Patent Application
20230250180
This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “2022-11-18_2870-0804PUS1_ST25.txt” created on Nov. 18, 2022 and is 10,401 bytes in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
The present invention relates to a therapeutic drug for carcinomatous peritonitis which comprises an anti-transferrin receptor antibody.
Transferrin receptor (TfR) has been identified to be a receptor that is present on a reticulocyte as a cell membrane structure for incorporating transferrin (Tf)-bound iron into a cell. Thereafter, it is discovered that TfR is expressed in placental trophoblasts, in activated lymphocytes, and further, in various tumor cells. For example, the high expression of TfR in breast cancer, prostate cancer, lung cancer, pancreatic cancer, colon cancer, stomach cancer, bladder cancer, hepatic cancer, cervical cancer, brain tumor, chronic lymphocytic leukemia, non-Hodgkin’s lymphoma, and adult T-cell leukemia has been reported. Moreover, since TfR is expressed on the surface of various types of cancer cells at a high level and is expressed in normal cells at a low level, this receptor is recognized as a molecular target for cancer therapy. For example, Patent Document 1 discloses an antibody capable of specifically recognizing the transferrin receptor and an anti-cancer agent using the aforementioned antibody.
Carcinomatous peritonitis is a pathological condition, in which cancer has metastasized to the peritoneum. Carcinomatous peritonitis is likely to occur in all of cancer species, but the complication rate of carcinomatous peritonitis is high in gastrointestinal cancers occurring in the abdominal cavity (e.g. stomach cancer, pancreatic cancer, colon cancer, etc.) or in ovarian cancer.
Patent Document 1: International Publication WO2014/073641
It has been known that peritoneally metastasized cancer cells are special cells, which have acquired viability under hypoxic conditions. It is predicted that such peritoneally metastasized cancer cells have acquired resistance to anticancer agents, which target a normal cell growth mechanism. Even from a large number of clinical experiences or clinical trial results, it has been elucidated that intraperitoneal administration of ordinary anticancer agents is invalid to the treatment of carcinomatous peritonitis. Examples of the method for treating carcinomatous peritonitis may include immunotherapy and hyperthermic treatment, in addition to chemical treatment. However, the therapeutic effects obtained from these treatment methods are not sufficient and provide poor prognosis. Thus, it has been desired to develop a novel method for treating carcinomatous peritonitis. It is an object of the present invention to provide a therapeutic drug for carcinomatous peritonitis.
The present inventors have conducted studies directed towards achieving the aforementioned object. The present inventors have administered an antibody that recognizes the amino acid sequence at a certain position in TfR, to carcinomatous peritonitis mouse models. As a result, the inventors have found that carcinomatous peritonitis can be treated by administration of the aforementioned antibody, thereby completing the present invention.
Specifically, according to the present invention, the following inventions are provided.
A therapeutic drug for carcinomatous peritonitis which comprises an antibody which recognizes a transferrin receptor.
The therapeutic drug for carcinomatous peritonitis according to (1), wherein the antibody which recognizes a transferrin receptor is an antibody which recognizes a human transferrin receptor.
The therapeutic drug for carcinomatous peritonitis according to (2), wherein the antibody which recognizes a human transferrin receptor is an antibody which recognizes the amino acids at positions 629 to 633 of the human transferrin receptor.
The therapeutic drug for carcinomatous peritonitis according to any one of (1) to (3), wherein the antibody is an antibody having 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), which are as set forth in SEQ ID NOs: 1, 2, and 3, respectively, and also having 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), which are as set forth in SEQ ID NOs: 4, 5, and 6, respectively.
The therapeutic drug for carcinomatous peritonitis according to any one of (1) to (4), wherein the antibody is an antibody having a heavy chain as set forth in SEQ ID NO: 7 and a light chain as set forth in SEQ ID NO: 8.
The therapeutic drug for carcinomatous peritonitis according to any one of (1) to (5), wherein the antibody is a human antibody or a humanized antibody.
The therapeutic drug for carcinomatous peritonitis 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, a single-chain antibody (scFv), a bispecific antibody, a disulfide-stabilized V region (dsFv) and a peptide comprising CDR.
The therapeutic drug for carcinomatous peritonitis according to any one of (1) to (7), wherein the carcinomatous peritonitis occurs in combination with cancer which causes the carcinomatous peritonitis.
The therapeutic drug for carcinomatous peritonitis according to any one of (1) to (8), wherein the carcinomatous peritonitis occurs in combination with stomach cancer, pancreatic cancer, colon cancer, ovarian cancer, bile duct cancer, hepatic cancer, gastrointestinal stromal tumor (GIST), or small intestine cancer.
The therapeutic drug for carcinomatous peritonitis according to any one of (1) to (9), which is intraperitoneally administered.
(A) There is provided a method for therapy of carcinomatous peritonitis which comprises administering an antibody which recognizes a transferrin receptor to a patient.
