HK1139163A - Anti-muc17 antibody - Google Patents
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Abstract
Disclosed is an antibody capable of binding to Mucin-17 (Muc17). Preferably, the antibody can bind to a peptide depicted in SEQ ID NO:3 but cannot bind to peptides depicted in SEQ ID NO:4 and SEQ ID NO:5. Also disclosed is an anti-cancer agent, preferably an anti-cancer agent against pancreatic cancer, which comprises the antibody. Further disclosed is a method for the diagnosis of cancer, which is characterized by using the antibody, preferably the antibody which cannot bind to secreted Muc17.
Description
Technical Field
This application claims priority based on Japanese patent application 2007-176319 (application 7/4/2007), the contents of which are incorporated herein by reference.
The present invention relates to an anticancer agent and a method for diagnosing cancer.
Background
Muc17 is a novel mucin belonging to the family of membrane-type mucins, and expression in normal tissues is limited to the small and large intestine [ non-patent document 1 ]. The majority of the extracellular domain, comprising a consensus tandem repeat of a 59mer rich in serine, threonine, proline, is thought to be involved in cellular defense by the mucin structure which is modified by the sugar chain. Furthermore, it is predicted that secreted Muc17 exists by cleavage since it has an SEA domain outside the cell [ non-patent document 1 ]. In addition, it has been reported that secreted Muc17 without a transmembrane domain exists in the form of a splice variant
[ non-patent document 2 ].
As for the expression in cancer, it was confirmed that the Muc17 gene was expressed at the RNA level in pancreatic cancer cell line AsPc-1, colorectal cancer cell lines NCI-H498, Caco-2 and LS 174T. In addition, a polyclonal antibody against Pro-Thr-Thr-Ala-Glu-Gly-Thr-Ser-Met-Pro-Thr-Ser-Thr-Pro-Ser-Glu (SEQ ID NO: 38) corresponding to the tandem repeat sequence was prepared, and expression of Muc17 at the protein level was confirmed by immunostaining of clinical pancreatic cancer tissues [ non-patent document 2 ].
The references cited in this specification are as follows. The contents of these documents are incorporated herein by reference in their entirety. None of these documents is admitted by the applicant to constitute prior art to the present invention.
Non-patent document 1: j.r.gum, jr., s.c.crawley, j.w.hicks, d.e.szymkowski, and y.s.kim, MUC17, a novel membrane-treated mucin, biochem, biophys.res.commu., 291(2002)466-75.
Non-patent document 2: n.monaux, w.m.junker, a.p.singh, a.m.jones, and s.k.batar, charaterization of human mucin MUC 17. comparative sequencing and organization j.biol.chem., 281(2006)23676-85.
Disclosure of Invention
The purpose of the present invention is to provide a novel antibody, an anticancer agent using the same, and a method for diagnosing cancer.
The present inventors obtained a monoclonal antibody against the extracellular membrane region of Muc17, and found that the antibody exhibits ADCC activity and has an antitumor effect by conjugation to a toxin.
The present invention provides an antibody that binds to Mucin17(Muc 17). Preferably, the antibodies of the invention do not bind to secreted Muc 17. Furthermore, it is preferable that the antibody of the present invention binds to the peptide of SEQ ID NO. 3 (4176-4390) and does not bind to the peptides of SEQ ID NO. 4 (4244-4390) and SEQ ID NO. 5 (4115-4243).
In a preferred embodiment, the antibody of the present invention has ADCC activity. Preferably, the antibody of the present invention is a chimeric antibody or a humanized antibody. It is further preferred that the antibody of the invention is a low fucosylated antibody.
In other preferred embodiments, the antibody of the present invention recognizes the same epitope as that recognized by an antibody having a heavy chain variable region having the amino acid sequence shown in seq id No. 23 and a light chain variable region having the amino acid sequence shown in seq id No.25 (MQ 155).
The present invention also provides an anticancer agent, preferably an anticancer agent against pancreatic cancer, containing the antibody of the present invention. The present invention also provides a method for diagnosing cancer, which comprises using the antibody of the present invention, preferably an antibody that does not bind to secreted Muc 17.
Drawings
FIG. 1 shows the amount of Muc17mRNA in normal tissues and cancer cell lines.
FIG. 2 is a graph showing the evaluation of the binding activity of the chimeric anti-Muc 17 antibody by ELISA.
FIG. 3 is a view showing the evaluation of the binding activity of a chimeric anti-Muc 17 antibody by flow cytometry.
FIG. 4 shows ADCC activity by an anti-Muc 17 antibody against a pancreatic cancer cell line in FIG. 4.
FIG. 5 shows the antitumor effect of the anti-Muc 17 antibody using Hum-ZAP.
FIG. 6 shows the epitope analysis of the anti-Muc 17 antibody in FIG. 6.
Detailed Description
Muc17
Muc17(access No. nm 001040105) is a type 1 membrane protein comprising 4,493 amino acids. The gene sequence encoding Muc17 is shown in SEQ ID NO. 1, and the amino acid sequence of Muc17 is shown in SEQ ID NO. 2. Muc17 belongs to the mucin family of membrane types, and most of the extracellular domain contains repeating units of a 59mer tandem repeat rich in serine, threonine, and proline, and is modified with sugar chains. In addition, protein cleavage was predicted from the protein having SEA domain (4182Glu-4287Asn), and it is thought that a part of the protein may exist as a secreted protein. In addition, the existence of a secretory splice variant (1Met-4241Arg is the same sequence) has been reported (non-patent document 2).
anti-Muc 17 antibodies
The anti-Muc 17 antibody of the present invention may be bound to Muc17, regardless of its origin (mouse, rat, human, etc.), type (monoclonal antibody, polyclonal antibody), shape (altered antibody, reduced molecular weight antibody, modified antibody, etc.), and the like.
The anti-Muc 17 antibody used in the invention preferably specifically binds to Muc 17. In addition, the anti-Muc 17 antibody used in the present invention is preferably a monoclonal antibody.
The anti-Muc 17 antibodies of the invention preferably recognize and bind to the extracellular domain of the Muc17 protein. The extracellular domain of the Muc17 protein corresponds to amino acid sequence numbers 1-4389 in the amino acid sequence of SEQ ID NO. 1. More preferably, the Muc17 antibody of the present invention is an antibody that binds to the extracellular domain of the Muc17 protein and does not bind to secreted Muc 17. The secreted Muc17 corresponds to 1 st to 4241 th amino acid residues in the amino acid sequence of SEQ ID NO. 2.
In a preferred embodiment of the present invention, the anti-Muc 17 antibody is an antibody that binds to the peptide comprising the amino acid sequence of SEQ ID NO. 3 (4176-4390), does not bind to the peptide comprising the amino acid sequence of SEQ ID NO. 4 (4244-4390) and does not bind to the peptide comprising the amino acid sequence of SEQ ID NO. 5 (4115-4243).
Whether an antibody binds to a peptide of interest can be determined by well-known methods (antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring harbor Laboratory, 1988). For example, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), fluoroimmunoassay, or the like can be used. More specifically, whether or not the antibody binds to the target peptide can be confirmed by the method described in the following examples.
Specific examples of the anti-Muc 17 antibody of the present invention include the following antibodies.
(1) An antibody (MQ128) comprising a heavy chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO. 6, CDR2 comprising the amino acid sequence set forth in SEQ ID NO. 7, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO. 8;
(2) an antibody comprising a light chain variable region having CDR1 comprising the amino acid sequence set forth in sequence No. 9, CDR2 comprising the amino acid sequence set forth in sequence No. 10, and CDR3 comprising the amino acid sequence set forth in sequence No. 11;
(3) an antibody comprising the heavy chain variable region of (1) and the light chain variable region of (2);
(4) an antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in seq id No. 19;
(5) an antibody comprising a light chain variable region comprising the amino acid sequence set forth in seq id No. 21;
(6) an antibody comprising the heavy chain variable region of (4) and the light chain variable region of (5);
(7) an antibody (MQ155) comprising a heavy chain variable region having CDR1 comprising the amino acid sequence set forth in SEQ ID NO. 12, CDR2 comprising the amino acid sequence set forth in SEQ ID NO. 13, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO. 14;
(8) an antibody comprising a light chain variable region having CDR1 comprising the amino acid sequence set forth in sequence No. 15, CDR2 comprising the amino acid sequence set forth in sequence No. 16, and CDR3 comprising the amino acid sequence set forth in sequence No. 17;
(9) an antibody comprising the heavy chain variable region of (7) and the light chain variable region of (8);
(10) an antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in seq id No. 23;
(11) an antibody comprising a light chain variable region comprising the amino acid sequence set forth in seq id No. 25;
(12) an antibody comprising the heavy chain variable region of (10) and the light chain variable region of (11);
(13) an antibody that recognizes an epitope identical to an epitope recognized by the antibody of any one of (1) to (12).
The test antibody shares an epitope with a certain antibody, and can be confirmed by competition between the two antibodies for the same epitope. Competition between antibodies can be detected by cross-blocking assay (cross-blocking assay) or the like. For example, the competitive ELISA assay is preferably a cross-blocking assay. Specifically, in the cross-blocking assay, the anti-Muc 17 antibody of the present invention is added after pre-incubation of Muc17 protein coated on wells of a microtiter plate in the presence or absence of a candidate competitor antibody. The amount of anti-Muc 17 antibody of the invention that binds to Muc17 protein in the well is indirectly related to the binding energy of the candidate competitor antibody (test antibody) that competes for binding to the same epitope. That is, the greater the affinity of the test antibody for the same epitope, the lower the amount of the anti-Muc 17 antibody of the present invention bound to the Muc17 protein-coated well, and the higher the amount of the test antibody bound to the Muc17 protein-coated well
The amount of antibody bound to the wells can be readily determined by pre-labeling the antibody. For example, biotin-labeled antibodies can be assayed by using an avidin peroxidase conjugate and an appropriate matrix. Cross-blocking assays using enzymatic labels such as peroxidase are particularly referred to as competitive ELISA assays. The antibody may be labeled with other labels that can be detected or measured. Specifically, a radioactive label, a fluorescent label, or the like is known.
Furthermore, when the test antibody has a constant region derived from a species different from that of the anti-Muc 17 antibody of the present invention, the amount of the antibody bound to the well can also be measured by a labeled antibody recognizing the constant region of the antibody. Alternatively, if the antibodies are from the same species but the types are different, the amount of antibody bound to the well can be measured by recognizing the antibodies of the respective types.
A candidate competing antibody is an antibody that binds to substantially the same epitope as the anti-Muc 17 antibody of the invention or an antibody that competes for binding to the same epitope if the candidate antibody is capable of blocking the binding of anti-Muc 17 antibody by at least 20%, preferably at least 20-50%, more preferably at least 50%, compared to the binding activity obtained in a control assay performed in the absence of the candidate competing antibody.
In the present invention, the epitope may be any epitope, and examples thereof include a stereo epitope, a linear epitope, and the like.
Cytotoxicity
A preferable embodiment of the antibody of the present invention is an antibody having cytotoxicity. Examples of cytotoxicity in the present invention include antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and the like. In the present invention, CDC activity refers to cytotoxicity caused by the complement system. On the other hand, ADCC activity refers to an activity of imparting toxicity to a target cell by binding a Fc portion of a cell (e.g., an immune cell) having an Fc γ receptor to the Fc portion thereof via the Fc γ receptor when a specific antibody is attached to a cell surface antigen of the target cell.
In the present invention, whether an antibody has ADCC activity or CDC activity can be determined by a known method (for example, Current protocols in Immunology, Chapter7.immunologic studios in humans, Editor, John E, Coligan et al, John Wiley & Sons, Inc. (1993), etc.).
Specifically, first, effector cells, a complement solution, and target cells are prepared.
(1) Preparation of Effector cells
Spleens were excised from CBA/N mice and the like, and spleen cells were isolated in RPMI 1640 medium (Invitrogen). After washing with the same medium containing 10% fetal bovine serum (FBS, Hyclone Co., Ltd.), the cells were washedCell concentration was adjusted to 5X 106(ii) ml, from which effector cells can be formulated.
(2) Preparation of complement solution
The Complement solution can be prepared by diluting Baby Rabbit supplement (CEDARLANE) 10-fold in a medium containing 10% FBS (Invitrogen).
(3) Preparation of target cells
The cells expressing Muc17 protein were cultured in DMEM medium containing 10% FBS for 1 hour at 37 ℃ together with 0.2mCi of 51 Cr-sodium chromate (manufactured by GEHealthcare Biosciences), and the target cells were radiolabeled. As the cell expressing the Muc17 protein, a cell transformed with a gene encoding the Muc17 protein, a cancer cell (e.g., a pancreatic cancer cell, a colorectal cancer cell), or the like can be used. After radiolabeling, cells were washed 3 times with RPMI 1640 medium containing 10% FBS, and cell concentration was adjusted to 2X 105The target cells can be prepared in ml.
The ADCC activity or CDC activity can be measured by the following method. For the measurement of ADCC activity, 50. mu.l each of the target cells and the anti-Muc 17 antibody was added to a 96-well U-bottom plate (Becton, Dickinson and Company), and the mixture was reacted for 15 minutes on ice. Then, 100ul of effector cells were added and cultured in a carbon dioxide incubator for 4 hours. The final concentration of antibody was made to be 0 or 10. mu.g/ml. After the culture, 100. mu.l of the supernatant was collected, and the radioactivity was measured by a GAMMA counter (COBRAII AUTO-GAMMA, MODEL D5005, manufactured by Packard Instrument company). The cytotoxicity (%) was calculated according to the calculation formula (A-C)/(B-C). times.100 using the obtained values. A represents the radioactivity (cpm) of each sample, B represents the radioactivity (cpm) of a sample to which 1% NP-40 (manufactured by nacalai tesque) was added, and C represents the radioactivity (cpm) of a sample containing only target cells.
On the other hand, when the CDC activity was measured, 50. mu.l each of the target cells and the anti-Muc 17 antibody was added to a 96-well flat-bottom plate (Becton, Dickinson and Company), and the mixture was reacted for 15 minutes on ice. Then, 100. mu.l of complement solution was added thereto, and the mixture was incubated in a carbon dioxide incubator for 4 hours. The final concentration of antibody was 0 or 3. mu.g/ml. After the incubation, 100. mu.l of the supernatant was recovered and the radioactivity was measured by a gamma counter. Cytotoxicity can be calculated in the same manner as in ADCC activity assay.
Sugar chain-modified antibody
A preferable embodiment of the antibody of the present invention includes an antibody modified with a sugar chain. It is known that the cytotoxicity of an antibody can be enhanced by modifying the sugar chain of the antibody.
Examples of the antibody modified with a sugar chain include an antibody modified by glycosylation (e.g., WO 99/54342), an antibody lacking fucose attached to a sugar chain (e.g., WO00/61739, WO 02/31140, WO2006/067847, WO2006/067913), and an antibody having a sugar chain containing a truncated GlcNAc (e.g., WO 02/79255).
Preferred examples of the sugar chain-modified antibody of the present invention include fucose-deficient antibodies. The sugar chain bound to the antibody includes an N-glycosyl-binding sugar chain bound to the N atom of the side chain of asparagine of the antibody molecule and an O-glycosyl-binding sugar chain bound to the hydroxyl group of the side chain of serine or threonine of the antibody molecule, and in the present invention, the presence or absence of fucose is related to the N-glycosyl-binding sugar chain.
In the present invention, the term "fucose-deficient antibody" means that 20% or more, preferably 50% or more, more preferably 70% or more, and still more preferably 90% or more of the N-glycosyl-linked sugar chains of the antibody in the composition are fucose-deficient.
Fucose-deficient antibodies can be produced by methods known to those skilled in the art, and can be produced, for example, by expressing antibodies in host cells that do not have or have a low ability to attach alpha-1, 6core fucose (alpha-1, 6core fucose). Host cells having no or low fucose attachment ability are not particularly limited, and examples thereof include rat myeloma YB2/3 HL.P2.G11.1698g.20 cells (YB 2/0 cells for short) (deposited as ATCC CRL 1662), FTVIII knockout CHO cells (WO 02/31140), Lec 13 cells (WO 03/035835), and fucose transporter deficient cells (WO2006/067847, WO 2006/067913).
The sugar chain can be analyzed by a method known to those skilled in the art. For example, N-glycosidase F (Roche) or the like is allowed to act on an antibody to separate a sugar chain from the antibody. Then, after desalting by solid phase extraction using a cellulose column (Shimizu Y.et al, Carbohydrateresearch 332(2001), 381-388), concentration of the dried solid and fluorescence labeling with 2-aminopyridine were carried out (Kondo A.et al, Agricultural and BiologicalChemistry 54: 8(1990), 2169-2170). The resulting PA-modified sugar chain was subjected to reagent removal by solid phase extraction using a cellulose column, and then concentrated by centrifugation to form a purified PA-modified sugar chain. Then, reversed-phase HPLC analysis using ODS column was performed, whereby the measurement was possible. After the PA-formed sugar chains are prepared, the two-dimensional mapping may be performed by combining the reversed-phase HPLC analysis using an ODS column and the normal-phase HPLC analysis using an amine column.
Chimeric and humanized antibodies
As another preferred embodiment of the antibody of the present invention, a chimeric antibody or a humanized antibody can be mentioned. A chimeric antibody is an antibody in which regions derived from different sources are linked to each other. Generally, a chimeric antibody is composed of a V region of an antibody derived from an animal other than a human and a C region derived from a human antibody. For example, an antibody comprising the variable regions of the heavy chain and the light chain of a mouse antibody and the constant regions of the heavy chain and the light chain of a human antibody is a mouse-human xenochimeric antibody.
In contrast, a humanized antibody is composed of Complementarity Determining Regions (CDRs) of an antibody derived from an animal other than a human, Framework Regions (FRs) derived from a human antibody, and C regions derived from a human antibody. Since the humanized antibody has low antigenicity in humans, it is useful as an active ingredient of the therapeutic agent of the present invention. Humanized antibodies are also known as reshaped (reshaped) human antibodies. Specifically, humanized antibodies and the like in which CDRs of an animal other than a human, for example, a mouse antibody are grafted onto a human antibody are known. General genetic recombination methods for obtaining humanized antibodies are also known.
Specifically, as a method for grafting CDRs of a mouse antibody into human FRs, for example, overlap Extension pcr (overlap Extension pcr) is known. In overlap extension PCR, a base sequence encoding a CDR of a mouse antibody to be grafted is attached to a primer for synthesizing a FR of a human antibody. Primers were prepared for each of the 4 FRs. In general, in grafting mouse CDRs into human FRs, it is advantageous to select human FRs having high homology with mouse FRs for maintaining the CDR functions. That is, it is generally preferable to use a human FR comprising an amino acid sequence having high homology with the amino acid sequence of the FR adjacent to the mouse CDR to be grafted.
The joined nucleotide sequences are designed to be connected to each other in frame. Human FRs were synthesized separately using each primer. As a result, DNA encoding mouse CDRs was attached to each FR. The base sequences of each product encoding the mouse CDR were designed to overlap each other. Next, a product is synthesized using a human antibody gene as a template, and overlapping CDR portions of the product are annealed to each other, thereby carrying out a complementary strand synthesis reaction. Using this reaction, human FRs are linked by the sequence of mouse CDRs.
Finally, the 3 CDR and 4 FR linked V region genes were amplified over their full length by primers that anneal at the 5 'and 3' ends and attach appropriate restriction enzyme recognition sequences. The DNA obtained as described above and a DNA encoding a human antibody C region are inserted into an expression vector and fused in frame, whereby a human antibody expression vector can be prepared. After the recombinant vector is introduced into a host and recombinant cells are established, the recombinant cells are cultured, and the humanized antibody is produced in the culture of the cultured cells by expressing a DNA encoding the humanized antibody (see European patent publication EP239400, International publication WO 96/02576).
By qualitatively or quantitatively measuring and evaluating the binding activity of the humanized antibody produced as described above to the antigen, it is possible to appropriately select the human antibody FR in which the CDR forms a good antigen-binding site when the antibody is bound by the CDR. If necessary, the amino acid residues of the FR may be substituted so that the CDRs of the reshaped human antibody form an appropriate antigen-binding site. For example, a mutation of an amino acid sequence can be introduced into an FR by applying a PCR method used for grafting a mouse CDR into a human FR. Specifically, a mutation of a partial base sequence can be introduced into a primer annealing FR. Mutations of the nucleotide sequence were introduced into the FRs synthesized using the primers. The binding activity of a mutant antibody having substituted amino acids to an antigen is measured and evaluated by the above-described method, whereby a mutant FR sequence having desired properties can be selected (Sato, K.et al., Cancer Res, 1993, 53, 851-856).
Bivalent antibody, low-molecular-weight antibody, and modified antibody
The anti-Muc 17 antibody of the present invention may bind to the Muc17 protein, and includes not only bivalent antibodies represented by IgG but also monovalent antibodies or multivalent antibodies represented by IgM. The multivalent antibody of the present invention includes multivalent antibodies having all the same antigen binding sites, or multivalent antibodies having partially or all different antigen binding sites.
The antibody of the present invention is not limited to a full-length antibody, and may be a low-molecular antibody or a modified product thereof as long as it binds to Muc17 protein.
