CN115124627A - MSLN-CAR-NK cell capable of autocrine IL2R beta gamma agonist and application thereof - Google Patents

MSLN-CAR-NK cell capable of autocrine IL2R beta gamma agonist and application thereof Download PDF

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CN115124627A
CN115124627A CN202210697062.7A CN202210697062A CN115124627A CN 115124627 A CN115124627 A CN 115124627A CN 202210697062 A CN202210697062 A CN 202210697062A CN 115124627 A CN115124627 A CN 115124627A
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罗建华
郭猛
刘秋燕
刘艳芳
曹雪涛
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Second Military Medical University SMMU
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Abstract

The invention relates to the technical field of biological medicines, and particularly relates to a CAR-NK cell capable of autocrine IL2R beta gamma agonist and targeting tumor antigen MSLN and application thereof. Wherein the MSLN-targeting chimeric antigen receptor is connected in parallel with IL2R beta gamma-A to form a co-expressed polycistronic structure. IL2R beta gamma-A is an artificial polypeptide designed and synthesized by a computer based on IL2R beta gamma conformation, and the affinity of the artificial polypeptide with IL2R beta gamma is obviously improved compared with that of natural IL-2; and the polypeptide has no binding activity with IL-2R alpha, so that the activation effect of natural cytokines on regulatory T cells is avoided, and the killing activity of CAR-NK cells is obviously improved. The invention improves the in vitro and in vivo anti-tumor effect of the targeting MSLN CAR-NK cell, and can be used for treating malignant tumors such as MSLN positive pancreatic cancer, ovarian cancer, cervical cancer and the like.

Description

MSLN-CAR-NK cell capable of autocrine IL2R beta gamma agonist and application thereof
Technical Field
The invention relates to the technical field of biological medicines, and particularly relates to CAR-NK cells capable of secreting IL2R beta gamma specific agonist IL2R beta gamma-A and targeting tumor antigen MSLN and application thereof.
Background
Natural killer cells (NK) are a subset of the killer cells that make up the innate immune system and are important effector cells for the rapid clearance of both malignant transformed cells and virally infected cells. NK cells do not require pre-sensitization for killing of tumor cells, are not MHC (major histocompatibility complex) restricted, and are not at risk for graft versus host disease (GvHD). Therefore, in recent years, NK cells are being returned to patients after in vitro expansion and/or genetic modification (CAR-NK), and are attracting attention as a new immunotherapy. CD 19-targeted CAR-NK show good clinical efficacy in the treatment of hematologic malignancies, but there is limited evidence of clinical benefit from CAR-NK cell therapy against solid tumors. The tumor microenvironment is an important factor that contributes to the failure of NK adoptive reinfusion therapy, which impairs the phenotype, activation, function and persistence of NK cells. NK cell proliferation and antitumor activity in tumors are inhibited by secretion of various immunosuppressive factors by tumor cells, including prostaglandin E2, Indoleamine 2, 3-dioxygenase (IDO), interleukin 10(IL-10), transforming growth factor-beta (TGF-beta), and vascular endothelial growth factor; while cytokines necessary for NK cell activation, such as IL-2, IL-15 and IL-21, are relatively deficient in tumors.
Cytokines play an important role in immunoregulation of NK cell function. Genetically engineered feeder cells expressing cytokines (IL-2, IL-15, or IL-21) have been widely used in the in vitro expansion of NK cells. If IL-15 or IL-2 combination therapy is given simultaneously in NK-treated patients, the clinical efficacy of NK reinfusion can be improved to some extent. However, treatment-related increases in Tregs occur following IL-2 administration; although the IL-15 or IL-21 treatment does not expand Tregs, the systemic inflammatory response of the recipients is often caused, and more than three levels of adverse reactions such as acute pancreatitis and acute vasculitis can occur in part of the recipients. These factors greatly limit the use of natural cytokines in CAR-NK therapy.
Natural cytokines have pleiotropic effects, and one cytokine may exert different effects on different cells, so that cytokines injected exogenously into the body often cause severe side effects or off-target activation of unrelated immune cells. As the structural basis for cytokine-receptor interactions has been deeply elucidated, the design of artificial activation peptides for specific receptors by computational biological methods has become an increasingly feasible and powerful approach. These artificially designed cytokine activators can bind to specific cytokine receptors with higher affinity than natural cytokines, and thus have a more narrow target cell lineage.
Disclosure of Invention
The invention aims to provide a CAR-NK cell which autocrine IL2 beta gamma specific agonist IL2R beta gamma-A and target tumor antigen MSLN, and a construction method and application thereof.
IL-2 is an activated cytokine of T cells and NK cells, and the IL-2 receptor is a multi-subunit receptor complex consisting of IL2R alpha, IL2R beta and IL2R gamma. Wherein IL2R beta and IL2R gamma form an affinity receptor in IL-2, and IL2R alpha, IL2R beta and IL2R gamma form a high affinity receptor for IL-2. Only intermediate affinity receptors consisting of IL2R beta gamma are expressed in NK cells, so that an agonist of IL2R beta gamma (IL2R beta gamma-A) is artificially designed and is connected with a chimeric antigen receptor in series, so that the NK cells can be autocrine in tumors to provide cytokine signals for self-activation, and the clinical curative effect of CAR-NK can be improved.
In a first aspect of the invention, there is provided a MSLN-targeted chimeric antigen receptor (MSLN-CAR) comprising a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, and an intracellular domain;
the signal peptide is a signal peptide of a protein selected from the group consisting of: the amino acid sequence of the OSM signal peptide is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2; the amino acid sequence of the IL-2 signal peptide is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO. 4; the HSA signal peptide has an amino acid sequence shown as SEQ ID NO.5 and a nucleotide sequence shown as SEQ ID NO. 6; the amino acid sequence of the CD8 alpha signal peptide is shown as SEQ ID NO.7, and the nucleotide sequence is shown as SEQ ID NO. 8; the amino acid sequence of the insulin signal peptide is shown as SEQ ID NO.9, and the nucleotide sequence is shown as SEQ ID NO. 10; the amino acid sequence of the trypsinogen 2 signal peptide is shown as SEQ ID NO.11, and the nucleotide sequence is shown as SEQ ID NO. 12.
In a preferred embodiment of the invention, the signal peptide sequence is shown in SEQ ID No. 7.
The antigen binding structural domain is MSLN scFv capable of being specifically bound, the structure of the antigen binding structural domain can be one of VH-linker-VL or VL-linker-VH, and the amino acid sequence of the linker is a 3-time repetitive sequence (G) 4 S) 3 Or 4 repeats (G) 4 S) 4 . Wherein the amino acid sequences of VH are respectively shown as SEQ ID NO. 13-18, and the corresponding nucleotide sequences are respectively shown as SEQ ID NO. 19-24; the amino acid sequence of VL is shown in SEQ ID NO. 25-30.
In a preferred embodiment of the invention, the amino acid sequence of the antigen binding domain is as shown in SEQ ID NO.37, SEQ ID NO.38 or SEQ ID NO. 39.
The hinge region connects the extracellular domain of the chimeric antigen receptor to the transmembrane region, and is selected from the following proteins: CD8 alpha, the amino acid sequence of which is shown as SEQ ID NO.40, and the nucleotide sequence of which is shown as SEQ ID NO. 41; CD28, the amino acid sequence of which is shown as SEQ ID NO.42, and the nucleotide sequence of which is shown as SEQ ID NO. 43; IgG1, the amino acid sequence of which is shown as SEQ ID NO.44 and the nucleotide sequence of which is shown as SEQ ID NO. 45; the amino acid sequence of the IgG4 is shown as SEQ ID NO.46, and the nucleotide sequence is shown as SEQ ID NO. 47.
In a preferred embodiment of the invention, the hinge region sequence is shown in SEQ ID No. 40.
The transmembrane domain is selected from the following proteins: CD4, the amino acid sequence of which is shown as SEQ ID NO.48, and the nucleotide sequence of which is shown as SEQ ID NO. 49; CD8 alpha, the amino acid sequence of which is shown as SEQ ID NO.50, and the nucleotide sequence of which is shown as SEQ ID NO. 51; CD28, the amino acid sequence of which is shown as SEQ ID NO.52, and the nucleotide sequence of which is shown as SEQ ID NO. 53; the amino acid sequence of NKG2D is shown in SEQ ID NO.54, and the nucleotide sequence is shown in SEQ ID NO. 55.
In a preferred embodiment of the invention, the transmembrane domain has the sequence shown in SEQ ID No. 50.
The intracellular domain can be composed of 2-4 tandem signal transduction domains or co-stimulation domains of the following molecules, and comprises: CD28, the amino acid sequence of which is shown as SEQ ID NO.56, and the nucleotide sequence of which is shown as SEQ ID NO. 57; CD137, the amino acid sequence of which is shown as SEQ ID NO.58, and the nucleotide sequence of which is shown as SEQ ID NO. 59; CD3 zeta, the amino acid sequence is shown in SEQ ID NO.60, and the nucleotide sequence is shown in SEQ ID NO. 61; DAP10, the amino acid sequence of which is shown as SEQ ID NO.62, and the nucleotide sequence of which is shown as SEQ ID NO. 63; DAP12, the amino acid sequence of which is shown as SEQ ID NO.64, and the nucleotide sequence of which is shown as SEQ ID NO. 65; IL2R gamma, the amino acid sequence of which is shown as SEQ ID NO.66, and the nucleotide sequence of which is shown as SEQ ID NO. 67; ICOS, the amino acid sequence of which is shown as SEQ ID NO.68, and the nucleotide sequence of which is shown as SEQ ID NO. 69; OX40, wherein the amino acid sequence is shown as SEQ ID NO.70, and the nucleotide sequence is shown as SEQ ID NO. 71; the amino acid sequence of NCR3 is shown in SEQ ID NO.72, and the nucleotide sequence is shown in SEQ ID NO. 73.
In a preferred embodiment of the invention, the intracellular domain sequence consists of SEQ ID NO.58 in tandem with SEQ ID NO. 60.
In a preferred embodiment of the invention, the sequence of the MSLN-targeted chimeric antigen receptor (MSLN-CAR) comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, and an intracellular domain; wherein the sequence of the signal peptide is shown as SEQ ID No. 7; the sequence of the antigen binding domain is shown as SEQ ID NO. 37; the sequence of the hinge region is shown as SEQ ID No.40, and the sequence of the transmembrane domain is shown as SEQ ID No. 50; the intracellular domain sequence is composed of SEQ ID NO.58 and SEQ ID NO.60 in tandem.
In a second aspect of the invention, a computer-designed IL2R beta gamma agonist IL2R beta gamma-A is provided, the amino acid sequence of which is shown in SEQ ID No. 74-81, and the corresponding nucleotide sequences are respectively shown in SEQ ID No. 82-89.
In a preferred embodiment of the invention, the receptor affinity assay is performed using IL2R β γ -A having the amino acid sequence shown in SEQ ID NO. 76.
In a third aspect of the present invention, a polycistronic structure coexpressing the above-mentioned MSLN-targeting chimeric antigen receptor and IL2R β γ -A is provided, wherein the chimeric antigen receptor and IL2R β γ -A sequence can be directly linked through P2A, T2A, E2A or F2A peptide; the nucleotide sequence of the polycistronic structure is selected from any one of SEQ ID NO. 90-93.
