CN109467607B - Acid-sensitive fusion peptide targeting tumor and application thereof - Google Patents
Acid-sensitive fusion peptide targeting tumor and application thereof Download PDFInfo
- Publication number
- CN109467607B CN109467607B CN201811459427.2A CN201811459427A CN109467607B CN 109467607 B CN109467607 B CN 109467607B CN 201811459427 A CN201811459427 A CN 201811459427A CN 109467607 B CN109467607 B CN 109467607B
- Authority
- CN
- China
- Prior art keywords
- tumor
- fusion peptide
- leu
- pro
- ala
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
Abstract
The invention discloses an acid-sensitive fusion peptide targeting a tumor and application thereof, wherein the fusion peptide is formed by connecting a low-pH insertion peptide and a fourth structural domain of a tumor surface antigen Her 2. The fusion peptide can be inserted into a tumor cell membrane in an acidic environment and displays the fourth domain of Her2 on the cell surface, so that the fusion peptide is successfully recognized by a tumor medicament trastuzumab, and the fusion peptide can effectively treat tumors. The research result of the invention overcomes the defect that the existing tumor medicament can only treat one type of cancer or can only treat one type of cancer, and the trastuzumab has the treatment efficacy on all cancers by marking the surface of the tumor with the fusion peptide of the invention, which has milestone significance for clinically treating the tumor.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a fusion peptide and an effect of the fusion peptide in targeted therapy of tumors.
Background
In recent years, with the rapid development of economy in China, the material culture level of people is continuously improved, the life style is also greatly changed, and the living environment of people is also changed, such as water quality deterioration, air quality reduction and the like. Due to the change of life style and the reduction of environmental quality, the death reasons of the population in China are greatly changed, non-infectious diseases such as malignant tumors, cardiovascular diseases, chronic diseases and the like become the main causes of death of residents in China, and the death caused by the malignant tumors accounts for a large percentage and becomes a problem which cannot be ignored.
At present, the common treatment methods for malignant tumors include surgical treatment, chemotherapy, radiotherapy and the like, but because malignant tumors have the characteristics of low differentiation degree, far difference between cell morphology and normal tissue cells, disordered arrangement, frequent relapse or metastasis and the like, the diagnosis and treatment of malignant tumors still have a plurality of unsolved problems. Malignant tumor is usually free of any symptom in early stage, when physical symptoms appear, the malignant tumor is mainly caused by tumor infiltration, organ compression and distant metastasis, at this time, the malignant tumor is usually in middle and late stages, and the middle and late stage tumors are not ideal in treatment effect and can not be cured mostly. At present, only tumors which do not have metastasis can be treated by surgical resection, and the advanced malignant tumors are difficult to operate and relapse or metastasis still occur after several years of operation. The chemotherapy is an important means in tumor treatment, but the existing antitumor drugs have the defects of poor targeting property, poor treatment effect, large toxic and side effects and the like, and are easy to destroy the immunity of a human body. Therefore, today's anticancer drugs face a great challenge in enriching the drug in tumor tissue using an effective carrier while avoiding damage to other normal tissues.
In the middle of the 19 th century, Bernard, a french physiologist, first proposed the concept of "internal environment", i.e., the microenvironment in which cells survive is extracellular fluid. Under normal physiological conditions, the physicochemical properties of the cell microenvironment are in a relatively stable state, and when the homeostasis of the cell microenvironment is destroyed, various pathological changes of the cells can be caused. The induction and maintenance of an abnormal extracellular slightly acidic environment is considered to be a key element in tumor formation and progression. It has been found that tumor metastasis is the most fatal aspect of the tumor, and the tumor metastasis is the growth process of malignant tumor cells to sites other than the primary tumor, and is one of the leading causes of death of tumor patients. Tumor metastasis is positively correlated with the cell migration ability of the tumor, and researchers find that the acid discharge strength of tumor cells is positively correlated with the cell migration ability of the tumor cells, and the extracellular fluid of the tumor cells is slightly acidic, so that the slightly acidic environment of the tumor increasingly becomes one of the hot spots in the research of the anti-tumor field.