The low molecular weight antibody includes an antibody fragment in which a part of a full-length antibody (e.g., a whole IgG or the like) is deleted. So long as it has the ability to bind to the Muc17 antigen, partial deletion of the antibody molecule is allowed. The antibody fragment of the present invention preferably contains either or both of a heavy chain variable region (VH) and a light chain variable region (VL). The amino acid sequence of VH or VL may include substitutions, deletions, additions and/or insertions. Further, any one or both of VH and VL may be partially deleted as long as it has a binding ability to Muc17 antigen. In addition, the variable region may be chimeric or humanized. Specific examples of the antibody fragment include Fab, Fab ', F (ab') 2, Fv, and the like. Specific examples of the antibody having a reduced molecular weight include Fab, Fab ', F (ab') 2, Fv, scFv (single chain Fv), Diabody (Diabody), sc (Fv)2 (single chain (Fv)2), and the like. Multimers (e.g., dimers, trimers, tetramers, polymers) of the above-described antibodies are also included in the low molecular weight antibodies of the present invention.
The fragment of the antibody can be obtained by treating the antibody with an enzyme to produce an antibody fragment. As an enzyme for producing an antibody fragment, for example, papain, pepsin, plasmin, or the like is known. Alternatively, genes encoding the above antibody fragments can be constructed and, upon introduction into an expression vector, expressed in an appropriate host cell (see, e.g., Co, M.S. et al, J.Immunol, (1994)152, 2968-2976, Better, M. & Horwitz, A.H.methods in Enzymology (1989)178, 476-496, Plueckthun, A. & Skerra, A.methods in Enzymology (1989)178, 476-496, Lamori, E., Methods in Enzymology (1989)121, 652-663, Rousseaux, J.et al., Methods in Enzymology (1989)121, 663-669, 137-rd, R.E.et al, TISSeaux, J.et al., ECH (1991) 132).
A diabody is a bivalent (bivalent) antibody fragment constructed by gene fusion (Holliger P et al, Proc. Natl. Acad. Sci. USA 90: 6444-6448(1993), EP404,097, WO93/11161, etc.). Diabodies are dimers consisting of 2 polypeptide chains. Usually, the polypeptide chains constituting the dimer are each bound via a linker to VL and VH in the same chain. Generally, the linker in a diabody is short in the region where VL and VH cannot bind to each other. Specifically, the linker is composed of about 5 amino acid residues, for example. Thus, VL and VH, encoded on the same polypeptide chain, cannot form single chain variable fragments, and form dimers with other single chain variable fragments. As a result, the diabody has 2 antigen-binding sites.
scFv is obtained by linking the H chain V region and the L chain V region of an antibody. In scFv, the H chain V region and the L chain V region are linked by a linker, preferably a peptide linker (Huston, J.S.et., Proc.Natl.Acad.Sci.U.S.A, 1988, 85, 5879-. The H chain V region and the L chain V region in the scFv are described as antibodies in the present specification, but may be derived from any antibodies. The peptide linker connecting the V regions is not particularly limited. For example, any single-chain peptide containing about 3 to 25 residues can be used as a linker.
sc (fv)2 is a low molecular weight antibody in which 2 VH and 2 VL are bound to form a single chain by a linker or the like (Hudson et al, J immunol. methods 1999; 231: 177-189). sc (fv)2 can be produced by, for example, linking an scFv to a linker.
The antibody of the present invention may be used as a modified antibody bound to various molecules such as polyethylene glycol (PEG) and cytotoxic substances. The modified antibody can be obtained by chemically modifying the antibody of the present invention. Methods for modifying antibodies are well established in the art.
Examples of the cytotoxic substance that binds to the antibody of the present invention include toxins, radioactive substances, and chemotherapeutic agents (chemotherapeutic agents). Cytotoxic agents include prodrugs of cytotoxic agents that become active in vivo. The prodrug may be activated by enzymatic conversion (non-enzymatic conversion) or non-enzymatic conversion (enzymatic conversion). In the present invention, the toxin refers to various proteins or polypeptides derived from microorganisms, animals, or plants and exhibiting cytotoxicity. In the present invention, the radioactive substance refers to a substance containing a radioactive isotope. The radioisotope is not particularly limited, and any radioisotope can be used. In the present invention, the chemotherapeutic agent is a substance having cytotoxicity other than the above toxin or radioactive substance. Chemotherapeutic agents include cytokines, antineoplastic agents, enzymes, and the like.
The antibody of the present invention may be a bispecific antibody (bispecificantibody). A bispecific antibody is an antibody having variable regions that recognize different epitopes in the same antibody molecule, and the epitopes may be present in different molecules or may be present in the same molecule. That is, in the present invention, the bispecific antibody may have an antigen binding site that recognizes a different epitope on the Muc17 protein. In the bispecific antibody, 2 molecules of the antibody molecule can be bound to 1 molecule of Muc 17. As a result, stronger cytotoxicity can be expected. The "antibody" in the present invention also includes the above-mentioned antibodies.
In the present invention, a bispecific antibody recognizing an antigen other than Muc17 may be included. For example, a bispecific antibody recognizing an antigen different from Muc17, which is the same antigen specifically expressed on the cell surface of a target cancer cell as Muc17, may be included.
Methods for making bispecific antibodies are well known. For example, bispecific antibodies can be prepared by binding 2 antibodies recognizing different antigens. The antibody to be bound may be 1/2 molecules having an H chain and an L chain, or 1/4 molecules having only an H chain. Alternatively, a bispecific antibody-producing fused cell can be prepared by fusing hybridomas producing different monoclonal antibodies. Further, bispecific antibodies can be produced by genetic engineering methods.
Method for producing antibody
The anti-Muc 17 antibody of the present invention can be obtained by a known method. The anti-Muc 17 antibody of the present invention is particularly preferably a monoclonal antibody derived from a mammal. Monoclonal antibodies derived from mammals include monoclonal antibodies produced by hybridomas, monoclonal antibodies produced by hosts transformed with expression vectors containing antibody genes by genetic engineering methods, and the like.
Hybridomas that produce monoclonal antibodies can be produced by known techniques, for example, as described below. First, Muc17 protein was used as a sensitizing antigen, and the antigen was immunized by a general immunization method. An immune cell obtained from an immunized animal is fused with a known parent cell by a conventional cell fusion method to obtain a hybridoma. Furthermore, a hybridoma producing the anti-Muc 17 antibody can be selected by screening a cell producing the target antibody from the hybridoma by a usual screening method.
Specifically, the monoclonal antibody can be produced, for example, as follows. First, by expressing the Muc17 gene, the Muc17 protein can be obtained and used as a sensitizing antigen for obtaining an antibody. The nucleotide sequence of the human Muc17 gene is disclosed in GenBank accession No. NM 001040105 (SEQ ID NO: 1), etc. That is, the gene sequence encoding Muc17 is inserted into a known expression vector, and after transformation of an appropriate host cell, the target human Muc17 protein can be purified from the host cell or culture supernatant by a known method. In addition, purified native Muc17 protein can be used in the same manner. The formation can be carried out by using a combination of 1 or more times of a plurality of types of chromatography such as ordinary ion chromatography and affinity chromatography. In addition, a fusion protein obtained by fusing a desired partial polypeptide of the Muc17 protein with a different polypeptide may also be used as an immunogen as used in the present invention. For the preparation of a fusion protein as an immunogen, for example, an Fc fragment of an antibody, a peptide tag, or the like can be used. The vector for expressing the fusion protein can be prepared by in-frame fusion of genes encoding two or more desired polypeptide fragments and insertion of the fused genes into an expression vector as described above. A method for producing a fusion protein is described in Molecular Cloning 2nd ed (Sambrook, J.et. al., Molecular Cloning 2)nded.,9.47-9.58,Cold Spring Harbor Lab.Press,1989)。
The Muc17 protein purified as indicated above can be used as a sensitizing antigen for use in immunizing a mammal. In addition, a partial peptide of Muc17 can also be used as a sensitizing antigen. For example, peptides as described below can be used as sensitizing antigens.
A peptide obtained by chemical synthesis from the amino acid sequence of human Muc 17;
a peptide obtained by incorporating a part of the human Muc17 gene into an expression vector to express it;
a peptide obtained by decomposing a human Muc17 protein with a protease.
The region and size of Muc17 used as a partial peptide are not limited. Preferred regions may be selected from the amino acid sequences constituting the extracellular domain of Muc17 (1 st to 4389 in the amino acid sequence of SEQ ID NO. 2). The number of amino groups constituting the peptide forming the sensitizing antigen is preferably at least 3 or more, for example, 5 or more, or 6 or more. More specifically, a peptide having 8 to 50, preferably 10 to 30 residues can be used as the sensitizing antigen.
The mammal immunized with the sensitizing antigen is not particularly limited. In order to obtain a monoclonal antibody by the cell fusion method, it is preferable to select an immunized animal in view of compatibility with the parent cell used in cell fusion. In general, rodents are preferred as the immunized animals. Specifically, a mouse, rat, hamster, or rabbit may be used as the immunized animal. Furthermore, monkeys and the like may be used as the immunized animals.
The animal can be immunized by sensitizing antigen according to a known method. For example, as a general approach, a mammal may be immunized by intraperitoneal or subcutaneous injection of a sensitizing antigen. Specifically, the sensitizing antigen is administered to the mammal a plurality of times every 4 to 21 days. The sensitizing antigen can be diluted with PBS (Phosphate-Buffered Saline) or physiological Saline at an appropriate dilution ratio for immunization. Also, the sensitizing antigen may be administered with an adjuvant. For example, the antigen can be mixed with Freund's complete adjuvant (Freund's complete adjuvant) and emulsified to form a sensitized antigen. In addition, a suitable carrier may be used for immunization with the sensitizing antigen. In particular, when a partial peptide having a small molecular weight is used as a sensitizing antigen, it is preferable to immunize the antigen by binding the sensitizing antigen peptide to a carrier protein such as albumin or keyhole limpet hemocyanin (keyhole limpet hemocyanin).
After the mammal is immunized as described above and the increase in the amount of the desired antibody in the serum is confirmed, immune cells are collected from the mammal and cell fusion is performed. As the immune cell, spleen cells are particularly preferably used.
As the cells to be fused with the above immune cells, mammalian myeloma cells are used. Myeloma cells preferably have an appropriate selection marker for selection. The selection marker is a characteristic that it can survive (or cannot survive) under a specific culture condition. Known selectable markers include hypoxanthine guanine phosphoribosyl transferase kinase deletion (hereinafter abbreviated as HGPRT deletion) and thymidine kinase deletion (hereinafter abbreviated as TK deletion). HGPRT or TK-deleted cells have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter referred to as HAT sensitivity). HAT-sensitive cells cannot synthesize DNA in HAT selection medium, but die, and if fused with normal cells, can continue DNA synthesis using the salvage pathway (solution pathway) of normal cells, and therefore can proliferate in HAT selection medium.
HGPRT-deleted cells can be selected using a medium containing 6-mercaptoguanine, 8-azaguanine (hereinafter referred to as 8AG), while TK-deleted cells can be selected using a medium containing 5' -bromodeoxyuridine. Normal cells die by incorporating these pyrimidine analogs into DNA, but cells lacking these enzymes can survive in selective media because they do not incorporate the pyrimidine analogs. Another selection marker, called G418 resistance, confers resistance to 2-deoxystreptomycin (2-deoxystreptamine) antibiotics (gentamicin analogs) due to the presence of a neomycin resistance gene. A variety of myeloma cells suitable for cell fusion are known. For example, cells such as P3 (P3X 63Ag8.653) (J.Immunol. (1979)123, 1548-1550), P3X 63Ag8U.1(Current copies in Microbiology and Immunology (1978)81, 1-7), NS-1(Kohler, G.and Milstein, C.Eur.J.Immunol. (1976)6, 511-519), MPC-11(Margulies, D.H.et. et. Cell (1976)8, 405-415), SP2/0(Shulman, M.et. Nature (1978)276, 269-269), FO (Med.Groth, S.F.et. et. J.Immunol.methods (1980)35, 1-21), S194 (Trumb.194, S.277.63, J.Immunol.methods (1980)35, 1-21), myeloma cells such as Escherichia coli, 11, 1978, 11-11 (Gal. J.H.et. 11, etc., myeloma.
The above-mentioned cell fusion of the immunocyte and the myeloma cell can be carried out by a known method, for example, a method of Kohler and Milstein et al (Kohler.G.and Milstein, C., MethodsEnzymol (1981)73, 3-46).
More specifically, for example, the cell fusion can be carried out in a normal nutrient medium in the presence of a cell fusion promoter. Examples of fusion promoters that can be used include polyethylene glycol (PEG) and sendai virus (HVJ). If desired, an auxiliary agent such as dimethyl sulfoxide may be added to improve the fusion efficiency.
The ratio of the immune cells to the myeloma cells to be used can be arbitrarily set. For example, it is preferable to make the immune cells 1 to 10 times as large as the myeloma cells. The culture medium used for the cell fusion may be, for example, an RPMI 1640 culture medium or an MEM culture medium suitable for the proliferation of the myeloma cell line, or a common culture medium used for such cell culture. Furthermore, serum replacement (serum replacement) such as Fetal Calf Serum (FCS) may be added to the culture medium.
In the cell fusion, predetermined amounts of the above immune cells and myeloma cells are mixed well in the above culture solution, and a PEG solution heated to about 37 ℃ in advance is mixed to form target fused cells (hybridomas). In the cell fusion method, for example, PEG having an average molecular weight of about 1000 to 6000 may be added at a concentration of usually 30 to 60% (w/v). Then, the appropriate culture medium as mentioned above is added, centrifuged, and the supernatant is removed, and the above operation is repeated to remove the cell fusion agent and the like which are not preferable for hybridoma culture.
The hybridoma obtained as described above can be selected using a selection medium suitable for a selection marker possessed by myeloma cells used for cell fusion. For example, HGPRT or TK-deleted cells can be selected by culturing in HAT medium (medium containing hypoxanthine, aminopterin and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have successfully undergone cell fusion with normal cells can be selectively proliferated in HAT culture medium. The culture using the HAT medium is continued for a time sufficient to cause the death of cells other than the target hybridoma (non-fused cells). Specifically, in general, a target hybridoma can be selected by culturing for several days to several weeks. Then, by performing a commonly used limiting dilution method, it is possible to screen and monoclonal hybridomas producing the target antibody. Alternatively, an antibody recognizing Muc17 can be produced according to the method described in International publication WO 03/104453.
The screening and the monoclonal screening of the target antibody are preferably carried out by a screening method based on a known antigen-antibody reaction. For example, the antigen is bound to a carrier such as a bead made of polystyrene or a commercially available 96-well microtiter plate, and reacted with a culture supernatant of hybridoma. Subsequently, the carrier is washed, and then an enzyme-labeled secondary antibody or the like is reacted. If the culture supernatant contains the target antibody that reacts with the sensitizing antigen, the secondary antibody is bound to the carrier by this antibody. Finally, by detecting the secondary antibody bound to the carrier, it is possible to determine whether the target antibody is present in the culture supernatant. Hybridomas that produce a desired antibody having antigen-binding ability can be cloned by a limiting dilution method or the like. In this case, a protein for immunization and substantially the same Muc17 protein can be preferably used as the antigen. For example, an oligopeptide including the extracellular domain of Muc17 or a partial amino acid sequence constituting the region can be used as an antigen.
In addition to the method of obtaining the above-mentioned hybridoma by immunizing an animal other than a human with an antigen, a target antibody can be obtained by sensitizing human lymphocytes with an antigen. Specifically, human lymphocytes are first sensitized in vitro with Muc17 protein. The immunosensitized lymphocytes are then fused with an appropriate fusion partner (fusion partner). As the fusion partner, for example, myeloma cells derived from human and having the ability to divide permanently can be used (see Japanese patent publication No. Hei 1-59878). The anti-Muc 17 antibody obtained by this method is a human antibody having a binding activity to Muc17 protein.
Furthermore, an anti-Muc 17 human antibody can be obtained by administering Muc17 protein as an antigen to a transgenic animal having the entire gene pattern of human antibody genes. Antibody-producing cells of an immunized animal can be immortalized by fusion with an appropriate fusion partner cell or treatment with Epstein-Barr virus (EB virus) infection or the like. Human antibodies against the Muc17 protein can be isolated from immortalized cells obtained as described above (see International publications WO94/25585, WO93/12227, WO92/03918, WO 94/02602). Furthermore, by cloning immortalized cells, it is also possible to clone cells that produce antibodies specific to the desired reaction. When the transgenic animal is used as an immunized animal, the immune system of the animal recognizes human Muc17 as a foreign body. Therefore, a human antibody against human Muc17 can be easily obtained. The monoclonal antibody-producing hybridoma prepared as described above can be subcultured in a conventional culture medium. In addition, the hybridoma can be stored in liquid nitrogen for a long period of time.
In addition, a technique of obtaining a human antibody by panning using a human antibody library is also known. For example, a human antibody can be expressed on the surface of a phage by phage display method using the V region of a human antibody as a single-chain antibody (scFv), and a phage that binds to an antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined. After the DNA sequence of the scFv that binds to the antigen is determined, the V region sequence is fused in frame with the sequence of the desired human antibody C region, and then an appropriate expression vector is inserted, whereby an expression vector can be prepared. The human antibody can be obtained by introducing the expression vector into the above-mentioned preferred expression cells and expressing a gene encoding the human antibody. The above-mentioned methods are known (International publications WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO 95/15388).
The hybridoma is cultured by a usual method, and the desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridoma may be administered to a mammal compatible with the hybridoma to proliferate the hybridoma, and the monoclonal antibody may be obtained from ascites of the mammal. The former method is suitable for obtaining an antibody with high purity.
Recombinant antibodies
The antibody of the present invention may be a recombinant antibody that can be produced using an antibody gene cloned from an antibody-producing cell. The antibody gene thus cloned can be introduced into a suitable vector and introduced into a host to express the antibody. Methods for the isolation of antibody genes, introduction into vectors, and transformation of host cells have been established (see, e.g., Vandamme, a.m.et al, eur.j.biochem. (1990)192, 767-775).
For example, a cDNA encoding the variable region (V region) of the anti-Muc 17 antibody can be obtained from a hybridoma cell producing the anti-Muc 17 antibody. For this purpose, usually total RNA is first extracted from the hybridomas. As a method for extracting mRNA from cells, for example, guanidine ultracentrifugation (Chirgwin, J.M.et. al., Biochemistry (1979)18, 5294-.
The extracted mRNA can be purified using an mRNA Purification Kit (manufactured by GEHealthcare Biosciences) or the like. Alternatively, a kit for directly extracting total mRNA from cells, such as QuickPrep mRNA purification kit (manufactured by GE Healthcare Biosciences), is also commercially available. Total mRNA can also be obtained from hybridomas using the kit described above. A cDNA encoding the V region of the antibody can be synthesized from the resulting mRNA using reverse transcriptase. cDNA can be synthesized using, for example, the AMV Reverse Transcriptasefirst-strand cDNA Synthesis Kit (manufactured by Biochemical industries, Ltd.). In addition, for the synthesis and amplification of cDNA, 5 '-Ampli FINDER RACE Kit (manufactured by Clontech) and 5' -RACE method using PCR (Frohman, M.A.et. al., Proc.Natl.Acad.Sci.USA (1988)85, 8998-. Furthermore, in the above-mentioned cDNA synthesis process, the following appropriate restriction enzyme sites may be introduced at both ends of the cDNA.
The target cDNA fragment was purified from the resulting PCR product, and then ligated with a vector DNA. After the recombinant vector is prepared as described above and introduced into Escherichia coli or the like to select a colony, a desired recombinant vector can be prepared from the Escherichia coli forming the colony. Next, it can be confirmed by a known method, for example, the dideoxyribonucleic acid chain termination method, whether or not the recombinant vector has the nucleotide sequence of the cDNA of interest.
In order to obtain a gene encoding a variable region, a PCR method using a primer for variable region gene amplification may be used. First, cDNA is synthesized using the extracted mRNA as a template to obtain a cDNA library. For convenience, commercially available kits were used for the synthesis of cDNA libraries. In fact, since only a very small amount of mRNA is obtained from a small number of cells, the yield is low when the mRNA is directly purified. Therefore, usually, vector RNA which is clearly known to contain no antibody gene is added and then purified. Alternatively, when a certain amount of RNA can be extracted, even only RNA from antibody-producing cells can be efficiently extracted. For example, when RNA is extracted from 10 or more, or 30 or more, preferably 50 or more antibody-producing cells, it may not be necessary to add carrier RNA.
The antibody gene was amplified by PCR using the obtained cDNA library as a template. Primers for amplifying antibody genes by the PCR method are well known. For example, primers for amplifying human antibody genes can be designed according to the disclosure of the article (J.mol.biol. (1991)222, 581-597) and the like. The primer has a different base sequence depending on the subclass of immunoglobulin. Therefore, when the cDNA library of unknown subclass is used as a template, all possibilities need to be considered for performing the PCR method.