In a preferred embodiment of the invention, the polycistronic structure has the sequence shown in SEQ ID NO. 90.
In a fourth aspect of the invention, there is provided the use of the above-described MSLN-targeting chimeric antigen receptor construct, or IL2R β γ -A construct, or polycistronic construct co-expressing MSLN-targeting chimeric antigen receptor and IL2R β γ -A, in the preparation of a CAR-NK cell line that autocrine IL2 β γ -specific agonist and targets the tumor antigen MSLN.
In a fifth aspect of the invention, there is provided a lentiviral vector comprising the amino acid and nucleotide sequences of the above chimeric antigen receptor targeting MSLN and IL2R β γ -a.
In a sixth aspect of the present invention, there is provided a genetically modified natural killer cell, which has integrated into its genome the nucleotide sequence of the above-described MSLN-targeting chimeric antigen receptor and IL2R β γ -A, or a polycistronic nucleotide sequence comprising both sequences.
The seventh aspect of the present invention provides the use of the above-mentioned MSLN-targeting chimeric antigen receptor structure, the above-mentioned IL2R β γ -A structure, the above-mentioned polycistronic structure coexpressing MSLN-targeting chimeric antigen receptor and IL2R β γ -A, the above-mentioned lentiviral vector, and the above-mentioned natural killer cell in the preparation of a medicament or a preparation for treating tumor.
Preferably, the tumor is an MSLN positive tumor; such as pancreatic cancer, ovarian cancer, cervical cancer, etc.
In an eighth aspect of the present invention, the above-mentioned chimeric antigen receptor structure targeting MSLN, or IL2R β γ -a structure, or polycistronic structure coexpressing the chimeric antigen receptor targeting MSLN and IL2R β γ -a is used to establish a system using NK92 cell line as the host cell.
Preferably, the construction method comprises the following steps:
A. designing genetic elements
Designing a chimeric antigen receptor structure targeting MSLN according to the amino acid sequence; designing polycistronic structure of IL2R beta gamma-A; designing polycistronic structure of chimeric antigen receptor and IL2R beta gamma-A for coexpression targeting MSLN;
B. lentiviral vector construction
Respectively cloning a polycistronic structure gene fragment which expresses a chimeric antigen receptor structure targeting MSLN, an IL2R beta gamma-A structure or co-expresses the chimeric antigen receptor targeting MSLN and IL2R beta gamma-A into a pLVX plasmid, wherein pMD2.G and pCMVR8.74 are used as a slow virus packaging plasmid; culturing Lenti-X-293 cells transfected by plasmids for 48 hours, 60 hours or 72 hours (preferably 72 hours), collecting virus supernatant when the cells are cultured for 36 hours midway, preserving the virus supernatant at 4 ℃, supplementing a fresh culture medium, culturing the cells for 36 hours again, and collecting the virus supernatant; mixing the two virus supernatants, centrifuging at 4 ℃ and 20000rpm for 2.5 hours, then discarding the supernatant, collecting virus particle precipitates, and suspending the viruses by using 50-100 mu L of serum-free culture medium;
C. MSLN-CAR-NK cell establishment line targeting MSLN
Recovering the low generation NK-92 cells, and carrying out passage for 3-4 times; cell plating, optionally 5 × 10 4 48-hole plate with 1X 10 holes 5 24-hole plate of 5X 10 5 6-hole plate (preferably 1 × 10) per hole 5 24-well plates per well); 37 ℃ and 5% CO 2 Culturing for 2-4 hours (preferably culturing for 4 hours); polybrene is added into each hole, and the final concentration of the polybrene is 1 to 6 mu g/mL (1, 2, 4, 5 or 6 mu g/mL can be selected, and 2mg/mL polybrene is preferably used); mix thoroughlyAfter homogenizing, adding the coated virus according to the MOI of 10-50 (the MOI of 10, 20, 30, 40 and 50, preferably 30); after 12 hours of infection, sucking all cell sap containing cells and viruses, transferring the cell sap into a culture flask containing complete culture medium, culturing the cell sap to the 4 th day, detecting the proportion of NK positive cells expressing the MSLN-CAR by FACS (FACS) and sorting the MSLN-CAR-NK positive cells, wherein the sorting idea comprises the following steps: MSLN-CAR-NK positive cells and NK-92 cells expressing MSLN-CAR are marked by anti-MSLN-PerCP-cyanine5.5 flow antibody, and MSLN expression positive cells are selected for sorting; the sorted cells were propagated to 10 7 The anti-MSLN-PerCP-cyanine5.5 flow antibody is used again to mark MSLN-CAR-NK positive cells and NK-92 cells expressing MSLN-CAR; selecting a positive cell group with 20% of MSLN expression abundance after marking, carrying out amplification culture, culturing for 7 days after culturing, picking single cell clusters to a 24-pore plate for continuous culture, picking 24 single cell clusters in total, transferring the 24-pore plate cells after amplification culture to a 6-pore plate for continuous amplification; taking the amplified MSLN-CAR-NK cells and NK-92 cell marker flow antibody anti-MSLN-cyanine5.5, and identifying the expression condition of the MSLN CAR; and recording the doubling time of each monoclonal; and finally, selecting the monoclonal with the highest MSLN-CAR-NK positive proportion and the shortest doubling time, continuing to carry out amplification culture, and completing establishment of the system.
According to the construction method, the established cell groups are uniform, and the MSLN-CAR shows positive expression. FACS examined the stability of MSLN expression of serial passaged MSLN-CAR-NK cells (figure 1).
D. MSLN-CAR-NK-IL2R beta gamma-A cell establishment line coexpressing MSLN-targeted chimeric antigen receptor and IL2R beta gamma-A
Constructing MSLN-CAR-NK-IL2R beta gamma-A cells, wherein the low-generation NK cells are infected by lentiviruses carrying a polycistronic structure of a chimeric antigen receptor and IL2R beta gamma-A of a co-expression targeting MSLN, or the low-generation MSLN-CAR-NK cells are infected by lentiviruses carrying IL2R beta gamma-A structural genes (preferably the low-generation MSLN-CAR-NK cells are infected by lentiviruses carrying IL2R beta gamma-A structural genes) on the basis of the MSLN-CAR-NK cells; the method comprises the following steps: recovering low-generation MSLN-CAR-NK cells, and carrying out passage for 3-4 times; cell plating, optionally 5 × 10 4 48-hole plate with 1X 10 holes 5 24-hole plate of 5X 10 5 6-hole plate (preferably 1 × 10) per hole 5 24-well plates per well); 37 ℃ and 5% CO 2 Culturing for 2-4 hours (preferably culturing for 4 hours); polybrene is added into each hole, and the final concentration of the polybrene is 1 to 6 mu g/mL (1, 2, 4, 5 or 6 mu g/mL can be selected, and 2mg/mL polybrene is preferably used); mixing, and adding the coated virus according to the MOI of 10-50 (the MOI is 10, 20, 30, 40, and 50, preferably 30); after infection for 12 hours, sucking all cell sap containing cells and viruses, transferring the cell sap into a culture bottle containing a complete culture medium, culturing for 4 days, picking monoclonal cell clusters to a 24-pore plate for continuous culture, picking 24 single cell clusters in total, transferring the 24-pore plate cells to a 6-pore plate for continuous amplification after amplification culture; adding Brefeldin A into each hole 12 hours before flow verification, wherein the usable concentration is 1-50 mu M (preferably adding Brefeldin A according to the working concentration of 10 mu M); after MSLN-CAR-NK positive cells expressing IL2R beta gamma-A and MSLN-CAR-NK cells not expressing IL2R beta gamma-A are taken to be fixed and broken membranes, flow antibody anti-IL2R beta gamma-A-PE is marked, and the expression condition of IL2R beta gamma-A is identified; and recording the doubling time of each monoclonal; and finally, selecting the monoclonal with the highest MSLN-CAR-NK positive proportion and the shortest doubling time for secreting IL2R beta gamma-A, continuously carrying out amplification culture, transferring to a 6-well plate, taking the amplified MSLN-CAR-NK positive cells expressing IL2R beta gamma-A and MSLN-CAR-NK cells to mark a flow antibody anti-HA-PE (Brefeldin A is added before the marking antibody and the cells are fixed to break membranes), identifying the expression condition of IL2R beta gamma-A, selecting the monoclonal with the highest MSLN-CAR-NK positive proportion and the shortest doubling time for secreting IL2R beta gamma-A again, and carrying out amplification culture to complete the establishment of a system.
According to the construction method, the established cell groups are uniform, IL2R beta gamma-A presents positive expression, and FACS detects the stability of IL2R beta gamma-A expression of serial-passage MSLN-CAR-NK-IL2R beta gamma-A cells (figure 2).
The invention has the advantages that:
1. the invention provides a CAR-NK cell capable of autocrine IL2R beta gamma agonist IL2R beta gamma-aginst (IL2R beta gamma-A) and efficiently killing MSLN positive solid tumor. IL2R beta gamma-A is an artificial polypeptide designed and synthesized by a computer based on IL2R beta gamma conformation, and the affinity of the artificial polypeptide with IL2R beta gamma is obviously improved compared with that of natural IL-2; and has no binding activity with interleukin 2 receptor alpha (IL-2R alpha), and avoids the activation effect of natural cytokines on regulatory T cells (Tregs), thereby remarkably improving the killing activity of CAR-NK cells. The invention improves the in vitro and in vivo anti-tumor effect of the targeting MSLN CAR-NK cell, and can be used for treating malignant tumors such as MSLN positive pancreatic cancer, ovarian cancer, cervical cancer and the like.
2. According to the invention, the MSLN-targeted CAR-NK cell highly expresses the agonist of IL2R beta gamma through gene modification, so that the cytotoxic activity of the CAR-NK cell is promoted, the large-scale amplification of the CAR-NK cell without exogenous addition of cytokines is realized, the NK cell can be autocrine in tumors to provide cytokine signals for self-activation, the clinical curative effect of the CAR-NK can be improved, and a new strategy is provided for promoting the in vitro and in vivo killing efficacy of the CAR-NK cell.
Drawings
FIG. 1 FACS detection of MSLN-CAR-NK cell MSLN expression (FIG. 1A); FACS examined the stability of MSLN expression of serial passaged MSLN-CAR-NK cells (fig. 1B).
FIG. 2 FACS detection of expression of IL2R β γ -A by MSLN-CAR-NK-IL2R β γ -A cells (FIG. 2A); FACS checked the stability of IL2R β γ -a expression of serial passaged MSLN-CAR-NK-IL2R β γ -a cells (fig. 2B).
FIG. 3 lysis efficiency of effector cells NK-92, MSLN-CAR-NK and MSLN-CAR-NK-IL-2R β γ -A on target cells AspC-1 at an effective target ratio of 1:1 or 1:2 (FIG. 3A); FIG. 3B shows the secretion of IFN-gamma after killing target cells AsPC-1 by effector cells NK-92, MSLN-CAR-NK and MSLN-CAR-NK-IL2R beta gamma-A according to the effective target ratio of 1: 2.