The normal cell and its surrounding tissue environment maintain dynamic balance (such as ion distribution, protein and enzyme synthesis, air pressure and pH, etc.) for maintaining normal physiological activities, and the two combined actions provide a stable environment for cell proliferation, differentiation, apoptosis and secretion and expression of various factors on the cell surface. Once this homeostasis is disrupted, disease occurs. The obvious difference between the tumor tissue and the normal tissue is that the extracellular acid is too much, and the pH is acidic. The extracellular normal physiological pH of normal human tissue was maintained at 7.4 and the intracellular pH (pHi) at 7.2. The phenomenon is formed by the combined action of various mechanisms such as polysaccharide decomposition, Na + reverse synergy, Cl and bicarbonate ion pumps, exchange of sodium ions and potassium ions and the like. However, in most tumor tissues, the intracellular and extracellular pH gradient changes are reversed, i.e., pHi > pH. Researches show that the pH value inside and outside the tumor cell is detected by using methods such as a microelectrode, a magnetic resonance spectrum and the like, the pH value of the tumor cell is between 6.15 and 6.8, the tumor cell is acidic, and the pH value is about 7.2, neutral or even alkaline. This phenomenon of intracellular and extracellular pH reversal is mainly associated with the metabolism of tumor tissues. A large amount of nutrient substances are needed for the growth of the tumor, but due to the disturbance of tumor neovascularization and non-functional capillaries, the nutrient substance is not supplied enough, the oxygen requirement of high metabolism of the tumor cannot be met, and the partial tumor tissue is anoxic. The hypoxic state prevents the cells from gaining energy through the mitochondrial respiratory chain, and the tumor cells in the hypoxic state can adapt to the hypoxic environment to survive by activating hypoxia-inducible factors and thus downstream signaling cascades such as production of glycolytic enzymes and upregulation of glycolytic metabolism. It is found clinically that most malignant tumors have internal hypoxic regions during their growth and development, and these regions are often necrotic and more prone to tumor metastasis. Even if the tumor cells are still glycolyzed in aerobic environment, the unique metabolic process of the tumor cells maintains the steady state of intracellular pH and the basic physiological functions of the tumor cells on one hand, and also leads to the formation of slightly acidic environment outside the tumor cells on the other hand, which is also the result of natural selection in the process of tumor evolution. The results of the study show that even the mutant cells which do not require glycolysis, their extracellular matrix is still acidic. This finding suggests that the acidic phenomenon of tumor tissue may arise from the tumor cell nature. Therefore, the characteristics of tumor slightly acidic environment can be used as a target for research in the medical field of tumor cells, and the search for a targeting probe sensitive to acidity is the key point for solving the problem.
The low pH insertion peptide (pHLIP) of transmembrane helix protein C derived from bacteriorhodopsin has been the focus of recent research due to its special properties in acidic microenvironment. pHLIP is a water-soluble polypeptide that can be inserted into the lipid bilayer membrane of a cell to form a stable transmembrane alpha helix. Peptide folding and membrane insertion are driven by a neutral or basic (pH >7.4) pH drop to weakly acidic (pH 7.0-6.5 or lower). pHLIP has three main forms: form I, in which no structure is soluble in water at neutral pH, state II, in which no structure is present and binds to the cell membrane surface, state III, in which insertion and alpha-helix cross the cell membrane occurs at acidic pH. Thus, the binding force of the peptide chain to the cell membrane is several times higher at low pH than under neutral conditions, which provides a favorable basis for targeted targeting of pHLIP to acidic disease tissues. It was found that at low pH, pHLIP is free at the N-terminus and embedded and penetrated into the cell at the C-terminus. Therefore, the small molecule is covalently bound to the N-terminus of pHLIP, and can be transported to the surface of the tumor cell membrane by pHLIP at low pH. Davies and the like use pHLIP as an imaging probe in platelets to construct a rare earth element-coated gold nanoparticle system for cell imaging, and the key point of the experiment is that pHLIP can embed the C end into cells when the pH is less than or equal to 6.5, so that imaging small molecules such as fluorescent molecules and the like are transported into the cells.
The development of a targeting system beneficial to the treatment of malignant tumors by utilizing the acidic environment tropism of pHLIP is a future research hotspot.
Disclosure of Invention
The invention is completed based on the following conception: the tumor has heterogeneity, even if the surface of the tumor cell in the same tumor tissue may express different protein antigens, the drug aiming at a certain protein antigen can only kill the tumor cell expressing the antigen, but has no killing effect on the tumor cell not expressing the antigen, and the tumor cells survive to form growth advantage, so that the tumor patient generates drug resistance to the drug. If a protein antigen is expressed on the surface of all tumor cells, the drug aiming at the protein antigen can completely kill all tumor cells. The same is true for different tumor tissues. The fourth structural domain of a molecular typing marker Her2 expressed on the surface of a breast cancer cell is connected with a low-pH insertion peptide targeting a tumor to form a fusion peptide, the fusion peptide can target any solid tumor cell and is displayed on the surface of the tumor cell, and a medicament aiming at the fourth structural domain of Her2, such as trastuzumab, can kill any cancer cell including the breast cancer cell, so that the application range of the tumor medicament is expanded.
One of the objectives of the present invention is to provide an acid-sensitive fusion peptide targeting tumor.
The second object of the present invention is to provide a method for preparing the above fusion peptide.
The invention also aims to provide the application of the fusion peptide in tumor targeted therapy.
In order to achieve the purpose, the invention adopts the following technical scheme:
according to one aspect of the present invention, there is provided an acid-sensitive fusion peptide targeting a tumor, the fusion peptide comprising a low pH insertion peptide, a tumor surface antigen or a functional domain thereof, the functional domain of the tumor surface antigen being a domain recognized and bound by an antibody.
Further, the fusion peptide includes a low pH insertion peptide, a functional domain of a tumor surface antigen, which is a domain recognized and bound by an antibody.
Examples of tumor surface antigens that can be used to construct the fusion peptides of the invention include, but are not limited to, ER, PR, P53, EGFR, IGFR, Her2, CD20, CD25, CD117, CD34, CD138, CD33, VEGFR, BCMA, Mesothelin, CEA, PSCA, MUC1, EpCAM, S100, CD22, CD19, CD70, CD30, ALK, RANK, GPC2, GPC3, HER3, EGFRvIII, GD2, PD-L1, or PD-L2.
