Therapeutic agent for carcinomatous peritonitis

Abstract

An object of the present invention is to provide a therapeutic agent for carcinomatous peritonitis. According to the present invention, there can be provided a therapeutic agent for carcinomatous peritonitis containing an antibody that recognizes the transferrin receptor.

Description

Technical Field

[0001] The present invention relates to a therapeutic agent for carcinomatous peritonitis containing an anti-transferrin receptor antibody. Background Art

[0002] Transferrin receptor (TfR) was first identified on reticulocytes as a cell membrane structure for taking up iron bound to transferrin (Tf) into cells. Subsequently, TfR has been found to be expressed in trophoblast cells of the placenta, activated lymphocytes, and various tumor cells. For example, it has been reported that TfR is highly expressed in breast cancer, prostate cancer, lung cancer, pancreatic cancer, colorectal cancer, gastric cancer, bladder cancer, liver cancer, cervical cancer, brain tumors, chronic lymphocytic leukemia, non-Hodgkin lymphoma, and adult T-cell leukemia. Since TfR is highly expressed on the surface of various cancer cells and is expressed at low levels in normal cells, it is considered a molecular target for cancer treatment. For example, Patent Document 1 describes an antibody that can specifically recognize the transferrin receptor and an anticancer agent using the above antibody.

[0003] Carcinomatous peritonitis is a disease in which cancer metastasizes to the peritoneum. Carcinomatous peritonitis may be associated with all types of cancer, but has a high incidence in cancers of the digestive organs in the abdominal cavity (gastric cancer, pancreatic cancer, colorectal cancer, etc.) and ovarian cancer.

[0004] Prior Art Documents

[0005] Patent Documents

[0006] Patent Document 1: International Publication WO2014 / 073641 Summary of the Invention

[0007] Problems to be Solved by the Invention

[0008] It is known that cancer cells metastasized to the peritoneum are special cells that have acquired the ability to survive under hypoxic conditions. It is predicted that cancer cells metastasized to the peritoneum have developed resistance to anticancer agents targeting the normal cell proliferation mechanism. Based on a large amount of clinical experience and the results of clinical trials, it is also known that intraperitoneal administration of conventional anticancer agents is ineffective for the treatment of carcinomatous peritonitis. As treatment methods for carcinomatous peritonitis, in addition to chemotherapy, immunotherapy, hyperthermia therapy, etc. can be cited, but the treatment effect is insufficient and the prognosis is poor, and thus a new treatment method is desired. The problem to be solved by the present invention is to provide an agent for treating cancer peritonitis.

[0009] Means for Solving the Problems

[0010] The inventors of the present invention conducted research to solve the above problems, and as a result, found that administering an antibody that recognizes an amino acid sequence at a specific position in TfR to a model mouse with carcinomatous peritonitis could treat carcinomatous peritonitis, thereby completing the present invention.

[0011] That is, according to the present invention, the following inventions are provided.

[0012] (1) A therapeutic agent for carcinomatous peritonitis, which contains an antibody that recognizes transferrin receptor.

[0013] (2) The therapeutic agent for carcinomatous peritonitis according to (1), wherein the antibody that recognizes transferrin receptor is an antibody that recognizes human transferrin receptor.

[0014] (3) The therapeutic agent for carcinomatous peritonitis according to (2), wherein the antibody that recognizes human transferrin receptor is an antibody that recognizes the 629th to 633rd amino acids of human transferrin receptor.

[0015] (4) The therapeutic agent for carcinomatous peritonitis according to any one of (1) to (3), wherein the antibody has the heavy chain first complementary determining region (VH CDR1), heavy chain second complementary determining region (VH CDR2), and heavy chain third complementary determining region (VH CDR3) as SEQ ID NOs: 1, 2, and 3, respectively, and the light chain first complementary determining region (VL CDR1), light chain second complementary determining region (VL CDR2), and light chain third complementary determining region (VL CDR3) as SEQ ID NOs: 4, 5, and 6, respectively.

[0016] (5) The therapeutic agent for carcinomatous peritonitis according to any one of (1) to (4), wherein the antibody has a heavy chain with SEQ ID NO: 7 and a light chain with SEQ ID NO: 8.

[0017] (6) The therapeutic agent for carcinomatous peritonitis according to any one of (1) to (5), wherein the antibody is a human antibody or a humanized antibody.

[0018] (7) The therapeutic agent for carcinomatous peritonitis according to any one of (1) to (6), wherein the antibody is an antibody fragment selected from Fab, Fab', F(ab')2, single-chain antibody (scFv), bispecific antibody, disulfide-stabilized Fv antibody (dsFv), and peptide containing CDR.

[0019] (8) The therapeutic agent for carcinomatous peritonitis according to any one of (1) to (7), wherein the carcinomatous peritonitis is complicated with cancer as the cause of the carcinomatous peritonitis.

[0020] (9) The therapeutic agent for carcinomatous peritonitis according to any one of (1) to (8), wherein the carcinomatous peritonitis is complicated with gastric cancer, pancreatic cancer, colorectal cancer, ovarian cancer, biliary tract cancer, liver cancer, gastrointestinal stromal tumor (GIST) or small intestine cancer.

[0021] (10) The therapeutic agent for carcinomatous peritonitis according to any one of (1) to (9), which is a therapeutic agent for intraperitoneal administration.

[0022] (A) Provided is a method for treating carcinomatous peritonitis, which includes the step of administering an antibody that recognizes the transferrin receptor to a subject.

[0023] (B) An antibody that recognizes the transferrin receptor and is used for treating carcinomatous peritonitis.

[0024] (C) Use of an antibody that recognizes the transferrin receptor in the manufacture of a therapeutic agent for carcinomatous peritonitis.

[0025] Effects of the Invention

[0026] The therapeutic agent for carcinomatous peritonitis of the present invention is very useful for treating carcinomatous peritonitis. The therapeutic agent for carcinomatous peritonitis of the present invention is a molecularly targeted drug targeting a new mechanism that attacks the nutrient supply pathway necessary for cell metabolism. The therapeutic agent for carcinomatous peritonitis of the present invention is the best method for peritoneal implanting cells that survive in a hypoxic and low-nutrient environment and are resistant to existing anticancer agents, leading to an unprecedented and original new medicine. Description of the Drawings

[0027] Figure 1 Indicates the site where point mutation is performed for each TfR mutant fragment.

