A polypeptide targeting GPC3 and its application

Abstract

The present invention discloses a polypeptide targeting GPC3 and its application. The amino acid sequence of the polypeptide targeting GPC3 is shown in SEQ ID No. 1. Experimental results show that the polypeptide targeting GPC3 of the present invention has high affinity and good endocytosis on cells with high expression of GPC3, and can be used as a candidate targeting molecule for anti-tumor drugs, and is used for treating or diagnosing cancers related to abnormal activation of GPC3 targets.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a polypeptide targeting GPC3 and an application 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 targeting GPC3 and its application. The polypeptide targets and binds to 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 targeting GPC3 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, and the polypeptide comprises less than 25 amino acid residues.

[0009] GCINACSFLFPWGKEACAAECG (SEQ ID No. 1).

[0010] In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID No.1.

[0011] In some embodiments, the amino acid sequence of the polypeptide targeting GPC3 provided by the present invention or a pharmaceutically acceptable salt thereof 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; preferably, it has 95% sequence identity.

[0012] 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.

[0013] 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 a pharmaceutically acceptable salt thereof.

[0014] 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 a pharmaceutically acceptable salt thereof.

[0015] Preferably, the polypeptide derivative is an N-terminal or C-terminal modification product of the polypeptide shown in SEQ ID No. 1 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.

[0016] Further preferably, the modification is selected from: C-terminal amidation blocking and / or N-terminal modification of acetyl group.

[0017] Another aspect of the present invention provides a polynucleotide encoding any one of the above-mentioned GPC3 targeting polypeptides or polypeptide derivatives.

[0018] Another aspect of the present invention provides a pharmaceutical composition, which comprises the above-mentioned GPC3 targeting polypeptide or a pharmaceutically acceptable salt or polypeptide derivative or polynucleotide thereof and a drug carrier as an active ingredient.

[0019] Another aspect of the present invention provides use of the above-mentioned GPC3 targeting polypeptide 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.

[0020] 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.

[0021] 1. The present invention has developed a novel high-affinity polypeptide for GPC3, which can be used to target GPC3. Taking advantage of the high expression of GPC3 in malignant tumors, the polypeptide can be used for the prevention, diagnosis and / or treatment of malignant tumors with high expression of GPC3.

[0022] 2. These polypeptides are all low molecular weight polypeptides with low synthesis cost, and these polypeptides are obviously internalized in GPC3 cells. As used herein, "amino acid" refers to natural and non-natural amino acids. The stereo configuration of amino acids is indicated by a three-letter code containing the prefix "L-" or "D-" (except for achiral glycine). For example, 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").

[0023] 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.

[0024] As used herein, "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt of the compound described in the present invention, 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

[0025] Figure 1 This is a graph showing the HPLC detection result of the polypeptide shown in SEQ ID No.1;

[0026] Figure 2 This is a graph showing the MS detection results of the peptide shown in SEQ ID No.1;

[0027] Figure 3 This is a diagram of the cellular endocytosis of the polypeptide shown in SEQ ID No.1. DETAILED DESCRIPTION

[0028] 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.

[0029] 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.

[0030] Example 1 Solid Phase Synthesis of Peptides

[0031] 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.

[0032] Experimental reagents

[0033]

[0034]

[0035] (1) Solid phase synthesis of the polypeptide represented by SEQ ID No. 1

[0036] SEQ ID No.1: GCINACSFLFPWGKEACAAECG

[0037] Step 1: Coupling of the first amino acid Fmoc-Gly-OH

[0038] 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.

[0039] Step 2: Linear peptide chain synthesis

[0040] 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:

[0041] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0042] The resin was washed 6-8 times with DMF until neutral pH.

[0043] 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 to the resin and react at room temperature for 1 hour.

[0044] The resin was washed 4-6 times with DMF before coupling the next amino acid.

[0045] After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

[0046] Step 3: Cleavage of the linear precursor peptide chain

[0047] 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.

[0048] Step 4: Peptide purification

[0049] 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 the pure polypeptide.

[0050] The results of HPLC and MS identification of the polypeptide shown in SEQ ID No.1 are as follows Figure 1 , Figure 2 shown.

[0051] Example 2 Affinity of polypeptide samples with GPC3 protein

[0052] 1. Experimental materials:

[0053] name factory SeriesSSensorChipProteinA Cytiva Recombinant Human GPC3 (Cat. No.: CW66-1mg) Nearshore

[0054] 2. Experimental steps:

[0055] 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.

[0056] 3. Experimental results

[0057] Table 1 Affinity results of peptide samples and GPC3 protein

[0058]

[0059] The results show that the affinity of the polypeptide of the present invention to the GPC3 protein reaches the pm level, and the affinity is high.

[0060] Example 3 Cell endocytosis experiment

[0061] Experimental Materials:

[0062] Cells: HEK293-GPC3; Negative cells: HEK293.

[0063] (1) Main reagents

[0064]

[0065]

[0066] (2) Main consumables

[0067] name Production brand Part Number Storage environment 96WellTC-TreatedBlackMicroplates Agilent 204626-100 RT

[0068] (3) Cell lines

[0069] 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

[0070] Experimental steps:

[0071] 1. Prepare SA-Cy5 diluent: dilution ratio 1:50, diluent: cell growth medium.

[0072] 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.

[0073] 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.

[0074] 4. Fixation: Wash the cells 3 times with DPBS, add 60 μL / well fixative, incubate at 4°C in dark for 30 min.

[0075] 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.

[0076] 6. Experimental results.

[0077] The experimental results are as follows Figure 1 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.

[0078] In summary, the above results prove that the polypeptide of the present invention has targeting property of targeting GPC3, and at the same time has good penetration ability of highly expressed GPC3 cells, and can achieve high-sensitivity in vivo imaging of tiny 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.

[0079] 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 targeting GPC3, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID No.

1.

2. A polypeptide derivative, characterized in that: The polypeptide derivative is a product obtained by modifying the polypeptide according to claim 1 at the N-terminus and / or the C-terminus, wherein the modification is selected from the group consisting of N-terminal acetyl modification and / or C-terminal amino modification.

3. A polynucleotide, characterized in that The polynucleotide encodes the polypeptide of claim 1.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polypeptide according to claim 1 or the polypeptide derivative according to claim 2 or the polynucleotide according to claim 3 as an active ingredient.

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