Drugs for treating peritonitis carcinomatosis

Abstract

The present invention addresses the problem of providing a therapeutic agent for carcinomatous peritonitis. The present invention provides a therapeutic agent that is for carcinomatous peritonitis and that contains an antibody that recognizes a transferrin receptor.

Description

[Technical Field]

[0001] The present invention relates to a therapeutic agent for peritoneal carcinomatosis, which comprises an anti-transferrin receptor antibody. [Background technology]

[0002] Transferrin receptor (TfR) was first identified on reticulocytes as a cell membrane structure responsible for the intracellular uptake of iron bound to transferrin (Tf). TfR has since been found to be expressed in placental trophoblast cells, activated lymphocytes, and various tumor cells. For example, high TfR expression has been reported 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's lymphoma, and adult T-cell leukemia. Furthermore, because TfR is highly expressed on the surface of various cancer cells and low in normal cells, it has been recognized as a molecular target for cancer therapy. For example, Patent Document 1 describes an antibody that can specifically recognize the transferrin receptor and an anticancer agent using the antibody.

[0003] Peritoneal carcinomatosis is a condition in which cancer has metastasized to the peritoneum. Peritoneal carcinomatosis can occur with any type of cancer, but it is more common with intraperitoneal gastrointestinal cancers (such as stomach cancer, pancreatic cancer, and colon cancer) and ovarian cancer. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. WO2014 / 073641 Summary of the Invention [Problem to be solved by the invention]

[0005] It is known that cancer cells with peritoneal metastasis are special cells that have acquired the ability to survive in hypoxic conditions. It is expected that cancer cells with peritoneal metastasis have acquired resistance to anticancer drugs that target normal cell proliferation mechanisms. Numerous clinical experiences and clinical trial results have revealed that intraperitoneal administration of conventional anticancer drugs is ineffective in treating peritoneal carcinomatosis. Treatments for peritoneal carcinomatosis include chemotherapy, immunotherapy, and hyperthermia, but these treatments have insufficient therapeutic effects and poor prognosis, so new treatments are desired. The present invention aims to solve the problem of providing a therapeutic agent for peritoneal carcinomatosis. [Means for solving the problem]

[0006] As a result of investigations to solve the above-mentioned problems, the present inventors discovered that when an antibody that recognizes an amino acid sequence at a specific position in TfR was administered to a mouse model of cancerous peritonitis, cancerous peritonitis could be treated, leading to the completion of the present invention.

[0007] That is, according to the present invention, the following inventions are provided. (1) A therapeutic agent for peritonitis carcinomatosis, comprising an antibody that recognizes the transferrin receptor. (2) The therapeutic agent for peritonitis carcinomatous according to (1), wherein the antibody that recognizes the transferrin receptor is an antibody that recognizes the human transferrin receptor. (3) The therapeutic agent for peritonitis carcinomatous according to (2), wherein the antibody that recognizes the human transferrin receptor is an antibody that recognizes the amino acids 629 to 633 of the human transferrin receptor. (4) The therapeutic agent for peritonitis carcinomatous according to any one of (1) to (3), wherein the antibody has a heavy chain first complementarity determining region (VH CDR1), a heavy chain second complementarity determining region (VH CDR2), and a heavy chain third complementarity determining region (VH CDR3) represented by SEQ ID NOs: 1, 2, and 3, respectively, and a light chain first complementarity determining region (VL CDR1), a light chain second complementarity determining region (VL CDR2), and a light chain third complementarity determining region (VL CDR3) represented by SEQ ID NOs: 4, 5, and 6, respectively. (5) The therapeutic agent for peritonitis carcinomatous according to any one of (1) to (4), wherein the antibody has a heavy chain having SEQ ID NO: 7 and a light chain having SEQ ID NO: 8. (6) The therapeutic agent for peritonitis carcinomatous according to any one of (1) to (5), wherein the antibody is a human antibody or a humanized antibody. (7) A therapeutic agent for peritonitis carcinomatous according to any one of (1) to (6), wherein the antibody is an antibody fragment selected from the group consisting of Fab, Fab', F(ab')2, single-chain antibody (scFv), bispecific antibody, disulfide-stabilized V region (dsFv), and peptide containing CDR. (8) The therapeutic agent for peritonitis carcinomatous according to any one of (1) to (7), wherein the peritonitis carcinomatous is associated with a cancer that causes the peritonitis carcinomatous. (9) A therapeutic agent for peritonitis carcinomatosa according to any one of (1) to (8), wherein the peritonitis carcinomatosa is associated with gastric cancer, pancreatic cancer, colon cancer, ovarian cancer, biliary tract cancer, liver cancer, gastrointestinal stromal tumor (GIST), or small intestine cancer. (10) The therapeutic agent for peritonitis carcinomatous according to any one of (1) to (9), which is administered intraperitoneally.

