CN110713548B - Fusion peptide for targeted therapy of tumors and application thereof - Google Patents
Fusion peptide for targeted therapy of tumors and application thereof Download PDFInfo
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Abstract
The invention discloses a fusion peptide for targeted therapy of tumors and application thereof. The fusion peptide is formed by connecting a low-pH insertion peptide with a second structural domain of a tumor surface antigen Her2 through a Linker. The fusion peptide can be inserted into a tumor cell membrane in an acidic environment and displays a second structural domain of Her2 on the cell surface, and is successfully recognized by trastuzumab. The achievement of the invention enlarges the indication of a single tumor medicament and realizes the treatment effect of the single anti-tumor medicament on all cancers.
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 of 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 the 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 the 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) was 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. The research finds that the pH value inside and outside the tumor cell is detected by a microelectrode, a magnetic resonance spectrum and other methods, 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 based on the following idea: 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 invention connects partial fragments of tumor surface antigen HER2 with low pH insertion peptide of targeted tumor to form fusion peptide, the fusion peptide can target any solid tumor cell and display on the surface of the tumor cell, and can kill any cancer cell including breast cancer cell aiming at HER2 medicine such as pertuzumab, thus expanding the application range of the tumor medicine.
One of the purposes of the present invention is to provide a fusion peptide for targeted therapy of 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 a fusion peptide for targeted therapy of tumor 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-Var16.
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 = a natural number.
In a specific embodiment of the invention, the Linker sequence is GGGGS (SEQ ID NO. 18).
In a particular embodiment of the invention, the tumor surface antigen is Her2.
In a particular embodiment of the invention, the functional domain of the tumor surface antigen is the Her2 protein second domain or a functionally similar domain thereof, said functionally similar domain of the Her2 protein second domain retaining the activity of said Her2 protein second domain binding antibody.
Furthermore, the sequence of the second domain of the Her2 protein which can be used for the invention is shown as SEQ ID NO. 19.
The functional similarity structural domain of the second structural domain of the Her2 protein comprises a polypeptide which is derived from the amino acid sequence shown in SEQ ID NO.19, has the same function with the sequence shown in SEQ ID NO.19, and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the second structural domain of the Her2 protein with the sequence shown in SEQ ID NO. 19.
The functionally similar domain of the second domain of Her2 protein includes polypeptides consisting of amino acid sequences having at least 80% homology (also referred to as sequence identity) with the amino acid sequence shown in SEQ ID NO.19, more preferably at least about 90% to 95% homology with the amino acid sequence shown in SEQ ID NO.19, 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.
Functionally similar domains of the second domain of the Her2 protein also include non-conservative modifications of the amino acid sequence shown in SEQ ID No.19, 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 second domain of the Her2 protein, and the sequence is shown in SEQ ID NO. 19.
In a specific embodiment of the invention, a fusion peptide sequence constructed by the second domain of the Her2 protein and the low-pH insertion peptide is shown in SEQ ID NO. 20-22.
According to a further aspect of the invention, there is provided a pharmaceutical combination comprising a fusion peptide as hereinbefore described.
Preferably, the pharmaceutical combination further comprises an anti-tumor drug targeting the aforementioned tumor surface antigen or functional domain thereof; more preferably, the anti-tumor drug comprises an antibody to a tumor surface antigen.
Although in the specific embodiment of the present invention, the antibody used is pertuzumab, it is known to those skilled in the art that the specific embodiment is only pertuzumab as an example, and it is demonstrated that an antibody drug recognizing and binding to the second domain of HER2 having a sequence shown in SEQ ID No.19 can exert an anti-tumor effect together with the fusion peptide of the present invention, and thus an antibody drug recognizing and binding to the second domain of HER2 having a sequence shown in SEQ ID No.19 other than pertuzumab can also exert an anti-tumor effect.
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, 64 Cu-DOTA、 68 Ga-DOTA、 18 F-O-pyridine、 18 F-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 (class Li Tating, 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 marking system is inserted into a cell membrane under the action of the low pH insertion peptide, the tumor surface antigen or the functional structural domain thereof is on the surface of the tumor cell, the antibody drug in the tumor killing system recognizes the tumor cell surface antigen or the functional structural domain thereof, and the antigen and the antibody are combined, so that the tumor killing system is gathered in the tumor tissue, and the tumor cell is completely and specifically killed.
According to a further aspect of the present invention, there is provided the use of the fusion peptide as described above in the preparation of an anti-tumor medicament.