[0028] Figure 2 Indicates the reactivity of TfR436 and soluble wild-type TfR (sTfR) and TfR mutant fragments.

[0029] Figure 3 Indicates the Tf-TfR binding inhibitory activity of TfR436.

[0030] Figure 4 Indicates the TfR expression in the tumor tissue of the patient.

[0031] Figure 5 Indicates the analysis result of the survival time.

[0032] Figure 6 Indicates the HE specimen of the peritoneal tumor of the mouse in the TfR436 antibody administration group.

[0033] Figure 7 Indicates the measurement result of the ascites weight.

[0034] Figure 8Shows the analysis results of the survival rate of the peritoneal implantation model of pancreatic cancer cell line SUIT2.

[0035] Figure 9 Shows the TfR expression in patient samples. Detailed implementation manners

[0036] Next, the present invention will be described in more detail.

[0037] Definitions and General Techniques

[0038] In this specification, unless otherwise defined, the scientific and technical terms used in the present invention include the meanings commonly understood by those skilled in the art. Generally speaking, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described in this specification are well-known techniques in this technical field and are commonly used.

[0039] Generally speaking, unless otherwise defined, the methods and techniques of the present invention can be implemented in the manner described in various general reference documents and more specific reference documents cited and discussed throughout this specification according to the methods well-known in this technical field.

[0040] TfR

[0041] In the case of humans, the transferrin receptor (TfR) is a single-pass transmembrane protein composed of 760 amino acids encoded by human chromosome 3 (SEQ ID NO: 9). This protein is known as the CD71 antigen and is thought to be involved in the uptake of iron by cells and in cell proliferation. The TfR of the present invention is not particularly limited in structure and includes monomers, polymers, intact forms expressed on the cell membrane, soluble forms composed of extracellular regions, truncated forms, and also includes mutant forms caused by gene mutations, deletions, etc., forms that have undergone post-translational modifications such as phosphorylation, etc., all referring to TfR.

[0042] Carrying out Reactions and Reactivity

[0043] In this specification, unless otherwise specified, "performing a reaction" and "reactivity" have the same meaning. That is, an antibody recognizes an antigen. The antigen can be the complete TfR expressed on the cell membrane, or a truncated or soluble form. Additionally, it can be the TfR maintaining its three-dimensional structure, or a modified TfR. Examples of methods for studying reactivity include flow cytometry (FACS), enzyme-linked immunosorbent assay (ELISA), western blot, fluorescence microplate assay technology (FMAT), surface plasmon resonance (BIAcore), immunostaining, immunoprecipitation, etc.

[0044] As the antibody used in flow cytometry, it can be an antibody labeled with a fluorescent substance such as FITC or biotin, or an unlabeled antibody. Depending on whether the antibody used is labeled or not and the type of label, fluorescent-labeled avidin, fluorescent-labeled anti-human immunoglobulin antibody, etc. are used. Reactivity can be evaluated by the following method: adding a sufficient amount of anti-TfR antibody (usually with a final concentration of 0.01 - 10 μg / mL) to a sample and comparing the reactivity with that of a negative control antibody and a positive control antibody.

[0045] Antibody

[0046] In this specification, the following abbreviations (in parentheses) are used as needed and in accordance with common practice.

[0047] Heavy chain (H chain), light chain (L chain), variable region of heavy chain (VH), variable region of light chain (VL), complementarity-determining region (CDR), first complementarity-determining region (CDR1), second complementarity-determining region (CDR2), third complementarity-determining region (CDR3), first complementarity-determining region of heavy chain (VH CDR1), second complementarity-determining region of heavy chain (VH CDR2), third complementarity-determining region of heavy chain (VH CDR3), first complementarity-determining region of light chain (VL CDR1), second complementarity-determining region of light chain (VL CDR2), third complementarity-determining region of light chain (VL CDR3).

[0048] In this specification, the term "antibody" is synonymous with immunoglobulin and can be understood as is commonly known in the art. Specifically, the term "antibody" is not limited by any particular method for producing an antibody. For example, the term "antibody" includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies, but is not limited to these antibodies.

[0049] In this specification, the term "human antibody" refers to any antibody in which the sequences of the variable and constant regions are of human origin. The term also includes antibodies having sequences of human gene origin but modified, for example, to remove cysteine residues that may cause unwanted folding in a manner that reduces immunogenicity and increases affinity. The term also includes antibodies that can effect glycosylation not characteristic of human cells, such as those produced recombinantly in non-human cells. These antibodies can be prepared in various ways.

[0050] In this specification, the term "humanized antibody" refers to an antibody of non-human origin in which the characteristic amino acid residues in the antibody sequence of a non-human species are replaced with residues recognized at the corresponding positions in human antibodies. It is believed that this "humanization" process results in an antibody with reduced immunogenicity in the human body. It is understood that non-human origin antibodies can be humanized using techniques well known in the art. For example, reference can be made to Winter et al., Immunol. Today 14:43-46 (1993). The antibody to be targeted can be engineered by replacing the corresponding human sequences of CH1, CH2, CH3, the hinge region, and / or the framework region using recombinant DNA technology. For example, reference can be made to WO92 / 02190 and U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and 5,777,085. In this specification, the term "humanized antibody" includes chimeric human antibodies and CDR-grafted antibodies within the scope of its meaning.

[0051] The sequence of the framework region (FR) in the variable region of an antibody is not particularly limited as long as it has no substantial effect on the specific binding to the corresponding antigen. It is preferable to use the FR region of a human antibody, and the FR region of an animal other than human (e.g., mouse, rat) can also be used.

[0052] In one embodiment of the antibody, it further includes a constant region (e.g., an IgG-type antibody) in addition to the variable region. The sequence of the constant region is not particularly limited. For example, the well-known constant region of a human antibody can be used. As the heavy chain constant region (CH) of a human antibody, it is sufficient if it belongs to the heavy chain constant region of human immunoglobulin (hereinafter referred to as hIgG), preferably the heavy chain constant region of the hIgG cluster, and any one of the subclusters such as hIgG1, hIgG2, hIgG3, and hIgG4 belonging to the hIgG cluster can also be used. In addition, as the light chain constant region (CL), as long as it belongs to hIg, the light chain constant region of the κ cluster or λ cluster can be used. In addition, the constant region of an animal other than human (e.g., mouse, rat) can also be used.