[0008] (A) A method for treating peritonitis carcinomatous is provided, comprising administering to a subject an antibody that recognizes a transferrin receptor. (B) An antibody that recognizes the transferrin receptor for use in the treatment of peritonitis carcinomatosis. (C) Use of an antibody that recognizes a transferrin receptor for the manufacture of a therapeutic agent for peritonitis carcinomatosis. [Effects of the Invention]

[0009] The therapeutic agent for peritonitis carcinomatous of the present invention is useful in treating peritonitis carcinomatous. The therapeutic agent for peritonitis carcinomatous of the present invention is a molecular targeted drug that targets a new mechanism that attacks the supply route of nutrients necessary for cellular metabolism. The therapeutic agent for peritonitis carcinomatous of the present invention is an optimal approach for existing anticancer drug-resistant peritoneal disseminated cells that survive in a hypoxic and low-nutrient environment, and will lead to unprecedented, original new medical treatments. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 shows the sites where point mutations are made in each TfR mutant fragment. [Figure 2] Figure 2 shows the reactivity of TfR436 with soluble wild-type TfR (sTfR) and TfR mutant fragments. [Figure 3] FIG. 3 shows the Tf-TfR binding inhibitory activity of TfR436. [Figure 4] FIG. 4 shows the expression of TfR in patient tumor tissues. [Figure 5] FIG. 5 shows the results of the survival analysis. [Figure 6] FIG. 6 shows HE specimens of abdominal tumors from mice in the TfR436 antibody administration group. [Figure 7] FIG. 7 shows the results of measuring the weight of ascites. [Figure 8] FIG. 8 shows the results of analyzing the survival rate of a peritoneal dissemination model of the pancreatic cancer cell line SUIT2. [Figure 9] FIG. 9 shows the expression of TfR in patient samples. DETAILED DESCRIPTION OF THE INVENTION

[0011] Next, the present invention will be described in more detail. Definitions and General Techniques Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the nomenclature used in connection with and techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art.

[0012] The methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout the specification, unless otherwise indicated.

[0013] TfR In humans, transferrin receptor (TfR) is a single-pass transmembrane protein consisting of 760 amino acids encoded by human chromosome 3 (SEQ ID NO: 9). This protein, also known as the CD71 antigen, is thought to be involved in cellular iron uptake and cell proliferation. The TfR of the present invention is not particularly limited in structure, and refers to all TfRs, including monomers, multimers, intact forms expressed in the cell membrane, soluble forms composed in the extracellular domain, truncated forms, mutation forms due to gene mutations or deletions, and forms that have undergone post-translational modifications such as phosphorylation.

[0014] react and reactivity As used herein, "react" and "reactivity" mean the same thing unless otherwise specified. That is, the recognition of an antigen by an antibody. This antigen may be intact TfR expressed on the cell membrane, a truncated form, or a soluble form. It may also be TfR that retains its three-dimensional structure or a denatured TfR. Methods for examining reactivity include flow cytometry (FACS), enzyme-linked immunosorbent assay (ELISA), Western blotting, fluorescence microanalysis (FMAT), surface plasmon resonance (BIAcore), immunostaining, and immunoprecipitation.

[0015] Antibodies used in flow cytometry may be labeled with a fluorescent substance such as FITC, biotin, or the like, or may be unlabeled. Depending on the type and whether the antibody is labeled, fluorescently labeled avidin, fluorescently labeled anti-human immunoglobulin antibody, or the like may be used. Reactivity can be assessed by adding a sufficient amount of anti-TfR antibody (usually at a final concentration of 0.01-10 μg / mL) to the sample and comparing the reactivity with that of a negative control antibody and a positive control antibody.

[0016] antibody In this specification, the following abbreviations (in parentheses) are used according to convention where necessary: Heavy chain (H chain), light chain (L chain), heavy chain variable region (VH), light chain variable region (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 the heavy chain (VH CDR1), second complementarity-determining region of the heavy chain (VH CDR2), third complementarity-determining region of the heavy chain (VH CDR3), first complementarity-determining region of the light chain (VL CDR1), second complementarity-determining region of the light chain (VL CDR2), third complementarity-determining region of the light chain (VL CDR3).

[0017] As used herein, the term "antibody" is synonymous with immunoglobulin and should be understood as commonly known in the art. Specifically, the term antibody is not limited by any particular method of making the antibody. For example, the term antibody includes, but is not limited to, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.

[0018] As used herein, the term "human antibody" refers to any antibody in which the variable and constant region sequences are human sequences. The term encompasses antibodies that have sequences derived from human genes but that have been altered to, for example, reduce potential immunogenicity, increase affinity, or remove cysteines that may cause undesired folding. The term also encompasses such antibodies that have been recombinantly produced in non-human cells, which can provide glycosylation not typical of human cells. These antibodies can be prepared in a variety of ways.

[0019] As used herein, the term "humanized antibody" refers to an antibody of non-human origin in which amino acid residues characteristic of the antibody sequence of the non-human species are replaced with residues found at the corresponding positions in human antibodies. This "humanization" process is believed to reduce the immunogenicity of the resulting antibody in humans. It will be understood that antibodies of non-human origin can be humanized using techniques well known in the art. See, for example, Winter et al., Immunol. Today 14:43-46 (1993). The antibody of interest can be engineered by recombinant DNA technology to replace the CH1, CH2, CH3, hinge domain, and / or framework domain with the corresponding human sequence. See, for example, WO 92 / 02190 and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and 5,777,085. As used herein, the term "humanized antibody" includes within its meaning chimeric human antibodies and CDR-grafted antibodies.

