CN113817056A - Single-chain antibody targeting CD70 and application thereof - Google Patents

Single-chain antibody targeting CD70 and application thereof Download PDF

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CN113817056A
CN113817056A CN202010559366.8A CN202010559366A CN113817056A CN 113817056 A CN113817056 A CN 113817056A CN 202010559366 A CN202010559366 A CN 202010559366A CN 113817056 A CN113817056 A CN 113817056A
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齐亚男
赵文旭
陈军
赵永春
徐艳敏
黄霞
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Chongqing Jingzhun Biological Industrial Technology Institute Co ltd
Chongqing Precision Biotech Co ltd
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Chongqing Precision Biotech Co ltd
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Abstract

The invention belongs to the technical field of immunotherapy, and particularly relates to ScFv targeting CD70, a humanized single-chain antibody capable of recognizing CD70 and application thereof. The amino acid sequence of the light chain variable region of the ScFv targeting CD70 is shown as SEQ ID NO. 8 or a functional variant thereof, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7 or a functional variant thereof. The ScFv after humanized modification of the ScFv targeting CD70, a vector containing the humanized ScFv, a CAR and an immune cell can effectively eliminate tumor target cells expressing CD70 antigen.

Description

Single-chain antibody targeting CD70 and application thereof
Technical Field
The invention belongs to the technical field of immunotherapy, and particularly relates to ScFv targeting CD70, a humanized single-chain antibody capable of recognizing CD70 and application thereof.
Background
CD70 is a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily, a type ii transmembrane protein, expressed primarily in activated T cells, B cells, natural killer cells, and dendritic cells. CD70 is highly expressed in kidney cancer, blood-borne malignant tumor (including AML, NHL, HL and the like which are highly expressed), thymus tumor, ovarian cancer, glioblastomas, nasopharyngeal carcinoma and other tumor tissues.
Chimeric Antigen Receptors (CARs) are artificial receptors that mimic TCR function and include a tumor-associated antigen binding region, an extracellular spacer region, a transmembrane region, and an intracellular signaling region. The intracellular signaling domain is typically the CD3 zeta chain or FcR gamma, or is associated with one or more costimulatory molecules, such as 4-1BB (CD137), CD28, ICOS (CD 278). CD70 shows a high expression state in various tumors, is only expressed in normal activated T cells, B cells, natural killer cells and dendritic cells, and the CD 70-targeted CAR is designed to have wider tumor application range and higher safety.
Disclosure of Invention
It is an object of the present invention to provide an isolated antibody or antigen-binding fragment that recognizes CD 70.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the light chain CDR region of the ScFv comprises CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and CDR3 shown in SEQ ID NO. 3.
Further, the CDR region of the heavy chain of the ScFv comprises CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5 and CDR3 shown in SEQ ID NO. 6 in amino acid sequence.
Furthermore, the amino acid sequence of the light chain variable region of the ScFv targeting CD70 is shown as SEQ ID NO. 8 or a functional variant thereof, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7 or a functional variant thereof.
The second object of the present invention is to provide a humanized single-chain antibody that recognizes CD 70.
Although various humanized modification schemes are reported, particularly for stability and affinity optimization, after site-directed mutagenesis is carried out, a great amount of verification is needed to confirm the effectiveness of the humanized single-chain antibody obtained by modification; in theory, "single chain antibodies" may be used to make chimeric antigen receptors, but not every single chain antibody may actually be used to make chimeric antigen receptors. This requires creative efforts of the inventors to find products with unexpected effects among the numerous engineered single chain antibodies.
A method for modifying single-chain antibody includes such steps as searching human frame library to obtain the frame sequence with high similarity to mouse frame, implanting the mouse CDR (variable region) into the variable region of human frame to form humanized antibody, and mutating the humanized FR region (constant region) by multi-position or unit site-directed mutagenesis to restore the stability and antigen recognizing activity of antibody. The results of the method are random, and the humanized antibody structure can be kept stable only under the condition of proper configuration, and the affinity of the humanized antibody structure is possible to be suitable for recognizing the target antigen on the surface of the tumor so as to activate the CAR-T cells.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the ScFv targeting CD70 is subjected to humanized transformation, the amino acid sequence of the light chain variable region of the humanized single-chain antibody is shown in any one of SEQ ID NO 9-12, and the amino acid sequence of the heavy chain variable region of the humanized single-chain antibody is shown in any one of SEQ ID NO 13-16.
Further, the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment recognizing CD70 are linked by a Linker, in some embodiments, the Linker amino acid sequence is shown in SEQ ID NO. 17; in some embodiments, the Linker amino acid sequence is shown as SEQ ID NO 18 or SEQ ID NO 31 or SEQ ID NO 32 or SEQ ID NO 33; in certain embodiments the heavy chain variable region and the light chain variable region are linked in the manner of a VH-linker-VL, and in certain embodiments the heavy chain variable region and the light chain variable region are linked in the manner of a VL-linker-VH.
It is a further object of the invention to provide a CAR structure, expression vector and immune cell comprising the CAR structure of an ScFv that recognizes CD70 and a humanized single chain antibody that recognizes CD 70.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the CAR structure comprising the humanized single chain antibody that recognizes CD70 comprises an antigen recognition region that recognizes CD70 antigen, a hinge region, a transmembrane region, and an intracellular signal domain.
Further, the antigen-recognizing region recognizing the CD70 antigen was selected from ScFv characterized in that the light chain CDR region comprised of CDR1 having an amino acid sequence shown in SEQ ID NO. 1, CDR2 having an amino acid sequence shown in SEQ ID NO. 2 and CDR3 having an amino acid sequence shown in SEQ ID NO. 3, and the heavy chain CDR region comprised of CDR1 having an amino acid sequence shown in SEQ ID NO. 4, CDR2 having an amino acid sequence shown in SEQ ID NO. 5 and CDR3 having an amino acid sequence shown in SEQ ID NO. 6.
Further, the antigen-recognizing region recognizing the CD70 antigen comprises ScFv having a heavy chain variable region as shown in SEQ ID NO. 7, a light chain variable region as shown in SEQ ID NO. 8 or a heavy chain variable region as shown in SEQ ID NO. 13, a light chain variable region as shown in SEQ ID NO. 9 or a heavy chain variable region as shown in SEQ ID NO. 14, a light chain variable region as shown in SEQ ID NO. 10 or a heavy chain variable region as shown in SEQ ID NO. 15, a light chain variable region as shown in SEQ ID NO. 11 or a heavy chain variable region as shown in SEQ ID NO. 16, and a light chain variable region as shown in SEQ ID NO. 12.
Preferably, the single-chain antibody recognizing the CD70 antigen comprises an amino acid sequence shown in SEQ ID NO. 14 in the heavy chain variable region and SEQ ID NO. 10 in the light chain variable region or SEQ ID NO. 16 in the heavy chain variable region and SEQ ID NO. 12 in the light chain variable region.
In certain embodiments, the hinge region of the chimeric antigen receptor to the CD70 antigen may be derived from CD8, IgG4, CD7, and the like.
Further, the amino acid sequence of the hinge region is as set forth in SEQ ID NO 19 or SEQ ID NO 20 or SEQ ID NO 21, and in some embodiments the hinge region may be derived from the IgG4 hinge region; the transmembrane region is derived from the transmembrane region of a CD8 molecule or a CD28 molecule; the intracellular signal is derived from the CD28 or CD137 intracellular signaling region and the CD3 intracellular sequence.
Furthermore, the amino acid sequence of the transmembrane region is shown as SEQ ID NO. 26 or SEQ ID NO. 27.
Further, the intracellular signal amino acid sequence is shown as SEQ ID NO. 28 or SEQ ID NO. 29.
Furthermore, the amino acid sequence of the CD3 activation region is shown as SEQ ID NO. 30.
The inventors designed 4 different humanized ScFv and 3 different CAR structures to be combined randomly, and evaluated 5 aspects of ScFv affinity, stability, CAR expression stability, CAR-T cell efficiency and safety, to verify that the antibody or antigen-binding fragment recognizing CD70 and the engineered humanized single-chain antibody described by the inventors are functional in different CAR structures, and have applicability to all CAR combinations targeting CD 70.
Preferably, the amino acid sequence of the chimeric antigen receptor is SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24 or SEQ ID NO. 25.
Further, the expression vector containing the CAR structure is any one of a lentivirus expression vector, a retrovirus expression vector, an adenovirus expression vector, an adeno-associated virus expression vector, a DNA vector, an RNA vector and a plasmid.
Further, the expression vector Promoter containing the CAR gene used repeated 5HRE regulatory elements in combination with a weakly-initiated CMV mini Promoter to construct the 5HRE-CMVmini Promoter.
Preferably, the vector is a lentiviral vector characterized in that the vector comprises an RRE element and an oPRE element.
Further, the vector is characterized by comprising a prokaryotic replicon pUCOri sequence for plasmid replication; a kanamycin or ampicillin resistance gene sequence for mass amplification of a target strain; for enhancing replication of the viral replicon SV40Ori sequence in eukaryotic cells; a lentiviral packaging cis-element for lentiviral packaging; a promoter for eukaryotic transcription of a chimeric antigen receptor gene; and the CAR coding gene is used for combining target point recognition, signal transmission and signal initiation.
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 encompassed herein, and a right (3') retroviral LTR.
In certain embodiments, the CAR vector comprises a hepatitis b virus post-transcriptional regulatory element (HPRE) or woodchuck post-transcriptional regulatory element (WPRE); the promoter of the 5' LTR is replaced by a heterologous promoter, which is a Cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter or a Simian Virus 40(SV40) promoter.
When the lentiviral vector is used for T cell transduction, the positive expression rate of the CAR is high, the CAR is stable in the cell culture process of a patient, and the positive rate cannot be reduced along with the time. Thus, cells infected with the lentiviral vector have a function of killing the target cells.
The fourth purpose of the invention is to provide a pharmaceutical composition and the application of the pharmaceutical composition in preparing malignant tumor drugs.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the pharmaceutical composition comprises the ScFv capable of recognizing CD70 or the humanized single-chain antibody or the CAR structure or the immune cell.
In certain embodiments, the CAR-expressing cell may also express or be used in combination with additional active agents, which may be immune checkpoint inhibitors, such as PD1/PDL1, TIM3, CTLA4, LAG3, TGFR β inhibitors, and the like; in other embodiments, these agents may be polypeptides or fusion proteins, such as fusion proteins formed by the PD-1-CD137-CD3 molecule; in other embodiments, these active agents may be antibody drugs, such as monoclonal antibody drugs, bispecific antibody drugs, and the like; in other embodiments, the active agent may be a kinase inhibitor, such as the tyrosine kinase inhibitor dasatinib.
Further, the malignancy is a malignancy that expresses CD70, including but not limited to renal cell carcinoma, esophageal carcinoma, mesothelioma, gastric carcinoma, adenocystic carcinoma, ovarian carcinoma, endometrial carcinoma, breast carcinoma, head and neck squamous cell carcinoma, glioma, lung carcinoma, osteosarcoma, thyroid carcinoma, melanoma, pancreatic carcinoma, lymphoma, acute myeloid leukemia, or multiple myeloma.
In certain embodiments, the cell is activated and stimulated in the presence of an inhibitor of the PI3K pathway.
In certain embodiments, the application is performed by a composition comprising the cell, the route of administration of the composition comprising: oral, intravenous, peritoneal infusion, subcutaneous injection, and tumor or organ local administration. Preferably, the method of administering the composition is: by intravenous, intraperitoneal or subcutaneous injection.
In general, after the CD 70-targeting CAR (chimeric antigen receptor) disclosed by the invention is expressed in immune cells, the positive rate of the CD 70-targeting Chimeric Antigen Receptor (CAR) in a patient cell culture process can be maintained, the proliferation and tumor killing capacity of CAR-T can be enhanced, and the CD 70-targeting CAR can be used for targeted therapy of tumors.
The invention has the beneficial effects that:
1) the antibody or antigen-binding fragment for recognizing CD70 provided by the invention can efficiently recognize CD70 antigen;
2) the antibody or antigen-binding fragment recognizing CD70 provided by the invention can recognize CD70 positive tissues, cells and blood samples.
3) After the anti-CD 70 chimeric antigen receptor constructed by the antibody or antigen binding fragment for recognizing CD70 is expressed in immune cells, the chimeric antigen receptor can effectively eliminate tumor target cells expressing CD70 antigen, can maintain the positive rate of the Chimeric Antigen Receptor (CAR) targeting CD70 in the cell culture process of patients, and can be used for targeted therapy of tumors.
Drawings
FIG. 1 shows the expression of CD70 in tissues.
Figure 2 is a CD70 murine antibody and humanized design.
FIG. 3 shows the affinity detection of CD70 antibody.
FIG. 4 is a graphical representation of the ability of the CD70 antibody to recognize cell surface CD 70.
FIG. 5 shows the CAR structure constructed by ScFv of CD70 antibody.
Figure 6 is validation of targeted CD70 CAR expression.
Figure 7 is an in vitro effective killing targeting CD70 CART.
Figure 8 is targeting CD70 CART effective killing in vivo.
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: detection of CD70 expression in Normal tissues
Single PCR (one plate for amplifying one gene, one plate for an internal reference gene and one plate for a target gene) was used. Using cDNA of Skov3 cells as a template of a positive control; the negative control was the reaction mixture without cDNA template as dH2O as template, the same Skov3 cell cDNA from the same reverse transcription synthesis was used in this experiment to amplify GAPDH under the same primer pair, which serves to demonstrate that the assay plate andcomparability between internal reference plates. Classical calculation method 2 for relative expression levels with peripheral blood lymphocytes as a control group-ΔΔCtThe relative expression of CD70 in each tissue was calculated. The results are shown in FIG. 1: with peripheral blood lymphocytes as control, the expression of CD70 in lymph nodes and tonsils was slightly higher than that in the control sample, while small intestine, lung, spleen, stomach, thymus, prostate and trachea were slightly lower than that in the control sample, and other tissue samples were much lower than that in the control sample. It can be seen that CD70 is expressed in tissues other than lymphocyte-rich tissues, and is hardly expressed in other normal tissues.
Example 2: CD70 murine antibody and humanization design
The CDR structure of the murine antibody is shown in FIG. 2, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8.
Through molecular docking simulation, humanized modification and transformation are carried out on a framework sequence of a murine antibody to obtain 2 humanized ScFvs, and on the basis, the 2 ScFvs are respectively subjected to back mutation and immunogenicity optimization to obtain ScFvs with good activity and lower immunogenicity, so that 4 optimized humanized ScFvs are obtained.
Designing a humanized ScFv sequence, wherein the amino acid sequence of the light chain variable region is respectively shown as any one of SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12; the heavy chain variable region amino acid sequence is respectively shown in any one of SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16.
Example 3: CD70 antibody affinity detection
The kinetics of interaction of hCD70-hFc with the targeted CD70 ScFv was analyzed using HIS1K sensor immobilization. Analyte concentration gradient: the specific experimental procedures at 200nM, 100nM, 50nM, 25nM are described in the protocol of the instrument. As shown in fig. 3 and table 1, ScFv had a better affinity for CD70 with affinity constants KD of 16.4nM and 4.65 nM.
TABLE 1 CD70 antibody affinity assays
Conc.(nM) KD(M) Kon(1/Ms) Kdis(1/s) Full R 2
100 2.138E-08 73993.971 0.0016 0.9965
50 2.138E-08 73993.971 0.0016 0.9965
25 2.138E-08 73993.971 0.0016 0.9965
12.5 2.138E-08 73993.971 0.0016 0.9965
Example 4: verification of CD70 molecular ability to recognize cell surface CD70
ScFv targeting CD70 flow stained CD70 positive 786-O, exogenously constructed K562-CD70 and CD70 negative K562 cells. As a result, as shown in FIG. 4, ScFv targeting CD70 could recognize both CD 70-positive 786-O cells and CD70 on the surface of exogenously constructed K562-CD70 cells. Thus, we disclose murine and humanized anti-CD 70 antibodies or polypeptide fragments comprising the CDR regions of SEQ ID NO:1-SEQ ID NO:6 that can be used as CD70 detection reagents.
Example 5: lentivirus preparation for expression of chimeric antigen receptor targeting human CD70 antigen
(1) Preparation of chimeric antigen receptor constructs targeting human CD70 antigen
Chimeric antigen receptor sequences were synthesized comprising a single chain antibody ScFv against human CD70 antigen, a hFc hinge region, a transmembrane region, and an intracellular signal segment. Wherein the anti-human CD70 antigen humanized single chain antibody comprises heavy chain variable region shown as SEQ ID NO. 7, light chain variable region shown as SEQ ID NO. 8 or heavy chain variable region shown as SEQ ID NO. 13, light chain variable region shown as SEQ ID NO. 9 or heavy chain variable region shown as SEQ ID NO. 14, light chain variable region shown as SEQ ID NO. 10 or heavy chain variable region shown as SEQ ID NO. 15, light chain variable region shown as SEQ ID NO. 11 or heavy chain variable region shown as SEQ ID NO. 16, light chain variable region shown as amino acid sequence shown as SEQ ID NO. 12; the transmembrane region is derived from a CD8 or CD28 sequence; the intracellular signal segment is derived from CD28 or CD137 and CD3 sequences. The final synthetic humanized anti-CD 70 chimeric antigen receptor 12 group, murine control 1 group. The structure is shown in fig. 5.
The enzyme digestion reaction was carried out as described in the specification. The enzyme digestion product is separated and recovered, then the target fragment and the vector fragment are connected through T4 ligase (purchased from Promega company) to obtain a lentiviral vector for expressing the chimeric antigen receptor, and a plasmid is extracted by a plasmid extraction kit (Invitrogen company), wherein the specific method is shown in the specification.
(2) Packaging of lentiviruses
The slow virus is packaged by a calcium phosphate method, and the method comprises the following specific steps: culturing 293T cells to a better state by using DMEM medium containing 10% FBS (w/v), adding ddH2O, a lentiviral vector and 2.5mM CaCl2 into a 15mL centrifuge tube according to a certain proportion, uniformly mixing, dropwise adding 2 xHBS into the mixed solution, standing at room temperature for 15min after uniformly mixing, dropwise adding the mixed solution into the prepared 293T cells, and replacing fresh DMEM medium containing 10% FBS (w/v) after continuously culturing overnight. And (3) respectively collecting cell supernatants after culturing the cells for 48h and 72h, purifying and packaging.
Example 6: preparation of chimeric antigen receptor-modified T cells for CD70 antigen
(1) Lentiviral infection of T cells
1) Isolation of human peripheral blood mononuclear cells
Separating lymphocytes by using a gradient centrifugation method; after centrifugation, the second white lymphocyte layer was washed with physiological saline and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human peripheral blood mononuclear cells.
2) Lentiviral vector infection of T lymphocytes
Obtaining PBMC cells, activating by anti-CD 3 and CD28 monoclonal antibodies, and then infecting by lentivirus; CAR-T cells were cultured for about 10 days after infection for in vitro and in vivo biological characterization. The chimeric antigen receptor T cells obtained are named after the corresponding lentiviral vectors.
3) Chimeric Antigen Receptor (CAR) expression detection targeting human CD70 antigen
The CAR positive rate was tested for virus-infected T cells cultured up to day 6 and 9 during the culture. The detection method is flow detection, and the antibody is as follows: 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. Results the CAR was able to be normally expressed on the T cell surface after infection of T cells with different viruses as shown in figure 6.
Example 7: verification of anti-tumor effect of T lymphocytes expressing chimeric antigen receptor targeting CD70
CD70 positive 786-O (786-O-Luc for short) and negative A549-Luc cells which stably express the firefly luciferase are taken as target cells.
CART prepared using healthy donor peripheral blood mononuclear cells using 8:1/4:1/2: 1-effect target to target ratioThe cells are used. Use of
Figure BDA0002545713580000101
The killing effect is detected by a standard method provided by a Luciferase Assay System (Promega Cat. # E2520) kit, and the killing rate is calculated by the following formula:
Figure BDA0002545713580000102
the killing results are shown in fig. 7, and the constructed CART has better in-vitro killing effect.
Example 8: verification of anti-tumor effect of T lymphocyte expressing chimeric antigen receptor targeting CD70 in animal model
A mouse transplantation tumor model of a human CD70 positive tumor cell line is established for verifying the anti-tumor effect of the T lymphocyte expressing the chimeric antigen receptor targeting CD70 in an animal model.
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 validation 786-O cells were used as tumor target cells. After mouse tumorigenesis, CAR-T cells, virus-uninfected PBMC cells, were injected tail-vein separately. Tumor volumes were measured every 7 days after CAR-T cell injection, during which time mice survival was observed and recorded daily. The results are shown in FIG. 8: the tumor volume of the mouse treated by the constructed CD70 CAR-T is obviously reduced, and the in vivo killing activity is better.
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 accurate Biotechnology Co., Ltd
CHONGQING JINGZHUN BIOLOGICAL INDUSTRIAL TECHNOLOGY INSTITUTE Co.,Ltd.
<120> single-chain antibody targeting CD70 and application thereof
<160> 33
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Glu Ser Val Ser Ile His Gly Thr His Leu
1 5 10
<210> 2
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Ala Ala Ser
1
<210> 3
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Gln Gln Ser Ile Glu Asp Pro Arg Thr
1 5
<210> 4
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Gly Tyr Thr Phe Thr Asp Tyr Tyr
1 5
<210> 5
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Ile Asn Pro Tyr Asn Gly Gly Thr
1 5
<210> 6
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Ala Arg Ser Val Tyr Asp Tyr Pro Phe Asp Tyr
1 5 10
<210> 7
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Glu Val Gln Leu Gln Gln Ser Gly Pro Val Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Glu Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Thr Leu Glu Trp Ile
35 40 45
Gly Val Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 8
<211> 111
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Ile His
20 25 30
Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Phe Cys Gln Gln Ser Ile
85 90 95
Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 9
<211> 111
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His
20 25 30
Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
35 40 45
Val Leu Val Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser
65 70 75 80
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Ile
85 90 95
Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 10
<211> 111
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His
20 25 30
Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Val Leu Val Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile His
65 70 75 80
Pro Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Ile
85 90 95
Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 11
<211> 110
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His
20 25 30
Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Val Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Asn His Pro
65 70 75 80
Val Glu Glu Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Ile Glu
85 90 95
Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 12
<211> 111
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Ser Ile His
20 25 30
Gly Thr His Leu Met His Trp Tyr Gln Gln Leu Pro Gly Thr Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser
65 70 75 80
Pro Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Ile
85 90 95
Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 13
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Asn Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Val Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 14
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Asn Trp Val Arg Gln Met His Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Val Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 15
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Gln Val Gln Leu Val Gln Ser Gly Pro Val Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Glu Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Asn Trp Val Arg Gln Met His Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Val Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 16
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Gln Val Gln Leu Val Gln Ser Gly Pro Ala Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Asn Trp Val Arg Gln Ala His Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Val Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 17
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
1 5 10 15
Lys Gly
<210> 18
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Gly Gly Gly Gly Ser
1 5
<210> 19
<211> 47
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
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> 20
<211> 36
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
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> 21
<211> 45
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
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 Arg Pro Ala Ala
20 25 30
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Asp
35 40 45
<210> 22
<211> 473
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His
20 25 30
Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Val Leu Val Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile His
65 70 75 80
Pro Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Ile
85 90 95
Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly
100 105 110
Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys
115 120 125
Gly Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly
130 135 140
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
145 150 155 160
Tyr Tyr Met Asn Trp Val Arg Gln Met His Gly Lys Gly Leu Glu Trp
165 170 175
Met Gly Val Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys
180 185 190
Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala
195 200 205
Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
210 215 220
Cys Ala Arg Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly Gln Gly
225 230 235 240
Thr Leu Val Thr Val Ser Ser Lys Pro Thr Thr Thr Pro Ala Pro Arg
245 250 255
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
260 265 270
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
275 280 285
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
290 295 300
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Val Lys
305 310 315 320
Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg
325 330 335
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
340 345 350
Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser
355 360 365
Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu
370 375 380
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
385 390 395 400
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
405 410 415
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
420 425 430
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
435 440 445
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
450 455 460
Leu His Met Gln Ala Leu Pro Pro Arg
465 470
<210> 23
<211> 462
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His
20 25 30
Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Val Leu Val Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile His
65 70 75 80
Pro Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Ile
85 90 95
Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly
100 105 110
Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys
115 120 125
Gly Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly
130 135 140
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
145 150 155 160
Tyr Tyr Met Asn Trp Val Arg Gln Met His Gly Lys Gly Leu Glu Trp
165 170 175
Met Gly Val Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys
180 185 190
Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala
195 200 205
Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
210 215 220
Cys Ala Arg Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly Gln Gly
225 230 235 240
Thr Leu Val Thr Val Ser Ser Ala Pro Pro Arg Ala Ser Ala Leu Pro
245 250 255
Ala Pro Pro Thr Gly Ser Ala Leu Pro Asp Pro Gln Thr Ala Ser Ala
260 265 270
Leu Pro Asp Pro Pro Ala Ala Ser Ala Leu Pro Ile Tyr Ile Trp Ala
275 280 285
Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
290 295 300
Leu Tyr Cys Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
305 310 315 320
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
325 330 335
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
340 345 350
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
355 360 365
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
370 375 380
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
385 390 395 400
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
405 410 415
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
420 425 430
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
435 440 445
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
450 455 460
<210> 24
<211> 473
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Ser Ile His
20 25 30
Gly Thr His Leu Met His Trp Tyr Gln Gln Leu Pro Gly Thr Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser
65 70 75 80
Pro Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Ile
85 90 95
Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly
100 105 110
Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys
115 120 125
Gly Gln Val Gln Leu Val Gln Ser Gly Pro Ala Leu Val Lys Pro Gly
130 135 140
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
145 150 155 160
Tyr Tyr Met Asn Trp Val Arg Gln Ala His Gly Gln Gly Leu Glu Trp
165 170 175
Met Gly Val Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys
180 185 190
Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala
195 200 205
Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
210 215 220
Cys Ala Arg Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly Gln Gly
225 230 235 240
Thr Leu Val Thr Val Ser Ser Lys Pro Thr Thr Thr Pro Ala Pro Arg
245 250 255
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
260 265 270
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
275 280 285
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
290 295 300
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Val Lys
305 310 315 320
Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg
325 330 335
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
340 345 350
Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser
355 360 365
Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu
370 375 380
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
385 390 395 400
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
405 410 415
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
420 425 430
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
435 440 445
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
450 455 460
Leu His Met Gln Ala Leu Pro Pro Arg
465 470
<210> 25
<211> 473
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His
20 25 30
Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Val Leu Val Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile His
65 70 75 80
Pro Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Ile
85 90 95
Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly
100 105 110
Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys
115 120 125
Gly Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly
130 135 140
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
145 150 155 160
Tyr Tyr Met Asn Trp Val Arg Gln Met His Gly Lys Gly Leu Glu Trp
165 170 175
Met Gly Val Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys
180 185 190
Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala
195 200 205
Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
210 215 220
Cys Ala Arg Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly Gln Gly
225 230 235 240
Thr Leu Val Thr Val Ser Ser Lys Pro Thr Thr Thr Pro Ala Pro Arg
245 250 255
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
260 265 270
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
275 280 285
Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val
290 295 300
Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp
305 310 315 320
Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met
325 330 335
Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala
340 345 350
Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys Phe Ser Arg Ser
355 360 365
Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu
370 375 380
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
385 390 395 400
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
405 410 415
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
420 425 430
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
435 440 445
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
450 455 460
Leu His Met Gln Ala Leu Pro Pro Arg
465 470
<210> 26
<211> 24
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
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> 27
<211> 27
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
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> 28
<211> 41
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
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> 29
<211> 44
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 29
Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
1 5 10 15
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
20 25 30
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
35 40
<210> 30
<211> 111
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 30
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
1 5 10 15
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
20 25 30
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
35 40 45
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
50 55 60
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
65 70 75 80
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
85 90 95
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 31
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 31
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 32
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 32
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 33
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 33
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20

Claims (19)

1. An ScFv targeting CD70, wherein the light chain CDR region of the ScFv comprises the CDR1 with the amino acid sequence shown as SEQ ID NO. 1, the CDR2 with the amino acid sequence shown as SEQ ID NO. 2 and the CDR3 with the amino acid sequence shown as SEQ ID NO. 3; the heavy chain CDR region of the ScFv comprises CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5 and CDR3 shown in SEQ ID NO. 6.
2. The ScFv targeting CD70, according to claim 1, wherein the amino acid sequence of the light chain variable region of the ScFv targeting CD70 is shown as SEQ ID NO. 8 or a functional variant thereof, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7 or a functional variant thereof.
3. A humanized single chain antibody capable of recognizing CD70, which is characterized in that the ScFv targeting CD70 of any one of claims 1-2 is subjected to humanized transformation, wherein the amino acid sequence of the light chain variable region of the humanized single chain antibody is shown as SEQ ID NO. 9-12, and the amino acid sequence of the heavy chain variable region of the humanized single chain antibody is shown as SEQ ID NO. 13-16.
4. The humanized single chain antibody capable of recognizing CD70 according to claim 3, wherein the Linker amino acid sequence of the antibody is shown in SEQ ID NO 17 or 18 or 31 or 32 or 33.
5. A CAR structure comprising an ScFv of any one of claims 1-2 capable of recognizing CD 70.
6. A CAR structure comprising a humanized single chain antibody of any of claims 3-4 that recognizes CD 70.
7. The CAR structure of claim 6, wherein the amino acid sequence of the hinge region of the CAR structure is as set forth in SEQ ID NO 19 or SEQ ID NO 20 or SEQ ID NO 21.
8. The CAR structure of claim 6, wherein the amino acid sequence of the transmembrane region of the CAR structure is as set forth in SEQ ID NO 26 or SEQ ID NO 27.
9. The CAR structure of claim 6, wherein the intracellular signaling amino acid sequence of the CAR structure is as set forth in SEQ ID NO 28 or SEQ ID NO 29 or SEQ ID NO 30.
10. The CAR structure of claim 6, wherein the amino acid sequence of the CAR structure is as set forth in SEQ ID NO 22 or SEQ ID NO 23 or SEQ ID NO 24 or SEQ ID NO 25 or a functional variant thereof.
11. An expression vector comprising the CAR structure of any one of claims 6-10.
12. The expression vector of claim 11, wherein 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 DNA vector, an RNA vector, and a plasmid.
13. The expression vector of claim 12, wherein the expression vector is a lentiviral expression vector.
14. An immune cell comprising the expression vector of any one of claims 11-13.
15. The immune cell of claim 14, wherein the immune cell is a stem cell, a T cell, an NK cell, a monocyte, or a macrophage.
16. The immune cell of claim 15, wherein the immune cell is a T cell.
17. A pharmaceutical composition comprising a ScFv capable of recognising CD70 according to any one of claims 1-2 or a humanized single chain antibody capable of recognising CD70 according to any one of claims 3-4 or a CAR structure according to any one of claims 6-10 or an immune cell according to any one of claims 14-16.
18. The use of the pharmaceutical composition of claim 17 for the preparation of a medicament for the treatment of a malignant tumor.
19. The use of claim 18, wherein the malignancy is a CD 70-expressing malignancy, including but not limited to renal cell carcinoma, esophageal carcinoma, mesothelioma, gastric carcinoma, adenocystic carcinoma, ovarian carcinoma, endometrial carcinoma, breast carcinoma, head and neck squamous cell carcinoma, glioma, lung carcinoma, osteosarcoma, thyroid carcinoma, melanoma, pancreatic carcinoma, lymphoma, acute myeloid leukemia, or multiple myeloma.
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