(B) An antibody which recognizes a transferrin receptor for use in the therapy of carcinomatous peritonitis.
(C) Use of an antibody which recognizes a transferrin receptor for production of a therapeutic drug for carcinomatous peritonitis.
The therapeutic drug for carcinomatous peritonitis of the present invention is useful in the treatment of carcinomatous peritonitis. The therapeutic drug for carcinomatous peritonitis of the present invention is a molecular targeted drug, which targets a novel mechanism of attacking a root of supplying nutrients necessary for the metabolism of cells. The therapeutic drug for carcinomatous peritonitis of the present invention is an optimal approach method for existing anticancer agent-resistant peritoneal disseminated cells, which survive in a low oxygen/undernutrition environment, and the present therapeutic drug for carcinomatous peritonitis will lead to an original novel medical treatment.
Hereinafter, the present invention will be described in more detail.
Unless otherwise specified in the present description, scientific terms used regarding the present invention have meanings that are generally understood by a person skilled in the art. In general, nomenclatures and techniques applied to the cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization, which are described in the present description, are well known in the present technical field, and thus, are commonly used.
The methods and techniques of the present invention are carried out in accordance with conventional methods that are well known in the present technical field, in such ways as described in a variety of general reference documents cited and discussed throughout the present description and more specific reference documents, unless otherwise specified.
In the case of humans, a transferrin receptor (TfR) is a single-pass transmembrane protein (SEQ ID NO: 9) comprising 760 amino acids, and it is encoded by human chromosome 3. This protein has also been known as a CD71 antigen, and it is considered that this protein is associated with incorporation of iron into cells and cell growth. The TfR of the present invention is not particularly limited in terms of structure. Thus, human TfR includes all of a monomer, a polymer, an intact form expressed on a cell membrane, a soluble form constituted in an extracellular region, a truncated form, a mutation form caused by genetic mutation, deletion, etc., and a form which has undergone posttranslational modification by phosphorylation or the like.
The terms “react” and “reactivity” have the same meanings in the present description, unless otherwise specified. That is, these terms mean that an antibody recognizes an antigen. The antigen used herein may be any of an intact TfR expressed on a cell membrane, a truncated form, and a soluble form. In addition, the antigen may be either a TfR having a three-dimensional structure or a modified TfR. Examples of a means for examining reactivity include flow cytometry (FACS), enzyme-linked immunosorbent assay (ELISA), Western blotting, microfluorescence measuring technique (FMAT), surface plasmon resonance (Biacore), immunostaining, and immunoprecipitation.
The antibody used in flow cytometry may be either an antibody labeled with a fluorescent substance such as FITC or with biotin, or an unlabeled antibody. A fluorescently-labeled avidin, a fluorescently-labeled anti-human immunoglobulin antibody, or the like is used, depending on the presence or absence of labeling of the antibody used and the type thereof. Reactivity can be evaluated by adding a sufficient amount of anti-TfR antibody (generally having a final concentration of 0.01 to 10 µg/mL) to an analyte, and then by comparing the obtained reactivity with the reactivity with a negative control antibody or a positive control antibody.
In the present description, the following abbreviations (in the parentheses) are used in accordance with the customs, as 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), heavy chain first complementarity determining region (VH CDR1), heavy chain second complementarity determining region (VH CDR2), heavy chain third complementarity determining region (VH CDR3), light chain first complementarity determining region (VL CDR1), light chain second complementarity determining region (VL CDR2), and light chain third complementarity determining region (VL CDR3).
In the present description, the term “antibody” has the same definitions as immunoglobulin, and should be understood as generally known in the present technical field. Specifically, the term “antibody” is not limited by any given specific method for producing the antibody. For example, the term “antibody” includes, but is not limited to, a recombinant antibody, a monoclonal antibody, and a polyclonal antibody.
In the present description, the term “human antibody” is used to mean any given antibody, in which the sequences of a variable region and a constant region are human sequences. This term includes antibodies which have human sequences and are modified, for example, to remove cysteine which may cause a possible decrease in immunogenicity, an increase in affinity, and undesirable folding. This term also includes antibodies produced in non-human cells by recombination, which enable glycosylation which is not specific to human cells. These antibodies can be prepared in various ways.
In the present description, the term “humanized antibody” means a non-human-derived antibody, in which amino acid residues characteristic for a non-human antibody sequence are substituted with residues found in positions corresponding to those of a human antibody. This “humanization” process is considered to reduce the immunogenicity of the obtained antibody in human. It would be understood that a non-human-derived antibody can be humanized using a technique well known in the present technical field. Please refer to, for example, Winter et al., Immunol. Today 14: 43-46 (1993). The target antibody can be produced by an engineering approach via a recombination DNA technique of substituting CH1, CH2, CH3, a hinge domain, and/or a framework domain with those of the corresponding human sequence. For example, 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 can be referred. In the present description, the term “humanized antibody” includes a chimeric human antibody and a CDR-grafted antibody, within the definitions thereof.