[0020] The sequence of the framework region (FR) in the variable region of an antibody is not particularly limited, as long as it does not substantially affect the specific binding to the corresponding antigen. Although it is preferable to use the FR region of a human antibody, the FR region of an animal species other than human (e.g., mouse or rat) can also be used.

[0021] In one embodiment, the antibody comprises a constant region in addition to the variable region (e.g., an IgG-type antibody). The sequence of the constant region is not particularly limited. For example, the constant region of a known human antibody can be used. The heavy chain constant region (CH) of a human antibody may be any that belongs to human immunoglobulin (hereinafter referred to as hIgG), but those of the hIgG class are preferred, and any subclass within the hIgG class, such as hIgG1, hIgG2, hIgG3, or hIgG4, can also be used. The light chain constant region (CL) may be any that belongs to hIg, and those of the κ class or λ class can be used. Constant regions from animal species other than humans (e.g., mice and rats) can also be used.

[0022] As used herein, a "modified antibody" or "modified antibody" refers to an antibody in which one or more amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence of the variable region (CDR sequence and / or FR sequence) of a parent antibody. In the present invention, a "parent antibody" refers to a TfR436 antibody having the amino acid sequences shown in SEQ ID NO: 7 for VH and SEQ ID NO: 8 for VL. In the amino acid sequence, one or several (e.g., 1 to 8, preferably 1 to 5, more preferably 1 to 3, and particularly preferably 1 or 2) amino acids have been deleted, added, substituted, and / or inserted. Methods for preparing the amino acid sequence of an antibody that has binding activity to TfR are well known to those skilled in the art, including methods for introducing mutations into proteins.For example, those skilled in the art will be familiar with site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Takagawa, 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 to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456; Kramer, W, and Fritz, HJ (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456). Modified antibodies functionally equivalent to antibodies with TfR-binding activity can be prepared by appropriately introducing mutations into the amino acid sequence of an antibody with TfR-binding activity using techniques such as those described in (HJ, 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). In this way, antibodies with TfR-binding activity that have one or several amino acid mutations in the variable or constant region of the antibody can also be used.

[0023] As used herein, "having activity equivalent to that of a parent antibody" means that the binding activity to TfR is equivalent. "Equivalent" does not necessarily mean that the activity is at the same level; the activity may be enhanced, or the activity may be decreased as long as the antibody still has activity. Examples of antibodies with decreased activity include antibodies that have 30% or more of the activity of the original antibody, preferably 50% or more, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 95% or more.

[0024] The binding activity refers to the recognition of an antigen. This antigen may be intact TfR expressed on the cell membrane, a truncated form, or a soluble form. It may also be TfR that retains its three-dimensional structure or a denatured TfR. For example, methods for examining binding activity include flow cytometry (FACS), enzyme-linked immunosorbent assay (ELISA), western blotting, fluorescence microanalysis (FMAT), and surface plasmon resonance (BIAcore).

[0025] The Tf-TfR binding inhibitory activity of an antibody can be measured according to the method described below in "Example 2(2) Comparison of TfR436 antibody and antibodies from other companies in Tf-TfR binding inhibition." A TfR solution is dispensed onto a substrate (e.g., a 96-well plate) and allowed to stand for immobilization and blocking. An HRP-labeled Tf solution is then dispensed, followed by the addition of an antibody, and the reaction is allowed to proceed at room temperature. The substrate is then washed, and a color reagent (e.g., TMB) is added to allow the reaction, and the absorbance is measured using a plate reader. The above procedures allow the Tf-TfR binding inhibitory activity of the antibody to be evaluated.

[0026] The antibody is not limited by its origin and may be derived from any animal, such as a human antibody, a mouse antibody, or a rat antibody. It may also be a chimeric antibody or a humanized antibody. One preferred embodiment of the antibody of the present invention is a human antibody.

[0027] Antibodies may differ in amino acid sequence, molecular weight, isoelectric point, or the presence or absence or morphology of sugar chains depending on the antibody-producing cells, host, or purification method described below. For example, the present invention also includes cases in which the amino acid sequences described in the present invention are modified after translation. Furthermore, post-translational modifications at sites other than known post-translational modifications are also included in the present invention. Furthermore, when antibodies are expressed in prokaryotic cells, such as Escherichia coli, a methionine residue is added to the N-terminus of the amino acid sequence of the original antibody. Such antibodies may be used in the present invention. Post-translational modifications at sites other than known post-translational modifications are also included in the present invention.

[0028] Antibody production (1) Antigen-reactive scFv from a phage display library Antibodies can be prepared according to several methods known in the art. For example, phage display technology can be used to provide libraries containing a repertoire of antibodies with varying affinities for TfR. These libraries can then be screened to identify and isolate antibodies against TfR. Preferably, the phage library is an scFv phage display library generated using human VL and VH cDNAs prepared from mRNA isolated from human B cells. Methods for preparing and screening such libraries are known in the art. Genetic material is recovered from phage clones that show reactivity when screened using TfR as an antigen. By analyzing the genes of the selected phage, the DNA sequences of the VH and VL regions encoding the variable regions of the human antibody that binds to the antigen can be determined. Using the scFv sequence, the scFv can be converted into IgG to obtain a human antibody.