[0053] In this specification, "mutant" or "mutated antibody" means the substitution, deletion, addition, and / or insertion of one or more amino acids in the amino acid sequence of the variable region (CDR sequence and / or FR sequence) of the parental antibody.

[0054] In the present invention, the "parent antibody" refers to the TfR436 antibody in which VH has the amino acid sequence shown in SEQ ID NO: 7 and VL has the amino acid sequence shown in SEQ ID NO: 8. In the amino acid sequence, 1 or several (for example, 1 to 8, preferably 1 to 5, more preferably 1 to 3, particularly preferably 1 or 2) amino acids are deleted, added, substituted and / or inserted. As is well known to those skilled in the art for the method of amino acid sequence for preparing an antibody having binding activity to TfR, there is a method of introducing a mutation into a protein. For example, those skilled in the art can use site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 100, 468-500, Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, HJ (1984) The gapped duplex DNA approach tooligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456, Kramer W, and Fritz HJ (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367, Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 82, 488-492), etc., to introduce appropriate mutations into the amino acid sequence of an antibody having binding activity to TfR, thereby preparing a mutant antibody having the same function as the antibody having binding activity to TfR.Thus, it is also possible to use an antibody having one or several amino acid mutations in the variable region or constant region of the antibody and having binding activity to TfR.

[0055] In the present specification, "equivalent activity to the parental antibody" means equivalent binding activity to TfR. "Equivalent" does not necessarily mean the same level of activity, and the activity can also be enhanced, or as long as it has activity, the activity can also be reduced. As an antibody with reduced activity, for example, an antibody having 30% or more, preferably 50% or more, more preferably 80% or more, still more preferably 90% or more, and particularly preferably 95% or more activity compared to the original antibody can be cited.

[0056] Binding activity refers to the recognition of an antigen. The antigen can be the full-length type of TfR expressed on the cell membrane, or a truncated type or soluble type. In addition, it can be TfR with a maintained three-dimensional structure, or a modified TfR. In addition, as methods for studying binding activity, flow cytometry (FACS), enzyme-linked immunosorbent assay (ELISA), western blot, fluorescence microassay technology (FMAT), surface plasmon resonance (BIAcore), etc. can be cited.

[0057] The Tf-TfR binding inhibitory activity of the antibody can be measured according to the method described in "Example 2 (2) Comparison of TfR436 antibody with antibodies from other companies in Tf-TfR binding inhibition" described later. The TfR solution is dispensed into a substrate (such as a 96-well plate), left standing and solidified, and blocked. Then, the HRP-labeled Tf solution is dispensed, and then the antibody is added, and the reaction is carried out at room temperature. Thereafter, the substrate is washed, a chromogenic reagent (such as TMB) is added, and the reaction is carried out, and the absorbance is measured with a spectrophotometer. Through the above operations, the Tf-TfR binding inhibitory activity of this antibody can be evaluated.

[0058] The source of the antibody is not limited, and it can be an antibody from any animal such as a human antibody, a mouse antibody, or a rat antibody. In addition, it can also be a chimeric antibody, a humanized antibody, etc. As one of the preferred forms of the antibody in the present invention, it is a human antibody.

[0059] The antibody can be different in amino acid sequence, molecular weight, isoelectric point, presence or absence of sugar chains, morphology, etc. according to the antibody-producing cells, host, or purification method described later. For example, it also includes the case where the amino acid sequence described in the present invention is modified after translation. It also includes post-translational modifications at sites other than known post-translational modifications. In addition, when the antibody is expressed in a prokaryotic cell, such as Escherichia coli, a methionine residue is added to the N-terminus of the amino acid sequence of the original antibody. In the present invention, such an antibody can also be used. It also includes post-translational modifications at sites other than known post-translational modifications.

[0060] Production of Antibodies

[0061] (1) Obtaining scFv by reacting a phage display library with an antigen

[0062] The acquisition of antibodies can be prepared by various methods known in the art. For example, phage display technology can be used to provide a library including antibody libraries with different affinities for TfR. Then, these libraries are screened to identify and isolate antibodies against TfR. Preferably, the phage library is a scFv phage display library generated using human VL and VH cDNAs prepared from mRNA isolated from human B cells. Methods for preparing such a library and screening it are known in the art. Using TfR as an antigen, the genetic material is recovered from the phage clones showing reactivity obtained by screening. By analyzing the genes of the selected phages, the DNA sequences of VH and VL encoding the variable regions of human antibodies that bind to the antigen can be determined. Using the sequence of this scFv and IgGifying the scFv, a human antibody can be obtained. (2) IgGification of scFv (production of human antibodies)

[0063] An expression vector for the H chain or L chain is prepared to express it in a host cell, and a human antibody is obtained by recovering and purifying the secreted supernatant. Additionally, a human antibody can also be obtained by expressing VH and VL in the same vector (tandem type). These methods are well-known, and references can be made to WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, W093 / 19172, WO95 / 01438, WO95 / 15388, WO97 / 10354, etc.

[0064] Specifically, a full-length heavy chain gene can be obtained by linking DNA encoding VH to other DNA molecules encoding the heavy chain constant regions (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (e.g., Kabat, E A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be the constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD, and most preferably the constant region of IgG1 or IgG2. The IgG1 constant region sequence can be any of the various alleles or allotypes such as Gm(1), Gm(2), Gm(3), Gm(17), etc. that are known to occur among different individuals. These allotypes correspond to naturally occurring amino acid substitutions in the IgG1 constant region.

[0065] A full-length L chain gene (and Fab light chain gene) can be obtained by linking DNA encoding VL to other DNA molecules encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (e.g., Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be the constant region of κ or λ. The κ constant region can be any of the various alleles such as Inv(1), Inv

[0066] (2), Inv(3), etc. that are known to occur among different individuals. The λ constant region can be from any one of the three λ genes.

[0067] An expression vector is prepared by inserting the DNA encoding the H chain or L chain obtained as described above into an expression vector, expressing it in a host cell, and obtaining a human antibody by recovering and purifying the secreted supernatant. Examples of the expression vector include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, cosmids, YACs, episomes from EBV, etc. The expression vector and the expression regulatory sequence are selected to match the host cell used for expression. The antibody light chain gene and the antibody heavy chain gene can be inserted into different vectors, or the genes of both can be inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment to the vector, or blunt-end ligation when there are no restriction sites).

