WO2024048644A1 - Carrier, complex, pharmaceutical composition, and diagnostic drug - Google Patents

Abstract

This carrier for delivering a target substance to MUC6 expression-negative cancer tissues contains a molecule having the activity of binding to mannose. The cancer tissues may be MUC6 expression-negative gastric cancer tissues arising from MUC6 gene mutations, intestinal-type gastric cancer, or gastric cancer that is negative for the expression of MUC5AC, MUC6, MUC2, and CD10. The molecule having the activity of binding to mannose may be a saccharide-binding protein.

Description

Carriers, complexes, pharmaceutical compositions, and diagnostic agents

The present invention relates to carriers, complexes, pharmaceutical compositions, and diagnostic agents.
This application claims priority from U.S. Provisional Application No. 63/402,071, filed in the United States on August 30, 2022, the contents of which are hereby incorporated by reference.

Although there are various drugs conventionally used in chemotherapy for advanced gastric cancer, their effectiveness is insufficient and survival times have not yet been significantly extended. For some gastric cancer cases, therapeutic drugs have been developed that target receptors and surface markers that are highly expressed in cancer cells and cancer tissues, such as HER2 and PD-L1, and promising therapeutic effects have been observed (e.g. , see Patent Document 1).
However, such cancer-specific markers have not been identified in the majority of gastric cancers.

During the gastric carcinogenesis process, chronic inflammatory changes such as atrophy of the gastric mucosa and intestinal metaplasia are caused. As a result, changes occur in mucin and sugar chain composition in the gastric mucosa, and decreased expression of MUC6, a major gastric mucin, has been confirmed in about 40% of gastric cancers.
MUC6 gene mutations are observed in gastric cancer, and are known to be associated with poor prognosis and resistance to chemotherapy.

Patent No. 5885764

However, the role of the MUC6 gene in the development and progression of gastric cancer is unknown, and there is a need for new drug therapy for advanced gastric cancer that is negative for MUC6 expression and is known to have a poor prognosis.

The present invention has been made in view of the above circumstances, and provides a carrier, a complex, a pharmaceutical composition, and a diagnostic agent that can be utilized for new drug therapy against cancer.

The present invention includes the following aspects.
[1] A carrier for delivering a target substance to a cancer tissue negative for MUC6 expression, the carrier containing a molecule having binding activity to mannose.
[2] The carrier according to [1], wherein the cancer tissue is a stomach cancer tissue.
[3] The carrier according to [2], wherein the gastric cancer tissue is a gastric cancer tissue that is negative for MUC6 expression due to a MUC6 gene mutation.
[4] The carrier according to [2], wherein the gastric cancer tissue is intestinal type gastric cancer.
[5] The carrier according to [2], wherein the gastric cancer tissue is a gastric cancer in which the expression of MUC5AC, MUC6, MUC2, and CD10 is negative.
[6] The carrier according to any one of [1] to [5], wherein the molecule is a sugar-binding protein.
[7] The carrier according to [6], wherein the sugar-binding protein has a defective or reduced hemagglutinating ability.
[8] The carrier according to [6] or [7], wherein the sugar-binding protein is banana lectin.
[9] The carrier according to [8], wherein the banana lectin has an amino acid mutation of H84T.
[10] A complex of the carrier according to any one of [1] to [9] and a target substance.
[11] The complex according to [10], wherein the target substance is an anticancer drug.
[12] A pharmaceutical composition comprising the complex according to [10] or [11].
[13] A companion diagnostic agent used for cancer patients who use the pharmaceutical composition described in [12], which comprises an anti-clusterin antibody, a primer set for amplifying the clusterin gene, or the clusterin gene or its amplification product. A diagnostic agent that includes a probe that binds.
[14] A treatment for cancer comprising administering to a patient in need of treatment a pharmaceutical composition containing a complex of the carrier according to any one of [1] to [9] and a target substance. Method of treatment.
[15] The method for treating cancer according to [14], wherein the target substance is an anticancer drug.

The present invention can be utilized for new drug therapy for cancer.

FIG. 2 is a schematic diagram showing a state in which a FlpER sequence is inserted into the start codon within exon 1 of the mouse Muc6 gene. These are the results of RNA ISH in stomach tissue sections prepared from Muc6KO mice. It is a graph showing the expression level of the Muc6 gene in the gastric mucosa of WT mice and Muc6KO mice as measured by qPCR. These are a bright field image of the stomach of a Muc6KO mouse and a stained image of a tissue section of the stomach. These are electron microscopic images of stomach tissue sections of WT mice and Muc6KO mice. FIG. 2 is a fluorescent immunostaining image visualizing MUC5AC in stomach tissue sections of WT mice and Muc6KO mice. FIG. 2 is a fluorescent immunostaining image visualizing MUC2 and MUC4 in stomach tissue sections of WT mice and Muc6KO mice. This is the analysis result of O-type sugar chain by LC-MS analysis. This is the analysis result of N-type sugar chain by LC-MS analysis. FIG. 2 is a schematic diagram of lectin microarray analysis of a sugar chain component extract of gastric mucosal epithelial cells. It is a graph showing the binding property of rBanana lectin to sugar chain component extracts of gastric mucosal epithelial cells of WT mice and Muc6KO mice. It is a graph showing the binding property of rGRFT lectin to sugar chain component extracts of gastric mucosal epithelial cells of WT mice and Muc6KO mice. It is a graph showing the binding property of Heltuba lectin to sugar chain component extracts of gastric mucosal epithelial cells of WT mice and Muc6KO mice. This is a fluorescent staining image showing the results of binding FITC-labeled banana lectin to a stomach tissue section of a WT mouse. This is a fluorescent staining image showing the results of binding FITC-labeled GNL to a tissue section of the stomach of a WT mouse. This is a fluorescent staining image showing the results of binding FITC-labeled banana lectin to a tissue section of the stomach of a Muc6KO mouse. This is a fluorescent staining image showing the results of binding FITC-labeled GNL to a tissue section of the stomach of a Muc6KO mouse. FIG. 2 is a schematic diagram showing banana lectin blotting. These are the results of SDS-PAGE of proteins pulled down with banana lectin. These are the results of Western blotting of proteins in cell lysate and pulled-down lysate. This is a fluorescent immunostained image of a gastric mucosal tissue section of a WT mouse. FIG. 15 is a partially enlarged view of the fluorescent immunostaining image shown in FIG. 15A. This is a fluorescent immunostaining image of a gastric mucosal tissue section of an 8-month-old Muc6KO mouse. FIG. 15C is a partially enlarged view of the fluorescent immunostaining image shown in FIG. 15C. This is a fluorescent immunostaining image of a gastric mucosal tissue section of a 12-month-old Muc6KO mouse. FIG. 15 is a partially enlarged view of the fluorescent immunostaining image shown in FIG. 15E. 1 is an immunostaining image showing the expression of clusterin in a human tissue array. It is a graph showing the results of classifying samples according to the proportion of cells expressing clusterin in a human gastric cancer tissue array. 1 is an immunostaining image showing the presence of clusterin and mannose in a human gastric cancer tissue array. FIG. 2 is a graph showing the percentage of mannose-bound clusterin in a human gastric cancer tissue array. These are staining images and flow cytometry results obtained when FITC-labeled banana lectin was administered to MKN45, a MUC6-negative human gastric cancer cell line, and HUG1-P1, a MUC6-positive human gastric cancer cell line. These are the results of a hemagglutination test using banana lectin. These are the results of evaluating the cytotoxic activity of each dose of the banana lectin drug complex against HUG1-P1 and MKN45. These are the results of evaluating the cytotoxic activity of MKN45 over time during each period of exposure to the banana lectin drug complex. FIG. 3 is a diagram showing the administration schedule of banana lectin drug complex to Muc6KO mice. These are the results of immunohistochemical staining of stomach tissue sections of Muc6KO mice administered with banana lectin drug complex. 24 is a drawing showing the quantitative results of the signal intensity in FIG. 23, the tumor diameter, and the glandular duct height. FIG. 2 is a drawing showing the influence of banana lectin having the H84T mutation and banana lectin drug complex on a Xenograft prepared by transplanting MKN45 into both buttocks of a nude mouse.