The sequence of a framework region (FR) in a variable region of the antibody is not particularly limited, unless it substantially affects the specific binding ability of the antibody to the corresponding antigen. The FR region of a human antibody is preferably used, but it is also possible to use FR regions of animal species other than humans (e.g. a mouse or a rat).
In one aspect, the antibody comprises a constant region as well as a variable region (e.g. IgG antibody). The sequence of such a 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 is not particularly limited, as long as it belongs to a human immunoglobulin (hereinafter referred to as “hIg”). Those of hIgG class are preferable, and any one of subclasses belonging to hIgG class, such as hIgG1, hIgG2, hIgG3 or hIgG4, may be used. On the other hand, the light chain constant region (CL) of a human antibody is not particularly limited, as long as it belongs to hIg, and those of κ class or λ, class can be used. In addition, constant regions of animal species other than humans (e.g. a mouse or a rat) can also be used.
In the present description, the term “modified body” or “modified antibody” is used to mean that the amino acid sequence of the variable region (CDR sequences and/or FR sequences) of a parent antibody comprises a substitution, deletion, addition and/or insertion of one or multiple amino acids.
In the present description, the “parent antibody” means a TfR436 antibody which has a VH comprising the amino acid sequence as set forth in SEQ ID NO: 7 and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 8. In the amino acid sequence, one or several (for example, 1 to 8, preferably 1 to 5, more preferably 1 to 3, and particularly preferably 1 or 2) amino acids are deleted, added, substituted and/or inserted. As a method of preparing the amino acid sequence of an antibody having a binding ability to TfR, which has been well known to a person skilled in the art, a method of introducing a mutation into a protein has been known. For instance, such a skilled person could prepare a modified antibody functionally equivalent to an antibody having a TfR-binding activity by appropriately introducing a mutation into the amino acid sequence of the antibody having a TfR-binding activity according to a site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, an DNA kagawa, 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 (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 USA. 82, 488-492). Thus, an antibody comprising a mutation of one or several amino acids in the variable region or constant region of a parent antibody and having a binding activity to TfR can also be used.
In the present description, the phrase “an activity equivalent to the activity of the parent antibody” is used to mean that the TfR-binding activity of a certain antibody is equivalent to that of the parent antibody thereof. The term “equivalent” does not necessarily mean the same level of activity. The activity may be increased, or the activity may also be decreased, as long as the antibody has the activity. An antibody having a decreased activity may be an antibody having an activity that is, for example, 30% or more, preferably 50% or more, more preferably 80% or more, further preferably 90% or more, and particularly preferably 95% or more of the activity of the original antibody.
The term “binding activity” means the activity of an antibody to recognize an antigen. This antigen may be an intact TfR expressed on a cell membrane, a truncated form, or a soluble form. In addition, the antigen may be either a TfR having a three-dimensional structure or a modified TfR. Examples of a means for examining the binding activity include flow cytometry (FACS), enzyme-linked immunosorbent assay (ELISA), Western blotting, microfluorescence measuring technique (FMAT), and surface plasmon resonance (Biacore).
The Tf-TfR binding inhibitory activity of an antibody can be measured according to the method described in the after-mentioned “Example 2(2): Comparison between TfR436 antibody and antibodies of other companies in terms of Tf-TfR binding inhibition.” A TfR solution is dispensed into a substrate (a 96-well plate, etc.) and is then left at rest to obtain a solid phase, and thereafter, blocking is carried out. Subsequently, an HRP-labeled Tf solution is dispensed into the substrate, and an antibody is further added thereto, so that they are allowed to react with each other at room temperature. Thereafter, the substrate is washed, and a coloring reagent (TMB, etc.) is then added thereto, so that they are allowed to react with each other. After that, the absorbance is measured using a plate reader. By performing the above-described operations, the Tf-TfR binding inhibitory activity of the antibody can be evaluated.
The antibody is not limited by its origin, and it may be an antibody derived from any animal, such as a human antibody, a mouse antibody, or a rat antibody. Also, the present antibody may be a chimeric antibody or a humanized antibody. In a preferred aspect, the antibody of the present invention is a human antibody.
The antibodies may be different from one another in terms of amino acid sequence, molecular weight, isoelectric point, the presence or absence of a sugar chain or the form thereof, etc., depending on the after-mentioned cells or hosts which produce the antibodies, or a purification method. For example, an antibody which undergoes a posttranslational modification on the amino acid sequence described in the present description is included in the present invention. Moreover, an antibody which undergoes a posttranslational modification on a site other than those for the known posttranslational modification is also included in the present invention. Furthermore, when the antibody is allowed to express 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 an antibody may also be used in the present invention. An antibody which undergoes a posttranslational modification on a site other than those for the known posttranslational modification is also included in the present invention.