Specifically, for example, in order to obtain a gene encoding human IgG, primers capable of amplifying genes encoding γ 1 to γ 5 as a heavy chain, a κ chain and a λ chain as a light chain may be used. For amplifying the variable region gene of IgG, a primer annealing to a portion corresponding to the hinge region is generally used as the 3' -side primer. On the other hand, primers suitable for each subclass can be used as primers on the 5' side.
PCR products generated from the primers for gene amplification of each subclass of heavy and light chains form independent gene pools, respectively. Using the synthetic gene library described above, immunoglobulins comprising a combination of heavy and light chains can be reconstituted. The binding activity of the reconstituted immunoglobulin to MUC17 was used as an index to screen out a target antibody.
More preferably, the antibody of the invention binds specifically to Muc 17. The antibody that binds to Muc17 can be screened, for example, as follows.
(1) The method comprises the following steps: contacting an antibody comprising a V region encoded by cDNA obtained from a hybridoma with Muc 17;
(2) the method comprises the following steps: detecting the binding of Muc17 to the antibody; and
(3) the method comprises the following steps: antibodies were selected that bound to Muc 17.
Methods for detecting binding of antibodies to Muc17 are well known. Specifically, a test antibody is reacted with Muc17 immobilized on a carrier, and then a labeled antibody recognizing the antibody is reacted. After washing, if the labeled antibody on the carrier is detected, it can be confirmed that the test antibody binds to Muc 17. As the label, an enzymatically active protein such as peroxidase or β -galactosidase, or a fluorescent substance such as FITC can be used. For the evaluation of the binding activity of the antibody, a fixed sample of Muc 17-expressing cells may also be used.
As a method for screening an antibody using a binding activity as an index, a panning method using a phage vector can be used. When the antibody gene is obtained as a gene library of the subclasses of heavy and light chains as described above, a screening method using a phage vector is advantageous. The genes encoding the variable regions of the heavy and light chains may be joined by appropriate linker sequences to form a single chain fv (scFv). If a gene encoding scFv is inserted into a phage vector, a phage expressing scFv on the surface can be obtained. When this phage is brought into contact with a target antigen and the phage bound to the antigen is recovered, DNA encoding scFv having a target binding activity can be recovered. By repeating this operation as necessary, the scFv having the target binding activity can be concentrated.
After obtaining a cDNA encoding the V region of the desired anti-Muc 17 antibody, the cDNA is digested with restriction enzymes recognizing the restriction sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and digest nucleotide sequences that are less likely to occur in the nucleotide sequences constituting the antibody genes. Furthermore, in order to insert 1 copy of the digested fragment into the vector in the correct orientation, it is preferable to provide a restriction enzyme for the sticky ends. An antibody expression vector can be obtained by inserting the cDNA encoding the V region of the anti-Muc 17 antibody digested as described above into an appropriate expression vector. In this case, a chimeric antibody can be obtained by in-frame fusion of a gene encoding the constant region (C region) of an antibody and a gene encoding the V region. Herein, the term "chimeric antibody" refers to a product in which the constant region and the variable region are derived from different organisms. Therefore, in addition to mouse-human xenochimeric antibodies, human-human allochimeric antibodies are also included in the chimeric antibodies of the present invention. A chimeric antibody expression vector can also be constructed by inserting the V region gene into an expression vector having a constant region in advance.
Specifically, for example, a restriction enzyme recognition sequence of a restriction enzyme digesting the above-mentioned V region gene may be arranged on the 5' side of an expression vector holding a DNA encoding a desired antibody constant region (C region). Both were digested with restriction enzymes of the same combination and fused in frame to construct a chimeric antibody expression vector.
In order to produce the anti-Muc 17 antibody of the present invention, the antibody gene may be incorporated into an expression vector to be expressed under the control of an expression control region. The expression control region for expressing the antibody includes, for example, an enhancer and a promoter. Then, by transforming an appropriate host cell with this expression vector, a recombinant cell expressing a DNA encoding the anti-Muc 17 antibody can be obtained.
When the antibody gene is expressed, DNAs encoding the heavy chain (H chain) and the light chain (L chain) of the antibody may be incorporated into different expression vectors. Antibody molecules having both H chains and L chains can be expressed by simultaneous transformation (co-transfection) of vectors incorporating both H chains and L chains in the same host cell. Alternatively, the DNA encoding the H chain and the L chain may be incorporated into a single expression vector to transform the host cell (see International publication WO 94/11523).
Various combinations of hosts and expression vectors are known for producing antibodies by temporarily isolating antibody genes and introducing them into appropriate hosts. The above expression systems can be used in the present invention. When eukaryotic cells are used as the host, animal cells, plant cells, or fungal cells can be used. Specifically, examples of the animal cell that can be used in the present invention include a mammalian cell (CHO, COS, myeloma, bhk (baby hamster kidney), Hela, Vero, etc.), an amphibian cell (Xenopus laevis oocytes, etc.), an insect cell (sf9, sf21, Tn5, etc.), and the like.
Alternatively, an expression system of an antibody gene produced by a cell of the genus Nicotiana such as Nicotiana tabacum is known as a plant cell. Transformation of plant cells cultured from callus may be used.
Further, as the fungal cell, yeast (genus Saccharomyces such as Saccharomyces cerevisiae, genus Pichia such as Pichia methanolica), filamentous fungi (genus Aspergillus such as Aspergillus niger) and the like can be used.
Alternatively, expression systems using antibody genes from prokaryotic cells are also known. For example, when bacterial cells are used, bacterial cells such as Escherichia coli (E.coli) and Bacillus subtilis can be used in the present invention.
When mammalian cells are used, an expression vector can be constructed which functionally binds to a commonly used effective promoter, an expressed antibody gene, and a downstream polyA signal on the 3' side thereof. Examples of the promoter/enhancer include human cytomegalovirus immediate early promoter/enhancer (human cytomegalovirus immediate early promoter/enhancer).
In addition, as a promoter/enhancer that can be used for expression of the antibody of the present invention, a viral promoter/enhancer, a promoter/enhancer derived from mammalian cells such as human elongation factor 1 α (HEF1 α), or the like can be mentioned. Examples of viruses that can utilize promoters and enhancers include retroviruses, polyomaviruses, adenoviruses, and monkey kidney virus 40(SV 40).
When the SV40 promoter/enhancer is used, the method of Mullgan et al (Nature (1979)277, 108) can be used. In addition, the HEF1 α promoter/enhancer can be readily used for target gene expression according to the method of Mizushima et al (Nucleic Acids Res. (1990)18, 5322).
In the case of E.coli, the gene can be expressed by functionally combining a commonly used effective promoter, a signal sequence for antibody secretion, and an expressed antibody gene. Examples of the promoter include lacZ promoter and araB promoter. When the lacZ promoter is used, the method of Ward et al (Nature (1989)341, 544-242546; FASEBJ. (1992)6, 2422-2427) can be employed. Alternatively, the araB promoter can be used for expression of a target gene by the method of Better et al (Science (1988)240, 1041-1043).
As a signal sequence for antibody secretion, pelB signal sequence can be used when produced in the periplasm of escherichia coli (Lei, s.p.et al, j.bacteriol. (1987)169, 4379). Then, after the antibody produced in the periplasm is separated, a protein denaturing agent such as guanidine hydrochloride of urea is used, thereby reorganizing (refolding) the structure of the antibody to have a desired binding activity.
As the origin of replication of the inserted expression vector, those derived from SV40, polyoma virus, adenovirus, Bovine Papilloma Virus (BPV), etc. can be used. Furthermore, in order to amplify the gene copy number in the host cell system, a selection marker may be inserted into the expression vector. Specifically, selection markers such as aminoglycoside transferase (APH) gene, Thymidine Kinase (TK) gene, E.coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, and dihydrofolate reductase (dhfr) gene can be used.
The above expression vector is introduced into a host cell, and the transformed host cell is cultured in vitro or in vivo to produce the target antibody. The host cell can be cultured according to a known method. For example, DMEM, MEM, RPMI 1640, IMDM may be used as the culture medium, and a serum supplement such as Fetal Calf Serum (FCS) may be used in combination.
The antibody expressed and produced as described above can be purified by a known method used for the purification of a general protein, alone or in a suitable combination. For example, Antibodies can be isolated and purified by appropriately selecting and combining an affinity column such as a protein A column, a chromatography column, filtration, ultrafiltration, salting out, dialysis, and the like (Antibodies A Laboratory Manual. Ed Harbor, David Lane, Cold Spring Harbor Laboratory, 1988).
In addition, in the production of recombinant antibodies, transgenic animals may be used in addition to the above host cells. That is, the antibody can be obtained from an animal into which a gene encoding the target antibody has been introduced. For example, an antibody gene is inserted in-frame into a gene encoding a protein inherently produced in milk to construct a fusion gene. As the protein secreted in milk, for example, goat β casein or the like can be used. The DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into an embryo of a goat, and the injected embryo is introduced into a female goat. A transgenic goat (or its offspring) is produced from the goat which received the embryo, and the desired antibody can be obtained from the milk produced from the transgenic goat as a fusion protein with a milk protein. In addition, in order to increase the amount of milk containing the desired antibody produced by the transgenic goat, hormones may be appropriately administered to the transgenic goat (Ebert, K.M.et. al., Bio/Technology (1994)12, 699-702).
As the C region of the recombinant antibody of the present invention, a C region derived from an animal antibody can be used. For example, as the H chain C region of a mouse antibody, C.gamma.1, C.gamma.2 a, C.gamma.2 b, C.gamma.3, C.mu.C.delta.C.alpha.1, C.alpha.2 and C.epsilon.can be used, and as the L chain C region, C.kappa.C.lambda.can be used. As the animal antibody other than the mouse antibody, an animal antibody of rat, rabbit, goat, sheep, camel, monkey, or the like can be used. The above sequences are well known. In addition, the C region may be modified in order to improve the stability of the antibody or its production.
Pharmaceutical composition
The present invention provides a pharmaceutical composition comprising the anti-Muc 17 antibody as an active ingredient. The present invention also relates to an anticancer agent containing the anti-Muc 17 antibody as an active ingredient. The anticancer agent of the present invention is preferably administered to a subject suffering from cancer or a subject who may have relapses.
In addition, in the present invention, in addition to an anticancer agent containing an anti-Muc 17 antibody as an active ingredient, a method for preventing or treating cancer comprising the step of administering an anti-Muc 17 antibody to a subject to be treated, or the use of an anti-Muc 17 antibody in the manufacture of an anticancer agent is provided.
The type of cancer to be treated with the anticancer agent of the present invention is not particularly limited, and usually, cancer expressed by Muc17 protein is preferably pancreatic cancer or colorectal cancer.
In the present invention, the phrase "containing an anti-Muc 17 antibody as an active ingredient" means that the anti-Muc 17 antibody is contained as a main active ingredient, and the content of the monoclonal antibody is not limited.
In addition, a plurality of antibodies may be incorporated into the pharmaceutical composition or the anticancer agent of the present invention as needed. For example, by making a mixture of anti-Muc 17 antibodies, it may be possible to enhance cytotoxicity against Muc17 expressing cells. Alternatively, in addition to the anti-Muc 17 antibody, an antibody recognizing another tumor-associated antigen may be added to improve the therapeutic effect. In addition, a pharmaceutical composition containing an anti-Muc 17 antibody and an antibody recognizing an anti-Muc 17 antibody such as an anti-human IgG antibody may be used. Preferably, a cytotoxic substance such as a toxin, a radioactive substance, or a chemotherapeutic agent is bound to an antibody recognizing an anti-Muc 17 antibody such as an anti-human IgG antibody.
The pharmaceutical composition of the present invention or the anticancer agent can be administered to a patient by any of oral administration and non-oral administration. Non-oral administration is preferred. Specific examples of the administration method include injection administration, nasal administration, pulmonary administration, and transdermal administration. As an example of administration by injection, the pharmaceutical composition of the present invention can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like. In addition, an appropriate administration method can be selected according to the age and symptoms of the patient. As the dose, for example, the dose is selected in the range of 0.0001mg to 1000mg per 1kg body weight per one administration. Alternatively, for example, the amount to be administered is selected in the range of 0.001 to 100000mg/body per patient. However, the pharmaceutical composition of the present invention is not limited to the above dosage.
The Pharmaceutical composition of the present invention can be formulated according to a conventional method (for example, Remington's Pharmaceutical Science, last edition, Mark publishing company, Easton, U.S. A), and can also contain a carrier or an additive acceptable for the drug. Examples of the surfactant include a surfactant, an excipient, a colorant, a perfume, a preservative, a stabilizer, a buffer, a suspending agent, an isotonic agent, a binder, a disintegrating agent, a lubricant, a fluidity enhancer, a flavoring agent, and the like. Further, the present invention is not limited to this, and other commonly used carriers may be used as appropriate. Specific examples of the carrier include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate (polyvinyl acetate), polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, white sugar, carboxymethylcellulose, corn starch, inorganic salts, and the like.
In addition, the present invention provides a method of disrupting a Muc 17-expressing cell or a method of inhibiting proliferation of a cell by contacting a Muc 17-expressing cell with an anti-Muc 17 antibody. anti-Muc 17 antibodies were as described above. The cell to which the anti-Muc 17 antibody binds is not particularly limited as long as it is a cell expressed by Muc 17. Preferred Muc 17-expressing cells of the invention are cancer cells. Preferred cancer cells include pancreatic cancer cells and colorectal cancer cells.
In the present invention, "contacting" may be performed in vitro or in vivo. For example, the contact is performed by adding an antibody to a culture solution of a Muc 17-expressing cell cultured in a test tube. In this case, the shape of the antibody to be added may be appropriately a solution or a solid obtained by freeze drying or the like. When added as an aqueous solution, the solution may be an aqueous solution containing only the antibody, or may be a solution containing, for example, the above-mentioned surfactant, excipient, colorant, perfume, preservative, stabilizer, buffer, suspending agent, isotonic agent, binder, disintegrant, lubricant, fluidity promoter, flavoring agent, and the like. The concentration to be added is not particularly limited, but is preferably in the range of 1pg/ml to 1g/ml, more preferably 1ng/ml to 1mg/ml, and still more preferably 1. mu.g/ml to 1mg/ml, as the final concentration in the culture solution.
In the present invention, the "contact" may be performed by administering a non-human animal in which the Muc 17-expressing cells are transplanted in vivo or an animal having cancer cells inherently expressing Muc 17. The administration method may be carried out by any of oral administration and non-oral administration. Particularly preferred is a method of administration by parenteral administration, and specific examples of the method of administration include injection administration, nasal administration, transpulmonary administration, and transdermal administration. Examples of the administration by injection include systemic or local administration of the pharmaceutical composition of the present invention, such as intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection, as well as cell growth inhibitor and anticancer agent. In addition, an appropriate administration method can be selected according to the age and symptoms of the animal to be tested. When the antibody is administered as an aqueous solution, the antibody may be simply contained in the aqueous solution, or may be contained in a solution containing, for example, the above-mentioned surfactant, excipient, colorant, perfume, preservative, stabilizer, buffer, suspending agent, isotonic agent, binder, disintegrating agent, lubricant, fluidity promoter, taste corrigent, and the like. As the dose, for example, the dose can be selected in the range of 0.0001mg to 1000mg per 1kg body weight per administration. Alternatively, for example, the amount to be administered is selected in the range of 0.001 to 100000mg/body per patient. However, the amount of the antibody of the present invention to be administered is not limited to the above amount.
Diagnostic method
Further, the present invention provides a method for diagnosing cancer using the anti-Muc 17 antibody. The cancer diagnosed by the method of the present invention is not particularly limited as long as it expresses Muc17, but is preferably pancreatic cancer or colorectal cancer.
The diagnostic method of the present invention may be carried out in vitro or in vivo, but is preferably carried out in vitro.
The method for diagnosing cancer using the anti-Muc 17 antibody of the present invention includes the following steps, for example.
(a) A step of providing a sample taken from a subject;
(b) step (c) of detecting the Muc17 protein contained in the sample of (a).
In the present invention, the term "detection" includes quantitative or qualitative detection. The qualitative detection includes, for example, a measurement of the presence or absence of Muc17 protein, a measurement of the presence or absence of a certain amount or more of Muc17 protein, a measurement of comparing the amount of Muc17 protein with another sample (e.g., a control sample). The quantitative measurement includes, for example, measurement of the concentration of Muc17 protein, measurement of the amount of Muc17 protein, and the like.
The test sample of the present invention is not particularly limited as long as it may contain Muc17 protein. Specifically, a sample collected from the body of a living body such as a mammal is preferable. More preferably, the sample is a sample taken from a human body. Specific examples of the test sample include blood, interstitial fluid (interstitial fluid), plasma, extravascular fluid, cerebrospinal fluid, synovial fluid, pleural fluid, serum, lymph fluid, saliva, urine, and tissue. The sample is preferably a sample obtained from a test sample such as a tissue or cell-immobilized specimen or a cell culture solution collected from a living body.
The Muc17 protein can be detected by a method known to those skilled in the art, and for example, it can be detected by Radioimmunoassay (RIA), Enzyme Immunoassay (EIA), Fluorescence Immunoassay (FIA), Luminescence Immunoassay (LIA), Immunoprecipitation (IP), immunoturbidimetry (TIA), Western Blotting (WB), Immunohistochemistry (IHC), immunodiffusion (SRID), or the like.
The contents of all patents and references cited explicitly in this specification are incorporated herein by reference in their entirety.
Examples
The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.
Example 1 mRNA expression analysis of mucin17(Muc17) by Real-time PCR
Pancreatic cancer cell lines Aspc1, Panc1, Capan1, BXPC 3 cells were purchased from ATCC. Culturing under the conditions shown in the accompanying data, recovering the resultant7For the corresponding cells, Trizol (Invitrogen corporation) was used to prepare total RNA. Mu.g of total RNA was treated with DNaseI (Invitrogen), and then cDNA was synthesized using oligo (dT) as a primer using SuperScript III First Strand Synthesis System for RT-PCR (Invitrogen). For each normal tissue total RNA shown in Table 1, cDNA was synthesized in the same manner. Using the above-obtained product, Real-time PCR was performed by an intercalation method (intercalation method) using SYBR Green I. That is, 20. mu.l of a reaction solution containing SYBR (registered trademark) Premix Ex Taq (Takara), a sense primer (SEQ ID NO: 30) and an antisense primer (SEQ ID NO: 31) was subjected to 33 times using cDNA derived from 3.3ng of total RNA as a templateA cycle of 5 seconds at 95 ℃ and 30 seconds at 60 ℃ was included. A standard curve is prepared based on a sample using a PCR product purified in advance as a template, and the amount of cDNA in the sample is converted from the quantitative value. As shown in FIG. 1, the mRNA of Muc17 showed a high value in AsPc1, which is a pancreatic cancer strain, and the expression in normal tissues was limited to the small intestine.
[ Table 1]
TABLE 1 tissue used for analysis of Muc17 Gene expression
| Name (R) | Company(s) | CAT# |
| Brain, cerebrum | Ambion | 6000 |
| Ovary (LU) of human | Ambion | 6000 |
| Pancreas gland | Ambion | 7954 |
| Thyroid gland | Ambion | 6000 |
| Testis | Ambion | 7972 |
| Breast | Stratagene | 540045 |
| Spleen | Ambion | 6000 |
| Tonsil | Clontech | 636587 |
| Thymus | Ambion | 6000 |
| Bone marrow | Clontech | 636548 |
| Lung (lung) | Ambion | 7968 |
| Heart and heart | Stratagene | 540011 |
| Esophagus | Ambion | 6000 |
| Stomach (stomach) | Stratagene | 540037 |
| Small intestine | Ambion | 6000 |
| Colon | Clontech | 636553 |
| Liver disease | Ambion | 6000 |
| Name (R) | Company(s) | CAT# |
| Salivary gland | Clontech | 636552 |
| Kidney (Kidney) | Ambion | 7976 |
| Prostate gland | Ambion | 6000 |
| Uterus | Stratagene | 540043 |
| Neck | Ambion | 6000 |
| Skeletal muscle | Ambion | 6000 |
| Skin(s) | Stratagene | 540031 |
| Back root | Clontech | 636150 |
| Pericardium | Ambion | 6852 |
| Placenta hominis | Ambion | 6000 |
| Peripheral blood | Clontech | 636580 |
| Lymph gland | Stratagene | 540021 |
| Inferior vena cava | Stratagene | 540121 |
| Medulla oblongata | Clontech | 636562 |
| Fat | Ambion | 6000 |
| Bladder of urinary bladder | Ambion | 6000 |
| Trachea | Ambion | 6000 |
Example 2 preparation of anti-Muc 17 antibody
2-1 cloning of cDNA encoding partial sequence of human Muc17
Muc17(Access No. NM-001040105) is a type 1 membrane protein (SEQ ID NOS: 1 and 2) comprising 4,493 amino acids. Muc17 belongs to the mucin family of membrane types, and most of the extracellular domain contains repeating units of a 59mer tandem repeat rich in serine, threonine, and proline, and is modified with sugar chains. Further, it is presumed that the protein is cleaved and a part of the protein may be present as a secreted protein by having the SEA domain (4182Glu-4287 Asn). In addition, the presence of a secreted splice variant (1Met-4241Arg is the same sequence) has been reported (Maniaux et al, J.biol.chem. (2006)281, 23676-23685). It is considered that an antibody having specificity to Muc17 and not binding to secreted Muc17 may be a novel therapeutic agent for pancreatic cancer, and the preparation of an anti-Muc 17 antibody has been carried out.