≪Carrier≫
The present embodiment provides a carrier for delivering a target substance to a cancer tissue negative for MUC6 expression, which includes a molecule having binding activity to mannose.

MUC6 is a secreted mucin that is expressed in the central part of the gastric corpus glandular duct and the gastric antrum. Furthermore, MUC6 has a tandem repeat region rich in serine and threonine residues, and has a characteristic sugar chain structure in this region.
As described later in the Examples, the inventors discovered that Muc6-/- mice deficient in both alleles of the mouse Muc6 gene (hereinafter referred to as "Muc6KO mice") spontaneously develop gastric cancer in the antrum. . Furthermore, an increase in high-mannose type N-glycans was observed in the cancerous areas of Muc6KO mice. The carrier of this embodiment containing a molecule having binding activity to mannose exhibits migration into cancer tissues via mannose.

Examples of molecules having binding activity to mannose include sugar-binding proteins and antibodies.
Examples of sugar-binding proteins having mannose-binding activity include rPALa (recombinant Phlebodium aureum agglutinin a), rHeltuba (recombinant Helianthus tuberosus lectin), and rGRFT (recombinant Helianthus tuberosus lectin). combinant Griffithia sp. lectin), rBanana (recombinant Musa acuminata lectin), NPA (Narcissus pseudonarcissus agglutinin), ConA (Canavalia ensiformis agglutinin A), GNA (Galanthus nivalis agglutinin), HHL (Hippeastrum hybr id lectin), DBAI (Dioscorea batatas agglutinin I), CCA (Castanea crenata agglutinin), rOrysata (recombinant Oryza sativa lectin), rCalsepa (recombinant Calystegia sepium lectin) and rBC2LA (recombinant Burkholderia cenocepacia lectin), with rBanana (banana lectin) being preferred. Banana lectin functions as a 15 kDa protein dimer. Banana lectin includes, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 1, but is not limited to proteins containing the amino acid sequence. Further, the above-mentioned protein having mannose-binding activity does not necessarily need to be r (recombinant), and may be a natural protein.

Furthermore, from the viewpoint of reducing side effects, it is preferable that the sugar-binding protein has a defective or reduced hemagglutinating ability. By adding the H84T amino acid mutation to banana lectin, the hemagglutination reaction specific to lectin can be suppressed compared to wild-type banana lectin that does not have the mutation.

The target cancer to which the target substance is delivered is not particularly limited as long as the expression of MUC6 is negative and mannose is bound, and breast cancer (e.g., invasive ductal carcinoma, ductal carcinoma in situ) , inflammatory breast cancer, etc.), prostate cancer (e.g., hormone-dependent prostate cancer, hormone-independent prostate cancer, etc.), pancreatic cancer (e.g., pancreatic ductal cancer, etc.), gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, glandular squamous adenocarcinoma, etc.) epithelial cancer, etc.), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, etc.), colon cancer (e.g., gastrointestinal stromal tumor, etc.), rectal cancer (e.g., gastrointestinal stromal tumor, etc.) , colorectal cancer (e.g., familial colorectal cancer, hereditary non-polyposis colorectal cancer, gastrointestinal stromal tumor, etc.), small intestine cancer (e.g., non-Hodgkin's lymphoma, gastrointestinal stromal tumor, etc.), esophageal cancer, duodenal cancer, tongue cancer, pharyngeal cancer (e.g., nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, etc.), head and neck cancer, salivary gland cancer, brain tumor (e.g., pineal astrocytic tumor, pilocytic astrocytoma, diffuse astrocytoma) Cytoma, anaplastic astrocytoma, etc.), schwannoma, liver cancer (e.g., primary liver cancer, extrahepatic cholangiocarcinoma, etc.), kidney cancer (e.g., renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, etc.) ), gallbladder cancer, pancreatic cancer, endometrial cancer, cervical cancer, ovarian cancer (e.g., epithelial ovarian cancer, extragonadal germ cell tumor, ovarian germ cell tumor, ovarian low-grade tumor, etc.), bladder cancer, Urethral cancer, skin cancer (e.g. intraocular (ocular) melanoma, Merkel cell carcinoma, etc.), hemangioma, malignant lymphoma (e.g. reticular sarcoma, lymphosarcoma, Hodgkin's disease, etc.), melanoma (malignant melanoma), thyroid Cancer (e.g., medullary thyroid cancer, etc.), parathyroid cancer, nasal cavity cancer, sinus cancer, bone tumors (e.g., osteosarcoma, Ewing tumor, uterine sarcoma, soft tissue sarcoma, etc.), metastatic medulloblastoma, vascular fibrosis tumor, dermatofibrosarcoma protuberans, retinal sarcoma, penile cancer, testicular tumor, pediatric solid cancer (e.g. Wilms tumor, pediatric renal tumor, etc.), Kaposi's sarcoma, Kaposi's sarcoma caused by AIDS, maxillary sinus tumor, fibrous histiocytes cancer, leiomyosarcoma, rhabdomyosarcoma, chronic myeloproliferative disease, or leukemia (eg, acute myeloid leukemia, acute lymphoblastic leukemia, etc.). Among these, as the cancer tissue to which the target substance is to be delivered, gastric cancer tissue is particularly preferable because it has negative expression of MUC6 and binds more mannose than normal gastric tissue.

Examples of methods for determining whether mannose is bound to cancer tissue include, but are not limited to, immunostaining and flow cytometry.
When determining mannose binding by immunostaining, for example, GNL, banana lectin, antibodies, etc. that bind mannose are labeled with a labeling agent such as FITC and added to the cancer tissue to be determined. , the presence or absence of mannose can be visually determined.