The antibody can be prepared by several methods known in the present technical field. For example, using a phage display technique, a library comprising a repertoire of antibodies having various affinity for TfR can be provided. Subsequently, such a library can be screened to identify and isolate antibodies against TfR. Preferably, the phage library is a scFv phage display library which is generated using human VL and VH cDNA which has been prepared from mRNA isolated from human B cells. A method of preparing and screening such a library is known in the present technical field. A genetic substance is recovered from phage clones exhibiting reactivity which have been screened using TfR as an antigen. By analyzing the selected phage gene, the DNA sequences of VH and VL encoding the variable region of a human antibody binding to the antigen can be determined. Using this scFv sequence, IgG is prepared from scFv, so as to obtain a human antibody.
An H chain or L chain expression vector is produced, and it is then allowed to express in a host cell. Thereafter, the secreted supernatant is recovered and is then purified, so as to obtain a human antibody. Alternatively, such a human antibody can also be obtained by allowing VH and VL to express in a single vector (tandem type). These methods are well known, and can be carried out with reference to WO92/01047, WO92/20791, WO93/06213, WO93/11236, W093/19172, WO95/01438, WO95/15388, WO97/10354, etc.
Specifically, DNA encoding VH is ligated to another DNA molecule encoding a heavy chain constant region (CH1, CH2 and CH3), so as to obtain a full-length heavy chain gene. The sequence of a human heavy chain constant region gene is known in the present technical field (for example, Kabat, E. A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th edition, U. S. Department of Health and Human Services, NIH Publication No. 91-3242), and a DNA fragment including such a region can be obtained by standard PCR amplification. The heavy chain constant region may be the constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD. The most preferred constant region is that of IgGl or IgG2. The constant region sequence of IgG 1 may include any given various alleles or allotypes known to be generated among different individuals, such as Gm (1), Gm (2), Gm (3) or Gm (17). These allotypes correspond to a substitution of amino acids naturally-occurring in the constant region of IgGl.
DNA encoding VL is ligated to another DNA molecule encoding the light chain constant region CL, so as to obtain a full-length L chain gene (and a Fab light chain gene). The sequence of a human light chain constant region gene is known in the present technical field (for example, Kabat, E. A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th edition, U. S. Department of Health and Human Services, NIH Publication No. 91-3242), and a DNA fragment including such a region can be obtained by standard PCR amplification. The light chain constant region may be the constant region of κ or λ. The κ constant region may include any given various alleles known to be generated among different individuals, such as Inv (1), Inv (2) or Inv (3). The λ, constant region may be derived from any one of the three λ, genes.
The thus obtained DNA encoding an H chain or L chain is inserted into a vector to produce an expression vector, and the produced expression vector is then allowed to express in a host cell. Thereafter, the secreted supernatant is recovered and purified to obtain a human antibody. Examples of the expression vector include a plasmid, retrovirus, adenovirus, adeno-associated virus (AAV), plant viruses such as cauliflower mosaic virus or tobacco mosaic virus, a cosmid, YAC, and EBV-derived episome. An expression vector and an expression regulatory sequence are selected, so that they are suitable for a host cell used for expression. An antibody light chain gene and an antibody heavy chain gene can be inserted into different vectors, or the two genes can also be inserted into a single expression vector. An antibody gene is inserted into an expression vector by a standard method (for example, ligation of a complementary restriction site on an antibody gene fragment to a vector, or blunt-ended ligation applied when no restriction sites are present).
A favorable vector encodes a functionally completed human CH or CL immunoglobulin sequence having a suitable restriction site, which has been produced by an engineering approach such that any given VH or VL sequence can be easily inserted and then expressed therein, as described above. In such a vector, splicing generally takes place between a splice donor site in the inserted J region and a splice acceptor site preceding a human C domain, or such splicing also takes place in a splice region existing in a human CH exon. Polyadenylation and transcription termination take place in a natural chromosomal site downstream of a coding region. A recombinant expression vector can also encode a signal peptide which promotes the secretion of an antibody chain derived from a host cell. An antibody chain gene can be cloned into a vector, such that a signal peptide can be ligated in-frame to the amino terminus of an immunoglobulin chain. The signal peptide may be either an immunoglobulin signal peptide or a heterogeneous signal peptide (namely, it may be a non-immunoglobulin protein-derived signal peptide).
An expression vector used for the antibody of the present invention may also have sequences such as a sequence for regulating replication of the vector in a host cell (e.g. a replication origin) or a selective marker gene sequence, as well as an antibody gene and a regulatory sequence. The selective marker gene promotes selection of a host cell into which a vector has been introduced. For instance, the selective marker generally imparts resistance to drugs such as G418, hygromycin or methotrexate to a host cell into which the vector has been introduced. Preferred selective marker genes include a dihydrofolate reductase (DHFR) gene (used in selection/amplification of methotrexate as a dhfr-host cell), a neomycin phosphotransferase gene (used in selection of G418), and a glutamate synthase gene.