In order to make an antibody against the C-terminal part sequence (Muc17ct, 4115Thr-4390Leu) which is highly specific to Muc17 in the extracellular domain of Muc17 (1Met-4389Ser), the sequence was cloned. That is, using cDNA of AsPc1 as a template, a sense primer (SEQ ID NO: 32) containing a signal sequence of mouse antibody to which an EcoRI recognition sequence and a CpoI recognition sequence are added at the 5' end, an antisense primer (SEQ ID NO: 33) containing a CpoI recognition sequence, 10 XKOD-Plus buffer, 2mM dNTPs, and 25mM MgSO4KOD-Plus (manufactured by Takara corporation) was subjected to 5 cycles including 10 seconds at 98 ℃, 5 seconds at 72 ℃ and 4 minutes at 68 ℃, 5 cycles including 10 seconds at 98 ℃, 5 seconds at 70 ℃ and 4 minutes at 68 ℃ and 25 cycles including 10 seconds at 98 ℃ and 4 minutes at 68 ℃. The amplified product generated by the PCR reaction was inserted into pGEM-T Easy using pGEM-T Easy Vector System I (Promega). The sequence was confirmed by ABI 3730 DNA Analyzer.
2-2 preparation of soluble human Muc17 ct/mouse IgG2a Fc fusion protein
The Muc17ct gene cloned into pGEM-T easy vector was digested with EcoRI, CpoI and cloned into pMCDN _ mIgG2 aFc. pMCDN _ mIgG2aFc is derived from pMCDN which is an expression vector for mammalian cells, can induce expression under a mouse CMV promoter (Accession No. U68299), and is incorporated with a neomycin-resistant gene and a DHFR gene. In pMCDN _ mIgG2aFc, the Fc sequence under the hinge of mouse H chain IgG2a is inserted into the EcoRI and NotI recognition sites of pMCDN vector, and Muc17ct and mIgG2aFc sequences are linked by CpoI recognition sequences. The sequence represented by SEQ ID NO. 34 represents the nucleotide sequence of Muc17ct _ mIgG2aFc, and the sequence represented by SEQ ID NO. 35 represents the amino acid sequence of Muc17ct _ mIgG2 aFc.
pMCDN/Muc17ct _ mIgG2aFc was electroporated into DG44 cells (Invitrogen) and selected with 500. mu.g/mL Geneticin, thereby establishing that Muc17ct _ mIgG2aFc expresses CHO cells continuously. The continuous expressing cells were cultured in large quantities and the Muc17ct _ mIgG2aFc protein was purified from the culture supernatant. The culture supernatant was packed into a HiTrap rProte in A (manufactured by GE Co., Ltd.), washed with a binding buffer (20mM sodium phosphate (pH7.0)) and then eluted with an elution buffer (0.1M glycine-HCl (pH 2.7)). The eluate was neutralized by direct transfer into a tube to which a neutralization buffer (1M Tris-HCl (pH9.0)) was added. Then, gel filtration was performed using Superdex 200HR 26/60(GE Co.) and the solution was replaced with PBS. The purified protein was quantified by conversion using DC protein assay (BIO-RAD) using bovine IgG as a standard.
2-3 preparation of anti-Muc 17 antibody
Balb/c mice, or MRL/MpJUmmCrj-lpr/lpr mice (hereinafter, MRL/lpr mice, purchased from Charles River, Japan) were used as the immunized animals. Immunization was started at 6 weeks of age, and Muc17ct _ mIgG2aFc was prepared at the beginning of the immunization at 100. mu.g/head, emulsified with Freund's complete adjuvant (FCA, Becton, Dickinson and Company), and administered subcutaneously. After 2 weeks, the antibody formulated at 50. mu.g/head was emulsified with Freund's incomplete adjuvant (FIA, Becton, Dickinson and Company) and administered subcutaneously. Additional immunizations were performed 2 to 5 times at 1 week intervals thereafter. 4 days after the final immunization, spleen cells were removed, mixed with mouse myeloma cells P3-X63Ag8U1(P3U1, purchased from ATCC) at a ratio of 2: 1, and cell fusion was performed by slowly adding PEG1500(Roche Diagnostics Co.). The PEG1500 was removed by carefully adding RPMI 1640 medium (GIBCO BRL Co.), diluting the PEG1500, and centrifuging the mixture, and then, it was suspended in RPMI 1640 containing 10% FBS and seeded in a 96-well plate at 100. mu.L/well. The next day, RPMI 1640 (hereinafter referred to as HAT medium) containing 10% FBS, 1 XHAT media supplement (SIGMA corporation) and 0.5 XBM-conditioned H1 Hybridoma cloning supplement (Roche Diagnostics corporation) was added to make it 100. mu.L/well. 2. After 3 days, half of the culture broth was replaced with HAT medium, and after 7 days, screening was performed using the culture supernatant. Screening was performed by ELISA in which Muc17ct _ mIgG2aFc was immobilized. Positive clones were monocloned using limiting dilution. The evaluation was performed by ELISA immobilizing the control Fc fusion protein without the Muc17ct sequence, thereby establishing antibodies (MQ016, MQ128, MQ155, MQ 169) that specifically bind to Muc 17. The isotypes of the antibodies were determined using Isostrip (manufactured by Roche Co., Ltd.) and were IgG1 kappa.
Purification of the antibody was performed as follows: the hybridoma was cultured in HAT medium using fbs (ultra low igg) (GIBCO BRL) as serum, and the antibody was purified from the culture supernatant of the hybridoma using HiTrap Protein G HP in the same manner as described above. The elution buffer was replaced with PBS using a PD-10 column (Amersham), and the mixture was stored at 4 ℃. Quantification of purified antibody was performed by using DC protein assay (BIO-RAD) in terms of bovine IgG attached thereto
Example 3 determination of the Gene sequences of the variable regions of the anti-Muc 17 antibody
The sequences of the antibody variable region genes were determined for MQ128 and MQ 155. Total RNA was obtained from 1X 10 using RNeasy Plant Mini kit (QIAGEN Co.) to yield a Total RNA7Extracting from cell hybridoma.
Using 1. mu.g of Total RNA, the 5' -end side gene fragment was amplified using SMART RACE cDNA amplification kit (CLONTECH), synthetic oligonucleotide MHC-IgG1 (SEQ ID NO: 36) complementary to the constant region sequence of mouse IgG1, or synthetic oligonucleotide kappa (SEQ ID NO: 37) complementary to the base sequence of mouse kappa chain constant region. The reverse transcription reaction was carried out at 42 ℃ for 1 hour and 30 minutes. A50. mu.L PCR solution containing 5. mu.L of 10 XDavantage 2PCR buffer, 5. mu.L of 10 XDUniversal Primer A Mix, 0.2mM dNTPs (dATP, dGTP, dCTP, dTTP), 1. mu.L of Advantage2 Polymerase Mix (manufactured by CLONTECH), 2.5. mu.L of a reverse transcription reaction product, and 10pmole of synthetic oligonucleotide MHC-IgG1 or kappa was reacted at an initial temperature of 94 ℃ for 30 seconds, and then a cycle of reaction at 94 ℃ for 5 seconds and 72 ℃ for 3 minutes was repeated 5 times, a cycle of reaction at 94 ℃ for 5 seconds and 70 ℃ for 10 seconds and 72 ℃ for 3 minutes was repeated 5 times, and a cycle of reaction at 94 ℃ for 5 seconds, 68 ℃ for 10 seconds and 72 ℃ for 3 minutes was repeated 25 times. Finally, the reaction product was heated at 72 ℃ for 7 minutes. Each PCR product was purified from agarose Gel using QIAquick Gel Extraction Kit (QIAGEN Co., Ltd.), and then cloned into pGEM-T Easy vector to determine the nucleotide sequence. The nucleotide sequence of the H chain variable region of MQ128 is represented by SEQ ID NO. 18, the amino acid sequence is represented by SEQ ID NO. 19, the nucleotide sequence of the L chain variable region is represented by SEQ ID NO. 20, and the amino acid sequence is represented by SEQ ID NO. 21. The nucleotide sequence of the H chain variable region of MQ155 is represented by SEQ ID NO. 22, the amino acid sequence thereof is represented by SEQ ID NO. 23, the nucleotide sequence of the L chain variable region is represented by SEQ ID NO. 24, and the amino acid sequence thereof is represented by SEQ ID NO. 25.
Example 4 preparation of anti-Muc 17 mouse-human chimeric antibody
The H chain and L chain variable region sequences of each antibody were linked to human H chain and human L chain constant region sequences. For the H chain, PCR was performed using a synthetic oligonucleotide complementary to the base sequence on the 5 '-terminal side of the variable region and having a Kozak sequence and a HindIII site, and a synthetic oligonucleotide complementary to the base sequence on the 3' -terminal side and having a NheI site. For the L chain, PCR was performed using a synthetic oligonucleotide complementary to the base sequence on the 5 '-terminal side of the variable region and having a Kozak sequence and a BamHI site, and a synthetic oligonucleotide complementary to the base sequence on the 3' -terminal side of the variable region and having a BsiWI site. The resulting PCR product was cloned in the antibody expression plasmid pMCDN _ Glk. pMCDN _ Glk has the following structure: the human IgG1 constant region was cloned in a pMCDN vector, and the mouse H chain variable region and the human H chain (γ 1 chain) constant region were linked by a NheI site. In addition, it also has the following structure: an additional expression unit containing a mouse CMV promoter, and a human kappa chain constant region were inserted, and the mouse L chain variable region and the human L chain (kappa chain) constant region were linked via a BsiWI site. The plasmid expresses a neomycin resistance gene, a DHFR gene, and an anti-Muc 17 mouse-human chimeric antibody gene in animal cells. The nucleotide sequence of the H chain of the chimeric MQ155 is shown as SEQ ID NO. 26, the amino acid sequence is shown as SEQ ID NO. 27, the nucleotide sequence of the L chain of the chimeric MQ155 is shown as SEQ ID NO. 28, and the amino acid sequence is shown as SEQ ID NO. 29.
pMCDN _ G1k _ MQ128, pMCDN _ G1k _ MQ155 were introduced into DG44 cells by electroporation. By selection with 500. mu.g/mL Geneticin, it was established that anti-Muc 17 chimeric antibodies expressed CHO cells continuously. Then, purification was performed from the culture supernatant using a Hi Trap rProtein A column. The buffer was replaced with PBS using a PD-10 column, and purified antibodies (chi. mq128(DG), chi. mq155(DG)) were quantified by DCProtein Assay and stored at 4 ℃.
Example 5 preparation of Low fucose anti-Muc 17 mouse-human chimeric antibody
As a method for enhancing ADCC activity of an antibody, a method of modifying a sugar chain of an antibody is known. For example, WO 99/54342 discloses that ADCC activity is improved by performing antibody glycosylation. Further, WO00/61739 discloses that ADCC activity is modulated by the presence or absence of fucose in a sugar chain of an antibody. WO 02/31140 discloses the formulation of antibodies having sugar chains that do not contain alpha-1, 6core fucose by producing antibodies in YB2/0 cells. WO2005/017155 describes an example of a fucose transporter gene-knocked-out CHO cell (CHO _ FTKO), and it is possible to produce an antibody having a sugar chain free of alpha-1, 6core fucose by using the same method.
The anti-Muc 17 mouse-human chimeric antibody expression plasmid constructed as described above was introduced into CHO _ FTKO cells (WO2005/017155) by electroporation and selected with 500. mu.g/mLGneticin, thereby establishing that the anti-Muc 17 chimeric antibody continuously expresses CHO _ FTKO cells. Then, purification was performed from the culture supernatant using a Hi Trap rProtein A column. The buffer was replaced with PBS using a PD-10 column, and the purified antibodies (chi. mq128(FTKO), chi. mq155 (FTKO)) were quantified by DC Protein Assay and stored at 4 ℃.
Example 6 evaluation of the binding Activity of anti-Muc 17 antibody by ELISA
Muc17ct _ mIgG2afc protein was coated with coating buffer (0.1mol/L NaHCO)3(pH9.6),0.02%(w/v)NaN3) Diluted to 1. mu.g/mL, added to an enzyme plate (microplate), and left overnight at 4 ℃ for coating (coating). Diluted with a dilution buffer (50mM Tris-HCl (pH8.1), 1mM MgCl2,150mM NaCl,0.05%(v/v)Tween 20,0.02%(w/v)NaN31% (w/v) BSA), and then an anti-Muc 17 antibody was added thereto and left at room temperature for 1 hour. After washing with a rinsing buffer (Rinse buffer) (0.05% (v/v) Tween20, PBS), an alkaline phosphatase-labeled anti-human kappa chain antibody (Sigma, CAT # A3813) was added thereto, and the mixture was left at room temperature for 1 hour. After washing with the rinse buffer, the substrate buffer (50mM NaHCO) was added3(pH9.8),10mM MgCl2) SIGMA104 (Sigma) diluted to 1mg/mL was allowed to develop color at room temperature for 1 hour, and then absorbance (405nm, reference 655nm) was measured using Benchmark Plus (BIO-RAD). As shown in fig. 2, chi.mq128, chi.mq155 showed strong binding activity with respect to Muc17ct _ mIgG2aFc in a concentration-dependent manner. Specific binding to Muc17 can be determined from the lack of binding activity exhibited by the control Fc fusion protein.
Example 7 evaluation of binding Activity of anti-Muc 17 antibody by flow cytometry
Binding to the pancreatic cancer cell line AsPc1 was assessed by flow cytometry. Will be at 5 × 105cells/mL suspended in FACS buffer (1% FBS/PBS) were dispensed into Multiscreen-HV Filter Plates (Millipore Corp.) and the supernatant removed by centrifugation. anti-Muc 17 antibody diluted to an appropriate concentration was added and allowed to react for 30 minutes on ice. The cells were washed 1 time with FACS buffer, added with FITC-labeled anti-human IgG antibody, and allowed to react for 30 minutes on ice. After the reaction, the supernatant was removed by centrifugation, suspended in 100. mu. LFACS buffer, and used in a flow cytometer. Flow cytometry used a FACS Calibur (Becton, Dickinson and Company). Histogram using forward scatter (forward scatter) and side scatter (side scatter)A gate (gate) is provided in the living cell population. As shown in fig. 3, chi.mq128, chi.mq155 and AsPc1 cells were firmly bound. Specific binding to Muc17 was concluded from the lack of binding to HepG2 cells that did not express Muc 17.
EXAMPLE 8 determination of antibody-dependent cellular cytotoxicity (ADCC) of anti-Muc 17 antibody
8-1) establishment of full-Length human CD16 constantly expressing cells
Full-length human CD16(RefSeq ID, NM — 000569) was cloned into a mammalian cell expression vector (pMCDN) (pMCDN/CD 16). NK-92 cell lines (CD16-NK92) that continuously express full-length human CD16 were established by electroporation of pMCDN/CD16 into NK-92 cells (purchased from ATCC, CRL-2407) and selection with 500. mu.g/ml geneticin. For the culture of CD16-NK92 cells, alpha-MEM (alpha-MEM) containing 500. mu.g/ml geneticin, penicillin/streptomycin (Invitrogen), 0.2mM myo-inositol (Sigma), 0.1mM 2-mercaptoethanol (Invitrogen), 0.02mM folic acid (Sigma), 100U/ml recombinant human interleukin-2 (Peprotech), 10% horse serum (Invitrogen) and 10% fetal bovine serum (Invitrogen) was used (non-nucleotide and deoxynucleotide, L-glutamine-containing) (Invitrogen).
8-2) measurement of ADCC Activity of anti-Muc 17 antibody
50. mu.l of 8X 10 cells were added to each well of a 96-well flat-bottomed plate4Cells/ml of AspC-1 cells, were cultured in a 5% carbon dioxide incubator at 37 ℃ for 2 days. To each well, 10. mu.l of a solution containing 10% fetal calf serum, penicillin/streptomycin in RPMI 1640 medium (hereinafter, referred to as medium.) was added 240. mu. Ci/ml Cr51(Code No. CJS4, GE Healthcare Biosciences), and the culture was continued for 1 hour. Each well was washed with 300. mu.l of the medium, and then 100. mu.l of the medium was added. Then, 50. mu.L each of an anti-Muc 17 antibody or a control human IgG1 antibody (Cat. No. PHP010, Serotec Co., Ltd.) was added. By starting from 10. mu.g/ml, 3 successive dilutions were carried out with a ratio of 10To adjust the final concentration of the antibody. Then, 50. mu.l of each was added at 1X 106Cells/ml CD16-NK92 cells suspended in culture medium. After the plate was incubated at 37 ℃ for 4 hours in a 5% carbon dioxide incubator, the radioactivity of 100. mu.l of the supernatant was measured by a gamma counter (1480WIZARD 3 ", Wallac). Specific chromium release rates were determined according to the following formula.
Specific chromium release rate (%) - (A-C). times.100/(B-C)
A represents the radioactivity (cpm) of each well, B represents the average value of the radioactivity (cpm) of wells to which 100. mu.l of a 2% NonidetP-40 solution (Code No.252-23, Nacalai Tesque Co., Ltd.) was added, and C represents the average value of the radioactivity (cpm) of wells to which 100. mu.l of a medium was added. The test was performed twice and the mean and standard deviation of the specific chromium release rate was calculated.
ADCC activities of chi.mq128(DG), chi.mq155(DG), chi.mq128(FTKO), and chi.mq155 (FTKO) were measured, and only chi.mq155 (FTKO) showed ADCC activity (fig. 4).
Example 9 antitumor Effect from anti-Muc 17 antibody Using Hum-ZAP
Next, whether or not an immunotoxin targeting Muc17 could exhibit an antitumor effect was evaluated using Hum-ZAP. Hum-ZAP is a substance in which saporin, a protein synthesis inhibitory toxin, is bound to an anti-human IgG antibody, and is manufactured by Advanced Targeting Systems. On the previous day, cells of the cancer cell line Aspc1 expressing Muc17 were seeded at 2000 cells/100. mu.L/well in 96-well culture plates, and a mixture of 100ng of Hum-ZAP and 0, 1, 10, 100ng of anti-Muc 17 chimeric antibody (chi. MQ155) was added. After 72 hours of culture, 10. mu.L of Cell Count Reagent SF (Nacalai Tesque Co.) was added thereto, and absorbance at 450nm was measured after 2 hours. As shown in fig. 5, Hum-Zap alone exhibited no inhibition of proliferation, and the concentration-dependent anti-Muc 17 antibody exhibited anti-tumor effects.
Example 10 epitope resolution of anti-Muc 17 antibody
In order to analyze the epitope of the anti-Muc 17 antibody prepared, a fusion protein of the partial sequence of Muc17 and GST was prepared. PCR amplification was performed, and an EcoRI recognition sequence was added upstream and a SalI recognition sequence was added downstream of the 4115Thr-4390Leu region of Muc17 gene, and the resulting product was cloned into pGEX4T-3 (TAKARA). This was introduced into BL21 to prepare a transformant. The cells were cultured in LB medium, IPTG was added to the cells at 1mM in the logarithmic growth phase, and the cells were cultured at room temperature for 4 hours. Then, the cells were collected and dissolved in B-PER (PIERCE) to prepare an inclusion fraction. Refolding was performed by dissolving the cells in a denaturing buffer (8M urea, 300mM NaCl, 50mM Tris-HCl (pH8.0), 5mM DTT) and replacing the solution with a refolding buffer (50mM Tris-HCl (pH8.0), 300mM NaCl, 1mM EDTA, 5mM DTT). The solubilized GST fusion protein was subjected to affinity purification using a Glutathione Sepharose FF column (GE Co.). After the column was exchanged for PBS using PD-10 column (GE Co.), quantification was performed by DC Protein Assay. GST _ Muc17ct _ del1(4176Ile-4390Leu), GST _ Muc17ct _ del2(4244Ser-4390Leu), GST _ Muc17ct _ del3(4115Thr-4243Gly) were prepared in the same manner (FIG. 6). The purified GST fusion protein was immobilized on a microplate at 1. mu.g/mL. After the blocking treatment, anti-Muc 17 antibody was added at a concentration of 3. mu.g/mL, and ELISA was performed according to the method described above to analyze the epitope. Although MQ155 showing an antitumor effect strongly bound to GST _ Muc17ct, GST _ Muc17ct _ del1, GST _ Muc17ct _ del2, GST _ Muc17ct _ del3 showed no binding activity (fig. 6). From this, it was considered that the epitope of MQ155 was located in a region surrounding the cleavage prediction site in the SEA domain. The secreted form of Muc17 is predicted to exist as a cleaved or spliced variant of the SEA domain. Since MQ155 is not expected to bind to secreted Muc17, it is expected that MQ155 will not be secreted and trapped when administered as a therapeutic antibody and reach cancer cells. Antibodies having such epitopes are considered to be promising therapeutic antibodies.
By the same analysis, it was determined that the epitope of MQ016 is present in the region of 4115-4243 and the epitope of MQ169 is present in the region of 4244-4390.