When determining mannose binding by flow cytometry, for example, GNL, banana lectin, etc. that bind to mannose are labeled with a labeling agent such as FITC and added to cancer cells. Thereafter, the presence or absence of mannose can be visually determined by subjecting it to flow cytometry.

On the other hand, “Yoshiro Otsuka: Do gastric-type and intestinal-type traits affect the prognosis in advanced gastric cancer? Dokkyo Journal of Medical Sciences 35(1):T59~T64, 2008.” and “Tajima Y., et al., Gastric and intestinal phenotypic marker expression in gastric carcinomas and recurrence pattern after surgery-immunohistochemical analysis of 213 lesions. British Journal of Cancer, 91, 1342-1348, 2004. Based on the expression of contributing genes, gastric cancer is generally classified into four types: gastric-type gastric cancer, intestinal-type gastric cancer, mixed-type gastric cancer, and non-expressing type gastric cancer. Among these gastric cancers, there are two types of MUC6 expression-negative gastric cancer: intestinal type gastric cancer and non-expressing type gastric cancer.

Intestinal-type gastric cancer is gastric cancer that is positive for at least one of MUC2 and CD10 and negative for MUC5AC and MUC6.

Non-expressing gastric cancer is gastric cancer in which the expression of MUC5AC, MUC6, MUC2, and CD10 is negative.

In this specification, "negative" refers to the results of immunostaining of proteins (MUC5AC, MUC6, MUC2, CD10, etc.) expressed from genes in the advanced tumor region (site at the tumor margin that does not contain necrotic tissue). "Positive" refers to a state in which the proportion of tumor cells stained is less than 5%, and "positive" refers to a state in which the proportion of tumor cells stained is 5% or more.

The target gastric cancer tissue to which the target substance is delivered by the carrier of this embodiment may be intestinal type gastric cancer or non-expressing type gastric cancer. In addition, the causes of negative MUC6 expression in gastric tissues include mutations in the MUC6 gene and disappearance of MUC6-expressing cells; however, the gastric cancer tissue to which the target substance is delivered is due to MUC6 gene mutations. It may be a gastric cancer tissue that is negative for MUC6 expression, or it may be a gastric cancer tissue that is negative for MUC6 expression due to disappearance of MUC6-expressing cells.

Furthermore, the target gastric cancer tissue to which the target substance is delivered may be a gastric cancer tissue that has a mutation in the MUC6 gene or has decreased expression of the MUC6 gene.

"Having a mutation in the MUC6 gene" means that the original function of the MUC6 protein is at least partially lost due to a genetic mutation in the MUC6 gene. "The expression of the MUC6 gene is reduced" means that the amount of the MUC6 gene product is reduced compared to a control wild type (hereinafter also referred to as "WT") individual. .

Furthermore, the target gastric cancer tissue to which the target substance is delivered may be a gastric cancer tissue in which clusterin is increased compared to normal gastric tissue. As shown in the Examples below, it has been confirmed that gastric cancer tissues negative for MUC6 expression have a high proportion of cells expressing clusterin.

≪Complex≫
This embodiment provides a complex of a carrier and a target substance. The target substance is not particularly limited as long as it is to be delivered to cancer tissue, and examples thereof include anticancer agents, contrast agents, light absorption agents, and the like, with anticancer agents being preferred.
Examples of anticancer drugs include low molecular drugs, nucleic acids, proteins, peptides, and the like. Examples of low-molecular drugs include antimetabolites such as 5-FU or methotrexate, alkylating drugs such as cyclofosmid, taxane compounds such as paclitaxel or docetaxel, and VcMMAE, which is one of the MMAE derivatives. It will be done.
Nucleic acids include cDNA, mRNA, siRNA, shRNA, miRNA, antisense RNA, and the like.
Examples of proteins include toxins, enzymes, and antibodies. Toxic proteins are proteins that have cytotoxic properties. Toxin proteins include diphtheria toxin, ricin, saporin, cholera toxin, enterotoxin, elorizin, abrin, or pertussis toxin. Furthermore, a 38 kDa partial region of the cell killing domain (ETA) region derived from Pseudomonas aeruginosa exotoxin A (see SEQ ID NO: 2) can be suitably used as the toxin protein. The above carrier such as banana lectin and a protein as an anticancer agent (for example, a toxin protein) may be connected, for example, with a linker consisting of the amino acid sequence Leu-Glu-Leu-Glu (see SEQ ID NO: 3); -Ser-Gly-Gly-Gly may be connected by a linker consisting of a twice-repeated sequence (see SEQ ID NO: 4). However, the sequence of the linker that connects the carrier and the protein is not limited to these, and other sequences may be used.
Examples of peptides include ligands, vaccines, and the like.
Anticancer agents also include genome editing-related factors. For example, in the CRISPR-Cas9 system, a complex is formed between a Cas9 expression vector, an expression vector encoding a guide RNA that induces Cas9 to a target cancer gene, and a carrier, and this complex is used for gene therapy. Can be done.

When a contrast agent is used as the target substance, the composite of this embodiment is made of barium sulfate, bismuth subcarbonate, bismuth oxide, zirconium oxide, ytterbium fluoride, iodoform, barium apatite, barium titanate, lanthanum glass, barium glass, or X-ray contrast agents such as strontium glass; contrast agents for computed tomography (CT) such as iodine contrast agents; contrast agents for MRI such as gadolinium agents or superparamagnetic iron oxide (SPIO); or technetium It is preferable to include radioisotopes for single photon emission computed tomography (SPECT) such as 99m (99mTc) and molybdenum 99 (99Mo).

Examples of the light absorber include near-infrared absorbers such as IR700. By administering such a light-absorbing agent as a complex bound to a carrier such as banana lectin, the light-absorbing agent can be bound to the target cancer tissue, and cancer cells can be stimulated by light irradiation. can be destroyed (photoimmunotherapy).

The method of bonding the carrier and the target substance is not particularly limited, and examples thereof include covalent bonding, hydrogen bonding, bonding methods using a divalent crosslinking agent, biotin-streptavidin bonding, and the like.

For example, a fusion protein of a lectin such as banana lectin and streptavidin or Halotag (registered trademark) can be produced, and a biotinylated drug or a Halotag ligand drug can be bound to the fusion protein as a target substance.
Furthermore, a low molecular drug such as VcMMAE can be bonded to the amino group or cysteine of a lectin such as banana lectin via a linker.
Further, as in the examples described later, the complex may be configured as a fusion protein in which a lectin as a carrier and a protein as an anticancer agent are fused.
Although the composites have been described in detail above, these composites can be manufactured by methods known to those skilled in the art. The same applies to the pharmaceutical compositions described below.

<<Pharmaceutical composition>>
This embodiment provides a pharmaceutical composition comprising a complex of a carrier and a target substance. As the carrier and the target substance, those similar to those described above can be used. The pharmaceutical composition of the present embodiment can be administered orally in the form of a tablet, coated tablet, pill, powder, granule, capsule, solution, suspension, or emulsion, or as an injection or suppository. Alternatively, it can also be administered parenterally in the form of a skin external preparation.

