Polypeptide specific for phosphatidyl alcohol proteoglycan 3 and application thereof

Abstract

The invention discloses a polypeptide specific to phosphatidyl alcohol proteoglycan 3 and application of the polypeptide. The amino acid sequence of the polypeptide is as shown in SEQ ID No. 1 to SEQ ID No. 3. Experimental results prove that the polypeptide disclosed by the invention has relatively high affinity to phosphatidyl alcohol proteoglycan 3 (GPC3), has a good endocytosis effect on high-expression GPC3 cells, can be used as a candidate targeting molecule of an anti-tumor drug, and is used for treating or diagnosing cancers related to abnormal activation of a GPC3 target spot.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a polypeptide specific to phosphatidyl alcohol proteoglycan 3 and a use thereof. Background Art

[0002] Glypican is a family of heparan sulfate (HS) glycoproteins that participate in the regulation of individual development, cell proliferation and differentiation. Glypican-3 (GPC3) is a member of the glypican family of heparan sulfate (HS) proteoglycans and can be attached to the cell surface through a glycosylphosphatidylinositol (GPI) anchor. GPC3 plays an important role in cell growth, differentiation and migration. Studies have found that GPC3 is expressed in many human malignant tumor cells and serum, especially in hepatocellular carcinoma. GPC3 is highly expressed on the membrane of more than 80% of human hepatocellular carcinoma cells, but not in normal adult tissues. A large number of studies have confirmed that GPC3 can be a therapeutic target for hepatocellular carcinoma. At present, antibodies and small molecules for GPC3 receptors have been successfully synthesized and have entered the preclinical experimental research stage. For example, patent CN200880103166.9 discloses a monoclonal antibody against Glypican-3, which is a monoclonal antibody prepared from the extracellular domain of Glypican-3 and is used as a tumor diagnostic drug; patent CN201210152819.0 discloses a monoclonal antibody prepared from a full-length fragment of Glypican-3 and is used as a tumor diagnostic drug, etc.

[0003] At present, there are many treatment methods for early hepatocellular carcinoma, such as surgery, radiofrequency ablation, chemotherapy and biological immunotherapy, but there is no effective treatment for advanced liver cancer. Targeted therapy targeting specific targets is an important development direction for the future treatment of advanced hepatocellular carcinoma. Targeted therapy targeting the GPC3 receptor is expected to change the current passive situation of advanced hepatocellular carcinoma treatment. And with the in-depth study of the mechanism of GPC-3 and malignant tumor formation, researchers have also found that it is overexpressed in a variety of malignant tumors, such as squamous cell carcinoma of the lung, breast cancer, gastric cancer, colorectal cancer, neuroblastoma, Wilm's sarcoma, liposarcoma, testicular non-seminoma, ovarian cancer, melanoma and other tumors. Therefore, the development of peptides targeting GPC3 for the treatment / diagnosis of malignant tumors with specific high expression of GPC3 has extremely high application value.

[0004] References:

[0005] Bhave, VS, et al., Regulation of liver growth by glypican3, CD81, hedgehog, and Hhex. Am J Pathol, 2013.183(1):p.153-9.

[0006] Capurro, MI, et al., Glypican-3inhibits Hedgehog signaling during development by competingwithpatched for Hedgehog binding. Dev Cell, 2008.14(5):p.700-11. Summary of the invention

[0007] The present invention aims to provide a polypeptide specific to glycan 3 and its application. The polypeptide targets and binds to glycan 3 (GPC3) overexpressed in tumor cells and has important application value in tumor molecular diagnosis and targeted therapy.

[0008] To achieve the above object, the present invention provides a polypeptide specific for glycan 3 or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises more than 8 consecutive amino acid residues in the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, and the polypeptide comprises no more than 25 amino acid residues.

[0009] GCPGHCKIFWQRIECMTFCG(SEQ ID No.1);

[0010] GNWCCNINVAWIHWCEDGHG (SEQ ID No. 2).

[0011] In some embodiments, the amino acid sequence of the polypeptide is shown as SEQ ID No.1 or SEQ ID No.2.

[0012] In some embodiments, the amino acid sequence of the polypeptide specific for glycan 3 or its pharmaceutically acceptable salt provided by the present invention has at least 70% or 75% or 80% or 85% or 90% or 95% sequence identity with the amino acid sequence of SEQ ID No.1 or SEQ ID No.2; preferably, it has 95% sequence identity.

[0013] In a second aspect, the present invention provides a polypeptide derivative, which is a modified product of the polypeptide described in the first aspect or a variant obtained by adding and / or replacing one or more amino acids.