Preferably, the antineoplastic drug comprises a pharmaceutical composition as previously 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 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 particular, the targeted tumor therapy system may comprise two subsystems, one being a tumor marker system comprising the fusion peptide of the 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, pembromumab, bannatuzumab, nivolumab, daratumumab, dinumuzumab, alemtuzumab, erlotintuzumab Zhu Shankang, avizumab, denosumab, gimumab, necitumumab, atezumab ozolizumab, 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 the diabodies can be divided into two types, i.e., T cell recruiting types, comprising tumor cell target-T cell recruiting sites, which are the majority of the proportion of diabodies, wherein T cell recruiting sites refer 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 and VEGF-Ang 2) and inhibit 2 signal paths, thereby reducing the possibility of drug resistance.
The sequences of the invention are listed in order from the N-terminus to the C-terminus.
The invention has the following advantages and beneficial effects:
the invention connects the second structural domain of the tumor surface antigen Her2 with the low pH insertion peptide for the first time to form the target tumor acid-sensitive fusion peptide which can mark 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 fluorescence image of Her2D2-WT localization on breast cancer cells using cofocal;
FIG. 2 fluorescence image of Her2D2-var7 localization on breast cancer cells using cofocal;
FIG. 3 fluorescence mapping of Her2D2-var3 localization on breast cancer cells using cofocal;
fig. 4 shows a graph of the effect of Her2 second domain-pHLIP in combination with Her2 mab pertuzumab on tumor cell growth, where a: her2D2-WT, B: her2D2-var7, C: her2D2-var3;
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
Designing a DNA sequence for whole gene synthesis according to an amino acid sequence SEQ ID NO.20 of a fusion peptide (Her 2D 2-WT) formed by the Her2 second domain and a wild-type low-pH insertion peptide, an amino acid sequence SEQ ID NO.21 of a fusion peptide (Her 2D2-var 7) formed by the Her2 second domain and a variant 7 of the wild-type low-pH insertion peptide, an amino acid sequence SEQ ID NO.22 of a fusion peptide (Her 2D2-var 3) formed by the Her2 second domain and a variant 3 of the wild-type low-pH insertion peptide, connecting enzyme cutting sites Nde I and XhoI sequences at two ends during synthesis, respectively cutting the fusion peptide coding nucleic acid and a PET28a vector by using the two enzymes, recovering a cut fragment and the vector, connecting by using T4 ligase, transforming a recipient bacterium BL21 (DE 3), carrying out flat plate culture, taking clones and sending the clones for sequencing.
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: 2mM Tris,1M urea, pH8.0, 1 wash.
(2) Renaturation, nickel column purification
Extraction: the inclusion bodies were extracted with 8M urea, 5mM beta-Me, 0.3M NaCl, 2mM Tris, pH8.0, at a ratio of 1:20.
renaturation and dialysis: the target protein was supplemented with 10 mM. Beta. -Me, reduced at 40 ℃ for 15 minutes, diluted to 0.2mg/ml with 10mM PB, 50. Mu.M CuCl 2 The 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 into 20mM PB,0.1M NaCl.
Example 2 localization of fusion peptides on tumor cells cultured in vitro
1. Cell culture
MBA-MD-231 cells were subjected to 5% CO at 37 ℃ in DMEM medium containing 10% calf serum and 160 ten thousand units gentamicin/ml 2 Cultured in a cell culture box. After cell confluence, passage was performed as 1.
2. confocal Observation localization
MBA-MD-231 cells (5X 10) 5 Per 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 expressed in example 1 (60. Mu.g/ml) was added, incubation was carried out 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, and pertuzumab-PE or IgG-PE (control antibody) (both at 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 (Her 2D2-pHLIP (WT/var 7/var 3)) + IgG-PE; (3) pH6.3 fusion peptide (Her 2D2-pHLIP (WT/var 7/var 3)) + pertuzumab-PE; (4) pH7.4 fusion peptide (Her 2D2-pHLIP (WT/var 7/var 3)) + pertuzumab-PE.
3. Results
The results are shown in FIGS. 1-3: MBA-MD-231 does not express Her2; in a neutral solution environment, the fusion peptide of the invention cannot be inserted into the cell membrane of MBA-MD-231 and cannot display the Her2 second domain on the cell membrane; in an acidic solution environment, the fusion peptide of the invention can be inserted into the cell membrane of MBA-MD-231, and the Her2 second domain displayed on the cell membrane can be recognized by pertuzumab.