The pharmaceutical composition of this embodiment may include a pharmaceutically acceptable delivery vehicle. As the pharmaceutically acceptable delivery vehicle, those used in conventional formulations can be used without particular limitation. More specifically, for example, binders such as gelatin, corn starch, gum tragacanth, or gum arabic; excipients such as starch or crystalline cellulose; leavening agents such as alginic acid; injections such as water, ethanol, or glycerin. and adhesives such as rubber-based adhesives and silicone-based adhesives. Pharmaceutically acceptable delivery vehicles can be used alone or in combination of two or more.

The pharmaceutical composition of this embodiment may further contain additives. Additives include lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin, and maltitol; flavoring agents such as peppermint and red oil; stabilizers such as benzyl alcohol and phenol; phosphoric acid. Examples include buffering agents such as salts and sodium acetate; solubilizing agents such as benzyl benzoate and benzyl alcohol; antioxidants; and preservatives.
The additives can be used alone or in combination of two or more.

(Administration method)
The method of administering the pharmaceutical composition of this embodiment is not particularly limited, and may be determined as appropriate depending on the patient's symptoms, weight, age, sex, etc. For example, tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, or emulsions are administered orally. Injections are administered intravenously alone or mixed with normal replacement fluids such as glucose and amino acids, and further intraarterially, intramuscularly, intradermally, subcutaneously, or intraperitoneally as necessary.

(Dose)
The dosage of the pharmaceutical composition of the present embodiment varies depending on the patient's symptoms, body weight, age, sex, etc., and cannot be determined unconditionally, but in the case of oral administration, for example, 1 μg to 10 g per day, for example, 1 μg to 10 g per day. 0.01 to 2000 mg of the active ingredient may be administered per dose. In the case of an injection, the active ingredient may be administered in an amount of, for example, 0.1 μg to 1 g per day, for example 0.001 to 200 mg per day. In the case of suppositories, the active ingredient may be administered in an amount of, for example, 1 μg to 10 g per day, for example 0.01 to 2000 mg per day.

[Method of treatment]
This embodiment provides a method for treating cancer, which includes administering the above-described pharmaceutical composition to a patient in need of treatment. The dosage of the pharmaceutical composition includes, for example, the dosage described above, but the dosage is not particularly limited as long as it is an effective amount of the pharmaceutical composition.
This embodiment involves introducing a chimeric antigen receptor gene (CAR gene) that recognizes mannose as a target molecule into T cells taken from a patient in need of treatment, and T cells into which the chimeric antigen receptor gene has been introduced. A method of treating cancer is provided, the method comprising administering cells (CAR-T cells) to a patient.

[Other embodiments]
This embodiment provides the use of a pharmaceutical composition for manufacturing a therapeutic agent for cancer. As the pharmaceutical composition, those similar to those mentioned above can be used.
This embodiment provides a pharmaceutical composition for the treatment of cancer, particularly for the treatment of MUC6 expression-negative gastric cancer, which comprises a complex of a carrier and a target substance. As the carrier, target substance, and pharmaceutical composition, those similar to those described above can be used.

≪Companion diagnostic agent≫
This embodiment is a companion diagnostic agent used for cancer patients using the above pharmaceutical composition, which includes an anti-clusterin antibody, a primer set for amplifying the clusterin gene, and/or binds to the clusterin gene or its amplification product. The present invention provides a diagnostic agent containing a probe for

Clusterin is a glycoprotein with a molecular weight of 75-80 kDa and is found in biological fluid samples. As described later in Examples, it has been confirmed that in MUC6-negative human gastric cancer tissue, the proportion of cells expressing clusterin is high, and the proportion of mannose-binding clusterin is high. Therefore, in identifying a patient who will use the pharmaceutical composition of this embodiment, it is preferable to use the expression level of the clusterin gene or clusterin protein as an index. In addition, mannose may be bound to clusterin or other glycoproteins in cancer tissues other than gastric cancer.

Furthermore, in addition to the above antibodies, primer sets, and/or probes, an ELISA kit for detecting clusterin protein, a kit for extracting nucleic acids (e.g., total RNA) from body fluids, cells, tissues, etc. , a fluorescent substance for labeling, a reagent for nucleic acid amplification, or the like.
Since clusterin is found in biological fluid samples, specimens include blood, urine, saliva, sweat, or tissue exudates, with blood or urine being preferred. As a method for detecting clusterin in patient-derived specimens, the expression level of clusterin protein may be analyzed using ELISA, etc., or clusterin DNA fragments are amplified by PCR using primers, and the amplified products are analyzed. Alternatively, the analysis may be performed by a method using hybridization using a probe complementary to a specific clusterin DNA or clusterin mRNA.
Although the companion diagnostics have been explained above, these companion diagnostics can be manufactured by methods known to those skilled in the art.

Hereinafter, the present invention will be explained with reference to examples, but the present invention is not limited to the following examples.

[Preparation of Muc6KO mouse]
Muc6KO mice were produced under contract to the University of Tsukuba Life Science Animal Resource Center.
Specifically, as shown in FIG. 1, a sequence encoding DsRED, a 2A sequence, a FlpER sequence, and a STOP sequence are knocked into the start codon present in exon 2 of the 33 exons of the mouse Muc6 gene. The design was such that the endogenous Muc6 gene would be deleted if the individual had the FlpER sequence and STOP sequence knocked into both alleles.

By incorporating the sequence encoding DsRED, mouse Muc6-expressing cells can be made to glow. The 2A sequence is a base sequence that encodes the 2A peptide that separates proteins during translation. The FlpER sequence is a sequence in which the Flpo sequence and the ERT2 sequence are connected with a linker (see SEQ ID NO: 5).
On the other hand, WT mice were purchased from CLEA Japan. The background of each mouse described in this example is C57BL/6N.

[Analysis of Muc6KO mouse]
A tissue section of the stomach containing Dysplasia was prepared from the 3-month-old MucKO mouse prepared as described above, and the expression of MUC6 was confirmed by RNA in situ hybridization (ISH).

Specifically, first, mouse stomach tissue was fixed with 10% formalin and embedded in paraffin, and then sections were prepared. In situ hybridization was performed on this section using RNAscope(R) 2.5 HD Duplex Detection Reagents Kit (ACD) and a probe that specifically recognizes mouse Muc6 and Gif. As a probe for mouse Muc6, ACD's RNAscope Probe-Mm-Muc6 (blue) was used. As the Gif probe, ACD's RNAscope Probe-Mm-Gif (red) was used.

FIG. 2 is a stained image visualizing Muc6 and Gif mRNA in the stomachs of Muc6KO mice and WT mice by in situ hybridization. The scale bar in FIG. 2 is 100 μm.

