WO2011074621A1 - Mesothelin (msln) antibody and use thereof - Google Patents

Abstract

Disclosed is a tumor cell imaging agent containing anti-mesothelin antibody. As a result of the research of the inventors focusing on the aforementioned mesothelin, cultured cells derived from cancer cells inoculated subcutaneously into mice were imaged using labeled mesothelin antibodies.

Description

Antibodies against mesothelin (MSLN) and uses thereof

The present invention relates to a tumor cell imaging agent comprising an anti-mesothelin antibody.

Any cancer is desirably diagnosed at an early stage, but early cancers with poor physical findings and subjective symptoms are only detected accidentally in medical examinations. In particular, mesothelioma that spreads rapidly along the pleura and peritoneum, or pancreatic cancer that is morphologically unclear is particularly difficult to detect and diagnose and is often too late.
In recent years, diagnostic imaging such as MRI and CT has become widely used in Japan, but it has the merit of visualizing the inside of the body, but it is necessary to read the state of the blood vessel wall from the density and shape of the image. Detection of early mesothelioma and pancreatic cancer is difficult. It also depends on the skills of the doctors and laboratory technicians who perform the tests. In addition, paramagnetic gadolinium and iron used in MRI and iodine contrast media used in CT have side effects and nephrotoxicity, and CT is accompanied by radiation exposure. Therefore, development of a new diagnostic method with less patient burden is desired.

Mesothelin, a kind of cell membrane glycoprotein, is expressed exclusively in the mesothelial cells that make up the pleura, peritoneum, pericardium and the like that wrap around organs such as the lung, heart, gastrointestinal tract, and liver. However, in cancerous tissues, not only mesothelial cells (epithelial mesothelioma) but also mesothelin is high in various cancer tissues such as pancreatic cancer, ovarian cancer, lung cancer, head and neck cancer, colon cancer, and breast cancer. It is known to be expressed (Eur. J. Cancer, 44, 46-53, 2008, Clin. Cancer Res. 10, 3937-3942, 2004).
Mesothelin is first synthesized as a precursor protein having a total length of 71 kDa, and then a polypeptide of about 31 kDa called megakaryocyte potentiating factor (MPF) corresponding to positions 34-286 of the extracellular domain by a proteolytic enzyme such as furin, The GPI anchor breaks down into a 40 kDa polypeptide (mature mesothelin) left on the cell membrane. Furthermore, it is known that part of the GPI-anchored membrane mesothelin is separated from the cell membrane and released (soluble mesothelin).
Against the backdrop of these molecular features, MPF and soluble mesothelin have been recognized as tumor markers, and serum measurement systems using the respective antibodies have been developed. However, tests using serum tumor markers are easy to operate, but the identification of the primary lesion and the presence or absence of metastasis cannot be confirmed. In addition, the amount of MPF and mesothelin secreted in the early stages of cancer is small, and early detection using serum as a specimen must be difficult in principle. Although a methodology for cancer imaging diagnosis using a monoclonal antibody against mesothelin labeled with indium 111 has been reported, the cancer cells used are cells in which the mesothelin gene has been artificially introduced. There is no guarantee that physiological conditions in actual cancer patients will be met (Int J Cancer. 80, 559-63, 1999).

JP2007-71849A Patent 3490125

An object of the present invention is to provide an imaging agent for tumor cells containing an anti-mesothelin antibody.

In order to solve the above-mentioned problems, the present inventors paid attention to the above-mentioned mesothelin and conducted intensive research. As a result, we successfully imaged cultured cells derived from cancer cells transplanted under the skin of mice using labeled mesothelin antibodies.
This application provides the following invention based on this knowledge.
[1] An imaging agent for tumor cells, comprising an anti-mesothelin antibody.
[2] The imaging agent according to [1], wherein the antibody is the antibody according to any of (a) to (e) below:
(A) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 4 as CDR1, the amino acid sequence of SEQ ID NO: 6 as CDR2, and the amino acid sequence of SEQ ID NO: 8 as CDR3;
(B) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 2 as the heavy chain variable region;
(C) an antibody comprising a light chain having the amino acid sequence of SEQ ID NO: 12 as CDR1, the amino acid sequence of SEQ ID NO: 14 as CDR2, and the amino acid sequence of SEQ ID NO: 16 as CDR3;
(D) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 as the light chain variable region,
(E) An antibody having the heavy chain pair described in (a) or (b) above and the light chain pair described in (c) or (d) above.
[3] The imaging agent according to [1] or [2], which is used in vivo.
[4] A tumor cell imaging kit comprising an anti-mesothelin antibody.
[5] The imaging kit according to [4], wherein the antibody is the antibody according to any of (a) to (e) below:
(A) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 4 as CDR1, the amino acid sequence of SEQ ID NO: 6 as CDR2, and the amino acid sequence of SEQ ID NO: 8 as CDR3;
(B) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 2 as the heavy chain variable region;
(C) an antibody comprising a light chain having the amino acid sequence of SEQ ID NO: 12 as CDR1, the amino acid sequence of SEQ ID NO: 14 as CDR2, and the amino acid sequence of SEQ ID NO: 16 as CDR3;
(D) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 as the light chain variable region,
(E) An antibody having the heavy chain pair described in (a) or (b) above and the light chain pair described in (c) or (d) above.
[6] The imaging kit according to [4] or [5], which is used in vivo.
[7] A method for imaging tumor cells using an anti-mesothelin antibody.
[8] The imaging method according to [7], wherein the antibody is the antibody according to any of (a) to (e) below:
(A) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 4 as CDR1, the amino acid sequence of SEQ ID NO: 6 as CDR2, and the amino acid sequence of SEQ ID NO: 8 as CDR3;
(B) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 2 as the heavy chain variable region;
(C) an antibody comprising a light chain having the amino acid sequence of SEQ ID NO: 12 as CDR1, the amino acid sequence of SEQ ID NO: 14 as CDR2, and the amino acid sequence of SEQ ID NO: 16 as CDR3;
(D) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 as the light chain variable region,
(E) An antibody having the heavy chain pair described in (a) or (b) above and the light chain pair described in (c) or (d) above.
[9] Use of an anti-mesothelin antibody in the production of an imaging agent for tumor cells.
[10] The use according to [9], wherein the antibody is the antibody according to any one of the following (a) to (e);
(A) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 4 as CDR1, the amino acid sequence of SEQ ID NO: 6 as CDR2, and the amino acid sequence of SEQ ID NO: 8 as CDR3;
(B) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 2 as the heavy chain variable region;
(C) an antibody comprising a light chain having the amino acid sequence of SEQ ID NO: 12 as CDR1, the amino acid sequence of SEQ ID NO: 14 as CDR2, and the amino acid sequence of SEQ ID NO: 16 as CDR3;
(D) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 as the light chain variable region,
(E) An antibody having the heavy chain pair described in (a) or (b) above and the light chain pair described in (c) or (d) above.
[11] An anti-mesothelin antibody for use in a method for imaging tumor cells.
[12] The antibody according to [11], wherein the antibody is the antibody according to any one of the following (a) to (e);
(A) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 4 as CDR1, the amino acid sequence of SEQ ID NO: 6 as CDR2, and the amino acid sequence of SEQ ID NO: 8 as CDR3;
(B) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 2 as the heavy chain variable region;
(C) an antibody comprising a light chain having the amino acid sequence of SEQ ID NO: 12 as CDR1, the amino acid sequence of SEQ ID NO: 14 as CDR2, and the amino acid sequence of SEQ ID NO: 16 as CDR3;
(D) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 as the light chain variable region,
(E) An antibody having the heavy chain pair described in (a) or (b) above and the light chain pair described in (c) or (d) above.

It is an immuno-staining photograph of BxPC-3 pancreatic cancer cells cultured in an 8-well chamber. Observed with a confocal laser scanning microscope. It is an immuno-staining photograph of CFPAC-1 pancreatic cancer cells cultured in a 2-well chamber. Observed with a confocal laser scanning microscope. It is an immuno-staining photograph of PANC-1 pancreatic cancer cells cultured in an 8-well chamber. Observed with a confocal laser scanning microscope. It is an immuno-staining photograph of a mesothelioma cell (211H). Observed with a confocal laser scanning microscope. It is an immuno-staining photograph with 11-25 anti-mesothelin antibody of PC3 prostate cancer cells, 211H mesothelioma cells, H520 and H226 lung cancer cells. Observed with a confocal laser scanning microscope. It is the in-vivo and in-vitro imaging photograph of the cancer transplanted to the nude mouse subcutaneously.