The low pH insertion peptide useful for constructing the fusion peptide of the present invention includes a polypeptide having the sequence shown in SEQ ID NO.1 or a variant thereof.
The polypeptide with the sequence shown as SEQ ID NO.1 is abbreviated as WT in the invention, and the variant of WT comprises Var1-Var 16.
The sequences of WT and its variants are as follows:
WT:ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT(SEQ ID NO.1)；
Var1:ACEDQNPYWARYADWLFTTPLLLLDLALLVDG(SEQ ID NO.2)；
Var2:ACEDQNPYWRAYADLFTPLTLLDLLALWDG(SEQ ID NO.3)；
Var3:ACDDQNPWRAYLDLLFPTDTLLLDLLW(SEQ ID NO.4)；
Var4:ACEEQNPWRAYLELLFPTETLLLELLW(SEQ ID NO.5)；
Var5:ACDDQNPWARYLDWLFPTDTLLLDL(SEQ ID NO.6)；
Var6:CDNNNPWRAYLDLLFPTDTLLLDW(SEQ ID NO.7)；
Var7:ACEEQNPWARYLEWLFPTETLLLEL(SEQ ID NO.8)；
Var8:CEEQQPWAQYLELLFPTETLLLEW(SEQ ID NO.9)；
Var9:CEEQQPWRAYLELLFPTETLLLEW(SEQ ID NO.10)；
Var10:ACEDQNPWARYADWLFPTTLLLLD(SEQ ID NO.11)；
Var11:ACEEQNPWARYAEWLFPTTLLLLE(SEQ ID NO.12)；
Var12:ACEDQNPWARYADLLFPTTLAW(SEQ ID NO.13)；
Var13:ACEEQNPWARYAELLFPTTLAW(SEQ ID NO.14)；
Var14:TEDADVLLALDLLLLPTTFLWDAYRAWYPNQECA(SEQ ID NO.15)；
Var15:CDDDDDNPNYWARYANWLFTTPLLLLNGALLVEAEET(SEQ ID NO.16)；
Var16:CDDDDDNPNYWARYAPWLFTTPLLLLPGALLVEAEET(SEQ ID NO.17)。
the tumor surface antigen or the functional domain thereof of the present invention is linked to the N-terminus of the low pH insertion peptide by Linker.
The Linker is conventionally used in the art, and the sequence may be (GGGS) m or (GGGGS) m, where m is a natural number.
In a specific embodiment of the invention, the Linker sequence is GGGGS (SEQ ID NO. 19).
In a specific embodiment of the invention, the sequence of the low pH insertion peptide of the invention is shown in SEQ ID NO. 1.
In a particular embodiment of the invention, the tumor surface antigen is Her 2.
In a particular embodiment of the invention, the functional domain of the tumor surface antigen is the fourth domain of the Her2 protein or a functionally similar domain thereof, and the functionally similar domain of the fourth domain of the Her2 protein retains the activity of the fourth domain of the Her2 protein in binding to antibodies.
Further, the fourth domain sequence of the Her2 protein used in the invention is shown in SEQ ID NO. 18.
The functional similarity structural domain of the fourth structural domain of the Her2 protein comprises a polypeptide which is derived from the amino acid sequence shown in SEQ ID NO.18, has the same function with the sequence shown in SEQ ID NO.18 and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the fourth structural domain of the Her2 protein with the sequence shown in SEQ ID NO. 18.
The functionally similar domain of the fourth domain of the Her2 protein includes a polypeptide consisting of an amino acid sequence having at least 80% homology (also referred to as sequence identity) with the amino acid sequence shown in SEQ ID NO.18, more preferably at least about 90% to 95% homology with the amino acid sequence shown in SEQ ID NO.18, and often 96%, 97%, 98%, 99% homology.
In general, it is known that modification of one or more amino acids in a protein or polypeptide does not affect the function of the protein. One skilled in the art will recognize that individual amino acid changes or small percentage amino acids or individual additions, deletions, insertions, substitutions to an amino acid sequence are conservative modifications, wherein a change in a protein polypeptide results in a protein or polypeptide with similar function. Conservative substitution tables providing functionally similar amino acids are well known in the art.
The functionally similar domain of the fourth domain of the Her2 protein also includes non-conservative modifications of the amino acid sequence shown in SEQ ID No.18, as long as the modified polypeptide still retains the biological activity of the binding antibody.
Preferably, the functional domain of the tumor surface antigen is the fourth domain of the Her2 protein, and the sequence is shown in SEQ ID NO. 18.
In a particular embodiment of the invention, the fusion peptide of the invention has the sequence shown in SEQ ID NO. 20.
According to a further aspect of the invention there is provided a tumour marker system comprising a fusion peptide as hereinbefore described.
Further, the tumor marker system may further include Cyanine 5.5, Alexa Flour 750, Alexa Fluor 647, Alexa Flour 488, Alexa Flour 546,64Cu-DOTA、68Ga-DOTA、18F-O-pyridine、18F-liposomes、liposomal Rhodamine、Nanogold、TAMRA。
according to a further aspect of the present invention there is provided a targeted tumour therapy system comprising a tumour marker system as hereinbefore described.
Further, the targeted tumor therapy system may also include a tumor killing system comprising antibodies against tumor surface antigens.