As shown in FIG. 2, in the stomach corpus and antrum of WT mice, region R1 where gastric accessory cells are located is stained blue, and region R2 where principal cells are located is stained red. Expression of mouse Muc6 was confirmed in accessory cells.
On the other hand, in the Muc6KO mouse, a red-stained region R3 was observed in the gastric corpus and gastric antrum, but no blue region was observed.
From this, it was confirmed that the expression of mouse Muc6 disappeared in the gastric corpus and gastric antrum of Muc6KO mice, and the generated Muc6KO mice lacked the mouse Muc6 gene. Note that no expression of murine Muc6 or Gif was confirmed in the duodenum.

FIG. 3 is a graph showing the expression level of the Muc6 gene in the gastric mucosa of WT mice and Muc6KO mice, as measured by qPCR.

In addition to in situ hybridization, deletion of Muc6 gene was also confirmed by qPCR expression level.
Specifically, qPCR was performed on cDNA obtained by reverse transcription of RNA extracted from the gastric mucosa of WT mice and Muc6KO mice. For reverse transcription, the forward primer shown in SEQ ID NO: 6 and the reverse primer shown in SEQ ID NO: 7 were used.

As shown in FIG. 3, in WT mice, expression of mouse Muc6 was confirmed in the gastric mucosa, whereas in Muc6KO mice, no expression of mouse Muc6 was observed. Note that the N number of WT mice was 4, and the N number of Muc6KO mice was 8.
From the above, it was revealed that the mouse Muc6 gene is definitely deleted in Muc6KO mice.

After producing MucKO mice, they were observed over time to see if they developed gastric cancer (from 4 weeks of gestation to December months). FIG. 4 shows a bright field image of the stomach of a Muc6KO mouse and a stained image of a tissue section of the stomach. HE staining was used for staining. In the explanation at the top of the figure, the embryonic week (w) or month (m) of the Muc6KO mouse is indicated.

Dysplasia was confirmed in 6 out of 6 MucKO mice at 3 months of age, as indicated by the marks in the figure. Furthermore, Adenocarcinoma was confirmed in 4 out of 4 mice at 8 months old and in 3 out of 3 mice at 12 months old.
Thus, it was confirmed that Muc6KO mice spontaneously develop cancer in the gastric antrum.

[Mucin analysis]
On the other hand, FIG. 5 is an electron microscope image of stomach tissue sections of WT mice and Muc6KO mice.
When tissue sections of the stomachs of WT mice and Muc6KO mice were observed using an electron microscope, the formation of a mucin layer (a linear structure in a 300,000x magnification) was confirmed in both, as shown in FIG. 5. Note that both the WT mouse and the Muc6KO mouse were 3 months old, and the sectioned stomach tissue of the Muc6KO mouse was Dysplasia.

FIG. 6 is a fluorescent immunostaining image visualizing MUC5AC in stomach tissue sections of WT mice and Muc6KO mice. The scale bar in FIG. 6 is 100 μm.
For fluorescent immunostaining, first, mouse stomach tissue was fixed with 10% formalin and embedded in paraffin, and sections were prepared. Note that both the WT mouse and the Muc6KO mouse were 3 months old, and the sectioned stomach tissue of the Muc6KO mouse was Dysplasia.
Next, the tissue section was subjected to deparaffinization treatment and antigen retrieval treatment, washed with PBS (Phosphate-buffered saline), and then blocked for 1 hour with PBS mixed with 10% goat serum.
Thereafter, after washing with PBS again, anti-Muc5AC antibody (manufactured by Abcam) was diluted 2000 times as a primary antibody in 2.5% PBS and reacted overnight at 4°C.
Next, after washing with PBS, a secondary antibody (Alexa Fluor 488, manufactured by Invitrogen) was diluted 200 times in 2.5% PBS and reacted for 1 hour at room temperature.
Thereafter, the cells were washed again with PBS, and the nuclei were stained using Hoechst (registered trademark) 33342 (Dojindo Laboratories), sealed, and observed under a microscope.

As shown in FIG. 6, the expression of MUC5AC protein (staining shown in bright color in the figure) was decreased in Muc6KO mice compared to WT mice.

FIG. 7 is a fluorescent immunostaining image visualizing MUC2 and MUC4 in stomach tissue sections of WT mice and Muc6KO mice. The scale bar in FIG. 7 is 100 μm.
In the staining shown in FIG. 7, Mouse monoclonal anti-MUC4 Santa Curuz Sc-33654 from Santa Cruz Biotechnology, Inc. and Rabbit monoclonal anti-MUC2 Abcam Ab9007 from Abcam were used as primary antibodies. Furthermore, GOAT-Rabbit 488 and GOAT-Mouse 555 were used as secondary antibodies, and Alexa Fluor 488 and Alexa Fluor 555 (both manufactured by Invitrogen) were used as fluorescent groups. Other conditions were the same as those for staining shown in FIG. 6.
In FIG. 7, the red stained area representing the mouse MUC6 protein is shown as R4, and the green stained area representing the MUC2 protein is shown as R5.
As shown in FIG. 7, the expression of Muc2 and Muc4 was increased in Muc6KO mice compared to WT mice. Furthermore, in Muc6KO mice, gastric parietal cells and principal cells decreased, and tuft cells increased. Note that the stomach tissue of the 3-month-old Muc6KO mouse that was sectioned was Dysplasia, and the stomach tissue of the 8-month-old Muc6KO mouse was cancerous tissue.

As described above, the mucin composition of Muc6KO mice was different from that of WT mice, and the formation of Metaplasia was confirmed.

[Analysis of new drug therapy in Muc6KO mouse gastric cancer]
Mouse MUC6 protein has a characteristic sugar chain structure, and mucin composition was changed in Muc6KO mice. From this, it was thought that if the cancer region-specific sugar chain structure could be identified in gastric cancer induced in Muc6KO mice, it could become a target for drug delivery systems (DDS). Therefore, LC-MS analysis and lectin microarray analysis were performed.

First, LC-MS analysis was performed on sugar chains bound to the vestibular normal tissue of WT mice and the vestibular cancer tissue of Muc6KO mice.
Whole lysates extracted from WT mice and Muc6KO mice using Lipa buffer were adjusted to approximately 3 mg/mL, desalted, and LC-MS analysis of O-glycans and N-glycans was entrusted to Sumitomo Bakelite. did.

Specifically, regarding O-glycans, first, the sugar chains in the sample were released using EZGlyco (registered trademark) O-Glycan Prep Kit, and purified and labeled.
Next, 50 μL of the obtained sugar chain solution was dried, redissolved in 10 μL of ultrapure water, and 1 μL of it was subjected to LC-MS analysis.

Regarding N-type sugar chains, first, the sample was concentrated to dryness using a centrifugal concentrator, and the sugar chains were released by reductive alkylation treatment, trypsin digestion, and PNGase F.
Thereafter, free sugar chains were purified and labeled using BlotGlyco (registered trademark). Next, 50 μL of the obtained sugar chain solution was concentrated and redissolved in 10 μL of pure water, of which 1 μL was subjected to LC-MS analysis.