[0029] (2) Conversion of scFv to IgG (production of human antibodies) Human antibodies are obtained by constructing expression vectors for the H or L chain, expressing them in host cells, and recovering and purifying the secreted supernatant. Human antibodies can also be obtained by expressing VH and VL in the same vector (tandem). These methods are well known, and reference can be made to WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, WO97 / 10354, etc.

[0030] Specifically, a full-length heavy chain gene can be obtained by ligating the VH-encoding DNA with 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, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDapartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be that of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD, with IgG1 or IgG2 constant regions being most preferred. The IgG1 constant region sequence may be any of the various alleles or allotypes known to occur between different individuals, such as Gm(1), Gm(2), Gm(3), and Gm(17). These allotypes correspond to naturally occurring amino acid substitutions in the IgG1 constant region.

[0031] A full-length L chain gene (as well as a Fab light chain gene) can be obtained by ligating the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDapartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. The kappa constant region can be any of the various alleles known to occur between different individuals, such as Inv(1), Inv(2), or Inv(3). The lambda constant region can be derived from any of the three lambda genes.

[0032] The DNA encoding the H or L chain obtained as described above is inserted into an expression vector to produce an expression vector, which is then expressed in host cells. The secreted supernatant is then collected and purified to obtain human antibodies. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, cosmids, YACs, and EBV-derived episomes. Expression vectors and expression regulatory sequences are selected to be compatible with the host cells used for expression. The antibody light chain gene and antibody heavy chain gene can be inserted into separate vectors, or both genes can be inserted into the same expression vector. The antibody genes are inserted into the expression vector using standard methods (e.g., ligation of the vector with complementary restriction sites on the antibody gene fragment, or blunt-end ligation if no restriction sites are present).

[0033] Convenient vectors encode functionally complete human CH or CL immunoglobulin sequences with appropriate restriction sites engineered to facilitate the insertion and expression of any VH or VL sequence, as described above. In such vectors, splicing typically occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C domain, as well as at splice regions present within the human CH exon. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding region. The recombinant expression vector can also encode a signal peptide that facilitates secretion of the antibody chain from host cells. The antibody chain gene can be cloned into the vector such that the signal peptide is linked 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 from a non-immunoglobulin protein).

[0034] In addition to the antibody genes and control sequences, antibody expression vectors may contain additional sequences, such as sequences that control replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced. For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on host cells into which the vector has been introduced. Preferred selectable marker genes include the dehydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection / amplification), the neomycin phosphotransferase gene (for G418 selection), and the glutamate synthetase gene.

[0035] Host cells are transformed with the antibody gene expression vector constructed by the above method. Host cells can be any cell capable of producing antibodies, including bacteria, yeast, animal cells, insect cells, and plant cells, but animal cells are preferred. Examples of animal cells include Chinese hamster ovary cells (CHO / dhfr(-) cells, CHO / DG44 cells), monkey-derived COS cells (A. Wright & S.L. Morrison, J. Immunol. 160, 3393-3402 (1998)), and SP2 / O cells (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)). For transformation, the lipofectin method (R.W. Marone et al., Proc. Natl. Acad. Sci. USA 86, 6007 (1989), P.L. Felgner et al., Proc. Natl. Acad. Sci. USA 84, 7413 (1987)), electroporation, the calcium phosphate method (F.L. Graham & A.J. van der Eb, Virology 52, 456-467 (1973)), the DEAE-Dextran method, etc. are preferably used.

[0036] After culturing the transformant, human antibodies are isolated from the cells of the transformant or from the culture medium. Antibody isolation and purification can be achieved by using an appropriate combination of methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, affinity chromatography, ion exchange chromatography, and gel filtration chromatography.

[0037] antibody fragment Antibody fragments can be produced based on antibodies or on the sequence information of the genes encoding the antibodies, including Fab, Fab', F(ab')2, scFv, and dsFv antibodies.

[0038] Fab is a fragment with a molecular weight of approximately 50,000, which is obtained by papain digestion of IgG in the presence of cysteine ​​and is composed of an L chain, an H chain variable region, and an H chain fragment consisting of a CH1 domain and part of the hinge region. In the present invention, it can be obtained by papain digestion of the above antibody. Alternatively, Fab can be prepared from a transformant transformed with an appropriate vector by incorporating DNA encoding a part of the H chain and L chain of the above antibody into the vector.

[0039] Fab' is a fragment with a molecular weight of approximately 50,000 that can be obtained by cleaving the disulfide bond between the H chains of F(ab')2, as described below. In the present invention, it can be obtained by digesting the above-mentioned antibody with pepsin and cleaving the disulfide bond using a reducing agent. Alternatively, like Fab, Fab' can also be prepared by genetic engineering using DNA encoding it.

[0040] F(ab')2 is a fragment with a molecular weight of approximately 100,000, obtained by digesting IgG with pepsin, in which fragments (Fab') consisting of an L chain, an H chain variable region, and an H chain fragment consisting of a CH1 domain and part of the hinge region are linked by disulfide bonds. In the present invention, it is obtained by digesting the above-mentioned antibody with pepsin. Furthermore, like Fab, it can also be prepared by genetic engineering using DNA encoding F(ab')2.