[0068] A suitable vector is an engineered vector with appropriate restriction sites that can easily insert and express any VH or VL sequence as described above and encodes a fully functional human CH or CL immunoglobulin sequence. Such vectors usually undergo splicing between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, or in the splice region present within the human CH exon. Polyadenylation and transcription termination occur at the natural chromosomal site downstream of the coding region. The recombinant expression vector can also encode a signal peptide for the secretion of the antibody chain derived from the host cell. The antibody chain gene can be cloned into the vector in such a way that the signal peptide is ligated in-frame to the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0069] In addition to the antibody gene and control sequences, the antibody expression vector can also have other sequences such as a sequence for controlling the replication of the vector in the host cell (e.g., origin of replication) and a selectable marker gene. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced. For example, generally, the selectable marker gene confers resistance to drugs such as G418, hygromycin, and methotrexate on the host cells into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (used in combination with methotrexate selection / amplification in dhfr - host cells), the neomycin phosphotransferase gene (for G418 selection), and the glutamine synthetase gene.

[0070] The host cell is transformed with the antibody gene expression vector prepared by the above method. As the host cell, it can be bacteria, yeast, animal cells, insect cells, plant cells, etc., as long as it can produce antibodies, and can be any cell, preferably animal cells. As animal cells, Chinese hamster ovary cells CHO / dhfr(-) cells, CHO / DG44 cells, cells from monkeys such as COS cells (A. Wright & S. L. Morrison, J. Immunol. 160, 3393 - 3402 (1998)), SP2 / O (mouse myeloma) (K. Motmans et al., Eur. J. Cancer Prev. 5, 512 - 5199 (1996), R. P. Junghans et al., Cancer Res. 50, 1495 - 1502 (1990)), etc. can be cited. In addition, transformation can be appropriately carried out using the liposome method (R. W. Malone et al., Proc. Natl. Acad. Sci. USA 86, 6007 (1989), P. L. Felgner et al., Proc. Natl. Acad. Sci. USA 84, 7413 (1987)), electroporation method, calcium phosphate method (F. L. Graham & A. J. van der Eb, Virology 52, 456 - 467 (1973)), DEAE - Dextran method, etc.

[0071] After culturing the transformant, the human antibody is separated from the inside of the transformant cell or the culture solution. For the separation and purification of the antibody, methods such as centrifugation, ammonium sulfate fractionation, salting - out, ultrafiltration, affinity chromatography, ion - exchange chromatography, gel - filtration chromatography, etc. can be appropriately combined and utilized.

[0072] Antibody Fragments

[0073] Antibody fragments can be prepared based on the sequence information of the antibody or the gene encoding the antibody. As antibody fragments, Fab, Fab', F(ab')2, scFv, dsFv antibodies can be cited.

[0074] Fab is a fragment with a molecular weight of about 50,000, which is obtained by digesting IgG with papain in the presence of cysteine and consists of an L chain and an H chain fragment including the variable region of the H chain, the CH1 region, and a part of the hinge region. In the present invention, it can be obtained by digesting the above - mentioned antibody with papain. In addition, it can also be prepared by recombining the DNA of a part of the H chain and the L chain encoding the above - mentioned antibody into an appropriate vector and using the transformant obtained by transforming with this vector.

[0075] Fab' is a fragment with a molecular weight of approximately 50,000 obtained by cleaving the disulfide bond between the H chains of the following F(ab')2. In the present invention, it is obtained by digesting the above antibody with pepsin and cleaving the disulfide bond using a reducing agent. Additionally, similar to Fab, it can also be prepared by genetic engineering using DNA encoding Fab'.

[0076] F(ab')2 is a fragment with a molecular weight of approximately 100,000 obtained by binding the fragments (Fab') composed of L chains and H chain fragments including the variable region of the H chain, the CH1 region, and a part of the hinge region, which are obtained by digesting IgG with pepsin, through disulfide bonds. In the present invention, it is obtained by digesting the above antibody with pepsin. Additionally, similar to Fab, it can also be prepared by genetic engineering using DNA encoding F(ab')2.

[0077] scFv is a single-chain antibody fragment in which the Fv including the variable region of the H chain and the variable region of the L chain is linked by an appropriate peptide linker to the C-terminus of one chain and the N-terminus of the other chain. As the peptide linker, for example, (GGGGS)3 with high flexibility can be used. For example, DNA encoding the scFv antibody can be constructed using DNA encoding the variable region of the H chain and the variable region of the L chain of the above antibody and DNA encoding the peptide linker, which is recombined into an appropriate vector, and scFv is prepared from the transformant transformed with this vector.

[0078] dsFv is an Fv fragment in which Cys residues are introduced at appropriate positions in the variable region of the H chain and the variable region of the L chain, and the variable region of the H chain and the variable region of the L chain are stabilized by disulfide bonds. The positions for introducing Cys residues in each chain can be determined based on the three-dimensional structure predicted by molecular simulation. In the present invention, for example, the three-dimensional structure is predicted from the amino acid sequences of the variable region of the H chain and the variable region of the L chain of the above antibody, DNA encoding the variable region of the H chain and the variable region of the L chain into which mutations are introduced based on this prediction is constructed, which is recombined into an appropriate vector, and dsFv is prepared from the transformant transformed with this vector.

[0079] In addition, antibody fragments can also be polymerized by linking scFv antibodies, dcFv antibodies, etc. using an appropriate linker, or by fusing streptavidin.

[0080] Bispecific Antibodies

[0081] The antibody can be a bispecific antibody.

[0082] As bispecific antibodies, examples include bispecific (mab)2 obtained by chemically crosslinking two molecules of monoclonal antibodies, bispecific F(ab’)2 obtained by chemically crosslinking two molecules of Fab fragments, quadroma, bsDb (bispecific diabody), scBsDb (single-chain bispecific diabody), scBsTaFv (single-chain bispecific tandem variable domain), Bite (Bispecific T cell Engager antibody), DNL-F(ab)3 (docl-and-lock trivalent Fab), etc. (Shim, H. Bispecific Antibodies and Antibody-Drug Comjugates for Cancer Therapy: Technological Considerations. Biomolecules 2020, 10, 360).