FIG. 8 shows the analysis results of O-type sugar chains by LC-MS analysis, and FIG. 9 shows the analysis results of N-type sugar chains by LC-MS analysis.
As shown in FIG. 8, the amount of O-glycan binding in Muc6KO mice was decreased overall compared to WT.
On the other hand, as shown in Figure 9, N-glycans in Muc6KO mice are found to have a core fucose structure, a mannose-rich sugar chain, and a sialic acid bond, compared to WT. It was revealed that there is an increase in

Next, changes in lectin binding to sugar chains in vestibular normal tissues of WT mice and vestibular cancer tissues of Muc6KO mice were analyzed using a lectin microarray.
Specifically, gastric mucosal cells from normal tissues of WT mice and gastric mucosal cells from cancerous tissues from Muc6KO mice were detached using collagenase and dispase, respectively, and then subjected to FACS (Fluorescence-Activated Cell Sorting) using Epicam antibody. gastric mucosal epithelial cells were sorted.
Next, an extract of sugar chain components extracted from gastric mucosal epithelial cells was labeled with Cy3, and a lectin microarray (manufactured by National Institute of Advanced Industrial Science and Technology, J Biol Chem. 2011 Jun 10; 96 types of lectins immobilized on a slide glass) was prepared. 286(23):20345-53. doi: 10.1074/jbc.M111.231274.). Then, the binding properties of various lectins to each mucosal epithelial cell of WT mice and Muc6KO mice were quantitatively evaluated.

FIG. 10A is a schematic diagram of lectin microarray analysis of a sugar chain component extract of gastric mucosal epithelial cells.
Moreover, FIG. 10B is a graph showing the binding of rBanana lectin to the sugar chain component extract of each gastric mucosal epithelial cell of WT mouse and Muc6KO mouse, and FIG. 10C is a graph showing the binding property of rBanana lectin to each gastric mucosal epithelial cell of WT mouse and Muc6KO mouse. FIG. 10D is a graph showing the binding property of rGRFT lectin to the sugar chain component extract of each gastric mucosal epithelial cell of WT mice and Muc6KO mice. .
The WT mice and Muc6KO mice are 8 months old, and the sugar chain component extract of Muc6KO mice was extracted from cancer tissue.
As shown in FIGS. 10B, 10C, and 10D, lectin microarray analysis revealed that in Muc6KO mice, lectins that bind to mannose (Heltuba, rGRFT, and rBanana) were more abundant in gastric mucosal epithelial cells than in WT mice. It was revealed that it easily binds to sugar chain components. Additionally, data indicating that rBC2LA easily binds was obtained.

Banana lectin was selected from among the above lectins that have high binding properties to mannose and used for subsequent analysis.

FIG. 11A is a fluorescent staining image showing the results of binding FITC-labeled banana lectin to a tissue section of the stomach of a WT mouse, and FIG. 11B is a fluorescent staining image showing the binding of FITC-labeled GNL to a tissue section of the stomach of a WT mouse. This is a fluorescent staining image showing the results.
Moreover, FIG. 11C is a fluorescent staining image showing the result of binding FITC-labeled banana lectin to a tissue section of the stomach of a Muc6KO mouse, and FIG. 11D is a fluorescent staining image showing the result of binding FITC-labeled GNL to a tissue section of the stomach of a Muc6KO mouse. This is a fluorescent staining image showing the result of combining . In FIGS. 11A-11D, the scale bar is 100 μm.
The mice in FIGS. 11A to 11D were 8 months old, and the sectioned stomach tissue of the Muc6KO mouse was cancerous tissue.

In the staining shown in FIGS. 11A to 11D, FITC-labeled banana lectin or FITC-labeled GNL (Galanthus nivalis lectin) was incubated with tissue sections. GNL, like banana lectin, is a lectin that binds to mannose.

In the Muc6KO mouse shown in FIGS. 11C and 11D, there are more areas that glow green (shown in bright colors in the figure) than in the WT mouse shown in FIGS. 11A and 11B, and therefore it is a mannose-binding lectin. It became clear that the binding of banana lectin was increasing (in other words, the amount of mannose was increasing).

FIG. 12 is a schematic diagram showing banana lectin blotting.
As described above, the protein to which banana lectin, which increases binding properties in Muc6KO mice, binds was confirmed by banana lectin blotting described in detail below.
Specifically, first, proteins were extracted from gastric mucosal cells of 8-month-old WT mice and Muc6KO mice using IP lysis buffer. Thereafter, the following experiments were carried out outsourced to Nippon Proteomics. Cancer tissue was formed in the gastric mucosa of Muc6KO mice, and the gastric mucosal cells were collected from the cancer tissue.

First, the gastric mucosal lysate was centrifuged at 18,000 G for 10 minutes to collect the supernatant, 50 μL of streptavidinated beads and 10 μg of biotinylated banana lectin were added, and the mixture was incubated at 4° C. for 4 hours. Streptavidinated beads are magnetic beads.
Next, it was washed twice with washing buffer (0.1M HEPES pH 7.5, 0.15M NaCl, 0.2% NP-40) using a magnet, and further washed once with PBS.
Then, after centrifuging once to remove the remaining buffer, add 40 μL of 0.1M pH 2.0 glycin buffer, stir the beads thoroughly by tapping, leave on ice for 2 minutes, and centrifuge. The supernatant was collected.
Next, 4uL of 1M pH 9.5 Tris-HCl buffer was added to the collected supernatant to neutralize it, and then Sample buffer for SDS-PAGE was added, boiled at 98°C for 4 minutes, and proteins were separated by SDS-PAGE. .

FIG. 13 shows the results of SDS-PAGE of proteins pulled down with banana lectin.
Focusing on the differences in bands between WT mice and Muc6KO mice, which are shown in boxes in Figure 13, seven types of bands whose expression was increased in Muc6KO mice were cut out from the gel, treated with trypsin, and then analyzed by LC-MS analysis. We attempted to identify the internal proteins.
As a result, the 65 kDa protein was identified as secreted clusterin protein precursor (psClu). Furthermore, psClu was also detected in Western blotting, which will be explained below.

Figure 14 shows the results of Western blotting of proteins in the cell lysate and the pulled-down lysate.

For Western blotting, first, gastric mucosal cells from 8-month-old WT mice and Muc6KO mice were incubated with cell lysate buffer (50mM Tris HCl, 1% Triton-X, 5mM EDTA, 1mM Na3VO4, 1mM Na3VO4, .25mM NaF, protease inhibitor WT mouse-derived protein (hereinafter referred to as "WT-derived protein") and Muc6 KO mouse-derived protein (hereinafter referred to as "KO-derived protein") were recovered. did. Cancer tissue is formed in the gastric mucosa of Muc6KO mice, and the gastric mucosal cells of Muc6KO mice were collected from the cancer tissue.