[0041] An scFv is an antibody fragment in which an Fv consisting of an H-chain variable region and an L-chain variable region is linked via a suitable peptide linker at the C-terminus of one chain to the N-terminus of the other to form a single chain. Examples of peptide linkers that can be used include the highly flexible (GGGGS)3. For example, DNA encoding an scFv antibody can be constructed using DNA encoding the H-chain and L-chain variable regions of the antibody and DNA encoding the peptide linker, and this can be inserted into an appropriate vector. The vector can then be used to transform the resulting transformant, from which the scFv can be prepared.

[0042] dsFv is an Fv fragment in which Cys residues have been introduced at appropriate positions in the H-chain variable region and L-chain variable region, stabilizing the H-chain variable region and the L-chain variable region through disulfide bonds. The positions of the Cys residues to be introduced in each chain can be determined based on the three-dimensional structure predicted by molecular modeling. In the present invention, for example, the three-dimensional structure is predicted from the amino acid sequences of the H-chain variable region and L-chain variable region of the above-mentioned antibody, and DNA encoding the H-chain variable region and L-chain variable region into which mutations have been introduced based on such prediction is constructed. This DNA is then inserted into an appropriate vector, and a dsFv can be prepared from a transformant transformed with the vector. Furthermore, antibody fragments can also be multimerized by linking scFv antibodies, dcFv antibodies, etc. using an appropriate linker, or by fusing streptavidin.

[0043] bispecific antibody The antibody may be a bispecific antibody. Bispecific antibodies include bispecific (mab)2, which is obtained by chemically crosslinking two monoclonal antibody molecules; bispecific F(ab')2, which is obtained by chemically crosslinking two Fab fragment molecules; quadroma; bispecific diabody (bsDb); single-chain bispecific diabody (scBsDb); single-chain bispecific tandem variable domain (scBsTaFv); bispecific T cell engager antibody (Bite); and docl-and-lock trivalent Fab (DNL-F(ab)3) (Shim, H. Bispecific Antibodies and Antibody-Drug Comjugates for Cancer Therapy: Technological Considerations. Biomolecules 2020, 10, 360).

[0044] Pharmaceutical Compositions and Formulations Pharmaceutical compositions and preparations containing the therapeutic agent for peritoneal carcinomatosis of the present invention are also included within the scope of the present invention. The therapeutic agent for peritoneal carcinomatosis of the present invention can be used for the treatment of peritoneal carcinomatosis.

[0045] Peritoneal carcinomatosis is a pathological condition in which cancer metastasizes to the peritoneum. Examples of peritoneal carcinomatosis include, but are not limited to, those associated with cancers that cause peritoneal carcinomatosis, such as gastric cancer, pancreatic cancer, colon cancer, ovarian cancer, biliary tract cancer, liver cancer, gastrointestinal stromal tumor (GIST), or small intestine cancer. In peritoneal carcinomatosis, cancer cells are scattered throughout the peritoneal cavity, causing numerous sand-like masses in the peritoneum and the accumulation of ascites.

[0046] Pharmaceutical compositions and formulations containing the therapeutic agent for peritonitis carcinomatosis of the present invention preferably contain a physiologically acceptable diluent or carrier in addition to the antibody, and may be a mixture with other drugs. Suitable carriers include, but are not limited to, physiological saline, phosphate-buffered saline, phosphate-buffered saline glucose solution, and buffered saline. Alternatively, the antibody may be lyophilized (freeze-dried) and reconstituted when needed by adding an aqueous buffer solution such as those described above. Examples of administration routes include parenteral administration via intraperitoneal administration (e.g., intraperitoneal injection).

[0047] The dosage of the therapeutic agent for peritonitis carcinomatosis of the present invention varies depending on symptoms, age, body weight, etc., but typically, for oral administration, the amount of antibody is about 0.01 mg to 1000 mg per day for adults, which can be administered once or in divided doses. For parenteral administration, about 0.01 mg to 3000 mg can be administered intraperitoneally.

[0048] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. [Example]

[0049] In the following examples, the TfR436 antibody described in paragraphs 0090 and 0091 of International Publication WO2014 / 073641 was used.

[0050] The CDR sequences of the TfR436 antibody are shown below. VH CDR1: SYGMH (SEQ ID NO: 1) VH CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 2) VH CDR3: DSNFWSGYYSPVDV (SEQ ID NO: 3) VL CDR1: TRSSGSIASNSVQ (SEQ ID NO: 4) VL CDR2: YEDTQRPS (SEQ ID NO: 5) VL CDR3: QSYDSAYHWV (SEQ ID NO: 6)

[0051] The VH and VL sequences of the TfR436 antibody are shown below. TfR436 VH (SEQ ID NO: 7) DVQLVQSGGGVVQPGRSLRLSCAASGFPFKSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRGEDTAVYYCARDSNFWSGYYSPVDVWGQGTTVTVSS

[0052] TfR436 VL (SEQ ID NO: 8) NFMLTQPHSVSESPGKTVTISCTRSSGSIASNSVQWYQQRPGSAPITVIYEDTQRPSGVPDRFSGSIDSSSNSASLTISGLQTEDEADYYCQSYDSAYHWVFGGGTKLAVL

[0053] Example 1: Identification of the binding site of the TfR436 antibody The TfR436 antibody did not cross-react with mouse TfR, but did cross-react with hamster TfR. We performed amino acid sequence alignment of the transferrin (TF) binding site (amino acids 569-760) in TfR. We selected amino acids in the human TfR sequence that were identical to those in the hamster TfR but different from those in the mouse TfR. We then performed point mutations at the selected amino acids as shown in Figure 1 to generate soluble TfR mutant fragments.