The present invention provides a tumor cell imaging agent comprising an anti-mesothelin antibody. The present invention also provides a method for imaging tumor cells, comprising the step of administering an anti-mesothelin antibody to a subject.

Mesothelin (“MSLN”) is observed on the cell surface of mesothelial cells, mesothelioma, certain squamous cell carcinomas, ovarian cancers, pancreatic cancers, and lung cancers (Chang et al., 1996, Proc. Natl). Acid. Sci. USA 93: 136-140; US Pat. No. 6,083,502), under investigation as a target for therapeutic antibodies against the cancer (eg, US clinical trials by Morphotek) NCT00325494). Mesothelin has a molecular weight of about 40 kDa and (i) at least 10 consecutive amino acids of SEQ ID NO: 17 or the known variants listed in Swiss-plot accession number Q13421, or (ii) A polypeptide having an amino acid sequence comprising any of a portion of at least 20 contiguous amino acid residues, wherein the portion of the amino acid residue is the 20 contiguous residues of SEQ ID NO: 17, or the Swiss -Match at least 90% (preferably at least 95% or 100%) with the known variants listed in the accession number Q13421 of the plot. The mesothelin variant is a detected 8-amino acid variant, PQAPRRPL (SEQ ID NO: 18), and the C-terminal VQGGRGGGQARAGGRAGGVEVGALSHPSLCRGPLGDALPPRTWTCSHRRPTAPPSLHPGLRAPLPC (SEQ ID NO: 19), MQEILS (SEQ ID NO: 19) It is known that it includes, but is not limited to, a variant substituted with (GTPCLLGPGPVVLTVALLALLLASTA) (SEQ ID NO: 21). The mesothelin may contain glycophosphatidylinositol (GPI) anchors as post-translational modifications and many N-linked sugar chains. The GPI anchor binds mesothelin to the cell membrane, but may be cleaved by a phospholipase to provide a soluble form of mesothelin.
The anti-mesothelin antibody included in the present invention is characterized by binding to the mesothelin.

Specific examples of antibodies included in the present invention include, but are not limited to, the following antibodies.
(A) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 4 as CDR1, the amino acid sequence of SEQ ID NO: 6 as CDR2, and the amino acid sequence of SEQ ID NO: 8 as CDR3;
(B) an antibody comprising a heavy chain having CDR1, CDR2, CDR3 derived from a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2;
(C) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 2 as the heavy chain variable region,
(D) an antibody comprising a light chain having the amino acid sequence of SEQ ID NO: 12 as CDR1, the amino acid sequence of SEQ ID NO: 14 as CDR2, and the amino acid sequence of SEQ ID NO: 16 as CDR3;
(E) an antibody comprising a light chain having CDR1, CDR2, CDR3 derived from a light chain variable region having the amino acid sequence of SEQ ID NO: 10;
(F) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 as the light chain variable region,
(G) An antibody having the heavy chain according to any of (a) to (c) above and the light chain pair according to any of (d) to (f) above.

The antibody of the present invention includes both polyclonal and monoclonal antibodies. Methods for preparing and purifying monoclonal and polyclonal antibodies are known in the art and are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1988).

The antibodies of the present invention also include recombinant antibodies such as humanized antibodies or chimeric antibodies. “Humanized antibody” refers to an antibody whose structure is similar to that of a human antibody. Such humanized antibodies or chimeric antibodies include human chimeric antibodies (for example, antibodies in which part of the antibody is humanized, antibodies in which the CH2 region is humanized, antibodies in which the Fc region is humanized, constant regions) Humanized antibodies), and human CDR-grafted antibodies (PTJohons et al., Nature 321,522 () other than the CDRs (complementarity determining regions) present in the constant and variable regions) 1986)), fully humanized antibodies and the like. In order to enhance the antigen binding activity of human CDR-grafted antibody, a method of selecting human antibody FR having high homology with mouse antibody, a method of producing humanized antibody having high homology, and further after transplanting mouse CDR to human antibody Techniques for improving the method of substituting amino acids in FR have already been developed (US Patent No. 5585089, US Patent No. 5693761, US Patent No. 5693762, US Patent No. 6180370, European Patent No. 451216, European Patent No. 682040) , Refer to Japanese Patent No. 2828340, etc.) and can also be used to prepare the human CDR-grafted antibody of the present invention.
A human chimeric antibody can be prepared, for example, by substituting the constant region of an antibody having the structure of the H chain variable region and / or the structure of the L chain variable region with a constant region of a human antibody. Known constant regions of human antibodies can be employed.

As a humanized antibody of the present invention, for example, the CDR is a CDR derived from an anti-mesothelin antibody, the FR is a FR derived from a human immunoglobulin, and the constant region is a constant region derived from a human immunoglobulin. And humanized antibodies. Specifically, a heavy chain variable region-derived CDR having the amino acid sequence set forth in SEQ ID NO: 2, a heavy chain variable region FR derived from a human immunoglobulin, and a heavy chain having a heavy chain constant region derived from a human immunoglobulin. A chain, and a CDR derived from a light chain variable region having the amino acid sequence of SEQ ID NO: 10, and an FR derived from a light chain variable region of a human immunoglobulin, and a light chain constant region derived from a human immunoglobulin Mention may be made of humanized antibodies having a pair of light chains.
Examples of the chimeric antibody of the present invention include a chimeric antibody characterized in that the variable region is a variable region derived from an anti-mesothelin antibody and the constant region is a constant region derived from human immunoglobulin. . Specifically, it has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 2, a heavy chain having a heavy chain constant region derived from human immunoglobulin, and the amino acid sequence set forth in SEQ ID NO: 10. A chimeric antibody having a pair of a light chain variable region and a light chain having a light chain constant region derived from human immunoglobulin can be mentioned.

An example of a method for producing a human chimeric antibody is shown below.
First, mRNA is extracted from a hybridoma producing a mouse antibody against a specific target antigen, and cDNA is synthesized according to a conventional method. The synthesized cDNA is incorporated into a vector to construct a cDNA library. From this cDNA library, a vector containing the H chain gene and the L chain gene is selected by using the H chain gene fragment and the L chain gene fragment as probes. By sequencing the insertion sequence of the selected vector, the sequences of the H chain variable region and L chain variable region genes are determined. Based on the sequence data thus obtained, DNA encoding the H chain variable region is prepared by chemical synthesis, biochemical cleavage / recombination, or the like. The resulting DNA encoding the H chain variable region is ligated with DNA encoding the human H chain constant region and incorporated into an expression vector to prepare an H chain expression vector. Examples of expression vectors include, but are not limited to, SV40 virus based vectors, EB virus based vectors, BPV (papilloma virus) based vectors, and the like. On the other hand, an L chain expression vector is prepared by the same method. Host cells are cotransformed with these H chain expression vector and L chain expression vector. As host cells, CHO cells (Chinese hamster ovary) (A. Wright & SL Morrison, J. Immunol. 160, 3393-3402 (1998)), SP2 / 0 cells (mouse myeloma) (K. Motmans et al., Eur. J. Cancer Prev. 5, 512-519 (1996), RP Junghans et al., Cancer Res. 50, 1495-1502 (1990)) and the like are preferably used. For transformation, the lipofectin method (RWMalone et al., Proc.Natl.Acad.Sci.USA 86,6077 (1989), PLFelgner et al., Proc.Natl.Acad.Sci.USA 84,7413 (1987) ), Electroporation method, calcium phosphate method (FL Graham & AJvan der Eb, Virology 52, 456-467 (1973)), DEAE-Dextran method and the like are preferably used.

After culturing the transformant, the human chimeric antibody is separated from the cells of the transformant or from the culture solution. For separation and purification of antibodies, methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, affinity chromatography, ion exchange chromatography, gel filtration chromatography and the like can be used in appropriate combination.

On the other hand, a human CDR-grafted antibody can be prepared, for example, by the following method. First, the amino acid sequences of the H chain variable region and the L chain variable region of an antibody against a specific antigen and the base sequence encoding the same are determined by the method described in the column of the method for producing a chimeric antibody. In addition, the amino acid sequence and base sequence of each CDR region are determined.