The tumor killing system of the invention may be a CAR-T or TCR-T system, expressing antibodies or TCRs against tumor surface antigens by immune cells, such as T cells. The tumor killing system can also be an adc (antibody drug conjugates) system, i.e. antibody-conjugated toxins (pseudomonas aeruginosa exotoxin PE38, diphtheria toxin, duocarmycin, staphylococcus aureus enterotoxin a/E-120, shiga toxin, ricin toxin), chemotherapeutic drugs (irinotecan, adriamycin), small molecule inhibitors (auristatins, calicheamicins, maytansinoids, tubulisin, antibacterial drugs, urease), liposomes, gold nanoparticles, etc. Alternatively, the tumor killing system may be Immunocytokines, i.e., certain Immunocytokines are linked to antibodies, such as IL-2, IL-12, TNF- α, IL-10, TGF- β, and the like. Also included are bispecific antibody killing systems, i.e., one antibody recognizes the fusion peptide linked antigen or antigenic domain and the other antibody recognizes the other antigen.
The action principle of the targeted tumor therapy system of the invention is as follows: the fusion peptide in the tumor marker system is inserted into a cell membrane under the action of the low-pH insertion peptide, the Her2 fourth structural domain is displayed on the surface of a tumor cell, an antibody drug in the tumor killing system recognizes the surface antigen of the tumor cell, and the antigen and the antibody are combined, so that the tumor killing system is gathered in a tumor tissue, and the tumor cell is completely and specifically killed.
According to a further aspect of the invention, there is provided the use of a fusion peptide as hereinbefore described for the construction of a tumour marker system as hereinbefore described.
According to a further aspect of the invention, there is provided the use of a fusion peptide as hereinbefore described for the construction of a targeted tumour therapy system as hereinbefore described.
According to a further aspect of the present invention there is provided the use of a tumour marker system as hereinbefore described in the construction of a targeted tumour therapy system as hereinbefore described.
In detail, the targeted tumor therapy system may comprise two sub-systems, one being a tumor marker system comprising the fusion peptide of the present invention as described above, and the other being a tumor killing system comprising antibodies against tumor surface antigens.
The antibody against a tumor surface antigen of the present invention may be any antibody against a tumor surface antigen. The antibody includes monoclonal antibody and bispecific antibody.
The antibodies of the present invention directed to a tumor surface antigen also include antigen binding portions of antibodies directed to a tumor surface antigen, and further, antigen binding fragments of the antibodies include Fab, Fab ', F (ab') 2, Fv, or single chain antibodies.
Fab refers to the portion of an antibody molecule that contains one light chain variable and constant region and one heavy chain variable and constant region that are disulfide bonded.
Fab' refers to a Fab fragment that contains part of the hinge region.
F (ab ') 2 refers to a dimer of Fab'.
Fv refers to the smallest antibody fragment containing the variable regions of the antibody heavy and light chains and having all antigen binding sites.
The single-chain antibody refers to an engineered antibody formed by connecting a light chain variable region and a heavy chain variable region directly or through a peptide chain.
The antibodies of the invention also include variants of the antibodies, such as variants derived from similar amino acid substitutions, deletion of amino acids, addition of amino acids, as are well known in the art.
The antibodies of the invention directed to tumor surface antigens may comprise one or more glycosylation sites in the heavy and light chain variable regions, as is well known in the art, the presence of one or more glycosylation sites in the variable region may result in enhanced immunogenicity of the antibody, or alter the pharmacokinetics of the antibody due to altered antigen binding.
Antibodies of the invention directed to tumor surface antigens can be designed to include modifications in the Fc region, typically to alter 1 or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. In addition, the antibodies of the invention may be chemically modified (e.g., one or more chemical groups may be attached to the antibody), or modified to alter glycosylation thereof, thereby altering one or more functional properties of the antibody.
Another modification that the antibodies of the invention against tumor surface antigens can be designed to be is pegylation. The antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To PEGylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as an active ester or aldehyde derivative of polyethylene glycol, under conditions suitable for one or more PEG groups to be attached to the antibody or antibody fragment. Preferably, the pegylation is achieved by acylation or alkylation with a reactive PEG molecule (or similar reactive water-soluble polymer).
Examples of antibodies include, but are not limited to: molecule-targeted monoclonal antibody drugs, targeted antibody conjugate drugs, bispecific antibody drugs, targeted immune checkpoint drugs, and the like. Examples of such antibodies are: rituximab, trastuzumab, gemtuzumab, alemtuzumab, ibritumomab tiuxetan, tositumomab, bevacizumab, cetuximab, panitumumab, ofatumumab, dinomumab, ipilimumab, bentuximab, pertuzumab, ado-trastuzumab, atrozumab, ramucirumab, pembrolizumab, bonatuzumab, nivolumab, darumamab, dinumuzumab, rituximab, elotuzumab, alemtuzumab, avizumab, denosumab, Necitumumab, Atezolizumab, and Atezolizumab.
The term "CAR-T" is used herein to refer collectively to the Chimeric Antigen Receptor T-Cell Immunotherapy. Based on the characteristics of the tumor microenvironment, scientists have optimized a series of CART sequences with completely different affinities for antigen at different pH values, thereby activating at different pH values.
