CN111686128A

CN111686128A - Application of hypoxia-controllable promoter in CAR-T

Info

Publication number: CN111686128A
Application number: CN201910184426.XA
Authority: CN
Inventors: 张巍; 朱秀秀; 赵永春; 赵文旭; 陈军; 黄霞
Original assignee: Chongqing Precision Biotech Co ltd
Current assignee: Chongqing Precision Biotech Co ltd
Priority date: 2019-03-12
Filing date: 2019-03-12
Publication date: 2020-09-22

Abstract

The invention belongs to the technical field of immunotherapy, and particularly relates to application of an hypoxia-controllable promoter in CAR-T, a CAR structure of the hypoxia-controllable promoter, an expression vector, CAR-T cells and application thereof, a method for improving the capability of the CAR-T cells to secrete IFN-gamma and/or IL-2 factors under a hypoxia environment, and a method for improving the killing capability of the CAR-T cells under the hypoxia environment. The CAR structure comprises an hypoxia-controllable promoter which is formed by connecting a Hif1a regulatory element and a mini-promoter, wherein the Hif1a regulatory element has the repetition number of 3-5. The CAR structure initiated by the hypoxia microenvironment induction can be effectively expressed in T lymphocytes, can enhance the activation of CAR-T cells in the hypoxia environment, enhances the effectiveness and safety of CAR-T, and can be used for the targeted therapy of tumors.

Description

Application of hypoxia-controllable promoter in CAR-T

Technical Field

The invention belongs to the technical field of immunotherapy, and particularly relates to application of an hypoxia-controllable promoter in CAR-T, a CAR structure of the hypoxia-controllable promoter, an expression vector, CAR-T cells and application thereof, a method for improving the capability of the CAR-T cells to secrete IFN-gamma and/or IL-2 factors under a hypoxia environment, and a method for improving the killing capability of the CAR-T cells under the hypoxia environment.

Background

CAR-T is known as chimeric antigen receptor T cell immunotherapy and achieves better performance in hematological tumors, but CAR-T is less effective in solid tumors than hematological tumors because CAR-T is less likely to enter solid tumors on the one hand, and because CAR-T cells cannot function normally even if they enter solid tumors because of the tumor microenvironment, both of which affect the efficacy of CAR-T cells in solid tumor therapy; and due to the high heterogeneous degree of the solid tumor, the target of the solid tumor is often expressed in normal tissues, so that the safety problems of off-target risk and the like also exist. Although there are currently structures for constructing fourth generation CARs, for constructing dual CARs (constructing CAR-T with dual antigens) or icars with activation inhibition, in the hope of improving the efficacy and safety of tumor CAR-T therapy, these CAR structures still suffer from safety or are more difficult to activate. Therefore, there is a need to construct a CAR structure that specifically initiates activation in the tumor microenvironment.

Acidity and hypoxia are two major physical factors in the tumor microenvironment, most cells in the tumor are in the hypoxic environment, and hypoxia changes the sugar metabolic pathway of tumor cells, generates a large amount of lactic acid, causes the characteristics of acidity in the tumor microenvironment, and the acidity can reduce tumor cell apoptosis, enhance cell proliferation and growth, and help the migration of tumor cells. The research of hypoxia in a solid tumor microenvironment is clear, and if a controllable promoter which is activated under the hypoxia condition can be designed, the purpose of specifically activating the expression of downstream protein in the tumor microenvironment so as to improve the effectiveness and safety of the CAR-T cell can be achieved, so that the designed and constructed hypoxia-controllable promoter and CAR-T cell technology are creatively applied to the immunotherapy of tumors.

Disclosure of Invention

In view of the above, an object of the present invention is to provide an application of an hypoxia-controllable promoter in CAR-T, wherein the hypoxia-controllable promoter can enhance the expression of a target gene, enhance the expression of a target factor (protein, nucleic acid, etc.), and improve the therapeutic effect of an anti-tumor drug in an anaerobic environment.

The invention starts from the hypoxia microenvironment of the tumor, regulates the expression of the specific regulatory protein by constructing the hypoxia-activated regulatory element, and shows the success of constructing the hypoxia environment by using the in vitro drugs and the subsequent CAR-T killing result in CoCl₂The CAR-T under the induced hypoxia condition has stronger killing effect than that of the non-induced CAR-T, and simultaneously, the constructed CAR structure and the CAR-T have the passive targeting characteristic of a hypoxia microenvironment, have stronger activity and lethality in the hypoxia microenvironment, and have safety characteristics in a non-hypoxia microenvironment. The method has important guiding significance for the clinical application of CAR-T and the development of new strategies of tumor combination therapy.

It is a further object of the invention to provide a CAR construct comprising a hypoxia-controllable promoter.

In order to achieve the purpose, the invention adopts the following scheme:

the CAR structure comprises an hypoxia-controllable promoter which is formed by connecting a Hif1 alpha regulatory element and a mini promoter, wherein the Hif1 alpha regulatory element has the repetition number of 3-5.

Preferably, the number of HREs is 5.

Further, the Mini-promoter is selected from a Cytomegalovirus (CMV) promoter (miniCMV), a promoter of HSV Thymidine Kinase (TK) (Mini-TK), a promoter of simian virus 40(SV40) (minSV40), an adenovirus late promoter (MLP), or a synthetic promoter.

Preferably, the mini-promoter is selected for miniCMV.

Further, the nucleic acid sequence of the hypoxia controllable Promoter is shown in SEQ ID NO.1 and SEQ ID NO.2, the SEQ ID NO.1 is formed by combining 5 repeated HRE control elements and a CMV mini Promoter which is weakly started, and the structure is 5HRE-CMVmini Promoter; the SEQ ID NO.2 is used for optimizing both ends of the SEQ ID NO.1 sequence, so that the matching of a promoter and a vector is facilitated.

Preferably, the nucleic acid sequence of the hypoxia controllable promoter is shown as SEQ ID NO. 2.

Further, the ScFv comprises a heavy chain variable region shown by an amino acid sequence SEQ ID NO.7 and a light chain variable region shown by an amino acid sequence SEQ ID NO.8 or comprises a heavy chain variable region shown by an amino acid sequence SEQ ID NO.9 and a light chain variable region shown by an amino acid sequence SEQ ID NO.10 or comprises a heavy chain variable region shown by an amino acid sequence SEQ ID NO.40 and a light chain variable region shown by an amino acid sequence SEQ ID NO. 41.

In certain embodiments, the amino acid sequence of the ScFv domain is as set forth in SEQ ID No.32 or SEQ ID No.33 or SEQ ID No.34 or SEQ ID No.35 or SEQ ID No. 36.

The CAR structure can be a conventional first generation, second generation and third generation CAR structure, and can also be a novel CAR structure such as a modified double CAR and a controllable CAR structure (such as FRB/FKBP12 regulation).

Further, the antigen recognized by the antigen recognition region is any one or more of CD19, CD20, CD123, CD22, BCMA, ROR1, mesothelin, PSCA, PSMA, c-Met, GPC-3, Her2, EGFRvIII, GD-2, NY-ESO-1TCR and MAGE A3 TCR.

Further, the CDR1, CDR2 and CDR3 of the heavy chain amino acid sequence SEQ ID No.7 and the light chain amino acid sequence SEQ ID No.8 are respectively the 26 th to 33 th, 51 th to 58 th and 97 th to 101 th positions of the heavy chain amino acid sequence SEQ ID No. 7; 23 st to 31 th, 49 th to 51 th, and 88 th to 96 th of SEQ ID NO. 8.

Further, the CDR1, CDR2 and CDR3 of the heavy chain amino acid sequence SEQ ID NO.9 and the light chain amino acid sequence SEQ ID NO.10 are respectively the 26 th to 33 th, 51 th to 57 th and 96 th to 109 th positions of the SEQ ID NO. 9; 27 to 32, 50 to 52 and 89 to 97 of SEQ ID NO. 10.

Further, the amino acid sequence of the CAR structure is shown as SEQ ID NO.5 or SEQ ID NO. 6.

The hinge region sequence in the CAR structure comprising the hypoxia promoter may be derived from: IgG, CD8, CD7, CD 4.

The transmembrane region in the CAR structure comprising the hypoxic promoter can be derived from: CD8, CD28, CD3, CD4, CD16, CD137, CD80, and CD 86.

The intracellular signaling region in the CAR structure comprising the hypoxic promoter can be derived from: CD3, CD137, CD28, CD27, OX40, ICOS, GITR, CD2, CD40, PD-1, PD1L, B7-H3, lymphocyte function-associated antigen-1 (LFA-1), ICAM-1, CD7, NKG2C, CD83, CD86 and CD 127.

In certain embodiments, the CAR structure comprising the hypoxic promoter carries a truncated EGFRt regulatory tag; in certain embodiments, the CAR comprising the hypoxic promoter is a universal CAR structure; in certain embodiments, the CAR structure comprising the hypoxic promoter carries a suicide gene such as iCasp 9.

In certain embodiments, the antigen recognition region can be a ligand/receptor that recognizes a target antigen; in certain embodiments, the ScFv may be a murine or fully human or human chimeric ScFv, a single domain antibody such as a shark, alpaca or camel antibody, a bispecific antibody, or a combination of target-specific fibronectin type III (FN3) domains designed to recognize a specific target.

In certain embodiments, the CAR structure comprises one or more components of a natural killer cell receptor (NKR), thus forming an NKR-CAR. The NKR component may be a transmembrane, hinge or cytoplasmic domain from any of the following natural killer cell receptors: killer immunoglobulin-like receptors (KIRs), such as KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP 1; natural Cytotoxic Receptors (NCRs), e.g., NKp30, NKp44, NKp 46; the Signaling Lymphocyte Activating Molecule (SLAM) family of immune cell receptors, e.g., CD48, CD229, 2B4, CD84, NTB-A, CRA, BLAME, and CD 2F-10; fc receptors (fcrs), e.g., CD16, and CD 64; and Ly49 receptors, e.g., Ly49A, Ly 49C. The NKR-CAR molecule can interact with an adaptor molecule or an intracellular signaling domain (e.g., DAP 12).

It is a further object of the invention to provide an expression vector for such a CAR structure.

In order to achieve the purpose, the invention adopts the following scheme:

the expression vector is any one of a lentivirus expression vector, a retrovirus expression vector, an adenovirus expression vector, an adeno-associated virus expression vector, a herpes simplex virus vector, a DNA vector, an RNA vector and a plasmid.

Preferably, the vector is a lentiviral vector.

In certain embodiments, the lentiviral vector is selected from the group consisting essentially of: human immunodeficiency virus 1(HIV-1), human immunodeficiency virus 2(HIV-2), visna-meidi virus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV).

In certain embodiments, the vector comprises a left (5') retroviral LTR, a Psi (Ψ) packaging signal, a central polypurine tract/DNA FLAP (cPPT/FLAP), a retroviral export element, a promoter operably linked to a polynucleotide encoding a CAR contemplated herein, and a right (3') retroviral LTR.

In certain embodiments, the CAR comprises a hepatitis b virus post-transcriptional regulatory element (HPRE) or woodchuck post-transcriptional regulatory element (WPRE) and an optimized woodchuck post-transcriptional regulatory element (oPRE).

In certain embodiments, the promoter of the 5' LTR is replaced with a heterologous promoter.

In certain embodiments, the heterologous promoter is a Cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, or an simian Virus 40(SV40) promoter.

In certain embodiments, the 5'LTR or 3' LTR is a lentiviral LTR.

In certain embodiments, the 3' LTR is a self-inactivating (SIN) LTR.

In certain embodiments, a polynucleotide encoding a CAR encompassed herein comprises an optimized Kozark sequence.

In certain embodiments, the promoter operably linked to a polynucleotide encoding a CAR encompassed herein is selected from the group consisting of table one and: cytomegalovirus immediate early gene promoter (CMV), elongation factor 1 alpha promoter (EF 1-alpha), phosphoglycerate kinase-1 Promoter (PGK), ubiquitin-C promoter (UBQ-C), cytomegalovirus enhancer/chicken beta-actin promoter (CAG), polyoma enhancer/herpes simplex thymidine kinase promoter (MC1), beta actin promoter (beta-ACT), simian virus 40 promoter (SV40), and myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substituted (MND) promoter.

Watch 1

WTPSK promoter	SEQ ID NO:12
		Truncated PGK promoter 1	SEQ ID NO:13
Truncated PGK promoter 2	SEQ ID NO:14
		Truncated PGK promoter 3	SEQ ID NO:15
Truncated PGK promoter 4	SEQ ID NO:16
		EF1 α promoter	SEQ ID NO:17
MND promoter	SEQ ID NO:18

In certain embodiments, the CAR-containing vector can comprise a secreted anti-PD-1 ScFv; in certain embodiments, the vector comprising the CAR comprises a PD-1 conjugated transduction peptide (e.g., PD-1-CD28-CD137-CD3 signaling structure); in certain embodiments, the vector comprising the CAR comprises a plurality of CAR combinations, such as 2 CAR combinations targeting different antigens or different recognition sites of the same antigen.

Preferably, the carrier is PBKL1-5H1P-CAR/anti-PSCA-OPRE, and comprises a CAR structure with a nucleic acid sequence shown as SEQ ID NO. 3; the vector is PBKL1-5H1P-CAR/anti-CD19-OPRE and comprises a CAR structure with a nucleic acid sequence shown as SEQ ID NO. 4; the vector is pBKL1-5H1P-CAR/anti-CEA-oPRE, comprises a CAR structure with a nucleic acid sequence shown as SEQ ID NO.43, and has an amino acid sequence shown as SEQ ID NO. 42.

The fourth purpose of the invention is to provide a CAR-T cell containing the expression vector, wherein the CAR-T cell can induce activation under the anoxic environment and in the presence of a target antigen, the CAR expression capacity and abundance are improved after the stimulation of the target antigen under the anoxic environment, the CAR-T effectiveness is enhanced, and the CAR-T cell has high IFN-gamma and IL-2 factor secretion capacity; the expression capacity and abundance of the CAR after the stimulation of the target antigen in a non-hypoxic environment are lower than those of the conventional CAR-T, and the safety is higher.

Further, the CAR-T cells can be applied to hematological diseases and solid tumor treatment.

Such hematological tumors include, but are not limited to: acute Lymphoid Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), non-Hodgkin's lymphoma, and Hodgkin's lymphoma.

Such solid tumors include, but are not limited to: prostate cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, lung cancer, kidney cancer, liver cancer, brain cancer, and skin cancer.

In particular, the cells can be used in combination with other agents and/or treatments that enhance the activity of CAR expression.

In particular, the active agent and/or treatment may be surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immune scavengers (immunoablativeagents) such as CAMPATH, anti-CD 3 antibody or other antibody therapy, cyclophosphamide (cytoxan), fludarabine (fludarabine), cyclosporine, FK506, rapamycin (rapamycin), mycophenolic acid (mycophenolic acid), steroids (steroids), 90fr 1228, cytokines and radiation.

In certain embodiments, the cell can express other agents, e.g., agents that enhance the activity of the CAR-expressing cell. The active agent may be an active agent that blocks inhibitory molecules. Inhibitory molecules such as PD1 may, in some embodiments, reduce the ability of CAR-expressing cells to mount an immune effector response. Inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (CEACAM-1, CEACAM-3, CEACAM-5), LAG3, VISTA, BTLA, TIG, LAIR1, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, GALMII, MHC class II, 9, adenosine, TGFR (TGFR β) and TGFR β. The extracellular domain (amino acid sequence shown in SEQ ID NO. 37) of the inhibitory molecule can be fused to a transmembrane domain and an intracellular signaling domain, such as a PD1CAR (nucleotide sequence shown in SEQ ID NO.38 or SEQ ID NO. 39).

The fifth object of the invention is a method of increasing the ability of CAR-T cells to secrete IFN- γ and/or IL-2 factor in hypoxic environments.

In order to achieve the purpose, the invention adopts the following scheme:

specifically, a CAR structure containing an hypoxia-controllable promoter is constructed, wherein the hypoxia-controllable promoter is formed by connecting a Hif1 alpha regulatory element and a mini promoter, and the repetition number of the Hif1 alpha regulatory element is 3-5; the mini-promoter is selected from a cellular viral promoter, a promoter of HSV thymidine kinase, a promoter of simian virus 40, an adenovirus late promoter or a synthetic promoter.

Preferably, the number of HREs is 5.

Preferably, the mini-promoter is selected for miniCMV.

The invention also aims to provide a method for improving killing capacity of the CAR-T cells in an anoxic environment.

In order to achieve the purpose, the invention adopts the following scheme:

specifically, a CAR structure containing an hypoxia-controllable promoter is constructed, wherein the hypoxia-controllable promoter is formed by connecting a Hif1a regulatory element and a mini promoter, and the repetition number of the Hif1 alpha regulatory element is 3-5; the mini-promoter is selected from a cellular viral promoter, a promoter of HSV thymidine kinase, a promoter of simian virus 40, an adenovirus late promoter or a synthetic promoter.

Preferably, the number of HREs is 5.

Preferably, the mini-promoter is selected for miniCMV.

Furthermore, the nucleic acid sequence of the hypoxia-controllable Promoter is shown in SEQ ID NO.1 and SEQ ID NO.2, the SEQ ID NO.1 is formed by combining 5 repeated HRE regulatory elements and a weakly-started CMV mini Promoter, and the structure is 5HRE-CMVmini Promoter.

The seventh purpose of the invention is to provide the application of the CAR structure and/or the expression vector and/or the CAR-T cell, in particular to the application in preparing medicaments for treating tumors.

The tumor highly expresses any one or more of CD19, CD20, CD123, CD22, BCMA, ROR1, mesothelin, PSCA, PSMA, c-Met, GPC-3, Her2, EGFRvIII, GD-2, NY-ESO-1TCR, MAGE A3 TCR.

The invention has the beneficial effects that:

1) the invention provides an application of an hypoxia-controllable promoter in CAR-T, wherein the promoter induced by an hypoxia microenvironment can enhance the expression of factors such as target genes, proteins and the like in the hypoxia environment, so that the drug curative effect of tumors and the effectiveness and safety of CAR-T treatment are improved;

2) the CAR structure induced and started by the hypoxic microenvironment can be effectively expressed in T lymphocytes, can enhance the activation of CAR-T cells in the hypoxic environment, enhances the effectiveness of CAR-T, enables the CAR-T cells to have high IFN-gamma and IL-2 factor secretion capacity, improves the killing of the CAR-T cells on tumor target cells, and can be used for the targeted therapy of tumors;

3) the CAR-T cells induced and started by the hypoxic microenvironment have lower CAR expression capacity and abundance than conventional CAR-T cells after stimulation of target antigens in a non-hypoxic environment, and are higher in safety;

4) the CAR-T cell provided by the invention has enhanced activity in an anoxic environment, can be used for adoptive cell therapy in preparation of tumor treatment drugs, and has important guiding significance for the clinical application of CAR-T and the development of a new tumor combination therapy strategy.

Drawings

FIG. 1 is a diagram of the structure of pBKL1-5H1P-CAR vector.

FIG. 2 shows the vector identification results.

Fig. 3A is a histogram of GFP positive rate validated by the hypoxia induction protocol and fig. 3B is a histogram of GFP expression fluorescence density validated by the hypoxia induction protocol.

Fig. 4A is a bar graph (Hela) of the effect of hypoxia-inducible drugs on cell proliferation, and fig. 4B is a bar graph (PBMC) of the effect of hypoxia-inducible drugs on cell proliferation.

FIG. 5 is flow chart of CAR positivity.

FIG. 6A shows the expression of PSCA on Hela in target cells; FIG. 6B is the killing efficiency of CAR-T on target cell Hela.

FIG. 7A is a test of IFN-gamma factor secretion after CAR-T activation, and FIG. 7B is a test of IL-2 factor secretion after CAR-T activation.

FIG. 8A is a demonstration of the effectiveness of CAR-T in lymphoma-bearing mice after modification of the hypoxic promoter, and FIG. 8B is a graph of the corresponding tumor growth.

FIG. 9A shows that CAR-T inhibits tumor proliferation in human colorectal cancer tumor-bearing mice after modification of the hypoxic promoter.

FIG. 9B is a graph of the effect of CAR-T on the size of the tumor volume in human colorectal cancer tumor-bearing mice after modification of the hypoxic promoter.

Detailed Description

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The experimental procedures, in the preferred embodiments, which do not specify specific conditions, are generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (third edition, sambrook et al), or according to the conditions recommended by the manufacturers. The examples are provided for better illustration of the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.

Example 1 plasmid construction

Plasmids were constructed according to the conditions shown in table two for subsequent experiments, taking mini promoter miniCMV, lentiviral expression vector, antigen recognition region CD19 and PSCA as examples for plasmid construction.

The nucleic acid sequence of the synthetic hypoxia initiation sequence 5HRE-CMVmini promoter is shown as SEQ ID NO.1, the promoter EcoRV-5HRE-CMVmini promoter-NheI (5H1P) matched with a lentiviral vector is further constructed, the nucleic acid sequence is shown as SEQ ID NO.2, the nucleic acid sequence of the targeting PSCA-CAR is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 5; the nucleic acid sequence of the targeting CD19-CAR is shown as SEQ ID NO.4, and the amino acid sequence is shown as SEQ ID NO. 6; the amino acid sequence of the targeting CEA-CAR is shown as SEQ ID NO. 42. Respectively cutting and recovering fragments by using double enzyme digestion, connecting the gene fragments, converting and selecting single clone to obtain recombinant vectors PBKL1-5H1P-PSCA (1) -OPRE, PBKL1-5H1P-CD19-OPRE and PBKL1-5H 1P-GFP-OPRE; PBKL1-5H1P-GFP-OPRE is a control vector that does not contain the CAR gene.

The nucleic acid sequences of the hypoxia promoter and plasmid constructs are shown in SEQ ID NO 11, and FIG. 1 is a diagram of the CAR construct and hypoxia inducible vector comprising the CAR. The obtained plasmid is identified by double enzyme digestion of NheI + SalI and is sequenced to verify the correctness, and the result is shown in FIG. 2: m is DL10000DNA molecular weight standard; 1: plasmid PBKL1-5H1P-PSCA (1) -OPRE (8325 bp); 2: plasmid PBKL1-5H1P-GFP-OPRE (7443 bp); 3: NheI + SalI double enzyme digestion plasmid PBKL1-5H1P-PSCA (1) -OPRE to obtain 6717 and 1608bp fragments; 4: the plasmid PBKL1-5H1P-GFP-OPRE was digested with NheI + SalI to obtain 6717 and 726bp fragments.

Construction conditions for Table two plasmids

Example 2 in vitro hypoxia model validation

CoCl₂The principle of induced hypoxia: cobalt dichloride (CoC)l₂) The divalent cobalt ion (Co2+) in (b) can replace the divalent iron ion of the PHD cofactor, because Co2+ has a low affinity for oxygen, so that heme cannot bind to oxygen and cannot be transformed into an oxidized state, thus forming an anoxic state.

1) And (3) verification of a hypoxia induction scheme:

PBMC activated by PBKL1-5HRE-GFP virus infection is used for constructing an in-vitro anoxic cell model, and the culture solution is changed after 12-18 h. Using CoCl₂Inducing an anoxic environment, culturing until day 4, verifying whether the anoxic model is constructed by green fluorescence detection, and respectively setting a control group (without adding CoCl)₂) Experimental group (with CoCl)₂)，CoCl₂The expression intensity of GFP was examined by fluorescent microscopy after 24 hours of drug treatment, and the positive rate and fluorescence expression intensity of GFP were examined by flow-assay.

As a result, as shown in FIGS. 3A and 3B, it was found that CoCl was added₂The GFP positive rate of the induction group (i.e. microenvironment hypoxia group) is obviously higher than that of the group without CoCl₂Group and the fluorescence intensity is also significantly higher than without CoCl₂Group, indicating that under hypoxic environment, the hypoxic promoter of PBKL1-5HRE-GFP is able to normally promote the expression of downstream GFP.

2) Effect of hypoxia-inducible drugs on cell proliferation

Spreading a 96-well plate with appropriate amount of Hela and PBMC cells in the morning, and allowing the cells to adhere for 5h without adding CoCl₂Group sum

final concentration

0, 100, 500, 1000. mu.M CoCl₂And (3) processing the groups, adding the incubated CellTiter-Glo One Solution Assay reagent according to the volume of 1:1 after 24 hours of processing, incubating for 10 minutes at room temperature, and then putting into an enzyme-linked immunosorbent Assay to detect absorbance. The results are shown in FIGS. 4A and 4B, CoCl₂Does not affect the proliferation of the primary target cells, so that the killing of CAR-T cells is not affected by the exogenous addition of CoCl₂Influence is only related to whether the hypoxia promoter is included or not.

Example 3 preparation of lentiviruses and infection of T lymphocytes

The slow virus package in this example adopts a calcium phosphate method, which specifically comprises: the 293T cells were cultured in DMEM medium to a preferred state, and the cells were replaced with a liquid. The packaging plasmid (RRE:REV:2G) and the expression plasmid are added into a centrifugal tube of 1.5 according to a certain ratio, and 2.5mol/LCaCl is added₂Supplement ddH₂And O until the total volume is 600 mu L, uniformly mixing, dropwise adding 2 × HBS into the mixed solution by using a liquid transfer device, uniformly mixing, standing at room temperature for 15min, adding into the treated 293T cell culture solution, changing the solution to 10mL of DMEM medium containing 10% FBS after 3-5h, collecting cell supernatant after 48h or 72h, purifying the virus, and measuring the titer.

Separating lymphocytes by using a gradient centrifugation method; after centrifugation, the second white lymphocyte layer was taken, washed with physiological saline, and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human PBMC cells. After the obtained PBMC cells are activated by anti-CD 3 and CD28 monoclonal antibodies, the activated PBMC cells are infected according to a certain multiplicity of infection (MOI), the PBMC cells are cultured overnight, the culture solution is changed, the PBMC cells are continuously cultured in an incubator at 37 ℃, the positive rate of CAR-T is detected on the 12 th day of virus infection, the detection method is flow detection, and the antibodies are: Protein-L-PE, Protein-L can recognize the antibody light chain, and the light chain of ScFv sequence of the CAR antigen recognition region can be recognized by Protein-L, so that the CAR positivity can be detected by using Protein-L.

The results are shown in FIG. 5: hypoxia-induced 5H1P-PSCA CAR-T group is CoCl₂The induced group of CAR-T containing hypoxia promoter is CoCl₂The inducible non-hypoxia promoter-free CAR-T group and the non-inducible 5H 1P-PSCACAAR-T group are not added with CoCl₂The induced group contains the hypoxia promoter CAR-T, and BLANK is BLANK control group. The CAR structure modified by the hypoxia promoter has higher expression abundance of CAR in a hypoxia environment, and CAR molecules are expressed on the surface of a T cell at higher density.

EXAMPLE 4 detection of CoCl₂CAR-T effectivity comprising a hypoxic promoter after induction

The target cells were Hela cells positive to PSCA and 293T cells negative to PSCA. After carrying out hypoxia treatment on effector cells (conventional CAR-T cells and CAR-T cells expressed by CAR containing a hypoxia promoter), paving the effector cells in target cells according to a certain effective target ratio, detecting the killing capacity of different CAR-T on the target cells by using an ACEA xCELLigence RTCA MP instrument, and carrying out experimental steps according to the instruction of the instrument. The ACEA xCELLigence RTCA MP principle is that the resistance index is taken as data to record tumor cells attached to the bottom of a hole every 15 minutes, and the proliferation or death condition of the attached target cells is judged through the resistance index. The result formula of the resistance index analysis is as follows: CAR-T cell killing rate-baseline resistance index-real-time resistance index.

The results are shown in FIGS. 6A and 6B: FIG. 6A shows the expression of PSCA on Hela as a target cell; FIG. 6B is a graph showing the killing efficiency of each group of CAR-T on target cell Hela 24h after the addition of CAR-T. Wherein the Medium is a culture Medium only, the Medium + is a culture Medium inducing an anoxic environment, the 5H1P-PSCA is CAR-T containing an anoxic promoter 5H1P, the 5H1P-PSCA + is CAR-T containing an anoxic promoter 5H1P in the anoxic environment, the CAG-PSCA is CAR-T containing no anoxic promoter, and the 5H1P-GFP + negative control. Under the hypoxia environment induced by adding drugs, the killing effect of the 5H1P-PSCA + protected by the patent is obviously enhanced and is larger than that of a CAG-PSCA group without introducing a hypoxia promoter.

Cell supernatants were collected 24 hours after killing and assayed for IFN-. gamma.and IL-2 secretion capacity following stimulation of CAR-T cells by target cells.

Example 5 Elisa assay for IFN-. gamma.and IL-2 assay

The supernatants collected in example 4 were assayed for secretion of IFN-. gamma.and IL-2 by ELISA (enzyme-linked immunosorbent assay). IFN-gamma detection adopts a BD IFN-gamma kit for detection, the product number is 555142, and the experimental steps are carried out according to the product instruction; IL-2 detection was carried out using the Inritrogen IL-2 kit, cat # 88-7025-88, with the experimental procedures performed according to the product instructions.

As shown in FIGS. 7A and 7B, 5H1P-PSCA is CAR-T containing the hypoxic promoter 5H1P, 5H1P-PSCA + is CAR-T containing the hypoxic promoter 5H1P in an hypoxic environment, CAG-PSCA is CAR-T without the hypoxic promoter, and 5H1P-GFP + is a negative control. The secretory capacity of IFN-gamma and IL-2, especially IL-2 after the CAR-T cells containing the hypoxia promoter are stimulated by antigens in a hypoxia environment is far higher than that in a non-hypoxia environment; the secretion of IFN-gamma after antigen stimulation by CAR-T containing the hypoxia promoter 5H1P in a non-hypoxic environment is much lower than that of CAR-T CAG-PSCA without the hypoxia promoter. Therefore, in the non-hypoxic environment of normal tissues, the secretion of IFN-gamma of 5H1P-PSCA after antigen stimulation is far lower than that of the IFN-gamma without a hypoxic promoter, the higher safety is achieved, and the enhancement of the IFN-gamma secretion capacity of 5H1P-PSCA after antigen stimulation in the hypoxic environment of tumor indicates that 5H1P plays a role in the hypoxic environment; under the hypoxic environment of the tumor, the IL-2 secretion capacity of 5H1P-PSCA is equivalent to that of CAG-PSCA, and is far greater than that of 5H1P-PSCA in the non-hypoxic environment, and the 5HRE-CMVmini promoter can improve the safety and effectiveness of CAR-T under the hypoxic environment.

Example 6CAR-T cell in vivo efficacy validation comprising a hypoxic promoter

Establishing a mouse tumor-bearing model of human lymphoma cells (Raji, CD19 positive) and a mouse tumor-bearing model of human colorectal cancer, and verifying the effect of the addition of the hypoxia promoter on the anti-tumor curative effect of the CAR-T cells.

Cg-PrkdcscidiI2rgtm1Sug/Jiccrl, NOG mouse for short, is bred by Mamoru Ito of the Japanese institute of Experimental animals (CIEA), and is the most common strain for CAR-T related in vivo tumor formation experiments internationally. In vivo verification, Raji cells are selected from the used tumor-forming target cells, and a human lymphoma tumor-bearing mouse model is constructed. Female NOD/SCID mice of 6-8 weeks of age were selected, labeled with ear number, and then injected subcutaneously with LoVo or Raji cells at 1X 106/cell count on the back of the mice. The tumor volume of the mice was measured on day 6 of tumor formation, and the experiments were performed in two parts, i.e., Control T group, hypoxia promoter-modified CAR-T (5H1P CD19CAR-T), non-modified promoter CAR-T group (CD19CAR-T), hypoxia promoter-modified CAR-T (5H1P CEA CAR-T), and Control group, according to the tumor volume. Injecting corresponding CAR-T cells 3 x 10^6CAR-T cells/mouse to tail vein of mice of different groups on 8 days of tumor formation in a human lymphoma bearing mouse group; the Control T group returned the same total number of T lymphocytes on day 8. Human colorectal cancer-bearing mouse group mice were injected intravenously with 5H1P CEA CAR-T and CONTROL T cells 1 x 10^6CAR-T cells/mouse at day 7 of neoplasia in different groups; 5H1P CEA CAR-T and CONTROL T cells were re-transfused once more at day 14 with 1 x 10^6CAR-T cells/cell.

Tumor volumes were measured weekly in each group of mice, and the results are shown in fig. 8A, 8B and fig. 9A, 9B. FIG. 8A is an experiment conducted on Raji tumor-bearing mice, and it can be seen that 5H1P group (5H1P CD19CAR-T) subjected to hypoxia promoter modification has significantly improved in vivo efficacy compared to non-modified CD19CAR-T group mice. In conclusion, the results of in vivo animal experiments show that the effectiveness of the CAR-T cells modified by the CAR structure of the 5HRE-CMVmini promoter is remarkably improved for tumor-bearing mice.

Fig. 9A and 9B are experiments performed using a human colorectal cancer mouse tumor-bearing model, fig. 9A is tumor proliferation curves after reinfusion of 5H1P group with modified hypoxic promoter and CAR-T group without modification, and fig. 9B is statistical results of tumor volumes of mice after different modification groups; as a result, the 5H1P group (5H1P CEA CAR-T) which is modified by the hypoxia promoter has obvious curative effect compared with Control.

The result shows that the effectiveness of the CAR-T cell which is modified by the CAR structure of the 5HRE-CMVmini promoter is obviously improved aiming at tumor-bearing mice.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

Sequence listing

<110> Chongqing precision Biotechnology Co., Ltd

<120> application of hypoxia-controllable promoter in CAR-T

<160>43

<170>SIPOSequenceListing 1.0

<210>1

<211>274

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>1

ccacagtgca tacgtgggct ccaacaggtc ctcttgtcga gccacagtgc atacgtgggc 60

tccaacaggt cctcttgtcg agccacagtg catacgtggg ctccaacagg tcctcttgtc 120

gagccacagt gcatacgtgg gctccaacag gtcctcttgt cgagccacag tgcatacgtg 180

ggctccaaca ggtcctcttg tcgagatctg gtaggcgtgt acggtgggag gtctatataa 240

gcagagctcg tttagtgaac cgtcagatca ctag 274

<210>2

<211>292

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>2

gtcgacgata tcccacagtg catacgtggg ctccaacagg tcctcttgtc gagccacagt 60

gcatacgtgg gctccaacag gtcctcttgt cgagccacag tgcatacgtg ggctccaaca 120

ggtcctcttg tcgagccaca gtgcatacgt gggctccaac aggtcctctt gtcgagccac 180

agtgcatacg tgggctccaa caggtcctct tgtcgagatc tggtaggcgt gtacggtggg 240

aggtctatat aagcagagct cgtttagtga accgtcagat cactaggcta gc 292

<210>3

<211>1599

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>3

atggccctgc cagtgaccgc cctgctgctg cccctggccc tgctgctgca cgcagcacgg 60

cccgacatcc aggatattca gctgacccag agcccgagca gcctgagcgc gagcgtgggc 120

gatcgcgtga ccattacctg cagcgcgagc agcagcgtgc gctttattca ttggtatcag 180

cagaaaccgg gcaaagcgcc gaaacgcctg atttatgata ccagcaaact ggcgagcggc 240

gtgccgagcc gctttagcgg cagcggcagc ggcaccgatt ttaccctgac cattagcagc 300

ctgcagccgg aagattttgc gacctattat tgccagcagt ggagcagcag cccgtttacc 360

tttggccagg gcaccaaagt ggaaattaaa ggcagcacca gcggcagcgg caaaccgggc 420

agcggcgaag gcagcaccaa aggcagcgaa gtgcagctgg tggaaagcgg cggcggcctg 480

gtgcagccgg gcggcagcct gcgcctgagc tgcgcggcga gcggctttaa cattaaagat 540

tattatattc attgggtgcg ccaggcgccg ggcaaaggcc tggaatgggt ggcgtggatt 600

gatccggaaa acggcgatac cgaatttgtg ccgaaatttc agggccgcgc gaccattagc 660

gcggatacca gcaaaaacac cgcgtatctg cagatgaaca gcctgcgcgc ggaagatacc 720

gcggtgtatt attgcaaaac cggcggcttt tggggccagg gcaccctggt gaccgtgagc 780

tctctcgaga ccaccacccc ggcgccgcgc ccgccgaccc cggcgccgac cattgcgagc 840

cagccgctga gcctgcgccc ggaagcgtgc cgcccggcgg cgggcggcgc ggtgcatacc 900

cgcggcctgg attttgcgtg cgatgaattc ttttgggtgc tggtggtggt gggcggcgtg 960

ctggcgtgct atagcctgct ggtgaccgtg gcgtttattattttttgggt gcgcagcaaa 1020

cgcagccgcg gcggccatag cgattatatg aacatgaccc cgcgccgccc gggcccgacc 1080

cgcaaacatt atcagccgta tgcgccgccg cgcgattttg cggcgtatcg cagcgtgaaa 1140

cgcggccgca aaaaactgct gtatattttt aaacagccgt ttatgcgccc ggtgcagacc 1200

acccaggaag aagatggctg cagctgccgc tttccggaag aagaagaagg cggctgcgaa 1260

ctgcgcgtga aatttagccg cagcgcggat gcgccggcgt atcagcaggg ccagaaccag 1320

ctgtataacg aactgaacct gggccgccgc gaagaatatg atgtgctgga taaacgccgc 1380

ggccgcgatc cggaaatggg cggcaaaccg cgccgcaaaa acccgcagga aggcctgtat 1440

aacgaactgc agaaagataa aatggcggaa gcgtatagcg aaattggcat gaaaggcgaa 1500

cgccgccgcg gcaaaggcca tgatggcctg tatcagggcc tgagcaccgc gaccaaagat 1560

acctatgatg cgctgcatat gcaggcgctg ccgccgcgc 1599

<210>4

<211>1473

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>4

atggcgctgc cggtgaccgc gctgctgctg ccgctggcgc tgctgctgca tgcggcgcgc 60

ccggatattc agatgaccca gagcccgagc agcctgagcg cgagcgtggg cgatcgcgtg 120

accattacct gccgcgcgag ccaggatatt agcaaatatc tgaactggta tcagcagaaa 180

ccgggcaaag cgccgcgcct gctgatttat cataccagcc gcctgcatag cggcgtgccg 240

agccgcttta gcggcagcgg cagcggcacc gattataccc tgaccattag cagcctgcag 300

ccggaagatt ttgcgaccta ttattgccag cagggcaaca ccctgccgta tacctttggc 360

ggcggcaccc gcctggaaat taaaggcagc accagcggca gcggcaaacc gggcagcggc 420

gaaggcagca ccaaaggcca ggtgcagctg caggaaagcg gcccgggcct ggtgaaaccg 480

agccagaccc tgagcctgac ctgcaccgtg agcggcgtga gcctgccgga ttatggcgtg 540

agctggattc gccagccgcc gggcaaagcg ctggaatggc tgggcgtgat ttggggcagc 600

gaaaccacct attataacag cagcctgaaa acccgcctga ccattagcaa agataacagc 660

aaaaaccagg tggtgctgac catgaccaac atggatccgg tggataccgc gacctattat 720

tgcgcgaaac attattatta tggcggcagc tatgcgatgg attattgggg ccagggcagc 780

agcgtgaccg tgagcagcct ggaaaccacc accccggcgc cgcgcccgcc gaccccggcg 840

ccgaccattg cgagccagcc gctgagcctg cgcccggaag cgtgccgccc ggcggcgggc 900

ggcgcggtgc atacccgcgg cctggatttt gcgtgcgata tttatatttg ggcgccgctg 960

gcgggcacct gcggcgtgct gctgctgagc ctggtgatta ccctgtattg caaacgcggc 1020

cgcaaaaaac tgctgtatat ttttaaacag ccgtttatgc gcccggtgca gaccacccag 1080

gaagaagatg gctgcagctg ccgctttccg gaagaagaag aaggcggctg cgaactgcgc 1140

gtgaaattta gccgcagcgc ggatgcgccg gcgtataaac agggccagaa ccagctgtat 1200

aacgaactga acctgggccg ccgcgaagaa tatgatgtgc tggataaacg ccgcggccgc 1260

gatccggaaa tgggcggcaa accgcgccgc aaaaacccgc aggaaggcct gtataacgaa 1320

ctgcagaaag ataaaatggc ggaagcgtat agcgaaattg gcatgaaagg cgaacgccgc 1380

cgcggcaaag gccatgatggcctgtatcag ggcctgagca ccgcgaccaa agatacctat 1440

gatgcgctgc atatgcaggc gctgccgccg cgc 1473

<210>5

<211>507

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>5

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Arg Phe Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Ser Pro Phe Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly Ser

100 105 110

Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Ser Glu Val Gln

115120 125

Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

130 135 140

Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr Tyr Ile His

145 150 155 160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Trp Ile

165 170 175

Asp Pro Glu Asn Gly Asp Thr Glu Phe Val Pro Lys Phe Gln Gly Arg

180 185 190

Ala Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Lys Thr Gly

210 215 220

Gly Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Lys Pro Thr

225 230 235 240

Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser

245 250 255

Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly

260 265 270

Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu

275 280285

Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val

290 295 300

Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His

305 310 315 320

Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys

325 330 335

His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

340 345 350

Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

355 360 365

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

370 375 380

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

385 390 395 400

Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr

405 410 415

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

420 425 430

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

435 440445

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

450 455 460

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

465 470 475 480

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

485 490 495

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505

<210>6

<211>470

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>6

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln

115 120 125

Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser

130 135 140

Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

145 150 155 160

Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly Val Ile

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Thr Arg Leu

180 185 190

Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Val Leu Thr Met Thr

195 200 205

Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Lys His Tyr

210 215 220

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Ser Ser

225 230 235 240

Val Thr Val Ser Ser Leu Glu Thr Thr Thr Pro Ala Pro Arg Pro Pro

245 250 255

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

260 265 270

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

275 280 285

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

290 295 300

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

305 310 315 320

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

325 330 335

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

340 345 350

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

355 360 365

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

370 375 380

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

385390 395 400

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

405 410 415

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

420 425 430

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

435 440 445

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

450 455 460

Gln Ala Leu Pro Pro Arg

465 470

<210>7

<211>112

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>7

Glu Val Gln Leu Val Glu 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 Asn Ile Lys Asp Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Phe Val Pro Lys Phe

50 55 60

Gln Gly Arg Ala Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Lys Thr Gly Gly Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210>8

<211>106

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>8

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Arg Phe Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Ser Pro Phe Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>9

<211>120

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>9

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys

50 55 60

Thr Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Val Leu

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100105 110

Gly Ser Ser Val Thr Val Ser Ser

115 120

<210>10

<211>107

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>10

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys

100 105

<210>11

<211>272

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>11

ccacagtgca tacgtgggct ccaacaggtc ctcttgtcga gccacagtgc atacgtgggc 60

tccaacaggt cctcttgtcg agccacagtg catacgtggg ctccaacagg tcctcttgtc 120

gagccacagt gcatacgtgg gctccaacag gtcctcttgt cgagccacag tgcatacgtg 180

ggctccaaca ggtcctcttg tcgagatctg gtaggcgtgt acggtgggag gtctatataa 240

gcagagctcg magtgaaccg tcagatcact ag 272

<210>12

<211>399

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>12

tttatttagt ctccagaaaa aggggggaat gaaagacccc acctgtaggt ttggcaagct 60

aggatcaagg ttaggaacag agagacagca gaatatgggc caaacaggat atctgtggta 120

agcagttcct gccccggctc agggccaaga acagttggaa cagcagaata tgggccaaac 180

aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag 240

atgcggtccc gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag 300

gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt 360

tcgcgcgctt ctgctccccg agctcaataa aagagccca 399

<210>13

<211>118

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>13

acccctctct ccagccacta agccagttgc tccctcggct gacggctgca cgcgaggcct 60

ccgaacgtct tacgccttgt ggcgcgcccg tccttgtccc gggtgtgatg gcggggtg 118

<210>14

<211>221

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>14

acccctctct ccagccacta agccagttgc tccctcggct gacggctgca cgcgaggcct 60

ccgaacgtct tacgccttgt ggcgcgcccg tccttgtccc gggtgtgatg gcggggtgtg 120

gggcggaggg cgtggcgggg aagggccggc gacgagagcc gcgcgggacg actcgtcggc 180

gataaccggt gtcgggtagc gccagccgcg cgacggtaac g 221

<210>15

<211>324

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>15

acccctctct ccagccacta agccagttgc tccctcggct gacggctgca cgcgaggcct 60

ccgaacgtct tacgccttgt ggcgcgcccg tccttgtccc gggtgtgatg gcggggtgtg 120

gggcggaggg cgtggcgggg aagggccggc gacgagagcc gcgcgggacg actcgtcggc 180

gataaccggt gtcgggtagc gccagccgcg cgacggtaac gagggaccgc gacaggcaga 240

cgctcccatg atcactctgc acgccgaagg caaatagtgc aggccgtgcg gcgcttggcg 300

ttccttggaa gggctgaatc cccg 324

<210>16

<211>422

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>16

acccctctct ccagccacta agccagttgc tccctcggct gacggctgca cgcgaggcct 60

ccgaacgtct tacgccttgt ggcgcgcccg tccttgtccc gggtgtgatg gcggggtgtg 120

gggcggaggg cgtggcgggg aagggccggc gacgagagcc gcgcgggacg actcgtcggc 180

gataaccggt gtcgggtagc gccagccgcg cgacggtaac gagggaccgc gacaggcaga 240

cgctcccatg atcactctgc acgccgaagg caaatagtgc aggccgtgcg gcgcttggcg 300

ttccttggaa gggctgaatc cccgcctcgt ccttcgcagc ggccccccgg gtgttcccat 360

cgccgcttct aggcccactg cgacgcttgc ctgcacttct tacacgctct gggtcccagc 420

cg 422

<210>17

<211>1184

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>17

cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 60

tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 120

aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa 180

gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa 240

gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt 300

gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg 360

ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg 420

cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg 480

ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt 540

tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg 600

gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc 660

tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg 720

tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg 780

caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat 840

ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct 900

ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc 960

tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg 1020

cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga 1080

tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc 1140

agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtga 1184

<210>18

<211>399

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>18

tttatttagt ctccagaaaa aggggggaat gaaagacccc acctgtaggt ttggcaagct 60

aggatcaagg ttaggaacag agagacagca gaatatgggc caaacaggat atctgtggta 120

agcagttcct gccccggctc agggccaaga acagttggaa cagcagaata tgggccaaac 180

aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag 240

atgcggtccc gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag 300

gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt 360

tcgcgcgctt ctgctccccg agctcaataa aagagccca 399

<210>19

<211>41

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>19

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>20

<211>42

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>20

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210>21

<211>35

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>21

Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr

1 5 10 15

Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp

20 25 30

Val Thr Leu

35

<210>22

<211>48

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>22

Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro

1 5 10 15

Ala Glu Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr

20 25 30

Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro

35 4045

<210>23

<211>112

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>23

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys 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>24

<211>47

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>24

Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

1 5 10 15

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

20 25 30

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210>25

<211>36

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>25

Ala Pro Pro Arg Ala Ser Ala Leu Pro Ala Pro Pro Thr Gly Ser Ala

1 5 10 15

Leu Pro Asp Pro Gln Thr Ala Ser Ala Leu Pro Asp Pro Pro Ala Ala

20 25 30

Ser Ala Leu Pro

35

<210>26

<211>45

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>26

Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

1 5 10 15

Ile Ala Ser Gln ProLeu Ser Leu Arg Pro Glu Ala Arg Pro Ala Ala

20 25 30

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Asp

35 40 45

<210>27

<211>228

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>27

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val

1 5 10 15

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

20 25 30

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

35 40 45

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

50 55 60

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr

65 70 75 80

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

85 90 95

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

100105 110

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

115 120 125

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

130 135 140

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

145 150 155 160

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

165 170 175

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

180 185 190

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

195 200 205

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

210 215 220

Ser Leu Gly Lys

225

<210>28

<211>229

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>28

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 1015

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210>29

<211>282

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>29

Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala

1 5 10 15

Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala

20 25 30

Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys

35 40 45

Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro

50 55 60

Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala Val Gln

65 70 75 80

Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val Val Gly

85 90 95

Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly Lys Val

100 105 110

Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser Asn Gly

115 120 125

Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu Trp Asn

130 135 140

Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu Pro Pro

145 150 155 160

Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro Val Lys

165 170 175

Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala Ala Ser

180 185 190

Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile Leu Leu

195 200 205

Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe Ala Pro

210 215 220

Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala Trp Ser

225 230 235 240

Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr Tyr Thr

245 250 255

Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala Ser Arg

260 265 270

Ser Leu Glu Val Ser Tyr Val Thr Asp His

275 280

<210>30

<211>27

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>30

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210>31

<211>24

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>31

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210>32

<211>243

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>32

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ser Ile Asn Trp Val Lys Arg Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp Phe

50 55 60

Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Tyr Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala

130 135 140

Met Ser LeuGly Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser

145 150 155 160

Val Ser Val Ile Gly Ala His Leu Ile His Trp Tyr Gln Gln Lys Pro

165 170 175

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Thr

180 185 190

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

195 200 205

Leu Thr Ile Asp Pro Val Glu Glu Asp Asp Val Ala Ile Tyr Ser Cys

210 215 220

Leu Gln Ser Arg Ile Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu

225 230 235 240

Glu Ile Lys

<210>33

<211>245

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>33

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys

115 120 125

Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser

130 135 140

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

145 150 155 160

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Arg

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

180 185 190

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn

195 200 205

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

210 215 220

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

225 230 235 240

Val Thr Val Ser Ser

245

<210>34

<211>243

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>34

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Thr Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Ile Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly

100 105 110

Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Gln

115 120 125

Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Thr

130 135 140

Cys Thr Val Ser Gly Phe Thr Ile Ser Ser Gly Tyr Ser Trp His Trp

145 150 155 160

Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile Gly Tyr Ile Gln

165 170 175

Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr

180 185 190

Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser

195 200 205

Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Asp Tyr

210 215 220

Asp Tyr His Trp Tyr Phe Asp Val Trp Gly Gln Gly Ser Thr Val Thr

225 230 235 240

Val Ser Ser

<210>35

<211>240

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>35

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Ile Ser Ser Gly

20 25 30

Tyr Ser Trp His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Gln Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Tyr Asp Tyr His Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Ser Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser

130 135 140

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Thr

145 150 155 160

Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys

165 170 175

Ala Pro Arg Leu Leu Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val

180 185 190

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr

195 200 205

Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys His Gln

210 215 220

Trp Ser Ser Tyr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

225 230 235 240

<210>36

<211>237

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>36

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Arg Phe Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Ser Pro Phe Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly Ser

100 105 110

Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Ser Glu Val Gln

115 120 125

Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

130 135 140

Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr Tyr Ile His

145 150 155 160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Trp Ile

165 170 175

Asp Pro Glu Asn Gly Asp Thr Glu Phe Val Pro Lys Phe Gln Gly Arg

180 185 190

Ala Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Lys Thr Gly

210 215 220

Gly Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

225 230 235

<210>37

<211>150

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>37

Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr

1 5 10 15

Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

8590 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210>38

<211>717

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>38

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg ttttgggtgc tggtggtggt tggtggagtc 540

ctggcttgct atagcttgct agtaacagtg gcctttatta ttttctgggt gaggagtaag 600

aggagcaggc tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc 660

cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg ctcctaa 717

<210>39

<211>833

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>39

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccactgtcc aagtccccta 480

tttcccggac cttctaagcc cttttgggtg ctggtggtgg ttggtggagt cctggcttgc 540

tatagcttgc tagtaacagt ggcctttatt attttctggg tgaggagtaa gaggagcagg 600

ctcctgcaca gtgactacat gaacatgact ccccgccgcc ccgggcccac ccgcaagcat 660

taccagccct atgccccacc acgcgacttc gcagcctatc gctccgttaa acggggcaga 720

aagaaactcc tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag 780

gaagatggct tagctgccga tttccagaag aagaagaagg aggatgtgaa ctg 833

<210>40

<211>120

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>40

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Ile Ser Ser Gly

20 25 30

Tyr Ser Trp His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Gln Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Tyr Asp Tyr His Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Ser Thr Val Thr Val Ser Ser

115 120

<210>41

<211>105

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>41

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Thr Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Ile Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>42

<211>660

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>42

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Thr Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Ile Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly

100 105 110

Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Gln

115 120 125

Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Thr

130 135 140

Cys Thr Val Ser Gly Phe Thr Ile Ser Ser Gly Tyr Ser Trp His Trp

145 150 155 160

Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile Gly Tyr Ile Gln

165 170 175

Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr

180 185 190

Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser

195 200 205

Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Asp Tyr

210 215 220

Asp Tyr His Trp Tyr Phe Asp Val Trp Gly Gln Gly Ser Thr Val Thr

225 230 235 240

Val Ser Ser Gly Ala Ala Ala Leu Glu Glu Ser Lys Tyr Gly Pro Pro

245 250 255

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

260 265 270

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

275 280 285

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

290 295 300

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

305 310 315 320

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

325 330 335

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

340 345 350

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

355 360 365

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

370 375 380

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

385 390 395 400

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

405 410 415

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

420 425 430

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

435 440 445

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

450 455 460

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Glu Phe

465 470 475 480

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

485 490 495

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser

500 505 510

Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly

515 520 525

Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala

530 535 540

Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

545 550 555 560

Tyr Gln Gln Gly Gln Ser Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

565 570 575

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu

580 585 590

Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

595 600 605

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

610 615 620

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

625 630 635 640

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

645 650 655

Leu Pro Pro Arg

660

<210>43

<211>1980

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>43

gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60

attacctgca gcaccagcag cagcgtgagc tatatgcatt ggtatcagca gaaaccgggc 120

aaagcgccgc gcctgctgat ttatagcacc agcaacctgg cgagcggcgt gccgagccgc 180

tttagcggca gcggcagcgg caccgatttt acctttacca ttagcagcct gcagccggaa 240

gatattgcga cctattattg ccatcagtgg agcagctatc cgacctttgg ccagggcacc 300

aaagtggaaa ttaaaggcag caccagcggc agcggcaaac cgggcagcgg cgaaggcagc 360

accaaaggcc aggtgcagct gcaggaaagc ggcccgggcc tggtgcgccc gagccagacc 420

ctgagcctga cctgcaccgt gagcggcttt accattagca gcggctatag ctggcattgg 480

gtgcgccagc cgccgggccg cggcctggaa tggattggct atattcagta tagcggcatt 540

accaactata acccgagcct gaaaagccgc gtgaccatgc tggtggatac cagcaaaaac 600

cagtttagcc tgcgcctgag cagcgtgacc gcggcggata ccgcggtgta ttattgcgcg 660

cgcgaagatt atgattatca ttggtatttt gatgtgtggg gccagggcag caccgtgacc 720

gtgagcagcg gcgcggcggc gctggaagaa agcaaatatg gcccgccgtg cccgccgtgc 780

ccggcgccgg aatttctggg cggcccgagc gtgtttctgt ttccgccgaa accgaaagat 840

accctgatga ttagccgcac cccggaagtg acctgcgtgg tggtggatgt gagccaggaa 900

gatccggaag tgcagtttaa ctggtatgtg gatggcgtgg aagtgcataa cgcgaaaacc 960

aaaccgcgcg aagaacagtt taacagcacc tatcgcgtgg tgagcgtgct gaccgtgctg 1020

catcaggatt ggctgaacgg caaagaatat aaatgcaaag tgagcaacaa aggcctgccg 1080

agcagcattg aaaaaaccat tagcaaagcg aaaggccagc cgcgcgaacc gcaggtgtat 1140

accctgccgc cgagccagga agaaatgacc aaaaaccagg tgagcctgac ctgcctggtg 1200

aaaggctttt atccgagcga tattgcggtg gaatgggaaa gcaacggcca gccggaaaac 1260

aactataaaa ccaccccgcc ggtgctggat agcgatggca gcttttttct gtatagccgc 1320

ctgaccgtgg ataaaagccg ctggcaggaa ggcaacgtgt ttagctgcag cgtgatgcat 1380

gaagcgctgc ataaccatta tacccagaaa agcctgagcc tgagcctggg caaagaattt 1440

ttttgggtgc tggtggtggt gggcggcgtg ctggcgtgct atagcctgct ggtgaccgtg 1500

gcgtttatta ttttttgggt gcgcagcaaa cgcagccgcg gcggccatag cgattatatg 1560

aacatgaccc cgcgccgccc gggcccgacc cgcaaacatt atcagccgta tgcgccgccg 1620

cgcgattttg cggcgtatcg cagccgcgtg aaatttagcc gcagcgcgga tgcgccggcg 1680

tatcagcagg gccagagcca gctgtataac gaactgaacc tgggccgccg cgaagaatat 1740

gatgtgctgg ataaacgccg cggccgcgat ccggaaatgg gcggcaaacc gcgccgcaaa 1800

aacccgcagg aaggcctgta taacgaactg cagaaagata aaatggcgga agcgtatagc1860

gaaattggca tgaaaggcga acgccgccgc ggcaaaggcc atgatggcct gtatcagggc 1920

ctgagcaccg cgaccaaaga tacctatgat gcgctgcata tgcaggcgct gccgccgcgc 1980

Claims

1. CAR-T cells comprising a hypoxia-regulatable promoter are useful in a therapeutic agent for tumors.

2. A CAR structure comprising a hypoxia-controllable promoter, wherein the CAR structure comprises the hypoxia-controllable promoter consisting of a Hif1a regulatory element linked to a mini-promoter, wherein the Hif1a regulatory element has a number of repeats in the range of 3 to 5; the mini-promoter is selected from any one of a cellular viral promoter, a promoter of HSV thymidine kinase, a promoter of simian virus 40, an adenovirus late promoter, and a synthetic promoter.

3. The CAR structure of claim 2, wherein the hypoxia controllable promoter has a nucleic acid sequence as set forth in SEQ ID No.1 and SEQ ID No. 2.

4. The CAR structure of claim 2, wherein the antigen recognized by the antigen recognition region is any one or more of CD19, CD20, CD123, CD22, BCMA, ROR1, mesothelin, PSCA, PSMA, c-Met, GPC-3, Her2, EGFRvIII, GD-2, NY-ESO-1TCR, MAGE A3 TCR.

5. A CAR structure according to claim 4, wherein the antigen recognition region of the CAR structure is an ScFv comprising the heavy chain variable region having an amino acid sequence as shown in SEQ ID No.7 and the light chain variable region as shown in SEQ ID No.8 or comprising the heavy chain variable region having an amino acid sequence as shown in SEQ ID No.9 and the light chain variable region as shown in SEQ ID No.10 or comprising the heavy chain variable region having an amino acid sequence as shown in SEQ ID No.40 and the light chain variable region as shown in SEQ ID No. 41.

6. The CAR structure of claim 2, wherein the amino acid sequence of the CAR structure is as shown in SEQ ID No.5 or SEQ ID No.6 or SEQ ID No. 42.

7. An expression vector comprising the CAR construct of any one of claims 2 to 6, wherein the expression vector is any one of a lentiviral expression vector, a retroviral expression vector, an adenoviral expression vector, an adeno-associated viral expression vector, a herpes simplex viral vector, a DNA vector, an RNA vector, and a plasmid.

8. The expression vector according to claim 7, wherein the vector is pBKL1-5H1P-CAR/anti-PSCA-oPRE comprising a CAR structure having a nucleic acid sequence shown in SEQ ID No. 3; the carrier is pBKL1-5H1P-CAR/anti-CD19-oPRE and comprises a CAR structure with a nucleic acid sequence shown as SEQ ID NO. 4; the vector is pBKL1-5H1P-CAR/anti-CEA-oPRE and comprises a CAR structure with a nucleic acid sequence shown as SEQ ID NO. 43.

9. A CAR-T cell comprising the expression vector of any one of claims 7-8.

10. A method for improving the ability of CAR-T cells to secrete IFN- γ and/or IL-2 factors in an anoxic environment, comprising constructing a CAR construct comprising an hypoxia-controllable promoter consisting of a Hif1a regulatory element linked to a mini-promoter, wherein the Hif1 α regulatory element has a number of repeats of 3 to 5; the mini-promoter is selected from a cellular viral promoter, a promoter of HSV thymidine kinase, a promoter of simian virus 40, an adenovirus late promoter or a synthetic promoter.

11. The method of claim 10, wherein the hypoxia controllable promoter has a nucleic acid sequence as set forth in SEQ ID No.1 and SEQ ID No. 2.

12. A method of increasing killing ability of CAR-T cells in an anoxic environment, constructing a CAR construct comprising an hypoxia controllable promoter comprising a Hif1 α regulatory element linked to a mini-promoter, wherein the Hif1 α regulatory element has a number of repeats of 3 to 5; the mini-promoter is selected from a cellular viral promoter, a promoter of HSV thymidine kinase, a promoter of simian virus 40, an adenovirus late promoter or a synthetic promoter.

13. The method of claim 12, wherein the hypoxia controllable promoter has a nucleic acid sequence as set forth in SEQ ID No.1 and SEQ ID No. 2.

14. Use of a CAR structure according to claim 2 and/or an expression vector according to claim 7 and/or a CAR-T cell according to claim 9 in the manufacture of a medicament for the treatment of a tumour.

15. The use of claim 14, wherein said tumors comprise hematological tumors including but not limited to acute lymphoid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, non-hodgkin lymphoma and hodgkin lymphoma, and solid tumors.

16. The use according to claim 15, wherein the solid tumors include, but are not limited to, prostate cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, lung cancer, kidney cancer, liver cancer, brain cancer and skin cancer.

17. The use according to claim 14, wherein the tumour expresses any one or more of CD19, CD20, CD123, CD22, BCMA, ROR1, mesothelin, PSCA, PSMA, c-Met, GPC-3, Her2, EGFRvIII, GD-2, NY-ESO-1TCR, MAGEA3 TCR.