[0014] Preferably, the polypeptide derivative is a variant obtained by adding and / or replacing one, two or three amino acids to the polypeptide shown in SEQ ID No. 1 or SEQ ID No. 2 or a pharmaceutically acceptable salt thereof.

[0015] Further preferably, the polypeptide derivative is a variant obtained by adding and / or replacing one amino acid to the polypeptide shown in SEQ ID No. 1 or SEQ ID No. 2 or a pharmaceutically acceptable salt thereof.

[0016] Preferably, the polypeptide derivative is an N-terminal or C-terminal modification product of the polypeptide shown in SEQ ID No.1 or SEQ ID No.2 or a pharmaceutically acceptable salt thereof, and the modification group is selected from one or more of an acetyl group, an amino group, an alkyl group, an aromatic group, a fatty acid group, a sugar group, a phosphate group, a sulfate group, a polyethylene glycol (PEG) group or a peptide linking group.

[0017] Further preferably, the modification is selected from: C-terminal amidation blocking and / or N-terminal modification of acetyl group.

[0018] Another aspect of the present invention provides a polynucleotide encoding any of the above-mentioned polypeptides or polypeptide derivatives specific to glycan 3.

[0019] Another aspect of the present invention provides a pharmaceutical composition, which comprises as active ingredients the above-mentioned polypeptide specific to glycan 3 or its pharmaceutically acceptable salt or polypeptide derivative or polynucleotide and a drug carrier.

[0020] Another aspect of the present invention provides the use of the above-mentioned polypeptide specific for GPC3 or its pharmaceutically acceptable salt, polypeptide derivative, polynucleotide or pharmaceutical preparation in the preparation of a drug for preventing, diagnosing and / or treating cancer associated with abnormal activation of GPC3.

[0021] Preferably, the cancer is selected from the group consisting of liver cancer, prostate cancer, breast cancer, pancreatic cancer, lung cancer, sarcoma, colorectal cancer, cholangiocarcinoma, chordoma, small intestinal cancer, pheochromocytoma, gastric cancer, kidney cancer, ovarian cancer, bladder cancer, esophageal cancer, head and neck cancer, thymic cancer, cervical cancer, endometrial cancer, neuroendocrine tumors, thyroid cancer, intestinal cancer and solid tumors such as bone metastasis. Further, the cancer is liver cancer.

[0022] Beneficial effects:

[0023] 1. The present invention has developed a novel polypeptide specific for GPC3, which has high affinity and can be used to target GPC3. GPC3 is highly expressed in malignant tumors, and can be used for the prevention, diagnosis and / or treatment of malignant tumors with high expression of GPC3.

[0024] 2. These peptides are all low molecular weight peptides with low synthesis cost, and these peptides are obviously internalized in GPC3 cells.

[0025] As used herein, "amino acid" refers to natural and non-natural amino acids. The stereo configuration of the amino acid is indicated by a three-letter code containing the prefix "L-" or "D-" (except for achiral glycine), for example, the L-amino acids are: alanine ("L-Ala" or "A"), arginine ("L-Arg" or "R"), asparagine ("L-Asn" or "N"), aspartic acid ("L-Asp" or "D"), cysteine ​​("L-Cys" or "C"), glutamine ("L-Gln" or "Q"), glutamic acid ("L-Glu" or "E"), glycine ("Gly" or "G"), histidine ("L- The amino acids in the formula (a) are 'His' or 'H'), 'Isoleucine' or 'L-Ile' or 'I'), 'L-Leu' or 'L'), 'Lysine' or 'L-Lys' or 'K'), 'Methionine' or 'L-Met' or 'M'), 'Phenylalanine' or 'L-Phe' or 'F'), 'Proline' or 'P'), 'Serine' or 'S'), 'Threonine' or 'T'), 'Tryptophan' or 'W'), 'Tyrosine' or 'L-Tyr' or 'Y'), and 'Valine' or 'V'. L-Norleucine and L-Norvaline can be represented as (NLeu) and (NVal), respectively. Nineteen naturally occurring chiral amino acids have corresponding D-isomers, which are designated by three-letter codes with the prefix "D-": alanine ("D-Ala" or "a"), arginine ("D-Arg" or "r"), asparagine ("D-Asn" or "a"), aspartic acid ("D-Asp" or "d"), cysteine ​​("D-Cys" or "c"), glutamine ("D-Gln" or "q"), glutamic acid ("D-Glu" or "e"), histidine ("D-His" or "h"), isoleucine ("D- The invention also includes the following: amino acid ("D-Ile" or "i"), leucine ("D-Leu" or "l"), lysine ("D-Lys" or "k"), methionine ("D-Met" or "m"), phenylalanine ("D-Phe" or "f"), proline ("D-Pro" or "p"), serine ("D-Ser" or "s"), threonine ("D-Thr" or "t"), tryptophan ("D-Trp" or "w"), tyrosine ("D-Tyr" or "y"), and valine ("D-Val" or "v").