As used herein, "tumor surface antigen" refers broadly to antigenic material that is newly present or overexpressed on the cell surface during tumorigenesis, development, etc.
The term "targeted antibody conjugated drug" or immunoconjugate is used herein. The immune conjugate molecule consists of a monoclonal antibody and a warhead drug. There are three main classes of substances that can be used as "warheads", namely radionuclides, drugs and toxins; and the monoclonal antibody is connected with the monoclonal antibody to respectively form a radioimmunoconjugate, a chemoimmunoconjugate and an immunotoxin.
The term "bispecific antibody drug" as used herein refers to an antibody that binds to two epitopes simultaneously, and diabodies can be divided into two types, i.e., T cell recruiting, comprising a tumor cell target-T cell recruiting site, which accounts for a majority of the proportion of diabodies, wherein T cell recruiting site refers to CD3(T cells), CD16 target (NK cells), and target is normally located on tumor cells; in addition, double antibodies may bind to double target sites (such as VEGF-PDGF, VEGF-Ang2) and inhibit 2 signaling pathways, thereby reducing the possibility of drug resistance.
The invention has the following advantages and beneficial effects:
the fourth structural domain of a tumor surface antigen Her2 is connected with the low-pH insertion peptide for the first time to form the target tumor acid-sensitive fusion peptide capable of marking the tumor. The research result of the invention greatly expands the indication of the existing tumor medicament aiming at one kind of cancer or one kind of cancer specific typing, and has very important significance for clinically treating the tumor.
Drawings
FIG. 1 shows an electrophoretogram identifying a fusion peptide of the present invention expressed and purified using SDS-PAGE;
FIG. 2 shows a fluorescence image of the expression of Her2 protein in A549 cells using cofocal;
FIG. 3 shows a fluorescence image of the localization of the fusion peptide of the invention on A549 cells in neutral environment using cofocal;
FIG. 4 shows a fluorescence image of the localization of the fusion peptide of the present invention on A549 cells in an acidic environment using cofocal;
FIG. 5 is a graph showing the effect of Her2 fourth domain-pHLIP on tumor growth.
Detailed Description
The invention is further illustrated by the following examples. It should be understood that the examples of the present invention are for illustrative purposes and not intended to limit the present invention. Simple modifications of the invention in accordance with its spirit fall within the scope of the claimed invention.
EXAMPLE 1 Synthesis of fusion peptides
1. Prokaryotic expression
The method comprises the following steps:
strain construction
The DNA sequence designed according to the amino acid sequence (SEQ ID NO.20) of the fusion peptide is subjected to whole gene synthesis, the sequences of enzyme cutting sites Nde I and XhoI are connected at two ends during synthesis, the fusion peptide coding nucleic acid and the PET28a vector are respectively cut by the two enzymes, enzyme cutting fragments and the vector are recovered, T4 ligase is used for connection and transformation of a recipient bacterium BL21(DE3), the mixture is cultured in a spread plate mode, and the clone is selected and sequenced.
Culture induction
And (3) carrying out clone culture (LB culture medium, 37 ℃) with correct sequencing, inducing by adding IPTG when the OD value reaches 0.6-0.8, carrying out centrifugal strain collection after the final concentration of IPTG is 0.5mM and 4-6 hours.
2. Inclusion body purification
The method comprises the following steps:
(1) inclusion body washing
Solution A: 50mM Tris, 2mM EDTA, pH8.0, washing 2 times;
and B, liquid B: 50mM Tris, 2mM EDTA, 0.1% Triton, pH8.0, washing 1 time;
and C, liquid C: 20mM Tris, 1M urea, pH8.0, 1 wash.
(2) Renaturation, nickel column purification
Extraction: the inclusion bodies were extracted with 8M urea, 5mM β -Me, 0.3M NaCl, 20mM Tris, pH8.0, extraction ratio 1: 20.
renaturation and dialysis: the target protein was supplemented with 10mM β -Me, reduced at 40 ℃ for 15 minutes, diluted to 0.2mg/ml with 10mM PB, 50. mu.M CuCl2The sample was dialyzed at pH8.0, the dialysate was changed 3 times, and the supernatant was collected by centrifugation.
And (3) Ni column purification: the column was equilibrated with 0.3M NaCl, 10mM PB, pH8.0, and after loading, the column was washed with an equilibration buffer containing 40mM imidazole, and the target protein was eluted with an equilibration buffer containing 300mM imidazole. The purity of the target protein is more than 95%.
Desalting: desalted to 20mM PB, 0.1M NaCl.
(3)SDS-PAGE
And (3) carrying out SDS-PAGE on the sample treated in the step (2). The results are shown in FIG. 1, and the present experiment enables the isolation and purification of the fusion peptide of the present invention, note: in FIG. 1, lane 1: renaturation of GJ inclusion bodies; lane 2: sample loading and flowing; lanes 3 and 4: balancing the buffer solution; lane 5: elution with 40mM imidazole; lane 6: 300mM imidazole; 7. and (5) Marker.
Example 2 localization of fusion peptides on tumor cells cultured in vitro
1. Cell culture
A549 cells were cultured in DMEM medium containing 10% calf serum and 160 ten thousand units of gentamicin/ml at 37 ℃ in 5% CO2In a cell culture incubatorAnd (5) nourishing. After the cells were confluent, the cells were passaged at a ratio of 1: 10.