A host cell is transformed with an antibody gene expression vector produced by the above-described method. Any type of cell may be used as a host cell, as long as it can produce the present antibody. Examples of such a host cell include bacteria, yeast, animal cells, insect cells, and plant cells. Among these cells, animal cells are preferable. Examples of the animal cells include Chinese hamster ovary cells CHO/dhfr(-) and CHO/DG44, monkey-derived cells COS (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, a lipofectin 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)), an electroporation method, a calcium phosphate method (F. L. Graham & A. J. van der Eb, Virology 52, 456-467 (1973)), a DEAE-Dextran method, and the like are preferably applied.
A transformant is cultured, and a human antibody is then separated from the cells of the transformant or a culture medium thereof. For separation/purification of the antibody, methods such as centrifugation, ammonium sulfate fractionation, salting-out, ultrafiltration, affinity chromatography, ion exchange chromatography and gel filtration chromatography can be used by appropriately combining them.
An antibody fragment can be produced based on the present antibody, or based on the sequence information of a gene encoding the present antibody. Examples of the antibody fragment include Fab, Fab′, F(ab′)2, scFv, and dsFv antibodies.
Fab is obtained by digesting IgG by papain in the presence of cysteine. It is an antibody fragment with a molecular weight of approximately 50,000, which is constituted with L chain and H chain variable regions, and an H chain fragment consisting of a CH1 domain and a portion of a hinge region. In the present invention, the above-described antibody can be obtained by papain digestion. In addition, Fab can also be prepared by incorporating DNA encoding a portion of the H chain and the L chain of the above-described antibody into a suitable vector, then performing transformation with the resulting vector, and then obtaining it from the transformant .
Fab′ is an antibody fragment with a molecular weight of approximately 50,000, which is obtained by cleaving a disulfide bond between the H chains of the below-mentioned F(ab′)2. In the present invention, Fab′ can be obtained by digesting the above-described antibody by pepsin, and then cleaving a disulfide bond with a reducing agent. In addition, as with Fab, Fab′ can also be prepared by genetic engineering using DNA encoding the Fab′.
F(ab′)2 is an antibody fragment with a molecular weight of approximately 100,000, which is obtained by binding, via a disulfide bond, one fragment (Fab′) constituted with L chain and H chain variable regions and an H chain fragment consisting of a CH1 domain and a portion of a hinge region, to the other fragment (Fab′). In the present invention, F(ab′)2 can be obtained by digesting the above-described antibody by pepsin. In addition, as with Fab, F(ab′)2 can also be prepared by genetic engineering using DNA encoding the F(ab′)2.
scFv is an antibody fragment obtained by ligating the C-terminus of one chain of Fv consisting of an H chain variable region and an L chain variable region to the N-terminus of the other chain thereof, using a suitable peptide linker, so as to form a single chain. (GGGGS)3 having high flexibility can be used, for example, as such a peptide linker. For instance, DNA encoding the H chain variable region and L chain variable region of the above-described antibody and DNA encoding a peptide linker are used to construct DNA encoding a scFv antibody, and the thus constructed DNA is then incorporated into a suitable vector. Thereafter, scFv can be prepared from a transformant obtained by transformation with the aforementioned vector.
dsFv is an Fv fragment obtained by introducing a Cys residue into a suitable site in each of an H chain variable region and an L chain variable region, and then stabilizing the H chain variable region and the L chain variable region by a disulfide bond. The site in each chain, into which the Cys residue is to be introduced, can be determined based on a conformation predicted by molecular modeling. In the present invention, for example, a conformation is predicted from the amino acid sequences of the H chain variable region and L chain variable region of the above-described antibody, and DNA encoding each of the H chain variable region and the L chain variable region, into which a mutation has been introduced based on such prediction, is then constructed. The thus constructed DNA is incorporated into a suitable vector. Thereafter, dsFv can be then prepared from a transformant obtained by transformation with the aforementioned vector.
Further, it is also possible to ligate the scFv antibody to the dcFv antibody or the like using a suitable linker, or to fuse such an antibody fragment with streptavidin, so as to multimerize the antibody fragment.
The antibody may be a bispecific antibody.
Examples of the bispecific antibody may include bispecific (mab)2 obtained by chemically crosslinking two molecules of monoclonal antibodies to each other, bispecific F(ab′)2 obtained by chemically crosslinking two molecules of Fab fragments to each other, quadroma, bsDb (a bispecific diabody), scBsDb (a single-chain bispecific diabody), scBsTaFv (a single-chain bispecific tandem variable domain), Bite (a bispecific T cell Engager antibody), and DNL-F(ab)3 (docl-and-lock trivalent Fab) (Shim, H. Bispecific Antibodies and Antibody-Drug Conjugates for Cancer Therapy: Technological Considerations. Biomolecules 2020, 10, 360).