Sequence listing
<110> future institute for drug creation of Kabushiki Kaisha
<120> anti-Muc 17 antibody
<130>PCG-9021WO
<150>JP 2007-176319
<151>2007-07-04
<160>38
<170>PatentIn version 3.1
<210>1
<211>14360
<212>DNA
<213> human (homo sapiens)
<400>1
tttcgccagc tcctctgggg gtgacaggca agtgagacgt gctcagagct ccgatgccaa 60
ggccagggac catggcgctg tgtctgctga ccttggtcct ctcgctcttg cccccacaag 120
ctgctgcaga acaggacctc agtgtgaaca gggctgtgtg ggatggagga gggtgcatct 180
cccaagggga cgtcttgaac cgtcagtgcc agcagctgtc tcagcacgtt aggacaggtt 240
ctgcggcaaa caccgccaca ggtacaacat ctacaaatgt cgtggagcca agaatgtatt 300
tgagttgcag caccaaccct gagatgacct cgattgagtc cagtgtgact tcagacactc 360
ctggtgtctc cagtaccagg atgacaccaa cagaatccag aacaacttca gaatctacca 420
gtgacagcac cacacttttc cccagttcta ctgaagacac ttcatctcct acaactcctg 480
aaggcaccga cgtgcccatg tcaacaccaa gtgaagaaag catttcatca acaatggctt 540
ttgtcagcac tgcacctctt cccagttttg aggcctacac atctttaaca tataaggttg 600
atatgagcac acctctgacc acttctactc aggcaagttc atctcctact actcctgaaa 660
gcaccaccat acccaaatca actaacagtg aaggaagcac tccattaaca agtatgcctg 720
ccagcaccat gaaggtggcc agttcagagg ctatcaccct tttgacaact cctgttgaaa 780
tcagcacacc tgtgaccatt tctgctcaag ccagttcatc tcctacaact gctgaaggtc 840
ccagcctgtc aaactcagct cctagtggag gaagcactcc attaacaaga atgcctctca 900
gcgtgatgct ggtggtcagt tctgaggcta gcaccctttc aacaactcct gctgccacca 960
acattcctgt gatcacttct actgaagcca gttcatctcc tacaacggct gaaggcacca 1020
gcataccaac ctcaacttat actgaaggaa gcactccatt aacaagtacg cctgccagca 1080
ccatgccggt tgccacttct gaaatgagca cactttcaat aactcctgtt gacaccagca 1140
cacttgtgac cacttctact gaacccagtt cacttcctac aactgctgaa gctaccagca 1200
tgctaacctc aactcttagt gaaggaagca ctccattaac aaatatgcct gtcagcacca 1260
tattggtggc cagttctgag gctagcacca cttcaacaat tcctgttgac tccaaaactt 1320
ttgtgaccac tgctagtgaa gccagctcat ctcccacaac tgctgaagat accagcattg 1380
caacctcaac tcctagtgaa ggaagcactc cattaacaag tatgcctgtc agcaccactc 1440
cagtggccag ttctgaggct agcaaccttt caacaactcc tgttgactcc aaaactcagg 1500
tgaccacttc tactgaagcc agttcatctc ctccaactgc tgaagttaac agcatgccaa 1560
cctcaactcc tagtgaagga agcactccat taacaagtat gtctgtcagc accatgccgg 1620
tggccagttc tgaggctagc accctttcaa caactcctgt tgacaccagc acacctgtga 1680
ccacttctag tgaagccagt tcatcttcta caactcctga aggtaccagc ataccaacct 1740
caactcctag tgaaggaagc actccattaa caaacatgcc tgtcagcacc aggctggtgg 1800
tcagttctga ggctagcacc acttcaacaa ctcctgctga ctccaacact tttgtgacca 1860
cttctagtga agctagttca tcttctacaa ctgctgaagg taccagcatg ccaacctcaa 1920
cttacagtga aagaggcact acaataacaa gtatgtctgt cagcaccaca ctggtggcca 1980
gttctgaggc tagcaccctt tcaacaactc ctgttgactc caacactcct gtgaccactt 2040
caactgaagc cacttcatct tctacaactg cggaaggtac cagcatgcca acctcaactt 2100
atactgaagg aagcactcca ttaacaagta tgcctgtcaa caccacactg gtggccagtt 2160
ctgaggctag caccctttca acaact cctg tgacaccag cacacctgtg accacttcaa 2220
ctgaagccag ttcctctcct acaactgctg atggtgccag tatgccaacc tcaactccta 2280
gtgaaggaag cactccatta acaagtatgc ctgtcagcaa aacgctgttg accagttctg 2340
aggctagcac cctttcaaca actcctcttg acacaagcac acatatcacc acttctactg 2400
aagccagttg ctctcctaca accactgaag gtaccagcat gccaatctca actcctagtg 2460
aaggaagtcc tttattaaca agtatacctg tcagcatcac accggtgacc agtcctgagg 2520
ctagcaccct ttcaacaact cctgttgact ccaacagtcc tgtgaccact tctactgaag 2580
tcagttcatc tcctacacct gctgaaggta ccagcatgcc aacctcaact tatagtgaag 2640
gaagaactcc tttaacaagt atgcctgtca gcaccacact ggtggccact tctgcaatca 2700
gcaccctttc aacaactcct gttgacacca gcacacctgt gaccaattct actgaagccc 2760
gttcgtctcc tacaacttct gaaggtacca gcatgccaac ctcaactcct ggggaaggaa 2820
gcactccatt aacaagtatg cctgacagca ccacgccggt agtcagttct gaggctagaa 2880
cactttcagc aactcctgtt gacaccagca cacctgtgac cacttctact gaagccactt 2940
catctcctac aactgctgaa ggtaccagca taccaacctc gactcctagt gaaggaacga 3000
ctccattaac aagcacacct gtcagccaca cgctggtggc caattctgag gctagcaccc 3060
tttcaacaac tcctgttgac tccaacactc ctttgaccac ttctactgaa gccagttcac 3120
ctcctcccac tgctgaaggt accagcatgc caacctcaac tcctagtgaa ggaagcactc 3180
cattaacacg tatgcctgtc agcaccacaa tggtggccag ttctgaaacg agcacacttt 3240
caacaactcc tgctgacacc agcacacctg tgaccactta ttctcaagcc agttcatctt 3300
ctacaactgc tgacggtacc agcatgccaa cctcaactta tagtgaagga agcactccac 3360
taacaagtgt gcctgtcagc accaggctgg tggtcagttc tgaggctagc accctttcca 3420
caactcctgt cgacaccagc atacctgtca ccacttctac tgaagccagt tcatctccta 3480
caactgctga aggtaccagc ataccaacct cacctcccag tgaaggaacc actccgttag 3540
caagtatgcc tgtcagcacc acgctggtgg tcagttctga ggctaacacc ctttcaacaa 3600
ctcctgtgga ctccaaaact caggtggcca cttctactga agccagttca cctcctccaa 3660
ctgctgaagt taccagcatg ccaacctcaa ctcctggaga aagaagcact ccattaacaa 3720
gtatgcctgt cagacacacg ccagtggcca gttctgaggc tagcaccctt tcaacatctc 3780
ccgttgacac cagcacacct gtgaccactt ctgctgaaac cagttcctct cctacaaccg 3840
ctgaaggtac cagcttgcca acctcaacta ctagtgaagg aagtactcta ttaacaagta 3900
tacctgtcag caccacgctg gtgaccagtc ctgaggctag caccctttta acaactcctg 3960
ttgacactaa aggtcctgtg gtcacttcta atgaagtcag ttcatctcct acacctgctg 4020
aaggtaccag catgccaacc tcaacttata gtgaaggaag aactccttta acaagtatac 4080
ctgtcaacac cacactggtg gccagttctg caatcagcat cctttcaaca actcctgttg 4140
acaacagcac acctgtgacc acttctactg aagcctgttc atctcctaca acttctgaag 4200
gtaccagcat gccaaactca aatcctagtg aaggaaccac tccgttaaca agtatacctg 4260
tcagcaccac gccggtagtc agttctgagg ctagcaccct ttcagcaact cctgttgaca 4320
ccagcacccc tgggaccact tctgctgaag ccacttcatc tcctacaact gctgaaggta 4380
tcagcatacc aacctcaact cctagtgaag gaaagactcc attaaaaagt atacctgtca 4440
gcaacacgcc ggtggccaat tctgaggcta gcaccctttc aacaactcct gttgactcta 4500
acagtcctgt ggtcacttct acagcagtca gttcatctcc tacacctgct gaaggtacca 4560
gcatagcaat ctcaacgcct agtgaaggaa gcactgcatt aacaagtata cctgtcagca 4620
ccacaacagt ggccagttct gaaatcaaca gcctttcaac aactcctgct gtcaccagca 4680
cacctgtgac cacttattct caagccagtt catctcctac aactgctgac ggtaccagca 4740
tgcaaacctc aacttatagt gaaggaagca ctccactaac aagtttgcct gtcagcacca 4800
tgctggtggt cagttctgag gctaacaccc tttcaacaac ccctattgac tccaaaactc 4860
aggtgaccgc ttctactgaa gccagttcat ctacaaccgc tgaaggtagc agcatgacaa 4920
tctcaactcc tagtgaagga agtcctctat taacaagtat acctgtcagc accacgccgg 4980
tggccagtcc tgaggctagc accctttcaa caactcctgt tgactccaac agtcctgtga 5040
tcacttctac tgaagtcagt tcatctccta cacctgctga aggtaccagc atgccaacct 5100
caacttatac tgaaggaaga actcctttaa caagtataac tgtcagaaca acaccggtgg 5160
ccagctctgc aatcagcacc ctttcaacaa ctcccgttga caacagcaca cctgtgacca 5220
cttctactga agcccgttca tctcctacaa cttctgaagg taccagcatg ccaaactcaa 5280
ctcctagtga aggaaccact ccattaacaa gtatacctgt cagcaccacg ccggtactca 5340
gttctgaggc tagcaccctt tcagcaactc ctattgacac cagcacccct gtgaccactt 5400
ctactgaagc cacttcgtct cctacaactg ctgaaggtac cagcatacca acctcgactc 5460
ttagtgaagg aatgactcca ttaacaagca cacctgtcag ccacacgctg gtggccaatt 5520
ctgaggctag caccctttca acaactcctg ttgactctaa cagtcctgtg gtcacttcta 5580
cagcagtcag ttcatctcct acacctgctg aaggtaccag catagcaacc tcaacgccta 5640
gtgaaggaag cactgcatta acaagtatac ctgtcagcac cacaacagtg gccagttctg 5700
aaaccaacac cctttcaaca actcccgctg tcaccagcac acctgtgacc acttatgctc 5760
aagtcagttc atctcctaca actgctgacg gtagcagcat gccaacctca actcctaggg 5820
aaggaaggcc tccattaaca agtatacctg tcagcaccac aacagtggcc agttctgaaa 5880
tcaacaccct ttcaacaact cttgctgaca ccaggacacc tgtgaccact tattctcaag 5940
ccagttcatc tcctacaact gctgatggta ccagcatgcc aaccccagct tatagtgaag 6000
gaagcactcc actaacaagt atgcctctca gcaccacgct ggtggtcagt tctgaggcta 6060
gcactctttc cacaactcct gttgacacca gcactcctgc caccacttct actgaaggca 6120
gttcatctcc tacaactgca ggaggtacca gcatacaaac ctcaactcct agtgaacgga 6180
ccactccatt agcaggtatg cctgtcagca ctacgcttgt ggtcagttct gagggtaaca 6240
ccctttcaac aactcctgtt gactccaaaa ctcaggtgac caattctact gaagccagtt 6300
catctgcaac cgctgaaggt agcagcatga caatctcagc tcctagtgaa ggaagtcctc 6360
tactaacaag tatacctctc agcaccacgc cggtggccag tcctgaggct agcacccttt 6420
caacaactcc tgttgactcc aacagtcctg tgatcacttc tactgaagtc agttcatctc 6480
ctatacctac tgaaggtacc agcatgcaaa cctcaactta tagtgacaga agaactcctt 6540
taacaagtat gcctgtcagc accacagtgg tggccagttc tgcaatcagc accctttcaa 6600
caactcctgt tgacaccagc acacctgtga ccaattctac tgaagcccgt tcatctccta 6660
caacttctga aggtaccagc atgccaacct caactcctag tgaaggaagc actccattca 6720
caagtatgcc tgtcagcacc atgccggtag ttacttctga ggctagcacc ctttcagcaa 6780
ctcctgttga caccagcaca cctgtgacca cttctactga agccacttca tctcctacaa 6840
ctgctgaagg taccagcata ccaacttcaa ctcttagtga aggaacgact ccattaacaa 6900
gtatacctgt cagccacacg ctggtggcca attctgaggt tagcaccctt tcaacaactc 6960
ctgttgactc caacactcct ttcactactt ctactgaagc cagttcacct cctcccactg 7020
ctgaaggtac cagcatgcca acctcaactt ctagtgaagg aaacactcca ttaacacgta 7080
tgcctgtcag caccacaatg gtggccagtt ttgaaacaag cacactttct acaactcctg 7140
ctgacaccag cacacctgtg actacttatt ctcaagccgg ttcatctcct acaactgctg 7200
acgatactag catgccaacc tcaacttata gtgaaggaag cactccacta acaagtgtgc 7260
ctgtcagcac catgccggtg gtcagttctg aggctagcac ccattccaca actcctgttg 7320
acaccagcac acctgtcacc acttctactg aagccagttc atctcctaca actgctgaag 7380
gtaccagcat accaacctca cctcctagtg aaggaaccac tccgttagca agtatgcctg 7440
tcagcaccac gccggtggtc agttctgagg ctggcaccct ttccacaact cctgttgaca 7500
ccagcacacc tatgaccact tctactgaag ccagttcatc tcctacaact gctgaagata 7560
tcgtcgtgcc aatctcaact gctagtgaag gaagtactct attaacaagt atacctgtca 7620
gcaccacgcc agtggccagt cctgaggcta gcaccctttc aacaactcct gttgactcca 7680
acagtcctgt ggtcacttct actgaaatca gttcatctgc tacatccgct gaaggtacca 7740
gcatgcctac ctcaacttat agtgaaggaa gcactccatt aagaagtatg cctgtcagca 7800
ccaagccgtt ggccagttct gaggctagca ctctttcaac aactcctgtt gacaccagca 7860
tacctgtcac cacttctact gaaaccagtt catctcctac aactgcaaaa gataccagca 7920
tgccaatctc aactcctagt gaagtaagta cttcattaac aagtatactt gtcagcacca 7980
tgccagtggc cagttctgag gctagcaccc tttcaacaac tcctgttgac accaggacac 8040
ttgtgaccac ttccactgga accagttcat ctcctacaac tgctgaaggt agcagcatgc 8100
caacctcaac tcctggtgaa agaagcactc cattaacaaa tatacttgtc agcaccacgc 8160
tgttggccaa ttctgaggct agcacccttt caacaactcc tgttgacacc agcacacctg 8220
tcaccacttc tgctgaagcc agttcttctc ctacaactgc tgaaggtacc agcatgcgaa 8280
tctcaactcc tagtgatgga agtactccat taacaagtat acttgtcagc accctgccag 8340
tggccagttc tgaggctagc accgtttcaa caactgctgt tgacaccagc atacctgtca 8400
ccacttctac tgaagccagt tcctctccta caactgctga agttaccagc atgccaacct 8460
caactcctag tgaaacaagt actccattaa ctagtatgcc tgtcaaccac acgccagtgg 8520
ccagttctga ggctggcacc ctttcaacaa ctcctgttga caccagcaca cctgtgacca 8580
cttctactaa agccagttca tctcctacaa ctgctgaagg tatcgtcgtg ccaatctcaa 8640
ctgctagtga aggaagtact ctattaacaa gtatacctgt cagcaccacg ccggtggcca 8700
gttctgaggc tagcaccctt tcaacaactc ctgttgatac cagcatacct gtcaccactt 8760
ctactgaagg cagttcttct cctacaactg ctgaaggtac cagcatgcca atctcaactc 8820
ctagtgaagt aagtactcca ttaacaagta tacttgtcag caccgtgcca gtggccggtt 8880
ctgaggctag caccctttca acaactcctg ttgacaccag gacacctgtc accacttctg 8940
ctgaagctag ttcttctcct acaactgctg aaggtaccag catgccaatc tcaactcctg 9000
gcgaaagaag aactccatta acaagtatgt ctgtcagcac catgccggtg gccagttctg 9060
aggctagcac cctttcaaga actcctgctg acaccagcac acctgtgacc acttctactg 9120
aagccagttc ctctcctaca actgctgaag gtaccggcat accaatctca actcctagtg 9180
aaggaagtac tccattaaca agtatacctg tcagcaccac gccagtggcc attcctgagg 9240
ctagcaccct ttcaacaact cctgttgact ccaacagtcc tgtggtcact tctactgaag 9300
tcagttcatc tcctacacct gctgaaggta ccagcatgcc aatctcaact tatagtgaag 9360
gaagcactcc attaacaggt gtgcctgtca gcaccacacc ggtgaccagt tctgcaatca 9420
gcaccctttc aacaactcct gttgacacca gcacacctgt gaccacttct actgaagccc 9480
attcatctcc tacaacttct gaaggtacca gcatgccaac ctcaactcct agtgaaggaa 9540
gtactccatt aacatatatg cctgtcagca ccatgctggt agtcagttct gaggatagca 9600
ccctttcagc aactcctgtt gacaccagca cacctgtgac cacttctact gaagccactt 9660
catctacaac tgctgaaggt accagcattc caacctcaac tcctagtgaa ggaatgactc 9720
cattaactag tgtacctgtc agcaacacgc cggtggccag ttctgaggct agcatccttt 9780
caacaactcc tgttgactcc aacactcctt tgaccacttc tactgaagcc agttcatctc 9840
ctcccactgc tgaaggtacc agcatgccaa cctcaactcc tagtgaagga agcactccat 9900
taacaagtat gcctgtcagc accacaacgg tggccagttc tgaaacgagc accctttcaa 9960
caactcctgc tgacaccagc acacctgtga ccacttattc tcaagccagt tcatctcctc 10020
caattgctga cggtactagc atgccaacct caacttatag tgaaggaagc actccactaa 10080
caaatatgtc tttcagcacc acgccagtgg tcagttctga ggctagcacc ctttccacaa 10140
ctcctgttga caccagcaca cctgtcacca cttctactga agccagttta tctcctacaa 10200
ctgctgaagg taccagcata ccaacctcaa gtcctagtga aggaaccact ccattagcaa 10260
gtatgcctgt cagcaccacg ccggtggtca gttctgaggt taacaccctt tcaacaactc 10320
ctgtggactc caacactctg gtgaccactt ctactgaagc cagttcatct cctacaatcg 10380
ctgaaggtac cagcttgcca acctcaacta ctagtgaagg aagcactcca ttatcaatta 10440
tgcctctcag taccacgccg gtggccagtt ctgaggctag caccctttca acaactcctg 10500
ttgacaccag cacacctgtg accacttctt ctccaaccaa ttcatctcct acaactgctg 10560
aagttaccag catgccaaca tcaactgctg gtgaaggaag cactccatta acaaatatgc 10620
ctgtcagcac cacaccggtg gccagttctg aggctagcac cctttcaaca actcctgttg 10680
actccaacac ttttgttacc agttctagtc aagccagttc atctccagca actcttcagg 10740
tcaccactat gcgtatgtct actccaagtg aaggaagctc ttcattaaca actatgctcc 10800
tcagcagcac atatgtgacc agttctgagg ctagcacacc ttccactcct tctgttgaca 10860
gaagcacacc tgtgaccact tctactcaga gcaattctac tcctacacct cctgaagtta 10920
tcaccctgcc aatgtcaact cctagtgaag taagcactcc attaaccatt atgcctgtca 10980
gcaccacatc ggtgaccatt tctgaggctg gcacagcttc aacacttcct gttgacacca 11040
gcacacctgt gatcacttct acccaagtca gttcatctcc tgtgactcct gaaggtacca 11100
ccatgccaat ctggacgcct agtgaaggaa gcactccatt aacaactatg cctgtcagca 11160
ccacacgtgt gaccagctct gagggtagca ccctttcaac accttctgtt gtcaccagca 11220
cacctgtgac cacttctact gaagccattt catcttctgc aactcttgac agcaccacca 11280
tgtctgtgtc aatgcccatg gaaataagca cccttgggac cactattctt gtcagtacca 11340
cacctgttac gaggtttcct gagagtagca ccccttccat accatctgtt tacaccagca 11400
tgtctatgac cactgcctct gaaggcagtt catctcctac aactcttgaa ggcaccacca 11460
ccatgcctat gtcaactacg agtgaaagaa gcactttatt gacaactgtc ctcatcagcc 11520
ctatatctgt gatgagtcct tctgaggcca gcacactttc aacacctcct ggtgatacca 11580
gcacaccttt gctcacctct accaaagccg gttcattctc catacctgct gaagtcacta 11640
ccatacgtat ttcaattacc agtgaaagaa gcactccatt aacaactctc cttgtcagca 11700
ccacacttcc aactagcttt cctggggcca gcatagcttc gacacctcct cttgacacaa 11760
gcacaacttt taccccttct actgacactg cctcaactcc cacaattcct gtagccacca 11820
ccatatctgt atcagtgatc acagaaggaa gcacacctgg gacaaccatt tttattccca 11880
gcactcctgt caccagttct actgctgatg tctttcctgc aacaactggt gctgtatcta 11940
cccctgtgat aacttccact gaactaaaca caccatcaac ctccagtagt agtaccacca 12000
catctttttc aactactaag gaatttacaa cacccgcaat gactactgca gctcccctca 12060
catatgtgac catgtctact gcccccagca cacccagaac aaccagcaga ggctgcacta 12120