Next, select the FR (framework area) that exists across the CDR area. There are approximately three methods for selecting FR. The first method is a method using a human antibody frame, such as NEWM and REI, whose three-dimensional structure has already been clarified (Riechmann L. et al., Nature 332, 323-3Z7 (1988); Tempst, PR. Et al., Protein Engineering) 7, 1501-1507 (1994); Ellis JH. et al., J. Immunol 155, 925-937 (1995)). The second method is to select a human antibody variable region having the highest homology with the mouse antibody variable region of interest from the database and use the FR (Queen C. et al., Proc Natl Acad SciUSA 86, 10029). -10033 (1989); Rozak MJ. Et al., J Biol Chem 271, 22611-22618 (1996); Shearman CW. Et al., J.Immunol 147, 4366-4373 (1991)). The third method is to select the most commonly used amino acid in human antibody FRs (Sato K. et al., Mol Immunol 31, 371-381 (1994); Kobinger F. et al., Protein Engineering 6, 971-980 (1993); ttleKettleborough CA. et al., Protein Engineering 4, 773-783 (1991)). Any of these methods can be used in the present invention.

Even if the amino acid sequence of the selected human FR is modified, it should be used as the FR amino acid sequence as long as the finally obtained human CDR-grafted antibody has a specific binding property to the target antigen. Can do. In particular, when a part of the amino acid of the selected human FR is changed to the FR amino acid of the antibody derived from CDR, it is highly possible that the characteristics of the antibody are maintained. The number of amino acids to be modified is preferably 30% or less of the entire FR, more preferably 20% or less of the entire FR, and further preferably 10% or less of the entire FR.

Next, a DNA encoding the H chain variable region and the L chain variable region is designed by combining the FR selected by any of these methods and the CDR. Based on this design, DNA encoding the H chain variable region and DNA encoding the L chain variable region are respectively prepared by chemical synthesis, biochemical cleavage / recombination, and the like. Then, the DNA encoding the H chain variable region is incorporated into an expression vector together with the DNA encoding the human immunoglobulin H chain constant region to construct an H chain expression vector. Similarly, an L chain expression vector is constructed by incorporating DNA encoding the L chain variable region into an expression vector together with DNA encoding the human immunoglobulin L chain constant region. Examples of expression vectors include, but are not limited to, SV40 virus-based vectors, EB virus-based vectors, BPV (papilloma virus) based vectors, and the like.

Host cells are cotransformed with the H chain expression vector and L chain expression vector prepared by the above method. As host cells, CHO cells (Chinese hamster ovary) (A. Wright & SL Morrison, J. Immunol. 160, 3393-3402 (1998)), SP2 / 0 cells (mouse myeloma) (K. Motmans et al., Eur. J. Cancer Prev. 5, 512-519 1996 (1996), RP Junghans et al., Cancer. Res. 50, 1495-1502 (1990)) and the like are preferably used. For transformation, the lipofectin method (RWMalone et al., Proc.Natl.Acad.Sci.USA 86,6077 (1989), PLFelgner et al., Proc.Natl.Acad.Sci.USA 84,7413 ( 1987)), electroporation method, calcium phosphate method (FL GrahamGra & AJvan der Eb, Virology 52,456-467 (1973)), DEAE-Dextran method and the like are preferably used.

After culturing the transformant, the human CDR-grafted antibody is isolated from the cells of the transformant or the culture solution. Separation and purification of the antibody can be performed by appropriately combining methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, affinity chromatography, ion exchange chromatography, and gel filtration chromatography.

In addition, a method for obtaining a human antibody is also known. For example, human lymphocytes are sensitized with a desired antigen or cells expressing the desired antigen in vitro, the sensitized lymphocytes are fused with human myeloma cells such as U266, and the desired human antibody having binding activity to the antigen is obtained. It can also be obtained (see Japanese Patent Publication No. 1-59878). Further, a desired human antibody can be obtained by immunizing a transgenic animal having all repertoires of human antibody genes with a desired antigen (International Patent Application Publication Nos. WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735).

In a further embodiment, antibodies or antibody fragments can be separated from antibody phage libraries produced using the techniques described in McCafferty et al. (Nature, 348: 552-554 (1990)). Clackson et al. (Nature, 352: 624-628 (1991)) and Marks et al. (J. Mol. Biol., 222: 581-597 (1991)) describe the separation of mouse and human antibodies using phage libraries. is doing. Subsequent publications generate high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10: 779-783 [1992]), as well as to construct very large phage libraries. Combinatorial infection and in vivo recombination (Waterhouse et al., Nuc. Acids. Res., 21: 2265-2266 [1993]) have been described as measures for this. These techniques are therefore viable alternatives to traditional monoclonal antibody hybridoma methods for the separation of monoclonal antibodies.

In this regard, bacteriophage (phage) display is a well-known technique that allows large oligopeptide libraries to be searched to identify members of those libraries capable of specifically binding to a polypeptide target. one of. Phage display is a technique by which various polypeptides are presented as fusion proteins to coat proteins on the surface of bacteriophage particles (Scott, J.K. and SmithSG. P. (1990) Science 249: 386). The usefulness of phage display can quickly and effectively classify large libraries of selectively randomized protein variants (or random clone cDNAs) for those sequences that bind to target molecules with high affinity. In the point. Peptides on phage (Cwirla, SE et al. (1990) Proc. Tl Natl. Acad. Sci. USA, 87: 6378) or proteins (Lowman, HB et al (1991) Biochemistry, 30: 10832; Clackson, T. et al (1991) Nature, 352: 624; Marks, JD et al. (1991), J. Mol. Biol., 222: 581; Kang, AS et al. (1991) Proc. Natl. Acad. Sci. USA, 88: 8363) Library display Has been used to screen a myriad of polypeptides or oligopeptides for specific binding properties (Smith, GP (1991) Current Opin. Biotechnol., 2: 668). Classification of phage libraries of random mutants requires methods to construct and propagate a large number of variants, methods of affinity purification using target receptors, and means to evaluate the results of enhanced binding (US) Nos. 5223409, 5,403,484, 5,571,689, and 5,663,143).

Although most phage display methods used filamentous phage, the λ phage display system (WO95 / 34683; US Pat. No. 5,627,024), the T4 phage display system (RenJ. Et al. Gene 215: 439 (1998); Zhu et al. Cancer Researc, 58 (15): 3209-3214 (1998); Jiang et al.Infection & Immunity, 65 (11): 4770-4777 (1997); Ren et al., Gene, 195 (2): 303-311 (1997); Ren, Protein Sci. 5: 1833 (1996); Efimov et al. Virus Genes 10: 173 (1995)) and T7 phage display system (Smith and Scott Methods Enzymology, 217, 228-257 (1993); US Pat. No. 5,766,905 ) Is also known.

Currently, many improvements and variations of the basic phage display method have been developed. These improvements have improved the method of screening from peptide libraries and protein libraries based on properties, abilities, etc., such as binding to selected target molecules. Recombination reaction means for the phage display method is described in WO98 / 14277. Phage display libraries have been used to analyze and control bimolecular interactions (WO 98/20169; WO 98/20159) and restricted helix peptide properties (WO 98/20036). In WO97 / 35196, a phage display library is contacted with a first solution in which a ligand can bind to a target molecule and a second solution in which an affinity ligand does not bind to the target molecule to selectively isolate the bound ligand. A method for isolating affinity ligands is described. WO 97/46251 describes a method of biopanning a random phage display library with affinity purified antibodies, then isolating bound phage, followed by micropanning in the wells of a microplate to isolate high affinity binding phage. To do. The use of Staphlylococcus aureus protein A as an affinity tag has also been reported (Li et al., (1998) Mol Biotech., 9: 187). WO 97/47314 describes the use of a substrate subtraction library to identify enzyme specificity using a combinatorial library that may be a phage display library. A method for selecting enzymes suitable for use in detergents used for phage display is described in WO 97/09446. Additional methods for selecting proteins that specifically bind are described in US Pat. Nos. 5,498,538, 5,432,018, and WO 98/15833. Methods for preparing peptide libraries and screening these libraries are described in US Pat. Nos. 5,723,286, 5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434 and 5,734,018. Nos. 5,698,426, 5762192, and 5723323.