A pharmaceutical composition and a preparation, which comprises the therapeutic drug for carcinomatous peritonitis of the present invention, are also included in the scope of the present invention.
The therapeutic drug for carcinomatous peritonitis of the present invention can be used to treat carcinomatous peritonitis.
Carcinomatous peritonitis is a pathological condition, in which cancer has metastasized to the peritoneum. Such carcinomatous peritonitis is not particularly limited, but may be carcinomatous peritonitis which occurs in combination with cancer which causes the carcinomatous peritonitis. More specifically, it may be carcinomatous peritonitis which occurs in combination with stomach cancer, pancreatic cancer, colon cancer, ovarian cancer, bile duct cancer, hepatic cancer, gastrointestinal stromal tumor (GIST), or small intestine cancer. In the case of carcinomatous peritonitis, cancer cells are dispersed in the abdominal cavity, a large number of sand grain tumor mass are formed in the peritoneum, and ascites fluid is accumulated.
A pharmaceutical composition and a preparation, which comprises the therapeutic drug for carcinomatous peritonitis of the present invention, preferably comprises a physiologically acceptable diluent or carrier, in addition to the antibody. The pharmaceutical composition or the preparation may also be a mixture with other drugs. Examples of a suitable carrier used herein may include a normal saline, a phosphate buffered saline, a phosphate buffered saline with glucose, and a buffered saline, but the examples are not limited thereto. Otherwise, the antibody is freeze-dried, and when needed, the aforementioned buffered aqueous solution may be added thereto to reconstitute the antibody, and the thus reconstituted antibody may be then used. The dosage form may be, for example, parenteral administration such as intraperitoneal administration (e.g. intraperitoneal injection, etc.).
The applied dose of the therapeutic drug for carcinomatous peritonitis of the present invention is different depending on symptom, age, body weight, etc. In general, in the case of oral administration, the present pharmaceutical composition is administered at a dose of approximately 0.01 mg to 1,000 mg per day per adult, in terms of the amount of an antibody contained therein. Such a dose can be administered once or divided over several administrations per day. On the other hand, in the case of parenteral administration, the present pharmaceutical composition can be intraperitoneally administered at a dose of approximately 0.01 mg to 3,000 mg for a single administration.
The present invention will be described in more detail in the following examples. However, these examples are not intended to limit the scope of the present invention.
In the following examples, the TfR436 antibody described in paragraphs 0090 and 0091 of International Publication WO2014/073641 was used.
The CDR sequences of the TfR436 antibody will be shown below.
The VH sequence and VL sequence of the TfR436 antibody will be shown below.
The TfR436 antibody did not cross-react with mouse TfR, but it exhibited cross-reactivity with hamster TfR. The amino acid sequence of a transferrin (TF)-binding site (the amino acids at positions 569 to 760) in TfR was aligned. The amino acids of a human TfR sequence, which were the same as hamster but were different from mouse, were picked up. The picked amino acids were subjected to point mutation, as shown in
A human TfR extracellular domain (the amino acids at positions 89 to 760) or each TfR mutant fragment (MF1 to MF7) as shown in
The thus purified sTfR or MF1 to MF7 were diluted with PBST (Phosphate Buffered Saline with Tween 20, TaKaRa), and the diluted solutions were prepared at 7 stages by 3-fold dilution from 600 ng/mL. Thereafter, the diluted solution was dispensed into Ni-NTA HisSorb Strips 96-well plate (QIAGEN) in an amount of 100 µL/well, and the plate was then placed on a shaker, followed by performing a reaction at room temperature. One hour later, the reaction mixture was washed with PBST Buffer 5 times, and the TfR436 antibody (1 µg/mL) was dispensed into the 96-well plate in an amount of 100 µL/well. The plate was placed on a shaker, and a reaction was then performed at room temperature for 1 hour. Thereafter, the reaction mixture was washed with PBS-T Buffer 5 times, and a 50,000-fold diluted secondary antibody, F(ab′)2 Fragment Anti-Human IgG Fcy (Jackson Immuno Research), was then dispensed into the 96-well plate in an amount of 100 µL/well, followed by performing a reaction at room temperature for 1 hour. The reaction mixture was washed with PBST Buffer 5 times, and TMB Soluble Reagent (High Sensitivity) (Scy Tek) was then dispensed into the plate in an amount of 100 µL/well, followed by performing a reaction at room temperature in a dark place for 3 minutes. After that, TMB Stop Buffer (Scy Tek) was added to the plate in an amount of 100 µL/well, and the obtained mixture was then shaken using a shaker for 1 minute. Thereafter, the absorbance at 450 nm (ref. 620 nm) was measured using a plate reader.