[0026] As used herein, the meaning of "peptide" or "polypeptide" is well known to those skilled in the art. Generally, a peptide or polypeptide is two or more amino acids linked by an amide bond, which is formed by the amino group of one amino acid and the carboxyl group of the adjacent amino acid. The polypeptide described herein may contain naturally occurring amino acids or non-naturally occurring amino acids. It may be modified into its analogs, derivatives, functional mimetics, pseudopeptides, and the like, which are compounds containing at least two amino acids.

[0027] As used herein, "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt of the compound described in the present invention, or a salt of the drug conjugate, which is safe and effective when used in mammals. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The cellular endocytosis diagram of the polypeptide shown in SEQ ID No.1;

[0029] Figure 2 The cellular endocytosis diagram of the polypeptide shown in SEQ ID No.2;

[0030] Figure 3 is the mass spectrum of the polypeptide shown in SEQ ID No.1;

[0031] Figure 4 is the HPLC chart of the polypeptide shown in SEQ ID No.1;

[0032] Figure 5 is the mass spectrum of the polypeptide shown in SEQ ID No.2;

[0033] Figure 6 is the HPLC chart of the polypeptide shown in SEQ ID No.2; Specific embodiments

[0034] The present invention is further described in detail below in conjunction with specific embodiments, but the embodiments of the present invention are not limited thereto. Various changes may be made to the form and details without departing from the spirit and scope of the present invention as defined in the appended claims.

[0035] The polypeptides of the present invention can be prepared using methods well known to those skilled in the art, including well-known chemical synthesis methods. The polypeptides can be expressed in an organism and purified using known purification techniques.

[0036] Example 1 Solid Phase Synthesis of Peptides

[0037] The polypeptide compound and its derivative disclosed in the present invention adopt a solid phase synthesis method to synthesize its linear precursor, and DMSO is used for oxidation to form two pairs of disulfide bonds in the molecule. The synthetic carrier is Fmoc-Gly-Wang Resin resin. In the synthesis process, the Fmoc-Gly-Wang Resin resin is first fully swollen in N,N-dimethylformamide (DMF), and then the solid phase carrier and the activated amino acid derivative are repeatedly condensed → washed → Fmoc protection → washed → the next round of amino acid condensation to achieve the desired length of the synthesized polypeptide chain, and finally a mixed solution of trifluoroacetic acid: water: triisopropylsilane: anisyl thioether (90:2.5:2.5:5, v:v:v:v) is reacted with the resin to cleave the polypeptide from the solid phase carrier, and then the solid crude product of the linear precursor is obtained after precipitation by frozen methyl tert-butyl ether. The cut linear precursor crude product is subjected to disulfide bond oxidation in a weak alkaline solution to obtain the target polypeptide crude product. The crude polypeptide was purified and separated by C-18 reverse phase preparative chromatography in 0.1% trifluoroacetic acid acetonitrile / water system to obtain pure polypeptide and its derivatives.

[0038] Experimental reagents

[0039]

[0040]

[0041] (1) Solid phase synthesis of the polypeptide represented by SEQ ID No. 1

[0042] SEQ ID No.1: GCPGHCKIFWQRIECMTFCG

[0043] Step 1: Coupling of the first amino acid Fmoc-Gly-OH

[0044] 0.2mmol of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1h. Fmoc-Gly-OH (0.16mmol) and diisopropylethylamine (DIEA, 0.64mmol) were weighed and dissolved in 8mL of DCM and added to the resin, and reacted at room temperature for 2h. After the reaction was completed, blocking solution (10mL) DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

[0045] Step 2: Linear peptide chain synthesis

[0046] The resin obtained in step 1 was fully swollen in DMF for 1 hour. Then, the linear precursor sequence was synthesized in the order from the second amino acid at the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0047] · Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0048] • Rinse the resin 6-8 times with DMF until neutral pH.

[0049] Dissolve 0.5 mmol of Fmoc-AA, 0.5 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol of 4-methylmorpholine (NMM) in DMF, add the resin and react at room temperature for 1 h.