2. confocal Observation localization
A549 cells (5x 10)5Per well) was cultured on a coverslip dish overnight, the culture supernatant was discarded and PBS of pH6.3 and 7.4 was added, respectively, the fusion peptide (60. mu.g/ml) expressed in example 1 was added, incubated at 37 ℃ for 1 hour, the supernatant was discarded and washed 3 times with PBS of the corresponding pH, PBS of pH6.3 and 7.4 was added, trastuzumab-FITC or TGLA-FITC (control antibody) (concentrations were 1: 400 dilution) was incubated at 37 ℃ for 30 minutes, the supernatant was discarded and washed 3 times with PBS of the corresponding pH, PBS of pH7.4 was added, and confocal was observed. Grouping: (1) untreated group ph 6.3; (2) pH6.3 fusion peptide (her2 fourth domain-pHLIP); (3) pH6.3 fusion peptide (her2 fourth domain-pHLIP) + trastuzumab-FITC; (4) pH6.3 fusion peptide (her2 fourth domain-pHLIP) + TGLA-FITC; (5) pH7.4 fusion peptide (her2 fourth domain-pHLIP) + trastuzumab-FITC; (6) (iii) Trastuzumab-FITC at pH 7.4.
3. Results
Figure 2 shows that human lung cancer cell line a549 does not express Her 2.
FIG. 3 shows that, in a neutral solution environment, the fusion peptide of the present invention cannot be inserted into the cell membrane of A549, and cannot display the Her2 fourth domain on the cell membrane.
Fig. 4 shows that, in an acidic solution environment, the fusion peptide of the present invention can be inserted into the cell membrane of a549, and the Her2 fourth domain displayed on the cell membrane can be recognized by trastuzumab.
The above results indicate that the low pH insertion peptide linked to the fourth domain of Her2 does not affect its cell membrane insertion properties, while the Her2 fourth domain linked to the low pH insertion peptide does not affect its conformation.
Example 3 evaluation of the Effect of the fourth Domain of Her 2-pHLIP and herceptin on tumor treatment
Experimental materials: a549 cells, purchased from ATCC; the fourth domain of Her2, pHLIP (Her 2D 4-pHLIP), is prokaryotic; herceptin purchased from roche; male nude mice at 6-8 weeks of age were purchased from Witonglihua.
The experimental steps are as follows:
a549 inoculating to nude mouse, and treating tumor with diameter of up toAt 1cm, aseptically stripping tumor, shearing, homogenizing, filtering to obtain single cell suspension, culturing and amplifying in 1640 complete culture medium, injecting cells subcutaneously into lateral abdomen of nude mouse, 1x106And (3) removing oversize and undersize tumors from each cell/mouse when the diameter of the tumor is 0.5-1cm, and grouping mice with the tumors basically consistent in size. And (4) components in total: her2D4-pHLIP single injection group, 10; her2D4-pHLIP combined herceptin injection group, 10; 10 groups of Her2D4-pHLIP combined IgG1 were injected; saline N.S was injected into groups, 10. Tumor size was measured every 3 days.
The administration method comprises the following steps: her2D4-pHLIP administration: injecting each vein with 40 μ M/100 μ l, starting from the day after grouping, and injecting once a day and once every 2 days; antibody administration: intraperitoneal injection is carried out at the dose of 10mg/kg, the injection frequency is the same as Her2D4-pHLIP, and the injection time is 6-12 hours after the administration of Her2D4-pHLIP until the end.
And (4) analyzing results:
the results in figure 5 show that Herceptin significantly inhibits tumor growth in lung cancer mice.
Although only specific embodiments of the present invention have been described above, it will be understood by those skilled in the art that these are by way of illustration only, and that the scope of the invention is defined by the appended claims. Various changes or modifications to these embodiments may be made by those skilled in the art without departing from the principle and spirit of the invention, and these changes or modifications are within the scope of the invention.