[0083] Pharmaceutical Compositions and Formulations

[0084] The pharmaceutical compositions and preparations containing the therapeutic agent for carcinomatous peritonitis of the present invention are also included within the scope of the present invention.

[0085] The therapeutic agent for carcinomatous peritonitis of the present invention can be used in the treatment of carcinomatous peritonitis.

[0086] Carcinomatous peritonitis refers to a disease in which cancer metastasizes to the peritoneum. As carcinomatous peritonitis, there is no particular limitation, and examples include carcinomatous peritonitis complicated by cancer that is the cause of carcinomatous peritonitis. More specifically, examples include carcinomatous peritonitis complicated by gastric cancer, pancreatic cancer, colorectal cancer, ovarian cancer, biliary tract cancer, liver cancer, gastrointestinal stromal tumor (GIST), or small intestine cancer. In carcinomatous peritonitis, cancer cells are scattered in the abdominal cavity, and a large number of granular tumors are produced on the peritoneum, and ascites accumulates.

[0087] The pharmaceutical compositions and preparations containing the therapeutic agent for carcinomatous peritonitis of the present invention preferably contain a physiologically acceptable diluent or carrier in addition to the antibody, and may also be a mixture with other drugs. Suitable carriers include normal saline, phosphate-buffered saline, phosphate-buffered saline glucose solution, and buffered saline, but are not limited to these. Alternatively, the antibody can be freeze-dried and, when needed, the above-mentioned buffered aqueous solution can be added for reconstitution and use. As the administration method, parenteral administration by intraperitoneal administration (intraperitoneal injection, etc.) can be cited.

[0088] The dosage of the therapeutic agent for carcinomatous peritonitis of the present invention varies depending on symptoms, age, body weight, etc. Generally, in the case of oral administration, as the amount of the antibody, for adults, it is about 0.01 mg to 1000 mg per day, and it can be administered once or in several divided doses. In addition, in the case of non-oral administration, it can be intraperitoneally administered at about 0.01 mg to 3000 mg per time.

[0089] The present invention will be described in further detail by the following examples, but the present invention is not limited to the examples.

[0090] Examples

[0091] In the following examples, the TfR436 antibody described in paragraphs 0090 and 0091 of International Publication WO2014 / 073641 was used.

[0092] The CDR sequences of the TfR436 antibody are shown below.

[0093] VH CDR1: SYGMH (SEQ ID NO: 1)

[0094] VH CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 2)

[0095] VH CDR3: DSNFWSGYYSPVDV (SEQ ID NO: 3)

[0096] VL CDR1: TRSSGSIASNSVQ (SEQ ID NO: 4)

[0097] VL CDR2: YEDTQRPS (SEQ ID NO: 5)

[0098] VL CDR3: QSYDSAYHWV (SEQ ID NO: 6)

[0099] The VH sequence and VL sequence of the TfR436 antibody are shown below.

[0100] TfR436 VH (SEQ ID NO: 7)

[0101] DVQLVQSGGGVVQPGRSLRLSCAASGFPFKSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRGEDTAVYYCARDSNFWSGYYSPVDVWGQGTTVTVSS

[0102] TfR436 VL (SEQ ID NO: 8)

[0103] NFMLTQPHSVSESPGKTVTISCTRSSGSIASNSVQWYQQRPGSAPITVIYEDTQRPSGVPDRFSGSIDSSSNSASLTISGLQTEDEADYYCQSYDSAYHWVFGGGTKLAVL

[0104] Example 1: Identification of the binding site of the TfR436 antibody

[0105] The TfR436 antibody does not cross-react with mouse TfR and shows cross-reactivity with hamster TfR. An amino acid sequence alignment of the transferrin (TF) binding site (amino acids 569 - 760) in TfR was performed. In the human TfR sequence, amino acids that are the same as those in hamsters and different from those in mice were selected. The selected amino acids were subjected to point mutations as shown below to produce soluble TfR mutant fragments. Figure 1 as shown below, and soluble TfR mutant fragments were prepared.

[0106] (1) Preparation of soluble wild-type TfR (sTfR) and TfR mutant fragments (MF1 - MF7)

[0107] Fully synthesized the base sequences encoding the extracellular region of human TfR (amino acids 89 - 760), or Figure 1 each of the TfR mutant fragments (MF1 - MF7) shown below and AAARGGPEQKLISEED LNSAVDHHHHHH (SEQ ID NO: 10). The neomycin resistance gene and the DHFR gene were recombined into the expression vector pCAGGS (Non-Patent Document 2: Niwa et al. 1991), and various synthesized genes were inserted into the multiple cloning site of this vector to produce the pCAGGS-Neo-DHFR-sTFR-myc-his expression plasmid. The above plasmid was transfected into Expi293 cells (Invitrogen) using Expifectamine (Invitrogen), and the cells were cultured at 37°C, 8% CO2, and 135 rpm for 5 days. Thereafter, the culture supernatant was recovered by centrifugation, and sTfR or MF1 - MF7 was purified by connecting a HisTrapHP column (GE Healthcare) to AKTA prime (GE Healthcare). The binding buffer used was 20 mM imidazole / DPBS, and the elution buffer used was 500 mM imidazole / DPBS. The eluted protein was replaced with a buffer of 30 mM HEPES, 5% trehalose, and pH 7.2 using a Zeba spin column (Thermoscientific).

[0108] (2) Identification of the binding site of the TfR436 antibody

[0109] The sTfR or MF1 - MF7 obtained through the above refinement was diluted using PBST (Phosphate Buffered Saline with Tween20, TaKaRa), and seven levels were prepared by 3-fold dilution starting from 600 ng / mL. Thereafter, the diluted solution was dispensed at 100 μL / well into a Ni-NTA HisSorb Strips 96-well plate (QIAGEN), placed on an oscillator, and reacted at room temperature. After 1 hour, it was washed 5 times with PBST Buffer, and the TfR436 antibody (1 μg / mL) was dispensed at 100 μL / well, placed on an oscillator, and reacted at room temperature for 1 hour. Thereafter, it was washed 5 times with PBS-T Buffer, and the secondary antibody F(ab’)2 Fragment Anti-Human IgG Fcγ (Jackson Immuno Research) diluted 50,000-fold was dispensed at 100 μL / well and reacted at room temperature for 1 hour. After washing 5 times with PBST Buffer, the TMB Soluble Reagent (High Sensitivity) (Scy Tek) was dispensed at 100 μL / well, reacted in the dark at room temperature for 3 minutes, then TMB Stop Buffer (Scy Tek) was added at 100 μL / well, oscillated on the oscillator for 1 minute, and the absorbance at 450 nm (ref. 620 nm) was measured using a spectrophotometer.

