Diagnostic aid method for pancreatic cancer and pharmaceutical composition for treating pancreatic cancer

Abstract

To provide a method for diagnosing pancreatic cancer early, and a pharmaceutical composition capable of treating pancreatic cancer.SOLUTION: A pancreatic cancer diagnosis assist method comprises (1) a step of contacting a sample derived from a subject suspected of having pancreatic cancer with an antibody specific for ganglioside GM2 and / or an antibody specific for ganglioside GD1a, to obtain an antigen-antibody complex, and (2) a step of detecting the antigen-antibody complex. A pharmaceutical composition for the treatment of pancreatic cancer contains a GM2 immune complex in which an anticancer drug or a photosensitizer is bound to the antibody specific for ganglioside GM2, and / or a GD1a immune complex in which an anticancer drug or photosensitizer is bound to the antibody specific for ganglioside GD1a.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for assisting in the diagnosis of pancreatic cancer and a pharmaceutical composition for treating pancreatic cancer. According to the present invention, pancreatic cancer can be diagnosed and treated. [Background technology]

[0002] Pancreatic cancer is rapidly increasing, especially among the elderly, and by the time it is discovered, the cancer has often already infiltrated and metastasized, with only about 20% of patients able to undergo surgery (Non-Patent Documents 1 and 2). Furthermore, the survival rate is less than 10% (Non-Patent Documents 3 and 4). Therefore, there is a need for early diagnostic methods and new treatments for pancreatic cancer. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Future Oncology (UK) 2016, Vol. 12, p669-685 [Non-patent document 2] Future Oncology (UK) 2016, Vol. 12, pp. 1929-1946 [Non-patent document 3] A Cancer Journal for Clinicians (USA) 2019, Vol. 69, p7-34 [Non-patent document 4] World Journal of Gastroenterology (USA) 2018, Vol. 24, pp. 4846-4861 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a method for early diagnosis of pancreatic cancer and a pharmaceutical composition capable of treating pancreatic cancer. [Means for solving the problem]

[0005] The present inventors have conducted extensive research into methods for early diagnosis of pancreatic cancer and pharmaceutical compositions for treating pancreatic cancer, and as a result, have surprisingly found that ganglioside GM2 or GD1a is expressed in pancreatic cancer. They have also found that the expression of GM2 or GD1a in cells and cancer tissues correlates with the malignancy of the cancer, such as rapid proliferation rate and stage. Furthermore, they have found that pancreatic cancer can be treated by conjugating an anticancer drug or photosensitizer to an antibody targeting ganglioside GM2 or GD1a. The present invention is based on this finding. Therefore, the present invention provides [1] A diagnostic aid method for pancreatic cancer, comprising: (1) contacting a specimen from a subject suspected of having pancreatic cancer with an antibody specific to ganglioside GM2 and / or an antibody specific to ganglioside GD1a to obtain an antigen-antibody complex; and (2) detecting the antigen-antibody complex. [2] A method for assisting in the prediction of the malignancy of pancreatic cancer, comprising: (1) contacting a specimen derived from a pancreatic cancer patient with an antibody specific to ganglioside GM2 and / or an antibody specific to ganglioside GD1a to obtain an antigen-antibody complex; and (2) detecting the antigen-antibody complex. [3] A kit for diagnosing or predicting the malignancy of pancreatic cancer, comprising an antibody specific to ganglioside GM2 and / or an antibody specific to ganglioside GD1a, and a reagent for detecting an antigen-antibody complex between the antibody and ganglioside GM2 and / or ganglioside GD1a. [4] A pharmaceutical composition for treating pancreatic cancer, comprising a GM2 immune complex in which an anticancer drug or a photosensitive substance is bound to an antibody specific to ganglioside GM2, and / or a GD1a immune complex in which an anticancer drug or a photosensitive substance is bound to an antibody specific to ganglioside GD1a, [5] The pharmaceutical composition for treating pancreatic cancer according to [4], wherein the photosensitizer is phthalocyanine. [6] The pharmaceutical composition for treating pancreatic cancer according to [4] or [5], wherein the antibody is a humanized antibody; and [7] A method for determining the therapeutic effect of the pharmaceutical composition for treating pancreatic cancer according to any one of [4] to [6]. Regarding. This specification [8] A method for assisting in the diagnosis of pancreatic cancer, comprising: (1) contacting a specimen from a subject suspected of having pancreatic cancer with an antibody specific to ganglioside GD1a to obtain an antigen-antibody complex; and (2) detecting the antigen-antibody complex. [9] A method for assisting in the prediction of the malignancy of pancreatic cancer, comprising: (1) contacting a specimen derived from a pancreatic cancer patient with an antibody specific to ganglioside GD1a to obtain an antigen-antibody complex; and (2) detecting the antigen-antibody complex.

