CN110713547A - Application of CD 47-low pH insertion peptide in marking or treating tumor - Google Patents
Application of CD 47-low pH insertion peptide in marking or treating tumor Download PDFInfo
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- 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
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- 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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- 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 application of CD 47-low pH insertion peptide in marking or treating tumor. The CD 47-low pH insertion peptide can mark tumor cells which do not express or express CD47 on the cell surface, and simultaneously, the CD47 antibody medicine is applied to ensure that the tumor cells marked with the CD 47-low pH insertion peptide are attacked by immune cells so as to achieve the aim of eliminating the tumor cells. The invention provides a novel tumor cell marker and a treatment method, and has wide clinical application prospect.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to application of a tumor-targeted CD 47-low pH insertion peptide in marking or treating 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 invention connects partial fragment of tumor surface antigen CD47 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, the antibody drug aiming at CD47 can kill any cancer cell including breast cancer cell, and the application range of the tumor drug is enlarged.
One of the objectives of the present invention is to provide a fusion peptide of CD47 and a low pH insertion peptide.
The second object of the present invention is to provide an antitumor agent comprising the 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 of a low pH insertion peptide, the fusion peptide comprising the 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, PD-L2, CD 47.
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.
Preferably, the Linker sequence is GGGS (SEQ ID NO. 18).
In a specific embodiment of the invention, the tumor surface antigen used is CD47, and linked to the low pH insertion peptide sequence is the functional domain of CD47, the functional domain sequence of CD47 is shown in SEQ ID No. 19. The polypeptide derived from the amino acid sequence shown in SEQ ID NO.19, wherein the amino acid sequence shown in SEQ ID NO.19 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, has the same function as the sequence shown in SEQ ID NO.19, also belongs to the functional domain of CD47, namely the functional domain of CD47 of the invention comprises wild type and variants thereof.
The functional domain variant of CD47 has at least 80% homology (also called sequence identity) with the amino acid sequence shown in SEQ ID NO.19, more preferably at least about 90% to 95% homology, often 96%, 97%, 98%, 99% homology with the amino acid sequence shown in SEQ ID NO. 19.
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.
Functional domain variants of CD47 also include non-conservative modifications to 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.
Fusion peptides constructed using the functional domain of CD47 with a low pH insertion peptide were as follows: the fusion peptide constructed by the functional domain of CD47 and the wild-type low pH insertion peptide is represented as CD 47-WT; the fusion peptide constructed from the functional domain of CD47 and variant 7 of the wild-type low pH insertion peptide is denoted CD47-var 7; the fusion peptide constructed from the functional domain of CD47 and variant 3 of the wild-type low pH insertion peptide is designated CD47-var 3.
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 embodiment of the present invention, the antibody used is 5F9, it is known to those skilled in the art that the embodiment is only 5F9 as an example, and it was confirmed that an antibody drug recognizing and binding to the domain of CD47 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 therefore an antibody drug recognizing and binding to the domain of CD47 having a sequence shown in SEQ ID No.19 other than 5F9 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 546,64Cu-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 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 for the preparation of an anti-tumor medicament.
Preferably, the anti-tumor drug comprises the pharmaceutical composition as described above.
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, alemtuzumab, rituximab, erlotuzumab, alemtuzumab, denosumimab, Necitumumab, Atezolizumab, 5F 9.
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 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 partial structural domain of tumor surface antigen CD47 with low pH insertion peptide for the first time, and forms targeting tumor acid sensitive fusion peptide which can mark 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 CD47-WT fusion peptide localization on breast cancer cells using cofocal;
FIG. 2 fluorescence image of CD47-var7 fusion peptide localization on breast cancer cells using cofocal;
FIG. 3 fluorescence view of CD47-var3 fusion peptide localization on breast cancer cells using cofocal;
FIG. 4 is a graph showing the effect of CD47-pHLIP in combination with CD47 monoclonal antibody 5F9 on tumor cell growth, wherein A: CD47-WT, B: CD47-var 7; c: CD47-var 3.