FIG. 4 shows the Western Blot detection of the activation of the PLC, Syk-Erk, NF- κ B signaling pathway, PI3K-Akt signaling pathway, JAK-STAT signaling pathway after 30min of interaction of NK-92, MSLN-CAR-NK-IL2R β γ -A cells and AsPC-1 cells (FIG. 4A-FIG. 4E, FIG. 4F is internal reference); NK-92, MSLN-CAR-NK and MSLN-CAR-NK-IL2R β γ -A cells are abbreviated as NK-92, C-NK-IL, respectively, in the figures.
FIG. 5. tumor volume size display shows inhibitory effect on AsPC-1 subcutaneous transplantable tumors by NK-92, MSLN-CAR-NK and MSLN-CAR-NK-IL2R β γ -A cells.
FIG. 6. determination of the affinity of IL2R β γ -A for interleukin 2 receptor β γ and interleukin 2 receptor α.
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
Example 1: construction and stability detection of MSLN-CAR-NK cell line
The experimental method comprises the following steps:
MSLN-CAR lentiviral packaging: the MSLN-CAR sequence was cloned into a lentiviral vector, transfected with the packaging plasmid into a Lenti-X-293 cell line, cultured continuously for 72 hours (during which time the cell culture broth was collected once, stored at 4 ℃ and cultured on a cell culture dish with fresh cell culture broth added), and the viral supernatant was collected and filtered. The virus was concentrated by centrifugation at 20000rpm at 4 ℃ for 2.5 hours, and the titer was quantified after resuspension of the virus particles.
Establishing a MSLN-CAR-NK cell line: infecting NK92 cells with lentivirus at MOI of 30, and sorting MSLN-CAR positive cells; after the cells are expanded and cultured after the first sorting, the cells are sorted for the second time. And after the secondary sorting cell is expanded and cultured, selecting a monoclonal establishment system, and performing expanded culture.
MSLN-CAR-NK cell stability assay: MSLN-CAR-NK cells are continuously cultured in complete culture medium for passage, and the positive rate of the lineage establishing cells MSLN-CAR is detected by using anti-MSLN-PerCP-cyanine5.5 flow antibody every 7 days.
The experimental results are as follows:
the established MSLN-CAR-NK cell lines are clustered uniformly, and MSLN-CAR shows positive expression and better expression abundance (FIG. 1A). In a culture period of continuous 8 weeks, the stability of the MSLN-CAR-NK cells after establishment is better, and the positive rate of the MSLN-CAR is stabilized to be more than 95% (FIG. 1B).
Example 2: construction and stability detection of MSLN-CAR-NK-IL2R beta gamma-A cell line
The experimental method comprises the following steps:
IL2R β γ -a lentivirus packaging: the IL 2R. beta. gamma. -A sequence was cloned into a lentiviral vector, transfected with a packaging plasmid into a Lenti-X-293 cell line, cultured continuously for 72 hours (during which the cell culture broth was collected once, stored at 4 ℃ and cultured by adding fresh cell culture broth to the cell culture dish), and the viral supernatant was collected and filtered. The virus was concentrated by centrifugation at 20000rpm at 4 ℃ for 2.5 hours, and the titer was quantified after resuspension of the virus particles.
Establishing a MSLN-CAR-NK-IL2R beta gamma-A cell line: infecting MSLN-CAR-NK cells by lentivirus according to MOI (30), selecting monoclonal after amplification culture, performing amplification culture to a 24-well plate, marking the cells by a flow antibody anti-IL2R beta gamma-A-PE (after Brefeldin A is added 12 hours before marking, the cells are fixed and membrane is broken), and identifying the expression condition of IL2R beta gamma-A by flow; and recording the doubling time for each monoclonal. And finally, selecting the monoclonal with the highest MSLN-CAR-NK positive proportion and the shortest doubling time for secreting IL2R beta gamma-A, continuously carrying out amplification culture, transferring to a 6-well plate, picking out the monoclonal again, verifying the expression condition of IL2R beta gamma-A after amplification, selecting the monoclonal with the highest MSLN-CAR-NK positive proportion and the shortest doubling time for secreting IL2R beta gamma-A again, and completing establishment of the system by amplification culture. According to the construction method, the established cell groups are uniform, IL2R beta gamma-A presents positive expression, and FACS detects the stability of IL2R beta gamma-A expression of serial-passage MSLN-CAR-NK-IL2R beta gamma-A cells (figure 2).
MSLN-CAR-NK-IL2R β γ -a cell stability assay: MSLN-CAR-NK-IL2R beta gamma-A cells are continuously cultured and passaged in a complete culture medium, and the positive rate of the established cell IL2R beta gamma-A is detected by using anti-HA-PE flow antibodies every 7 days.
The experimental results are as follows:
the MSLN-CAR-NK-IL2R beta gamma-A cell line after establishment is uniform in grouping, and IL2R beta gamma-A shows positive expression and is better in expression abundance (figure 2A). In a continuous culture period of 8 weeks, the stability of the MSLN-CAR-NK-IL2R beta gamma-A cells after line establishment is better, and the positive rate of IL2R beta gamma-A is stabilized to be more than 95% (fig. 2B).
Example 3: IL2R beta gamma-A remarkably improves killing activity of MSLN-CAR-NK cells
The experimental method comprises the following steps:
cell killing experiment: setting NK-92, MSLN-CAR-NK and MSLN-CAR-NK-IL2R beta gamma-A cells and MSLN high expression pancreasThe cancer cell line AspC-1 target cells had effective target ratios (E: T) of 1:1 and 1:2 and killing time of 2 hrs. Preparing single cell suspension of tumor cells, 10 times 6 10 μ L of 50 μ M CSFE was added to each tumor cell, and the cells were incubated at 37 ℃ for 15min in the absence of light. Ice cold PBS washing 2 times, using killing culture medium to suspend cells, 24 hole plate per hole add 4X 10 4 (250. mu.L) AsPC-1 cells. Effector cell count: adding 4X 10 per hole according to effective target ratio 4 Or 8X 10 4 (250 μ L) effector cells; effector cells are mixed with cells and incubated in a cell culture incubator. After 2 hours, the cell suspension was collected, centrifuged at 350G for 5min, washed once with 1mL PBS, centrifuged at 350G for 5min, the supernatant was discarded, the cell pellet was resuspended, a portion of the cells was separated and added to 5. mu.L of 7-AAD (Beyotime, C1053S), incubated at room temperature in the dark for 15min, washed once with ice-cold PBS, and the tumor cell death rate was determined by FACS. Another part of the cells was fixed to rupture the membranes and then labeled with flow antibody (Biolegend, 308504) to detect IFN-. gamma.secretion.
The experimental results are as follows:
the results show that both MSLN-CAR-NK and MSLN-CAR-NK-IL2R beta gamma-A cells can kill AsPC-1 target cells; however, compared to MSLN-CAR-NK cells that did not express IL2R β γ -a, IL2R β γ -a significantly increased the killing activity of MSLN-CAR-NK cells (fig. 3A), FACS detected the expression of IL2R β γ -a, and IFN- γ expression of MSLN-CAR-NK-IL2R β γ -a cells was significantly increased (fig. 3B).
Example 4: study of IL2R betagamma-A on activation of MSLN-CAR-NK cell killing-associated signaling pathway
The experimental method comprises the following steps:
western-blot: respectively co-culturing NK-92, MSLN-CAR-NK and MSLN-CAR-NK-IL2R beta gamma-A cells and tumor cells according to the ratio of 2:1 for 30min, and respectively collecting NK-92, MSLN-CAR-NK and MSLN-CAR-NK 2R beta gamma-A cells 5 x 10 6 Cells, protein extraction reagent (Thermo Scientific, 78503) extracted protein and protein quantification was performed using BCA protein quantification kit (Thermo Scientific, 23222). After quantification, the protein concentrations of all groups of samples are adjusted to be consistent, and activation conditions of PLC, Syk-Erk, NF-kB signal channels, PI3K-Akt signal channels and JAK-STAT channel activation are verified by Western Blot. The following were confirmed: detected activation of NK cells but not tumor cellsRoad surface
The experimental results are as follows:
comparing the differences in activation of NK-92, MSLN-CAR-NK and MSLN-CAR-NK-IL-2R β γ -A cells, including PLC, Syk-Erk, NF- κ B and PI 3K-Akt-associated signals. The results indicate that the PLC- γ phosphorylation level of MSLN-CAR-NK-IL2R β γ -a cells was significantly elevated compared to MSLN-CAR-NK (fig. 4A); significantly promotes the activation of IKK alpha/beta in NF-kB pathway (FIG. 4B); STAT3, STAT5, STAT6 phosphorylation levels were significantly elevated (fig. 4C); phosphorylation levels of Syk and ERK1/2 were significantly increased (fig. 4D); akt phosphorylation in the PI3K-Akt signaling pathway was also significantly enhanced (fig. 4E); beta-actin was used as the internal reference (FIG. 4F).
Example 5: killing effect of MSLN-CAR-NK-IL2R beta gamma-A cells on AsPC-1 in-vivo in-situ tumor
The experimental method comprises the following steps: NSG mice were injected subcutaneously with MSLN high-expression AsPC-1 cells in logarithmic growth phase 1X 10 7 Tumor-bearing mouse models are constructed, and NK-92, MSLN-CAR-NK and MSLN-CAR-NK-IL2R beta gamma-A cells are respectively given for treatment after 7 days (tail vein injection is 1 x 10) 7 Once every 5 days for a total of 3 times). Tumors were harvested on day 25 of molding and the therapeutic effects of each group were compared.
The experimental results are as follows:
the therapeutic effect of MSLN-CAR-NK-IL2R beta gamma-A cells was significantly better than that of MSLN-CAR-NK cells, and the tumor volume was significantly reduced (FIG. 5).
Example 6: affinity detection of IL2R beta gamma-A for interleukin 2 receptor
The experimental method comprises the following steps: the interleukin 2 receptor beta gamma subunit mixture or interleukin 2 receptor alpha subunit coating enzyme label plate. IL2R β γ -A was added in a dilution of two fold to an ELISA plate and incubated at 37 ℃ for 2 hours, and IL2 was added in an equal concentration in a dilution of two fold to serve as a control. After washing the plate three times, add enzyme-labeled secondary antibody, incubate for 30 minutes. The plate was washed again and then developed and the absorbance measured.
The experimental results are as follows:
artificially designed IL2R β γ -a has a higher affinity for IL2 β γ than native IL 2; but no binding to IL2 alpha (fig. 6).
