CN118909052B - A polypeptide targeting GPC3 and its application - Google Patents

Abstract

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

Description

Polypeptide targeting GPC3 and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a polypeptide targeting GPC3 and application thereof.

Background

Glypican is a family of Heparan Sulfate (HS) glycoproteins involved in the regulation of processes such as ontogenesis, cell proliferation and differentiation. Wherein glypican-3 (GPC 3) is a member of the family of Heparan Sulfate (HS) proteoglycans glypican, which can be attached to the cell surface via a Glycosyl Phosphatidylinositol (GPI) anchor. GPC3 plays an important role in cell growth, differentiation and migration. GPC3 was found to be expressed in many human malignant cells and serum, especially in hepatocellular carcinoma, with GPC3 specificity being highly expressed on more than 80% of human hepatocellular carcinoma cell membranes, but not in normal tissues of adults. Numerous studies have demonstrated that GPC3 can be a therapeutic target for hepatocellular carcinoma, and antibodies and small molecules to the GPC3 receptor have been successfully synthesized at present and have entered the preclinical experimental research stage. For example, patent CN200880103166.9 discloses a monoclonal antibody of glypican-3, which is a monoclonal antibody prepared from the extracellular domain of glypican-3 and used for tumor diagnosis drugs, and patent CN201210152819.0 discloses a monoclonal antibody prepared from the full-length fragment of glypican-3 and used for tumor diagnosis drugs, etc.

At present, early hepatocytes have more treatment means such as surgery, radiofrequency ablation, chemotherapy and biological immunotherapy, but no effective treatment method for advanced liver cancer exists. Targeted therapies targeting specific targets are an important direction of development for future treatment of advanced hepatocellular carcinoma. Targeted therapies targeting the GPC3 receptor are expected to alter the very passive scenario of current advanced hepatocellular carcinoma therapies. And with the intensive research on the mechanism of GPC-3 and malignant tumor formation, researchers have also found that it is overexpressed in various malignant tumors, such as lung squamous carcinoma, breast carcinoma, gastric carcinoma, colorectal carcinoma, neuroblastoma, wilm's sarcoma, liposarcoma, testicular non-seminoma, ovarian carcinoma, melanoma, and the like. Therefore, the development of the polypeptide targeting GPC3 for the treatment/diagnosis of malignant tumors with high expression of GPC3 specificity has extremely high application value.

Reference is made to:

Bhave,V.S.,et al.,Regulation of liver growth by glypican3,CD81,hedgehog,and Hhex.Am JPathol,2013.183(1):p.153-9.

Capurro,M.I.,et al.,Glypican-3inhibits Hedgehog signaling during development by competingwithpatched for Hedgehog binding.Dev Cell,2008.14(5):p.700-11.

Disclosure of Invention

The invention aims to provide a polypeptide targeting GPC3 and application thereof, wherein the polypeptide targets and binds to phosphatidylinositol proteoglycan 3 (GPC 3) overexpressed in tumor cells, and has important application value in tumor molecular diagnosis and targeted therapy.

In order to achieve the above object, the present invention provides a polypeptide targeting GPC3, which contains more than 8 consecutive amino acid residues in the amino acid sequence shown in SEQ ID No.1 and which contains less than 25 amino acid residues, or a pharmaceutically acceptable salt thereof,

GCINACSFLFPWGKEACAAECG(SEQ ID No.1)。

In some embodiments, the amino acid sequence of the polypeptide is set forth in SEQ ID No. 1.

In some embodiments, the amino acid sequence of a polypeptide targeting GPC3 or a pharmaceutically acceptable salt thereof provided herein has at least 70% or 75% or 80% or 85% or 90% or 95% sequence identity, preferably 95% sequence identity, to the amino acid sequence of SEQ ID No. 1.

In a second aspect, the present invention provides a polypeptide derivative which is a modified product of a polypeptide as described in the first aspect or a variant obtained by addition and/or substitution of one or more amino acids.

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

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

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, wherein the modification group is selected from one or more of acetyl, amino, alkyl, aryl, fatty acid, glycosyl, phosphate, sulfate, polyethylene glycol (PEG) group or peptide linking group.

Further preferably, the modification is selected from the group consisting of C-terminal amidation blocking and/or N-terminal modified acetyl.

In another aspect, the invention provides a polynucleotide encoding any one of the GPC 3-targeting polypeptides or polypeptide derivatives described above.

In another aspect, the present invention provides a pharmaceutical composition comprising as an active ingredient the above-described GPC 3-targeting polypeptide or a pharmaceutically acceptable salt or polypeptide derivative or polynucleotide thereof and a pharmaceutical carrier.

In another aspect, the present invention provides the use of a GPC 3-targeting polypeptide as defined above, or a pharmaceutically acceptable salt, polypeptide derivative, polynucleotide or pharmaceutical formulation thereof, in the manufacture of a medicament for the prevention, diagnosis and/or treatment of cancer associated with abnormal activation of GPC 3.