[0054] (1) Preparation of soluble wild-type TfR (sTfR) and TfR mutant fragments (MF1-MF7) The nucleotide sequences encoding the human TfR extracellular domain (amino acids 89-760) and the TfR mutant fragments (MF1-MF7) and AAARGGPEQKLISEEDLNSAVDHHHHHH (SEQ ID NO: 10) shown in Figure 1 were synthesized. The synthesized genes were inserted into the multicloning site of the expression vector pCAGGS (Niwa et al., 1991), which contained a neomycin resistance gene and a DHFR gene, to generate the pCAGGS-Neo-DHFR-sTFR-myc-his expression plasmid. The plasmids were transfected into Expi293 cells (Invitrogen) using Expifectamine (Invitrogen) and cultured at 37°C, 8% CO2, and 135 rpm for 5 days. The culture supernatant was then collected by centrifugation, and sTfR or MF1–MF7 was purified using a HisTrapHP (GE Healthcare) column connected to an AKTA prime (GE Healthcare) with 20 mM imidazole / DPBS as the binding buffer and 500 mM imidazole / DPBS as the elution buffer. The eluted proteins were buffer-exchanged to 30 mM HEPES, 5% trehalose, pH 7.2 using a Zeba spin column (Thermo Scientific).

[0055] (2) Identification of the binding site of the TfR436 antibody The purified sTfR or MF1-MF7 was diluted with PBST (Phosphate Buffered Saline with Tween 20, TaKaRa) to prepare seven 3-fold dilutions starting from 600 ng / mL. The diluted solutions were then dispensed at 100 μL / well into a Ni-NTA HisSorb Strips 96-well plate (QIAGEN) and incubated at room temperature on a shaker. After 1 hour, the plate was washed five times with PBST buffer, and TfR436 antibody (1 μg / mL) was dispensed at 100 μL / well. The plate was then incubated at room temperature for 1 hour on a shaker. The plate was then washed five times with PBS-T buffer, and 100 μL / well of the secondary antibody F(ab')2 Fragment Anti-Human IgG Fcγ (Jackson Immuno Research) diluted 50,000 times was dispensed. The plate was then incubated at room temperature for 1 hour. After washing five times with PBST buffer, 100 μL of TMB Soluble Reagent (High Sensitivity) (ScyTek) was dispensed into each well and allowed to react at room temperature in the dark for 3 minutes. Then, 100 μL of TMB Stop Buffer (ScyTek) was added to each well, and the plate was shaken for 1 minute. The absorbance at 450 nm (ref. 620 nm) was measured using a plate reader.

[0056] As shown in Figure 2, the TfR436 antibody showed reduced reactivity with the TfR mutant fragment MF5, but not with other mutant fragments. In other words, if the amino acids at positions 629, 630, and 633 of TfR were replaced with other amino acids, the TfR436 antibody could no longer recognize TfR. This suggests that amino acids 629 to 633 are the epitope recognized by the TfR436 antibody.

[0057] Example 2: Comparison of TfR436 antibody and comparative antibody in inhibiting Tf-TfR binding (1) Preparation of comparative antibody A24 Patent document US2008 / 0193453 describes the A24 antibody against human TfR. To compare this antibody with the TfR436 antibody, the deposited hybridoma was obtained and the antibody was produced. Specifically, the hybridoma was cultured in RPMI1640 (GIBCO) medium containing 10% FBS at a cell concentration of 1 to 2 x 10 cells. 5 The cells were mixed to a final volume of 1 / mL and cultured in a 37°C, 5% CO2 incubator. After expansion, the cells were collected by centrifugation, washed twice with PBS, and then further expanded to 550 mL in serum-free medium cosmedium005 (Cosmo Bio), 0.5% Nutridoma-CS (Roche). Five days after the cells reached confluence, the culture supernatant was collected by centrifugation.

[0058] The collected supernatant was applied to a Protein A carrier (Ab-Capcher Extra, Proteinnova), and the antibody bound to Protein A was eluted with 0.1 M glycine-HCl buffer (pH 2.7) and immediately neutralized with 1 M Tris-HCl buffer (pH 8.5). The buffer was then exchanged with PBS using an Ultracel ultrafiltration disk (Merck Millipore).