[10] A kit for diagnosing or predicting the malignancy of pancreatic cancer, comprising an antibody specific to ganglioside GD1a and a reagent for detecting an antigen-antibody complex between the antibody and ganglioside GD1a.

[11] A pharmaceutical composition for treating pancreatic cancer, comprising a GD1a immune complex in which an antibody specific to ganglioside GD1a is bound to an anticancer drug or a photosensitizer;

[12] The pharmaceutical composition for treating pancreatic cancer according to

[11] , wherein the photosensitizer is phthalocyanine.

[13] The pharmaceutical composition for treating pancreatic cancer according to

[11] or

[12] , wherein the antibody is a humanized antibody; and

[14] A method for determining the therapeutic effect of the pharmaceutical composition for treating pancreatic cancer according to any one of

[11] to

[13] . Disclose. [Effects of the Invention]

[0006] The diagnostic aid method for pancreatic cancer of the present invention allows early diagnosis of pancreatic cancer. The predictive aid method for the malignancy of pancreatic cancer of the present invention allows prediction of the malignancy of pancreatic cancer. Furthermore, the pharmaceutical composition for treating pancreatic cancer of the present invention allows effective treatment of pancreatic cancer. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a diagram showing the sugar chain structures of ganglioside GM2 (A) and ganglioside GD1a (B). [Figure 2] FIG. 1 is a diagram schematically illustrating a diagnostic aid method for pancreatic cancer that detects ganglioside-positive cells, and pancreatic cancer treatment by photoimmunotherapy. [Figure 3] FIG. 1 shows flow cytometric analysis of GM2-positive MIA PaCa-2 cells (A) and cell sorting (B). [Figure 4] 1 is a graph showing the expression levels of GD1a in pancreatic cancer-derived cells PANC-1, T3M-4, PK-59, PK-45P, MIA PaCa-2, PK-8, PK-1, and KP-4. [Figure 5] 1 is a graph showing the proliferation rates of GM2-positive and GM2-negative MIA PaCa-2 cells. [Figure 6] 1 is a graph showing the anticancer effects of gemcitabine, fluorouracil [5-FU], and Abraxane, which are anticancer drugs for pancreatic cancer, on GM2-positive cells or GM2-negative cells. [Figure 7] FIG. 10 is a diagram examining the effect of GM2 expression in MIA PaCa-2 cells in three-dimensional culture. [Figure 8] FIG. 1 shows the results of examining the effect of N-(5′-adamantane-1′-yl-methoxy)-pentyl-1-deoxynojirimycin (AMP-dNM) or a MAP kinase inhibitor on GM2 expression in MIA PaCa-2 cells. [Figure 9] 1 is a graph showing the proliferation of GM2-expressing cells in nude mice. [Figure 10] 1 shows photographs of immunohistochemical staining examining the expression of GM2 in human pancreatic cancer tissue. [Figure 11] Photographs of immunohistochemical staining examining the expression of GD1a in human pancreatic cancer tissue. [Figure 12]Graphs showing the induction of cell death by anti-GM2 or anti-GD1a antibodies and saporin-labeled secondary antibodies targeting GM2 (A) or GD1a (B) expressing cells. DETAILED DESCRIPTION OF THE INVENTION

[0008] [1] Diagnostic aid for pancreatic cancer The diagnostic support method for pancreatic cancer of the present invention includes: (1) contacting a specimen from a subject suspected of having pancreatic cancer with an antibody specific to ganglioside GM2 and / or an antibody specific to ganglioside GD1a to obtain an antigen-antibody complex; and (2) detecting the antigen-antibody complex. As shown in Figure 2, early diagnosis of pancreatic cancer can be aided by detecting GM2 or GD1a from pancreatic tissue or serum of a patient suspected of having pancreatic cancer.

[0009] (Pancreatic cancer) More than 90% of pancreatic cancers are ductal carcinomas that develop in the cells of the pancreatic duct, and the term "pancreatic cancer" usually refers to this type. Pancreatic cancers include neuroendocrine tumors and intraductal papillary mucinous neoplasms. Risk factors for pancreatic cancer include chronic pancreatitis, diabetes, obesity, and smoking.

[0010] (Subjects suspected of having pancreatic cancer) The diagnostic support method for pancreatic cancer of the present invention tests a sample from a subject suspected of having pancreatic cancer. A subject suspected of having pancreatic cancer is, for example, a patient with symptoms suggestive of pancreatic cancer, such as abdominal pain, loss of appetite, abdominal distension, jaundice, and pain in the lower back or back. Pancreatic cancer may also develop diabetes. These symptoms can be used to distinguish pancreatic cancer from other cancers. Subjects suspected of having pancreatic cancer include patients who have been diagnosed with pancreatic cancer by a doctor, etc. This is because the diagnostic aid method for pancreatic cancer of the present invention can be used as an aid in the diagnosis of patients diagnosed with pancreatic cancer.