Preferably, the cancer is selected from the group consisting of liver cancer, prostate cancer, breast cancer, pancreatic cancer, lung cancer, sarcoma, colorectal cancer, cholangiocellular carcinoma, chordoma, small intestine cancer, pheochromocytoma, gastric cancer, renal cancer, ovarian cancer, bladder cancer, esophageal cancer, head and neck cancer, thymus cancer, cervical cancer, endometrial cancer, neuroendocrine tumor, thyroid cancer, intestinal cancer, and solid tumors such as bone metastasis. Further, the cancer is liver cancer.

1. The present invention develops a novel high affinity polypeptide of GPC3, which can be used to target GPC3. The GPC3 is highly expressed in malignant tumors, and can be used for the prevention, diagnosis and/or treatment of malignant tumors in which GPC3 is highly expressed.

2. The polypeptides are all low molecular weight polypeptides, the synthesis cost is low, and the endocytosis of the polypeptides in GPC3 cells is obvious. As used herein, "amino acid" refers to both natural and unnatural amino acids. The three-letter code containing the prefix "L-" or "D-" (except for achiral glycine) indicates the three-letter configuration of an amino acid, e.g., L-form amino acid such as 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-His" or "H"), isoleucine ("L-Ile" or "I"), leucine ("L-Leu" or "L"), lysine ("L-Lys" or "K"), methionine ("L-Met" or "M"), phenylalanine ("L-Phe" or "F"), proline ("L-Pro" or "P"), serine ("L-Asp" or "S"), threonine ("L-Thr" or "T"), glycine ("L-P" or "Tyr" or "L-Tyr" and Val "L-Tyr" or "L-Val". L-norleucine and L-norvaline can be represented as (NLeu) and (NVal), respectively. Nineteenth naturally occurring chiral amino acids have the corresponding D-isomer indicated by the three letter code containing 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-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 ("D-Val" v ").

As used herein, the meaning of "peptide" or "polypeptide" is well known to those skilled in the art. Typically, a peptide or polypeptide is one in which two or more amino acids are linked by an amide bond, which is formed by the amino group of one amino acid and the carboxyl group of an adjacent amino acid. The polypeptides described herein may comprise naturally occurring amino acids or non-naturally occurring amino acids. May be modified to include at least two amino acids such as analogs, derivatives, functional mimics, pseudopeptides, and the like.

As used herein, "pharmaceutically acceptable salts" or "pharmaceutically acceptable salts" refer to salts of the compounds described herein which are safe and effective for use in a mammal, non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pear ate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate.

Drawings

FIG. 1 is a diagram of the HPLC detection result of the polypeptide shown in SEQ ID No. 1;

FIG. 2 is a diagram showing the detection result of polypeptide MS shown in SEQ ID No. 1;

FIG. 3 shows the endocytosis of the polypeptide shown in SEQ ID No. 1.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but embodiments of the present invention are not limited thereto. Various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

The polypeptides of the invention may be prepared using methods well known to those skilled in the art, including well known methods of chemical synthesis. The polypeptide may be expressed in an organism and purified by well known purification techniques.

EXAMPLE 1 solid phase Synthesis of Polypeptides

The polypeptide compound and its derivative are synthesized into straight-chain precursor through solid phase synthesis and oxidized with DMSO to form two pairs of disulfide bonds inside molecule. The synthetic carrier is Fmoc-Gly-WANG RESIN resin. In the synthesis process, fmoc-Gly-WANG RESIN resin is fully swelled in N, N-Dimethylformamide (DMF), then the solid-phase carrier and the activated amino acid derivative are repeatedly condensed, washed, deprotected Fmoc, washed and subjected to the next round of amino acid condensation so as to reach the length of the polypeptide chain to be synthesized, and finally trifluoroacetic acid is used for: the mixed solution of water and triisopropylsilane and phenyl sulfide (90:2.5:2.5:5, v: v) reacts with resin to crack the polypeptide from the solid carrier, and then the solid crude product of the linear precursor is obtained after the solid crude product is settled by freezing methyl tertiary butyl ether. And performing disulfide bond oxidation on the cut linear precursor crude product in a weak alkaline solution to obtain a target polypeptide crude product. Purifying and separating the crude polypeptide in acetonitrile/water system of 0.1% trifluoroacetic acid by C-18 reversed phase preparative chromatographic column to obtain pure product of polypeptide and its derivative.

Experimental reagent

(1) Solid phase synthesis of the polypeptide shown in SEQ ID No.1

SEQ ID No.1:GCINACSFLFPWGKEACAAECG

Step 1, coupling Fmoc-Gly-OH of first amino acid

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

Step 2, synthesis of straight-chain peptide chain

The resin obtained in step 1 was fully swollen in DMF for 1h. The following linear precursor sequences were then synthesized in the order from the second amino acid at the carboxy terminus to the amino terminus. Each coupling cycle proceeds as follows:

fmoc-deprotection was performed twice for 8min each with 20% piperidine/DMF (20% v/v,10 mL).