cttctgcatc aacgctttct gcaaccagta cacctcacac ctctacttct gtcaccaccc 12180
gtcctgtgac cccttcatca gaatccagca ggccgtcaac aattacttct cacaccatcc 12240
cacctacatt tcctcctgct cactccagta cacctccaac aacctctgcc tcctccacga 12300
ctgtgaaccc tgaggctgtc accaccatga ccaccaggac aaaacccagc acacggacca 12360
cttccttccc cacggtgacc accaccgctg tccccacgaa tactacaatt aagagcaacc 12420
ccacctcaac tcctactgtg ccaagaacca caacatgctt tggagatggg tgccagaata 12480
cggcctctcg ctgcaagaat ggaggcacct gggatgggct caagtgccag tgtcccaacc 12540
tctattatgg ggagttgtgt gaggaggtgg tcagcagcat tgacataggg ccaccggaga 12600
ctatctctgc ccaaatggaa ctgactgtga cagtgaccag tgtgaagttc accgaagagc 12660
taaaaaacca ctcttcccag gaattccagg agttcaaaca gacattcacg gaacagatga 12720
atattgtgta ttccgggatc cctgagtatg tcggggtgaa catcacaaag ctacgtcttg 12780
gcagtgtggt ggtggagcat gacgtcctcc taagaaccaa gtacacacca gaatacaaga 12840
cagtattgga caatgccacc gaagtagtga aagagaaaat cacaaaagtg accacacagc 12900
aaataatgat taatgatatt tgctcagaca tgatgtgttt caacaccact ggcacccaag 12960
tgcaaaacat tacggtgacc cagtacgacc ctgaagagga ctgccggaag atggccaagg 13020
aatatggaga ctacttcgta gtggagtacc gggaccagaa gccatactgc atcagcccct 13080
gtgagcctgg cttcagtgtc tccaagaact gtaacctcgg caagtgccag atgtctctaa 13140
gtggacctca gtgcctctgc gtgaccacgg aaactcactg gtacagtggg gagacctgta 13200
accagggcac ccagaagagt ctggtgtacg gcctcgtggg ggcaggggtc gtgctgatgc 13260
tgatcatcct ggtagctctc ctgatgctcg ttttccgctc caagagagag gtgaaacggc 13320
aaaagtacag attgtctcag ttatacaagt ggcaagaaga ggacagtgga ccagctcctg 13380
ggaccttcca aaacattggc tttgacatct gccaagatga tgattccatc cacctggagt 13440
ccatctatag taatttccag ccctccttga gacacataga ccctgaaaca aagatccgaa 13500
ttcagaggcc tcaggtaatg acgacatcat tttaaggcat ggagctgaga agtctgggag 13560
tgaggagatc ccagtccggc taagcttggt ggagcatttt cccattgaga gccttccatg 13620
ggaactcaat gttcccattg taagtacagg aaacaagccc tgtacttacc aaggagaaag 13680
aggagagaca gcagtgctgg gagattctca aatagaaacc cgtggacgct ccaatgggct 13740
tgtcatgata tcaggctagg ctttcctgct catttttcaa agacgctcca gatttgaggg 13800
tactctgact gcaacatctt tcaccccatt gatcgccagg attgatttgg ttgatctggc 13860
tgagcaggcg ggtgtccccg tcctccctca ctgccccata tgtgtccctc ctaaagctgc 13920
atgctcagtt gaagaggacg agaggacgac cttctctgat agaggaggac cacgcttcag 13980
tcaaaggcat acaagtatct atctggactt ccctgctagc acttccaaac aagctcagag 14040
atgttcctcc cctcatctgc ccgggttcag taccatggac agcgccctcg acccgctgtt 14100
tacaaccatg accccttgga cactggactg catgcacttt acatatcaca aaatgctctc 14160
ataagaatta ttgcatacca tcttcatgaa aaacacctgt atttaaatat agagcattta 14220
ccttttggta tataagattg tgggtatttt ttaagttctt attgttatga gttctgattt 14280
tttccttagt aaatattata atatatattt gtagtaacta aaaataataa agcaatttta 14340
ttacaatttt aaaaaaaaaa 14360
<210>2
<211>4493
<212>PRT
<213> human
<400>2
Met Pro Arg Pro Gly Thr Met Ala Leu Cys Leu Leu Thr Leu Val Leu
1 5 10 15
Ser Leu Leu Pro Pro Gln Ala Ala Ala Glu Gln Asp Leu Ser Val Asn
20 25 30
Arg Ala Val Trp Asp Gly Gly Gly Cys Ile Ser Gln Gly Asp Val Leu
35 40 45
Asn Arg Gln Cys Gln Gln Leu Ser Gln His Val Arg Thr Gly Ser Ala
50 55 60
Ala Asn Thr Ala Thr Gly Thr Thr Ser Thr Asn Val Val Glu Pro Arg
65 70 75 80
Met Tyr Leu Ser Cys Ser Thr Asn Pro Glu Met Thr Ser Ile Glu Ser
85 90 95
Ser Val Thr Ser Asp Thr Pro Gly Val Ser Ser Thr Arg Met Thr Pro
100 105 110
Thr Glu Ser Arg Thr Thr Ser Glu Ser Thr Ser Asp Ser Thr Thr Leu
115 120 125
Phe Pro Ser Ser Thr Glu Asp Thr Ser Ser Pro Thr Thr Pro Glu Gly
130 135 140
Thr Asp Val Pro Met Ser Thr Pro Ser Glu Glu Ser Ile Ser Ser Thr
145 150 155 160
Met Ala Phe Val Ser Thr Ala Pro Leu Pro Ser Phe Glu Ala Tyr Thr
165 170 175
Ser Leu Thr Tyr Lys Val Asp Met Ser Thr Pro Leu Thr Thr Ser Thr
180 185 190
Gln Ala Ser Ser Ser Pro Thr Thr Pro Glu Ser Thr Thr Ile Pro Lys
195 200 205
Ser Thr Asn Ser Glu Gly Ser Thr Pro Leu Thr Ser Met Pro Ala Ser
210 215 220
Thr Met Lys Val Ala Ser Ser Glu Ala Ile Thr Leu Leu Thr Thr Pro
225 230 235 240
Val Glu Ile Ser Thr Pro Val Thr Ile Ser Ala Gln Ala Ser Ser Ser
245 250 255
Pro Thr Thr Ala Glu Gly Pro Ser Leu Ser Asn Ser Ala Pro Ser Gly
260 265 270
Gly Ser Thr Pro Leu Thr Arg Met Pro Leu Ser Val Met Leu Val Val
275 280 285
Ser Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Ala Ala Thr Asn Ile
290 295 300
Pro Val Ile Thr Ser Thr Glu Ala Ser Ser Ser Pro Thr Thr Ala Glu
305 310 315 320
Gly Thr Ser Ile Pro Thr Ser Thr Tyr Thr Glu Gly Ser Thr Pro Leu
325 330 335
Thr Ser Thr Pro Ala Ser Thr Met Pro Val Ala Thr Ser Glu Met Ser
340 345 350
Thr Leu Ser Ile Thr Pro Val Asp Thr Ser Thr Leu Val Thr Thr Ser
355 360 365
Thr Glu Pro Ser Ser Leu Pro Thr Thr Ala Glu Ala Thr Ser Met Leu
370 375 380
Thr Ser Thr Leu Ser Glu Gly Ser Thr Pro Leu Thr Asn Met Pro Val
385 390 395 400
Ser Thr Ile Leu Val Ala Ser Ser Glu Ala Ser Thr Thr Ser Thr Ile
405 410 415
Pro Val Asp Ser Lys Thr Phe Val Thr Thr Ala Ser Glu Ala Ser Ser
420 425 430
Ser Pro Thr Thr Ala Glu Asp Thr Ser Ile Ala Thr Ser Thr Pro Ser
435 440 445
Glu Gly Ser Thr Pro Leu Thr Ser Met Pro Val Ser Thr Thr Pro Val
450 455 460
Ala Ser Ser Glu Ala Ser Asn Leu Ser Thr Thr Pro Val Asp Ser Lys
465 470 475 480
Thr Gln Val Thr Thr Ser Thr Glu Ala Ser Ser Ser Pro Pro Thr Ala
485 490 495
Glu Val Asn Ser Met Pro Thr Ser Thr Pro Ser Glu Gly Ser Thr Pro
500 505 510
Leu Thr Ser Met Ser Val Ser Thr Met Pro Val Ala Ser Ser Glu Ala
515 520 525
Ser Thr Leu Ser Thr Thr Pro Val Asp Thr Ser Thr Pro Val Thr Thr
530 535 540
Ser Ser Glu Ala Ser Ser Ser Ser Thr Thr Pro Glu Gly Thr Ser Ile
545 550 555 560
Pro Thr Ser Thr Pro Ser Glu Gly Ser Thr Pro Leu Thr Asn Met Pro
565 570 575
Val Ser Thr Arg Leu Val Val Ser Ser Glu Ala Ser Thr Thr Ser Thr
580 585 590
Thr Pro Ala Asp Ser Asn Thr Phe Val Thr Thr Ser Ser Glu Ala Ser
595 600 605
Ser Ser Ser Thr Thr Ala Glu Gly Thr Ser Met Pro Thr Ser Thr Tyr
610 615 620
Ser Glu Arg Gly Thr Thr Ile Thr Ser Met Ser Val Ser Thr Thr Leu
625 630 635 640
Val Ala Ser Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val Asp Ser
645 650 655
Asn Thr Pro Val Thr Thr Ser Thr Glu Ala Thr Ser Ser Ser Thr Thr
660 665 670
Ala Glu Gly Thr Ser Met Pro Thr Ser Thr Tyr Thr Glu Gly Ser Thr
675 680 685
Pro Leu Thr Ser Met Pro Val Asn Thr Thr Leu Val Ala Ser Ser Glu
690 695 700
Ala Ser Thr Leu Ser Thr Thr Pro Val Asp Thr Ser Thr Pro Val Thr
705 710 715 720
Thr Ser Thr Glu Ala Ser Ser Ser Pro Thr Thr Ala Asp Gly Ala Ser
725 730 735
Met Pro Thr Ser Thr Pro Ser Glu Gly Ser Thr Pro Leu Thr Ser Met
740 745 750
Pro Val Ser Lys Thr Leu Leu Thr Ser Ser Glu Ala Ser Thr Leu Ser
755 760 765
Thr Thr Pro Leu Asp Thr Ser Thr His Ile Thr Thr Ser Thr Glu Ala
770 775 780
Ser Cys Ser Pro Thr Thr Thr Glu Gly Thr Ser Met Pro Ile Ser Thr
785 790 795 800
Pro Ser Glu Gly Ser Pro Leu Leu Thr Ser Ile Pro Val Ser Ile Thr
805 810 815
Pro Val Thr Ser Pro Glu Ala Ser Thr Leu Ser Thr Thr Pro Val Asp
820 825 830
Ser Asn Ser Pro ValTh r Thr Ser Thr Glu Val Ser Ser Ser Pro Thr
835 840 845
Pro Ala Glu Gly Thr Ser Met Pro Thr Ser Thr Tyr Ser Glu Gly Arg
850 855 860
Thr Pro Leu Thr Ser Met Pro Val Ser Thr Thr Leu Val Ala Thr Ser
865 870 875 880
Ala Ile Ser Thr Leu Ser Thr Thr Pro Val Asp Thr Ser Thr Pro Val
885 890 895
Thr Asn Ser Thr Glu Ala Arg Ser Ser Pro Thr Thr Ser Glu Gly Thr
900 905 910
Ser Met Pro Thr Ser Thr Pro Gly Glu Gly Ser Thr Pro Leu Thr Ser
915 920 925
Met Pro Asp Ser Thr Thr Pro Val Val Ser Ser Glu Ala Arg Thr Leu
930 935 940
Ser Ala Thr Pro Val Asp Thr Ser Thr Pro Val Thr Thr Ser Thr Glu
945 950 955 960
Ala Thr Ser Ser Pro Thr Thr Ala Glu Gly Thr Ser Ile Pro Thr Ser
965 970 975
Thr Pro Ser Glu Gly Thr Thr Pro Leu Thr Ser Thr Pro Val Ser His
980 985 990
Thr Leu Val Ala Asn Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val
995 1000 1005
Asp Ser Asn Thr Pro Leu Thr Thr Ser Thr Glu Ala Ser Ser Pro
1010 1015 1020
Pro Pro Thr Ala Glu Gly Thr Ser Met Pro Thr Ser Thr Pro Ser
1025 1030 1035
Glu Gly Ser Thr Pro Leu Thr Arg Met Pro Val Ser Thr Thr Met
1040 1045 1050
Val Ala Ser Ser Glu Thr Ser Thr Leu Ser Thr Thr Pro Ala Asp
1055 1060 1065
Thr Ser Thr Pro Val Thr Thr Tyr Ser Gln Ala Ser Ser Ser Ser
1070 1075 1080
Thr Thr Ala Asp Gly Thr Ser Met Pro Thr Ser Thr Tyr Ser Glu
1085 1090 1095
Gly Ser Thr Pro Leu Thr Ser Val Pro Val Ser Thr Arg Leu Val
1100 1105 1110
Val Ser Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val Asp Thr
1115 1120 1125
Ser Ile Pro Val Thr Thr Ser Thr Glu Ala Ser Ser Ser Pro Thr
1130 1135 1140
Thr Ala Glu Gly Thr Ser Ile Pro Thr Ser Pro Pro Ser Glu Gly
1145 1150 1155
Thr Thr Pro Leu Ala Ser Met Pro Val Ser Thr Thr Leu Val Val
1160 1165 1170
Ser Ser Glu Ala Asn Thr Leu Ser Thr Thr Pro Val Asp Ser Lys
1175 1180 1185
Thr Gln Val Ala Thr Ser Thr Glu Ala Ser Ser Pro Pro Pro Thr
1190 1195 1200
Ala Glu Val Thr Ser Met Pro Thr Ser Thr Pro Gly Glu Arg Ser
1205 1210 1215
Thr Pro Leu Thr Ser Met Pro Val Arg His Thr Pro Val Ala Ser
1220 1225 1230
Ser Glu Ala Ser Thr Leu Ser Thr Ser Pro Val Asp Thr Ser Thr
1235 1240 1245
Pro Val Thr Thr Ser Ala Glu Thr Ser Ser Ser Pro Thr Thr Ala
1250 1255 1260
Glu Gly Thr Ser Leu Pro Thr Ser Thr Thr Ser Glu Gly Ser Thr
1265 1270 1275
Leu Leu Thr Ser Ile Pro Val Ser Thr Thr Leu Val Thr Ser Pro
1280 1285 1290
Glu Ala Ser Thr Leu Leu Thr Thr Pro Val Asp Thr Lys Gly Pro
1295 1300 1305
Val Val Thr Ser Asn Glu Val Ser Ser Ser Pro Thr Pro Ala Glu
1310 1315 1320
Gly Thr Ser Met Pro Thr Ser Thr Tyr Ser Glu Gly Arg Thr Pro
1325 1330 1335
Leu Thr Ser Ile Pro Val Asn Thr Thr Leu Val Ala Ser Ser Ala
1340 1345 1350
Ile Ser Ile Leu Ser Thr Thr Pro Val Asp Asn Ser Thr Pro Val
1355 1360 1365
Thr Thr Ser Thr Glu Ala Cys Ser Ser Pro Thr Thr Ser Glu Gly
1370 1375 1380
Thr Ser Met Pro Asn Ser Asn Pro Ser Glu Gly Thr Thr Pro Leu
1385 1390 1395
Thr Ser Ile Pro Val Ser Thr Thr Pro Val Val Ser Ser Glu Ala
1400 1405 1410
Ser Thr Leu Ser Ala Thr Pro Val Asp Thr Ser Thr Pro Gly Thr
1415 1420 1425
Thr Ser Ala Glu Ala Thr Ser Ser Pro Thr Thr Ala Glu Gly Ile
1430 1435 1440
Ser Ile Pro Thr Ser Thr Pro Ser Glu Gly Lys Thr Pro Leu Lys
1445 1450 1455
Ser Ile Pro Val Ser Asn Thr Pro Val Ala Asn Ser Glu Ala Ser
1460 1465 1470
Thr Leu Ser Thr Thr Pro Val Asp Ser Asn Ser Pro Val Val Thr
1475 1480 1485
Ser Thr Ala Val Ser Ser Ser Pro Thr Pro Ala Glu Gly Thr Ser
1490 1495 1500
Ile Ala Ile Ser Thr Pro Ser Glu Gly Ser Thr Ala Leu Thr Ser
1505 1510 1515
Ile Pro Val Ser Thr Thr Thr Val Ala Ser Ser Glu Ile Asn Ser
1520 1525 1530
Leu Ser Thr Thr Pro Ala Val Thr Ser Thr Pro Val Thr Thr Tyr
1535 1540 1545
Ser Gln Ala Ser Ser Ser Pro Thr Thr Ala Asp Gly Thr Ser Met
1550 1555 1560
Gln Thr Ser Thr Tyr Ser Glu Gly Ser Thr Pro Leu Thr Ser Leu
1565 1570 1575
Pro Val Ser Thr Met Leu Val Val Ser Ser Glu Ala Asn Thr Leu
1580 1585 1590
Ser Thr Thr Pro Ile Asp Ser Lys Thr Gln Val Thr Ala Ser Thr
1595 1600 1605
Glu Ala Ser Ser Ser Thr Thr Ala Glu Gly Ser Ser Met Thr Ile
1610 1615 1620
Ser Thr Pro Ser Glu Gly Ser Pro Leu Leu Thr Ser Ile Pro Val
1625 1630 1635
Ser Thr Thr Pro Val Ala Ser Pro Glu Ala Ser Thr Leu Ser Thr
1640 1645 1650
Thr Pro Val Asp Ser Asn Ser Pro Val Ile Thr Ser Thr Glu Val
1655 1660 1665
Ser Ser Ser Pro Thr Pro Ala Glu Gly Thr Ser Met Pro Thr Ser
1670 1675 1680
Thr Tyr Thr Glu Gly Arg Thr Pro Leu Thr Ser Ile Thr Val Arg
1685 1690 1695
Thr Thr Pro Val Ala Ser Ser Ala Ile Ser Thr Leu Ser Thr Thr
1700 1705 1710
Pro Val Asp Asn Ser Thr Pro Val Thr Thr Ser Thr Glu Ala Arg
1715 1720 1725
Ser Ser Pro Thr Thr Ser Glu GIy Thr Ser Met Pro Asn Ser Thr
1730 1735 1740
Pro Ser Glu Gly Thr Thr Pro Leu Thr Ser Ile Pro Val Ser Thr
1745 1750 1755
Thr Pro Val Leu Ser Ser Glu Ala Ser Thr Leu Ser Ala Thr Pro
1760 1765 1770
Ile Asp Thr Ser Thr Pro Val Thr Thr Ser Thr Glu Ala Thr Ser
1775 1780 1785
Ser Pro Thr Thr Ala Glu Gly Thr Ser Ile Pro Thr Ser Thr Leu
1790 1795 1800
Ser Glu Gly Met Thr Pro Leu Thr Ser Thr Pro Val Ser His Thr
1805 1810 1815
Leu Val Ala Asn Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val
1820 1825 1830
Asp Ser Asn Ser Pro Val Val Thr Ser Thr Ala Val Ser Ser Ser
1835 1840 1845
Pro Thr Pro Ala Glu Gly Thr Ser Ile Ala Thr Ser Thr Pro Ser
1850 1855 1860
Glu Gly Ser Thr Ala Leu Thr Ser Ile Pro Val Ser Thr Thr Thr
1865 1870 1875
Val Ala Ser Ser Glu Thr Asn Thr Leu Ser Thr Thr Pro Ala Val
1880 1885 1890
Thr Ser Thr Pro Val Thr Thr Tyr Ala Gln Val Ser Ser Ser Pro
1895 1900 1905
Thr Thr Ala Asp Gly Ser Ser Met Pro Thr Ser Thr Pro Arg Glu
1910 1915 1920
Gly Arg Pro Pro Leu Thr Ser Ile Pro Val Ser Thr Thr Thr Val
1925 1930 1935
Ala Ser Ser Glu Ile Asn Thr Leu Ser Thr Thr Leu Ala Asp Thr
1940 1945 1950
Arg Thr Pro Val Thr Thr Tyr Ser Gln Ala Ser Ser Ser Pro Thr
1955 1960 1965
Thr Ala Asp Gly Thr Ser Met Pro Thr Pro Ala Tyr Ser Glu Gly
1970 1975 1980
Ser Thr Pro Leu Thr Ser Met Pro Leu Ser Thr Thr Leu Val Val
1985 1990 1995
Ser Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val Asp Thr Ser
2000 2005 2010
Thr Pro Ala Thr Thr Ser Thr Glu Gly Ser Ser Ser Pro Thr Thr
2015 2020 2025
Ala Gly Gly Thr Ser Ile Gln Thr Ser Thr Pro Ser Glu Arg Thr
2030 2035 2040
Thr Pro Leu Ala Gly Met Pro Val Ser Thr Thr Leu Val Val Ser
2045 2050 2055
Ser Glu Gly Asn Thr Leu Ser Thr Thr Pro Val Asp Ser Lys Thr
2060 2065 2070
Gln Val Thr Asn Ser Thr Glu Ala Ser Ser Ser Ala Thr Ala Glu
2075 2080 2085
Gly Ser Ser Met Thr Ile Ser Ala Pro Ser Glu Gly Ser Pro Leu
2090 2095 2100
Leu Thr Ser Ile Pro Leu Ser Thr Thr Pro Val Ala Ser Pro Glu
2105 2110 2115
Ala Ser Thr Leu Ser Thr Thr Pro Val Asp Ser Asn Ser Pro Val
2120 2125 2130
Ile Thr Ser Thr Glu Val Ser Ser Ser Pro Ile Pro Thr Glu Gly
2135 2140 2145
Thr Ser Met Gln Thr Ser Thr Tyr Ser Asp Arg Arg Thr Pro Leu
2150 2155 2160
Thr Ser Met Pro Val Ser Thr Thr Val Val Ala Ser Ser Ala Ile
2165 2170 2175
Ser Thr Leu Ser Thr Thr Pro Val Asp Thr Ser Thr Pro Val Thr
2180 2185 2190
Asn Ser Thr Glu Ala Arg Ser Ser Pro Thr Thr Ser Glu Gly Thr
2195 2200 2205
Ser Met Pro Thr Ser Thr Pro Ser Glu Gly Ser Thr Pro Phe Thr
2210 2215 2220
Ser Met Pro Val Ser Thr Met Pro Val Val Thr Ser Glu Ala Ser
2225 2230 2235
Thr Leu Ser Ala Thr Pro Val Asp Thr Ser Thr Pro Val Thr Thr
2240 2245 2250
Ser Thr Glu Ala Thr Ser Ser Pro Thr Thr Ala Glu Gly Thr Ser
2255 2260 2265
Ile Pro Thr Ser Thr Leu Ser Glu Gly Thr Thr Pro Leu Thr Ser
2270 2275 2280
Ile Pro Val Ser His Thr Leu Val Ala Asn Ser Glu Val Ser Thr
2285 2290 2295
Leu Ser Thr Thr Pro Val Asp Ser Asn Thr Pro Phe Thr Thr Ser
2300 2305 2310
Thr Glu Ala Ser Ser Pro Pro Pro Thr Ala Glu Gly Thr Ser Met
2315 2320 2325
Pro Thr Ser Thr Ser Ser Glu Gly Asn Thr Pro Leu Thr Arg Met
2330 2335 2340
Pro Val Ser Thr Thr Met Val Ala Ser Phe Glu Thr Ser Thr Leu
2345 2350 2355
Ser Thr Thr Pro Ala Asp Thr Ser Thr Pro Val Thr Thr Tyr Ser
2360 2365 2370
Gln Ala Gly Ser Ser Pro Thr Thr Ala Asp Asp Thr Ser Met Pro
2375 2380 2385
