the intracellular signaling region further comprises one or more of the following proteins or intracellular signaling regions thereof: CD28, 4-1BB, OX40, ICOS, CD27, CD40-MyD88, DAP12, DAP10, and 2B4.
7. The use according to any one of claims 1 to 6, wherein the amino acid sequence of the chimeric antigen receptor is selected from the group consisting of SEQ ID NOs: 5 to 7.
8. The use of any one of claims 1-6, wherein the antigen presenting cell expresses the amino acid sequence of SEQ ID NO:4, and a protein shown in the formula 4.
9. A cytopharmaceutical formulation comprising:
An antigen presenting cell and a CAR-T cell, packaged separately or mixed together, wherein the antigen presenting cell and the CAR-T cell are as defined in any one of claims 1 to 8; and
Pharmaceutically acceptable auxiliary materials.
CN202210475661.4A 2022-04-29 2022-04-29 Application of antigen presenting cells and CAR-T cell combination in anti-tumor Active CN114949190B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202210475661.4A CN114949190B (en) 2022-04-29 2022-04-29 Application of antigen presenting cells and CAR-T cell combination in anti-tumor
CN202410324943.3A CN118178626A (en) 2022-04-29 2022-04-29 Application of antigen presenting cells and CAR-T cell combination in anti-tumor

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210475661.4A CN114949190B (en) 2022-04-29 2022-04-29 Application of antigen presenting cells and CAR-T cell combination in anti-tumor

Related Child Applications (1)

Application Number Title Priority Date Filing Date
CN202410324943.3A Division CN118178626A (en) 2022-04-29 2022-04-29 Application of antigen presenting cells and CAR-T cell combination in anti-tumor

Publications (2)

Publication Number Publication Date
CN114949190A CN114949190A (en) 2022-08-30
CN114949190B true CN114949190B (en) 2024-04-30

Family

ID=82980068

Family Applications (2)

Application Number Title Priority Date Filing Date
CN202210475661.4A Active CN114949190B (en) 2022-04-29 2022-04-29 Application of antigen presenting cells and CAR-T cell combination in anti-tumor
CN202410324943.3A Pending CN118178626A (en) 2022-04-29 2022-04-29 Application of antigen presenting cells and CAR-T cell combination in anti-tumor

Family Applications After (1)

Application Number Title Priority Date Filing Date
CN202410324943.3A Pending CN118178626A (en) 2022-04-29 2022-04-29 Application of antigen presenting cells and CAR-T cell combination in anti-tumor

Country Status (1)

Country Link
CN (2) CN114949190B (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106119193A (en) * 2016-07-28 2016-11-16 上海闪锦生物科技有限公司 A kind of preparation method of the T cells with antigenic specificity having NK cell speciality concurrently
CN108025024A (en) * 2015-07-28 2018-05-11 宾夕法尼亚大学董事会 Express modification monocyte/macrophage of Chimeric antigen receptor and application thereof
CN109593726A (en) * 2018-12-26 2019-04-09 重庆精准生物技术有限公司 Enhancing CAR-T cell is gone back to the nest reagent and its application to solid tumor tissue ability
CN110804594A (en) * 2019-11-21 2020-02-18 启辰生生物科技(珠海)有限公司 Engineered antigen presenting cells and their use for activating CD3+ immune cells
CN113354739A (en) * 2021-01-11 2021-09-07 上海莱馥医疗科技有限公司 Chimeric antigen receptor for targeted expression of Claudin18.2 cell and application thereof
CN113528455A (en) * 2021-07-19 2021-10-22 广州百暨基因科技有限公司 Gamma delta T cells expressing chimeric antigen receptors
CN113754780A (en) * 2020-06-04 2021-12-07 四川科伦博泰生物医药股份有限公司 Chimeric antigen receptor targeting CLDN18.2, compositions and uses thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108025024A (en) * 2015-07-28 2018-05-11 宾夕法尼亚大学董事会 Express modification monocyte/macrophage of Chimeric antigen receptor and application thereof
CN106119193A (en) * 2016-07-28 2016-11-16 上海闪锦生物科技有限公司 A kind of preparation method of the T cells with antigenic specificity having NK cell speciality concurrently
CN109593726A (en) * 2018-12-26 2019-04-09 重庆精准生物技术有限公司 Enhancing CAR-T cell is gone back to the nest reagent and its application to solid tumor tissue ability
CN110804594A (en) * 2019-11-21 2020-02-18 启辰生生物科技(珠海)有限公司 Engineered antigen presenting cells and their use for activating CD3+ immune cells
CN113754780A (en) * 2020-06-04 2021-12-07 四川科伦博泰生物医药股份有限公司 Chimeric antigen receptor targeting CLDN18.2, compositions and uses thereof
CN113354739A (en) * 2021-01-11 2021-09-07 上海莱馥医疗科技有限公司 Chimeric antigen receptor for targeted expression of Claudin18.2 cell and application thereof
CN113528455A (en) * 2021-07-19 2021-10-22 广州百暨基因科技有限公司 Gamma delta T cells expressing chimeric antigen receptors

Also Published As

Publication number Publication date
CN114949190A (en) 2022-08-30
CN118178626A (en) 2024-06-14

Similar Documents

Publication Publication Date Title
KR102557834B1 (en) Expression of novel cell tags
JP6161098B2 (en) CAR expression vector and CAR-expressing T cell
CN110462040A (en) The expression of polypeptide is adjusted by new gene switch expression system
EP3331913A1 (en) Treatment of cancer using chimeric cd3 receptor proteins
KR20170068598A (en) Methods and compositions for dosing in adoptive cell therapy
EP3778649A1 (en) Method and composition for treating tumors
JP7252135B2 (en) gene therapy
JP6884697B2 (en) Compositions and Methods for Stimulating and Expanding T Cells
WO2022174498A1 (en) Chimeric antigen receptor fusion protein co-expressing il-7 and ccr2b, and application thereof
CN113402620B (en) Fusion protein of cytokine combined chimeric antigen receptor and application thereof
CN113490689A (en) TCR fusion proteins and cells expressing TCR fusion proteins
CN110404061B (en) Improved T cell therapy
JP2022513372A (en) Chimeric antigen receptor targeting Sialyl Lewis A and its use
WO2021027785A1 (en) Immune effector cell for co-expressing chemokine receptor
WO2022033483A1 (en) Multifunctional immune effector cell and use thereof
KR20240000529A (en) CAR NKT expressing artificial microRNA embedded shRNA for downregulation of MHC class I and II expression
KR20240024047A (en) Cancer treatment method using NK cells and CD38 targeting antibodies
US20230256093A1 (en) Method
US20220370558A1 (en) Combination cancer immunotherapy
CN114949190B (en) Application of antigen presenting cells and CAR-T cell combination in anti-tumor
WO2023235440A2 (en) Compositions and methods comprising chimeric adaptor polypeptides
CN115806625B (en) Chimeric antigen receptor expressed by T cell limitation and application thereof
AU2022379538A1 (en) Chimeric adaptor polypeptides
CN113528455A (en) Gamma delta T cells expressing chimeric antigen receptors
RU2776890C9 (en) Cell therapy based on improved natural killer cells

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant