Targeting B7H3 co-expression IL-21 fully human chimeric antigen receptor, iNKT cell and application thereof
1. A fully human chimeric antigen receptor targeted to B7H3, comprising an antibody scFv that specifically binds to B7H 3;
preferably, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain;
preferably, the chimeric antigen receptor further comprises a hinge region;
preferably, the chimeric antigen receptor further comprises a costimulatory signal domain;
preferably, the chimeric antigen receptor further comprises a signal peptide;
preferably, the chimeric antigen receptor further comprises a self-cleaving peptide;
preferably, the chimeric antigen receptor further comprises an immunomodulatory molecule or cytokine;
more preferably, the amino acid sequence of the scFv comprises the amino acid sequence shown as SEQ ID NO. 1, the amino acid sequence with at least 90% homology with the amino acid sequence shown as SEQ ID NO. 1, the amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids with the amino acid sequence shown as SEQ ID NO. 1;
most preferably, the amino acid sequence of the scFv is the amino acid sequence shown in SEQ ID NO. 1;
more preferably, the transmembrane domain and hinge region comprises the transmembrane domain and hinge region of the following molecules: CD8 α, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3 ε;
most preferably, the transmembrane domain and hinge region is a CD 8a transmembrane domain and CD 8a hinge region;
most preferably, the amino acid sequence of the transmembrane domain of CD8 alpha is shown in SEQ ID NO 2;
most preferably, the amino acid sequence of the CD 8a hinge region is shown in SEQ ID NO 3;
more preferably, the intracellular signaling domain comprises the intracellular signaling domains of the following molecules: CD3 ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 epsilon, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, fcepsilon RI, DAP10, DAP12, CD66d, DAP10, DAP12, FYN;
most preferably, the intracellular signaling domain is a CD3 ζ intracellular signaling domain;
most preferably, the amino acid sequence of the intracellular signaling domain of CD3 ζ is set forth in SEQ ID NO 4;
more preferably, the costimulatory signal domain comprises the costimulatory signal domain of the following molecule: CD28, ICOS, CD27, CD19, CD4, CD8 α, CD8 β, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, CD 278;
most preferably, the costimulatory signaling domain is a CD28 costimulatory signaling domain;
most preferably, the amino acid sequence of the CD28 co-stimulatory signaling domain is as shown in SEQ ID NO 5;
more preferably, the signal peptide comprises the signal peptide of the following molecule: the α and β chains of the T cell receptor, CD3 ζ, CD3 ∈, CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS, IgG 6;
most preferably, the signal peptide is an IgG6 signal peptide;
most preferably, the amino acid sequence of the IgG6 signal peptide is shown in SEQ ID NO 6;
more preferably, the self-cleaving peptide is T2A;
most preferably, the amino acid sequence of T2A is shown as SEQ ID NO. 7;
more preferably, the immune modulatory molecule or cytokine comprises: b7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL-12, IL-2, IL-18, IL-21, LEC, OX 40L;
most preferably, the immunomodulatory molecule or cytokine is IL-21;
most preferably, the amino acid sequence of the IL-21 is shown as SEQ ID NO 8;
most preferably, the chimeric antigen receptor is selected from any one of the following groups:
(1) a fusion protein comprising an IgG6 signal peptide, scFv, a CD8 α hinge region, a CD8 α transmembrane domain, a CD28 costimulatory signal domain, a CD3 ζ intracellular signaling domain, T2A, and IL-21, linked in sequence;
(2) derivative fusion protein formed by substituting, deleting or adding one or more amino acids in the amino acid sequence of the fusion protein in (1);
most preferably, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO 9.
2. A polynucleotide having a sequence comprising, connected in series, a coding sequence for a chimeric antigen receptor targeting B7H3, or a complement thereof;
preferably, the coding sequence of the chimeric antigen receptor targeting B7H3 comprises the coding sequence of IgG6 signal peptide, scFv, CD 8a hinge region, CD 8a transmembrane domain, CD28 costimulatory signal domain, CD3 ζ intracellular signaling domain, T2A, IL-21, connected in sequence;
preferably, the coding sequence of the IgG6 signal peptide is shown as SEQ ID NO. 10;
preferably, the coding sequence of the scFv is shown as SEQ ID NO. 11;
preferably, the coding sequence for the CD 8a hinge region is as shown in SEQ ID NO 12;
preferably, the coding sequence of the transmembrane domain of CD8 alpha is shown in SEQ ID NO 13;
preferably, the coding sequence of the CD28 co-stimulatory signaling domain is shown in SEQ ID NO: 14;
preferably, the coding sequence for the intracellular signaling domain of CD3 ζ is set forth in SEQ ID NO: 15;
preferably, the coding sequence of T2A is shown as SEQ ID NO: 16;
preferably, the coding sequence of IL-21 is shown in SEQ ID NO 17;
preferably, the coding sequence of the chimeric antigen receptor targeting B7H3 is shown in SEQ ID NO 18.
3. A vector comprising the polynucleotide of claim 2, wherein said vector comprises a cloning vector, an expression vector;
preferably, the vector includes a DNA vector, an RNA vector, a plasmid, a virus-derived vector;
more preferably, the viral derived vector includes a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector, a herpesvirus vector;
most preferably, the vector is a retroviral vector.
4. An engineered host cell comprising the polynucleotide of claim 2, the vector of claim 3;
preferably, the host cell comprises a eukaryotic cell, a prokaryotic cell;
more preferably, the host cell is a eukaryotic cell;
most preferably, the eukaryotic cells include mammalian cells, plant cells, yeast cells;
most preferably, the eukaryotic cell is an immune cell;
most preferably, the immune cells comprise T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof;
most preferably, the immune cell is an iNKT cell.
5. A derivative comprising a detectably labeled chimeric antigen receptor of claim 1 and/or a polynucleotide of claim 2, a chimeric antigen receptor of claim 1 and/or a polynucleotide of claim 2 that confers antibiotic resistance, a chimeric antigen receptor of claim 1 and/or a polynucleotide of claim 2 bound or coupled to a therapeutic agent, a chimeric antigen receptor of claim 1 and/or a polynucleotide of claim 2;
preferably, the detectable label comprises a fluorescent dye, colloidal gold, a chemiluminescent label, a chemiluminescent catalyst;
more preferably, the chemiluminescent label comprises luminol and derivatives thereof, isoluminol and derivatives thereof, acridinium ester and derivatives thereof, adamantane, rare earth elements, bipyridine ruthenium complex;
more preferably, the chemiluminescent catalyst comprises horseradish peroxidase, alkaline phosphatase;
preferably, the antibiotic resistance gene comprises a penicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, a kanamycin resistance gene;
preferably, the therapeutic agent comprises a radionuclide, cytokine, gold nanoparticle, viral particle, liposome, nanomagnet, prodrug-activating enzyme, chemotherapeutic agent;
more preferably, the cytokines include IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN- γ, TNF- β, TNF- α, G-CSF, M-CSF;
more preferably, the chemotherapeutic agent comprises cisplatin, paclitaxel, vincristine, asparaginase, oxaliplatin, platinum oxalate, levofloxacin.
6. A pharmaceutical composition comprising the polynucleotide of claim 2, the vector of claim 3, the engineered host cell of claim 4;
preferably, the pharmaceutical composition further comprises one or more pharmaceutically or physiologically acceptable carriers, diluents or excipient combinations.
7. A kit comprising the polynucleotide of claim 2, the vector of claim 3;
preferably, the kit further comprises reagents for introducing the polynucleotide, vector into a host cell;
preferably, the kit further comprises instructions for introducing the polynucleotide, vector, into a host cell.
8. A biological agent comprising the engineered host cell of claim 4.
9. Any of the following methods, wherein the method comprises:
(1) a method of stimulating an immune response to a target cell population or tissue in a mammal, comprising the steps of: administering to a mammal an effective amount of the engineered host cell of claim 4;
(2) a method of making the engineered host cell of claim 4, comprising the steps of: introducing the polynucleotide of claim 2, the vector of claim 3 into a host cell;
preferably, the method of introduction comprises lipofection, microinjection, electroporation, DNA vectors, RNA vectors, retroviral vectors, lentiviral vectors, poxvirus vectors, herpes simplex virus vectors, adenoviral vectors, adeno-associated virus vectors;
more preferably, the method of introduction is using a retroviral vector.
10. The use of any one of the following aspects, wherein said use comprises:
(1) use of the chimeric antigen receptor of claim 1, the polynucleotide of claim 2, the vector of claim 3, the engineered host cell of claim 4, the derivative of claim 5, the pharmaceutical composition of claim 6, the kit of claim 7, the biological agent of claim 8 in the manufacture of a medicament for the prevention and/or treatment of a tumor;
(2) use of the chimeric antigen receptor of claim 1, the polynucleotide of claim 2, the vector of claim 3, the engineered host cell of claim 4, the derivative of claim 5 for the preparation of a kit for the prevention and/or treatment of a tumor;
(3) use of the chimeric antigen receptor of claim 1, the polynucleotide of claim 2, the vector of claim 3, the engineered host cell of claim 4, the derivative of claim 5, the pharmaceutical composition of claim 6, the kit of claim 7 for the preparation of a biological agent for the prevention and/or treatment of a tumor;
(4) the pharmaceutical composition of claim 6 for use in the prevention and/or treatment of tumors;
(5) use of a kit according to claim 7 for the preparation of immune cells for the prevention and/or treatment of tumors;
(6) use of the biological agent of claim 8 for the prevention and/or treatment of tumors;
(7) use of the chimeric antigen receptor of claim 1 in the preparation of polynucleotides, vectors, engineered host cells, derivatives;
(8) use of the polynucleotide of claim 2 in the preparation of vectors, engineered host cells, derivatives;
(9) use of the vector of claim 3 in the preparation of an engineered host cell, derivative;
(10) use of the engineered host cell of claim 4 in the preparation of a derivative;
(11) use of IL-21 for the preparation of chimeric antigen receptor modified immune cells for the prevention and/or treatment of tumors;
preferably, the tumor is a B7H 3-expressing tumor;
more preferably, the tumor comprises ovarian cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, esophageal cancer, prostate cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, liver cancer, bladder cancer, osteosarcoma, glioma, myeloid leukemia;
most preferably, the tumor is a renal cancer.
Background
Tumor cell immunotherapy (Tumor cell immunotherapy) is the fourth major Tumor treatment technology after surgery, radiotherapy and chemotherapy, and is a novel treatment method for separating Tumor specific or non-specific killer cells activated in vitro and infused back to patients themselves or allogeneic patients by using biotechnology and biological preparation. Traditional therapies for tumors, including surgery, chemotherapy and radiation therapy, all have limitations: surgery is often not eradicated by infiltration of cancer cells into adjacent or metastatic tissue; chemotherapy and radiation therapy are limited by toxicity and damage to other normal tissues in the body. In recent years, the popular targeted therapy can design corresponding therapeutic drugs aiming at the definite carcinogenic sites on the cellular molecular level, and the drugs enter the body and specifically select the carcinogenic sites to combine and act so as to cause the specific death of tumor cells. The tumor cell immunotherapy is different from the traditional therapy, the immune system of a normal human body can recognize and eliminate tumor cells, but cancer patients, particularly late-stage cancer patients, are often accompanied by the damage of the immune system, so that the capacity of eliminating the tumor cells is lost, in this case, the aim of controlling and killing the tumor cells can be achieved by exciting and enhancing the immune function of an organism, and the treatment method is the tumor cell immunotherapy.
In the tumor cell immunotherapy, Chimeric antigen receptor modified T cell (CAR-T) and Chimeric antigen receptor modified NK cell (CAR-NK) immunotherapy are the two therapies that are the most rapidly developed at present, and the principle thereof is that T cells or NK cells modified by Chimeric Antigen Receptor (CAR) can specifically recognize tumor-associated antigens on the surface of tumor cells, so that the targeting, killing activity and persistence of effector T cells or NK cells are higher than those of conventionally applied immune cells, and the microenvironment of tumor local immunosuppression can be overcome, and the host immune tolerance state can be broken. Natural killer T cells (NKTs), which are immune cells at the same time, are different from conventional T cells or NK cells, but are special T cells with innate immune response functions, have both NK cell functions and T cell characteristics, and are divided into type I NKT cells, type II NKT cells, and type III NKT cells, wherein type I NKT cells are also called constant Natural killer T cells (inkts), which are the most extensive and most deep NKT cells in current research, and a large number of studies indicate that iNKT cells have a better anti-tumor effect and have a great potential application value in tumor immunotherapy.
B7H3(CD276) belongs to B7 superfamily, is a transmembrane glycoprotein, and its extracellular region structure is 2, one is monovalent 2Ig-B7-H3, one is bivalent 4Ig-B7-H3 composed of 2 repeating unit structures. Related studies have shown that B7H3 inhibits T Cell proliferation and cytokine release by interacting with a receptor of unknown structure (Suh W K, Gajewska B U, Okada H, et al. the B7 family member B7-H3 presenting a dominant down-regulation T half type 1-mediated immunity [ J ]. Nature immunity, 2003,4(9):899-906.) although the receptor of B7H3 is unknown, more and more reports have been made in recent years about negative regulation of B7H3 and receptor action in tumor immunity, and tumor cells express B7H3 to evade immune surveillance of CD8+ T cells, and that B7H3 knockout mice or antibodies against B7H3 all inhibit tumor growth significantly in response to the receptor function of Cell growth inhibition (March H J-expressing Cell activation) and Cell growth inhibition by the receptor of growth of tumor Cell activation T + T Cell (March H23. J.: immune activation) 2017,27(8): 1034-1045), which all indicate that B7H3 can be used as an effective therapeutic target for tumor immunotherapy.
In view of the above, the invention provides a fully human chimeric antigen receptor targeting B7H3 and iNKT cells thereof based on the high expression characteristic of B7H3 in tumor cells and the characteristic of the inhibition activity of B7H3 on immune cells, and has an important application prospect in the field of tumor cell immunotherapy.
Disclosure of Invention
The invention aims to provide a fully human chimeric antigen receptor targeting B7H3 co-expressing IL-21, an iNKT cell and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, a fully human chimeric antigen receptor is provided that targets B7H3.
Further, the chimeric antigen receptor comprises an antibody scFv that specifically binds to B7H 3;
preferably, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain;
preferably, the chimeric antigen receptor further comprises a hinge region;
preferably, the chimeric antigen receptor further comprises a costimulatory signal domain;
preferably, the chimeric antigen receptor further comprises a signal peptide;
preferably, the chimeric antigen receptor further comprises a self-cleaving peptide;
preferably, the chimeric antigen receptor further comprises an immunomodulatory molecule or cytokine;
more preferably, the amino acid sequence of the scFv comprises the amino acid sequence shown as SEQ ID NO. 1, the amino acid sequence with at least 90% homology with the amino acid sequence shown as SEQ ID NO. 1, the amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids with the amino acid sequence shown as SEQ ID NO. 1;
most preferably, the amino acid sequence of the scFv is the amino acid sequence shown in SEQ ID NO. 1;
more preferably, the transmembrane domain and hinge region comprises the transmembrane domain and hinge region of the following molecules: CD8 α, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3 ε;
most preferably, the transmembrane domain and hinge region is a CD 8a transmembrane domain and CD 8a hinge region;
most preferably, the amino acid sequence of the transmembrane domain of CD8 alpha is shown in SEQ ID NO 2;
most preferably, the amino acid sequence of the CD 8a hinge region is shown in SEQ ID NO 3;
more preferably, the intracellular signaling domain comprises the intracellular signaling domains of the following molecules: CD3 ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 epsilon, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, fcepsilon RI, DAP10, DAP12, CD66d, DAP10, DAP12, FYN;
most preferably, the intracellular signaling domain is a CD3 ζ intracellular signaling domain;
most preferably, the amino acid sequence of the intracellular signaling domain of CD3 ζ is set forth in SEQ ID NO 4;
more preferably, the costimulatory signal domain comprises the costimulatory signal domain of the following molecule: CD28, ICOS, CD27, CD19, CD4, CD8 α, CD8 β, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, CD 278;
most preferably, the costimulatory signaling domain is a CD28 costimulatory signaling domain;
most preferably, the amino acid sequence of the CD28 co-stimulatory signaling domain is as shown in SEQ ID NO 5;
more preferably, the signal peptide comprises the signal peptide of the following molecule: the α and β chains of the T cell receptor, CD3 ζ, CD3 ∈, CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS, IgG 6;
most preferably, the signal peptide is an IgG6 signal peptide;
most preferably, the amino acid sequence of the IgG6 signal peptide is shown in SEQ ID NO 6;
more preferably, the self-cleaving peptide is T2A;
most preferably, the amino acid sequence of T2A is shown as SEQ ID NO. 7;
more preferably, the immune modulatory molecule or cytokine comprises: b7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL-12, IL-2, IL-18, IL-21, LEC, OX 40L;
most preferably, the immunomodulatory molecule or cytokine is IL-21;
most preferably, the amino acid sequence of the IL-21 is shown as SEQ ID NO 8;
most preferably, the chimeric antigen receptor is selected from any one of the following groups:
(1) a fusion protein comprising an IgG6 signal peptide, scFv, a CD8 α hinge region, a CD8 α transmembrane domain, a CD28 costimulatory signal domain, a CD3 ζ intracellular signaling domain, T2A, and IL-21, linked in sequence;
(2) derivative fusion protein formed by substituting, deleting or adding one or more amino acids in the amino acid sequence of the fusion protein in (1);
most preferably, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO 9.
In a second aspect, the present invention provides a polynucleotide.
The sequence of the polynucleotide is a coding sequence of a chimeric antigen receptor containing target B7H3 which are connected in sequence, or a complementary sequence thereof;
preferably, the coding sequence of the chimeric antigen receptor targeting B7H3 comprises the coding sequence of IgG6 signal peptide, scFv, CD 8a hinge region, CD 8a transmembrane domain, CD28 costimulatory signal domain, CD3 ζ intracellular signaling domain, T2A, IL-21, sequentially linked;
preferably, the coding sequence of the IgG6 signal peptide is shown as SEQ ID NO. 10;
preferably, the coding sequence of the scFv is shown as SEQ ID NO. 11;
preferably, the coding sequence for the CD 8a hinge region is as shown in SEQ ID NO 12;
preferably, the coding sequence of the transmembrane domain of CD8 alpha is shown in SEQ ID NO 13;
preferably, the coding sequence of the CD28 co-stimulatory signaling domain is shown in SEQ ID NO: 14;
preferably, the coding sequence for the intracellular signaling domain of CD3 ζ is set forth in SEQ ID NO: 15;
preferably, the coding sequence of T2A is shown as SEQ ID NO: 16;
preferably, the coding sequence of IL-21 is shown in SEQ ID NO 17;
preferably, the coding sequence of the chimeric antigen receptor targeting B7H3 is shown in SEQ ID NO 18.
In a third aspect, the invention provides a vector comprising a polynucleotide according to the second aspect of the invention.
Further, the vector comprises a cloning vector and an expression vector;
preferably, the vector includes a DNA vector, an RNA vector, a plasmid, a virus-derived vector;
more preferably, the viral derived vector includes a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector, a herpesvirus vector;
most preferably, the vector is a retroviral vector.
In a fourth aspect of the invention, an engineered host cell is provided.
Further, the engineered host cell comprises the polynucleotide of the second aspect of the invention, the vector of the third aspect of the invention;
preferably, the host cell comprises a eukaryotic cell, a prokaryotic cell;
more preferably, the host cell is a eukaryotic cell;
most preferably, the eukaryotic cells include mammalian cells, plant cells, yeast cells;
most preferably, the eukaryotic cell is an immune cell;
most preferably, the immune cells comprise T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof;
most preferably, the immune cell is an iNKT cell.
Further, the host cell may be obtained from a subject or a commercially available culture (e.g., American Type Culture Collection (ATCC));
preferably, the host cell may be obtained from a number of sources in the subject, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, tumors of the subject.
A fifth aspect of the invention provides a derivative.
Further, the derivative comprises a detectably labeled chimeric antigen receptor of the first aspect of the invention and/or a polynucleotide of the second aspect of the invention, a chimeric antigen receptor of the first aspect of the invention and/or a polynucleotide of the second aspect of the invention that confers antibiotic resistance, a chimeric antigen receptor of the first aspect of the invention and/or a polynucleotide of the second aspect of the invention bound or coupled to a therapeutic agent;
preferably, the detectable label comprises a fluorescent dye, colloidal gold, a chemiluminescent label, a chemiluminescent catalyst;
more preferably, the chemiluminescent label comprises luminol and derivatives thereof, isoluminol and derivatives thereof, acridinium ester and derivatives thereof, adamantane, rare earth elements, bipyridine ruthenium complex;
more preferably, the chemiluminescent catalyst comprises horseradish peroxidase, alkaline phosphatase;
preferably, the antibiotic resistance gene comprises a penicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, a kanamycin resistance gene;
preferably, the therapeutic agent comprises a radionuclide, cytokine, gold nanoparticle, viral particle, liposome, nanomagnet, prodrug-activating enzyme, chemotherapeutic agent;
more preferably, the cytokines include IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN- γ, TNF- β, TNF- α, G-CSF, M-CSF;
more preferably, the chemotherapeutic agent comprises cisplatin, paclitaxel, vincristine, asparaginase, oxaliplatin, platinum oxalate, levofloxacin.
In a sixth aspect of the invention, a pharmaceutical composition is provided.
Further, the pharmaceutical composition comprises a polynucleotide of the second aspect of the invention, a vector of the third aspect of the invention, an engineered host cell of the fourth aspect of the invention;
preferably, the pharmaceutical composition further comprises one or more pharmaceutically or physiologically acceptable carriers, diluents or excipient combinations.
Further, such combinations may include: 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, such as glycine; an antioxidant; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative.
The pharmaceutical compositions disclosed herein may be formulated for oral, intravenous, topical, enteral and/or parenteral administration as desired.
The seventh aspect of the present invention provides a kit.
Further, the kit comprises the polynucleotide of the second aspect of the invention, the vector of the third aspect of the invention;
preferably, the kit further comprises reagents for introducing the polynucleotide, vector into a host cell;
preferably, the kit further comprises instructions for introducing the polynucleotide, vector, into a host cell.
In an eighth aspect, the invention provides a biological agent comprising an engineered host cell according to the fourth aspect of the invention.
Further, the biological agent may be used in combination with other therapeutic agents.
A ninth aspect of the present invention provides any one of the following methods:
(1) a method of stimulating an immune response to a target cell population or tissue in a mammal, comprising the steps of: administering to a mammal an effective amount of an engineered host cell according to the fourth aspect of the invention;
(2) a method of producing an engineered host cell according to the fourth aspect of the invention, comprising the steps of: introducing the polynucleotide of the second aspect of the invention, the vector of the third aspect of the invention, into a host cell;
preferably, the method of introduction comprises lipofection, microinjection, electroporation, DNA vectors, RNA vectors, retroviral vectors, lentiviral vectors, poxvirus vectors, herpes simplex virus vectors, adenoviral vectors, adeno-associated virus vectors;
more preferably, the method of introduction is using a retroviral vector.
The invention also provides a method of treating a subject having a B7H 3-expressing cancer.
Further, the method comprises administering the engineered host cell of the fourth aspect of the invention, the pharmaceutical composition of the sixth aspect of the invention, the biological agent of the eighth aspect of the invention to a subject in need thereof.
A tenth aspect of the invention provides the use of any one of the following aspects:
(1) use of a chimeric antigen receptor according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, a vector according to the third aspect of the invention, an engineered host cell according to the fourth aspect of the invention, a derivative according to the fifth aspect of the invention, a pharmaceutical composition according to the sixth aspect of the invention, a kit according to the seventh aspect of the invention, a biological agent according to the eighth aspect of the invention for the manufacture of a medicament for the prevention and/or treatment of a tumour;
(2) use of a chimeric antigen receptor according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, a vector according to the third aspect of the invention, an engineered host cell according to the fourth aspect of the invention, a derivative according to the fifth aspect of the invention for the preparation of a kit for the prevention and/or treatment of a tumor;
(3) use of a chimeric antigen receptor according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, a vector according to the third aspect of the invention, an engineered host cell according to the fourth aspect of the invention, a derivative according to the fifth aspect of the invention, a pharmaceutical composition according to the sixth aspect of the invention, a kit according to the seventh aspect of the invention for the preparation of a biological agent for the prevention and/or treatment of a tumor;
(4) the use of a pharmaceutical composition according to the sixth aspect of the invention for the prevention and/or treatment of tumours;
(5) use of a kit according to the seventh aspect of the invention for the preparation of immune cells for the prevention and/or treatment of tumours;
(6) the biological agent according to the eighth aspect of the present invention is used for preventing and/or treating tumors;
(7) use of a chimeric antigen receptor according to the first aspect of the invention in the preparation of a polynucleotide, a vector, an engineered host cell, a derivative;
(8) use of a polynucleotide according to the second aspect of the invention in the preparation of vectors, engineered host cells, derivatives;
(9) use of a vector according to the third aspect of the invention in the preparation of an engineered host cell, derivative;
(10) use of an engineered host cell according to the fourth aspect of the invention in the preparation of a derivative;
(11) use of IL-21 for the preparation of chimeric antigen receptor modified immune cells for the prevention and/or treatment of tumors;
preferably, the tumor is a B7H 3-expressing tumor;
more preferably, the tumor comprises ovarian cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, esophageal cancer, prostate cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, liver cancer, bladder cancer, osteosarcoma, glioma, myeloid leukemia;
most preferably, the tumor is a renal cancer.
The invention has the following advantages and beneficial effects:
based on the high expression characteristic of B7H3 in tumor cells and the characteristic of the high expression characteristic of B7H3 in immune cells, the invention provides a B7H 3-targeted fully human chimeric antigen receptor and iNKT cells thereof, and experiments prove that the B7H 3-targeted B7H3.CAR/IL-21-iNKT cells prepared by the invention have strong cytokine release capacity and tumor cell killing capacity, can effectively eliminate tumor cells, and have important application prospects in the field of tumor cell immunotherapy.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
fig. 1 shows a graph of the results of CAR positivity detection using flow cytometry, wherein, a-graph: UT-iNKT, Panel B: b7h3.car-iNKT, panel C: b7h3. car/IL-21-iNKT;
FIG. 2 shows a graph of statistical results of CAR transduction rates measured using flow cytometry;
FIG. 3 is a graph showing the results of examining cytokine release capacity of B7H3.CAR/IL-21-iNKT cells in coculture with different renal cancer cells, wherein, A is a graph: IFN-. gamma.B Panels: IL-2;
FIG. 4 shows a graph of the results of flow cytometry detection of B7H3.CAR/IL-21-iNKT cell apoptosis;
fig. 5 is a graph showing the results of RTCA assay to examine the killing effect of b7h3.car/IL-21-iNKT cells on renal cancer cells 786-O, wherein a: 2/1, B: E/T1/1, C: 1/2;
FIG. 6 is a graph showing the results of an RTCA assay for detecting the killing effect of B7H3.CAR/IL-21-iNKT cells on OSRC-2 in renal cancer cells, wherein A: 5/1, B: E/T1/1, C: 1/5;
FIG. 7 is a graph showing the results of the clearance of B7H3.CAR/IL-21-iNKT from mouse renal cancer subcutaneous transplantation tumors, wherein A is a graph: experimental flow chart, panel B: statistical plot of tumor volumes in mice, panel C: statistical plot of CAR-iNKT cell number in peripheral blood.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.
Example 1 preparation of fully human B7H3.CAR/IL-21-iNKT targeting B7H3 Co-expression of IL-21
1. Experimental methods
(1) Preparation of iNKT
1) Isolation of PBMCs: collecting peripheral blood of a donor, diluting whole blood with equivalent physiological saline, adding lymphocyte separation liquid and diluted blood into a centrifugal tube according to the ratio of 1:2, centrifuging at 2000rpm/min for 20 minutes, collecting leucocyte layer cells, cleaning twice with physiological saline, and centrifuging at 1500rpm/min for 8 minutes to obtain peripheral blood mononuclear cells PBMCs;
2) induction of iNKT cells: resuspending PBMCs in lymphocyte culture medium at 2X 106/mL, add α -Galcer, IL-2, IL-21, IL-4 and GM-CSF, inoculate cells in 24-well plates, place at 37 ℃ with 5% CO2An incubator, which observes the cell state every day and changes the liquid half every other day;
3) magnetic bead sorting iNKT cells: collecting the induced cells on the 10 th day, re-suspending the cells by using 500 mu L of MACS buffer, adding Anti-iNKT MicroBeads according to the dosage of the instruction, uniformly mixing, incubating at 4 ℃ for 30 minutes, adding 5mL of MACS buffer for washing, centrifuging at 400 Xg for 5 minutes, and discarding the supernatant; resuspend with 500 μ L MACS buffer, load LS sorting column, and wash MACS buffer 3 times, 3mL each time; finally, placing the sorting column in a collecting tube, and adding 500 mu L of MACS buffer for elution to obtain iNKT positive cells;
4) activation expanded iNKT cells: on day 10, the cells obtained were further purified by resuspension in lymphocyte cultures containing IL-7 and IL-15, seeded on CD3Ab and CD28Ab pre-coated plates, placed at 37 ℃ and 5% CO2The incubator performs a large number of amplifications.
(2) Construction of CAR expression vectors
1) Synthesizing an scFv coding sequence targeted to human B7H3, said scFv comprising a heavy chain VH and a light chain VL linked by a short G4S peptide;
2) carrying out double enzyme digestion on a reverse transcription virus vector MSCV and a synthesized scFv targeting human B7H3 through Nco I and Mlu I, recovering fragments, connecting the recovered target fragments with T4 ligase, and then transforming Stbl3 competent cells;
3) selecting a single clone to carry out plasmid extraction, carrying out enzyme digestion identification, and then sending to sequencing for confirmation, wherein the correct plasmid is the B7H3. CAR;
in the construction method, the coding sequence of the fully human chimeric antigen receptor of the target B7H3 coexpression IL-21 is shown as SEQ ID NO. 18.
(3) Retroviral packaging
Mixing 6 mu g of shuttle plasmid MSCV-B7H3.CAR containing CAR structure and 4 mu g of helper plasmid pCL-Ampho in 300 mu L of opti-MEM culture medium, dropwise adding 30 mu L of Genejuice transfection reagent in another 300 mu L of opti-MEM culture medium, flicking and uniformly mixing, standing at room temperature for 5 minutes, dropwise adding the mixture containing the transfection reagent into the plasmid mixture, shaking and uniformly mixing, standing at room temperature for 15 minutes, then dropwise adding PEI and plasmid mixture into a pre-paved 293T cell culture dish, gently shaking and uniformly mixing, collecting supernatant after 48-72 hours, filtering through a 0.45 mu m needle filter, and storing at ultra-low temperature for later use.
(4) Viral infection of iNKT cells
Adding B7H3.CAR virus solution into 10 μ M HEPES and 6-8 μ g/mL polybrene, mixing, resuspending activated iNKT cells with the virus solution, adding into a 24-well plate pre-coated with RetroNectin, centrifuging at 1500g and 30 deg.C for 2H, removing supernatant, adding X-Vivo culture medium containing 5% fetal calf serum, 200U/mL IL-2, 10ng/mL IL-7 and 5ng/mL IL-15, continuing amplification culture, and obtaining the whole human B7H3. CAR-NKT cells B7H3.CAR/IL-21-iNKT which target B7H3 co-expression IL-21.
(5) Detection of CAR expression efficiency
Taking 2X 10 after 48-72 hours of virus infection5Cells were stained by first adding 1. mu.g/mL B7H3-Fc protein (R)&D, 1027-B3-100), incubating at 4 ℃ for 30 minutes, washing the cells, adding AF647-anti-human IgG antibody (Jackson, 109-.
2. Results of the experiment
The results of CAR transfection efficiency of b7h3.CAR/IL-21-iNKT cells prepared above using flow assay are shown in fig. 1A-C and fig. 2, and the results show: the CAR transfection efficiency of the B7H3.CAR/IL-21-iNKT cells is as high as 75-92%, and is obviously higher than that of the B7H3.CAR-iNKT cells and the iNKT cells, which indicates that the CAR transfection efficiency of the B7H3.CAR/IL-21-iNKT cells prepared by the invention is high.
Example 2 detection of apoptosis and Activity of B7H 3-Targeted fully human B7H3 CAR/IL-21-iNKT cytokines
1. Experimental methods
(1) Detection of cytokine Release ability Using ELISA kit
Collecting 2X 105B7H3.CAR/IL-21-iNKT cells separately from 2X 105Uniformly mixing renal cancer cells 786-O, OSRC-2, adding into a 24-pore plate for co-incubation, setting multiple pores, and collecting culture supernatant after 24 h; and detecting the contents of IFN-gamma and IL-2 by using an ELISA kit.
(2) Detection of B7H3.CAR-iNKT apoptosis Using flow cytometry
The B7H3.CAR-iNKT and B7H3.CAR/IL-21-iNKT cells prepared in this example were collected, resuspended in T cell medium without cytokines (IL-2/IL-7/IL-15), and placed in CO2An incubator. The washed cells were collected at 0 hour and 72 hours, resuspended with 1 × Annexin V Binding Buffer, FITC-Annexin V and PI were added and incubated for 15 minutes at room temperature in the dark, washed, resuspended on the machine for detection, and the effect of co-expressed IL-21 on apoptosis of B7H3.CAR-iNKT cells was analyzed.
2. Results of the experiment
The results are shown in FIGS. 3A and B and show that: compared with iNKT and B7H3.CAR-iNKT, the B7H3.CAR/IL-21-iNKT cell prepared by the invention can secrete high-level cell factor IL-2, and the secretion of the cell factor IFN-gamma has no significant difference, thereby indicating that the B7H3.CAR/IL-21-iNKT cell prepared by the invention has stronger expansion capability and survival capability.
The results of fig. 4 show that: the B7H3.CAR-iNKT cells are subjected to massive apoptosis after being cultured for 72 hours by starvation, and the apoptosis ratio of the B7H3.CAR/IL-21-iNKT cells is obviously less than that of the B7H3.CAR-iNKT cells, thereby indicating that the B7H3.CAR/IL21-iNKT cells prepared by the invention have enhanced anti-apoptosis capability.
Example 3B 7H3 CAR/IL-21-iNKT cells ability to kill renal carcinoma cells in vitro
1. Experimental methods
Firstly, adding 50 mu L DMEM complete culture medium into an E-Plate detection Plate of an xCELLigence cell function analyzer, and measuring a background impedance value; collecting renal cancer cells 786-O, OSRC-2 at log phase, adjusting cell suspension concentration to 1 × 105Adding 100 mu L of cell suspension into an E-Plate detection Plate, standing for 30 minutes at room temperature, and then placing on a detection table; real-time dynamic observation of cells to be targetedWhen proliferation is in the plateau phase, 50 μ L of effector cells iNKT, b7h3.car-iNKT and b7h3.car/IL-21-iNKT were added to the experimental wells at effective target ratios 5/1, 1/1, 1/5, while cell-mediated killing effect curves were observed in real time using individual tumor cells as control groups.
2. Results of the experiment
The results are shown in FIGS. 5A-C and FIGS. 6A-C, showing that: both the B7H3.CAR/IL-21-iNKT cell and the B7H3.CAR-iNKT cell can efficiently kill B7H3 high-expression tumor target cells, and the in-vitro killing activities of the B7H3 high-expression tumor target cells are not obviously different.
Example 4 validation of in vivo depletion of renal carcinoma transplanted tumors by B7H3 CAR/IL-21-iNKT cells
1. Experimental methods
Male NCG mice of 6 weeks of age were purchased and injected subcutaneously at 4X 106786-O-Luc-GFP cells to construct a model of subcutaneous transplantation tumor of kidney cancer of mice, and the mice were randomly grouped into a Blank group, a B7H3.CAR-iNKT group and a B7H3.CAR/IL-21-iNKT group after tumor formation on day 10, and each group had 5 mice, and 3 mice were divided into 3 groups; treatment by tail vein infusion of B7H3.CAR-iNKT and B7H3.CAR/IL-21-iNKT cells on days 11 and 18, respectively, 5X 106A/only; treatment effect was observed 2 times per week by measuring tumor volume and CAR-iNKT survival in vivo was examined by submaxillary venous bleeding and survival of mice was recorded.
2. Results of the experiment
The results of the experiment are shown in FIGS. 7A-C, and the results of FIG. 7A show: establishment of models of subcutaneous transplantation tumor of kidney cancer in NCG mice and treatment pattern maps using b7h3.car-iNKT and b7h3.car/IL-21-iNKT cells; the results in FIG. 7B show that: b7h3.car/IL-21-iNKT cells have better ability to inhibit tumor growth compared to Blank and b7h3.car-iNKT groups; fig. 7C results show: the number of CAR-iNKT cells in peripheral blood of mice in the b7h3.CAR/IL-21-iNKT group was significantly higher than those in the Blank and b7h3.CAR-iNKT groups at 14, 21 days after treatment, indicating that the b7h3.CAR/IL-21-iNKT cells were more survivable in vivo.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.
Sequence listing
<110> Xuzhou university of medicine
<120> fully human chimeric antigen receptor targeting B7H3 to co-express IL-21, iNKT cell and application thereof
<141> 2021-07-12
<150> 2021107393054
<151> 2021-06-30
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 240
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Glu Val Gln Leu Phe Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Val Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr
100 105 110
Val Thr Val Ser Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser
130 135 140
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
145 150 155 160
Ile Ser Ile Tyr Leu Asn Trp Tyr Arg Gln Gln Pro Gly Lys Ala Pro
165 170 175
Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser
180 185 190
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
195 200 205
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Thr Tyr
210 215 220
Ser Thr Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys
225 230 235 240
<210> 2
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
1 5 10 15
Val Ile Thr Leu Tyr Cys
20
<210> 3
<211> 47
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
35 40 45
<210> 4
<211> 112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 5
<211> 41
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 6
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys
<210> 7
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu
1 5 10 15
Glu Asn Pro Gly Pro
20
<210> 8
<211> 162
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Met Arg Ser Ser Pro Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met
1 5 10 15
Val Ile Phe Leu Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly Gln
20 25 30
Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln
35 40 45
Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro
50 55 60
Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln
65 70 75 80
Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile
85 90 95
Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala
100 105 110
Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr
115 120 125
Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu
130 135 140
Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu
145 150 155 160
Asp Ser
<210> 9
<211> 670
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Glu Val Gln Leu Phe Gln Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45
Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala
65 70 75 80
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
85 90 95
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Gly Val Gly Arg Gly Phe Asp Tyr Trp Gly Gln
115 120 125
Gly Thr Thr Val Thr Val Ser Ser Ser Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser
145 150 155 160
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
165 170 175
Ser Gln Ser Ile Ser Ile Tyr Leu Asn Trp Tyr Arg Gln Gln Pro Gly
180 185 190
Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly
195 200 205
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
210 215 220
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln
225 230 235 240
Gln Thr Tyr Ser Thr Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val
245 250 255
Asp Ile Lys Thr Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro
260 265 270
Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys
275 280 285
Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala
290 295 300
Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu
305 310 315 320
Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser Lys Arg Ser Arg
325 330 335
Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro
340 345 350
Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala
355 360 365
Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
370 375 380
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
385 390 395 400
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
405 410 415
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
420 425 430
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
435 440 445
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
450 455 460
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
465 470 475 480
Pro Pro Arg Ala Cys Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr
485 490 495
Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Arg Thr Met Arg Ser Ser
500 505 510
Pro Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met Val Ile Phe Leu
515 520 525
Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly Gln Asp Arg His Met
530 535 540
Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr
545 550 555 560
Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp Val Glu
565 570 575
Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu
580 585 590
Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val Ser Ile
595 600 605
Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg Arg Gln
610 615 620
Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys Pro
625 630 635 640
Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys Met Ile
645 650 655
His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser
660 665 670
<210> 10
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atggaatttg gcctgagctg gctgtttctg gtggcgattc tgaaaggcgt gcagtgc 57
<210> 11
<211> 720
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gaggtgcagc tgttccagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc caggggtgtt 300
ggccggggct ttgactactg gggccagggg accacggtca ccgtctcctc aagtggcggt 360
ggctctggcg gtggtgggtc gggtggcggc ggatcagaca tccagttgac ccagtctcca 420
tcctccctgt ctgcatctgt aggagacaga gtcaccatca cttgccgggc aagtcagagc 480
attagcatct atttaaattg gtatcggcag caaccaggga aagcccctaa gctcctgatc 540
tatgctgcat ccagtttgca aagtggggtc ccatcaaggt tcagtggcag tggatctggg 600
acagatttca ctctcaccat cagcagtctg caacctgaag attttgcaac ttacttctgt 660
caacagactt acagtacccc tccgtggacg ttcggccaag ggaccaaagt ggatatcaaa 720
<210> 12
<211> 141
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgatatcta c 141
<210> 13
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 60
tactgc 66
<210> 14
<211> 123
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60
gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120
tcc 123
<210> 15
<211> 336
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 16
<211> 63
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ggatctggag agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc 60
cct 63
<210> 17
<211> 489
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
atgagatcca gtcctggcaa catggagagg attgtcatct gtctgatggt catcttcttg 60
gggacactgg tccacaaatc aagctcccaa ggtcaagatc gccacatgat tagaatgcgt 120
caacttatag atattgttga tcagctgaaa aattatgtga atgacttggt ccctgaattt 180
ctgccagctc cagaagatgt agagacaaac tgtgagtggt cagctttttc ctgctttcag 240
aaggcccaac taaagtcagc aaatacagga aacaatgaaa ggataatcaa tgtatcaatt 300
aaaaagctga agaggaaacc accttccaca aatgcaggga gaagacagaa acacagacta 360
acatgccctt catgtgattc ttatgagaaa aaaccaccca aagaattcct agaaagattc 420
aaatcacttc tccaaaagat gattcatcag catctgtcct ctagaacaca cggaagtgaa 480
gattcctga 489
<210> 18
<211> 2013
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
atggaatttg gcctgagctg gctgtttctg gtggcgattc tgaaaggcgt gcagtgcgag 60
gtgcagctgt tccagtctgg gggaggcttg gtacagcctg gggggtccct gagactctcc 120
tgtgcagcct ctggattcac ctttagcagc tatgccatga gctgggtccg ccaggctcca 180
gggaaggggc tggagtgggt ctcagctatt agtggtagtg gtggtagcac atactacgca 240
gactccgtga agggccggtt caccatctcc agagacaatt ccaagaacac gctgtatctg 300
caaatgaaca gcctgagagc cgaggacacg gccgtatatt actgtgccag gggtgttggc 360
cggggctttg actactgggg ccaggggacc acggtcaccg tctcctcaag tggcggtggc 420
tctggcggtg gtgggtcggg tggcggcgga tcagacatcc agttgaccca gtctccatcc 480
tccctgtctg catctgtagg agacagagtc accatcactt gccgggcaag tcagagcatt 540
agcatctatt taaattggta tcggcagcaa ccagggaaag cccctaagct cctgatctat 600
gctgcatcca gtttgcaaag tggggtccca tcaaggttca gtggcagtgg atctgggaca 660
gatttcactc tcaccatcag cagtctgcaa cctgaagatt ttgcaactta cttctgtcaa 720
cagacttaca gtacccctcc gtggacgttc ggccaaggga ccaaagtgga tatcaaaacg 780
cgtaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccatcgc gtcgcagccc 840
ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgaggggg 900
ctggacttcg cctgtgatat ctacatctgg gcgcccttgg ccgggacttg tggggtcctt 960
ctcctgtcac tggttatcac cctttactgc aggagtaaga ggagcaggct cctgcacagt 1020
gactacatga acatgactcc ccgccgcccc gggcccaccc gcaagcatta ccagccctat 1080
gccccaccac gcgacttcgc agcctatcgc tccagagtga agttcagcag gagcgcagac 1140
gcccccgcgt accagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 1200
gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 1260
agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 1320
gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 1380
taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 1440
ccccctcgcg catgcggatc tggagagggc agaggaagtc ttctaacatg cggtgacgtg 1500
gaggagaatc ccggccctcg tacgatgaga tccagtcctg gcaacatgga gaggattgtc 1560
atctgtctga tggtcatctt cttggggaca ctggtccaca aatcaagctc ccaaggtcaa 1620
gatcgccaca tgattagaat gcgtcaactt atagatattg ttgatcagct gaaaaattat 1680
gtgaatgact tggtccctga atttctgcca gctccagaag atgtagagac aaactgtgag 1740
tggtcagctt tttcctgctt tcagaaggcc caactaaagt cagcaaatac aggaaacaat 1800
gaaaggataa tcaatgtatc aattaaaaag ctgaagagga aaccaccttc cacaaatgca 1860
gggagaagac agaaacacag actaacatgc ccttcatgtg attcttatga gaaaaaacca 1920
cccaaagaat tcctagaaag attcaaatca cttctccaaa agatgattca tcagcatctg 1980
tcctctagaa cacacggaag tgaagattcc tga 2013