The above results indicate that the low pH insertion peptide linked to Her2 second domain does not affect its low pH insertion peptide insertion into cell membranes, while the Her2 second domain linked to the low pH insertion peptide does not affect its conformation.
Example 3 evaluation of the Effect of fusion peptide in combination with antibody drug on tumor treatment
1. Reagent material
4T1 cells (purchased from ATCC); 6-8 week old female Balb/c mice (Witonglihua); pertuzumab was purchased from roche.
2. Step (ii) of
4T1 cells seeded in mouse mammary glands, 2X10 6 Individual cells/individual, 50 μ l volume.
When the tumor grows to 50mm 3 And treatment is started. The method comprises the following four groups: her2D2-pHLIP ((WT/var 7/var 3))/pertuzumab group (10), pertuzumab group (5), her2D2-pHLIP ((WT/var 7/var 3)) group (5), PBS group (10). The administration method comprises the following steps: her2D2-pHLIP ((WT/var 7/var 3)), 1mg/kg, administered intravenously every other day; pertuzumab, 30mg/kg, i.p. injection, 2 times a week. Tumor volume and mouse body weight were measured every 3 days.
The tumor volume of the control group is up to 1000mm 3 When so, the administration is terminated.
3. Results
The results are shown in fig. 4, and the her2 second domain-pHLIP combined with pertuzumab can obviously inhibit the growth of tumor cells.
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
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Claims (7)
1. The fusion peptide for targeted therapy of tumors is formed by connecting a low-pH insertion peptide and a Her2 protein second domain; the Her2 protein second domain is linked to the N-terminus of the low pH insertion peptide by Linker; the sequence of the second structural domain of the Her2 protein is shown as SEQ ID NO. 19; the sequence of the low pH insertion peptide is shown in SEQ ID NO.1, SEQ ID NO.4 and SEQ ID NO. 8.
2. The fusion peptide of claim 1, wherein the Linker sequence is (GGGS) m or (GGGGS) m, wherein m = a natural number.
3. The fusion peptide of claim 2, wherein the Linker sequence is GGGGS.
4. A tumor marker system comprising the fusion peptide of any one of claims 1-3.
5. A targeted tumor therapy system comprising the tumor marker system of claim 4 and a tumor killing system; the tumor killing system comprising an antibody to the Her2 protein second domain in the fusion peptide of any one of claims 1-3, which antibody is pertuzumab.
6. The use of a fusion peptide according to any of claims 1 to 3, comprising the use of any of:
(1) Use in the preparation of a tumor marker system according to claim 4;
(2) Use in the preparation of a targeted tumor therapy system according to claim 5.
7. Use of a tumor marker system according to claim 4 for the preparation of a targeted tumor therapy system according to claim 5.
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| CN201911208044.2A Pending CN110698565A (en) | 2018-11-30 | 2019-11-30 | Tumor-targeting CD38-pHLIP fusion peptides |
| CN201911207888.5A Active CN110713548B (en) | 2018-11-30 | 2019-11-30 | Fusion peptide for targeted therapy of tumors and application thereof |
| CN201911207884.7A Active CN110713547B (en) | 2018-11-30 | 2019-11-30 | Application of CD 47-low pH insertion peptide in marking or treating tumor |
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| 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 |
| CN112300264A (en) * | 2020-11-09 | 2021-02-02 | 青岛大学附属医院 | Synthetic labeling method of novel radioactive labeling anticancer drug |
| CN113372412B (en) * | 2021-04-09 | 2022-10-11 | 上海交通大学医学院附属第九人民医院 | Cell-targeted polypeptide for treating bone tumor and preparation method and application thereof |
| CN114149510B (en) * | 2021-10-29 | 2024-01-30 | 上海鑫湾生物科技有限公司 | Condition-controlled splicing chimeric antigen receptor molecule and application thereof |
| CN114181319A (en) * | 2021-11-26 | 2022-03-15 | 北京双赢科创生物科技有限公司 | Polypeptide conjugate for targeting tumor cells and preparation method and application thereof |
| 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 |
| CN115043907A (en) * | 2022-06-02 | 2022-09-13 | 中山大学 | Parent peptide or derived peptide with CD47/SIRP alpha blocking effect and application thereof |
| CN116769717B (en) * | 2023-04-18 | 2024-06-11 | 河南中医药大学第一附属医院 | Targeted exosome, and preparation method and application thereof |
| CN116676265B (en) * | 2023-04-18 | 2024-05-24 | 河南中医药大学第一附属医院 | Acid-sensitive fusion peptide targeted exosome and preparation method and application thereof |
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