Sequence listing
<110> Beijing Zeqin biopharmaceutical Co., Ltd
<120> tumor-targeting acid-sensitive fusion peptide and application thereof
<150>2017114647646
<151>2017-12-28
<160>20
<170>SIPOSequenceListing 1.0
<210>1
<211>36
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>1
Ala Cys Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu
1 5 10 15
Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala
20 25 30
Asp Glu Gly Thr
35
<210>2
<211>32
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>2
Ala Cys Glu Asp Gln Asn Pro Tyr Trp Ala Arg Tyr Ala Asp Trp Leu
1 5 10 15
Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Gly
20 25 30
<210>3
<211>30
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>3
Ala Cys Glu Asp Gln Asn Pro Tyr Trp Arg Ala Tyr Ala Asp Leu Phe
1 5 10 15
Thr Pro Leu Thr Leu Leu Asp Leu Leu Ala Leu Trp Asp Gly
20 25 30
<210>4
<211>27
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>4
Ala Cys Asp Asp Gln Asn Pro Trp Arg Ala Tyr Leu Asp Leu Leu Phe
1 5 10 15
Pro Thr Asp Thr Leu Leu Leu Asp Leu Leu Trp
20 25
<210>5
<211>27
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>5
Ala Cys Glu Glu Gln Asn Pro Trp Arg Ala Tyr Leu Glu Leu Leu Phe
1 5 10 15
Pro Thr Glu Thr Leu Leu Leu Glu Leu Leu Trp
20 25
<210>6
<211>25
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>6
Ala Cys Asp Asp Gln Asn Pro Trp Ala Arg Tyr Leu Asp Trp Leu Phe
1 5 10 15
Pro Thr Asp Thr Leu Leu Leu Asp Leu
20 25
<210>7
<211>24
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>7
Cys Asp Asn Asn Asn Pro Trp Arg Ala Tyr Leu Asp Leu Leu Phe Pro
1 5 10 15
Thr Asp Thr Leu Leu Leu Asp Trp
20
<210>8
<211>25
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>8
Ala Cys Glu Glu Gln Asn Pro Trp Ala Arg Tyr Leu Glu Trp Leu Phe
1 5 10 15
Pro Thr Glu Thr Leu Leu Leu Glu Leu
20 25
<210>9
<211>24
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>9
Cys Glu Glu Gln Gln Pro Trp Ala Gln Tyr Leu Glu Leu Leu Phe Pro
1 5 10 15
Thr Glu Thr Leu Leu Leu Glu Trp
20
<210>10
<211>24
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>10
Cys Glu Glu Gln Gln Pro Trp Arg Ala Tyr Leu Glu Leu Leu Phe Pro
1 5 10 15
Thr Glu Thr Leu Leu Leu Glu Trp
20
<210>11
<211>24
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>11
Ala Cys Glu Asp Gln Asn Pro Trp Ala Arg Tyr Ala Asp Trp Leu Phe
1 5 10 15
Pro Thr Thr Leu Leu Leu Leu Asp
20
<210>12
<211>24
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>12
Ala Cys Glu Glu Gln Asn Pro Trp Ala Arg Tyr Ala Glu Trp Leu Phe
1 5 10 15
Pro Thr Thr Leu Leu Leu Leu Glu
20
<210>13
<211>22
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>13
Ala Cys Glu Asp Gln Asn Pro Trp Ala Arg Tyr Ala Asp Leu Leu Phe
1 5 10 15
Pro Thr Thr Leu Ala Trp
20
<210>14
<211>22
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>14
Ala Cys Glu Glu Gln Asn Pro Trp Ala Arg Tyr Ala Glu Leu Leu Phe
1 5 10 15
Pro Thr Thr Leu Ala Trp
20
<210>15
<211>34
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>15
Thr Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu Pro
1 5 10 15
Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Pro Asn Gln Glu
20 25 30
Cys Ala
<210>16
<211>37
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>16
Cys Asp Asp Asp Asp Asp Asn Pro Asn Tyr Trp Ala Arg Tyr Ala Asn
1 5 10 15
Trp Leu Phe Thr Thr Pro Leu Leu Leu Leu Asn Gly Ala Leu Leu Val
20 25 30
Glu Ala Glu Glu Thr
35
<210>17
<211>37
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>17
Cys Asp Asp Asp Asp Asp Asn Pro Asn Tyr Trp Ala Arg Tyr Ala Pro
1 5 10 15
Trp Leu Phe Thr Thr Pro Leu Leu Leu Leu Pro Gly Ala Leu Leu Val
20 25 30
Glu Ala Glu Glu Thr
35
<210>18
<211>138
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>18
His His His His His His Pro His Gln Ala Leu Leu His Thr Ala Asn
1 5 10 15
Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His Gln Leu
20 25 30
Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val Asn
35 40 45
Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg Val
50 55 60
Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro
65 70 75 80
Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly
85 90 95
Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp Pro Pro
100 105 110
Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr
115 120 125
Met Pro Ile Trp Lys Phe Pro Asp Glu Glu
130 135
<210>19
<211>5
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>19
Gly Gly Gly Gly Ser
1 5
<210>20
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>20
His His His His His His Pro His Gln Ala Leu Leu His Thr Ala Asn
1 5 10 15
Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His Gln Leu
20 25 30
Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val Asn
35 40 45
Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg Val
50 55 60
Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro
65 70 75 80
Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly
85 90 95
ProGlu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp Pro Pro
100 105 110
Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr
115 120 125
Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Gly Gly Gly Ser Ala
130 135 140
Cys Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu Phe
145 150 155 160
Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala Asp
165 170 175
Glu Gly
Claims (7)
1. An acid-sensitive fusion peptide targeted to tumors, which consists of a low-pH insertion peptide, a Linker and a Her2 protein fourth domain, wherein the Her2 protein fourth domain is connected to the N-terminal of the low-pH insertion peptide through the Linker; the sequence of the low-pH insertion peptide is shown as SEQ ID NO.1, the sequence of the Linker is GGGGS, and the sequence of the fourth structural domain of the Her2 protein is shown as SEQ ID NO. 18.
2. A tumor labeling agent comprising the fusion peptide of claim 1.
3. A targeted tumor therapy agent comprising the tumor labeling agent of claim 2.
4. The targeted tumor therapy agent according to claim 3, wherein the targeted tumor therapy agent further comprises a tumor killing agent.
5. The targeted tumor therapy agent according to claim 4, wherein said tumor killing agent comprises an antibody that binds to the fourth domain of Her2 protein.