Furthermore, a technique for obtaining a human antibody by panning using a human antibody library is also known. For example, the variable region of a human antibody is expressed as a single chain antibody (scFv) on the surface of the phage by the phage display method, and a phage that binds to the antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. If the DNA sequence of scFv that binds to the antigen is clarified, a human antibody can be obtained by preparing an appropriate expression vector having the sequence and introducing it into an appropriate host for expression. These methods are already well known and can be carried out with reference to WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, and WO 95/15388. it can.

Alternatively, human antibodies and antibody fragments can be generated in vitro from the immunoglobulin variable (V) domain gene repertoire of non-immunized donors using phage display technology (McCafferty et al., Nature 348: 552-553 [1990]). Can be produced. According to this technique, antibody V domain genes are cloned in frame units with either filamentous bacteriophage, eg, M13 or fd coat protein genes, and displayed as functional antibody fragments on the surface of phage particles. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, the selection of a gene encoding an antibody exhibiting these properties results as a result, even based on selection based on the functional properties of the antibody. Thus, this phage mimics some properties of B cells. Phage display can be performed in a variety of formats; see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3: 564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolated a large number of diverse anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. The V gene repertoire of non-immunized human donors is configurable, and antibodies against a wide variety of antigens (including self-antigens) can be found in Marks et al., J. Mol. Biol. 222: 581-597 (1991), or Griffith. Etc., EMBO J. 12: 725-734 (1993). See also U.S. Pat. Nos. 5,565,332 and 5,573,905.

The antibody of the present invention includes functional fragments of antibodies such as Fab, Fab ′, F (ab ′) ₂ , Fv, scFv, dsFv, Diabody, sc (Fv) 2, and minibody. In addition, multimers (for example, dimers, trimers, tetramers, polymers) of these functional fragments are also included in the antibody of the present invention.
Fab has a molecular weight of about 50,000 composed of L chain and H chain variable region obtained by digesting IgG with papain in the presence of cysteine, and H chain fragment consisting of C _H 1 domain and part of hinge part. It is a fragment. In the present invention, the antibody can be obtained by digesting with papain. Alternatively, Fab can be prepared from a transformant obtained by incorporating a DNA encoding part of the H chain and L chain of the above antibody into an appropriate vector and transforming using the vector.
Fab ′ is a fragment having a molecular weight of about 50,000 obtained by cleaving a disulfide bond between the H chains of F (ab ′) ₂ described later. In the present invention, the antibody is obtained by digesting with pepsin and cleaving disulfide bonds using a reducing agent. Similarly to Fab, it can also be prepared by genetic engineering using DNA encoding Fab ′.
F (ab ′) ₂ is a fragment (Fab) composed of an L chain and an H chain variable region obtained by digesting IgG with pepsin, and an H chain fragment consisting of a C _H 1 domain and a part of the hinge region. ') Is a fragment having a molecular weight of about 100,000 bonded by a disulfide bond. In the present invention, the antibody is obtained by pepsin digestion. Similarly to Fab, it can be prepared by genetic engineering using DNA encoding F (ab ′) ₂ .
Fv can be expressed in an appropriate host cell after the antibody is treated with an enzyme such as papain or pepsin to generate antibody fragments, or a gene encoding these antibody fragments is constructed and introduced into an expression vector. (For example, Co, MS et al., J. Immunol. (1994) 152, 2968-2976, Better, M. &; Horwitz, AH Methods in Enzymology (1989) 178, 476-496, Plueckthun, A .; Skerra , A. Methods in Enzymology (1989) 178, 476-496, Lamoyi, E., Methods in Enzymology (1989) 121, 652-663, Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663- 669, Bird, RE et al., TIBTECH (1991) 9, 132-137).
scFv is an antibody fragment in which an Fv comprising an H chain variable region and an L chain variable region is made into a single chain by linking the C terminus of one chain and the other N terminus with an appropriate peptide linker. As the peptide linker, for example, (GGGGS) ₃ having high flexibility can be used. For example, a DNA encoding an scFv antibody is constructed using DNA encoding the H chain variable region and L chain variable region of the above antibody and DNA encoding a peptide linker, and this is incorporated into an appropriate vector, and the vector is used. ScFv can be prepared from the transformant transformed as described above.
dsFv is an Fv fragment in which a Cys residue is introduced at an appropriate position in the H chain variable region and the L chain variable region, and the H chain variable region and the L chain variable region are stabilized by disulfide bonds. The position of Cys residue introduction in each chain can be determined based on the three-dimensional structure predicted by molecular modeling. In the present invention, for example, a three-dimensional structure is predicted from the amino acid sequences of the H chain variable region and the L chain variable region of the above-described antibody, and DNAs encoding the H chain variable region and L chain variable region into which mutations are introduced are constructed based on such prediction Then, this is incorporated into an appropriate vector, and dsFv can be prepared from a transformant transformed with the vector.
It is also possible to multimerize antibody fragments by linking scFv antibody, dsFv antibody or the like using an appropriate linker, or by fusing streptavidin. A fusion antibody or a labeled antibody can be constructed by fusing or binding a low molecular compound, protein, labeling substance or the like to the antibody (including antibody fragment) of the present invention. As the labeling substance, a radioactive substance such as ¹²⁵ I can be used.

Diabody refers to a bivalent antibody fragment constructed by gene fusion (Holliger P et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP 404,097, WO 93 / 11161 etc.). Diabody is a dimer composed of two polypeptide chains. Normally, each polypeptide chain is short to a position where VL and VH cannot bind to each other in the same chain, for example, by a linker of about 5 residues. Are combined. Diabody has two antigen-binding sites because VL and VH encoded on the same polypeptide chain cannot form a single-chain variable region fragment due to the short linker between them, and form a dimer. It will be. Diabody treats an antibody with an enzyme such as papain or pepsin to generate antibody fragments or constructs DNA encoding these antibody fragments and introduces them into an expression vector, and then in an appropriate host cell. (Eg, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476-496; Pluckthun , A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. 1986) 121, 663-669; Bird, RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137).
sc (Fv) 2 is a low molecular weight antibody in which two VHs and two VLs are combined with a linker or the like to form a single chain (Hudson et al, J Immunol. Methods 1999; 231: 177-189). sc (Fv) 2 can be prepared, for example, by linking scFv with a linker.
A minibody (low molecular weight antibody) is a full-length antibody (whole antibody, such as whole IgG), which is a parent antibody and includes an antibody fragment in which a part of the full-length antibody is missing, and has a binding ability to an antigen. If it does not specifically limit.

The antibody of the present invention includes a fusion protein in which the antibody of the present invention is fused with another peptide or protein. In the method for producing a fusion protein, a polynucleotide encoding an antibody of the present invention and a polynucleotide encoding another peptide or polypeptide are linked so that the frames coincide with each other, introduced into an expression vector, and expressed in a host. Any technique known to those skilled in the art can be used. Other peptides or polypeptides to be subjected to fusion with the antibody of the present invention include, for example, FLAG (Hopp, T. P. et al., BioTechnology (1988) 6, 1204-1210), 6 His ( (Histidine) 6xHis, 10xHis, influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, Known peptides such as the SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, Protein C fragment and the like can be used. Further, examples of other polypeptides subjected to fusion with the antibody of the present invention include GST (glutathione-S-transferase), HA (influenza agglutinin), β-galactosidase, MBP (maltose binding protein) and the like. Can be mentioned.

The antibody of the present invention includes an antibody bound with a labeling substance.
Examples of the labeling substance include, but are not limited to, luminescence by enzyme luminescence (luciferase), a substance using a luminescent low molecule, a substance using a fluorescent protein or a fluorescent small molecule, and a radionuclide. Radionuclides include gamma ray emitting nuclides such as ⁵¹ Cr, ⁵⁹ Fe, ⁵⁷ Co, ⁶⁷ Ga, ⁷⁵ Se, ^{81 m} Kr, ^{99 m} Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹³³ Xe, ²⁰¹ Tl, ¹¹ C, Positron emitting nuclides such as ¹³ N, ¹⁵ O, ¹⁸ F, ^{35 m} Cl, ⁷⁶ Br, ⁴⁵ Ti, ⁴⁸ V, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁶ Ga, ⁸⁹ Zr, ^{94 m} Tc, ¹²⁴ I However, it is not limited to these. As is obvious to those skilled in the art, “m” represents a nuclear isomer.
As fluorescent labels and luminescent labels, those using light emission by enzyme luminescence (luciferase) and fluorescence (fluorescent proteins such as GFP, DsRed, and wedge orange, and small fluorescent molecules such as FITC, Cy5.5, Alexa Fluor 750) Can be used.
In the case of luminescence by enzyme luminescence (luciferase), administration of a substrate is required separately.
In particular, those that reduce the influence of the animal's natural autofluorescence are preferred, and labels that emit signals with high skin permeability are preferred.