As a result, as shown in
Patent Document US2008/0193453 discloses an A24 antibody reacting against human TfR. In order to compare the TfR436 antibody with the A24 antibody, the deposited hybridomas were acquired, and the antibody was produced. Specifically, the hybridomas were seeded into RPMI1640 (GIBCO) supplemented with 10% FBS at a cell concentration of 1 to 2 × 105/mL, and were then cultured in a 5% CO2 incubator at 37° C. After completion of an expansion culture, the cells were recovered by centrifugation, and were then washed with PBS twice. Thereafter, the resulting cells were further subjected to an expansion culture in a serum-free medium Cosmedium 005 (Cosmo Bio Co., Ltd.) and 0.5% Nutridoma-CS (Roche) to result in a volume of 550 mL. Five days after the cells became confluent, a culture supernatant was recovered by centrifugation.
The recovered supernatant was applied onto a protein A carrier (Ab-Capcher ExTra: ProteNova), and an antibody binding to protein A was eluted with a 0.1 M glycine-HCl buffer (pH 2.7), and then, was rapidly neutralized with a 1 M Tris-HCl buffer (pH 8.5). Thereafter, using an Ultracell Ultrafiltration Disk (Merck Millipore), the buffer was exchanged with PBS.
The sTfR described in Example 1 was adjusted to a concentration of 5.0 µg/mL by addition of PBST, and the diluted solution was then dispensed into MaxiSorp 96-well plate (Nunc) in an amount of 100 µL/well. Thereafter, it was left at rest at 4° C. overnight to obtain a solid phase. On the following day, the solid phase solution was discarded, and 100% Block Ace (DS Pharma Biomedical) was added to the plate in an amount of 200 µL/well, followed by leaving the obtained mixture at rest at room temperature so as to carry out blocking. One hour later, the reaction mixture was washed with PBST Buffer 5 times, and HRP-labeled Tf (2 µg/mL) was then dispensed into the plate in an amount of 50 µL/well. Thereafter, the TfR436 antibody, the A24 antibody (2-fold dilution series from 10 µg/mL), or holo-Tf (Sigma) (2-fold dilution series from 300 µg/mL) was further added to the mixture in an amount of 50 µL/well. The obtained mixture was reacted at room temperature for 1 hour, and was then washed with PBST Buffer 5 times. Thereafter, TMB Soluble Reagent (High Sensitivity) was dispensed into the plate in an amount of 100 µL/well, and the obtained mixture was then reacted at room temperature in a dark place. Twenty five minutes later, TMB Stop Buffer was added to the plate in an amount of 100 µL/well, and the obtained mixture was shaken with a shaker for 1 minute. Thereafter, the absorbance at 450 nm (ref. 620 nm) was measured.
As a result, as shown in
The expression of TfR1 in the tumor tissues of a primary focus and a peritoneal dissemination focus collected from patients with carcinomatous peritonitis which was derived from stomach cancer as primary cancer was studied by immunostaining. A tumor tissue slide was deparaffinized, and was then subjected to antigen activation in an activation buffer with pH 6.0 at 121° C. for 15 minutes using an autoclave. The resultant was subjected to endogenous peroxidase treatment with 0.3% H2O2 water, and was then reacted with a primary antibody (0.4 µg/mL Anti TFRC Rabbit, ATLAS ANTIBODIES, HPA028598) at 4° C. overnight. Thereafter, the resultant was washed with PBS, and was then reacted with a polymer reagent (Histofine Simple Stain MAX-PO MULTI, NICHIREI CORPORATION, 424152) at room temperature for 30 minutes, followed by washing with PBS. The resultant was colored with a DAB reagent (Histofine Simple Stain DAB, NICHIREI CORPORATION, 415172). The nucleus was stained with hematoxylin, and the resultant was then subjected to dehydration, penetration, and enclosing, so as to obtain an immunostained specimen.
As a result, as shown in
Using stomach cancer cell line MKN45-Luc peritoneal dissemination models, the antitumor effects of a TfR436 antibody was studied.
The MKN45-Luc cell line (JCRB1379) was cultured in a culture medium (RPMI-1640, 10% FBS, 1% P/S). The culture medium was removed, and the cells were then washed with PBS once. Thereafter, the cells were peeled using TrypLE Express. The cells were recovered by addition of the culture medium, and were then washed with PBS once. The resulting cells were suspended in PBS again, and the number of the cells was then counted using 0.4% Trypan blue. After completion of the cell counting, the cell density was adjusted to 1 × 107/mL with PBS. This cell suspension was drawn into a 1 mL tuberculin syringe, and a two-step needle was then equipped into the syringe. The cell suspension was transplanted into the abdominal cavity of twenty C.B-17/IcrHsd-Prkdcscid mice (female, 6-week-old at the arrival) in an amount of 200 µL/mouse. The number of the transplanted cells was 2 × 106 cells/mouse.