While the preferred embodiments of the present invention have been illustrated and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Figure BDA0003703064540000121
Figure BDA0003703064540000131
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Sequence listing
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acctgcaccg ccagcggcat cgacttcagc cgctactaca tgtgctgggt gcgccaggcc 120
cccggcaagg gcctggaggg catcgcctgc atctacatcg gcggcagcgg cagcacctac 180
tacgccagct gggccaaggg ccgcttcacc atcagcaagg ccagcagcac caccgtgacc 240
ctgcagatga ccagcctgac cgccgccgac accgccacct acttctgcgc ccgcggcacc 300
aacctgaact acatcttccg cctgtggggc cccggcaccc tggtgaccgt gagcagc 357
<210> 23
<211> 345
<212> DNA
<213> Artificial sequence (Artificial)
<400> 23
cagagcctgg aggagagcgg cggcgacctg gtgaagcccg gcgccagcct gaccctgacc 60
tgcaaggcca gcggcttcga cttcagcagc aacgccatgt gctgggtgcg ccaggccccc 120
ggcaagggcc tggagtggat cgcctgcatc tacgtgggcg acggcaacac ctactacgcc 180
agctgggcca agggccgctt caccatcagc aagaccagca gcaccaccgt gaccctgcag 240
atgaccagcc tgaccgccgc cgacaccgcc acctacttct gcgcccgcgg ctacgccagc 300
tacggcagcg actactactg ggactacttc aagctgtggg gcccc 345
<210> 24
<211> 360
<212> DNA
<213> Artificial sequence (Artificial)
<400> 24
cagagcctgg aggagagcgg cggcgacctg gtgaagcccg gcgccagcct gaccctgacc 60
tgcaccgcca gcggcttcag cttcagcggc gactactaca tgtgctgggt gcgccaggcc 120
cccggcaagg gcctggagtg gatcgcctgc atcggcggcg gcagcaacac cgccacctac 180
tacgccacct gggccaaggg ccgcttcacc atcagcaaga ccagcagcac caccgtgacc 240
ctgcagatga ccagcctgac cgccgccgac accgccacct acttctgcgc ccgcgacctg 300
ggcttcgtgg actacgccct ggagctgtgg ggccccggca ccctggtgac cgtgagcagc 360
<210> 25
<211> 113
<212> PRT
<213> Artificial sequence (Artificial)
<400> 25
Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln
85 90 95
Tyr Ala Ser Tyr Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Met
100 105 110
Lys
<210> 26
<211> 110
<212> PRT
<213> Artificial sequence (Artificial)
<400> 26
Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly
1 5 10 15
Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile
35 40 45
Phe Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly
50 55 60
Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Asp Leu Glu Cys
65 70 75 80
Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser
85 90 95
Asn Asn Trp His Ala Phe Gly Gly Gly Thr Glu Val Val Val
100 105 110
<210> 27
<211> 109
<212> PRT
<213> Artificial sequence (Artificial)
<400> 27
Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Ser Ala Ala Val Gly
1 5 10 15
Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Asn Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
35 40 45
Tyr Gln Ala Ser Thr Leu Ala Pro Gly Val Ser Ser Arg Phe Lys Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Gly Val Glu Cys
65 70 75 80
Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Ser Ser Asn
85 90 95
Val Glu Asn Val Phe Gly Gly Gly Thr Gly Val Val Val
100 105
<210> 28
<211> 80
<212> PRT
<213> Artificial sequence (Artificial)
<400> 28
Asp Val Val Met Thr Gln Thr Pro Ser Pro Val Ser Ala Ala Val Gly
1 5 10 15
Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Asn Asn Gly
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Lys Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys
65 70 75 80
<210> 29
<211> 109
<212> PRT
<213> Artificial sequence (Artificial)
<400> 29
Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Ser Ala Ala Val Gly
1 5 10 15
Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Thr Ala
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Val Leu Ile
35 40 45
Tyr Ala Ala Ser Asn Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys
65 70 75 80
Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ala Ala Thr Ile Ile Asn
85 90 95
Val Asp Asn Val Phe Gly Gly Gly Thr Glu Val Val Val
100 105
<210> 30
<211> 111
<212> PRT
<213> Artificial sequence (Artificial)
<400> 30
Asp Ile Val Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly
1 5 10 15
Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Glu Asn Met Tyr Asn Ser
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
35 40 45
Tyr Arg Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Lys Gly
50 55 60
Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Asp Leu Glu Cys
65 70 75 80
Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Cys Thr Phe Tyr Ser His Asn
85 90 95
Asn Asn Tyr Gly Gly Ala Phe Gly Gly Gly Thr Glu Val Val Val
100 105 110
<210> 31
<211> 339
<212> DNA
<213> Artificial sequence (Artificial)
<400> 31
gacatcgtga tgagccagag ccccagcagc ctggccgtga gcgtgggcga gaaggtgacc 60
atgagctgca agagcagcca gagcctgctg tacagcagca accagaagaa ctacctggcc 120
tggtaccagc agaagcccgg ccagagcccc aagctgctga tctactgggc cagcacccgc 180
gagagcggcg tgcccgaccg cttcaccggc agcggcagcg gcaccgactt caccctgacc 240
atcagcagcc tggagagcga ggacttcgcc gactactact gcctgcagta cgccagctac 300
ccccgcacct tcggcggcgg caccaagctg gagatgaag 339
<210> 32
<211> 330
<212> DNA
<213> Artificial sequence (Artificial)
<400> 32
gacgtcgtga tgacccagac tccagcctcc gtgtctgaac ctgtgggagg cacagtcacc 60
atcaagtgcc aggccagtca gaggattagt agttacttat cctggtatca gcagaaacca 120
gggcagcgtc ccaagctcct gatctttggt gcatccactc tggcatctgg ggtcccctcg 180
cggttcaaag gcagtggatc tgggacagaa tacactctca ccatcagcga cctggagtgt 240
gccgatgctg ccacttacta ctgtcagagt tatgcttatt ttgatagtaa taattggcat 300
gctttcggcg gagggaccga ggtggtggtc 330
<210> 33
<211> 327
<212> DNA
<213> Artificial sequence (Artificial)
<400> 33
gcctacgaca tgacccagac ccccgccagc gtgagcgccg ccgtgggcgg caccgtgacc 60
atcaagtgcc aggccagcca gagcatcagc aactacctgg cctggtacca gcagaagccc 120
ggccagcccc ccaagctgct gatctaccag gccagcaccc tggcccccgg cgtgagcagc 180
cgcttcaagg gcagcggcag cggcaccgag ttcaccctga ccatcagcgg cgtggagtgc 240
gccgacgccg ccacctacta ctgccagcag ggctacacca gcagcaacgt ggagaacgtg 300
ttcggcggcg gcaccggcgt ggtggtg 327
<210> 34
<211> 240
<212> DNA
<213> Artificial sequence (Artificial)
<400> 34
gacgtggtga tgacccagac ccccagcccc gtgagcgccg ccgtgggcgg caccgtgacc 60
atcaagtgcc aggccagcca gagcatcaac aacggcctgg cctggtacca gcagaagccc 120
ggccagcccc cccgcctgct gatctacagc gccagcaacc tggagagcgg cgtgcccagc 180
cgcttcaagg gcagcggcag cggcaccgag ttcaccctga ccatcagcga cctggagtgc 240
<210> 35
<211> 327
<212> DNA
<213> Artificial sequence (Artificial)
<400> 35
gcctacgaca tgacccagac ccccgccagc gtgagcgccg ccgtgggcgg caccgtgacc 60
atcaagtgcc aggccagcca gagcatcagc accgccctgg cctggtacca gcagaagccc 120
ggccagcccc ccaaggtgct gatctacgcc gccagcaacc tggccagcgg cgtgagcagc 180
cgcttcaagg gcagcggcag cggcaccgag ttcaccctga ccatcagcga cctggagtgc 240
gccgacgccg ccacctacta ctgccagcag gccgccacca tcatcaacgt ggacaacgtg 300
ttcggcggcg gcaccgaggt ggtggtg 327
<210> 36
<211> 333
<212> DNA
<213> Artificial sequence (Artificial)
<400> 36
gacatcgtga tgacccagac ccccgccagc gtggaggtgg ccgtgggcgg caccgtgacc 60
atcaagtgcc aggccagcga gaacatgtac aacagcctgg cctggtacca gcagaagccc 120
ggccagcccc ccaagctgct gatctaccgc gccagcaccc tggagagcgg cgtgcccagc 180
cgcttcaagg gcagcggcag cggcaccgag tacaccctga ccatcagcga cctggagtgc 240
gccgacgccg ccacctacta ctgccagtgc accttctaca gccacaacaa caactacggc 300
ggcgccttcg gcggcggcac cgaggtggtg gtg 333
<210> 37
<211> 247
<212> PRT
<213> Artificial sequence (Artificial)
<400> 37
Glu Val Arg Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Gly Trp Tyr Ile Asp Phe Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Thr Pro
130 135 140
Ala Ser Val Glu Val Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln
145 150 155 160
Ala Ser Glu Asn Met Tyr Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro
165 170 175
Gly Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Thr Leu Glu Ser
180 185 190
Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Tyr Thr
195 200 205
Leu Thr Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys
210 215 220
Gln Cys Thr Phe Tyr Ser His Asn Asn Asn Tyr Gly Gly Ala Phe Gly
225 230 235 240
Gly Gly Thr Glu Val Val Val
245
<210> 38
<211> 246
<212> PRT
<213> Artificial sequence (Artificial)
<400> 38
Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Ser Ala Ala Val Gly
1 5 10 15
Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Asn Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
35 40 45
Tyr Gln Ala Ser Thr Leu Ala Pro Gly Val Ser Ser Arg Phe Lys Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Gly Val Glu Cys
65 70 75 80
Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Ser Ser Asn
85 90 95
Val Glu Asn Val Phe Gly Gly Gly Thr Gly Val Val Val Gly Gly Gly
100 105 110
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Glu Gln Leu
115 120 125
Glu Glu Ser Gly Gly Asp Leu Val Gln Pro Glu Gly Ser Leu Thr Leu
130 135 140
Thr Cys Lys Ala Ser Gly Leu Asp Phe Ser Ser Ser Tyr Trp Ile Cys
145 150 155 160
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Cys Arg
165 170 175
His Thr Phe Thr Ala Asn Thr Trp Ser Ala Ser Trp Val Asn Gly Arg
180 185 190
Phe Thr Ile Ser Arg Ser Thr Ser Leu Gly Thr Val Asp Leu Lys Met
195 200 205
Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp
210 215 220
Glu Ser Asn Asn Asp Gly Trp Asp Phe Lys Leu Trp Gly Pro Gly Thr
225 230 235 240
Leu Val Thr Val Ser Ser
245
<210> 39
<211> 248
<212> PRT
<213> Artificial sequence (Artificial)
<400> 39
Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly
1 5 10 15
Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile
35 40 45
Phe Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly
50 55 60
Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Asp Leu Glu Cys
65 70 75 80
Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser
85 90 95
Asn Asn Trp His Ala Phe Gly Gly Gly Thr Glu Val Val Val Gly Gly
100 105 110
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gln Gln
115 120 125
Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Glu Gly Ser Leu Thr
130 135 140
Leu Thr Cys Lys Ala Ser Gly Phe Asp Leu Gly Phe Tyr Phe Tyr Ala
145 150 155 160
Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Ala Cys
165 170 175
Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys
180 185 190
Gly Arg Phe Thr Ile Ser Lys Ala Ser Ser Thr Thr Val Thr Leu Gln
195 200 205
Met Thr Ser Leu Ala Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg
210 215 220
Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn Leu Trp Gly Pro
225 230 235 240
Gly Thr Leu Val Thr Val Ser Ser
245
<210> 40
<211> 45
<212> PRT
<213> Artificial sequence (Artificial)
<400> 40
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
35 40 45
<210> 41
<211> 135
<212> DNA
<213> Artificial sequence (Artificial)
<400> 41
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 42
<211> 21
<212> PRT
<213> Artificial sequence (Artificial)
<400> 42
Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro
1 5 10 15
Gly Pro Ser Lys Pro
20
<210> 43
<211> 63
<212> DNA
<213> Artificial sequence (Artificial)
<400> 43
atcatccacg tgaagggcaa gcacctgtgc cccagccccc tgttccccgg ccccagcaag 60
ccc 63
<210> 44
<211> 21
<212> PRT
<213> Artificial sequence (Artificial)
<400> 44
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly
20
<210> 45
<211> 63
<212> DNA
<213> Artificial sequence (Artificial)
<400> 45
gagcccaaga gctgcgacaa gacccacacc tgccccccct gccccgcccc cgagctgctg 60
ggc 63
<210> 46
<211> 19
<212> PRT
<213> Artificial sequence (Artificial)
<400> 46
Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val
1 5 10 15
Ala Gly Pro
<210> 47
<211> 57
<212> DNA
<213> Artificial sequence (Artificial)
<400> 47
gagcgcaagt gctgcgtgga gtgccccccc tgccccgccc cccccgtggc cggcccc 57
<210> 48
<211> 25
<212> PRT
<213> Artificial sequence (Artificial)
<400> 48
Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile
1 5 10 15
Gly Leu Gly Ile Phe Phe Cys Val Arg
20 25
<210> 49
<211> 75
<212> DNA
<213> Artificial sequence (Artificial)
<400> 49
atggccctga tcgtgctggg cggcgtggcc ggcctgctgc tgttcatcgg cctgggcatc 60
ttcttctgcg tgcgc 75
<210> 50
<211> 24
<212> PRT
<213> Artificial sequence (Artificial)
<400> 50
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> 51
<211> 72
<212> DNA
<213> Artificial sequence (Artificial)
<400> 51
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 52
<211> 30
<212> PRT
<213> Artificial sequence (Artificial)
<400> 52
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 Arg Ser Lys
20 25 30
<210> 53
<211> 90
<212> DNA
<213> Artificial sequence (Artificial)
<400> 53
ttctgggtgc tggtggtggt gggcggcgtg ctggcctgct acagcctgct ggtgaccgtg 60
gccttcatca tcttctgggt gcgcagcaag 90
<210> 54
<211> 24
<212> PRT
<213> Artificial sequence (Artificial)
<400> 54
Asn Ala Ser Pro Phe Phe Phe Cys Cys Phe Ile Ala Val Ala Met Gly
1 5 10 15
Ile Arg Phe Ile Ile Met Val Thr
20
<210> 55
<211> 72
<212> DNA
<213> Artificial sequence (Artificial)
<400> 55
aacgccagcc ccttcttctt ctgctgcttc atcgccgtgg ccatgggcat ccgcttcatc 60
atcatggtga cc 72
<210> 56
<211> 38
<212> PRT
<213> Artificial sequence (Artificial)
<400> 56
Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg
1 5 10 15
Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp
20 25 30
Phe Ala Ala Tyr Arg Ser
35
<210> 57
<211> 114
<212> DNA
<213> Artificial sequence (Artificial)
<400> 57
cgcagccgcc tgctgcacag cgactacatg aacatgaccc cccgccgccc cggccccacc 60
cgcaagcact accagcccta cgcccccccc cgcgacttcg ccgcctaccg cagc 114
<210> 58
<211> 42
<212> PRT
<213> Artificial sequence (Artificial)
<400> 58
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> 59
<211> 126
<212> DNA
<213> Artificial sequence (Artificial)
<400> 59
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 60
<211> 112
<212> PRT
<213> Artificial sequence (Artificial)
<400> 60
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> 61
<211> 336
<212> DNA
<213> Artificial sequence (Artificial)
<400> 61
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> 62
<211> 24
<212> PRT
<213> Artificial sequence (Artificial)
<400> 62
Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly Lys Val
1 5 10 15
Tyr Ile Asn Met Pro Gly Arg Gly
20
<210> 63
<211> 72
<212> DNA
<213> Artificial sequence (Artificial)
<400> 63
ctgtgcgccc gcccccgccg cagccccgcc caggaggacg gcaaggtgta catcaacatg 60
cccggccgcg gc 72
<210> 64
<211> 51
<212> PRT
<213> Artificial sequence (Artificial)
<400> 64
Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala
1 5 10 15
Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu
20 25 30
Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro
35 40 45
Tyr Tyr Lys
50
<210> 65
<211> 153
<212> DNA
<213> Artificial sequence (Artificial)
<400> 65
tacttcctgg gccgcctggt gccccgcggc cgcggcgccg ccgaggccac ccgcaagcag 60
cgcatcaccg agaccgagag cccctaccag gagctgcagg gccagcgcag cgacgtgtac 120
agcgacctga acacccagcg cccctactac aag 153
<210> 66
<211> 86
<212> PRT
<213> Artificial sequence (Artificial)
<400> 66
Glu Arg Thr Met Pro Arg Ile Pro Thr Leu Lys Asn Leu Glu Asp Leu
1 5 10 15
Val Thr Glu Tyr His Gly Asn Phe Ser Ala Trp Ser Gly Val Ser Lys
20 25 30
Gly Leu Ala Glu Ser Leu Gln Pro Asp Tyr Ser Glu Arg Leu Cys Leu
35 40 45
Val Ser Glu Ile Pro Pro Lys Gly Gly Ala Leu Gly Glu Gly Pro Gly
50 55 60
Ala Ser Pro Cys Asn Gln His Ser Pro Tyr Trp Ala Pro Pro Cys Tyr
65 70 75 80
Thr Leu Lys Pro Glu Thr
85
<210> 67
<211> 258
<212> DNA
<213> Artificial sequence (Artificial)
<400> 67
gagcgcacca tgccccgcat ccccaccctg aagaacctgg aggacctggt gaccgagtac 60
cacggcaact tcagcgcctg gagcggcgtg agcaagggcc tggccgagag cctgcagccc 120
gactacagcg agcgcctgtg cctggtgagc gagatccccc ccaagggcgg cgccctgggc 180
gagggccccg gcgccagccc ctgcaaccag cacagcccct actgggcccc cccctgctac 240
accctgaagc ccgagacc 258
<210> 68
<211> 38
<212> PRT
<213> Artificial sequence (Artificial)
<400> 68
Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn
1 5 10 15
Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg
20 25 30
Leu Thr Asp Val Thr Leu
35
<210> 69
<211> 114
<212> DNA
<213> Artificial sequence (Artificial)
<400> 69
tgctggctga ccaagaagaa gtacagcagc agcgtgcacg accccaacgg cgagtacatg 60
ttcatgcgcg ccgtgaacac cgccaagaag agccgcctga ccgacgtgac cctg 114
<210> 70
<211> 42
<212> PRT
<213> Artificial sequence (Artificial)
<400> 70
Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His
1 5 10 15
Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln
20 25 30
Ala Asp Ala His Ser Thr Leu Ala Lys Ile
35 40
<210> 71
<211> 126
<212> DNA
<213> Artificial sequence (Artificial)
<400> 71
gccctgtacc tgctgcgccg cgaccagcgc ctgccccccg acgcccacaa gccccccggc 60
ggcggcagct tccgcacccc catccaggag gagcaggccg acgcccacag caccctggcc 120
aagatc 126
<210> 72
<211> 45
<212> PRT
<213> Artificial sequence (Artificial)
<400> 72
Gly Ser Thr Val Tyr Tyr Gln Gly Lys Cys Leu Thr Trp Lys Gly Pro
1 5 10 15
Arg Arg Gln Leu Pro Ala Val Val Pro Ala Pro Leu Pro Pro Pro Cys
20 25 30
Gly Ser Ser Ala His Leu Leu Pro Pro Val Pro Gly Gly
35 40 45
<210> 73
<211> 135
<212> DNA
<213> Artificial sequence (Artificial)
<400> 73
ggcagcaccg tgtactacca gggcaagtgc ctgacctgga agggcccccg ccgccagctg 60
cccgccgtgg tgcccgcccc cctgcccccc ccctgcggca gcagcgccca cctgctgccc 120
cccgtgcccg gcggc 135
<210> 74
<211> 142
<212> PRT
<213> Artificial sequence (Artificial)
<400> 74
Arg Lys Arg Val Glu Trp Asp Phe Glu Arg Pro Ser Gln Val Gly Asp
1 5 10 15
Leu Arg Gly Val Gln Pro Trp Thr Pro Lys Lys Lys Ile Met Leu His
20 25 30
Ala Gly His Ala Leu Tyr Asp Ala Leu Met Ile Thr Asn Ile Val Lys
35 40 45
Thr Asn Ser Pro Pro Ala Tyr Glu Lys Leu Glu Asp Tyr Ala Phe Ala
50 55 60
Phe Glu Leu Ile Leu Glu Glu Ile Ala Arg Leu Phe Glu Ser Ala Asp
65 70 75 80
Gln Lys Glu Lys Ala Cys Arg Cys Lys Arg Trp Met Lys Arg Ile Lys
85 90 95
Thr Thr Ala Ser Glu Asp Gln Gln Glu Glu Met Ala Asn Ala Ile Ile
100 105 110
Asp Ile Leu Gln Ser Trp Ile Pro Pro Arg Lys Arg Ser Tyr Leu Gln
115 120 125
Trp Ala Glu Ala Gly Trp Arg Arg Gly Gly Glu Pro Lys Thr
130 135 140
<210> 75
<211> 145
<212> PRT
<213> Artificial sequence (Artificial)
<400> 75
Arg Lys Arg Ser Glu Thr Asp Phe Glu Phe Pro Ser Gln Val Gly Gly
1 5 10 15
Arg Arg Gly Val Gln Pro Trp Thr Pro Lys Lys Lys Ile Gln Leu His
20 25 30
Ala Glu His Ala Leu Tyr Asp Ala Leu Met Ile Leu Asn Ile Val Lys
35 40 45
Thr Asn Ser Pro Pro Ala Glu Glu Lys Leu Glu Asp Tyr Ala Phe Asn
50 55 60
Phe Glu Leu Ile Leu Glu Glu Ile Ala Arg Leu Phe Glu Ser Gly Asp
65 70 75 80
Gln Lys Asp Glu Ala Glu Lys Ala Lys Arg Met Lys Glu Trp Met Lys
85 90 95
Arg Ile Lys Thr Thr Ala Ser Glu Asp Glu Gln Glu Glu Met Ala Asn
100 105 110
Ala Ile Ile Thr Ile Leu Gln Ser Trp Ile Phe Ser Arg Lys Arg Ser
115 120 125
Tyr Leu Gln Pro Ala Glu Ala Gly Trp Arg Arg Gly Gly Glu Pro Trp
130 135 140
Thr
145
<210> 76
<211> 144
<212> PRT
<213> Artificial sequence (Artificial)
<400> 76
Arg Lys Asn Ser Glu Thr Asp Phe Glu Phe Pro Ser Gln Asp Gly Gly
1 5 10 15
Arg Arg Gly Val Leu Cys Trp Thr Pro Pro Lys Lys Ile Gln Leu His
20 25 30
Glu Cys Asp Ala Pro Tyr Asp Ala Leu Met Ile Leu Asn Ile Val Lys
35 40 45
Thr Asn Ser Trp Pro Ala Glu Glu Cys Leu Gly His Tyr Ala Phe Asn
50 55 60
Phe Asp Leu Ile Leu Pro His Ile Val Arg Leu Glu Glu Ser Asp Asp
65 70 75 80
Gln Lys Asp Glu Trp Glu Ile Ala Met Arg Met Leu Trp Met Lys Arg
85 90 95
Ile Lys Thr Thr Ala Ser Glu Asp Glu Gln Glu Glu Met Ala Asn Ala
100 105 110
Ile Ile Ala Ile Leu Gln Ser Trp Ile Arg Ile Leu Lys Arg Ser Tyr
115 120 125
Leu Gln Pro Ile Glu Ala Gly Trp Arg Arg Gly Gly Ile Arg Trp Val
130 135 140
<210> 77
<211> 141
<212> PRT
<213> Artificial sequence (Artificial)
<400> 77
Arg Lys Arg Ser Glu Thr Asp Phe Glu Phe Pro Asp Val Gly Gly Arg
1 5 10 15
Arg Gly Gln Pro Trp Thr Thr Lys Tyr Ile Gln Leu His Ala Glu His
20 25 30
Val Leu Tyr Asp Ala Leu Met Ile Leu Asn Ile Val Lys Thr Asn Ser
35 40 45
Pro Pro Ala Glu Glu Lys Leu Met Asp Tyr Phe Phe Asn Phe Glu Leu
50 55 60
Ile Leu Glu Glu Ile Ala Arg Leu Phe Glu Ser Gly Asp Gln Lys Ile
65 70 75 80
Glu Ala Glu Lys Ala Lys Arg Met Glu Trp Met Lys Arg Ile Lys Thr
85 90 95
Thr Ala Ser Thr Asp Glu Gln Glu Glu Met Ala Asn Ala Ile Ile Thr
100 105 110
Ile Leu Gln Ser Trp Ile Phe Ser Arg Lys Arg Ser Tyr Leu Gln Pro
115 120 125
Ala Glu Ala Gly Trp Arg Arg Gly Gly Glu Pro Trp Thr
130 135 140
<210> 78
<211> 143
<212> PRT
<213> Artificial sequence (Artificial)
<400> 78
Arg Lys Ile Ser Glu Thr Asp Phe Glu Phe Pro Ser Gln Val Gly Gly
1 5 10 15
Arg Arg Gly Trp Gln Pro Trp Thr Pro Lys Lys Lys Ile Gln Met His
20 25 30
Ala Ser Trp Ala Leu Tyr Leu Met Ile Leu Asn Tyr Val Lys Thr Asn
35 40 45
Ser Pro Pro Ala Glu Glu Lys Leu Glu Trp Tyr Ala His Asn Phe Lys
50 55 60
Leu Ile Leu Glu Glu Ile Ala Arg Leu Phe Glu Ser Gly Asp Glu Lys
65 70 75 80
Tyr Glu Ala Glu Gly Ala Lys Arg Met Lys Glu Trp Met Ile Lys Arg
85 90 95
Ile Lys Thr Gln Asp Ser Glu Ala Glu Thr Glu Glu Met Ala Asn Ala
100 105 110
Val Phe Thr Ile Leu Gln Ser Trp Ile Phe Ser Arg Tyr Arg Gly Tyr
115 120 125
Leu Gln Pro Leu Glu Ala Trp Glu Trp Arg Gly Glu Pro Trp Thr
130 135 140
<210> 79
<211> 145
<212> PRT
<213> Artificial sequence (Artificial)
<400> 79
Arg Lys Ser Glu Glu Asp Asp Phe Glu Phe Pro Ala Gln Val Val Gly
1 5 10 15
Glu Arg Gly Val Gln Ile Trp Thr Pro Lys Lys Lys Ile Gln Leu His
20 25 30
Ala Glu Ala Ala Leu Cys Asp Ala Leu Lys Ile Leu Glu Ile Phe Lys
35 40 45
Leu Glu Ser Pro Pro Ala Glu Tyr Lys Leu Glu Asp Tyr Ala Phe Asn
50 55 60
Phe Glu Leu Ile Leu Ile Glu Ile Ala Thr Leu Phe Glu Ser Gly Asp
65 70 75 80
Gln Lys Asp Glu Ala His Lys Ala Lys Arg Met Lys Glu Trp Met Pro
85 90 95
Arg Ile Lys Thr Thr Ala Ser Glu Asp Glu Gln Glu Glu Met Ala Asn
100 105 110
Ala Ile Ile Phe Ile Leu Gln Ser Lys Ile Thr Ser Arg Lys Arg Ser
115 120 125
Tyr Leu Gln Pro Ala Pro Phe Gly Trp Arg Arg Gly Gly Glu Pro Trp
130 135 140
Gly
145
<210> 80
<211> 145
<212> PRT
<213> Artificial sequence (Artificial)
<400> 80
Arg Lys Arg Ser Glu Thr Gly Phe Glu Phe Pro Ser Gln Val Gly Gly
1 5 10 15
Arg Cys Gly Gln Asn Ile Gly Thr Ala Asn Lys Lys Ile Gln Leu His
20 25 30
Ala Glu His Ala Leu Tyr Asp Ala Leu His Asp Leu Asn Ile Val Lys
35 40 45
Thr Asn Ser Pro Pro Ala Glu Gly Lys Leu Glu His Tyr Ala Phe Asn
50 55 60
Phe Glu Leu Arg Leu Asn Glu His Ala Trp Leu Lys Glu Ser Gly Asp
65 70 75 80
Cys Lys Asp Glu Phe Glu Ile Val Tyr Arg His Tyr Glu Trp Leu Lys
85 90 95
Arg Ile Asp Thr Gln Ala Val Glu Asp Glu Gln Glu His Asp Ala Asn
100 105 110
Ala Phe Ile Trp Ile Gly Ala Ser Trp Glu Phe Ser Asp Ser Arg Ser
115 120 125
Tyr Leu Arg Pro Ala Glu Ala Gly Trp Arg Arg Gly Gly Glu Pro Trp
130 135 140
Thr
145
<210> 81
<211> 145
<212> PRT
<213> Artificial sequence (Artificial)
<400> 81
Arg Pro Arg Ser Glu Thr Thr Phe Ser Asn Pro Ser Gln Leu Gly Gly
1 5 10 15
Lys Arg Gly Val Gln Pro His Thr Asp Lys Lys Lys Ile Asn Leu His
20 25 30
Val Pro His Ala Ser Tyr Asp Ala Leu Met Tyr Leu Asn Ile Val Lys
35 40 45
Thr Asn Ser Pro Pro Ala Glu Glu Lys Pro Glu Gly Tyr Ala Ile Asn
50 55 60
Phe Glu Leu Ile Leu Glu Glu Ile Met Arg Arg Phe Glu Ser His Asp
65 70 75 80
Gln Lys Lys Glu Ala Glu Lys Leu Lys Arg Ser Glu Glu Trp Cys Lys
85 90 95
Arg Cys Lys Thr Thr Ala Thr Glu Asp Glu Gln Glu Glu Met Ala Asn
100 105 110
Ile Gln Ile Ser Ile Leu Gln Ser Trp Ile Ala Ser Arg Arg Arg Ser
115 120 125
Tyr Leu Gln Pro Ala Glu Val Gly Met Arg Arg Gly Gly Cys Pro Trp
130 135 140
Thr
145
<210> 82
<211> 426
<212> DNA
<213> Artificial sequence (Artificial)
<400> 82
cgcaagcgcg tggagtggga cttcgagcgc cccagccagg tgggcgacct gcgcggcgtg 60
cagccctgga cccccaagaa gaagatcatg ctgcacgccg gccacgccct gtacgacgcc 120
ctgatgatca ccaacatcgt gaagaccaac agcccccccg cctacgagaa gctggaggac 180
tacgccttcg ccttcgagct gatcctggag gagatcgccc gcctgttcga gagcgccgac 240
cagaaggaga aggcctgccg ctgcaagcgc tggatgaagc gcatcaagac caccgccagc 300
gaggaccagc aggaggagat ggccaacgcc atcatcgaca tcctgcagag ctggatcccc 360
ccccgcaagc gcagctacct gcagtgggcc gaggccggct ggcgccgcgg cggcgagccc 420
aagacc 426
<210> 83
<211> 435
<212> DNA
<213> Artificial sequence (Artificial)
<400> 83
cgcaagcgca gcgagaccga cttcgagttc cccagccagg tgggcggccg ccgcggcgtg 60
cagccctgga cccccaagaa gaagatccag ctgcacgccg agcacgccct gtacgacgcc 120
ctgatgatcc tgaacatcgt gaagaccaac agcccccccg ccgaggagaa gctggaggac 180
tacgccttca acttcgagct gatcctggag gagatcgccc gcctgttcga gagcggcgac 240
cagaaggacg aggccgagaa ggccaagcgc atgaaggagt ggatgaagcg catcaagacc 300
accgccagcg aggacgagca ggaggagatg gccaacgcca tcatcaccat cctgcagagc 360
tggatcttca gccgcaagcg cagctacctg cagcccgccg aggccggctg gcgccgcggc 420
ggcgagccct ggacc 435
<210> 84
<211> 432
<212> DNA
<213> Artificial sequence (Artificial)
<400> 84
cgcaagaaca gcgagaccga cttcgagttc cccagccagg acggcggccg ccgcggcgtg 60
ctgtgctgga ccccccccaa gaagatccag ctgcacgagt gcgacgcccc ctacgacgcc 120
ctgatgatcc tgaacatcgt gaagaccaac agctggcccg ccgaggagtg cctgggccac 180
tacgccttca acttcgacct gatcctgccc cacatcgtgc gcctggagga gagcgacgac 240
cagaaggacg agtgggagat cgccatgcgc atgctgtgga tgaagcgcat caagaccacc 300
gccagcgagg acgagcagga ggagatggcc aacgccatca tcgccatcct gcagagctgg 360
atccgcatcc tgaagcgcag ctacctgcag cccatcgagg ccggctggcg ccgcggcggc 420
atccgctggg tg 432
<210> 85
<211> 423
<212> DNA
<213> Artificial sequence (Artificial)
<400> 85
cgcaagcgca gcgagaccga cttcgagttc cccgacgtgg gcggccgccg cggccagccc 60
tggaccacca agtacatcca gctgcacgcc gagcacgtgc tgtacgacgc cctgatgatc 120
ctgaacatcg tgaagaccaa cagccccccc gccgaggaga agctgatgga ctacttcttc 180
aacttcgagc tgatcctgga ggagatcgcc cgcctgttcg agagcggcga ccagaagatc 240
gaggccgaga aggccaagcg catggagtgg atgaagcgca tcaagaccac cgccagcacc 300
gacgagcagg aggagatggc caacgccatc atcaccatcc tgcagagctg gatcttcagc 360
cgcaagcgca gctacctgca gcccgccgag gccggctggc gccgcggcgg cgagccctgg 420
acc 423
<210> 86
<211> 429
<212> DNA
<213> Artificial sequence (Artificial)
<400> 86
cgcaagatca gcgagaccga cttcgagttc cccagccagg tgggcggccg ccgcggctgg 60
cagccctgga cccccaagaa gaagatccag atgcacgcca gctgggccct gtacctgatg 120
atcctgaact acgtgaagac caacagcccc cccgccgagg agaagctgga gtggtacgcc 180
cacaacttca agctgatcct ggaggagatc gcccgcctgt tcgagagcgg cgacgagaag 240
tacgaggccg agggcgccaa gcgcatgaag gagtggatga tcaagcgcat caagacccag 300
gacagcgagg ccgagaccga ggagatggcc aacgccgtgt tcaccatcct gcagagctgg 360
atcttcagcc gctaccgcgg ctacctgcag cccctggagg cctgggagtg gcgcggcgag 420
ccctggacc 429
<210> 87
<211> 435
<212> DNA
<213> Artificial sequence (Artificial)
<400> 87
cgcaagagcg aggaggacga cttcgagttc cccgcccagg tggtgggcga gcgcggcgtg 60
cagatctgga cccccaagaa gaagatccag ctgcacgccg aggccgccct gtgcgacgcc 120
ctgaagatcc tggagatctt caagctggag agcccccccg ccgagtacaa gctggaggac 180
tacgccttca acttcgagct gatcctgatc gagatcgcca ccctgttcga gagcggcgac 240
cagaaggacg aggcccacaa ggccaagcgc atgaaggagt ggatgccccg catcaagacc 300
accgccagcg aggacgagca ggaggagatg gccaacgcca tcatcttcat cctgcagagc 360
aagatcacca gccgcaagcg cagctacctg cagcccgccc ccttcggctg gcgccgcggc 420
ggcgagccct ggggc 435
<210> 88
<211> 435
<212> DNA
<213> Artificial sequence (Artificial)
<400> 88
cgcaagcgca gcgagaccgg cttcgagttc cccagccagg tgggcggccg ctgcggccag 60
aacatcggca ccgccaacaa gaagatccag ctgcacgccg agcacgccct gtacgacgcc 120
ctgcacgacc tgaacatcgt gaagaccaac agcccccccg ccgagggcaa gctggagcac 180
tacgccttca acttcgagct gcgcctgaac gagcacgcct ggctgaagga gagcggcgac 240
tgcaaggacg agttcgagat cgtgtaccgc cactacgagt ggctgaagcg catcgacacc 300
caggccgtgg aggacgagca ggagcacgac gccaacgcct tcatctggat cggcgccagc 360
tgggagttca gcgacagccg cagctacctg cgccccgccg aggccggctg gcgccgcggc 420
ggcgagccct ggacc 435
<210> 89
<211> 435
<212> DNA
<213> Artificial sequence (Artificial)
<400> 89
cgcccccgca gcgagaccac cttcagcaac cccagccagc tgggcggcaa gcgcggcgtg 60
cagccccaca ccgacaagaa gaagatcaac ctgcacgtgc cccacgccag ctacgacgcc 120
ctgatgtacc tgaacatcgt gaagaccaac agcccccccg ccgaggagaa gcccgagggc 180
tacgccatca acttcgagct gatcctggag gagatcatgc gccgcttcga gagccacgac 240
cagaagaagg aggccgagaa gctgaagcgc agcgaggagt ggtgcaagcg ctgcaagacc 300
accgccaccg aggacgagca ggaggagatg gccaacatcc agatcagcat cctgcagagc 360
tggatcgcca gccgccgccg cagctacctg cagcccgccg aggtgggcat gcgccgcggc 420
ggctgcccct ggacc 435
<210> 90
<211> 1998
<212> DNA
<213> Artificial sequence (Artificial)
<400> 90
atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgcccgc 60
cccgactaca aggacgacga cgacgacaag gcctacgaca tgacccagac ccccgccagc 120
gtgagcgccg ccgtgggcgg caccgtgacc atcaagtgcc aggccagcca gagcatcagc 180
aactacctgg cctggtacca gcagaagccc ggccagcccc ccaagctgct gatctaccag 240
gccagcaccc tggcccccgg cgtgagcagc cgcttcaagg gcagcggcag cggcaccgag 300
ttcaccctga ccatcagcgg cgtggagtgc gccgacgccg ccacctacta ctgccagcag 360
ggctacacca gcagcaacgt ggagaacgtg ttcggcggcg gcaccggcgt ggtggtgggc 420
ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gccaggagca gctggaggag 480
agcggcggcg acctggtgca gcccgagggc agcctgaccc tgacctgcaa ggccagcggc 540
ctggacttca gcagcagcta ctggatctgc tgggtgcgcc aggcccccgg caagggcctg 600
gagtggatcg gctgccgcca caccttcacc gccaacacct ggagcgccag ctgggtgaac 660
ggccgcttca ccatcagccg cagcaccagc ctgggcaccg tggacctgaa gatgaccagc 720
ctgaccgccg ccgacaccgc cacctacttc tgcgcccgcg acgagagcaa caacgacggc 780
tgggacttca agctgtgggg ccccggcacc ctggtgaccg tgagcagcac cacgacgcca 840
gcgccgcgac caccaacacc ggcgcccacc atcgcgtcgc agcccctgtc cctgcgccca 900
gaggcgtgcc ggccagcggc ggggggcgca gtgcacacga gggggctgga cttcgcctgt 960
gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 1020
atcacccttt actgcaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt 1080
atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 1140
gaagaaggag gatgtgaact gagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1200
cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1260
gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 1320
cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 1380
attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 1440
agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgcggc 1500
agcggcgcca ccaacttcag cctgctgaag caggccggcg acgtggagga gaaccccggc 1560
ccccgcaaga acagcgagac cgacttcgag ttccccagcc aggacggcgg ccgccgcggc 1620
gtgctgtgct ggaccccccc caagaagatc cagctgcacg agtgcgacgc cccctacgac 1680
gccctgatga tcctgaacat cgtgaagacc aacagctggc ccgccgagga gtgcctgggc 1740
cactacgcct tcaacttcga cctgatcctg ccccacatcg tgcgcctgga ggagagcgac 1800
gaccagaagg acgagtggga gatcgccatg cgcatgctgt ggatgaagcg catcaagacc 1860
accgccagcg aggacgagca ggaggagatg gccaacgcca tcatcgccat cctgcagagc 1920
tggatccgca tcctgaagcg cagctacctg cagcccatcg aggccggctg gcgccgcggc 1980
ggcatccgct gggtgtaa 1998
<210> 91
<211> 2001
<212> DNA
<213> Artificial sequence (Artificial)
<400> 91
atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgcccgc 60
cccgactaca aggacgacga cgacgacaag gcctacgaca tgacccagac ccccgccagc 120
gtgagcgccg ccgtgggcgg caccgtgacc atcaagtgcc aggccagcca gagcatcagc 180
aactacctgg cctggtacca gcagaagccc ggccagcccc ccaagctgct gatctaccag 240
gccagcaccc tggcccccgg cgtgagcagc cgcttcaagg gcagcggcag cggcaccgag 300
ttcaccctga ccatcagcgg cgtggagtgc gccgacgccg ccacctacta ctgccagcag 360
ggctacacca gcagcaacgt ggagaacgtg ttcggcggcg gcaccggcgt ggtggtgggc 420
ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gccaggagca gctggaggag 480
agcggcggcg acctggtgca gcccgagggc agcctgaccc tgacctgcaa ggccagcggc 540
ctggacttca gcagcagcta ctggatctgc tgggtgcgcc aggcccccgg caagggcctg 600
gagtggatcg gctgccgcca caccttcacc gccaacacct ggagcgccag ctgggtgaac 660
ggccgcttca ccatcagccg cagcaccagc ctgggcaccg tggacctgaa gatgaccagc 720
ctgaccgccg ccgacaccgc cacctacttc tgcgcccgcg acgagagcaa caacgacggc 780
tgggacttca agctgtgggg ccccggcacc ctggtgaccg tgagcagcac cacgacgcca 840
gcgccgcgac caccaacacc ggcgcccacc atcgcgtcgc agcccctgtc cctgcgccca 900
gaggcgtgcc ggccagcggc ggggggcgca gtgcacacga gggggctgga cttcgcctgt 960
gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 1020
atcacccttt actgcaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt 1080
atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 1140
gaagaaggag gatgtgaact gagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1200
cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1260
gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 1320
cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 1380
attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 1440
agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgcggc 1500
agcggcgcca ccaacttcag cctgctgaag caggccggcg acgtggagga gaaccccggc 1560
ccccgcaaga gcgaggagga cgacttcgag ttccccgccc aggtggtggg cgagcgcggc 1620
gtgcagatct ggacccccaa gaagaagatc cagctgcacg ccgaggccgc cctgtgcgac 1680
gccctgaaga tcctggagat cttcaagctg gagagccccc ccgccgagta caagctggag 1740
gactacgcct tcaacttcga gctgatcctg atcgagatcg ccaccctgtt cgagagcggc 1800
gaccagaagg acgaggccca caaggccaag cgcatgaagg agtggatgcc ccgcatcaag 1860
accaccgcca gcgaggacga gcaggaggag atggccaacg ccatcatctt catcctgcag 1920
agcaagatca ccagccgcaa gcgcagctac ctgcagcccg cccccttcgg ctggcgccgc 1980
ggcggcgagc cctggggcta a 2001
<210> 92
<211> 2004
<212> DNA
<213> Artificial sequence (Artificial)
<400> 92
atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgcccgc 60
cccgactaca aggacgacga cgacgacaag gacgtggtga tgacccagac ccccgccagc 120
gtgagcgagc ccgtgggcgg caccgtgacc atcaagtgcc aggccagcca gcgcatcagc 180
agctacctga gctggtacca gcagaagccc ggccagcgcc ccaagctgct gatcttcggc 240
gccagcaccc tggccagcgg cgtgcccagc cgcttcaagg gcagcggcag cggcaccgag 300
tacaccctga ccatcagcga cctggagtgc gccgacgccg ccacctacta ctgccagagc 360
tacgcctact tcgacagcaa caactggcac gccttcggcg gcggcaccga ggtggtggtg 420
ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagccagca gcagctggag 480
gagagcggcg gcggcctggt gaagcccgag ggcagcctga ccctgacctg caaggccagc 540
ggcttcgacc tgggcttcta cttctacgcc tgctgggtgc gccaggcccc cggcaagggc 600
ctggagtgga tcgcctgcat ctacaccgcc ggcagcggca gcacctacta cgccagctgg 660
gccaagggcc gcttcaccat cagcaaggcc agcagcacca ccgtgaccct gcagatgacc 720
agcctggccg ccgccgacac cgccacctac ttctgcgccc gcagcaccgc caacacccgc 780
agcacctact acctgaacct gtggggcccc ggcaccctgg tgaccgtgag cagcaccacg 840
acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 900
cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 960
gcctgtgata tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca 1020
ctggttatca ccctttactg caaacggggc agaaagaaac tcctgtatat attcaaacaa 1080
ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca 1140
gaagaagaag aaggaggatg tgaactgaga gtgaagttca gcaggagcgc agacgccccc 1200
gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1260
tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 1320
aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1380
agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1440
ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 1500
cgcggcagcg gcgccaccaa cttcagcctg ctgaagcagg ccggcgacgt ggaggagaac 1560
cccggccccc gcaagaacag cgagaccgac ttcgagttcc ccagccagga cggcggccgc 1620
cgcggcgtgc tgtgctggac cccccccaag aagatccagc tgcacgagtg cgacgccccc 1680
tacgacgccc tgatgatcct gaacatcgtg aagaccaaca gctggcccgc cgaggagtgc 1740
ctgggccact acgccttcaa cttcgacctg atcctgcccc acatcgtgcg cctggaggag 1800
agcgacgacc agaaggacga gtgggagatc gccatgcgca tgctgtggat gaagcgcatc 1860
aagaccaccg ccagcgagga cgagcaggag gagatggcca acgccatcat cgccatcctg 1920
cagagctgga tccgcatcct gaagcgcagc tacctgcagc ccatcgaggc cggctggcgc 1980
cgcggcggca tccgctgggt gtaa 2004
<210> 93
<211> 2007
<212> DNA
<213> Artificial sequence (Artificial)
<400> 93
atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgcccgc 60
cccgactaca aggacgacga cgacgacaag gacgtggtga tgacccagac ccccgccagc 120
gtgagcgagc ccgtgggcgg caccgtgacc atcaagtgcc aggccagcca gcgcatcagc 180
agctacctga gctggtacca gcagaagccc ggccagcgcc ccaagctgct gatcttcggc 240
gccagcaccc tggccagcgg cgtgcccagc cgcttcaagg gcagcggcag cggcaccgag 300
tacaccctga ccatcagcga cctggagtgc gccgacgccg ccacctacta ctgccagagc 360
tacgcctact tcgacagcaa caactggcac gccttcggcg gcggcaccga ggtggtggtg 420
ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagccagca gcagctggag 480
gagagcggcg gcggcctggt gaagcccgag ggcagcctga ccctgacctg caaggccagc 540
ggcttcgacc tgggcttcta cttctacgcc tgctgggtgc gccaggcccc cggcaagggc 600
ctggagtgga tcgcctgcat ctacaccgcc ggcagcggca gcacctacta cgccagctgg 660
gccaagggcc gcttcaccat cagcaaggcc agcagcacca ccgtgaccct gcagatgacc 720
agcctggccg ccgccgacac cgccacctac ttctgcgccc gcagcaccgc caacacccgc 780
agcacctact acctgaacct gtggggcccc ggcaccctgg tgaccgtgag cagcaccacg 840
acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 900
cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 960
gcctgtgata tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca 1020
ctggttatca ccctttactg caaacggggc agaaagaaac tcctgtatat attcaaacaa 1080
ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca 1140
gaagaagaag aaggaggatg tgaactgaga gtgaagttca gcaggagcgc agacgccccc 1200
gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1260
tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 1320
aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1380
agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1440
ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 1500
cgcggcagcg gcgccaccaa cttcagcctg ctgaagcagg ccggcgacgt ggaggagaac 1560
cccggccccc gcaagagcga ggaggacgac ttcgagttcc ccgcccaggt ggtgggcgag 1620
cgcggcgtgc agatctggac ccccaagaag aagatccagc tgcacgccga ggccgccctg 1680
tgcgacgccc tgaagatcct ggagatcttc aagctggaga gcccccccgc cgagtacaag 1740
ctggaggact acgccttcaa cttcgagctg atcctgatcg agatcgccac cctgttcgag 1800
agcggcgacc agaaggacga ggcccacaag gccaagcgca tgaaggagtg gatgccccgc 1860
atcaagacca ccgccagcga ggacgagcag gaggagatgg ccaacgccat catcttcatc 1920
ctgcagagca agatcaccag ccgcaagcgc agctacctgc agcccgcccc cttcggctgg 1980
cgccgcggcg gcgagccctg gggctaa 2007

Claims (9)

1. A chimeric antigen receptor targeting MSLN comprising a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, and an intracellular domain;
the signal peptide is a signal peptide of a protein selected from the group consisting of: the amino acid sequence of the OSM signal peptide is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2; the amino acid sequence of the IL-2 signal peptide is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO. 4; the HSA signal peptide has an amino acid sequence shown as SEQ ID NO.5 and a nucleotide sequence shown as SEQ ID NO. 6; the amino acid sequence of the CD8 alpha signal peptide is shown as SEQ ID NO.7, and the nucleotide sequence is shown as SEQ ID NO. 8; the amino acid sequence of the insulin signal peptide is shown as SEQ ID NO.9, and the nucleotide sequence is shown as SEQ ID NO. 10; the trypsinogen 2 signal peptide has an amino acid sequence shown as SEQ ID NO.11 and a nucleotide sequence shown as SEQ ID NO. 12;
the antigen binding domain can be specifically bound with MSLN scFv and is constructed into one of two configurations of VH-linker-VL or VL-linker-VH, and the amino acid sequence of the linker is a 3-time repetitive sequence (G) 4 S) 3 Or 4 repeats (G) 4 S) 4 (ii) a Wherein the amino acid sequences of VH are respectively shown as SEQ ID NO. 13-18, and the corresponding nucleotide sequences are respectively shown as SEQ ID NO. 19-24; the amino acid sequence of VL is shown in SEQ ID NO. 25-30, and the corresponding nucleotide sequences are respectively shown in SEQ ID NO. 31-36;
the hinge region connects the extracellular domain of the chimeric antigen receptor to the transmembrane region, and is selected from the following proteins: CD8 alpha, the amino acid sequence of which is shown as SEQ ID NO.40, and the nucleotide sequence of which is shown as SEQ ID NO. 41; CD28, the amino acid sequence of which is shown as SEQ ID NO.42, and the nucleotide sequence of which is shown as SEQ ID NO. 43; IgG1, the amino acid sequence of which is shown as SEQ ID NO.44 and the nucleotide sequence of which is shown as SEQ ID NO. 45; IgG4, the amino acid sequence of which is shown as SEQ ID NO.46, and the nucleotide sequence of which is shown as SEQ ID NO. 47;
the transmembrane domain is selected from the following proteins: CD4, the amino acid sequence of which is shown as SEQ ID NO.48, and the nucleotide sequence of which is shown as SEQ ID NO. 49; CD8 alpha, the amino acid sequence of which is shown as SEQ ID NO.50, and the nucleotide sequence of which is shown as SEQ ID NO. 51; CD28, the amino acid sequence of which is shown as SEQ ID NO.52, and the nucleotide sequence of which is shown as SEQ ID NO. 53; NKG2D, wherein the amino acid sequence is shown as SEQ ID NO.54, and the nucleotide sequence is shown as SEQ ID NO. 55;
the intracellular structural domain is composed of 2-4 tandem connection of signal transduction structural domains or co-stimulation structural domains of the following molecules and comprises: CD28, the amino acid sequence of which is shown as SEQ ID NO.56, and the nucleotide sequence of which is shown as SEQ ID NO. 57; CD137, the amino acid sequence of which is shown as SEQ ID NO.58, and the nucleotide sequence of which is shown as SEQ ID NO. 59; CD3 zeta, the amino acid sequence is shown in SEQ ID NO.60, and the nucleotide sequence is shown in SEQ ID NO. 61; DAP10, the amino acid sequence of which is shown as SEQ ID NO.62, and the nucleotide sequence of which is shown as SEQ ID NO. 63; DAP12, the amino acid sequence of which is shown as SEQ ID NO.64, and the nucleotide sequence of which is shown as SEQ ID NO. 65; IL2R gamma, the amino acid sequence of which is shown as SEQ ID NO.66, and the nucleotide sequence of which is shown as SEQ ID NO. 67; ICOS, the amino acid sequence of which is shown as SEQ ID NO.68, and the nucleotide sequence of which is shown as SEQ ID NO. 69; OX40, wherein the amino acid sequence is shown as SEQ ID NO.70, and the nucleotide sequence is shown as SEQ ID NO. 71; the amino acid sequence of NCR3 is shown in SEQ ID NO.72, and the nucleotide sequence is shown in SEQ ID NO. 73.
2. The MSLN-targeting chimeric antigen receptor of claim 1, wherein the signal peptide sequence is set forth in SEQ ID No. 7; the antigen binding domain sequence is shown as SEQ ID NO.37, SEQ ID NO.38 or SEQ ID NO. 39; the sequence of the hinge region is shown as SEQ ID No.40, and the sequence of the transmembrane domain is shown as SEQ ID No. 50; the intracellular domain sequence is composed of SEQ ID NO.58 and SEQ ID NO.60 in tandem.
3. An IL2R beta gamma agonist IL2R beta gamma-A is characterized in that the amino acid sequence is shown as SEQ ID NO. 74-81, and the corresponding nucleotide sequences are respectively shown as SEQ ID NO. 82-89.
4. A polycistronic structure co-expressing the MSLN-targeted chimeric antigen receptor of claim 1 and IL2R β γ -a of claim 3, wherein the chimeric antigen receptor is linked directly to the IL2R β γ -a sequence via the P2A, T2A, E2A or F2A peptides; the nucleotide sequence of the polycistronic structure is selected from any one of SEQ ID NO. 90-93.
5. Use of the MSLN-targeting chimeric antigen receptor structure of claim 1, or the IL2R β γ -a structure of claim 2, or the polycistronic structure of co-expressing the MSLN-targeting chimeric antigen receptor of claim 4 with IL2R β γ -a for the preparation of a CAR-NK cell line that autocrine for IL2 β γ -specific agonists and targets the tumor antigen MSLN.
6. A lentiviral vector comprising the MSLN-targeting chimeric antigen receptor of claim 1 and the amino acid and nucleotide sequence of IL2R β γ -a of claim 3.
7. A genetically modified natural killer cell having integrated into its genome the MSLN-targeting chimeric antigen receptor of claim 1 and a nucleotide sequence secreting IL2R β γ -a of claim 3, or the polycistronic nucleotide sequence of claim 4.
8. Use of the MSLN-targeting chimeric antigen receptor structure of claim 1, the IL2R β γ -A structure of claim 3, the polycistronic structure of the MSLN-targeting chimeric antigen receptor co-expressed of claim 4 and IL2R β γ -A, the lentiviral vector of claim 6, the natural killer cell of claim 7, for the manufacture of a medicament or formulation for the treatment of a tumor.
9. The method of claim 7, wherein the NK92 cell line is established as a host cell using the MSLN-targeting chimeric antigen receptor structure of claim 1, the IL2R β γ -a structure of claim 3, or the polycistronic structure of the co-expressed MSLN-targeting chimeric antigen receptor of claim 4 and IL2R β γ -a.
CN202210697062.7A 2022-06-20 2022-06-20 MSLN-CAR-NK cell capable of autocrine IL2R beta gamma agonist and application thereof Pending CN115124627A (en)

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