The present invention also provides DNA encoding the antibody of the present invention, a vector into which the DNA has been inserted, and a transformed cell into which the vector has been introduced. Examples of vectors include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, and the like. In addition, for the purpose of subcloning and excision of cDNA, in addition to the above vector, for example, pGEM-T, pDIRECT, pT7 and the like can be mentioned. A DNA encoding the antibody of the present invention, a vector into which the DNA has been inserted, and a transformed cell into which the vector has been introduced are prepared using known techniques.

Examples of the DNA encoding the anti-mesothelin antibody of the present invention include the following DNAs.
(A) DNA encoding the heavy chain having the amino acid sequence described in SEQ ID NO: 4 as CDR1, the amino acid sequence described in SEQ ID NO: 6 as CDR2, and the amino acid sequence described in SEQ ID NO: 8 as CDR3;
(B) DNA encoding a heavy chain having the amino acid sequence set forth in SEQ ID NO: 2 as the heavy chain variable region,
(C) DNA encoding a light chain having the amino acid sequence set forth in SEQ ID NO: 12 as CDR1, the amino acid sequence set forth in SEQ ID NO: 14 as CDR2, and the amino acid sequence set forth in SEQ ID NO: 16 as CDR3;
(D) DNA encoding a light chain having the amino acid sequence set forth in SEQ ID NO: 10 as the light chain variable region.

As an expression vector, for example, for the purpose of expression in E. coli, in addition to the above characteristics that the vector is amplified in E. coli, the host is E. coli such as JM109, DH5α, HB101, XL1-Blue, etc. In some cases, promoters that can be efficiently expressed in E. coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427), araB promoter (Better et al. , (Science (1988) (240), (1041-1043)), or having a T7 promoter or the like. In addition to the above vectors, such vectors include pGEX-5X-1 (Pharmacia), “QIAexpress® system” (Qiagen), pEGFP, or pET (in this case, the host expresses T7 RNA polymerase). BL21 is preferred).

The vector may also contain a signal sequence for polypeptide secretion. As a signal sequence for protein secretion, the pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379) may be used when the periplasm of E. coli is used for production. Introduction of a vector into a host cell can be performed using, for example, a calcium chloride method or an electroporation method.

In addition to E. coli, for example, mammalian-derived expression vectors (for example, pcDNA3 (manufactured by Invitrogen), pEGF-BOS (Nucleic Acids. Res.1990, 18 (17), p5322), pEF, pCDM8), insects Expression vectors derived from cells (eg, “Bac-to-BAC baculovairus expression system” (Gibco BRL), pBacPAK8), plant-derived expression vectors (eg, pMH1, pMH2), animal virus-derived expression vectors (eg, pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (for example, pZIPneo), yeast-derived expression vectors (for example, “Pichia Expression Kit” (manufactured by Invitrogen), pNV11, SP-Q01), Bacillus subtilis-derived expression vectors (For example, pPL608, pKTH50).

For the purpose of expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, etc., promoters necessary for expression in cells, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), It is essential to have MMTV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter, etc., and genes for selecting transformation into cells (for example, More preferably, it has a drug resistance gene that can be discriminated by a drug (neomycin, G418, etc.). Examples of such a vector include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

Furthermore, when the gene is stably expressed and the purpose is to amplify the copy number of the gene in the cell, a vector having a DHFR gene complementary to the CHO cell lacking the nucleic acid synthesis pathway (for example, , PCHOI, etc.), and amplifying with methotrexate (MTX). For the purpose of transient expression of the gene, COS with a gene expressing SV40 T antigen on the chromosome An example is a method of transforming with a vector (such as pcD) having an SV40 replication origin using cells. As the replication origin, those derived from polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can also be used. Furthermore, for gene copy number amplification in host cell systems, the expression vectors are selectable markers: aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase ( dhfr) gene and the like.

The host cell into which the vector is introduced is not particularly limited, and for example, E. coli or various animal cells can be used. The host cell can be used, for example, as a production system for production and expression of the antibody of the present invention. Production systems for polypeptide production include in vitro and in vivo production systems. Examples of in vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells.

When eukaryotic cells are used, for example, animal cells, plant cells, and fungal cells can be used as the host. Animal cells include mammalian cells such as CHO (J. Exp. Med. (1995) 108, 945), COS, 3T3, myeloma, BHK (baby hamster kidney), HeLa, Vero, amphibian cells such as Xenopus eggs Mother cells (Valle, et al., Nature (1981) 291, 358-340) or insect cells such as Sf9, Sf21, and Tn5 are known. In the present invention, CHO-DG44, CHO-DXB11, COS7 cells, and BHK cells are preferably used. In animal cells, CHO cells are particularly preferred for mass expression purposes. Introduction of a vector into a host cell can be performed by, for example, a calcium phosphate method, a DEAE dextran method, a method using a cationic liposome DOTAP (Boehringer Mannheim), an electroporation method, a lipofection method, or the like.

As plant cells, for example, cells derived from Nicotiana tabacum are known as protein production systems, and these may be cultured in callus. Fungal cells include yeasts such as the genus Saccharomyces, such as Saccharomyces cerevisiae, Saccharomyces pombe, filamentous fungi such as the genus Aspergillus, such as Aspergillus. Aspergillus niger) is known.

When using prokaryotic cells, there is a production system using bacterial cells. Bacterial cells include E. coli (eg, JM109, DH5α, HB101, etc.), and Bacillus subtilis is also known. The antibody of the present invention can be obtained by culturing cells transformed with the DNA of the present invention in vitro and purifying the cells by a method commonly used by those skilled in the art.

The present invention also provides a host organism having a vector containing a nucleic acid encoding the antibody of the present invention. The host organisms of the present invention are useful for the production of recombinant antibodies. Examples of host organisms in the present invention include goats. For example, the transgenic goat of the present invention can be produced as follows. That is, a fusion gene is constructed in which an antibody gene is inserted in-frame into a gene encoding a protein (such as goat β-casein) that is uniquely produced in milk. When a DNA fragment containing a fusion gene into which an antibody gene has been inserted is injected into a goat embryo, the injected embryo is introduced into a female goat. The antibody of the present invention can be obtained from the milk produced by a transgenic goat born from a goat that has received the embryo or its offspring. In order to increase the amount of milk containing the antibody of the present invention produced from a transgenic goat, hormones can also be used in the transgenic goat as appropriate (Ebert, KM et al., Bio / Technology (1994) 12 , 699-702).

The imaging agent of the present invention is administered to a mammal in order to image tumor cells.
In this specification, “imaging” refers to measuring and visualizing an object by adding a labeling substance to an antibody. A method of observing with a CCD camera using a fluorescent / radioisotope label, PET, SPECT, or CT using MRI is also included.
Examples of mammals include human and non-human mammals (eg, mouse, rat, hamster, rabbit, pig, monkey, etc.). The imaging agent of the present invention is useful in the diagnosis of the presence or absence of tumor cells. The imaging agent of the present invention can be used for both in vivo use and in vitro use.

The tumor cells imaged by the imaging agent of the present invention are cells expressing mesothelin. Cells expressing mesothelin, that is, cells imaged by the imaging agent of the present invention include lung cancer, mesothelioma, ovarian cancer, colon cancer, squamous cell carcinoma, pancreatic cancer, uterine cancer, head and neck cancer, breast cancer, etc. However, it is not limited to these.

The imaging agent of the present invention is obtained by binding an imaging label or probe that can be directly or indirectly tracked to an anti-mesothelin antibody.
After the probe is administered to a living body in vivo (for example, intravenously administered), the amount and distribution of tumor cells can be measured using an image measuring device such as PET, SPECT, or CCD camera.