Three days after the transplantation, 500 µg of luciferin was intraperitoneally administered to the mice, and light emission from the transplanted cells was then measured. Among the 20 transplanted mice, the survival of the transplanted cells was confirmed in 17 mice by detection of the light emission. The 17 mice were divided into groups (control group: 8 mice; and antibody administration group: 9 mice) depending on the intensity of the emitted light. Thereafter, the drug was intraperitoneally administered to the mice once a week, a total of 4 administrations. PBS was administered to the control group, whereas 15 mg/kg TfR436 antibody was administered to the antibody administration group.
The mice were observed during the test period, and viability was examined. On Day 77 after the transplantation, the test was terminated, and statistical analysis was then carried out based on the survival time of the mice. As a result, it was confirmed that the TfR436 antibody administration group exhibited significant life-extending effects (Log-Rank (p = 0.0002) and Wilcoxon (p = 0.0004)), in comparison to the control group (
The SUIT-2 cell line (JCRB1094) was cultured in a culture medium (EMEM, 10% FBS, 1% P/S). The culture medium was removed, and the cells were then washed with PBS once. Thereafter, the cells were peeled using TrypLE Express. The cells were recovered by addition of the culture medium, and were then washed with PBS once. The resulting cells were suspended in PBS again, and the number of the cells was then counted using 0.4% Trypan blue. After completion of the cell counting, the cell density was adjusted to 1 × 107/mL with PBS. This cell suspension was drawn into a 1 mL tuberculin syringe, and a two-step needle was then equipped into the syringe. The cell suspension was transplanted into the abdominal cavity of thirty C.B-17/IcrHsd-Prkdcscid mice (female, 6-week-old at the arrival) in an amount of 200 µL/mouse. The number of the transplanted cells was 2 × 106 cells/mouse.
The cell survival rate after the transplantation was 100% in the present models (from the results of a preliminary test). Thus, 4 days after the cell transplantation, random assignment was carried out based on body weight (a control group and an antibody administration group; n = 10). Thereafter, the drug was intraperitoneally administered to the mice once a week, a total of 2 administrations. PBS was administered to the control group, whereas 15 mg/kg TfR436 antibody was administered to the antibody administration group. During the test period, the measurement of the body weight of the mice and observation thereof were carried out, and on Day 17 after the transplantation, on which deterioration of the systemic condition of the control group mice was confirmed due to an increase in peritoneal disseminated tumors, the test was terminated. The mice in the two groups were subjected to euthanasia, and thereafter, collection of ascites fluid and observation of intraperitoneal tumor mass were carried out. With regard to the weight of ascites fluid, the control group was compared with the TfR436 antibody administration group according to a t-test. The measurement results of the ascites fluid weight are shown in
The SUIT-2 cell line (JCRB1094) was cultured in a culture medium (EMEM, 10% FBS, 1% P/S). The culture medium was removed, and the cells were then washed with PBS once. Thereafter, the cells were peeled using TrypLE Express. The cells were recovered by addition of the culture medium, and were then washed with PBS once. The resulting cells were suspended in PBS again, and the number of the cells was then counted using 0.4% Trypan blue. After completion of the cell counting, the cell density was adjusted to 1 × 107/mL with PBS. This cell suspension was drawn into a 1 mL tuberculin syringe, and a two-step needle was then equipped into the syringe. The cell suspension was transplanted into the abdominal cavity of twenty-five C.B-17/IcrHsd-Prkdcscid mice (female, 7-week-old at the arrival) in an amount of 200 µL/mouse. The number of the transplanted cells was 2 × 106 cells/mouse.
The cell survival rate after the transplantation was 100% in the present models (from the results of a preliminary test). Thus, 4 days after the cell transplantation, random assignment was carried out based on body weight (a control group, an antibody intraperitoneal administration group, and an antibody intravenous administration group; n = 8). Thereafter, the drug was administered to the mice once a week, a total of 2 administrations. That is, PBS was administered to the control group, whereas 15 mg/kg TfR436 antibody was administered to the antibody administration groups. Until Day 55 after the cell transplantation, the measurement of the body weight of the mice and observation of the systemic condition thereof were carried out. At the time point, in which deterioration of the systemic condition due to an increase in tumors (e.g. excessive accumulation of ascites fluid, anemia, and weakness) was confirmed, the mice were subjected to euthanasia. The number of days elapsed from the cell transplantation until euthanasia was defined as a survival period. The survival period from Day 55 after the transplantation was subjected to Log-Rank analysis. As a result, it was confirmed that the TfR436 antibody administration groups exhibited significant life-extending effects (intraperitoneal administration group (p = 0.0003); and intravenous administration group (p = 0.007)), in comparison to the PBS group, and further that the medicinal effects obtained by intraperitoneal administration were superior to the medicinal effects obtained by intravenous administration (
Using the specimens of cancer patients shown in the following table, the TfR expression state was analyzed by immunostaining of only the peritoneal disseminated tumors according to the same method as that of Example 3. Determination criteria are shown in Table 1.
The results are shown in Table 2 and
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019-213812 | Nov 2019 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2020/044185 | 11/27/2020 | WO |