The WT-derived protein, KO-derived protein, and the protein pulled down with the banana lectin described above were subjected to SDS-PAGE using a 7.5% to 16% lysate gel, and then transferred to a PVDF membrane (manufactured by Pall Corporation). Transcribed. In the following, the pulled down protein derived from WT mouse is referred to as "WT pull down protein", and the pulled down protein derived from Muc6KO mouse is referred to as "KO pull down protein".

This membrane was blocked with 5% skim milk for 1 hour, washed with Tris Buffered Saline with Tween 20 (TBS-T), and then incubated overnight at 4°C with anti-clusterin antibody (RD bioscience) as a primary antibody. 2000 times diluted).

Thereafter, the membrane was washed three times with TBS-T and reacted with a secondary antibody (manufactured by GE Healthcare) labeled with horseradish peroxidase (HRP) for 1 hour. The HRP signal was detected using Hikari Junyaku Co., Ltd.).

As shown in FIG. 14, as a result of Western blotting, it was confirmed that the KO-derived protein contained more psClu than the WT-derived protein. Similarly, it was confirmed that the KO pulldown protein contained more psClu than the WT pulldown protein.
This revealed that psClu expression was enhanced in Muc6KO mice.
In addition, the results shown in FIGS. 13 and 14 revealed that banana lectin binds to clusterin via mannose.

FIG. 15 is a fluorescent immunostaining image visualizing nuclei, clusterin, and mannose in stomach tissue sections of WT mice and Muc6KO mice. The scale bar in FIG. 15 is 100 μm.
In detail, FIG. 15A is a fluorescent immunostained image of a gastric mucosal tissue section of a WT mouse, and FIG. 15B is a partially enlarged view of the fluorescent immunostained image shown in FIG. 15A. Further, FIG. 15C is a fluorescent immunostained image of a gastric mucosal tissue section of an 8-month-old Muc6KO mouse, and FIG. 15D is a partially enlarged view of the fluorescent immunostained image shown in FIG. 15C. Moreover, FIG. 15E is a fluorescent immunostained image of a gastric mucosal tissue section of a 12-month-old Muc6KO mouse, and FIG. 15F is a partially enlarged view of the fluorescent immunostained image shown in FIG. 15E. Note that the stomach tissues of 8-month-old and 12-month-old Muc6KO mice that were sectioned were cancerous tissues.
The staining shown in FIGS. 15A to 15F was performed in the same manner as the staining shown in FIG. 7, except that anti-clusterin antibody (RD bioscience, 2000-fold dilution), FITC-labeled GNL, and DAPI were used to stain the nucleus. went. In FIGS. 15A to 15F, the blue region indicating the nucleus is indicated as R6, the red region indicating clusterin is indicated as R7, and the green region indicating GNL (and thus mannose) is indicated as R8.

In particular, as shown in the enlarged views of FIGS. 15D and 15F, an increase in mannose-binding clusterin was confirmed in clusterin Muc6KO mice compared to WT mice.

FIG. 16A is an immunostaining image showing the expression of clusterin in a human gastric cancer tissue array, and FIG. 16B is a graph showing the results of classifying samples according to the proportion of cells expressing clusterin in the human gastric cancer tissue array.
Further, FIG. 17A is an immunostaining image showing the presence of clusterin and mannose in the human gastric cancer tissue array, and FIG. 17B is a graph showing the proportion of mannose-binding clusterin in the human gastric cancer tissue array. The scale bar in FIGS. 16A and 17A is 100 μm.

The human gastric cancer tissue array was obtained by plotting 72 human gastric cancer tissues for which written consent for research use had been obtained, and was purchased from US Biomax.
FIGS. 16A and 17A show the above-mentioned human gastric cancer tissue array with the above-mentioned anti-clusterin antibody (GOAT antibody) as a primary antibody, 555 donkey-GOAT (manufactured by Invitrogen) as a secondary antibody, and a dye as a dye. A stained image is shown in which staining was performed using the above-mentioned Alexa Fluor 555, FITC-labeled GNL, and Hoechst (registered trademark) 33342. In FIGS. 16A and 17A, the red region representing clusterin is shown as R9, the blue region representing the cell nucleus is shown as R10, and in FIG. 17A, the green region representing GNL (and mannose) is shown as R11. has been done.

In addition, specimens in which the percentage of stained clusterin-expressing cells was less than 5% were classified as grade 1, specimens in which the ratio was 5% to 50% were classified as grade 2, and specimens in which the ratio exceeded 50% were classified as grade 3.
As a result, as shown in FIG. 16A, it was revealed that the proportion of grades 2 and 3 was higher in MUC6-negative specimens than in MUC6-positive specimens. Note that whether the test sample was MUC6 negative or MUC6 positive was determined based on the results of immunostaining.

Furthermore, as shown in FIG. 17B, an increase in mannose-binding clusterin was observed in the MUC6-negative human gastric cancer tissue array compared to the MUC6-positive case.

FIG. 18 shows the staining images and flow cytometry results obtained when FITC-labeled banana lectin was administered to MKN45, a MUC6-negative human gastric cancer cell line, and HUG1-P1, a MUC6-positive human gastric cancer cell line. Banana lectin binding in each gastric cancer cell line was quantitatively evaluated using flow cytometry as follows. For MKN45 and HUG1-P1, cells purchased from Sumitomo Pharma International Co., Ltd. and RIKEN CELL BANK were used.

First, FITC-labeled banana lectin (final concentration 0.4 μg/mL) or unlabeled banana lectin as a control was added to 0.4×10 ⁶ cells of each gastric cancer cell line, and cultured for 24 hours.
Next, trypsin treatment was performed, and the cells were collected, washed, and centrifuged, suspended in 10% FBS (Fetal Bovine Serum), and passed through a 20 μL cell strainer to prepare a sample.
Thereafter, flow cytometry was performed using Guava EasyCyte Plus (manufactured by Merck & Co.). After one sample was run and fluorescence correction was performed, all samples were run under the same conditions.
Next, after gating was performed to remove dead cells, etc., the horizontal axis was plotted with the fluorescence intensity at a wavelength of 488 nm and the vertical axis was plotted with the number of cells for the control specimen and the sample administered with FITC-labeled banana lectin, as shown in the lower part of Figure 18. It is a graph.

In the stained image shown in FIG. 18, the MUC6-negative human gastric cancer cell line emits strong green fluorescence as a whole, and the MUC6-negative human gastric cancer cell line is more sensitive than the MUC6-positive human gastric cancer cell line. The green fluorescence was strong. This revealed that the expression level of mannose-binding clusterin was high in the MUC6-negative human gastric cancer cell line.
Furthermore, as shown in the lower part of Figure 18, the distance between the two waveforms is larger in the MUC6-negative gastric cancer cell line, indicating that more banana lectin is bound in the MUC6-negative gastric cancer cell line. I can see it.

As described above, banana lectin binding was high even in the MUC6-negative human gastric cancer cell line, so a hemagglutination reaction using banana lectin was performed as follows.