[0059] (2) Comparison of TfR436 antibody and other companies' antibodies in Tf-TfR binding inhibition The sTfR described in Example 1 was adjusted to 5.0 μg / mL with PBST, and the diluted solution was dispensed into a MaxiSorp 96-well plate (Nunc) at 100 μL / well and left to stand overnight at 4°C for immobilization. The next day, the immobilization solution was discarded, and 200 μL / well of 100% Block Ace (DS Pharma Biomedical) was added and left to stand at room temperature for blocking. After 1 hour, the plate was washed five times with PBST buffer, and 50 μL / well of HRP-labeled Tf (2 μg / mL) was dispensed. Furthermore, 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) was added at 50 μL / well. After incubation at room temperature for 1 hour, the plate was washed five times with PBST buffer. 100 μL of TMB Soluble Reagent (High Sensitivity) was added to each well and incubated in the dark at room temperature. After 25 minutes, 100 μL of TMB Stop Buffer was added to each well and the plate was shaken for 1 minute. The absorbance at 450 nm (ref. 620 nm) was measured using a plate reader.

[0060] As shown in Figure 3, the TfR436 antibody completely inhibited Tf-TfR binding at a much lower dose (100 ng / mL). On the other hand, the A24 antibody was unable to completely inhibit Tf-TfR binding even at a dose of 10 μg / mL, inhibiting Tf-TfR binding by only 50%. This suggests that the TfR436 antibody is superior in inhibiting Tf-TfR binding.

[0061] Example 3: Expression of TfR in specimens from patients with peritonitis carcinomatosa (immunostaining of patient pathological specimens) TfR1 expression was examined by immunostaining in primary tumor tissues and peritoneal disseminations collected from patients with peritonitis carcinomatous from primary gastric cancer. Tumor tissue slides were deparaffinized and subjected to antigen retrieval by autoclaving at 121°C for 15 minutes in pH 6.0 retrieval buffer. After endogenous peroxidase treatment with 0.3% H2O2, the slides were incubated overnight at 4°C with a primary antibody (0.4 μg / mL Anti-TFRC Rabbit, ATLAS ANTIBODIES, HPA028598). After washing with PBS, the slides were incubated with a polymer reagent (Histofine Simple Stain MAX-PO MULTI, Nichirei, 424152) at room temperature for 30 minutes. After washing with PBS, the slides were developed with DAB reagent (Histofine Simple Stain DAB, Nichirei, 415172). After nuclear staining with hematoxylin, the specimens were dehydrated, cleared, and mounted to prepare immunostained specimens.

[0062] As a result, as shown in FIG. 4, TfR expression was confirmed in both tumor tissue derived from the primary lesion of a gastric cancer patient (FIG. 4A) and tumor tissue derived from peritoneal dissemination (FIG. 4B).

[0063] Example 4: Antitumor effect of gastric cancer cell line MKN45-Luc in a peritoneal dissemination model The antitumor effect of TfR436 antibody was examined using a gastric cancer cell line MKN45-Luc peritoneal dissemination model. The MKN45-Luc cell line (JCRB1379) was cultured in culture medium (RPMI-1640, 10% FBS, 1% P / S). The culture medium was removed, washed once with PBS, and then the cells were detached using TrypLE Express. Culture medium was added to recover the cells, washed once with PBS, and then resuspended in PBS. The number of cells was counted using 0.4% trypan blue. After counting, the cell concentration was 1 x 10 7 The cell suspension was then drawn up into a 1 mL tuberculin syringe and fitted with a double-step needle. scid 200 μL of the cells were transplanted into the abdominal cavity of 20 mice (female, 6 weeks old at the time of delivery). The number of cells transplanted was 2 × 10 6 Pieces / animal.

[0064] Three days after transplantation, 500 μg of luciferin was administered intraperitoneally and the luminescence of the transplanted cells was measured. Of the 20 transplanted mice, 17 mice in which engraftment of the transplanted cells was confirmed by luminescence detection were used and divided into groups based on luminescence intensity (8 mice in the control group and 9 mice in the antibody-treated group). The drugs were then administered intraperitoneally once a week for a total of four times. The control group received PBS, and the antibody-treated group received 15 mg / kg of TfR436 antibody.

[0065] Mice were observed throughout the study, and survival was assessed. The study was terminated 77 days after transplantation, and statistical analysis was performed based on the survival time of the mice. The results confirmed a significant survival benefit in the TfR436 antibody-treated group compared with the control group, based on Log-Rank (p=0.0002) and Wilcoxon (p=0.0004) measures (Figure 5). Furthermore, tumor formation was confirmed in the abdominal cavities of seven mice in the TfR436 antibody-treated group that survived at the end of the study. Pathological specimens were collected and found to have extensive necrosis within the tumor, with tumor cells retaining their proliferative capacity remaining only on the surface of the tumor and in a small area around blood vessels (Figure 6).

[0066] Example 5: Examination of antitumor effects in a peritoneal dissemination model of pancreatic cancer cell line SUIT-2 SUIT-2 cell line (JCRB1094) was cultured in culture medium (EMEM, 10% FBS, 1% P / S). The culture medium was removed, washed once with PBS, and then the cells were detached using TrypLE Express. Culture medium was added to recover the cells, washed once with PBS, and then resuspended in PBS. The number of cells was counted using 0.4% trypan blue. After counting, the cell concentration was 1 x 10 7 The cell suspension was then drawn up into a 1 mL tuberculin syringe and fitted with a double-step needle. scid 200 μL of the cells were transplanted into the abdominal cavity of 30 mice (female, 6 weeks old at the time of delivery). The number of cells transplanted was 2 × 10 6 Pieces / animal.