Thr Ser Thr Tyr Ser Glu Gly Ser Thr Pro Leu Thr Ser Val Pro
2390 2395 2400
Val Ser Thr Met Pro Val Val Ser Ser Glu Ala Ser Thr His Ser
2405 2410 2415
Thr Thr Pro Val Asp Thr Ser Thr Pro Val Thr Thr Ser Thr Glu
2420 2425 2430
Ala Ser Ser Ser Pro Thr Thr Ala Glu Gly Thr Ser Ile Pro Thr
2435 2440 2445
Ser Pro Pro Ser Glu Gly Thr Thr Pro Leu Ala Ser Met Pro Val
2450 2455 2460
Ser Thr Thr Pro Val Val Ser Ser Glu Ala Gly Thr Leu Ser Thr
2465 2470 2475
Thr Pro Val Asp Thr Ser Thr Pro Met Thr Thr Ser Thr Glu Ala
2480 2485 2490
Ser Ser Ser Pro Thr Thr Ala Glu Asp Ile Val Val Pro Ile Ser
2495 2500 2505
Thr Ala Ser Glu Gly Ser Thr Leu Leu Thr Ser Ile Pro Val Ser
2510 2515 2520
Thr Thr Pro Val Ala Ser Pro Glu Ala Ser Thr Leu Ser Thr Thr
2525 2530 2535
Pro Val Asp Ser Asn Ser Pro Val Val Thr Ser Thr Glu Ile Ser
2540 2545 2550
Ser Ser Ala Thr Ser Ala Glu Gly Thr Ser Met Pro Thr Ser Thr
2555 2560 2565
Tyr Ser Glu Gly Ser Thr Pro Leu Arg Ser Met Pro Val Ser Thr
2570 2575 2580
Lys Pro Leu Ala Ser Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro
2585 2590 2595
Val Asp Thr Ser Ile Pro Val Thr Thr Ser Thr Glu Thr Ser Ser
2600 2605 2610
Ser Pro Thr Thr Ala Lys Asp Thr Ser Met Pro Ile Ser Thr Pro
2615 2620 2625
Ser Glu Val Ser Thr Ser Leu Thr Ser Ile Leu Val Ser Thr Met
2630 2635 2640
Pro Val Ala Ser Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val
2645 2650 2655
Asp Thr Arg Thr Leu Val Thr Thr Ser Thr Gly Thr Ser Ser Ser
2660 2665 2670
Pro Thr Thr Ala Glu Gly Ser Ser Met Pro Thr Ser Thr Pro Gly
2675 2680 2685
Glu Arg Ser Thr Pro Leu Thr Asn Ile Leu Val Ser Thr Thr Leu
2690 2695 2700
Leu Ala Asn Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val Asp
2705 2710 2715
Thr Ser Thr Pro Val Thr Thr Ser Ala Glu Ala Ser Ser Ser Pro
2720 2725 2730
Thr Thr Ala Glu Gly Thr Ser Met Arg Ile Ser Thr Pro Ser Asp
2735 2740 2745
Gly Ser Thr Pro Leu Thr Ser Ile Leu Val Ser Thr Leu Pro Val
2750 2755 2760
Ala Ser Ser Glu Ala Ser Thr Val Ser Thr Thr Ala Val Asp Thr
2765 2770 2775
Ser Ile Pro Val Thr Thr Ser Thr Glu Ala Ser Ser Ser Pro Thr
2780 2785 2790
Thr Ala Glu Val Thr Ser Met Pro Thr Ser Thr Pro Ser Glu Thr
2795 2800 2805
Ser Thr Pro Leu Thr Ser Met Pro Val Asn His Thr Pro Val Ala
2810 2815 2820
Ser Ser Glu Ala Gly Thr Leu Ser Thr Thr Pro Val Asp Thr Ser
2825 2830 2835
Thr Pro Val Thr Thr Ser Thr Lys Ala Ser Ser Ser Pro Thr Thr
2840 2845 2850
Ala Glu Gly Ile Val Val Pro Ile Ser Thr Ala Ser Glu Gly Ser
2855 2860 2865
Thr Leu Leu Thr Ser Ile Pro Val Ser Thr Thr Pro Val Ala Ser
2870 2875 2880
Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val Asp Thr Ser Ile
2885 2890 2895
Pro Val Thr Thr Ser Thr Glu Gly Ser Ser Ser Pro Thr Thr Ala
2900 2905 2910
Glu Gly Thr Ser Met Pro Ile Ser Thr Pro Ser Glu Val Ser Thr
2915 2920 2925
Pro Leu Thr Ser Ile Leu Val Ser Thr Val Pro Val Ala Gly Ser
2930 2935 2940
Glu Ala Ser Thr Leu Ser Thr Thr Pro Val Asp Thr Arg Thr Pro
2945 2950 2955
Val Thr Thr Ser Ala Glu Ala Ser Ser Ser Pro Thr Thr Ala Glu
2960 2965 2970
Gly Thr Ser Met Pro Ile Ser Thr Pro Gly Glu Arg Arg Thr Pro
2975 2980 2985
Leu Thr Ser Met Ser Val Ser Thr Met Pro Val Ala Ser Ser Glu
2990 2995 3000
Ala Ser Thr Leu Ser Arg Thr Pro Ala Asp Thr Ser Thr Pro Val
3005 3010 3015
Thr Thr Ser Thr Glu Ala Ser Ser Ser Pro Thr Thr Ala Glu Gly
3020 3025 3030
Thr Gly Ile Pro Ile Ser Thr Pro Ser Glu Gly Ser Thr Pro Leu
3035 3040 3045
Thr Ser Ile Pro Val Ser Thr Thr Pro Val Ala Ile Pro Glu Ala
3050 3055 3060
Ser Thr Leu Ser Thr Thr Pro Val Asp Ser Asn Ser Pro Val Val
3065 3070 3075
Thr Ser Thr Glu Val Ser Ser Ser Pro Thr Pro Ala Glu Gly Thr
3080 3085 3090
Ser Met Pro Ile Ser Thr Tyr Ser Glu Gly Ser Thr Pro Leu Thr
3095 3100 3105
Gly Val Pro Val Ser Thr Thr Pro Val Thr Ser Ser Ala Ile Ser
3110 3115 3120
Thr Leu Ser Thr Thr Pro Val Asp Thr Ser Thr Pro Val Thr Thr
3125 3130 3135
Ser Thr Glu Ala His Ser Ser Pro Thr Thr Ser Glu Gly Thr Ser
3140 3145 3150
Met Pro Thr Ser Thr Pro Ser Glu Gly Ser Thr Pro Leu Thr Tyr
3155 3160 3165
Met Pro Val Ser Thr Met Leu Val Val Ser Ser Glu Asp Ser Thr
3170 3175 3180
Leu Ser Ala Thr Pro Val Asp Thr Ser Thr Pro Val Thr Thr Ser
3185 3190 3195
Thr Glu Ala Thr Ser Ser Thr Thr Ala Glu Gly Thr Ser Ile Pro
3200 3205 3210
Thr Ser Thr Pro Ser Glu Gly Met Thr Pro Leu Thr Ser Val Pro
3215 3220 3225
Val Ser Asn Thr Pro Val Ala Ser Ser Glu Ala Ser Ile Leu Ser
3230 3235 3240
Thr Thr Pro Val Asp Ser Asn Thr Pro Leu Thr Thr Ser Thr Glu
3245 3250 3255
Ala Ser Ser Ser Pro Pro Thr Ala Glu Gly Thr Ser Met Pro Thr
3260 3265 3270
Ser Thr Pro Ser Glu Gly Ser Thr Pro Leu Thr Ser Met Pro Val
3275 3280 3285
Ser Thr Thr Thr Val Ala Ser Ser Glu Thr Ser Thr Leu Ser Thr
3290 3295 3300
Thr Pro Ala Asp Thr Ser Thr Pro Val Thr Thr Tyr Ser Gln Ala
3305 3310 3315
Ser Ser Ser Pro Pro Ile Ala Asp Gly Thr Ser Met Pro Thr Ser
3320 3325 3330
Thr Tyr Ser Glu Gly Ser Thr Pro Leu Thr Asn Met Ser Phe Ser
3335 3340 3345
Thr Thr Pro Val Val Ser Ser Glu Ala Ser Thr Leu Ser Thr Thr
3350 3355 3360
Pro Val Asp Thr Ser Thr Pro Val Thr Thr Ser Thr Glu Ala Ser
3365 3370 3375
Leu Ser Pro Thr Thr Ala Glu Gly Thr Ser Ile Pro Thr Ser Ser
3380 3385 3390
Pro Ser Glu Gly Thr Thr Pro Leu Ala Ser Met Pro Val Ser Thr
3395 3400 3405
Thr Pro Val Val Ser Ser Glu Val Asn Thr Leu Ser Thr Thr Pro
3410 3415 3420
Val Asp Ser Asn Thr Leu Val Thr Thr Ser Thr Glu Ala Ser Ser
3425 3430 3435
Ser Pro Thr Ile Ala Glu Gly Thr Ser Leu Pro Thr Ser Thr Thr
3440 3445 3450
Ser Glu Gly Ser Thr Pro Leu Ser Ile Met Pro Leu Ser Thr Thr
3455 3460 3465
Pro Val Ala Ser Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val
3470 3475 3480
Asp Thr Ser Thr Pro Val Thr Thr Ser Ser Pro Thr Asn Ser Ser
3485 3490 3495
Pro Thr Thr Ala Glu Val Thr Ser Met Pro Thr Ser Thr Ala Gly
3500 3505 3510
Glu Gly Ser Thr Pro Leu Thr Asn Met Pro Val Ser Thr Thr Pro
3515 3520 3525
Val Ala Ser Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val Asp
35303535 3540
Ser Asn Thr Phe Val Thr Ser Ser Ser Gln Ala Ser Ser Ser Pro
3545 3550 3555
Ala Thr Leu Gln Val Thr Thr Met Arg Met Ser Thr Pro Ser Glu
3560 3565 3570
Gly Ser Ser Ser Leu Thr Thr Met Leu Leu Ser Ser Thr Tyr Val
3575 3580 3585
Thr Ser Ser Glu Ala Ser Thr Pro Ser Thr Pro Ser Val Asp Arg
3590 3595 3600
Ser Thr Pro Val Thr Thr Ser Thr Gln Ser Asn Ser Thr Pro Thr
3605 3610 3615
Pro Pro Glu Val Il e Thr Leu Pro MetSer Thr Pro Ser Glu Val
3620 3625 3630
Ser Thr Pro Leu Thr Ile Met Pro Val Ser Thr Thr Ser Val Thr
3635 3640 3645
Ile Ser Glu Ala Gly Thr Ala Ser Thr Leu Pro Val Asp Thr Ser
3650 3655 3660
Thr Pro Val Ile Thr Ser Thr Gln Val Ser Ser Ser Pro Val Thr
3665 3670 3675
Pro Glu Gly Thr Thr Met Pro Ile Trp Thr Pro Ser Glu Gly Ser
3680 3685 3690
Thr Pro Leu Thr Thr Met Pro Val Ser Thr Thr Arg Val Thr Ser
3695 3700 3705
Ser Glu Gly Ser Thr Leu Ser Thr Pro Ser Val Val Thr Ser Thr
3710 3715 3720
Pro Val Thr Thr Ser Thr Glu Ala Ile Ser Ser Ser Ala Thr Leu
3725 3730 3735
Asp Ser Thr Thr Met Ser Val Ser Met Pro Met Glu Ile Ser Thr
3740 3745 3750
Leu Gly Thr Thr Ile Leu Val Ser Thr Thr Pro Val Thr Arg Phe
3755 3760 3765
Pro Glu Ser Ser Thr Pro Ser Ile Pro Ser Val Tyr Thr Ser Met
3770 3775 3780
Ser Met Thr Thr Ala Ser Glu Gly Ser Ser Ser Pro Thr Thr Leu
3785 3790 3795
Glu Gly Thr Thr Thr Met Pro Met Ser Thr Thr Ser Glu Arg Ser
3800 3805 3810
Thr Leu Leu Thr Thr Val Leu Ile Ser Pro Ile Ser Val Met Ser
3815 3820 3825
Pro Ser Glu Ala Ser Thr Leu Ser Thr Pro Pro Gly Asp Thr Ser
3830 3835 3840
Thr Pro Leu Leu Thr Ser Thr Lys Ala Gly Ser Phe Ser Ile Pro
3845 3850 3855
Ala Glu Val Thr Thr Ile Arg Ile Ser Ile Thr Ser Glu Arg Ser
3860 3865 3870
Thr Pro Leu Thr Thr Leu Leu Val Ser Thr Thr Leu Pro Thr Ser
3875 3880 3885
Phe Pro Gly Ala Ser Ile Ala Ser Thr Pro Pro Leu Asp Thr Ser
3890 3895 3900
Thr Thr Phe Thr Pro Ser Thr Asp Thr Ala Ser Thr Pro Thr Ile
3905 3910 3915
Pro Val Ala ThrThr Ile Ser Val Ser Val Ile Thr Glu Gly Ser
3920 3925 3930
Thr Pro Gly Thr Thr Ile Phe Ile Pro Ser Thr Pro Val Thr Ser
3935 3940 3945
Ser Thr Ala Asp Val Phe Pro Ala Thr Thr Gly Ala Val Ser Thr
3950 3955 3960
Pro Val Ile Thr Ser Thr Glu Leu Asn Thr Pro Ser Thr Ser Ser
3965 3970 3975
Ser Ser Thr Thr Thr Ser Phe Ser Thr Thr Lys Glu Phe Thr Thr
3980 3985 3990
Pro Ala Met Thr Thr Ala Ala Pro Leu Thr Tyr Val Thr Met Ser
3995 4000 4005
Thr Ala Pro Ser Thr Pro Arg Thr Thr Ser Arg Gly Cys Thr Thr
4010 4015 4020
Ser Ala Ser Thr Leu Ser Ala Thr Ser Thr Pro His Thr Ser Thr
4025 4030 4035
Ser Val Thr Thr Arg Pro Val Thr Pro Ser Ser Glu Ser Ser Arg
4040 4045 4050
Pro Ser Thr Ile Thr Ser His Thr Ile Pro Pro Thr Phe Pro Pro
4055 4060 4065
Ala His Ser Ser Thr Pro Pro Thr Thr Ser Ala Ser Ser Thr Thr
4070 4075 4080
Val Asn Pro Glu Ala Val Thr Thr Met Thr Thr Arg Thr Lys Pro
4085 4090 4095
Ser Thr Arg Thr Thr Ser Phe Pro Thr Val Thr Thr Thr Ala Val
4100 4105 4110
Pro Thr Asn Thr Thr Ile Lys Ser Asn Pro Thr Ser Thr Pro Thr
4115 4120 4125
Val Pro Arg Thr Thr Thr Cys Phe Gly Asp Gly Cys Gln Asn Thr
4130 4135 4140
Ala Ser Arg Cys Lys Asn Gly Gly Thr Trp Asp Gly Leu Lys Cys
4145 4150 4155
Gln Cys Pro Asn Leu Tyr Tyr Gly Glu Leu Cys Glu Glu Val Val
4160 4165 4170
Ser Ser Ile Asp Ile Gly Pro Pro Glu Thr Ile Ser Ala Gln Met
4175 4180 4185
Glu Leu Thr Val Thr Val Thr Ser Val Lys Phe Thr Glu Glu Leu
4190 4195 4200
Lys Asn His Ser Ser Gln Glu Phe Gln Glu Phe Lys Gln Thr Phe
4205 4210 4215
Thr Glu Gln Met Asn Ile Val Tyr Ser Gly Ile Pro Glu Tyr Val
4220 4225 4230
Gly Val Asn Ile Thr Lys Leu Arg Leu Gly Ser Val Val Val Glu
4235 4240 4245
His Asp Val Leu Leu Arg Thr Lys Tyr Thr Pro Glu Tyr Lys Thr
4250 4255 4260
Val Leu Asp Asn Ala Thr Glu Val Val Lys Glu Lys Ile Thr Lys
4265 4270 4275
Val Thr Thr Gln Gln Ile Met Ile Asn Asp Ile Cys Ser Asp Met
4280 4285 4290
Met Cys Phe Asn Thr Thr Gly Thr Gln Val Gln Asn Ile Thr Val
4295 4300 4305
Thr Gln Tyr Asp Pro Glu Glu Asp Cys Arg Lys Met Ala Lys Glu
4310 4315 4320
Tyr Gly Asp Tyr Phe Val Val Glu Tyr Arg Asp Gln Lys Pro Tyr
4325 4330 4335
Cys Ile Ser Pro Cys Glu Pro Gly Phe Ser Val Ser Lys Asn Cys
4340 4345 4350
Asn Leu Gly Lys Cys Gln Met Ser Leu Ser Gly Pro Gln Cys Leu
4355 4360 4365
Cys Val Thr Thr Glu Thr His Trp Tyr Ser Gly Glu Thr Cys Asn
4370 4375 4380
Gln Gly Thr Gln Lys Ser Leu Val Tyr Gly Leu Val Gly Ala Gly
4385 4390 4395
Val Val Leu Met Leu Ile Ile Leu Val Ala Leu Leu Met Leu Val
4400 4405 4410
Phe Arg Ser Lys Arg Glu Val Lys Arg Gln Lys Tyr Arg Leu Ser
4415 4420 4425
Gln Leu Tyr Lys Trp Gln Glu Glu Asp Ser Gly Pro Ala Pro Gly
4430 4435 4440
Thr Phe Gln Asn Ile Gly Phe Asp Ile Cys Gln Asp Asp Asp Ser
4445 4450 4455
Ile His Leu Glu Ser Ile Tyr Ser Asn Phe Gln Pro Ser Leu Arg
4460 4465 4470
His Ile Asp Pro Glu Thr Lys Ile Arg Ile Gln Arg Pro Gln Val
4475 4480 4485
Met Thr Thr Ser Phe
4490
<210>3
<211>215
<212>PRT
<213> human
<400>3
Ile Asp Ile Gly Pro Pro Glu Thr Ile Ser Ala Gln Met Glu Leu Thr
1 5 10 15
Val Thr Val Thr Ser Val Lys Phe Thr Glu Glu Leu Lys Asn His Ser
20 25 30
Ser Gln Glu Phe Gln Glu Phe Lys Gln Thr Phe Thr Glu Gln Met Asn
35 40 45
Ile Val Tyr Ser Gly Ile Pro Glu Tyr Val Gly Val Ash Ile Thr Lys
50 55 60
Leu Arg Leu Gly Ser Val Val Val Glu His Asp Val Leu Leu Arg Thr
65 70 75 80
Lys Tyr Thr Pro Glu Tyr Lys Thr Val Leu Asp Asn Ala Thr Glu Val
85 90 95
Val Lys Glu Lys Ile Thr Lys Val Thr Thr Gln Gln Ile Met Ile Asn
100 105 110
Asp Ile Cys Ser Asp Met Met Cys Phe Asn Thr Thr Gly Thr Gln Val
115 120 125
Gln Asn Ile Thr Val Thr Gln Tyr Asp Pro Glu Glu Asp Cys Arg Lys
130 135 140
Met Ala Lys Glu Tyr Gly Asp Tyr Phe Val Val Glu Tyr Arg Asp Gln
145 150 155 160
Lys Pro Tyr Cys Ile Ser Pro Cys Glu Pro Gly Phe Ser Val Ser Lys
165 170 175
Asn Cys Asn Leu Gly Lys Cys Gln Met Ser Leu Ser Gly Pro Gln Cys
180 185 190
Leu Cys Val Thr Thr Glu Thr His Trp Tyr Ser Gly Glu Thr Cys Asn
195 200 205
Gln Gly Thr Gln Lys Ser Leu
210 215
<210>4
<211>147
<212>PRT
<213> human
<400>4
Ser Val Val Val Glu His Asp Val Leu Leu Arg Thr Lys Tyr Thr Pro
1 5 10 15
Glu Tyr Lys Thr Val Leu Asp Asn Ala Thr Glu Val Val Lys Glu Lys
20 25 30
Ile Thr Lys Val Thr Thr Gln Gln Ile Met Ile Asn Asp Ile Cys Ser
35 40 45
Asp Met Met Cys Phe Asn Thr Thr Gly Thr Gln Val Gln Asn Ile Thr
50 55 60
Val Thr Gln Tyr Asp Pro Glu Glu Asp Cys Arg Lys Met Ala Lys Glu
65 70 75 80
Tyr Gly Asp Tyr Phe Val Val Glu Tyr Arg Asp Gln Lys Pro Tyr Cys
85 90 95
Ile Ser Pro Cys Glu Pro Gly Phe Ser Val Ser Lys Asn Cys Asn Leu
100 105 110
Gly Lys Cys Gln Met Ser Leu Ser Gly Pro Gln Cys Leu Cys Val Thr
115 120 125
Thr Glu Thr His Trp Tyr Ser Gly Glu Thr Cys Asn Gln Gly Thr Gln
130 135 140
Lys Ser Leu
145
<210>5
<211>129
<212>PRT
<213> human
<400>5
Thr Asn Thr Thr Ile Lys Ser Asn Pro Thr Ser Thr Pro Thr Val Pro
1 5 10 15
Arg Thr Thr Thr Cys Phe Gly Asp Gly Cys Gln Ash Thr Ala Ser Arg
20 25 30
Cys Lys Asn Gly Gly Thr Trp Asp Gly Leu Lys Cys Gln Cys Pro Asn
35 40 45
Leu Tyr Tyr Gly Glu Leu Cys Glu Glu Val Val Ser Ser Ile Asp Ile
50 55 60
Gly Pro Pro Glu Thr Ile Ser Ala Gln Met Glu Leu Thr Val Thr Val
65 70 75 80
Thr Ser Val Lys Phe Thr Glu Glu Leu Lys Asn His Ser Ser Gln Glu
85 90 95
Phe Gln Glu Phe Lys Gln Thr Phe Thr Glu Gln Met Asn Ile Val Tyr
100 105 110
Ser Gly Ile Pro Glu Tyr Val Gly Val Asn Ile Thr Lys Leu Arg Leu
115 120 125
Gly
<210>6
<211>5
<212>PRT
<213> mouse (Mus musculus)
<400>6
Ser His Trp Met Asn
1 5
<210>7
<211>17
<212>PRT
<213> mice
<400>7
Gln Ile Tyr Pro Gly Asp Gly Asp Ile Asn Tyr Asn Gly Lys Phe Lys
1 5 10 15
Gly
<210>8
<211>8
<212>PRT
<213> mice
<400>8
His Tyr Asn Tyr Ala Met Asp Tyr
1 5
<210>9
<211>10
<212>PRT
<213> mice
<400>9
Ser Ala Ser Ser Ser Val Ser Tyr Met Cys
1 5 10
<210>10
<211>7
<212>PRT
<213> mice
<400>10
Arg Thr Ser Asn Leu Ala Ser
1 5
<210>11
<211>9
<212>PRT
<213> mice
<400>11
Gln Gln Phe His Ser Tyr Pro Arg Thr
1 5
<210>12
<211>5
<212>PRT
<213> mice
<400>12
Thr Tyr Trp Met Asn
1 5
<210>13
<211>17
<212>PRT
<213> mice
<400>13
Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys
1 5 10 15
Asp
<210>14
<211>12
<212>PRT
<213> mice
<400>14
Gly Pro Tyr Tyr Gly Thr Asn Pro Trp Phe Pro Tyr
1 5 10
<210>15
<211>11
<212>PRT
<213> mice
<400>15
Arg Ala Ser Lys Ser Ile Ser Lys Tyr Leu Ala
1 5 10
<210>16
<211>7
<212>PRT
<213> mice
<400>16
Ser Gly Ser Thr Leu Gln Ser
1 5
<210>17
<211>9
<212>PRT
<213> mice
<400>17
Gln Gln His His Glu Tyr Pro Tyr Thr
1 5
<210>18
<211>351
<212>DNA
<213> mice
<400>18
caggttcagc tgcagcagtc tggggctgag ctggtgaggc ctgggtcctc agcgaagatt 60
tcctgtaagg cttctggcta tgaattcagt agccactgga tgaactgggt gaagcagagg 120
cctggacagg gtcttgagtg gattggacag atctatcctg gagatggtga tattaactac 180
aatggaaagt tcaagggtaa agccacactg actgcagaca aatcctccag tacagtctac 240
atgcagctca gcagcctaac atctgaggac tctgcggtct atttctgtgc aagacattat 300
aactatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc a 351
<210>19
<211>117
<212>PRT
<213> mice
<400>19