[0050] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0051] After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

[0052] Step 3: Cleavage of the linear precursor peptide chain

[0053] Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 1 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 volumes of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

[0054] Step 4: Peptide purification

[0055] After filtering through a 0.45um membrane, the mixture was separated using a reverse phase high performance liquid chromatography system, with buffers A (0.1wt% trifluoroacetic acid, aqueous solution) and B (0.1wt% trifluoroacetic acid, acetonitrile). The product-related fractions were collected, and after HPLC identification of the purity, the fractions with a purity of >95% were combined and freeze-dried to obtain a pure polypeptide. The mass spectrometry and HPLC test results of the obtained polypeptides are shown below: Figure 3 , Figure 4 shown.

[0056] (2) Solid Phase Synthesis of the Peptide Represented by SEQ ID No. 2

[0057] SEQ ID No.2:GNWCCNINVAWIHWCEDGHG

[0058] Step 1: Coupling of the first amino acid Fmoc-Gly-OH

[0059] 0.2mmol of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1h. Fmoc-Gly-OH (0.16mmol) and diisopropylethylamine (DIEA, 0.64mmol) were weighed and dissolved in 8mL of DCM and added to the resin, and reacted at room temperature for 2h. After the reaction was completed, blocking solution (10mL) DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

[0060] Step 2: Linear peptide chain synthesis

[0061] The resin obtained in step 1 was fully swollen in DMF for 1 hour. Then, the linear precursor sequence was synthesized in the order from the second amino acid at the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0062] · Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0063] • Rinse the resin 6-8 times with DMF until neutral pH.

[0064] Dissolve 0.5 mmol of Fmoc-AA, 0.5 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol of 4-methylmorpholine (NMM) in DMF, add the resin and react at room temperature for 1 h.

[0065] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0066] After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

[0067] Step 3: Cleavage of the linear precursor peptide chain

[0068] Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 1 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 volumes of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

[0069] Step 4: Peptide purification

[0070] After filtering through a 0.45um membrane, the mixture was separated using a reverse phase high performance liquid chromatography system, with buffers A (0.1wt% trifluoroacetic acid, aqueous solution) and B (0.1wt% trifluoroacetic acid, acetonitrile). The product-related fractions were collected, and after HPLC identification of the purity, the fractions with a purity of >95% were combined and freeze-dried to obtain a pure polypeptide. The mass spectrometry and HPLC test results of the obtained polypeptides are shown below: Figure 5 , Figure 6 shown.

[0071] Example 2 Affinity of polypeptide samples with GPC3 protein

[0072] 1. Experimental materials:

[0073] name factory SeriesSSensorChipProteinA Cytiva Recombinant Human GPC3 (Cat. No.: CW66-1mg) Nearshore

[0074] 2. Experimental steps:

[0075] The affinity test of peptides and GPC3 was performed using Biacore T200. About 2000 RU of FAP protein was captured at 25°C using ProteinA chip, 1×HBS, pH 7.4 was used as running buffer, and the binding experiment was performed at 25°C. The flow rate of peptide analysis was 30 μL / min, Association 120s, Dissociation 600s, and single-cycle or multi-concentration cycle Kinetics / Affinity was selected to detect the binding of peptide samples to proteins. Gly-HCl pH=1.5, 30 μL / min, 30s were used for chip regeneration, and the data were fitted with a 1:1 binding model.

[0076] 3. Experimental results

[0077] Table 1 Affinity results of peptide samples and GPC3 protein

[0078] sample KD(μM) SEQ ID NO.1 1.2 SEQ ID NO.2 1.0

[0079] The results show that the affinity of the polypeptide of the present invention to the GPC3 protein reaches the μm level, and the affinity is high.

[0080] Example 3 Cell endocytosis experiment

[0081] Experimental Materials:

[0082] Cells: HEK293-GPC3; Negative cells: HEK293.

[0083] (1) Main reagents

[0084] Reagent name factory Part Number FBS Excell FSP500 Puromycin Invivogen ant-pr-1 4% paraformaldehyde fixative Beyotime P0099 DAPI solution (10ug / ml, ready to use) Solarbio C0065 Streptavidin-Cy5(SA-Cy5) ApexBio K1080 Streptomycin sulfate Aladdin S105491 Penicillin G Sodium Aladdin P105489

[0085] (2) Main consumables

[0086] name Production brand Part Number Storage environment 96WellTC-TreatedBlackMicroplates Agilent 204626-100 RT

[0087] (3) Cell lines

[0088] Cell lines Culture medium HEK293 (negative cells) DMEM+10%FBS+1%PS HEK293-GPC3 (positive cells) DMEM+10%FBS+1%P.S+0.75μg / mL Puromycin

[0089] Experimental steps:

[0090] 1. Prepare SA-Cy5 diluent: dilution ratio 1:50, diluent: cell growth medium.