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

0.5Mmol of Fmoc-AA, 0.5mmol of 6-chlorobenzotriazole-1, 3-tetramethyluronium Hexafluorophosphate (HCTU) and 1mmol of 4-methylmorpholine (NMM) were dissolved in DMF and the resin was added to react for 1h at room temperature.

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

The linear polypeptide was synthesized and the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 3 cleavage of the Linear precursor peptide chain

Freshly prepared cut cocktail (10 mL) of trifluoroacetic acid in water in triisopropylsilane in phenylsulfide (90:2.5:2.5:5, v: v) was added to the resin from step 1 and reacted for 2 hours with shaking at room temperature. After the reaction was completed, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4-fold volume of cold MTBE to obtain a crude product. The crude product was washed 3 times with MTBE and placed in vacuum for drying.

Step 4 purification preparation of the polypeptide

After filtration through a 0.45um membrane, separation was performed using a reverse phase high performance liquid chromatography system with buffers A (0.1 wt% trifluoroacetic acid, aqueous solution) and B (0.1 wt% trifluoroacetic acid, acetonitrile). And collecting the relevant fractions of the product, combining the fractions with the purity of more than 95% after the purity is identified by HPLC, and freeze-drying to obtain the polypeptide pure product.

The identification results of the polypeptide HPLC and MS shown in SEQ ID No.1 are shown in FIG. 1 and FIG. 2.

EXAMPLE 2 affinity of polypeptide samples with GPC3 protein

1. Experimental materials:

Name of the name	Manufacturer' s
		SeriesSSensorChipProteinA	Cytiva
RecombinantHumanGPC3 (goods number: CW66-1 mg)	Near shore

2. The experimental steps are as follows:

polypeptide affinity assays with GPC3 were performed with Biacore T200. FAP protein was captured at 25 ℃ using a protein a chip at about 2000RU, binding experiments were performed at 25 ℃ with 1 x HBS, pH 7.4 as runningbuffer, polypeptide analyte flow rates were 30 μl/min, association 120s,dissociation 600s, single-cycle or multi-concentration cycle kinetic/Affinity detection of binding of polypeptide samples to protein was selected, gly-HCl ph= 1.5,30 μl/min, chip regeneration was performed for 30s, and data were fitted using a 1:1 binding model.

3. Experimental results

Table 1 affinity results of polypeptide samples with GPC3 protein

The result shows that the affinity of the polypeptide provided by the invention and GPC3 protein reaches pm level, and the affinity is high.

EXAMPLE 3 endocytic assay

Experimental materials:

HEK293-GPC3, and HEK293 as a negative cell.

(1) Main reagent

(2) Main consumable

Name of the name	Production brands	Goods number	Storage environment
				96WellTC-TreatedBlackMicroplates	Agilent	204626-100	RT

(3) Cell lines

Cell lines	Culture medium
		HEK293 (negative cell)	DMEM+10%FBS+1%P.S
HEK293-GPC3 (Positive cells)	DMEM+10%FBS+1%P.S+0.75μg/mLPuromycin

The experimental steps are as follows:

1. SA-Cy5 dilutions were prepared at a dilution ratio of 1:50, and the dilutions were cell growth medium.

2. Sample preparation, namely directly diluting the mother solution to the required concentration (0.1-1 mu M), wherein the diluted solution is SA-Cy5 diluted solution prepared in the step 1. Premix for 1h at room temperature in dark.

3. And (3) sample adding, namely sucking out the culture medium in the hole, replacing the culture medium with a sample with the corresponding concentration (0.1-1 mu M), and incubating for 2h at the temperature of 37 ℃.

4. Fixation DPBS cells were washed 3 times, 60. Mu.L/well fixative was added, 4℃and protected from light for 30min.

5. The nuclear dyeing step, in which DPBS is used for washing cells for 3 times, 40-50 mu L/hole DAPI nuclear dyeing liquid is added, and the temperature is 37 ℃ and the light is prevented from being used for 20min.

6. Experimental results.

As shown in FIG. 1, the polypeptide of the invention can enter cells through endocytosis in cells with high expression of GPC3, and the polypeptide with corresponding sequence has good endocytosis.

In conclusion, the above results prove that the polypeptide of the invention has the targeting property of targeting GPC3, has better penetration capacity of cells expressing GPC3 in high degree, and can realize high-sensitivity in vivo imaging of micro tumor, so that in practical application, the polypeptide of the invention can be combined with anticancer drugs or imaging agents for targeted treatment and imaging of tumor.

It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the technical solution described in the above-mentioned embodiments may be modified or some technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solution of the embodiments of the present invention.

Claims (4)

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 N-terminus and/or C-terminus of the polypeptide according to claim 1, and the modification is selected from N-terminal acetyl modification and/or C-terminal amino modification. 3. A polynucleotide, characterized in that the polynucleotide encodes the polypeptide according to 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.