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser
1 5 10 15
Ser Ala Lys Ile Ser Cys Lys Ala Ser Gly Tyr Glu Phe Ser Ser His
20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gln Ile Tyr Pro Gly Asp Gly Asp Ile Asn Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg His Tyr Asn Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser
115
<210>20
<211>318
<212>DNA
<213> mice
<400>20
caaattgttc tcacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60
atatcctgca gtgccagctc aagtgtaagt tacatgtgct ggtatcagca gaagccagga 120
tcctctccca aaccctggat ttatcgcaca tccaacctgg cttctggagt ccctgctcgc 180
ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 240
gatgctgcca cttattactg ccagcagttt catagttacc cacggacgtt cggtggaggc 300
accaagctgg aaatcaaa 318
<210>21
<211>106
<212>PRT
<213> mice
<400>21
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Ile Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
Cys Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Phe His Ser Tyr Pro Arg Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210>22
<211>363
<212>DNA
<213> mice
<400>22
caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggggcttc agtgaagttg 60
tcctgcaagg cttctggcta cacgttcacc acctactgga tgaactgggt taagcagagg 120
cctgagcaag gccttgagtg gattggaagg attgatcctt acgatagtga aactcactac 180
aatcaaaagt tcaaggacaa ggccatattg actgtagaca aatcctccag cacagcctac 240
atgcaactca gcagcctgac atctgaggac tctgcggtct attactgtgc aaggggacct 300
tactacggta ctaacccctg gtttccttac tggggccaag ggactctggt cactgtctct 360
tca 363
<210>23
<211>121
<212>PRT
<213> mice
<400>23
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr
20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Tyr Gly Thr Asn Pro Trp Phe Pro Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210>24
<211>321
<212>DNA
<213> mice
<400>24
gatgtccaga taacccagtc tccatcttat cttgctgcat ctcctggaga aaccattact 60
attaattgca gggcaagtaa gagcattagc aaatatttag cctggtatca agagaaacct 120
gggaaaacta ataagcttct tatctactct ggatccactt tgcaatctgg aattccatca 180
aggttcagtg gcagtggatc tggtacagat ttcactctca ccatcagtag cctggagcct 240
gaagattttg caatgtatta ctgtcaacag catcatgaat acccgtacac gttcggaggg 300
gggaccaagc tggaaataaa a 321
<210>25
<211>107
<212>PRT
<213> mice
<400>25
Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly
1 5 10 15
Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His His Glu Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210>26
<211>1413
<212>DNA
<213> Artificial
<220>
<223> chimeric antibody
<400>26
atgggatgga gctatatcat cctcttcttg ttagcaacag ctacatgtgt ccactcccag 60
gtccaactgc agcagcctgg ggctgagctg gtgaggcctg gggcttcagt gaagttgtcc 120
tgcaaggctt ctggctacac gttcaccacc tactggatga actgggttaa gcagaggcct 180
gagcaaggcc ttgagtggat tggaaggatt gatccttacg atagtgaaac tcactacaat 240
caaaagttca aggacaaggc catattgact gtagacaaat cctccagcac agcctacatg 300
caactcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag gggaccttac 360
tacggtacta acccctggtt tccttactgg ggccaaggga ctctggtcac tgtctcttca 420
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 480
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 660
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 720
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 780
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 840
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 900
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 960
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1020
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1080
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1140
ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1200
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1260
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1320
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380
cagaagagcc tctccctgtc tccgggtaaa tga 1413
<210>27
<211>470
<212>PRT
<213> Artificial
<220>
<223> chimeric antibody
<400>27
Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Leu Ala Thr Ala Thr Cys
1 5 10 15
Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg
20 25 30
Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
Thr Thr Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Glu Gln Gly Leu
50 55 60
Glu Trp Ile Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn
65 70 75 80
Gln Lys Phe Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser
85 90 95
Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Gly Pro Tyr Tyr Gly Thr Asn Pro Trp Phe Pro
115 120 125
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
130 135 140
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
145 150 155 160
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
165 170 175
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
180 185 190
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
195 200 205
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
210 215 220
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
225 230 235 240
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
245 250 255
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
260 265 270
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
275 280 285
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
290 295 300
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
305 310 315 320
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
325 330 335
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
340 345 350
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
355 360 365
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
370 375 380
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
385 390 395 400
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
405 410 415
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
420 425 430
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
435 440 445
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
450 455 460
Ser Leu Ser Pro Gly Lys
465 470
<210>28
<211>705
<212>DNA
<213> Artificial
<220>
<223> chimeric antibody
<400>28
atgaggttcc aggttcaggt tctggggctc cttctgctct ggatatcagg tgcccagtgt 60
gatgtccaga taacccagtc tccatcttat cttgctgcat ctcctggaga aaccattact 120
attaattgca gggcaagtaa gagcattagc aaatatttag cctggtatca agagaaacct 180
gggaaaacta ataagcttct tatctactct ggatccactt tgcaatctgg aattccatca 240
aggttcagtg gcagtggatc tggtacagat ttcactctca ccatcagtag cctggagcct 300
gaagattttg caatgtatta ctgtcaacag catcatgaat acccgtacac gttcggaggg 360
gggaccaagc tggaaataaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 420
tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 540
gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccct gacg 600
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 660
ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttga 705
<210>29
<211>234
<212>PRT
<213> Artificial
<220>
<223> chimeric antibody
<400>29
Met Arg Phe Gln Val Gln Val Leu Gly Leu Leu Leu Leu Trp Ile Ser
1 5 10 15
Gly Ala Gln Cys Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala
20 25 30
Ala Ser Pro Gly Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser
35 40 45
Ile Ser Lys Tyr Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn
50 55 60
Lys Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
85 90 95
Ser Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His His
100 105 110
Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
115 120 125
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
130 135 140
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
145 150 155 160
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
165 170 175
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
180 185 190
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
195 200 205
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
210 215 220
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230
<210>30
<211>27
<212>DNA
<213> human
<400>30
tccaacaacc tctgcctcct ccacgac 27
<210>31
<211>27
<212>DNA
<213> human
<400>31
tccattcttg cagcgagagg ccgtatt 27
<210>32
<211>91
<212>DNA
<213> Artificial
<220>
<223> PCR primer
<400>32
taagaattcc accatggact ggacctggag gttcctcttt gtggtggcag cagctacagg 60
tgtccagtcc acgaatacta caattaagag c 91
<210>33
<211>41
<212>DNA
<213> Artificial
<220>
<223> PCR primer
<400>33
ttgtcggtcc gcgaggttcc agactcttct gggtgccctg g 41
<210>34
<211>1587
<212>DNA
<213> Artificial
<220>
<223> Muc17 fusion protein
<400>34
atggactgga cctggaggtt cctctttgtg gtggcagcag ctacaggtgt ccagtccacg 60
aatactacaa ttaagagcaa ccccacctca actcctactg tgccaagaac cacaacatgc 120
tttggagatg ggtgccagaa tacggcctct cgctgcaaga atggaggcac ctgggatggg 180
ctcaagtgcc agtgtcccaa cctctattat ggggagttgt gtgaggaggt ggtcagcagc 240
attgacatag ggccaccgga gactatctct gcccaaatgg aactgactgt gacagtgacc 300
agtgtgaagt tcaccgaaga gctaaaaaac cactcttccc aggaattcca ggagttcaaa 360
cagacattca cggaacagat gaatattgtg tattccggga tccctgagta tgtcggggtg 420
aacatcacaa agctacgtct tggcagtgtg gtggtggagc atgacgtcct cctaagaacc 480
aagtacacac cagaatacaa gacagtattg gacaatgcca ccgaagtagt gaaagggaaa 540
atcacaaaag tgaccacaca gcaaataatg attaatgata tttgctcaga catgatgtgt 600
ttcaacacca ctggcaccca agtgcaaaac attacggtga cccagtacga ccctgaagag 660
gactgccgga agatggccaa ggaatatgga gactacttcg tagtggagta ccgggaccag 720
aagccatact gcatcagccc ctgtgagcct ggcttcagtg tctccaagaa ctgtagcctc 780
ggcaagtgcc agatgtctct aagtggacct cagtgcctct gcgtgaccac ggaaactcac 840
tggtacagtg gggagacctg taaccagggc acccagaaga gtctggaacc tcgcggaccg 900
acaatcaagc cctgtcctcc atgcaaatgc ccagcaccta acctcttggg tggaccatcc 960
gtcttcatct tccctccaaa gatcaaggat gtactcatga tctccctgag ccccatagtc 1020
acatgtgtgg tggtggatgt gagcgaggat gacccagatg tccagatcag ctggtttgtg 1080
aacaacgtgg aagtacacac agctcagaca caaacccata gagaggatta caacagtact 1140
ctccgggtgg tcagtgccct ccccatccag caccaggact ggatgagtgg caaggagttc 1200
aaatgcaagg tcaacaacaa agacctgcca gcgcccatcg agagaaccat ctcaaaaccc 1260
aaagggtcag taagagctcc acaggtatat gtcttgcctc caccagaaga agagatgact 1320
aagaaacagg tcactctgac ctgcatggtc acagacttca tgcctgaaga catttacgtg 1380
gagtggacca acaacgggaa aacagagcta aactacaaga acactgaacc agtcctggac 1440
tctgatggtt cttacttcat gtacagcaag ctgagagtgg aaaagaagaa ctgggtggaa 1500
agaaatagct actcctgttc agtggtccac gagggtctgc acaatcacca cacgactaag 1560
agcttct ccc ggactccggg taaatga 1587
<210>35
<211>528
<212>PRT
<213> Artificial
<220>
<223> Muc17 fusion protein
<400>35
Met Asp Trp Thr Trp Arg Phe Leu Phe Val Val Ala Ala Ala Thr Gly
1 5 10 15
Val Gln Ser Thr Asn Thr Thr Ile Lys Ser Asn Pro Thr Ser Thr Pro
20 25 30
Thr Val Pro Arg Thr Thr Thr Cys Phe Gly Asp Gly Cys Gln Asn Thr
35 40 45
Ala Ser Arg Cys Lys Asn Gly Gly Thr Trp Asp Gly Leu Lys Cys Gln
50 55 60
Cys Pro Asn Leu Tyr Tyr Gly Glu Leu Cys Glu Glu Val Val Ser Ser
65 70 75 80
Ile Asp Ile Gly Pro Pro Glu Thr Ile Ser Ala Gln Met Glu Leu Thr
85 90 95
Val Thr Val Thr Ser Val Lys Phe Thr Glu Glu Leu Lys Asn His Ser
100 105 110
Ser Gln Glu Phe Gln Glu Phe Lys Gln Thr Phe Thr Glu Gln Met Asn
115 120 125
Ile Val Tyr Ser Gly Ile Pro Glu Tyr Val Gly Val Asn Ile Thr Lys
130 135 140
Leu Arg Leu Gly Ser Val Val Val Glu His Asp Val Leu Leu Arg Thr
145 150 155 160
Lys Tyr Thr Pro Glu Tyr Lys Thr Val Leu Asp Asn Ala Thr Glu Val
165 170 175
Val Lys Gly Lys Ile Thr Lys Val Thr Thr Gln Gln Ile Met Ile Asn
180 185 190
Asp Ile Cys Ser Asp Met Met Cys Phe Asn Thr Thr Gly Thr Gln Val
195 200 205
Gln Asn Ile Thr Val Thr Gln Tyr Asp Pro Glu Glu Asp Cys Arg Lys
210 215 220
Met Ala Lys Glu Tyr Gly Asp Tyr Phe Val Val Glu Tyr Arg Asp Gln
225 230 235 240
Lys Pro Tyr Cys Ile Ser Pro Cys Glu Pro Gly Phe Ser Val Ser Lys
245 250 255
Asn Cys Ser Leu Gly Lys Cys Gln Met Ser Leu Ser Gly Pro Gln Cys
260 265 270
Leu Cys Val Thr Thr Glu Thr His Trp Tyr Ser Gly Glu Thr Cys Asn
275 280 285
Gln Gly Thr Gln Lys Ser Leu Glu Pro Arg Gly Pro Thr Ile Lys Pro
290 295 300
Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser
305 310 315 320
Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu
325 330 335
Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro
340 345 350
Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala
355 360 365
Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val
370 375 380
Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe
385 390 395 400
Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr
405 410 415
Ile Ser Lys Pro Lys Gly SerVal Arg Ala Pro Gl n Val Tyr Val Leu
420 425 430
Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys
435 440 445
Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn
450 455 460
Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp
465 470 475 480
Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys
485 490 495
Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly
500 505 510
Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
515 520 525
<210>36
<211>21
<212>DNA
<213> mice
<400>36
gggccagtgg atagacagat g 21
<210>37
<211>23
<212>DNA
<213> mice
<400>37
gctcactgga tggtgggaag atg 23
<210>38
<211>16
<212>PRT
<213> human
<400>38
Pro Thr Thr Ala Glu Gly Thr Ser Met Pro Thr Ser Thr Pro Ser Glu
1 5 10 15
Claims (12)
1. An antibody that binds to Mucin17(Muc 17).
2. The antibody of claim 1, wherein said antibody does not bind secreted Muc 17.
3. The antibody of claim 1 or 2, wherein the antibody binds to the peptide of SEQ ID NO. 3 (4176) -4390) and does not bind to the peptides of SEQ ID NO. 4 (4244) -4390) and SEQ ID NO. 5 (4115-4243).
4. The antibody of any one of claims 1 to 3, wherein said antibody has ADCC activity.
5. The antibody of any one of claims 1 to 4, wherein the antibody is a chimeric antibody or a humanized antibody.
6. The antibody of any one of claims 1 to 5, wherein the antibody is a low fucosylated antibody.
7. An antibody that recognizes the same epitope as an epitope recognized by an antibody having a heavy chain variable region having the amino acid sequence represented by SEQ ID NO. 23 and a light chain variable region having the amino acid sequence represented by SEQ ID NO. 25.
8. An anticancer agent comprising the antibody according to any one of claims 1 to 7.
9. The anticancer agent of claim 8, wherein said cancer is pancreatic cancer.
10. A method for diagnosing cancer, comprising using an antibody that binds to Muc 17.
11. The diagnostic method of claim 10, wherein the cancer is pancreatic cancer.
12. Diagnostic method according to claim 10 or 11, wherein the antibody is an antibody which does not bind to secreted Muc 17.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2007-176319 | 2007-07-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| HK1139163A true HK1139163A (en) | 2010-09-10 |
Family
ID=
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