[0091] 2. Sample preparation: dilute directly from the mother solution to the required concentration (0.1-1 μM), diluent: the SA-Cy5 diluent prepared in 1 above. Premix at room temperature in the dark for 1 hour.

[0092] 3. Sample addition: Aspirate the culture medium in the well and replace it with samples of corresponding concentration (0.1-1 μM), incubate at 37°C for 2 h.

[0093] 4. Fixation: Wash the cells 3 times with DPBS, add 60 μL / well fixative, incubate at 4°C in dark for 30 min.

[0094] 5. Nuclear staining: Wash the cells 3 times with DPBS, add 40-50 μL / well DAPI nuclear staining solution, incubate at 37°C in dark for 20 min.

[0095] 6. Experimental results

[0096] The experimental results are as follows Figure 1 and Figure 2 As shown, the polypeptide of the present invention can enter cells through cellular endocytosis in cells that highly express GPC3, and the polypeptide corresponding to the sequence has good cellular endocytosis.

[0097] In summary, the above results prove that the polypeptide of the present invention has a high affinity for glycan 3, can target GPC3, and has good penetration ability in highly expressed GPC3 cells, and can achieve high-sensitivity in vivo imaging of micro-tumors. Therefore, in practical applications, the polypeptide of the present invention can be combined with anticancer drugs or imaging agents for targeted treatment and imaging of tumors.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polypeptide specific for glycan 3 or a pharmaceutically acceptable salt thereof, It is characterized in that The polypeptide contains more than 8 consecutive amino acid residues in the amino acid sequence shown in SEQ ID No.1 or SEQ ID No.2, and the polypeptide contains less than 25 amino acid residues, GCPGHCKIFWQRIECMTFCG(SEQ ID No.1); GNWCCNINVAWIHWCEDGHG (SEQ ID No. 2).

2. The polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof, It is characterized in that The amino acid sequence of the polypeptide is shown as SEQ ID No.1 or SEQ ID No.

2.

3. A polypeptide derivative, It is characterized in that The polypeptide derivative is a modified product of the polypeptide or a pharmaceutically acceptable salt thereof according to claim 1 or 2, or a variant obtained by adding and / or replacing one or more amino acids to the polypeptide or a pharmaceutically acceptable salt thereof according to claim 1 or 2.

4. The polypeptide derivative according to claim 3, It is characterized in that The polypeptide derivative is a modified product of the N-terminus or C-terminus of the polypeptide described in SEQ ID No.1 or SEQ ID No.2, and the modified group is selected from one or more of an acetyl group, an amino group, an alkyl group, an aromatic group, a fatty acid group, a sugar group, a phosphate group, a sulfate group, a PEG group or a peptide linking group; preferably, the modification is selected from: C-terminal amidation blocking and / or N-terminal modification acetyl group.

5. The polypeptide derivative according to claim 3, It is characterized in that The polypeptide derivative is a variant obtained by adding and / or replacing one, two or three amino acids to the polypeptide described in SEQ ID No.1 or SEQ ID No.

2.

6. A polynucleotide, It is characterized in that The polynucleotide encodes the polypeptide according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, or the polypeptide derivative according to any one of claims 3-5.

7. A pharmaceutical composition, It is characterized in that The pharmaceutical composition comprises as an active ingredient the polypeptide according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, the polypeptide derivative according to any one of claims 3 to 5, or the polynucleotide according to claim 6.

8. Use of the polypeptide according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, the polypeptide derivative according to any one of claims 3 to 5, the polynucleotide according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for preventing, diagnosing and / or treating cancer associated with abnormal activation of GPC3.

9. The use according to claim 8, wherein the cancer is selected from the group consisting of liver cancer, prostate cancer, breast cancer, pancreatic cancer, lung cancer, sarcoma, colorectal cancer, cholangiocarcinoma, chordoma, small intestine cancer, pheochromocytoma, gastric cancer, kidney cancer, ovarian cancer, bladder cancer, esophageal cancer, head and neck cancer, thymic cancer, cervical cancer, endometrial cancer, neuroendocrine tumors, thyroid cancer, intestinal cancer and solid tumors such as bone metastasis.

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