[0110] The results are as Figure 2 shown. It can be seen that the reactivity of the TfR436 antibody with the TfR mutant fragment MF5 decreased, however, no decrease in reactivity with other mutant fragments was found. That is, when the 629th, 630th, and 633rd amino acids of TfR were substituted with other amino acids, the TfR436 antibody became unable to recognize TfR. This implies that the 629 - 633 amino acids are the recognition epitope of the TfR436 antibody.

[0111] Example 2: Comparison of the inhibition of Tf-TfR binding by the TfR436 antibody and a comparative antibody

[0112] (1) Preparation of the comparative antibody A24

[0113] The A24 antibody against human TfR was described in the patent document US2008 / 0193453. To compare the TfR436 antibody with this antibody, a preserved hybridoma was obtained to produce the antibody. Specifically, the hybridoma was cultured in a medium of RPMI1640 (GIBCO) and 10% FBS at a cell concentration of 1 - 2×10 5Inoculate in the way of / mL and culture in a 5% CO₂ incubator at 37°C. After expansion culture, recover the cells by centrifugation, wash twice with PBS, and then further expand the culture to 550 mL in serum-free medium cosmedium005 (COSMOBIO) and 0.5% Nutridoma-CS (Roche). Recover the culture supernatant by centrifugation 5 days after cell confluence.

[0114] Load the recovered supernatant onto a protein A carrier (Ab-Capcher ExTra: Protenova), elute the antibody bound to protein A with 0.1M glycine hydrochloride buffer (pH 2.7), and quickly neutralize it with 1M Tris hydrochloride buffer (pH 8.5). Thereafter, exchange the buffer to PBS using an ultrafiltration disc (Merck Millipore).

[0115] (2) Comparison of the inhibition of Tf-TfR binding by TfR436 antibody and antibodies from other companies

[0116] Adjust the sTfR described in Example 1 to 5.0 μg / mL with PBST, dispense the dilution solution at 100 μL / well in a MaxiSorp 96-well plate (Nunc), and let it stand overnight at 4°C for solidification. Discard the solidification solution the next day, add 200 μL / well of 100% BlockACE (DS Pharma Biomedical) and let it stand at room temperature for blocking. After 1 hour, wash 5 times with PBST Buffer, then dispense HRP-labeled Tf (2 μg / mL) at 50 μL / well, and further add TfR436 antibody, A24 antibody (2-fold dilution series starting from 10 μg / mL), or holo-Tf (Sigma) (2-fold dilution series starting from 300 μg / mL) at 50 μL / well. After reacting at room temperature for 1 hour, wash 5 times with PBST Buffer, dispense TMB Soluble Reagent (HighSensitivity) at 100 μL / well, and let it react in the dark at room temperature. After 25 minutes, add TMB Stop Buffer at 100 μL / well, shake on an oscillator for 1 minute, and measure the absorbance at 450 nm (ref. 620 nm) with a spectrophotometer.

[0117] The results are as Figure 3 shown. The TfR436 antibody completely inhibited the binding of Tf-TfR at a very low dosage (100 ng / mL). On the other hand, the A24 antibody could not completely inhibit the binding of Tf-TfR even at a dosage of 10 μg / mL and could only inhibit the binding of Tf-TfR by 50%. It shows that the TfR436 antibody is excellent in inhibiting the binding of Tf-TfR.

[0118] Example 3: Expression of TfR in samples from patients with carcinomatous peritonitis (immunostaining of patient pathological specimens)

[0119] The expression of TfR1 in tumor tissues of primary lesions and peritoneal implants collected from patients with carcinomatous peritonitis originating from gastric cancer was studied by immunostaining. The tumor tissue sections were dewaxed, and antigen activation was performed using an autoclave at 121 °C for 15 minutes in an activation buffer at pH 6.0. After endogenous peroxidase treatment in 0.3% H2O2 water, the sections were reacted with the primary antibody (0.4 μg / mL Anti TFRC Rabbit, ATLAS ANTIBODIES, HPA028598) overnight at 4 °C. After washing with PBS, the sections were reacted with a polymer reagent (Histofine Simple Stain MAX-PO MULTI, NICHIREI, 424152) for 30 minutes at room temperature, washed with PBS, and developed with a DAB reagent (Histofine Simple Stain DAB, NICHIREI, 415172). After nuclear staining with hematoxylin, dehydration, clearing, and mounting were performed to obtain immunostained specimens.

[0120] The results are as Figure 4 shown. Expression of TfR was confirmed in tumor tissues derived from primary lesions ( Figure 4 A) and peritoneal implant tumor tissues ( Figure 4 B) of gastric cancer patients.

[0121] Example 4: Antitumor effect using a peritoneal implant model of gastric cancer cell line MKN45-Luc

[0122] The antitumor effect of the TfR436 antibody was studied using a peritoneal implant model of gastric cancer cell line MKN45-Luc.

[0123] The MKN45-Luc cell line (JCRB1379) was cultured in a culture medium (RPMI-1640, 10% FBS, 1% P / S). After removing the culture medium, the cells were washed once with PBS and then detached using TrypLE Express. Culture medium was added, the cells were recovered, washed once with PBS, resuspended in PBS again, and cell counting was performed using 0.4% trypan blue. After counting, the cell concentration was adjusted to 1×10 7 / mL with PBS. 1 mL of this cell suspension was aspirated using a tuberculin syringe, a secondary needle was attached, and 200 μL / rat was transplanted into the abdominal cavity of 20 C.B-17 / IcrHsd-Prkdc scid mice (female, 6 weeks old at the time of purchase). The number of transplanted cells was 2×10 6 cells / rat.