[0011] (Specimen) The specimen used in the present invention is not particularly limited as long as it is derived from a subject suspected of having pancreatic cancer, and examples thereof include pancreatic cells (including pancreatic-derived cultured cells), pancreatic tissue, urine, blood, serum, plasma, lymph, tissue fluid, cerebrospinal fluid, saliva, and sweat. That is, the specimen is not particularly limited as long as it may contain ganglioside GM2 or ganglioside GD1a.

[0012] (Ganglioside GM2) Gangliosides are sphingoglycolipids with one or more sialic acids (N-acetylneuraminic acid: Neu5Ac, NANA) attached to the sugar chain. More than 40 types of gangliosides have been found. GM2 is GalNAcβ1→4(NeuAcα2→3)Galβ1→4Glcβ1→1Cer Specifically, as shown in Figure 1(A), glucose, galactose, and N-acetylgalactosamine are bound in that order from the cell membrane, with sialic acid (N-acetylneuraminic acid) bound as a side chain of galactose.

[0013] (Ganglioside GD1a) GD1a is also a type of ganglioside, NeuAcα2→3Galβ1→3GalNAcβ1→4(NeuAcα2→3)Galβ1→4Glcβ1→1Cer Specifically, as shown in Figure 1(B), glucose, galactose, N-acetylgalactosamine, and galactose are bound in this order from the cell membrane, with two sialic acids (N-acetylneuraminic acid) bound as side chains of the galactose.

[0014] (antibody) The antibodies used in the diagnostic aid method for pancreatic cancer of the present invention are not particularly limited as long as they specifically bind to ganglioside GM2 or ganglioside GD1a, and include monoclonal antibodies, polyclonal antibodies, or antigen-binding fragments thereof. Monoclonal or polyclonal antibodies can be prepared by known methods, except for using ganglioside GM2 or ganglioside GD1a as the immunizing antigen. For example, monoclonal antibodies can be prepared according to the method of Koehler and Milstein (Nature 256:495-497, 1975). Polyclonal antibodies can be prepared by periodically intradermally injecting rabbits with ganglioside GM2 or ganglioside GD1a alone or conjugated with BSA, KLH, or the like, mixed with an adjuvant such as Freund's complete adjuvant. Blood samples are collected when the antibody titer in the blood increases, and the antiserum can be used directly, or the antibodies can be purified by known methods and used. Commercially available antibodies can be used as the antibody. Examples of commercially available antibodies include Anti-GM2 Monoclonal Antibody (TCI: A2576) and Anti-GD 1a Monoclonal Antibody (TCI: A2507) is an example.

[0015] Examples of the antigen-binding fragment of the antibody include F(ab')2, Fab', Fab, Fv, scFV, dsFV, and nanobody. These antigen-binding fragments can be obtained, for example, by digesting the antibody with a protease (e.g., pepsin or papain) using a standard method, followed by purification using a standard protein separation and purification method. Alternatively, the antibody gene can be inserted into a vector and then obtained by genetic recombination using Escherichia coli or the like. In this specification, the terms "antibody specific to ganglioside GM2" and "antibody specific to ganglioside GD1a" may refer to an antigen-binding fragment having the antigen-binding site of an antibody that specifically binds to GM2 or GD1a. Furthermore, when the diagnostic aid method for pancreatic cancer of the present invention is an immunological technique using a labeled antibody (e.g., enzyme immunoassay, chemiluminescent immunoassay, fluorescent antibody assay, radioimmunoassay, or immunohistochemical staining), the antibody can be included in the form of a labeled antibody or labeled antibody fragment labeled with a labeling substance. Specific examples of labeling substances include enzymes such as peroxidase, alkaline phosphatase, and β-D-galactosidase. In the case of enzymes or chemiluminescent substances, it is preferable to use an appropriate corresponding substrate, etc., since they cannot generate a measurable signal by themselves.

[0016] 《Contact process 1》 In the contacting step (1), a specimen from a subject suspected of having pancreatic cancer is contacted with an antibody specific to ganglioside GM2 and / or an antibody specific to ganglioside GD1a to obtain an antigen-antibody complex. The antigen-antibody complex is a complex in which GM2 or GD1a binds to the antibody via an epitope and the antigen-binding site of the antibody. Specific examples of the diagnostic aid for pancreatic cancer of the present invention include enzyme immunoassay, latex agglutination immunoassay, chemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, immunoprecipitation, immunohistochemical staining, and Western blot, and an antigen-antibody complex is formed in any of these methods.