In recent years, a computer computed tomography (hereinafter referred to as “CT”) that forms an internal image of an object by scanning the object with a transmissive radiation source and processing the data using a computer. (Also means Computed Tomography) and its clinical application.
Such CT techniques can be used to obtain cross-sectional images such as positron tomography (PET), single photon emission tomography (SPECT), and nuclear magnetic resonance imaging (MRI). Although it is a technology to obtain a three-dimensional cross-sectional image, these inspection techniques are not only used as a cross-sectional image, but are often displayed as three-dimensional graphics by integrating the two-dimensional image by improving computer image processing technology. It is a promising technique for specifying the location of three-dimensional lesions, making diagnoses, and determining surgical strategies.
For example, simple CT is to perform imaging such as X-ray irradiation without using a contrast agent, and can be sufficiently observed without using a contrast agent such as tissue edema, abnormal bone morphology, morphology, Contrast-enhanced CT refers to imaging performed after injecting a contrast agent or the like having a high X-ray absorption rate into a blood vessel, and the shape of a tissue rich in blood vessels and blood flow can be observed. In addition, so-called next-generation CT, for example, helical CT in which the radiation source moves in a spiral, multi-row detector CT (MDCT) (also called multi-slice CT (MSCT)) in which the detector itself is divided in the body axis direction are also developed. Any of them is not particularly limited and can be used alone or in combination for detection of the imaging agent of the present invention.
When the labeled probe for imaging (imaging agent of the present invention) is a radionuclide having a high X-ray absorption rate, it is possible to use CT alone as a detector.

Examples of the labeling substance include, but are not limited to, light emission by enzyme luminescence (luciferase), a substance using a light emitting small molecule, a substance using a fluorescent protein or a fluorescent small molecule, and a radionuclide. Radionuclides include gamma ray emitting nuclides such as ⁵¹ Cr, ⁵⁹ Fe, ⁵⁷ Co, ⁶⁷ Ga, ⁷⁵ Se, ^{81 m} Kr, ^{99 m} Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹³³ Xe, ²⁰¹ Tl, ¹¹ C, Positron emitting nuclides such as ¹³ N, ¹⁵ O, ¹⁸ F, ^{35 m} Cl, ⁷⁶ Br, ⁴⁵ Ti, ⁴⁸ V, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁶ Ga, ⁸⁹ Zr, ^{94 m} Tc, ¹²⁴ I However, it is not limited to these. As is obvious to those skilled in the art, “m” represents a nuclear isomer. In particular, indium-111, technetium-99m or iodine-131 can be used particularly preferably for plane scanning or single photon tomography (SPECT). A positron emission label such as fluorine-18, which can be particularly preferably used in positron emission tomography. Paramagnetic ions such as gadolinium (III) or manganese (II) are particularly preferably used in magnetic resonance imaging (MRI).

As fluorescent labels and luminescent labels, those using a luminescence system with an enzyme (luciferase), fluorescent proteins such as GFP, DsRed, and wedge orange, and fluorescent small molecules such as FITC, Cy5.5, Alexa Fluor 750, etc. ) Can be used.
In the case of luminescence by enzyme luminescence (luciferase), administration of a substrate is required separately.
In particular, those that reduce the influence of the animal's natural autofluorescence are preferred, and labels that emit signals with high skin permeability are preferred.

As the imaging detector, magnetic resonance imaging (MRI) PET or SPECT is used. In particular, in observation of a fluorescently labeled probe, from the viewpoint of low invasiveness, the measuring instrument is preferably a CCD camera.
Therefore, labels that emit light at a wavelength that can be captured by a CCD camera, for example, approximately 350-900 nm, are preferred. Furthermore, for measuring the intensity of the light source in the living body, a machine capable of measuring the intensity of the light source from the measurement value on the body surface of the animal to be measured by the CCD camera is preferable. In the case of a fluorescent label, either a reflected fluorescent image or a transmitted fluorescent image may be used, but it is preferable to capture both images. In addition, it is possible to superimpose fluorescent images in various directions (regardless of reflection and transmission), integrate the source information and process them, and capture them as a three-dimensional image (three-dimensional). Can be configured accurately, which is preferable. The three-dimensional image thus obtained can be further integrated with the CT image.

When the labeled probe for imaging binds a radionuclide having a high X-ray absorption rate, as described above, CT alone can be sufficiently used (for example, PET or SPECT), the position of the tumor cell, Accumulation amount and distribution can be measured.

On the other hand, after the labeled probe for imaging is administered to a living body in vivo (for example, intravenous administration), CT observation of the labeled probe or combined observation with CCD is also possible. As an example of combined observation, a simple CT (and / or superimposition with an image of contrast CT) can be used for a CCD image of a fluorescently labeled probe. In other words, CT images obtained by extraction of images of organs such as bones and lungs (and / or blood vessel and tissue images of contrast-enhanced CT) by simple CT and fluorescence probe images by a CCD camera are integrated, and tumor cells With regard to position, accumulation amount and distribution, it is possible to more accurately grasp the three-dimensional tissue, the relative positional relationship with blood vessels, and the three-dimensional (localized) image of the tumor cell.

The imaging agent of the present invention can be formulated by a known method by introducing a pharmaceutically acceptable carrier in addition to the antibody. For example, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection of suspension. For example, a pharmacologically acceptable carrier or medium, specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It is conceivable to prepare a pharmaceutical preparation by combining with a binder or the like as appropriate and mixing in a unit dosage form generally required for pharmaceutical practice. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.

Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.
Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride, and suitable solubilizers such as You may use together with alcohol, specifically ethanol, polyalcohol, for example, propylene glycol, polyethylene glycol, nonionic surfactant, for example, polysorbate 80 (TM), HCO-50.

Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Moreover, you may mix | blend with buffer, for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant. The prepared injection solution is usually filled into a suitable ampoule.

Administration is preferably parenteral administration, and specific examples include injection, nasal administration, pulmonary administration, and transdermal administration. As an example of the injection form, it can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.

The administration method can be appropriately selected depending on the age and symptoms of the patient. The dosage of the imaging agent of the present invention can be selected, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight at a time. Alternatively, for example, the dose can be selected in the range of 0.001 to 100,000 mg / body per subject, but is not necessarily limited to these values.
The dose and administration method vary depending on the weight, age, symptom, symptom of the subject, the intensity of the fluorescent label per mg antibody, the detection sensitivity of the detection machine, etc., but can be appropriately selected by those skilled in the art.

The present invention also provides a tumor cell imaging kit comprising an anti-mesothelin antibody. The kit of the present invention is characterized by imaging tumor cells by being administered to a subject. In addition to the antibody of the present invention, the kit includes, for example, a labeling substance, an injection (infusion) device for administration of an imaging agent, an auxiliary agent that suppresses non-adsorption (such as albumin), and the like, but is not limited thereto. It is not something.
In addition, the kit may include instructions included in a normal kit, such as instructions for use in imaging, appropriate containers, and control reagents.

The uses of the composition of the present invention described above can also be expressed as (1) to (3) below.
(1) Use of an anti-mesothelin antibody in the production of an imaging agent for tumor cells.
(2) An anti-mesothelin antibody for use in a tumor cell imaging method.
(3) A method for imaging tumor cells, comprising a step of administering an anti-mesothelin antibody to a subject.

The base sequences and amino acid sequences of the antibodies disclosed herein are described in the sequence listing according to the following SEQ ID NOs. The 11-25 antibody can also be referred to as # 11-25 antibody or clone 11-25.
<11-25 antibody>
SEQ ID NO: 1: base sequence of heavy chain variable region SEQ ID NO: 2: amino acid sequence of heavy chain variable region SEQ ID NO: 3: base sequence of heavy chain CDR1 SEQ ID NO: 4: amino acid sequence of heavy chain CDR1 SEQ ID NO: 5: heavy chain CDR2 Base sequence SEQ ID NO: 6: amino acid sequence of heavy chain CDR2 SEQ ID NO: 7: base sequence of heavy chain CDR3 SEQ ID NO: 8: amino acid sequence of heavy chain CDR3 SEQ ID NO: 9: base acid sequence of light chain variable region SEQ ID NO: 10: light chain Variable region amino acid sequence SEQ ID NO: 11: light chain CDR1 base sequence SEQ ID NO: 12: light chain CDR1 amino acid sequence SEQ ID NO: 13: light chain CDR2 base sequence SEQ ID NO: 14: light chain CDR2 amino acid sequence SEQ ID NO: 15: light Nucleotide sequence of chain CDR3 SEQ ID NO: 16: Amino acid sequence of light chain CDR3

Note that all prior art documents cited in the present specification are incorporated herein by reference.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the scope of the present invention is not limited to the aspect described in these Examples.
[Example 1]
The present inventors performed immunostaining on a plurality of cancer cells.

As the antibody anti-mesothelin antibody used, five types of antibodies, namely, 11-25 antibody, IC14-30, IC7-4, IC17-35, 2-9 antibody were used. As a control, one without added antibody was used. The method for obtaining and producing each antibody is as follows.
11-25 antibody: See Reference Examples 1 and 2, JP 2010-014691
IC14-30: Refer to JP2010-014691
IC7-4: See Reference Example 2
IC17-35: See Reference Example 2.
2-9 antibody: see Reference Example 2

The cell lines used are NCI-H226 (lung cancer), NCI-H520 (lung cancer), 211H (mesothelioma), PC3 (prostate cancer), PANC-1 (pancreatic cancer), BxPC-3 (pancreas) Cancer), CFPAC-1 (pancreatic cancer). The method for obtaining each cell line is as follows.
NCI-H226: Purchased from ATCC (search for CRL-5826).
NCI-H520: Purchased from ATCC (search for HTB-182).
211H: Yuji Kashiwakura, et al. Cancer Res 2008; 68: (20). October 15: 8333-8341, 2008, Yuji Kashiwakura, et al. Circulation April 29: 2078-2081, 2003 PC3: Obtained from Caliper Life Sciences (The name is PC-3M-luc-C6).
PANC-1: Purchased from ATCC (http://www.atcc.org/ATCCAdvancedCatalogSearch/tabid/112/Default.aspx and entered CRL-1469 in ATCC numbers category search) (Sumisho Pharma International Co., Ltd.), See Lieber M, et al. Establishment of a continuous tumor-cell line (panc-1) from a human carcinoma of the exocrine pancreas. Int. J. Cancer 15: 741-747, 1975.
BxPC-3: Purchased from ATCC (searched with CRL-1687), see Loor R, et al. Use of pancreas-specific antigen in immunodiagnosis of pancreatic cancer. Clin. Lab. Med. 2: 567-578, 1982.
CFPAC-1: Purchased from ATCC (searched by CRL-1918), see Schoumacher RA, et al. A cystic fibrosis pancreatic adenocarcinoma cell line. Proc. Natl. Acad. Sci. USA 87: 4012-4016, 1990.

Immunostaining respective cells were cultured for 3 days at Rabutekku II 8well (or 2Well) chamber slides were fixed with 10% formalin · PBS, washed with PBS (PBS-G) containing 10 mM glycine, containing 3% BSA Blocked with PBS and treated with anti-mesothelin antibody for 1 hour. After washing with PBS containing 0.1% BSA, TRITC-labeled donkey anti-mouse IgG (Santa Cruz, diluted 100-fold), DAPI (4000-fold diluted; nuclear staining), rhodamine phalloidin (molecular probe R415, diluted 200-fold) For 1 hour. After washing, it was sealed with a fluorescent mounting medium and a cover glass.
The images were observed with an Olympus IX71 fluorescence microscope or a confocal laser scanning microscope ZEISS LSM510.

Results BxPC-3 pancreatic cancer cells were cultured in an 8-well chamber, immunostained with five anti-mesothelin antibodies, and observed with a confocal laser microscope (FIG. 1). Green indicates binding of anti-mesothelin antibody, red indicates actin filaments, and blue indicates nuclei. The 11-25 antibody showed significant anti-mesothelin antibody binding near the cell surface. Binding to the cells was not detected with the other four anti-mesothelin antibodies.
CFPAC-1 pancreatic cancer cells were cultured in a 2-well chamber, immunostained with five anti-mesothelin antibodies, and observed with a confocal laser microscope (FIG. 2). Green indicates binding of anti-mesothelin antibody, red indicates actin filaments, and blue indicates nuclei. It was found that the binding of strong 11-25 anti-mesothelin antibody was distributed in small clumps near the cell surface. No binding to cells was detected with the other four types of anti-mesothelin antibodies.
PANC-1 pancreatic cancer cells were cultured in an 8-well chamber, immunostained with five anti-mesothelin antibodies, and observed with a confocal laser microscope (FIG. 3). Green indicates binding of anti-mesothelin antibody, red indicates actin filaments, and blue indicates nuclei. Binding of 11-25 anti-mesothelin antibody was observed in the cytoplasm. With the other four types of anti-mesothelin antibodies, binding to cells was hardly detected.
211H mesothelioma cells were cultured in an 8-well chamber, immunostained with two anti-mesothelin antibodies (11-25 antibody and # IC14-30 antibody), and observed with a confocal laser microscope (FIG. 4). 11-25 antibody binding was observed on the cell surface and cytoplasm, but # IC14-30 antibody binding was not observed.
Four types of PC3 prostate cancer cells, 211H mesothelioma cells, H520 and H226 lung cancer cells were stained with 11-25 anti-mesothelin antibody and observed with a confocal laser microscope (FIG. 5). PC3 cells showed binding of anti-mesothelin antibody to the cell membrane. In 211H cells, binding of anti-mesothelin antibody was observed in the cell membrane and cytoplasm. H520 cells showed weak antibody binding to the cytoplasm. In H226 cells, anti-mesothelin antibody binding was observed in the membrane and cytoplasm.

The reactivity of anti-mesothelin antibody 11-25 is summarized in Table 1.
[Table 1]
―――――――――――――――――――――――――
Immunostaining (positive site) In vivo imaging ―――――――――――――――――――――――――
211H (mesothelioma) + (cytoplasm & membrane) +
PC3 (prostate cancer) +/- (cell membrane) +/-
NCI-H226 (Lung cancer) + (Cytoplasm & membrane) +
NCI-H520 (lung cancer) +/- (cytoplasm)-
PANC-1 (pancreatic cancer) +/- (cytoplasm)-
BxPC-3 (pancreatic cancer) ++ (cell membrane) ++
CFPAC-1 (pancreatic cancer) ++ (cell membrane) ++++
―――――――――――――――――――――――――

For the 11-25 antibody that could be used for immunostaining, flow cytometry was performed in order to confirm the reactivity of living cells to the cell surface. NCI-H226, NCI-H520, BxPC-3, CFPAC-1 cells (1.0 x 10 ⁵ Cells / sample) stained with 50 µl of 11-25 antibody (1.0 µg / ml) for 1 hour at 4 ° C PE-labeled goat anti-mouse IgG antibody (IM0855 Beckman Coulter) was used as the secondary antibody. For flow cytometry, Beckman Coulter FC500 was used. As a result, the reactivity of the 11-25 antibody was confirmed in NCI-H226, BxPC-3, and CFPAC-1 cells.

[Example 2]
The cancer cells used in Example 1 were transplanted subcutaneously in the back of the back of nude mice, and after 3 weeks, 90 μg of Alexa 750 (Invitrogen) -labeled or Cy5.5 (GE Healthcare) -labeled 11-25 anti-mesothelin antibody was injected from the tail vein of the mice. After administration, the reflected fluorescence was measured with a biological fluorescence imaging apparatus IVIS200 (Xenogen) over time. When using Alexa 750-labeled antibody, excitation was performed at 745 nm and fluorescence at 800 nm, and when using Cy5.5-labeled antibody, measurement was performed at excitation at 675 nm and fluorescence at 740 nm.

Results Nude mice transplanted subcutaneously with PC3 prostate cancer cells and 211H mesothelioma cells were administered 90 μg of Alexa750-labeled 11-25 anti-mesothelin antibody via the tail vein, and fluorescence was measured 48 hours later. The PC3 tumor was somewhat weak and the 211H tumor was strongly fluorescent (FIG. 6 (A)).
Nude mice transplanted subcutaneously with NCI-H520 and NCI-H226 lung cancer cells were administered 90 μg of Cy5.5-labeled 11-25 antibody via the tail vein, and the reflected fluorescence was measured 24 hours later. The H520 tumor part did not show specific fluorescence, and the H226 tumor part emitted strong fluorescence (FIG. 6 (B)).
Nude mice transplanted subcutaneously with BxPC-3 and PANC-1 pancreatic cancer cells were administered 90 μg of Alexa750-labeled 11-25 anti-mesothelin antibody via the tail vein, and the reflected fluorescence was measured 24 hours later. The BxPC-3 tumor part showed strong fluorescence, but no fluorescence was observed in the PANC-1 tumor part (FIG. 6 (C)).
Nude mice transplanted with NCI-520 lung cancer cells and CFPAC-1 pancreatic cancer cells were administered with tail vein of 90 μg of Alexa750-labeled 11-25 anti-mesothelin antibody, and the reflected fluorescence was measured 24 hours later. The CFPAC-1 tumor part showed very strong fluorescence, but no specific fluorescence was observed in the H520 tumor part (FIG. 6 (D)).
FIG. 6 (E) is a photograph of the mouse tumor of FIG. 6 (B) taken out. H226 tumor showed strong fluorescence, while H520 tumor showed no specific fluorescence.
FIG. 6 (F) is a photograph of the mouse tumor of FIG. 6 (C) taken out. BxPC-3 tumors showed strong fluorescence while PANC-1 tumors did not show specific fluorescence.
FIG. 6 (G) is a photograph of the mouse tumor of (D) taken out. CFPAC-1 tumors showed strong fluorescence while H520 tumors did not show specific fluorescence.