[0124] On the 3rd day after transplantation, 500 μg of fluorescein was administered intraperitoneally, and the luminescence of the transplanted cells was measured. Among the 20 transplanted mice, 17 mice in which the survival of the transplanted cells was confirmed by luminescence detection were used, and grouped according to the luminescence intensity (8 mice in the control group and 9 mice in the antibody administration group). Thereafter, intraperitoneal administration of the drug was performed 4 times in total at intervals of once a week. PBS was administered to the control group, and 15 mg / kg of TfR436 antibody was administered to the antibody administration group.

[0125] During the test period, the mice were observed and the life and death determination was performed. The test was terminated on the 77th day after transplantation, and statistical analysis was performed based on the survival time of the mice. As a result, a significant survival prolongation effect of Log-Rank (p = 0.0002) and Wilcoxon (p = 0.0004) was confirmed in the TfR436 antibody administration group compared with the control group ( Figure 5 ). In addition, the formation of tumors was confirmed in the abdominal cavities of 7 mice in the TfR436 antibody administration group that survived at the end of the test. When collecting and preparing pathological specimens, extensive necrosis was confirmed in the tumors, and residual tumor cells with proliferative ability were confirmed only in a small area on the tumor surface and around blood vessels ( Figure 6 ).

[0126] Example 5: Study on the anti-tumor effect using a peritoneal implantation model of pancreatic cancer cell line SUIT-2

[0127] The SUIT-2 cell line (JCRB1094) was cultured in a culture medium (EMEM, 10% FBS, 1% P / S). After removing the culture medium and washing once with PBS, the cells were detached using TrypLE Express. The culture medium was added, the cells were recovered, washed once with PBS, and then resuspended in PBS again. Cell counting was performed using 0.4% trypan blue. After counting, the cell concentration was adjusted to 1×10 7 / mL with PBS. 1 mL of this cell suspension was aspirated with a tuberculin syringe, a secondary needle was attached, and 200 μL / mouse was transplanted into the abdominal cavities of 30 C.B-17 / IcrHsd-Prkdc scid mice (female, 6 weeks old at the time of purchase). The number of transplanted cells was 2×10 6 cells / mouse.

[0128] In this model, since the engraftment rate of the transplanted cells was 100% (based on the results of the preliminary experiment), random grouping (control group, antibody administration group, n = 10) was performed according to body weight 4 days after cell transplantation. Thereafter, intraperitoneal administration of the drug was performed a total of 2 times at intervals of once a week. PBS was administered to the control group, and 15 mg / kg of the TfR436 antibody was administered to the antibody administration group. During the test period, the body weight of the mice was measured and observed. In the control group mice, deterioration of the general condition was confirmed along with the increase in peritoneal implanted tumors, and the test was terminated 17 days after transplantation. After euthanizing the two groups of mice, ascites collection and observation of the tumors in the peritoneal cavity were performed. For the weight of the ascites, comparison between the control group and the TfR436 antibody administration group was performed by t-test. In Figure 7 the measurement results of the ascites weight are shown. As a result, a significant reduction in ascites weight with P = 0.0005 was confirmed in the TfR436 antibody administration group. In addition, in the observation of the tumors in the peritoneal cavity, multiple tumor formations were confirmed on the mesentery of the control group. However, in the TfR436 antibody administration group, 1 to 2 tumors were confirmed in 4 mice, and no obvious tumor formation was observed in 6 mice.

[0129] Example 6: Survival prolongation effect of the peritoneal implantation model of pancreatic cancer cell line SUIT2

[0130] The SUIT-2 cell line (JCRB1094) was cultured in a culture medium (EMEM, 10% FBS, 1% P / S). After removing the culture medium and washing once with PBS, the cells were detached using TrypLE Express. The culture medium was added, the cells were recovered, washed once with PBS, and then resuspended in PBS again. Cell counting was performed using 0.4% trypan blue. After counting, the cell concentration was adjusted to 1×10 7 / mL with PBS. 1 mL of this cell suspension was aspirated with a tuberculin syringe, a secondary needle was attached, and 200 μL / animal was transplanted into the peritoneal cavity of 25 C.B-17 / IcrHsd-Prkdc scid mice (female, 7 weeks old at the time of purchase). The number of transplanted cells was 2×10 6 cells / animal.

[0131] In this model, since the engraftment rate of the transplanted cells was 100% (based on the results of preliminary experiments), random grouping (control group, antibody administration group, n = 8) was performed according to body weight 4 days after cell transplantation. Thereafter, intraperitoneal administration of the drug was performed a total of 2 times at intervals of once a week. PBS was administered to the control group, and 15 mg / kg of the TfR436 antibody was administered to the antibody administration group. Mouse body weight was measured and the general condition was observed from cell transplantation until 55 days later. When deterioration of the general condition (excessive ascites retention, anemia, debilitation) accompanied by tumor growth was confirmed, euthanasia was performed, and the number of days from cell transplantation to euthanasia was taken as the survival period. Log-Rank analysis was performed on the survival period 55 days after transplantation. As a result, a significant survival prolongation effect was confirmed in the TfR436 administration group compared to the PBS group in the intraperitoneal administration group (p = 0.0003) and the intravenous administration group (p = 0.007), and the efficacy of intraperitoneal administration was better ( Figure 8 ).

[0132] Example 7: Analysis of TfR expression in patient samples

[0133] Using the samples of cancer patients shown in the following table, the TfR expression status was analyzed by immunostaining only peritoneal implant tumors in the same manner as in Example 3. The judgment criteria are shown in Table 1.

[0134] [Table 1]

[0135] Minute Judgment Criteria 0 Less positive staining on the cell membrane 1 The cell membrane is slightly stained 2 Weak to moderate cell membrane staining is present 3 Strong cell membrane staining is present

[0136] The results are shown in Table 2 and Figure 9 .