[0067] Because this model demonstrated a 100% cell engraftment rate after transplantation (based on preliminary results), randomization (control group, antibody-treated group, n = 10) was performed based on body weight four days after cell transplantation. Drugs were then administered intraperitoneally twice, once a week. The control group received PBS, while the antibody-treated group received 15 mg / kg of the TfR436 antibody. Mice were weighed and observed throughout the study. The study was terminated 17 days after transplantation, when the control group mice showed signs of deterioration in their overall condition due to increased peritoneal tumor dissemination. After euthanasia, mice from both groups were collected for ascites collection and abdominal tumor observation. A t-test was performed to compare ascites weight between the control group and the TfR436 antibody-treated group. The results of the ascites weight measurements are shown in Figure 7. A significant reduction in ascites weight was confirmed in the TfR436 antibody-treated group (P = 0.0005). In addition, when observing tumors in the abdominal cavity, multiple tumor formations were observed on the mesentery in the control group, but in the TfR436 antibody-administered group, one to two tumors were observed in four mice, but no clear tumor formation was observed in six mice.

[0068] Example 6: Survival prolongation effect of pancreatic cancer cell line SUIT2 peritoneal dissemination model SUIT-2 cell line (JCRB1094) was cultured in culture medium (EMEM, 10% FBS, 1% P / S). The culture medium was removed, washed once with PBS, and then the cells were detached using TrypLE Express. Culture medium was added to recover the cells, washed once with PBS, and then resuspended in PBS. The number of cells was counted using 0.4% trypan blue. After counting, the cell concentration was 1 x 10 7 The cell suspension was then drawn up into a 1 mL tuberculin syringe and fitted with a double-step needle. scid 200 μL of the cells were transplanted into the abdominal cavity of 25 mice (female, 7 weeks old at the time of delivery). The number of transplanted cells was 2 × 10 6 Pieces / animal.

[0069] Because this model demonstrated a 100% cell engraftment rate after transplantation (based on preliminary results), mice were randomized (control, intraperitoneal antibody, and intravenous antibody groups, n = 8) based on body weight four days after cell transplantation. Subsequently, the control group received PBS, while the antibody-treated group received 15 mg / kg TfR436 twice weekly. Mice were weighed and their general condition was monitored for 55 days after cell transplantation. Mice were euthanized when tumor growth-related deterioration in their general condition (excessive ascites, anemia, and weakness) was confirmed. The survival time was measured as the number of days from cell transplantation to euthanasia. Log-Rank analysis of survival time 55 days after transplantation revealed a significant survival benefit for the intraperitoneal TfR436 group (p = 0.0003) and intravenous TfR436 group (p = 0.007) compared to the PBS group, demonstrating the superior efficacy of intraperitoneal administration (Figure 8).

[0070] Example 7: TfR expression analysis of patient samples Using specimens from cancer patients shown in the table below, the TfR expression state was analyzed by immunostaining only the peritoneal disseminated tumors in the same manner as in Example 3. The evaluation criteria are shown in Table 1.

[0071] [Table 1]

[0072] The results are shown in Table 2 and FIG.

[0073] [Table 2]

Claims

1. A therapeutic agent for carcinomatous peritonitis, comprising an antibody which recognizes a transferrin receptor, the antibody recognizing amino acids 629 to 633 of the human transferrin receptor.

2. The therapeutic agent for cancerous peritonitis according to claim 1, wherein the antibody has a heavy chain first complementarity determining region (VH CDR1), a heavy chain second complementarity determining region (VH CDR2), and a heavy chain third complementarity determining region (VH CDR3) represented by SEQ ID NOs: 1, 2, and 3, respectively, and a light chain first complementarity determining region (VL CDR1), a light chain second complementarity determining region (VL CDR2), and a light chain third complementarity determining region (VL CDR3) represented by SEQ ID NOs: 4, 5, and 6, respectively.

3. The therapeutic agent for cancerous peritonitis according to claim 1 or 2, wherein the antibody has a heavy chain having SEQ ID NO: 7 and a light chain having SEQ ID NO:

8.

4. The therapeutic agent for cancerous peritonitis according to any one of claims 1 to 3, wherein the antibody is a human antibody or a humanized antibody.

5. The antibody is Fab, Fab', F(ab') ２ The therapeutic agent for cancerous peritonitis according to any one of claims 1 to 4, which is an antibody fragment selected from the group consisting of a single-chain antibody (scFv), a bispecific antibody, a disulfide-stabilized V region (dsFv) and a peptide containing CDR.

6. The therapeutic agent for carcinomatous peritonitis according to any one of claims 1 to 5, wherein the carcinomatous peritonitis is associated with a cancer that causes the carcinomatous peritonitis.

7. The therapeutic agent for carcinomatous peritonitis according to any one of claims 1 to 6, wherein the carcinomatous peritonitis is associated with gastric cancer, pancreatic cancer, colon cancer, ovarian cancer, biliary tract cancer, liver cancer, gastrointestinal stromal tumor (GIST), or small intestine cancer.

8. The therapeutic agent for cancerous peritonitis according to any one of claims 1 to 7, which is administered intraperitoneally.

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