6. Use of the fusion peptide of claim 1 for the preparation of a tumor labeling reagent of claim 2.
7. Use of the fusion peptide of claim 1 or the tumor labeling agent of claim 2 in the preparation of the targeted tumor therapy agent of claim 3.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711464764 | 2017-12-28 | ||
| CN2017114647646 | 2017-12-28 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109467607A CN109467607A (en) | 2019-03-15 |
| CN109467607B true CN109467607B (en) | 2020-10-30 |
Family
ID=65674586
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811459427.2A Active CN109467607B (en) | 2017-12-28 | 2018-11-30 | Acid-sensitive fusion peptide targeting tumor and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109467607B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109517073A (en) * | 2018-11-30 | 2019-03-26 | 北京泽勤生物医药有限公司 | A kind of fusogenic peptide of targeting therapy on tumor and its application |
| CN111285936A (en) * | 2020-03-11 | 2020-06-16 | 北京双赢科创生物科技有限公司 | Acid sensitive nano peptide segment of targeted tumor and application thereof |
| WO2021208106A1 (en) * | 2020-04-18 | 2021-10-21 | 北京泽勤生物医药有限公司 | Fusion peptide for treating autoimmune disease |
| CN111454371B (en) * | 2020-04-18 | 2023-01-06 | 北京泽勤生物医药有限公司 | PDL1-pHLIP, preparation method and application thereof in treatment of autoimmune disease |
| CN114716566A (en) * | 2022-02-22 | 2022-07-08 | 广东粤港澳大湾区国家纳米科技创新研究院 | Fusion protein and application thereof in preparing tumor medicine |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0209893D0 (en) * | 2002-04-30 | 2002-06-05 | Molmed Spa | Conjugate |
| WO2012047354A2 (en) * | 2010-07-13 | 2012-04-12 | Rhode Island Board Of Governors For Higher Education | Environmentally sensitive compositions |
| EP2603201B1 (en) * | 2010-08-13 | 2020-03-11 | Rhode Island Board of Governors for Higher Education | Liposome compositions and methods of use thereof |
| CN104045717B (en) * | 2014-07-08 | 2016-07-06 | 北京华安科创生物技术有限公司 | A kind of tumor vessel blocker polypeptide, gene, expression vector and application thereof |
| CA2965952A1 (en) * | 2014-10-27 | 2016-05-06 | University Of Central Florida Research Foundation, Inc. | Methods and compositions for natural killer cells |
-
2018
- 2018-11-30 CN CN201811459427.2A patent/CN109467607B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN109467607A (en) | 2019-03-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110713548B (en) | Fusion peptide for targeted therapy of tumors and application thereof | |
| CN109467607B (en) | Acid-sensitive fusion peptide targeting tumor and application thereof | |
| JP6780021B2 (en) | Anti-CD47 monoclonal antibody and its applications | |
| Kutova et al. | Targeted delivery to tumors: multidirectional strategies to improve treatment efficiency | |
| ES2889906T3 (en) | Trispecific binding proteins and medical uses | |
| Pastan et al. | Immunotoxin therapy of cancer | |
| US10106609B2 (en) | CD7 nanobodies, encoding sequence and use thereof | |
| FitzGerald et al. | Recombinant immunotoxins for treating cancer | |
| CN110494443A (en) | The sub- polypeptide of shiga toxin A Effect of subunit, shiga toxin effect submounts and cell-targeting molecule for locus specificity conjugation | |
| Shan et al. | Recombinant immunotoxin therapy of solid tumors: challenges and strategies | |
| CA2973538C (en) | Antibody-urease conjugates for therapeutic purposes | |
| CN102250245A (en) | Bispecific antibody capable of resisting B cell lymphoma and application thereof | |
| CN110790829B (en) | Application of antibody prepared by using pHLIP extracellular segment as antigen in preparation of antitumor drugs | |
| US20120195895A1 (en) | Fusion Protein of an Anti-CD20 Antibody Fab Fragment and Lidamycin, a Method for Preparing the Same, and the Use Thereof | |
| Guo et al. | HER2-targeted immunotoxins with low nonspecific toxicity and immunogenicity | |
| Kuo et al. | Antibody-drug conjugates with HER2-targeting antibodies from synthetic antibody libraries are highly potent against HER2-positive human gastric tumor in xenograft models | |
| CN101475643B (en) | Double single chain antibody strengthened fusion protein dFv-LDP-AE, preparation and use thereof | |
| US20210214399A1 (en) | Ph low insertion peptide and composition thereof | |
| JP7179855B2 (en) | Low pH insertion peptides and compositions thereof | |
| EP2414389A1 (en) | Tumor targeting peptides, therapeutic and diagnostic compositions comprising the peptides | |
| CN115724988A (en) | Polypeptide fusion molecule close to natural molecule | |
| CN109879964A (en) | Anti-EGFR single-chain antibody, anti-PD1 single-chain antibody and fusion protein | |
| Kreitman | Recombinant fusion toxins for cancer treatment | |
| CN101139399B (en) | Fused protein restructuring target lethal leukemia cells and preparation method and use thereof | |
| CN106519018A (en) | High-affinity T cell receptor targeted to RHAMM antigen short peptide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Wei Huawei Inventor after: Yang Chenggang Inventor before: Wang Ying Inventor before: Wei Huawei Inventor before: Yang Chenggang |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |