DNA, peptides, anti-mesothelin antibodies, tumor imaging agents and complexes

Abstract

The present invention provides a DNA comprising any one of the following (a) to (c): (a) a DNA comprising any one of the following base sequences: nucleotides 16 to 831 of sequence number 1, nucleotides 16 to 822 of sequence number 3, nucleotides 16 to 825 of sequence number 5, nucleotides 16 to 819 of sequence number 7, nucleotides 16 to 834 of sequence number 9, and nucleotides 16 to 828 of sequence number 11; (b) a DNA encoding a polypeptide comprising any one of the following amino acid sequences: nucleotides 1 to 272 of sequence number 2, nucleotides 1 to 269 of sequence number 4, nucleotides 1 to 270 of sequence number 6, nucleotides 1 to 268 of sequence number 8, nucleotides 1 to 273 of sequence number 10, and nucleotides 1 to 271 of sequence number 12; and (c) a complementary chain of the DNA of the above (a) or (b).

Description

Technical Field

[0001] The present invention relates to DNA, polypeptide, anti-mesothelin antibody, tumor imaging agent and complex. Background Art

[0002] Mesothelin (MSLN) is a 40 kDa glycoprotein that binds to the membrane via a GPI anchor protein. MSLN is usually expressed locally in the mesothelium of the pleura, peritoneum, and pericardium. However, it is known that in cancerous tissues, it is highly expressed in many cancer cells such as mesothelioma, ovarian cancer, pancreatic cancer, colorectal cancer, and breast cancer (Non-patent Documents 1-2).

[0003] MSLN is first synthesized as a full-length 71kDa precursor protein, which is then cut by protease such as furin to form a 40kDa polypeptide, i.e., mature MSLN, which is obtained by binding to the cell membrane via a 31kDa polypeptide called megakaryocyte potentiating factor (MPF) and a GPI-anchored protein. Furthermore, it has been reported that a portion of the GPI-anchored MSLN is separated from the cell membrane and becomes free (soluble MSLN). As a function of MSLN, there are reports that it participates in the adhesion and proliferation of cells.

[0004] Based on these backgrounds, MSLN is considered to be a promising target molecule for cancer diagnosis and treatment, and antibodies recognizing MSLN have been produced and reported in large quantities, such as MORAb-009 (amatuximab) and HN1 (Non-Patent Documents 3 to 5, Patent Documents 1 to 2).

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: JAPAN Patent Publication No. 2014-221064

[0008] Patent Document 2: JAPAN Patent List 2011-504372

[0009] Non-patent literature

[0010] Non-patent document 1: Eur. J. Cancer 44, 46-53, 2008

[0011] Non-patent document 2: Clin. Cancer Res. 10, 3937-3942, 2004

[0012] Non-patent document 3: Lung Cancer, 68: 455-459, 2010

[0013] Non-patent document 4: Int. J. Cancer, 128: 2020-30, 2011

[0014] Non-patent literature 5: Scientific Reports 5: 09928, 2015 Summary of the invention

[0015] (Problems to be solved by the invention)

[0016] The object of the present invention is to provide a novel polypeptide, DNA, anti-mesothelin antibody, complex, and tumor imaging agent that are effective for the diagnosis and treatment of cancer.

[0017] (Technical solutions to solve problems)

[0018] The present invention provides the following DNA, polypeptide, anti-mesothelin antibody, tumor imaging agent and complex.

[0019] Scheme 1. A DNA comprising any one of the following (a) to (c),

[0020] (a) a DNA comprising any of the following base sequences,

[0021] Sequence number 1, 16th to 831st bit,

[0022] Sequence number 3, 16th to 822nd bit,

[0023] The 16th to 825th bits of sequence number 5,

[0024] From the 16th to the 819th position of the sequence number 7,

[0025] The 16th to 834th bits of sequence number 9,

[0026] The 16th to 828th bits of sequence number 11;

[0027] (b) a DNA encoding a polypeptide comprising any one of the following amino acid sequences,

[0028] Sequence number 2, 1st to 272nd bit,

[0029] Sequence number 4, 1st to 269th bit,

[0030] The 1st to 270th digits of sequence number 6,

[0031] The 1st to 268th bits of sequence number 8,

[0032] Serial number 10, 1st to 273rd bit,

[0033] 1st to 271st bits of sequence number 12;

[0034] (c) The complementary strand of the DNA of (a) or (b) above.

[0035] Scheme 2. A polypeptide comprising any of the following amino acid sequences,

[0036] Sequence number 2, 1st to 272nd bit,

[0037] Sequence number 4, 1st to 269th bit,

[0038] The 1st to 270th digits of sequence number 6,

[0039] The 1st to 268th bits of sequence number 8,

[0040] Serial number 10, 1st to 273rd bit,

[0041] Serial number 12, digits 1 to 271.

[0042] Scheme 3. An anti-mesothelin antibody comprising any one of the following amino acid sequences,

[0043] Sequence number 2, 1st to 272nd bit,

[0044] Sequence number 4, 1st to 269th bit,

[0045] The 1st to 270th digits of sequence number 6,

[0046] The 1st to 268th bits of sequence number 8,

[0047] Serial number 10, 1st to 273rd bit,

[0048] Serial number 12, digits 1 to 271.

[0049] Scheme 4. A tumor imaging agent comprising the polypeptide described in Scheme 2.

[0050] Scheme 5. The tumor imaging agent according to Scheme 4, wherein the tumor imaging agent further comprises 89 Zr.

[0051] Embodiment 6. The tumor imaging agent according to Embodiment 4 or 5, wherein the tumor imaging agent is used for imaging a tumor expressing mesothelin.

[0052] Scheme 7. A complex obtained by linking the polypeptide described in Scheme 2 and an antitumor substance.

[0053] (Effects of the Invention)

[0054] Compared with conventional full-length antibodies, the anti-MSLN scFv of the present invention has a lower molecular weight and a faster blood clearance rate. Therefore, in PET based on radiolabeled scFv, tumors expressing MSLN can be visualized specifically in a shorter time than conventional methods.

[0055] Furthermore, since the antibody is reduced in molecular weight, it can also be modified for targeting antitumor agents such as high-molecular micelle-type DDS preparations.

[0056] In addition, by using PET nuclides that do not emit β-rays when decaying, 89 Zr, enabling safer cancer imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1: The structural diagram of the production of anti-human MSLN scFv antibodies and the structure of anti-human MSLN scFv-cp3 antibodies. a Scheme for the production of human natural antibody phage library. The library was produced from human tonsil lymphocytes. b Structure of scFv antibodies with cp3 sequence. It has a structure in which PelB and cp3 sequences are attached to the variable regions of the heavy and light chains and the constant region of the light chain bound by a linker. PT7 represents the T7 promoter. In addition, the variable region is represented by a black square and the constant region is represented by a gray square. c Scheme for the concentration and selection of scFv (cp3-scFv) antibodies with cp3 sequence and the production of scFv antibodies with His-tag sequence. The cp3-scFv antibodies were concentrated by 4-5 biopannings using a column (r-MSLN column) using beads (r-MSLN beads) bound to human recombinant MSLN via His-tag (Dynabeads (registered trademark) His-tag Isolation & Pull down: 10103D: Invitrogen) or mouse monoclonal anti-His-tag antibody (MBL: M136-3) and protein G (Dynabeads (registered trademark) protein G: 100.04D: Invitrogen). After concentration, each cp3-scFv antibody was cloned and its binding ability to MSLN was evaluated by solid phase enzyme immunoassay (ELISA). For scFv antibodies with high activity by ELISA, the ability to bind to cancer cells was confirmed again by flow cytometry (FCM), and 5 clones with high reactivity were selected. Based on the sequence information of the selected scFv, DNA of scFv antibody with His-tag sequence attached was synthesized and expressed in Chinese hamster ovary (CHO) cells, and the ability to bind to cancer cells was confirmed by FCM. ScFv antibody with His-tag sequence attached that was highly evaluated by FCM was evaluated by PET imaging. H represents the heavy chain variable region, V L represents the light chain variable region, C L In addition, the variable region is shown in black, and the constant region is shown in gray.

[0058] Figure 2:Selection of cp3-scFv clones and evaluation based on ELISA. In order to evaluate the specificity of cp3-scFv for r-MSLN, r-MSLN was fixed to a 96-well plate and used for ELISA. The Y-axis represents the absorbance, the X-axis represents the name of each clone (due to space constraints, every other clone name is marked with a name), the black column represents the fixed r-MSLN, and the white column represents the reaction of the cp3-scFv clone when EGFR was fixed to the plate as a negative control. In addition, the gray column represents the reaction of the cp3-scFv clone only with PBS in which r-MSLN was not fixed to the plate. In the figure, the 4 th , 5 th Indicates the number of washes, 4 th indicates clones obtained after four rounds of panning and concentration, 5 th indicates clones obtained after five rounds of panning. th After two rounds of panning with beads obtained by binding the antigen r-MSLN to Dynabeads (registered trademark) His-tag Isolation & Pulldown: 10103D (hereinafter referred to as His-tag beads), mouse monoclonal anti-His-tag antibody (MBL: M136-3) was bound to Dynabeads (registered trademark) protein G: 100.04D: invitrogen), and then two rounds of panning were performed with beads obtained by binding the antigen r-MSLN to the beads (hereinafter referred to as PG beads). th The lower panel shows clones obtained by further panning with PG beads after four rounds of panning. HR1 is a pc3-scFv specific to Protobothrops flavoviridis used as a control.

[0059] Figure 3:Evaluation of anti-human MSLN cp3-scFv antibody clones using flow cytometry (FCM). The reactivity of anti-human MSLN cp3-scFv antibody clones was analyzed by FCM using lung cancer cells NCI-H226, gastric cancer cells NCI-N87, pancreatic cancer cells BxPC-3 and PANC-1. The black color in the figure represents each anti-human MSLN cp3-scFv antibody clone, and the gray color represents the HR1 007 anti-Japanese agkistrodon toxin scFv antibody used as a control. The clones surrounded by black frames are clones selected based on the height of reactivity based on FCM. The numerical values ​​in the figure are the ratio of the mean fluorescence intensity (mean fluorescence intensity, MFI) of each anti-human MSLN cp3-scFv antibody clone to the MFI of the HR1 007 anti-Japanese agkistrodon toxin scFv antibody used as a control.

[0060] Figure 4 : The structure of the anti-human MSLNHis-tag-scFv antibody obtained and the analysis based on SDS-PAGE. a The structure of the anti-human MSLNHis-tag-scFv antibody. b The amino acid sequence of the anti-human MSLNHis-tag-scFv antibody. The complementary determining regions (CDRs) of the light chain variable region and the heavy chain variable region are shown in the box. (*) indicates the amino acid residues that are different between the clones, and the red text indicates the amino acid residues that do not change in the CDR region. The His-tag is composed of 6 residues of histidine. SS represents the signal sequence. The complementary determining region (CDR) was inferred based on the data of VBASE (The MRC Centre for ProteinEngineering, MRC Laboratory of Molecular Biology). c Reduced SDS-PAGE analysis of His-tag-scFv antibody clones. 100mM dithiothreitol (DTT) was used to reduce the sample. Lane 1: molecular size marker, Lane 2: His-Tag-scFv H1a050 (27064.24 Da), Lane 3: His-Tag-scFv H2a064 (26683.87 Da), Lane 4: His-Tag-scFv H2a021 (26708.96 Da), Lane 5: His-Tag-scFv H2a006 (26701.85 Da), Lane 6: His-Tag-scFv H2a059 (27124.32 Da), Lane 7: His-Tag-scFv H2b011 (27033.18 Da). Molecular weights were calculated from the amino acid sequence.

[0061] Figure 5: Evaluation of anti-human MSLN His-tag scFv antibody clones using various cancer cell lines. The reactivity of anti-human MSLN His-tag scFv antibody clones to 3 types of pancreatic cancer, 2 types of lung cancer, 1 type of prostate cancer, 1 type of uterine cancer, 2 types of cervical cancer, 2 types of ovarian cancer, and 3 types of gastric cancer was analyzed by flow cytometry. The black color in the figure represents each anti-human MSLN His-tag scFv antibody clone, and the gray color represents the use of only the secondary antibody, i.e., the anti-His-tag antibody, as a control without using the primary antibody, i.e., the anti-human MSLN His-tag scFv antibody clone. The lower section represents a graph of FCM using full-length IgG mouse anti-human MSLN antibodies 11-25. The black color in the figure represents the full-length IgG mouse anti-human MSLN antibody, and the gray color represents the result of using the anti-KLH antibody of the IgG isotype control as a negative control. The numerical values ​​in the figure are the ratios of the mean fluorescence intensity (MFI) of each anti-human MSLN cp3-scFv antibody clone and full-length IgG mouse anti-human MSLN antibody 11-25 to the MFI of the control.

[0062] Figure 6 Identification of human MSLM using mouse models 89 PET·CT images of Zr-DFO-scFv antibody. BALB / c nu / nu tumor-bearing mice have tumors caused by MSLN-high-expressing gastric cancer cell line MCI-N87 (right shoulder) (white arrow) and NSLN-low-expressing pancreatic cancer cell line PANC-1 (left shoulder) (blue arrow). 89 PET·CT images 3 hours after administration of Zr-DFO-scFv antibody. H2a064 and H1a050 are clone names of anti-human MSLN His-tag scFv antibodies.

[0063] Figure 7 : Distribution of 89Zr-DFO-scFv in vivo. 89 Distribution of Zr-DFO-scFv in vivo 3 hours after intravenous injection. Data are calculated as % of injected dose per 1 gram (%ID / g tissue). The black columns in the figure represent scFv clone H1a050, and the white columns represent scFv clone H2a064. Error bars represent SD. (*p<0.01)

[0064] Figure 8 It is calculated 89Graphs showing the accumulation of Zr-DFO-scFv antibodies in tumors and blood over time. (a) The Y axis represents the accumulation of antibodies in the blood. (b) represents the accumulation of antibodies in tumors from gastric cancer cell lines (NCI-N87) with high expression of MSLN and pancreatic cancer cell lines (PANC-1) with low expression of MSLN (%ID / g tissue). In addition, the error bars in the graphs represent the SD values.

[0065] Fig. 9 Shows the DNA sequence and amino acid sequence of MSLN H1a050LH scFv.

[0066] Fig.10 (a) is the identification of human MSLM using model mice 89 PET·CT images of Zr-DFO-1125-IgG, (b) shows 89 Graph showing the temporal changes in Zr radioactivity

[0067] Fig.11 is to use (a) 89 Zr-labeled trastuzumab and (b) 64 PET·CT images of Cu-labeled trastuzumab DETAILED DESCRIPTION

[0068] The present inventors have developed tumor cell imaging technology using IgG antibodies with high affinity and specificity for MSLN obtained by immunizing mice with mesothelin (MSLN) protein. However, mouse-type antibody injections are immunogenic when administered to humans and induce antibody production against the administered antibody, making it difficult to administer multiple times. Therefore, in order to reduce antigenicity, anti-mesothelin antibodies derived from human antibody genes were established.

[0069] The antibody used in the present invention is a low-molecular-weight antibody, namely, scFv, in which only the variable regions of the H chain and L chain of the antibody are linked by a short linker.

[0070] The anti-mesothelin scFv antibody of the present invention is a polypeptide comprising any one of the following amino acid sequences: positions 1 to 272 of sequence number 2, positions 1 to 269 of sequence number 4, positions 1 to 270 of sequence number 6, positions 1 to 268 of sequence number 8, positions 1 to 273 of sequence number 10, and positions 1 to 271 of sequence number 12. In order to facilitate purification, the polypeptide can also be bound to protein tags such as His-tag, GST, MBP, HA-tag, myc-tag, FLAG-tag, etc. In addition, polypeptides obtained by binding any peptide to the N-terminal and C-terminal sides are also included in the polypeptide of the present invention. The peptides of sequence numbers 2, 4, 6, 8, 10, and 12 of the present invention are shown in Figure 4 b.

[0071] Sequence numbers 1 and 2 are from scFv clone H1a050,

[0072] Sequence numbers 3 and 4 are from scFv clone H2a021,

[0073] Sequence numbers 5 and 6 are from scFv clone H2a064,

[0074] Sequence numbers 7 and 8 are from scFv clone H2a006,

[0075] Sequence numbers 9 and 10 are from scFv clone H2a059,

[0076] Sequence numbers 11 and 12 are from scFv clone H2b011.

[0077] The preferred scFv antibodies of the present invention are shown in Fig. 9 .

[0078] The DNA of the present invention is a DNA encoding the peptide of the present invention, which comprises:

[0079] (a) a DNA comprising any one of the base sequences of nucleotides 16 to 831 of SEQ ID NO: 1, 16 to 822 of SEQ ID NO: 3, 16 to 825 of SEQ ID NO: 5, 16 to 819 of SEQ ID NO: 7, 16 to 834 of SEQ ID NO: 9, and 16 to 828 of SEQ ID NO: 11;

[0080] (b) a DNA encoding a polypeptide comprising any one of the amino acid sequences of SEQ ID NO: 1 to 272, SEQ ID NO: 4, SEQ ID NO: 1 to 269, SEQ ID NO: 6, SEQ ID NO: 1 to 270, SEQ ID NO: 8, SEQ ID NO: 1 to 273, SEQ ID NO: 10, and SEQ ID NO: 1 to 271;

[0081] (c) The complementary strand of the DNA of (a) or (b) above.

[0082] Sequence numbers 1, 3, 5, 7, 9, and 11 have HindIII recognition sequences and Kozak sequences added to the 5' end and EcoRI recognition sequences added to the 3' end, but any sequence may be added in place of or in addition to them. For example, the DNA of the present invention includes a vector assembled with any base sequence of positions 16 to 831 of sequence number 1, positions 16 to 822 of sequence number 3, positions 16 to 825 of sequence number 5, positions 16 to 819 of sequence number 7, positions 16 to 834 of sequence number 9, and positions 16 to 828 of sequence number 11.

[0083] The polypeptide of the present invention can be obtained by transforming cells with a vector containing the DNA of the present invention and culturing the transformed cells. Cells used to produce the polypeptide of the present invention include eukaryotic organisms including yeast, insect cells (insect cell / baculovirus expression system) and mammalian cells (CHO, etc.), prokaryotes including Escherichia coli and Bacillus subtilis, and archaea, preferably CHO.

[0084] The polypeptide of the present invention can be labeled with a labeling substance. Examples of the labeling substance include 89 Zr, 99m Tc, 111 In, 113m In, 67 Ga, 68 Ga, 201 Tl, 51 Cr, 57 Co. 58 Co. 60 Co. 85 Sr. 197 Hg, 64 Cu, 123 I. 125 I. 124 I. 131 I. 90 Y. 177 Lu, 186 Re, 188 Re, 211 At 225 Ac, 213 Bi, 212 Pb, 166 Ho, 44 Sc, 47 Sc, 227 Radionuclides such as Th, fluorescein, rhodamine, cyanine dyes, Alexa Fluor, quantum dots, sulforhodamine 101 (Texas Red), indocyanine green (ICG) and other fluorescent substances, preferably 89 Zr.

[0085] The complex of the present invention comprises the polypeptide of the present invention and an antitumor substance. Examples of the antitumor substance include anticancer agents, polymer micelle-type DDS preparations, and the like. Examples of the anticancer agent include doxorubicin, daunorubicin, cisplatin, oxaliplatin, carboplatin, paclitaxel, irinotecan, SN-38, actinomycin D, vincristine, vinblastine, methotrexate, azathioprine, fluorouracil, mitomycin C, docetaxel, cyclophosphamide, capecitabine, epirubicin, gemcitabine, mitoxantrone, leucovorin, vinorelbine, trastuzumab, etoposide, estramustine, prednisone, interferon α, interleukin-2, bleomycin, ifosfamide, mesna, Hexamethylmelamine, topotecan, cytarabine, methylprednisolone, dexamethasone, mercaptopurine, thioguanine, fludarabine, gemtuzumab, idarubicin, mitoxantrone, retinoic acid, alemtuzumab, chlorambucil, cladribine, imatinib, epirubicin, dacarbazine, procarbazine, nitrogen mustard, rituximab, denileukin, trimethoprim / sulfamethoxazole, allopurinol, carmustine, tamoxifen, filgrastim, temozolomide, melphalan, vinorelbine, azacitidine, thalidomide and mitomycin, etc., but are not limited to them.

[0086] In the present invention, the tumor to be imaged is not particularly limited as long as it is a tumor expressing mesothelin, and examples thereof include small cell lung cancer, non-small cell lung cancer, pancreatic cancer, prostate cancer, cervical cancer, uterine cancer, ovarian cancer, breast cancer, and gastric cancer.

[0087] The tumor imaging agent of the present invention can be used for imaging using PET, SPECT, CT or MRI alone or in combination (eg, PET / CT, SPECT / CT, SPECT / CT / PET, etc.).

[0088] Example

[0089] The present invention is further illustrated by the following examples, which should not be construed as further limiting.

[0090] Example 1

[0091] I. Materials and Methods

[0092] (1) Reagents

[0093] Deferoxamine-p-SCN (DFO) was purchased from Macrocyclics (Dallas, TX). PD-10 desalting column was purchased from GE Healthcare (Uppsala, Sweden). Amicon Ultra 0.5 centrifugal filter unit was purchased from Merck Millipore (Billerica, MA). In addition, other reagents used special grade reagents.

[0094] (2) Anti-human MSLN-scFv production

[0095] Total RNA was extracted from the lymphocytes of the palatine tonsils of patients with tonsil hypertrophy and inflammation, and the RNA was expressed using a conventional method using a marker for each V H 、V L and V L -C L The obtained sequence was added with the expression signal PeIB and the phage cp3 sequence, and the cDNA was obtained by reverse transcription. 4 S) 3 Linker binding V H 、V L -C L Sequence and V H 、V L The sequences were introduced into Escherichia coli (DH12S) using the pTZ19R phagemid vector (Thermo Fisher scientific, Massachusetts, USA). The M13KO7 helper phage was used to infect Escherichia coli to create a human natural antibody phage library ( Figure 1 a, b). V H 、V L The scFv composed of the sequence is set as H1, and the V H 、V L -C L The sequence of scFv-CL is set as H2( Figure 1 c). The preparation of recombinant MSLN (hereinafter referred to as r-MSLN) for scFv concentration and enzyme-linked immunosorbent assay (ELISA) is shown in the literature of Iwahori et al. (Iwahori K. et al., Lung Cancer. 2008; 62(1): 45-54). The obtained scFv-cp3 of H1 and H2 was concentrated by washing 4 to 5 times using a column that allowed r-MSLN to bind to beads, and the specific affinity for MSLN was confirmed by ELISA using a plate with r-MSLN fixed on it ( Figure 2 ). The phage was infected with Escherichia coli to form colonies in a plate culture medium, and then cloned. The gene sequence of each clone was confirmed by a DNA sequencer. The obtained human MSLN-scFv-cp3 clones were selected based on their reactivity to r-MSLN by using ELISA with fixed r-MSLN. The scFv-cp3 selected by ELISA was further confirmed by FCM using cancer cell lines with high MSLN expression and cancer cell lines with low MSLN expression to determine their reactivity with MSLN expressed in cancer cells ( Figure 3). The scFv gene with a His-tag sequence was synthesized from the DNA sequence of the selected highly reactive scFv-cp3, and the scFv was expressed using mammalian cells. The overall process of humanized anti-human MSLN-scFv production is shown in Figure 1 c. This study was approved by the Ethics Committee of Okayama University and the Ethics Committee of the Institute of Medical Biology and was conducted in accordance with the ethical guidelines for human genome / gene research established by the JAPAN government and the Declaration of Helsinki.

[0096] (3) Selection using anti-human MSLNscFv-cp3 ELISA.

[0097] Human r-MSLN and epidermal growth factor receptor (EGFR) as a negative control were dissolved in PBS and dispensed into 96-well MAXIsorp plates at 5 μg / mL and 50 μL per well, respectively, and fixed by incubation at 4°C overnight. 2.5% BSA 200 μL was added and blocked by incubation at room temperature for 2 hours. 50 μL of each anti-human MSLN scFv-cp3 clone was added, and after incubation at room temperature for 1 hour, 50 μL of anti-cp3 rabbit polyclonal antibody (5 μg / mL) was added and incubated at room temperature for 1 hour. 50 μL of the tertiary antibody Anti-IgG (H+L chain) (rabbit) pAb-HRP (MBL458, Nagoya, JAPAN) diluted 5000 times with PBS was added, and after incubation at room temperature for 1 hour, 50 μL of 3,3',5,5'-tetramethylbenzidine was used to develop color, and the absorbance at 450 nm was measured using an enzyme reader.

[0098] (4) Preparation of anti-human MSLN-scFv-His tag

[0099] With clinical application in mind, scFvs expressed in mammalian cells were prepared. V was synthesized in the anti-MSLN scFv-cp3 selected by ELISA and FCM. H 、V L The artificial gene with a linker and His-tag attached to the sequence was inserted into the mammalian cell expression vector pCx17.4 (Lonza, San Francisco, CA, USA), and the gene was introduced into CHOK1-GSKO ( Figure 4a, b). Anti-human MSLN-scFv-His-tag was prepared by applying the culture supernatant to 1 mL of Ni-NTA agarose (QIAGEN) column, washing with PBS, eluting with 200 mM imidazole 3 mL x 4 times, dialyzing the total amount with PBS overnight, concentrating by ultrafiltration (Amicon), and then refining it, and confirming it by SDS-PAGE. In addition, the molecular weight marker used was Precision Plus Protein TM Pre-dyed standard Dual Color (Bio-rad, California, USA) Figure 4 c). Furthermore, the molecular weight was measured by MALDI-TOF-MS (AXIMA (registered trademark) Performance, SHIMADZU, Kyoto, JAPAN).

[0100] (5) Cell culture

[0101] Cancer cell lines established from various tissues are sold separately by the American Type Culture Collection (ATCC) and the JCRB cell bank. Culture dishes are based on RPMI-1640, EMEM, DMEM, and IMDM according to the data sheet of each cell, with 10-20% fetal bovine serum (FBS) added, and supplements such as insulin and non-essential amino acids (NEAA) added in necessary amounts according to the cells, and 1% penicillin / streptomycin is added to all culture media. Culture media, supplements, etc. are purchased from (Gibco / Life Technologies, CA, USA). The name of the cell line, the source of the tissue, the name of the disease, etc. are shown in Table 1. Culture is carried out in a humidified culture chamber at 37°C and a carbon dioxide concentration of 5%.

[0102] [Table 1]

[0103] Study of MSLN expression in various cancer cell types using flow cytometry

[0104]

[0105] Table 1 shows the cell lines, tissues, diseases and the expression level of MSLN. It includes 3 gastric cancer cell lines, 4 lung cancer cell lines (including bronchus), 3 pancreatic cancer cell lines, 2 ovarian cancer cell lines, 2 cervical cancer cell lines, 3 uterine cancer cell lines, 1 prostate cancer cell line, 1 colorectal cancer cell line, 1 skin cancer cell line, 1 neuroblastoma cell line, 1 glial cell line, 3 leukemia cell lines, 1 breast cancer cell line, 1 liver cancer cell line and 1 kidney cancer cell line.

[0106] A total of 28 cell lines were used for FCM analysis. "MSLN exp ref" indicates MSLN expression positivity as shown in previous studies. "MSLN exp FCM" indicates the ratio of the mean fluorescence intensity (MFI) of the scFv clone H1a050 in each MSLN-positive cancer cell line to the control MIF, with 5 or more being indicated as ++, 4.9 to 1.5 being indicated as +, and 1.4 or less being indicated as -.

[0107] (6) FCM analysis of antibodies

[0108] Various human cancer cell lines (Table 1) were treated with cell dissociation solution, enzyme-free, PBS (Gibco / Life Technologies, CA, USA) to prepare and obtain individual cell suspensions. 1×10 6The cells were washed once, and full-length anti-human MSLN antibody, each anti-human MSLN cp3-scFv clone or each His-tag scFv clone was used as the primary antibody. The full-length anti-human MSLN antibody used an anti-KLH antibody (IgG2 isotype control) as a negative control, and a goat anti-mouse IgG antibody labeled with Alexa Fluor 488 was used as the secondary antibody. Anti-human MSLN cp3-scFv used an antibody against the hemorrhagic factor HR1-007 of the Japanese proto-axillary toxin as a negative control, and a rabbit anti-cp3 polyclonal antibody (MBL, Nagoya, JAPAN) was used as the secondary antibody. In addition, a goat anti-rabbit polyclonal antibody labeled with Alexa Fluor 488 (Invitrogen: A11034, USA) was used as the tertiary antibody. For the anti-human MSLN His-tag scFv, a mouse anti-His-tag monoclonal antibody (No.D291-A48, MBL, Nagoya, JAPAN) labeled with Alexa Fluor 488 was used as the secondary antibody. As a control, the cells were treated without the His-tag scFv. Finally, in order to select dead cells, they were suspended in 100 μL of PBS containing 5 μL of 7-amino-actinomycin D (immunostep, Salamanca, Spain) (7AAD) and 1 mM EDTA, and then measured using a BD FACSAriaIII flow cytometer (BD Biosciences, NJ, USA). The antibody reactions were all carried out on ice for 1 hour. In addition, at each stage of the reaction with the antibody, the cells were washed twice with 500 μL of PBS containing 2% FBS and 1 mM EDTA. In addition, the average value of the fluorescence intensity excluding dead cell aggregates was obtained using BD FACSDiva software and calculated using Microsoft Excel.

[0109] (7) DFO modification and radiolabeling of His-tag scFv

[0110] DFO modification of His-tag scFv was performed by incubation in a ratio of 3:1 dissolved chelator deferoxamine (p-SCN-Bn-DFO):His-tag scFv in bicarbonate buffer at pH 9.0 at 37°C for 1 hour. 89 Zr was obtained by cyclotron (HM-12cyclotron, Sumitomo Heavy Industries Ltd., Tokyo, JAPAN) 89 Zr-oxalate. 89 Zr-oxalate:Na 2 CO3 The mixture was adjusted to pH 7.0 by mixing 2M:HEPES (0.5M) in a ratio of 2:1:10, and then mixed with the DFO-modified His-tag scFv clone in which the solvent was replaced with gentisic acid saline (5 mg / mL), and incubated at 37°C for 30 minutes to obtain 89 Zr-DFO-scFv. Unbound 89 Zr was removed by ultrafiltration using an Amicon Ultra 10K centrifugal filter. The radiochemical purity was determined by thin layer chromatography-based autoradiography (TLC-ARG) and HPLC (LC-20, Shimadzu Co., Kyoto, JAPAN). TLC-ARG was developed by spotting the sample on a silica gel plate (silica gel, 60RP-18F254S, Millipore) using 50 mM EDTA (pH 5.0) as the mobile phase. HPLC was performed using D-PBS (Wako) (pH 7.0) as the mobile phase at a flow rate of 0.75 mL / min and a Superdex 200 10 / 300 column (10 mm×30 cm, GE Healthcare, Buckinghamshire, England). The in vitro stability of the radiolabeled scFv after incubation at 37°C for 6 hours in 50% human plasma / PBS was also analyzed. 89 Zr-DFO-scFv or anti-KLH scFv (50 μL) was added to 450 μL of mouse plasma. Immediately after mixing and after 6 hours of incubation, a portion of the mixture of radiolabeled scFv and plasma was measured by HPLC using GABI star (Raytest, Straubenhardt, Germany) at 230 nm and radioactivity. 89 The change in the binding ability of the Zr label was measured by using the molecular interaction analysis device BLItz (ForteBio, Inc., CA, USA) using an amine-reactive second-generation (AR2G) biosensor probe to measure the equilibrium dissociation constant (KD) for each antigen, namely r-MSLN.

[0111] (8) Model Animals

[0112] All animal experiments were performed according to the guidelines of Okayama University and approved by the University Animal Experiment Committee (OKU-2013098). Male BALB / cnu / nu mice aged 5 weeks were purchased from Charp River (Tokyo, JAPAN) and maintained under specific pathogen-free conditions at the Animal Resources Department of the Natural Life Science Research Support Center, Okayama University before use. For PET imaging, the MSLN-positive culture cell line NCI-N87 and the MSLN-negative culture cell line PANC-1 were cultured separately, and 3 × 10 NCI-N87 cells were plated. 6 PANC-1 cells were transplanted into the right shoulder of nude mice. 7 The tumor was transplanted into the left shoulder to create a tumor-bearing mouse model. Imaging was performed when the tumor diameter was about 8 mm.

[0113] (9) Small Animal PET·CT Imaging

[0114] Each mouse bearing a tumor derived from the NCI-N87 or PANC-1 cell line was anesthetized by inhalation of isoflurane, and the drug was administered through the tail vein of the mouse under anesthesia. 89 Zr-DFO-scFv (n=3) was imaged using a PET / CT system for animals (ClairvivoPET, Shimadzu, Kyoto, JAPAN). Dynamic PET scanning was performed for 3 hours, and images were reconstructed using the 3D-DRAMA method. The average doses were H1a050 (6.0 MBq / 8.3 μg) and H2a064 (4.1 MBq / 11.5 μg), respectively. In addition, CT data were acquired using a CT scanner (Eminence Stargate, Shimadzu) before PET scanning. The PET and CT images converted to DICOM format were fused using PMOD software version 3.3 (PMOD Technologies Ltd., Zurich, Switzerland), and the three-dimensional volume region of interest (VOI) in the heart was depicted as the tumor and blood pool on the PET / CT image, and the average percentage of the injected dose per 1 gram of tissue (%ID / g) was determined. After CT scanning, all mice were euthanized to study the in vivo distribution. Tumors and major organs of mice were collected and weighed, and the radioactivity of organs was measured using a gamma counter (AccuFLEXγ7001, HitachiAloka Medical, Tokyo, JAPAN). The in vivo distribution data were expressed as %ID / g.

[0115] (10) Statistical analysis

[0116] Data are expressed as mean ± standard deviation. Statistical analysis was performed using an unpaired Student's t-test for comparison between two groups. P < 0.05 was considered statistically significant.

[0117] II Results

[0118] (1) Preparation and selection of anti-human MSLN-scFv

[0119] The cDNA of the lymphocytes of the palatine tonsils of patients with tonsil hypertrophy and inflammation was used to prepare an antibody phage library, and 120 anti-MSLN cp3-scFv clones were obtained by 4 to 5 biopannings. The reactivity of the anti-MSLN cp3-scFv obtained by ELISA study using fixed r-MSLN was studied, and 15 scFvs with high reactivity to r-MSLN were selected. In addition, when selecting these 15 species, the gene sequence of each scFv clone was confirmed, and clones with the same sequence were not selected. In addition, the reactivity of these anti-MSLNcp3-scFv clones was confirmed using cultured cancer cell lines. As the objects, the lung cancer cell line NCI-H226, which strongly expresses MSLN, the gastric cancer cell line NCI-N87, the pancreatic cancer cell line BxPC-3, and the pancreatic cancer cell line PANC-1, which expresses MSLN very weakly, were used for analysis using FCM. Through FCM analysis, six scFvs with high reactivity to cancer cells with strong MSLN expression and scFvs with low reactivity to cancer cells with very weak MSLN expression were selected ( Figure 3 ).

[0120] (2) Preparation and selection of anti-human MSLN-scFv-His tag

[0121] In order to prevent scFv itself from becoming an antigen, scFv is produced in mammalian cells. Specifically, starting from the DNA sequence of the anti-MSLNcp3-scFv clone selected in item (1) above, an anti-human MSLN-scFv-His-tag single-chain antibody that can be produced in mammalian cells is produced by total gene synthesis, and then inserted into a vector and introduced into CHO cells ( Figure 4 a, b). Six scFv clones were prepared and analyzed by FCM using various cancer cell lines. Clones H1a050 and H2a064 showed high reactivity to MSLN-expressing cell lines. In particular, clone H1a050 also showed good reactivity to cancer cell lines established from various tissues ( Figure 5 In addition, H1a050 scFv showed the same reactivity as the full-length anti-MSLN antibody 11-25 ( Figure 5 ). Anti-human MSLNHis-tag scFv ( Figure 4 a) and the structures of the amino acid sequences of the six scFv clones ( Figure 4b). The molecular weights estimated from the DNA sequence and the molar absorption coefficients calculated were His-tag scFv H1a050 (27063.24 Da, ε = 45130) and His-tag scFv H2a064 (26683.87 Da, ε = 49980), respectively. It was confirmed that the His-tag anti-human MSLN scFv clone produced by CHO cells was produced into a scFv of the target molecular weight by SDS-PAGE ( Figure 4 c). In addition, in the analysis of molecular weight using MALDI-TOF-MS, it was confirmed that although the calculated values ​​were His-tag scFv H1a050 (26926.58 Da) and His-tag scFv H2a064 (26547.88 Da), respectively, there was a difference in molecular weight of about 1 amino acid residue, but peptides of the target molecular weight were obtained.

[0122] (3) DFO modification and radiolabeling of His-tag scFv

[0123] DFO modified His-tag scFv 89 The specific activity after Zr labeling is H1a050 (0.496MBq / μg) and H2a064 (0.365MBq / μg). 89 The in vitro stabilities of Zr-labeled scFv in 50% human plasma after incubation at 37°C for 6 hours were H1a050 (98.3%) and H2a064 (100%). 89 The effect of Zr labeling on the binding ability of scFv is shown below: 89 The equilibrium dissociation constants (KD) of each scFv after Zr labeling are as follows: H1a050: (4.68E-09, 3.38E-09, 4.62E-08), H2a064 (5.96E-08, 1.14E-07, 7.82E-08), all in mol / L.

[0124] (4) Small Animal PET·CT Imaging I

[0125] Will give medication 89 The PET·CT imaging images of Zr-labeled DFO-His-tag-scFv 3 hours later are shown in Figure 6 The anti-human MSLN-scFv-His tag clones showed that both H1a050 and H2a064 were MSLN-strongly expressing cells, namely NCI-N87, which specifically aggregated to transplanted tumors. In particular, clone H1a050 showed higher aggregation than H2a064. 89The in vivo distribution of Z-labeled anti-human MSLN-scFv-His tag clone after PET / CT imaging is shown in Figure 7 . Can be confirmed 89 Z marker H1a050 was significantly more highly accumulated in tumors from NCI-N87 than in tumors from PANC-1. However, strong effects on kidney and liver were also confirmed. 89 Aggregation of Zr(DFO-His-tag-scFv).

[0126] Figure 8 The results of the PET images of the NCI-N87 and PANC-1 tumors in the blood showed that 89 The graph of the time-dependent change of Z radioactivity. From these data, it can be confirmed that the antibody is taken up into the blood and tumors expressing MSLN soon after administration of the labeled antibody. These data show that in contrast to the rapid excretion in the blood, the tumor 89 The Zr radioactivity gradually decreased, but remained at a high concentration even after 3 hours from administration.

[0127] exist Figure 8 The lower part shows the expression of MSLN weak expression and MSLN strong expression in tumors 89 Graph showing the accumulation of Zr-labeled scFv over time. Radiolabeled anti-human MSLN-scFv accumulates more in NCI-N87 tumors than in PANC-1 tumors. In addition, the radioactivity of scFv clone H1a050 in NCI-N87 tumors is gradually reduced compared to H2a064.

[0128] (5) Small Animal PET·CT Imaging II

[0129] (i) Methods: 89Zr-DFO-1125-IgG 1MBq / 15μg (n=3) was administered via the tail vein of each mouse bearing a tumor caused by the NCI-N87 cell line, and images were taken using a PET system (Clairvivo PET, Shimadzu, Kyoto, JAPAN) immediately after administration and 24, 48, 72, 96, and 144 hours later. The collected data were reconstructed using the 3D-DRAMA method. In addition, after the PET scan, CT data were acquired using a CT scanner (Eminence Stargate, Shimadzu). The PET and CT images converted into DICOM format were fused using PMOD software version 3.3 (PMOD Technologies Ltd., Zurich, Switzerland), and the three-dimensional volume region of interest (VOI) in the heart was depicted as the tumor and blood pool on the PET / CT image, and the average percentage of the injected dose per 1 gram of tissue (%ID / g) was determined.

[0130] (ii) Result: Drug administration 89 The PET·CT images of Zr-labeled DFO-1125-IgG are shown in Fig.10 a. It shows the specific accumulation of MSLN-strongly expressing cells, namely NCI-N87, into transplanted tumors. Fig.10 b represents the blood and NCI-N87 tumors obtained from PET images 89 Graph showing the temporal change of Zr radioactivity. From these data, it was confirmed that after administration of the labeled antibody, the antibody was cleared from the blood over time, while the antibody was taken up into the tumor expressing MSLN and its accumulation continued.

[0131] (6) Small Animal PET Imaging

[0132] (i) Method: 89Zr-DFO-trastuzumab 7.2 MBq / 100 μg was administered via the tail vein of each mouse bearing a tumor derived from the HER2-highly expressing SKOV3 cell line. Immediately after administration and 24, 48, 72, and 120 hours after administration, or 64 Cu-labeled NOTA-trastuzumab was imaged using a PET system (Clairvivo PET, Shimadzu, Kyoto, JAPAN) immediately after administration and 24 and 48 hours later. The collected data were reconstructed using the 3D-DRAMA method.

[0133] (ii) Results: Showing the administration of 89 Zr-labeled DFO-trastuzumab and 64 PET images after Cu-labeled NOTA-trastuzumab ( Fig.11 ). Both of them began to show high aggregation of HER2-strongly expressing cells, namely SKOV3, to transplanted tumors 24 hours after administration, and the aggregation increased with the passage of time. 64 Cu was also observed to accumulate significantly in the liver. 89 No accumulation of Zr in the liver was observed. Sequence Listing <110> Okayama University <120> DNA, peptides, anti-mesothelin antibodies, tumor imaging agents and complexes <130> P18-036WO <150> JP 2017-210508 <151> 2017-10-31 <160> 12 <170> PatentIn 3.5 <210> 1 <211> 861 <212> DNA <213> Artificial <220> <223> MSLNH1a050LHscFv <220> <221> CDS <222> (16)..(855) <400> 1 aagcttgccg ccacc atg gcc agc ttc cct ctc ctc ctc acc ctc ctc act 51 Met Ala Ser Phe Pro Leu Leu Leu Thr Leu Leu Thr 1 5 10 cac tgt gca gga tcc tgg gcc tcc tat gag ctg act cag cca ccc tcg 99 His Cys Ala Gly Ser Trp Ala Ser Tyr Glu Leu Thr Gln Pro Pro Ser 15 20 25 gtg tca gtg tcc cca gga cag acg gcc agg atc acc tgc tct gga aat 147 Val Ser Val Ser Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asn 30 35 40 gca ttg cca aag caa tat gct tat tgg tac cag cag aag cca ggc cag 195 Ala Leu Pro Lys Gln Tyr Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln 45 50 55 60 gcc cct gtg ttg atg ata tat aaa gac agt gag agg ccc tca ggg atc 243 Ala Pro Val Leu Met Ile Tyr Lys Asp Ser Glu Arg Pro Ser Gly Ile 65 70 75 cct gag cga ttc tct ggc tcc agc tca ggg aca aca gtc acg ttg acc 291 Pro Glu Arg Phe Ser Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr 80 85 90 atc agt gga gtc cag gca gaa gac gag gct gac tat tac tgt caa tca 339 Ile Ser Gly Val Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser 95 100 105 gca gac agc agt cat act tat aag gtg ttc ggc gga ggg acc aag ctg 387 Ala Asp Ser Ser His Thr Tyr Lys Val Phe Gly Gly Gly Thr Lys Leu 110 115 120 acc gtc cta ggc ggt ggc gga tca ggt ggc ggt gga agt ggc ggt ggt 435 Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 125 130 135 140 ggg tct gga ggt ggg ggc agt gag gtg cag ctg gtg cag tct ggg gga 483 Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Gly 145 150 155 ggc ttg gta cag cct gac agg tcc ctg aga ctc tcc tgt gca gcc tct 531 Gly Leu Val Gln Pro Asp Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser 160 165 170 gga ttc acc ttt gat gat tat gcc atg cac tgg gtc cgg caa gct cca 579 Gly Phe Thr Phe Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro 175 180 185 ggg aag ggc ctg gag tgg gtc tca ggt att agt tgg aat agt ggt agc 627 Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Ser Trp Asn Ser Gly Ser 190 195 200 ata ggc tat gcg gac tct gtg aag ggc cga ttc acc atc tcc aga gac 675 Ile Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 205 210 215 220 aac gcc aag aac tcc ctg tat ctg caa atg aac agt ctg aga gct gag 723 Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 225 230 235 gac acg gcc ttg tat tac tgt gca aaa gat ctg ggg tcc tac tat ggt 771 Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Asp Leu Gly Ser Tyr Tyr Gly 240 245 250 tcg ggg agt ggt atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc 819 Ser Gly Ser Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val 255 260 265 tcg agc ggg ggt cat cat cac cac cac cac tag tga gaattc 861 Ser Ser Gly Gly His His His His His His 270 275 <210> 2 <211> 278 <212> PRT <213> Artificial <220> <223> MSLNH1a050LHscFv <400> 2 Met Ala Ser Phe Pro Leu Leu Leu Thr Leu Leu Thr His Cys Ala Gly 1 5 10 15 Ser Trp Ala Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser 20 25 30 Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asn Ala Leu Pro Lys 35 40 45 Gln Tyr Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu 50 55 60 Met Ile Tyr Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe 65 70 75 80 Ser Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val 85 90 95 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser 100 105 110 His Thr Tyr Lys Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln 145 150 155 160 Pro Asp Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 165 170 175 Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 180 185 190 Glu Trp Val Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala 195 200 205 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 210 215 220 Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu 225 230 235 240 Tyr Tyr Cys Ala Lys Asp Leu Gly Ser Tyr Tyr Gly Ser Gly Ser Gly 245 250 255 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly 260 265 270 His His His His His His 275 <210> 3 <211> 852 <212> DNA <213> Artificial <220> <223> MSLNH2a021LHscFv <220> <221> CDS <222> (16)..(846) <400> 3 aagcttgccg ccacc atg gcc agc ttc cct ctc ctc ctc acc ctc ctc act 51 Met Ala Ser Phe Pro Leu Leu Leu Thr Leu Leu Thr 1 5 10 cac tgt gca gga tcc tgg gcc tct tct gag ctg act cag gac cct gct 99 His Cys Ala Gly Ser Trp Ala Ser Ser Glu Leu Thr Gln Asp Pro Ala 15 20 25 gtg tct gtg gcc ttg gga cag aca gtc agg atc aca tgc caa gga gac 147 Val Ser Val Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp 30 35 40 agc ctc aga agc tat tat gca agc tgg tac cag cag aag cca gga cag 195 Ser Leu Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln 45 50 55 60 gcc cct gta ctt gtc atc tat ggt aaa aac aac cgg ccc tca ggg atc 243 Ala Pro Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile 65 70 75 cca gac cga ttc tct ggc tcc agc tca gga aac aca gct tcc ttg acc 291 Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr 80 85 90 atc act ggg gct cag gcg gaa gat gag gct gac tat tac tgt cag gcg 339 Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ala 95 100 105 tgg gac agc agt act gca gtg gta ttc ggc gga ggg acc aag ctg acc 387 Trp Asp Ser Ser Thr Ala Val Val Phe Gly Gly Gly Thr Lys Leu Thr 110 115 120 gtc cta ggc ggt ggc gga tca ggt ggc ggt gga agt ggc ggt ggt ggg 435 Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 125 130 135 140 tct gga ggt ggg ggc agt cag gtg cag ctg gtg cag tct ggg gga ggc 483 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Gly Gly 145 150 155 ttg gta cag cct ggc agg tcc ctg aga ctc tca tgt gca gct tct gga 531 Leu Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 160 165 170 ttc acc ttt gat gat tat gcc atg cac tgg gtc cgg caa gct cca ggg 579 Phe Thr Phe Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly 175 180 185 aag ggc ctg gag tgg gtc tca ggt att agt tgg aat agt ggt atc ata 627 Lys Gly Leu Glu Trp Val Ser Gly Ile Ser Trp Asn Ser Gly Ile Ile 190 195 200 ggc tat gcg gac tct gtg aag ggc cga ttc acc atc tcc aga gac aac 675 Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 205 210 215 220 gcc aag aac tcc ctg tat ctg caa atg aac agt ctg aga gct gag gac 723 Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 225 230 235 acg gcc ttg tat tac tgt gca aaa gat ctg agc agc agc tgg tac ggt 771 Thr Ala Leu Tyr Tyr Cys Ala Lys Asp Leu Ser Ser Ser Trp Tyr Gly 240 245 250 gct ttt gat atc tgg ggc caa ggg aca atg gtc acc gtc tcg agc ggg 819 Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly 255 260 265 ggt cat cat cac cac cac cac tag tga gaattc 852 Gly His His His His His His 270 275 <210> 4 <211> 275 <212> PRT <213> Artificial <220> <223> MSLNH2a021LHscFv <400> 4 Met Ala Ser Phe Pro Leu Leu Leu Thr Leu Leu Thr His Cys Ala Gly 1 5 10 15 Ser Trp Ala Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala 20 25 30 Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser 35 40 45 Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu 50 55 60 Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe 65 70 75 80 Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala 85 90 95 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser 100 105 110 Thr Ala Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro 145 150 155 160 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp 165 170 175 Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 180 185 190 Trp Val Ser Gly Ile Ser Trp Asn Ser Gly Ile Ile Gly Tyr Ala Asp 195 200 205 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 210 215 220 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr 225 230 235 240 Tyr Cys Ala Lys Asp Leu Ser Ser Ser Trp Tyr Gly Ala Phe Asp Ile 245 250 255 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly His His His 260 265 270 His His His 275 <210> 5 <211> 855 <212> DNA <213> Artificial <220> <223> MSLNH2a064LHscFv <220> <221> CDS <222> (16)..(849) <400> 5 aagcttgccg ccacc atg gcc agc ttc cct ctc ctc ctc acc ctc ctc act 51 Met Ala Ser Phe Pro Leu Leu Leu Thr Leu Leu Thr 1 5 10 cac tgt gca gga tcc tgg gcc tcc tat gag ctg act cag cca ccc tcg 99 His Cys Ala Gly Ser Trp Ala Ser Tyr Glu Leu Thr Gln Pro Pro Ser 15 20 25 gtg tca gtg tcc cca gga cag acg gcc agg atc acc tgc tct gga gat 147 Val Ser Val Ser Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp 30 35 40 gca ttg cca aag cac tat gct tct tgg tac cag cag aag cca ggc cag 195 Ala Leu Pro Lys His Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln 45 50 55 60 gcc cct gtc ctg ctg ata tat aaa gac act gag agg ccc tca ggg atc 243 Ala Pro Val Leu Leu Ile Tyr Lys Asp Thr Glu Arg Pro Ser Gly Ile 65 70 75 cct gag cga ttc tct ggc tcc agc tca ggg aca aca gtc acg ttg acc 291 Pro Glu Arg Phe Ser Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr 80 85 90 atc agt gga gtc cag gca gaa gac gag gct gac tat tac tgt caa tca 339 Ile Ser Gly Val Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser 95 100 105 gcc gac agc agt gtt act gtg gta ttc ggc gga ggg acc aag ctg acc 387 Ala Asp Ser Ser Val Thr Val Val Phe Gly Gly Gly Thr Lys Leu Thr 110 115 120 gtc cta ggc ggt ggc gga tca ggt ggc ggt gga agt ggc ggt ggt ggg 435 Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 125 130 135 140 tct gga ggt ggg ggc agt cag gtg cag ctg gtg caa tct ggg gga ggc 483 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Gly Gly 145 150 155 ttg gta cag cct ggc agg tcc ctg aga ctc tcc tgt gca gcc tct gga 531 Leu Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 160 165 170 ttc acc ttt gat gat tat gcc atg cac tgg gtc cgg caa gct cca ggg 579 Phe Thr Phe Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly 175 180 185 aag ggc ctg gag tgg gtc tca ggt att agt tgg aat agt ggt agc ata 627 Lys Gly Leu Glu Trp Val Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile 190 195 200 ggc tat gcg gac tct gtg aag ggc cga ttc acc atc tcc aga gac aac 675 Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 205 210 215 220 gcc aag aac tcc ctg tat ctg caa atg aac agt ctg aga gct gag gac 723 Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 225 230 235 acg gcc ttg tat tac tgt gca aaa gat atc ggt agc agt ggc tgg tac 771 Thr Ala Leu Tyr Tyr Cys Ala Lys Asp Ile Gly Ser Ser Gly Trp Tyr 240 245 250 ggg gct ttt gat atc tgg ggc caa ggg acc acg gtc acc gtc tcg agc 819 Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 255 260 265 ggg ggt cat cat cac cac cac cac tag tga gaattc 855 Gly Gly His His His His His His 270 275 <210> 6 <211> 276 <212> PRT <213> Artificial <220> <223> MSLNH2a064LHscFv <400> 6 Met Ala Ser Phe Pro Leu Leu Leu Thr Leu Leu Thr His Cys Ala Gly 1 5 10 15 Ser Trp Ala Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser 20 25 30 Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys 35 40 45 His Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu 50 55 60 Leu Ile Tyr Lys Asp Thr Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe 65 70 75 80 Ser Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val 85 90 95 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser 100 105 110 Val Thr Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro 145 150 155 160 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp 165 170 175 Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 180 185 190 Trp Val Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp 195 200 205 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 210 215 220 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr 225 230 235 240 Tyr Cys Ala Lys Asp Ile Gly Ser Ser Gly Trp Tyr Gly Ala Phe Asp 245 250 255 Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly His His 260 265 270 His His His His 275 <210> 7 <211> 849 <212> DNA <213> 人工 <220> <223> MSLNH2a006LHscFv <220> <221> CDS <222> (16)..(843) <400> 7 aagcttgccg ccacc atg gcc agc ttc cct ctc ctc ctc acc ctc ctc act 51 Met Ala Ser Phe Pro Leu Leu Leu Thr Leu Leu Thr 1 5 10 cac tgt gca gga tcc tgg gcc tcc tat gag ctg aca cag cca ccc tcg 99 His Cys Ala Gly Ser Trp Ala Ser Tyr Glu Leu Thr Gln Pro Pro Ser 15 20 25 gtg tca gtg tcc cca gga cag acg gcc agg atc acc tgc tct gga gat 147 Val Ser Val Ser Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp 30 35 40 gca ttg cca aag caa tat gct tat tgg tac cag cag aag cca ggc cag 195 Ala Leu Pro Lys Gln Tyr Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln 45 50 55 60 gcc cct gtg ctg gtg ata tat aaa gac agt gag agg ccc tca ggg atc 243 Ala Pro Val Leu Val Ile Tyr Lys Asp Ser Glu Arg Pro Ser Gly Ile 65 70 75 cct gag cga ttc tct ggc tcc agc tca ggg aca aca gtc acg ttg acc 291 Pro Glu Arg Phe Ser Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr 80 85 90 atc agt gga gtc cag gca gaa gac gag gct gac tat tac tgt caa tca 339 Ile Ser Gly Val Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser 95 100 105 gca gac agc agt ggt act gtg gta ttc ggc gga ggg acc aag ctg acc 387 Ala Asp Ser Ser Gly Thr Val Val Phe Gly Gly Gly Thr Lys Leu Thr 110 115 120 gtc ctg ggc ggt ggc gga tca ggt ggc ggt gga agt ggc ggt ggt ggg 435 Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 125 130 135 140 tct gga ggt ggg ggc agt gag gtg cag ctg gtg cag tct ggg gga ggc 483 Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Gly Gly 145 150 155 ttg gta cag cct ggc agg tcc ctg aga ctc tcc tgt gca gcc tct gga 531 Leu Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 160 165 170 ttc acc ttt gat gat tat gcc atg cac tgg gtc cgg caa gct cca ggg 579 Phe Thr Phe Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly 175 180 185 aag ggc ctg gag tgg gtc tca ggt att agt tgg aat agt ggt agc ata 627 Lys Gly Leu Glu Trp Val Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile 190 195 200 gtc tat gcg gac tct gtg aag ggc cga ttc acc atc tcc aga gac aac 675 Val Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 205 210 215 220 gcc aag aac tcc ctg tat ctg caa atg aac agt ctg aga gct gag gac 723 Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 225 230 235 acg gcc ttg tat tac tgt gca aaa gac cgc agc agc tgg tac ggg ggg 771 Thr Ala Leu Tyr Tyr Cys Ala Lys Asp Arg Ser Ser Trp Tyr Gly Gly 240 245 250 ttt gac tac tgg ggc cag gga acc ctg gtc acc gtc tcg agc ggg ggt 819 Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 255 260 265 cat cat cac cac cac cac tag tga gaattc 849 His His His His His His 270 <210> 8 <211> 274 <212> PRT <213> Artificial <220> <223> MSLNH2a006LHscFv <400> 8 Met Ala Ser Phe Pro Leu Leu Leu Thr Leu Leu Thr His Cys Ala Gly 1 5 10 15 Ser Trp Ala Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser 20 25 30 Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys 35 40 45 Gln Tyr Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu 50 55 60 Val Ile Tyr Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe 65 70 75 80 Ser Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val 85 90 95 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser 100 105 110 Gly Thr Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro 145 150 155 160 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp 165 170 175 Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 180 185 190 Trp Val Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Val Tyr Ala Asp 195 200 205 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 210 215 220 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr 225 230 235 240 Tyr Cys Ala Lys Asp Arg Ser Ser Trp Tyr Gly Gly Phe Asp Tyr Trp 245 250 255 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly His His His His 260 265 270 His His <210> 9 <211> 864 <212> DNA <213> artificial <220> <223> MSLNH2a059LHscFv <220> <221> CDS <222> (16)..(858) <400> 9 aagcttgccg ccacc atg gcc agc ttc cct ctc ctc ctc acc ctc ctc act 51 Met Ala Ser Phe Pro Leu Leu Leu Thr Leu Leu Thr 1 5 10 cac tgt gca gga tcc tgg gcc tcc tat gag ctg atg cag cca ccc tcg 99 His Cys Ala Gly Ser Trp Ala Ser Tyr Glu Leu Met Gln Pro Pro Ser 15 20 25 gtg tca gtg tcc cca gga cag acg gcc agg atc acc tgc tct gga gat 147 Val Ser Val Ser Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp 30 35 40 gca ttg cca aag caa tat gct tat tgg tac cag cag aag cca ggc cag 195 Ala Leu Pro Lys Gln Tyr Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln 45 50 55 60 gcc cct gtg ctg gtg ata tat aaa gac agt gag agg ccc tca ggg atc 243 Ala Pro Val Leu Val Ile Tyr Lys Asp Ser Glu Arg Pro Ser Gly Ile 65 70 75 cct gag cga ttc tct ggc tcc agc tca ggg aca aca gtc acg ttg acc 291 Pro Glu Arg Phe Ser Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr 80 85 90 atc agt gga gtc cag gca gaa gac gag gct gac tat tac tgt caa tca 339 Ile Ser Gly Val Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser 95 100 105 gca gac agc agt ggt act tat aga gat gtg gta ttc ggc gga ggg acc 387 Ala Asp Ser Ser Gly Thr Tyr Arg Asp Val Val Phe Gly Gly Gly Thr 110 115 120 aag ctg acc gtc cta ggc ggt ggc gga tca ggt ggc ggt gga agt ggc 435 Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 125 130 135 140 ggt ggt ggg tct gga ggt ggg ggc agt gag gtg cag ctg gtg cag tct 483 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Gln Ser 145 150 155 ggg gga ggc ttg gta cag cct ggc agg tcc ctg aga ctc tcc tgt gca 531 Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala 160 165 170 gcc tct gga ttc acc ttt gat gat tat gcc atg cac tgg gtc cgg caa 579 Ala Ser Gly Phe Thr Phe Asp Asp Tyr Ala Met His Trp Val Arg Gln 175 180 185 gct cca ggg aag ggc ctg gag tgg gtc tca ggt att agt tgg aat agt 627 Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Ser Trp Asn Ser 190 195 200 ggt agc ata ggc tat gcg gac tct gtg aag ggc cga ttc acc atc tcc 675 Gly Ser Ile Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 205 210 215 220 aga gac aac gcc aag aac tcc ctg tat ctg caa atg aac agt ctg aga 723 Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg 225 230 235 gct gag gac acg gcc ttg tat tac tgt gca aaa ggt ctg ggt agt agt 771 Ala Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Gly Leu Gly Ser Ser 240 245 250 ggt tat tac gac gct ttt gat atc tgg ggc caa ggg acc acg gtc acc 819 Gly Tyr Tyr Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Thr Val Thr 255 260 265 gtc tcg agc ggg ggt cat cat cac cac cac cac tag tga gaattc 864 Val Ser Ser Gly Gly His His His His His His 270 275 <210> 10 <211> 279 <212> PRT <213> Artificial <220> <223> MSLNH2a059LHscFv <400> 10 Met Ala Ser Phe Pro Leu Leu Leu Thr Leu Leu Thr His Cys Ala Gly 1 5 10 15 Ser Trp Ala Ser Tyr Glu Leu Met Gln Pro Pro Ser Val Ser Val Ser 20 25 30 Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys 35 40 45 Gln Tyr Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu 50 55 60 Val Ile Tyr Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe 65 70 75 80 Ser Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val 85 90 95 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser 100 105 110 Gly Thr Tyr Arg Asp Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val 115 120 125 Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu 145 150 155 160 Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 165 170 175 Thr Phe Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys 180 185 190 Gly Leu Glu Trp Val Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly 195 200 205 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 210 215 220 Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 225 230 235 240 Ala Leu Tyr Tyr Cys Ala Lys Gly Leu Gly Ser Ser Gly Tyr Tyr Asp 245 250 255 Ala Phe Asp Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly 260 265 270 Gly His His His His His His 275 <210> 11 <211> 858 <212> DNA <213> Artificial <220> <223> MSLNH2b011LHscFv <220> <221> CDS <222> (16)..(852) <400> 11 aagcttgccg ccacc atg gcc agc ttc cct ctc ctc ctc acc ctc ctc act 51 Met Ala Ser Phe Pro Leu Leu Leu Thr Leu Leu Thr 1 5 10 cac tgt gca gga tcc tgg gcc cag gca ggg ctg act cag cca ccc tcg 99 His Cys Ala Gly Ser Trp Ala Gln Ala Gly Leu Thr Gln Pro Pro Ser 15 20 25 gtg tca gtg tcc cca gga cag acg gcc agg atc acc tgc tct gga gat 147 Val Ser Val Ser Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp 30 35 40 gca ttg cca aag caa ttt act tat tgg tac cag cag aag cca ggc cag 195 Ala Leu Pro Lys Gln Phe Thr Tyr Trp Tyr Gln Gln Lys Pro Gly Gln 45 50 55 60 gcc cct gtg ttg gtg ata tat caa gac cgt gag agg ccc tca ggg atc 243 Ala Pro Val Leu Val Ile Tyr Gln Asp Arg Glu Arg Pro Ser Gly Ile 65 70 75 cct gag cga ttc tct ggc tcc agc tca ggg aca aaa gtc acg ttg acc 291 Pro Glu Arg Phe Ser Gly Ser Ser Ser Gly Thr Lys Val Thr Leu Thr 80 85 90 atc agt gga gtc cag gca gaa gac gag gct gac tat tat tgt caa tca 339 Ile Ser Gly Val Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser 95 100 105 gca gac agc agt ggt act tat cgg gtg ttc ggc gga ggg acc aag ctg 387 Ala Asp Ser Ser Gly Thr Tyr Arg Val Phe Gly Gly Gly Thr Lys Leu 110 115 120 acc gtc cta ggc ggt ggc gga tca ggt ggc ggt gga agt ggc ggt ggt 435 Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 125 130 135 140 ggg tct gga ggt ggg ggc agt gag gtg cag ctg gtg gag tct ggg gga 483 Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly 145 150 155 ggc ttg gta cag cct ggc agg tcc ctg aga ctc tcc tgt gca gcc tct 531 Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser 160 165 170 gga ttc acc ttt gat gat tat gcc atg cac tgg gtc cgg caa gct cca 579 Gly Phe Thr Phe Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro 175 180 185 ggg aag ggc ctg gag tgg gtc tca ggt att agt tgg aat agt ggt agc 627 Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Ser Trp Asn Ser Gly Ser 190 195 200 ata ggc tat gcg gac tct gtg aag ggc cga ttc acc atc tcc aga gac 675 Ile Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 205 210 215 220 aac gcc aag aac tcc ctg tat ctg caa atg aac agt ctg aga gct gag 723 Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 225 230 235 gac acg gcc ttg tat tac tgt gca aaa gat gcg ggt agt agt ggt tat 771 Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Asp Ala Gly Ser Ser Gly Tyr 240 245 250 tgg agc tac ttt gac tac tgg ggc cag ggc acc ctg gtc acc gtc tcg 819 Trp Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 255 260 265 agc ggg ggt cat cat cac cac cac cac tag tga gaattc 858 Ser Gly Gly His His His His His His 270 275 <210> 12 <211> 277 <212> PRT <213> Artificial <220> <223> MSLNH2b011LHscFv <400> 12 Met Ala Ser Phe Pro Leu Leu Leu Thr Leu Leu Thr His Cys Ala Gly 1 5 10 15 Ser Trp Ala Gln Ala Gly Leu Thr Gln Pro Pro Ser Val Ser Val Ser 20 25 30 Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys 35 40 45 Gln Phe Thr Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu 50 55 60 Val Ile Tyr Gln Asp Arg Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe 65 70 75 80 Ser Gly Ser Ser Ser Gly Thr Lys Val Thr Leu Thr Ile Ser Gly Val 85 90 95 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser 100 105 110 Gly Thr Tyr Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 145 150 155 160 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 165 170 175 Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 180 185 190 Glu Trp Val Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala 195 200 205 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 210 215 220 Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu 225 230 235 240 Tyr Tyr Cys Ala Lys Asp Ala Gly Ser Ser Gly Tyr Trp Ser Tyr Phe 245 250 255 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly His 260 265 270 His His His His His 275

Claims

1. A DNA, which is any one of the following (a) to (c), (a) a DNA having a base sequence as shown in SEQ ID NO: 1, or a DNA having a base sequence as shown in positions 16 to 831 of SEQ ID NO: 1; (b) a DNA encoding a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2, or a polypeptide having an amino acid sequence as shown in positions 1 to 272 of SEQ ID NO: 2; (c) The complementary strand of the DNA described in (a) or (b).

2. A polypeptide whose amino acid sequence is as shown in SEQ ID NO: 2, or as shown in positions 1 to 272 of SEQ ID NO:

2.

3. Use of the polypeptide according to claim 2 for preparing a tumor imaging agent.

4. A tumor imaging agent, characterized in that: Comprising the polypeptide according to claim 2.

5. The tumor imaging agent according to claim 4, wherein The tumor imaging agent also comprises 89 Zr.

6. The tumor imaging agent according to claim 4 or 5, wherein: The tumor imaging agent is used for imaging tumors expressing mesothelin.

7. A composite, characterized in that The complex is obtained by linking the polypeptide according to claim 2 and an antitumor substance.

Images