[0017] <Detection step 2> In the detection step (2), the antigen-antibody complex is detected. The detection method is not particularly limited. For example, in immunohistochemical staining, an antibody bound to the GM2 or GD1a antigen in pancreatic cells or pancreatic tissue can be captured. Specifically, the antibody can be labeled with an enzyme such as peroxidase and reacted with a chromogenic or luminescent substrate to detect the signal. Furthermore, in fluorescent antibody techniques and radioimmunoassays, the antibody can be labeled with a fluorescent substance such as fluorescein or a radioactive substance to detect fluorescence or radioactivity. In Western blotting, the signal can also be detected by electrophoresis of the enzyme-labeled antibody of the antigen-antibody complex bound to a nitrocellulose membrane or the like, followed by reaction with a chromogenic or luminescent substrate. In either method, detection may be performed using a secondary antibody against the antibody in the antigen-antibody complex. In this case, the secondary antibody can be labeled with an enzyme, fluorescent substance, or radioactive substance to produce a signal for detection.

[0018] When the diagnostic aid method for pancreatic cancer of the present invention is carried out using a solid-phase enzyme immunoassay, which is one type of enzyme immunoassay, examples include, but are not limited to, the sandwich method and the competitive method, with the competitive method being preferred. In the sandwich method, an antigen is sandwiched between two antibodies for detection, but GM2 and GD1a are small molecules, so it may be difficult for the two antibodies to bind to the antigen simultaneously. In the competitive assay, an antibody that specifically binds to ganglioside GM2 or ganglioside GD1a is immobilized on an ELISA plate, beads, magnetic particles, or the like. Next, a sample containing GM2 or GD1a is mixed with competitive GM2 or GD1a (labeled competing antigen) labeled with an enzyme or the like, and the mixture is added to an ELISA plate or the like. GM2 or GD1a in the sample can be measured by detecting the signal from the enzyme after the reaction. When the sample contains a large amount of GM2 or GD1a, the signal becomes small, and when the sample contains a small amount of GM2 or GD1a, the signal becomes large; therefore, the concentration of GM2 or GD1a in the sample can be determined from a standard curve. The solid-phase enzyme immunoassay can be preferably used for liquid samples such as urine, blood, serum, plasma, lymph, tissue fluid, cerebrospinal fluid, saliva, or sweat.

[0019] In the diagnostic support method for pancreatic cancer of the present invention, pancreatic cancer can be detected by measuring the amount or concentration of ganglioside GM2 or ganglioside GD1a in a specimen (test sample) and comparing the measured value with a reference value established based on the amount or concentration of ganglioside GM2 or ganglioside GD1a in specimens (test samples) from healthy individuals. The reference value for healthy individuals or the cutoff value for pancreatic cancer is determined through controlled clinical trials. More specifically, if the amount or concentration of GM2 or GD1a in a specimen from a subject suspected of having pancreatic cancer is significantly higher than the amount or concentration of ganglioside GM2 or ganglioside GD1a in healthy individuals, the subject can be determined to have a high likelihood of having pancreatic cancer. For example, in immunohistochemical staining, the amount of color or luminescence in tissue can be analyzed as an image and compared. In Western blotting, the amount of color or luminescence of a band can be analyzed as an image and compared. In solid-phase enzyme immunoassay, the amount of color or luminescence in a liquid can be measured and compared.

[0020] [2] Method for predicting the malignancy of pancreatic cancer The method for assisting in the prediction of the malignancy of pancreatic cancer of the present invention comprises: (1) contacting a specimen derived from a pancreatic cancer patient with an antibody specific to ganglioside GM2 and / or an antibody specific to ganglioside GD1a to obtain an antigen-antibody complex; and (2) detecting the antigen-antibody complex.

[0021] (Malignancy) Pancreatic cancer cells expressing GM2 grow faster in vitro than those that do not. Therefore, it is believed that GM2-expressing pancreatic cancer cells will grow rapidly in vivo. Furthermore, when transplanted into nude mice, GM2-expressing pancreatic cancer cells increase in size more rapidly than those that do not express GM2. Therefore, it is believed that GM2-expressing pancreatic cancer cells are more malignant. Furthermore, when examining the correlation between the stage of pancreatic cancer and GM2, it is found that more advanced pancreatic cancers have higher GM2 expression. Similarly, GD1a-expressing cells have a high proliferation rate, grow rapidly in nude mice, and are frequently expressed in advanced pancreatic cancers, meaning that pancreatic cancers expressing GD1a are highly malignant.

[0022] (Samples from pancreatic cancer patients) In the method for predicting the malignancy of pancreatic cancer of the present invention, a sample from a pancreatic cancer patient is used to predict the malignancy. A doctor or other medical professional can comprehensively assess whether a patient has pancreatic cancer based on symptoms, GM2 or GD1a expression, CT scans, MRI scans, etc. However, samples from subjects suspected of having pancreatic cancer are not excluded, and the method of the present invention may be used to simultaneously assist in the diagnosis of pancreatic cancer and predict the malignancy of pancreatic cancer.

[0023] In the method for assisting in the prediction of the malignancy of pancreatic cancer, the terms "sample," "ganglioside GM2," "ganglioside GD1a," and "antibody" have the meanings described in the above "(1) Method for assisting in the diagnosis of pancreatic cancer," and the contacting step (1) and the detecting step (2) can also be carried out in accordance with the descriptions in "(1) Method for assisting in the diagnosis of pancreatic cancer."

[0024] When the GM2 and / or GD1a detected in the detection step (2) is above a predetermined value (cutoff value), it can be determined that the cancer is highly malignant or that the cancer is progressing. When the GM2 and / or GD1a is below the set value (cutoff value), it can be determined that the cancer is low malignant or that the cancer is not progressing. The set value (cutoff value) is determined by a controlled clinical trial. The cutoff value for the method for assisting in the prediction of the malignancy of pancreatic cancer may be set by comparing with the amount or concentration of ganglioside GM2 or ganglioside GD1a in a specimen (test sample) from a healthy person, or by comparing with the amount or concentration of ganglioside GM2 or ganglioside GD1a in a specimen (test sample) from a relatively benign pancreatic cancer.

[0025] [3] Pancreatic cancer diagnosis or malignancy prediction kit The pancreatic cancer diagnosis or malignancy prediction kit of the present invention comprises an antibody specific to ganglioside GM2 and / or an antibody specific to ganglioside GD1a, and a reagent for detecting an antigen-antibody complex between the antibody and ganglioside GM2 and / or ganglioside GD1a.

[0026] (Antibody specific to ganglioside GM2) The antibody specific to ganglioside GM2 contained in the kit of the present invention is not particularly limited as long as it can bind to ganglioside GM2, but the antibody specific to ganglioside GM2 described in the above section "[1] Diagnostic support method for pancreatic cancer" can be used.

[0027] (Antibody specific to ganglioside GD1a) The antibody specific to ganglioside GD1a contained in the kit of the present invention is not particularly limited as long as it can bind to ganglioside GD1a, but the antibody specific to ganglioside GD1a described in the above section "[1] Diagnostic support method for pancreatic cancer" can be used.

[0028] (reagent) The reagent contained in the kit of the present invention is a reagent for detecting an antigen-antibody complex between an antibody and ganglioside GM2 and / or ganglioside GD1a. Therefore, the reagent is not particularly limited as long as it is used to detect the antigen-antibody complex. For example, when the antibody is not labeled with a labeling substance such as an enzyme, the antigen-antibody complex may be detected with a secondary antibody labeled with an enzyme, biotin, or the like. Therefore, the reagent may be a reagent containing a secondary antibody. Alternatively, the reagent may contain avidin labeled with an enzyme or the like. Furthermore, the signal from enzymes such as peroxidase, alkaline phosphatase, or β-D-galactosidase bound to the antigen-antibody complex can be detected by reacting them with a chromogenic or luminogenic substrate. Therefore, the reagent may contain a chromogenic or luminogenic substrate. For example, a substrate for peroxidase is 3,3',5,5'-tetramethylbenzidine (TMB). A substrate for alkaline phosphatase is 5-bromo-4-chloro-3-indolyl phosphate (BCIP, X-Phos) and the oxidizing reagent nitro blue tetrazolium chloride (NBT). Substrates for β-D-galactosidase include 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) or fluorescein Di-β-D-galactopyranoside (FDG), and substrates for luciferase include luciferin. Furthermore, the reagents may include a stop solution for stopping the enzyme reaction, a washing solution for washing the solid phase, and the like.

[0029] [4] Pharmaceutical composition The pharmaceutical composition of the present invention comprises a GM2 immune complex in which an anticancer drug or a photosensitive substance is bound to an antibody specific to ganglioside GM2, and / or a GD1a immune complex in which an anticancer drug or a photosensitive substance is bound to an antibody specific to ganglioside GD1a. As shown in Figure 2, cancer cells can be destroyed by binding an immune complex containing, for example, a photosensitive substance to GM2-positive or GD1a-positive pancreatic cancer cells in vivo and irradiating them with non-thermal red light.

[0030] (immune complex) The immune complex contained in the pharmaceutical composition of the present invention is a complex in which an anticancer drug or a photosensitive substance is bound to an antibody specific to ganglioside GM2, or a complex in which an anticancer drug or a photosensitive substance is bound to an antibody specific to ganglioside GD1a. (antibody) The antibodies that can be used in the pharmaceutical compositions of the present invention are not particularly limited, as long as they are antibodies that specifically bind to GM2 or GD1a, or antibody fragments having the antigen-binding site thereof. However, mouse monoclonal antibodies, or chimeric antibodies thereof, humanized antibodies (CDR-grafted antibodies), or human antibodies are preferred.

[0031] Chimeric antibodies can be obtained, for example, by linking DNA encoding mouse heavy and light chain variable region domains to DNA encoding constant region polypeptides of a human antibody, incorporating the resultant into an expression vector, and introducing the vector into a host for production. The origin of the heavy and light chain variable region domains and constant region polypeptides used in chimeric antibodies is not particularly limited as long as they are human antibodies. For example, a chimeric antibody can be obtained using mouse IgG heavy and light chain variable region domains and human IgM or IgG constant region polypeptides.

[0032] Humanized antibodies (CDR-grafted antibodies) are antibodies in which the complementarity-determining regions (CDRs) of, for example, a mouse antibody are replaced and grafted with those of, for example, a human antibody. Specifically, a DNA sequence designed to link the CDRs of a mouse antibody with the framework regions (FRs) of a human antibody is synthesized by PCR from several oligonucleotides engineered to have overlapping ends. The resulting DNA is ligated to DNA encoding the C regions of a human antibody, then incorporated into an expression vector, which is then introduced into a host for production. Humanized antibodies can be obtained by grafting the complementarity-determining regions and the framework and constant region polypeptides used in humanized antibodies without particular limitation, as long as they are human antibodies. For example, a humanized antibody can be obtained by combining the complementarity-determining regions of a mouse IgG with polypeptides of human IgM or IgG framework and constant regions. Furthermore, an antigen-binding fragment of a humanized antibody can be obtained by combining the complementarity-determining regions of a mouse IgG with human IgM or IgG framework regions.

[0033] Human antibodies are monoclonal antibodies obtained from transgenic animals into which human antibody genes have been introduced, or from cell fusion of human antibody-producing cells with myeloma cells. Furthermore, methods for obtaining human antibodies include those from transgenic animals and those obtained by cell fusion of human antibody-producing cells, as well as techniques for obtaining human antibodies by panning using a human antibody library. For example, the variable regions of human antibodies can be expressed on the surface of phages as single-chain fragments (scFvs) using phage display, and phages that bind to antigens can be selected. Genetic analysis of the selected phages allows the DNA sequence encoding the variable regions of human antibodies that bind to antigens to be determined. Once the DNA sequence of the scFv that binds to an antigen has been determined, the sequence can be used to construct an appropriate expression vector to obtain human antibodies.

[0034] (Anticancer drug) The anticancer drug used in the immunoconjugate is not particularly limited as long as it is effective against pancreatic cancer, and examples thereof include tegafur, gimeracil, oteracil potassium, gemcitabine (Gemzar), fluorouracil [5-FU], levofolinate calcium, irinotecan, oxaliplatin, nab-paclitaxel (Abraxane), and erlotinib (Tarceva). Radioisotopes can also be bound to antibodies and used as immunoconjugates.

[0035] (photosensitive substance) Examples of the photosensitizer include porphyrin compounds (e.g., 5-aminolevulinic acid), proline compounds (e.g., proline), bacterioproline compounds, and phthalocyanine compounds (e.g., phthalocyanine). Other examples include Photofrin, Laserphyrin, aminolevulinic acid (ALA), silicon phthalocyanine Pc4, m-tetrahydroxyphenylchlorin (mTHPC), chlorin e6 (Ce6), Almela, Levran, Foscan, Metobix, Hexbix, Photochlor, Photosense, Photorex, Lumacan, Visonac, Amfinex, verteporfin, Purlitin, ATMPn, zinc phthalocyanine (ZnPc), protoporphyrin IX (PpIX), pyropheophorbide a (PPa), and pheophorbide (PhA). For example, phthalocyanine (IR700) undergoes a chemical change when exposed to near-infrared light (non-thermal red light) at 700 nm. This means that it absorbs light energy and generates heat, causing damage (swelling, destruction, necrosis) to cancer cells. Near-infrared irradiation can also be performed by guiding an optical fiber near the pancreatic cancer. Furthermore, it can be designed so that it is activated by near-infrared light only when the immunoconjugate (antibody-phthalocyanine conjugate) binds to the target molecule (GM2 or GD1a).

[0036] The cancer-specific proteins released from cancer cells destroyed by the pharmaceutical composition of the present invention serve as antigens that sensitize and proliferate anti-cancer cytotoxic T cells, thereby increasing the number of cytotoxic T cells that attack cancer. This mechanism also allows the pharmaceutical composition of the present invention to effectively treat pancreatic cancer.

[0037] The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include oral preparations such as powders, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts, or pills, or injections, with injections being preferred. For example, in preparing injections, in addition to the active ingredient, any of a water-soluble solvent such as physiological saline or Ringer's solution, a water-insoluble solvent such as vegetable oil or fatty acid ester, an isotonic agent such as glucose or sodium chloride, a solubilizer, a stabilizer, a preservative, a suspending agent, or an emulsifier may be used.

[0038] The pharmaceutical composition of the present invention may contain an active ingredient in an amount of, but is not limited to, 0.01 to 99% by weight, preferably 0.1 to 80% by weight. The dosage (therapeutically effective amount) when using the pharmaceutical composition of the present invention can be appropriately determined depending on, for example, the type of active ingredient used (anticancer agent or photosensitizer), the type or stage of pancreatic cancer, the age, sex, weight, severity of symptoms, or administration method of the patient, and can be administered orally or parenterally.

[0039] The pharmaceutical composition of the present invention can be used in a method for treating pancreatic cancer, which comprises administering a therapeutically effective amount of the pharmaceutical composition to a pancreatic cancer patient.

[0040] [5] Method for assessing therapeutic efficacy The method for assessing therapeutic efficacy of the present invention assesses the therapeutic efficacy of the pharmaceutical composition for treating pancreatic cancer. The method for assessing therapeutic efficacy can be carried out, for example, by measuring an anticancer agent or its degradation product, or a photosensitizer or its degradation product. For example, when a conjugate (immune complex) of an antibody and a photosensitizer is bound to GM2 or GD1a, in an immunoconjugate designed to be activated by near-infrared light, the therapeutic effect of the pharmaceutical composition for treating pancreatic cancer can be measured by measuring the released photosensitizer or its degradation products. [Example]

[0041] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0042] Example 1 In this example, the expression of ganglioside GM2 in cultured cells derived from eight types of pancreatic cancer was examined. Fluorescent antibody staining was performed using a mouse anti-GM2 antibody (TCI). After the GM2 antibody reaction, fluorescent staining was performed using a fluorescently labeled secondary antibody (Molecular Probes). The percentage of cells expressing ganglioside GM2 was calculated using a FACSAria Cell Sorter (Becton Dickinson). As shown in Table 1 and Figure 3(A), 21.4% of MIA PaCa-2 cells were GM2 positive. Furthermore, when the fluorescently stained cells were sorted using a cell sorter, they were separated into GM2-positive cells and GM2-negative cells, as shown in Figure 3(B).

[0043] [Table 1]

[0044] Example 2 In this example, the expression of ganglioside GD1a in cultured cells derived from eight types of pancreatic cancer was examined. Fluorescent antibody staining was performed using a mouse anti-GD1a antibody (TCI). After the GD1a antibody reaction, fluorescent staining was performed using a fluorescently labeled secondary antibody (Molecular Probes). The percentage of cells expressing ganglioside GD1a was calculated using a FACSAria Cell Sorter (Becton Dickinson). As shown in Figure 4, GD1a expression was high in seven cell types other than MIA PaCa-2 cells, with the expression rates being particularly high in PK-59 cells, PK-45P cells, and PK-1 cells.

[0045] Example 3 In this example, the proliferation rate of GM2-positive cells was examined. The GM2-positive MIA PaCa-2 cells and the GM2-negative MIA PaCa-2 cells obtained in Example 1 were individually cultured in a 96-well plate at 5 × 10 3 Cells were seeded in RPMI-1640 medium at a concentration of 100 cells / well and cultured for 72 hours. Adherent cells were cultured for 2 hours using WST-8 cell counting reagent (Wako), and the absorbance was measured at 450 nm. As shown in Figure 5, the proliferation rate of GM2-positive cells was higher than that of GM2-negative cells. Therefore, GM2-positive cells were considered to have a higher malignant potential.

[0046] Example 4 In this example, the sensitivity of GM2-expressing cells to anticancer drugs (gemcitabine, fluorouracil [5-FU], and Abraxane) was examined. MIA PaCa-2 cells (3.0×10 3 GM2-positive cells (cells / well) were seeded onto a 96-well plate. One day later, each anticancer drug was added at a concentration of 10 μM or 100 μM. After culturing for three days, the cell proliferation rate was measured by ATP assay. The proliferation rate of GM2-positive cells was lower with gemcitabine, fluorouracil [5-FU], and Abraxane, suggesting that the anticancer drugs are effective against GM2-positive cells (Figure 6).

[0047] Example 5 In this example, changes in GM2 expression due to three-dimensional culture were investigated. In ultra-low attachment plates, 1.0 x 10 MIA PaCa-2 cells were cultured. 4 The cells were cultured for 7 days at a density of 100 cells / well (24-well plate: Corning). Cell clusters were obtained. GM2 expression on the cell surface was examined, and as shown in Figure 7(A), 96.1% of the cells were GM2-positive. Therefore, 3D culture increased the number of GM2-positive cells. Furthermore, GM2-positive cells and GM2-negative cells were separated and subjected to three-dimensional culture in the same manner as in Example 1. As shown in Figure 7(B), three-dimensional culture resulted in an increase in GM2-positive cells from the GM2-negative cells. Furthermore, two-dimensional culture of the GM2-positive cells resulted in the proliferation of both GM2-negative and GM2-positive cells.

[0048] Example 6 In this example, the effect of AMP-dNM or a MAP kinase inhibitor on GM2 expression was examined. MIA PaCa-2 cells were cultured in three dimensions under the same conditions as in Example 5, and AMP-dNM (10 μM), a glycolipid synthesis inhibitor, or PD0325901 (1 μM), a MAP kinase inhibitor, was added, and GM2 expression was measured. As shown in Figure 8(A), the addition of AMP-dNM reduced GM2 expression. As shown in Figure 8(B), microvilli were hardly observed in the cell clusters, and the addition of AMP-dNM resulted in prominent vacuoles, as indicated by asterisks. As shown in Figure 8(C), the addition of PD0325901 reduced the expression of GM2, and as shown in Figure 8(D), the size of the cell clusters was clearly reduced.

[0049] Example 7 In this example, the proliferation of GM2-expressing cells in nude mice was examined. 1 × 10 5 GM2-positive or GM2-negative cells were inoculated at 1000 cells / animal. Tumor size was calculated every week for 5 weeks according to the following formula: Size (volume) = a x b² x 0.5 (where a is the longest diameter and b is the shortest diameter) As shown in FIG. 9, the tumor mass of GM2-positive cells grew approximately 3.5 times larger than that of GM2-negative cells in 5 weeks.

[0050] Example 8 In this example, we investigated the expression of GM2 in human pancreatic cancer tissues. Pathological tissue blocks were cut into 4-μm-thick sections and stained with anti-GM2 antibody (1:1000 dilution; TCI). As shown in Figure 10, the GM2 positivity rate was 7.9% in 38 specimens from stages IA-IB, but 24.1% in 79 specimens from stages IIA-IV, indicating that the GM2 positivity rate increased with increasing stage. GM2 expression was determined to be positive when 5% of the cancer cells in the tissue were stained.

[0051] Example 9: Expression of GD1a in pancreatic cancer tissues In this example, we investigated the expression of GD1a in human pancreatic cancer tissues. Pathological tissue blocks were cut into 4-μm-thick sections and stained with anti-GD1a antibody (1:1000 dilution; TCI). As shown in Figure 11, no GD1a expression was observed in normal human pancreatic tissue, but GD1a was positive in 45% (9 / 20 cases) of surgically treated pancreatic cancer tissues.

[0052] Example 10 In this example, cell death induction by anti-GM2 or anti-GD1a antibodies targeting GM2- or GD1a-expressing cells and a second immunotoxin in which the secondary antibody was labeled with saporin was investigated. GM2-expressing MIA PaCa-2 cells (2.0 × 10 3 Cells (1000 cells / well) were seeded onto a 96-well plate. One day later, anti-GM2 antibody was added at a concentration of 5 ng / ml and secondary antibody (anti-M-ZAP) at a concentration of 0.2 ng / ml. After culturing for 3 days, cell proliferation was measured by ATP assay. PK-8 cells expressing GD1a were reacted with anti-GD1a antibody, followed by secondary antibody (anti-M-ZAP), and 2.0 × 10 3 Cells were seeded onto a 96-well plate at 100 cells / well and cultured for 3 days, after which the cell proliferation rate was measured by ATP assay. In both MIA PaCa-2 and PK-8 cells, the proliferation rate of cells reacted with the primary and secondary antibodies was low, suggesting that cell death could be induced by targeting GM2 or GD1a, respectively (Figure 12). [Industrial Applicability]

[0053] The diagnostic aid method for pancreatic cancer of the present invention can be used to aid in the early diagnosis of pancreatic cancer. The predictive aid method for the malignancy of pancreatic cancer of the present invention can predict the malignancy of pancreatic cancer. Furthermore, the pharmaceutical composition of the present invention can be used for the treatment of pancreatic cancer.

Claims

1. (1) contacting a specimen from a subject suspected of having pancreatic cancer with an antibody specific to ganglioside GD1a to obtain an antigen-antibody complex; and (2) detecting the antigen-antibody complex; A method for aiding in the diagnosis of pancreatic cancer.

2. (1) contacting a specimen derived from a pancreatic cancer patient with an antibody specific to ganglioside GD1a to obtain an antigen-antibody complex; and (2) detecting the antigen-antibody complex; A method for assisting in predicting the malignancy of pancreatic cancer.

3. an antibody specific for ganglioside GD1a, and a reagent for detecting an antigen-antibody complex between the antibody and ganglioside GD1a; A kit for diagnosing or predicting malignancy of pancreatic cancer comprising:

4. A pharmaceutical composition for treating pancreatic cancer, comprising a GD1a immune complex in which an antibody specific to ganglioside GD1a is bound to an anticancer drug or a photosensitive substance.

5. The pharmaceutical composition for treating pancreatic cancer according to claim 4 , wherein the photosensitizer is a phthalocyanine.

6. The pharmaceutical composition for treating pancreatic cancer according to claim 4 or 5, wherein the antibody is a humanized antibody.

7. A method for assisting in the assessment of the therapeutic effect of the pharmaceutical composition for treating pancreatic cancer according to any one of claims 4 to 6.

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