[0137] [Table 2]

[0138] Sequence Listing <110> Protein Science Perseus Co., Ltd. <110> National University Corporation Gunma University <120> Therapeutic agent for carcinomatous peritonitis <130> F20827A-WO <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> human <400> 1 Ser Tyr Gly Met His 1 5 <210> 2 <211> 17 <212> PRT <213> Person <400> 2 Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 14 <212> PRT <213> Person <400> 3 Asp Ser Asn Phe Trp Ser Gly Tyr Tyr Ser Pro Val Asp Val 1 5 10 <210> 4 <211> 13 <212> PRT <213> Person <400> 4 Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn Ser Val Gln 1 5 10 <210> 5 <211> 8 <212> PRT <213> Person <400> 5 Tyr Glu Asp Thr Gln Arg Pro Ser 1 5 <210> 6 <211> 10 <212> PRT <213> Person <400> 6 Gln Ser Tyr Asp Ser Ala Tyr His Trp Val 1 5 10 <210> 7 <211> 123 <212> PRT <213> Human <400> 7 Asp Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Lys Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Gly Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Asn Phe Trp Ser Gly Tyr Tyr Ser Pro Val Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 8 <211> 111 <212> PRT <213> Human <400> 8 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30 Ser Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Ile Thr Val 35 40 45 Ile Tyr Glu Asp Thr Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Gln Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Ala Tyr His Trp Val Phe Gly Gly Gly Thr Lys Leu Ala Val Leu 100 105 110 <210> 9 <211> 760 <212> PRT <213> Human <400> 9 Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu 1 5 10 15 Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp 20 25 30 Asn Ser His Val Glu Met Lys Leu Ala Val Asp Glu Glu Glu Asn Ala 35 40 45 Asp Asn Asn Thr Lys Ala Asn Val Thr Lys Pro Lys Arg Cys Ser Gly 50 55 60 Ser Ile Cys Tyr Gly Thr Ile Ala Val Ile Val Phe Phe Leu Ile Gly 65 70 75 80 Phe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr 85 90 95 Glu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Val Arg Glu Glu Pro 100 105 110 Gly Glu Asp Phe Pro Ala Ala Arg Arg Leu Tyr Trp Asp Asp Leu Lys 115 120 125 Arg Lys Leu Ser Glu Lys Leu Asp Ser Thr Asp Phe Thr Gly Thr Ile 130 135 140 Lys Leu Leu Asn Glu Asn Ser Tyr Val Pro Arg Glu Ala Gly Ser Gln 145 150 155 160 Lys Asp Glu Asn Leu Ala Leu Tyr Val Glu Asn Gln Phe Arg Glu Phe 165 170 175 Lys Leu Ser Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val 180 185 190 Lys Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Arg 195 200 205 Leu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys 210 215 220 Ala Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys 225 230 235 240 Lys Asp Phe Glu Asp Leu Tyr Thr Pro Val Asn Gly Ser Ile Val Ile 245 250 255 Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu 260 265 270 Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe 275 280 285 Pro Ile Val Asn Ala Glu Leu Ser Phe Phe Gly His Ala His Leu Gly 290 295 300 Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln 305 310 315 320 Phe Pro Pro Ser Arg Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr 325 330 335 Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp 340 345 350 Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Arg Met Val Thr Ser 355 360 365 Glu Ser Lys Asn Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Ile 370 375 380 Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro Asp 385 390 395 400 His Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala 405 410 415 Ala Lys Ser Gly Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met 420 425 430 Phe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile 435 440 445 Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr 450 455 460 Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr 465 470 475 480 Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val 485 490 495 Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln Asn 500 505 510 Val Lys His Pro Val Thr Gly Gln Phe Leu Tyr Gln Asp Ser Asn Trp 515 520 525 Ala Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe 530 535 540 Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp 545 550 555 560 Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu 565 570 575 Ile Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu 580 585 590 Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Val Glu Leu Asn 595 600 605 Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Ser Phe Val Arg Asp 610 615 620 Leu Asn Gln Tyr Arg Ala Asp Ile Lys Glu Met Gly Leu Ser Leu Gln 625 630 635 640 Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu 645 650 655 Thr Thr Asp Phe Gly Asn Ala Glu Lys Thr Asp Arg Phe Val Met Lys 660 665 670 Lys Leu Asn Asp Arg Val Met Arg Val Glu Tyr His Phe Leu Ser Pro 675 680 685 Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser 690 695 700 Gly Ser His Thr Leu Pro Ala Leu Leu Glu Asn Leu Lys Leu Arg Lys 705 710 715 720 Gln Asn Asn Gly Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala 725 730 735 Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly Asp 740 745 750 Val Trp Asp Ile Asp Asn Glu Phe 755 760 <210> 10 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Recombinant Tag <400> 10 Ala Ala Ala Arg Gly Gly Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp 1 5 10 15 Leu Asn Ser Ala Val Asp His His His His His His 20 25

Claims

1. Use of an antibody that recognizes the transferrin receptor in the preparation of a therapeutic agent for carcinomatous peritonitis accompanied by peritoneal tumors, characterized in that: The carcinomatous peritonitis accompanied by peritoneal tumors is treated by inhibiting peritoneal seeding in a subject with peritoneal tumors. The antibody that recognizes the human transferrin receptor is an antibody that recognizes amino acids 629 to 633 of the human transferrin receptor. The antibody has a heavy chain first complementary determining region (VH CDR1), a heavy chain second complementary determining region (VH CDR2), and a heavy chain third complementary determining region (VH CDR3) that are respectively SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and a light chain first complementary determining region (VL CDR1), a light chain second complementary determining region (VL CDR2), and a light chain third complementary determining region (VL CDR3) that are respectively SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:

6.

2. The use according to claim 1, characterized in that: The antibody has a heavy chain that is SEQ ID NO: 7 and a light chain that is SEQ ID NO:

8.

3. The use according to claim 1 or 2, characterized in that: The antibody is a human antibody or a humanized antibody.

4. The use according to claim 1 or 2, characterized in that: The antibody is an antibody fragment selected from Fab, Fab', F(ab')2, single-chain antibody (scFv), bispecific antibody, disulfide-stabilized Fv antibody (dsFv), and a peptide containing a CDR.

5. The use according to claim 1 or 2, characterized in that: The carcinomatous peritonitis is complicated by a cancer that is the cause of the carcinomatous peritonitis.

6. The use according to claim 1 or 2, characterized in that: The carcinomatous peritonitis is complicated by gastric cancer, pancreatic cancer, colorectal cancer, ovarian cancer, biliary tract cancer, liver cancer, gastrointestinal stromal tumor (GIST), or small intestine cancer.

7. The use according to claim 1 or 2, characterized in that: The therapeutic agent for carcinomatous peritonitis is a therapeutic agent administered intraperitoneally.

Images