[Reference Example 1 Production of recombinant mesothelin protein]
Recombinant mesothelin protein is composed of 5′-AAATTTCCAAGCTTGTGGAGAAGACAGCCTGTCCTTCAGGCAAG-3 ′ (SEQ ID NO: 22) and 5′-AAGGAAAAAAGCGGCCCCGCCCTGTAGCCCCAGCCCCAGCGGTCCAG-3 ′ (SEQ ID NO: 23) using a primer called G It was created by amplifying the coding part of amino acids 297-580 derived from cDNA encoding transcription variant 1 of session number NM_005823). The amplified DNA was inserted into the HindIII / NotI sites of the expression vector pSecTag2B (Invitrogen). Amino acids 297-580 of the protein encoded by clone NM005823 are common to human mesothelin isoforms 1 and 3 described by Scholler et al. (Proc Natl Acad Sci US A. 1999; 96 (20): 11531-6). Note that this is an array of The mesothelin expression plasmid was transfected into HEK293T cells. Mesothelin protein was purified from the culture supernatant of the transfected cells using TALON resin. The purified mesothelin protein thus obtained was dialyzed twice with PBS and stored at −80 ° C. until use.

[Reference Example 2 Production of Mesothelin Antibody]
To produce monoclonal antibodies (mAbs) against mesothelin, 4-6 week old BALB / c mice were treated with purified recombinant mesothelin protein intraperitoneally on days 0, 7, 14, and 16. Immunized by injection (10 micrograms / injection). On day 18 after the fourth immunization, splenic lymphocytes were collected and fused with P3U1 melanoma cells using a 50% polyethylene glycol 4000 solution. The fused cells were seeded on 96-well plates using RPMI-1640 medium containing 15% fetal bovine serum, penicillin / streptomycin and HAT solution (Invitrogen). Culture supernatants were collected after 10 days of culture at 37 ° C., 5% CO ₂ , saturated water vapor and screened for the ability to bind to the immunogen by indirect ELISA using recombinant mesothelin protein. Selected positive hybridoma colonies were grown and subcloned by limiting dilution. Subcloned hybridomas were cultured and the subcloned antibody isotype was determined using Roche's isostrip kit. Antibody purification was performed by protein A affinity chromatography. After an immunogen competition assay between the resulting clones, the 11-25 antibody (IgG2b kappa) was selected to construct an ELISA for ELISA detection of MPF. The 11-25 antibody was biotinylated using the GE Healthcare ECL protein biotination module.
Similarly to the 11-25 antibody, IC7-4, IC17-35, 2-9 antibodies were prepared.

[Reference Example 3 Characterization of MSLN Antibody]
To determine the nucleic acid sequence of the CDR regions of the anti-MSLN monoclonal antibody, 11-25 antibody, total RNA was collected from hybridomas producing each of IC11-25 produced according to Reference Example 2. Messenger RNA in the total RNA collected was reverse transcribed into complementary DNA using oligo (dT) 12-18 primer (purchased from Invitrogen). From this DNA, the nucleotide sequence of the immunoglobulin variable region sequence is 5'-ATGGCTGTCTTGGGGCTGCTCTTCTGC-3 '(SEQ ID NO: 24) and 5'-CAGTGGATAGACTGATGGGGG-3' (SEQ ID NO: 25) for the heavy chain of 11-25. Amplified by PCR technique using primers and 5′-ATGAAGTTGCCCGTTAGGCTGTGTGTGCTG-3 ′ (SEQ ID NO: 26) and 5′-ACTGGATGGTGGAGAATGG-3 ′ (SEQ ID NO: 27) primers for 11-25 light chains. The amplified cDNA was inserted into the EcoRV site of the pBluescript II KS (+) plasmid (Stratagene). Next, the sequences of the VH and VL regions were analyzed by a gene sequencing device (3130 / 3100-Avant (R) Genetic Analyzer Capillary Arrays, Applied Biosystems). The determination of the region between FR1, 2, 3 and 4 and CDR1, 2 and 3 is as follows: IgG blast (NCBI database) and SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Volume 1, 5th edition, 1991; NIH Publication 91-3242 P. (Elvin A. Kabat et al.). The sequence of each region of the 11-25 antibody is as follows.
<11-25 antibody>
SEQ ID NO: 1: base sequence of heavy chain variable region SEQ ID NO: 2: amino acid sequence of heavy chain variable region SEQ ID NO: 3: base sequence of heavy chain CDR1 SEQ ID NO: 4: amino acid sequence of heavy chain CDR1 SEQ ID NO: 5: heavy chain CDR2 Base sequence SEQ ID NO: 6: amino acid sequence of heavy chain CDR2 SEQ ID NO: 7: base sequence of heavy chain CDR3 SEQ ID NO: 8: amino acid sequence of heavy chain CDR3 SEQ ID NO: 9: base acid sequence of light chain variable region SEQ ID NO: 10: light chain Variable region amino acid sequence SEQ ID NO: 11: light chain CDR1 base sequence SEQ ID NO: 12: light chain CDR1 amino acid sequence SEQ ID NO: 13: light chain CDR2 base sequence SEQ ID NO: 14: light chain CDR2 amino acid sequence SEQ ID NO: 15: light Nucleotide sequence of chain CDR3 SEQ ID NO: 16: Amino acid sequence of light chain CDR3

The present inventors have developed a reagent that can directly image cancer derived from an actual patient that highly expresses mesothelin membrane. The present invention is useful for imaging cancers (lung cancer, mesothelioma, ovarian cancer, colon cancer, squamous cell cancer, pancreatic cancer, uterine cancer, etc.) expressing mesothelin. .

Claims (6)

1. A tumor cell imaging agent comprising an anti-mesothelin antibody.
2. The imaging agent according to claim 1, wherein the antibody is an antibody according to any one of the following (a) to (e):
   (A) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 4 as CDR1, the amino acid sequence of SEQ ID NO: 6 as CDR2, and the amino acid sequence of SEQ ID NO: 8 as CDR3;
   (B) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 2 as the heavy chain variable region;
   (C) an antibody comprising a light chain having the amino acid sequence of SEQ ID NO: 12 as CDR1, the amino acid sequence of SEQ ID NO: 14 as CDR2, and the amino acid sequence of SEQ ID NO: 16 as CDR3;
   (D) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 as the light chain variable region,
   (E) An antibody having the heavy chain pair described in (a) or (b) above and the light chain pair described in (c) or (d) above.
3. The imaging agent according to claim 1 or 2, which is used in vivo.
4. A kit for imaging tumor cells, comprising an anti-mesothelin antibody.
5. The imaging kit according to claim 4, wherein the antibody is the antibody according to any of the following (a) to (e):
   (A) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 4 as CDR1, the amino acid sequence of SEQ ID NO: 6 as CDR2, and the amino acid sequence of SEQ ID NO: 8 as CDR3;
   (B) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 2 as the heavy chain variable region;
   (C) an antibody comprising a light chain having the amino acid sequence of SEQ ID NO: 12 as CDR1, the amino acid sequence of SEQ ID NO: 14 as CDR2, and the amino acid sequence of SEQ ID NO: 16 as CDR3;
   (D) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 as the light chain variable region,
   (E) An antibody having the heavy chain pair described in (a) or (b) above and the light chain pair described in (c) or (d) above.
6. The imaging kit according to claim 4 or 5, which is used in vivo.

Images