FIG. 19 shows the results of a hemagglutination test using banana lectin.
A hemagglutination reaction was performed using WT banana lectin whose amino acid sequence is shown in SEQ ID NO: 1 and H84T banana lectin (see SEQ ID NO: 8), which is obtained by adding the H84T mutation to the banana lectin in order to suppress the hemagglutination reaction specific to lectin. An inspection was conducted.

Furthermore, a banana lectin drug complex (H84T banana lectin-PE38, amino acid (See SEQ ID NO: 9) was prepared and a hemagglutination test was performed.
In the production of a banana lectin drug complex, first, a nucleic acid sequence encoding a linker shown in SEQ ID NO: 3 is attached to the 3' end of a nucleic acid sequence encoding banana lectin having the H84T mutation (see SEQ ID NO: 8). , a nucleic acid sequence (see SEQ ID NO: 10) fused with a nucleic acid sequence encoding a 38 kDa partial region of the cell killing domain (ETA) region of Pseudomonas aeruginosa toxin PE38 (see SEQ ID NO: 2 above) was inserted into the pET27b vector, and the E. coli transformed into. Next, E. coli was cultured at 37°C, and protein expression was induced with IPTG. Thereafter, Escherichia coli was collected by centrifugation, and the protein was extracted, followed by affinity purification with Sepharose CL-6B (Cytiva) on which D-mannose was immobilized, to obtain a banana lectin drug complex.

Regarding the hemagglutination test, first, blood from a mouse was collected, 4 to 5 times the volume of PBS was added, centrifugation was repeated 3 to 4 times, and the supernatant was discarded to obtain a red blood cell fraction.
PBS was then added to prepare a 2% v/v red blood cell suspension for 25 μL of lectin (0.12-30 μg/mL) with a concentration gradient on a 96-well U-shaped titer plate. 50 μL of red blood cell suspension was added and allowed to stand for about 1 hour, and the hemagglutination reaction was confirmed.

As shown by the broken line in Figure 19, the hemagglutination reaction was suppressed in the H84T mutant banana lectin and the banana lectin drug complex with the H84T mutation compared to WT banana lectin without the H84T mutation. It was getting worse.

Figure 20 shows the results of evaluating the cytotoxic activity of the banana lectin drug complex against HUG1-P1 and MKN45 at different doses. These are the results of evaluating the damage activity over time.

In the experiment shown in FIG. 20, when 1 to 1000 ng of the banana lectin drug complex was administered to each gastric cancer cell line, the number of viable cells for each dose was evaluated. As a control, a group in which PBS was administered instead of the banana lectin drug complex was also prepared.
In the experiment shown in FIG. 21, each gastric cancer cell line was exposed to the banana lectin drug complex for 1 to 3 days, and the number of viable cells was evaluated for each exposure period.
To evaluate the number of living cells, use cell counting kit-8 (manufactured by Dojindo Laboratories) and add the above reagents at a ratio of 1:10 to a culture solution containing RPMI1640 mixed with 10% FBS and 1% PC/SM. The absorbance was measured at 450 nm after 4 hours.

As shown in Figures 20 and 21, it is clear that when the banana lectin drug complex was administered to the MUC6-negative gastric cancer cell line MKN45, the cell number significantly decreased in a dose- and period-dependent manner. became.

FIG. 22 is a diagram showing the administration schedule of banana lectin drug complex to Muc6KO mice.
Furthermore, FIG. 23 shows the results of immunohistochemical staining of gastric tissue sections of Muc6KO mice administered with the banana lectin drug complex, and FIG. 24 shows the results of quantifying the signal intensity in FIG. FIG. The scale bar in FIG. 23 is 100 μm.

Is it possible to suppress tumor growth by intraperitoneally administering 1 μg/body of the banana lectin drug complex once a week to Muc6KO mice in which tumors have developed in the gastric ducts according to the administration schedule shown in Figure 22? It was confirmed.
In the staining shown in FIG. 23, hematoxylin and eosin, anti-Ki67 antibody (manufactured by Cell Signaling Technology, diluted 1:2000), and anti-cleaved caspase-3 antibody (manufactured by Cell Signaling Technology, diluted 1:2000) were used. This is basically the same as the staining shown in FIG.
Tumor shrinkage was observed in the banana lectin drug complex administered group compared to the non-administered group. Furthermore, in the banana lectin drug complex administration group, the number of Ki-67 positive cells, which indicates the proliferative ability of cancer cells, decreased, indicating that cell proliferation was suppressed. Furthermore, the number of cleaved caspase-3-positive cells, which is a marker for apoptosis, increased, confirming induction of apoptosis in tumor ducts.

FIG. 25 is a diagram showing the influence of banana lectin having the H84T mutation or banana lectin drug complex on a Xenograft prepared by transplanting MKN45 into both buttocks of a nude mouse.

In this experiment, a Xenograft was created by transplanting the human gastric cancer cell line MKN45 into a nude mouse. As a result of histopathological examination, it has been revealed that the Xenograft is a cancer cell.

Starting one week after the transplantation of MKN45, 2 μg of banana lectin having the H84T mutation or the banana lectin drug complex was intraperitoneally administered twice a week for 4 weeks. Then, one week after the last administration, the Xenograft was removed, and the tumor suppressing effect of the banana lectin drug complex on banana lectin having the H84T mutation was confirmed.

As shown in FIG. 25, the extracted Xenografts in the group administered with the banana lectin drug complex were smaller and lighter in weight than in the control group administered with banana lectin having the H84T mutation. From the above results, it was revealed that the banana lectin drug complex has the effect of suppressing the proliferation of human gastric cancer cells and shrinking tumors.

This embodiment can be utilized for new drug therapy for cancer.

R1 to R11...area

Claims (13)

A carrier for delivering a target substance to cancer tissues negative for MUC6 expression, the carrier containing a molecule having binding activity to mannose. The carrier according to claim 1, wherein the cancer tissue is a gastric cancer tissue. The carrier according to claim 2, wherein the gastric cancer tissue is a gastric cancer tissue that is negative for MUC6 expression due to a mutation in the MUC6 gene. The carrier according to claim 2, wherein the gastric cancer tissue is intestinal type gastric cancer. The carrier according to claim 2, wherein the gastric cancer tissue is a gastric cancer in which the expression of MUC5AC, MUC6, MUC2, and CD10 is negative. The carrier according to claim 1, wherein the molecule is a sugar-binding protein. The carrier according to claim 6, wherein the sugar-binding protein has a defective or reduced hemagglutinating ability. The carrier according to claim 6, wherein the sugar-binding protein is banana lectin. The carrier according to claim 8, wherein the banana lectin has an amino acid mutation of H84T. A complex of the carrier according to claim 1 and a target substance. The complex according to claim 10, wherein the target substance is an anticancer drug. A pharmaceutical composition comprising the complex according to claim 10 or 11. A companion diagnostic agent for use in cancer patients using the pharmaceutical composition according to claim 12,
A diagnostic agent comprising an anti-clusterin antibody, a primer set for amplifying the clusterin gene, or a probe that binds to the clusterin gene or its amplification product.

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