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
The fusion peptide prepared from the functional domain of CD47 and wild-type pHLIP is represented by CD47-WT in the present invention, the fusion peptide prepared from the functional domain of CD47 and wild-type pHLIP variant 7 is represented by CD47-var7 in the present invention, and the fusion peptide prepared from the functional domain of CD47 and wild-type pHLIP variant 3 is represented by CD47-var3 in the present invention. CD47-WT (SEQ ID NO.20), CD47-var7(SEQ ID NO.21), and CD47-var3(SEQ ID NO.22) (DMSO solubilized, synthesized by Hangzhou peptide Biochemical Co., Ltd.).
Example 2 localization of fusion peptides on tumor cells cultured in vitro
1. Cell culture
MBA-MD-231 cells were cultured in DMEM medium containing 10% calf serum and 160 ten thousand units gentamicin/ml at 37 ℃ in 5% CO2Cultured in a cell culture box. After the cells were confluent, the cells were passaged at a ratio of 1: 10.
2. confocal Observation localization
MBA-MD-231 cells (5x 10)5/well) was cultured overnight on coverslip dishes and discardedPBS of pH6.3 and 7.4 was added to the culture supernatant, respectively, the fusion peptide (60. mu.g/ml) expressed in example 1 was added thereto, the mixture was 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 thereto, 5F9-PE or IgG-PE (control antibody) (both at a concentration of 1: 400 dilution) was added thereto, the mixture 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 thereto, and they were observed by confocal. Grouping: (1) untreated group ph 6.3; (2) pH6.3 fusion peptide (CD47-pHLIP (WT/var7/var3)) + IgG-PE; (3) pH6.3 fusion peptide (CD47-pHLIP (WT/var7/var3)) +5F 9-PE; (4) pH7.4 fusion peptide (CD47-pHLIP (WT/var7/var3)) +5F 9-PE.
As a result:
the results are shown in FIGS. 1-3, and MBA-MD-231 does not express CD 47. In a neutral solution environment, CD47-WT, CD47-var7 and CD47-var3 cannot insert into a cell membrane and cannot display a CD47 domain on the cell membrane. In an acidic solution environment, CD47-WT, CD47-var7 and CD47-var3 can be inserted into a cell membrane, and a CD47 domain displayed on the cell membrane can be recognized by 5F 9. The above results indicate that the domain of CD47 does not affect the insertion of its low pH insertion peptide into the cell membrane, while the domain of CD47 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); 5F9 (Beijing Xin constant biotechnology Co., Ltd gift)
2. Step (ii) of
(1)CD47-WT
4T1 cells inoculated in mouse mammary glands, 2X106Individual cells/individual, 50 μ l volume.
When the tumor grows to 50mm3And treatment is started. The method comprises the following four groups: CD47-WT/5F9 group (10), 5F9 group (5), CD47-WT (5), PBS group (10). The administration method comprises the following steps: CD47-WT, 1mg/kg, was administered intravenously every other day; 5F9, 30mg/kg, i.p., 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 1000mm3When so, the administration is terminated.
(2)CD47-var3
4T1 cells inoculated in mouse mammary glands, 2X106Individual cells/individual, 50 μ l volume.
When the tumor grows to 50mm3And treatment is started. The method comprises the following four groups: CD47-var3/5F9 group (10), 5F9 group (5), CD47-var3 (5), PBS group (10). The administration method comprises the following steps: CD47-var3, 1mg/kg, intravenous injection, every other day administration; 5F9, 30mg/kg, i.p., 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 1000mm3When so, the administration is terminated.
(3)CD47-var7
4T1 cells inoculated in mouse mammary glands, 2X106Individual cells/individual, 50 μ l volume.
When the tumor grows to 50mm3And treatment is started. The method comprises the following four groups: CD47-var7/5F9 group (10), 5F9 group (5), CD47-var7 group (5), PBS group (10). The administration method comprises the following steps: CD47-var7, 1mg/kg, intravenous injection, every other day administration; 5F9, 30mg/kg, i.p., 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 1000mm3When so, the administration is terminated.
3. Results
The result is shown in fig. 4, the CD47-WT combined with CD47 monoclonal antibody 5F9 can significantly inhibit the growth of tumor cells; the CD47-var3 combined with the CD47 monoclonal antibody 5F9 can obviously inhibit the growth of tumor cells; the CD47-var7 combined with the CD47 monoclonal antibody 5F9 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
<110> Beijing Zeqin biopharmaceutical Co., Ltd
<120> CD 47-use of low pH insertion peptide for marking or treating tumor
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<151>2018-11-30
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Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala
20 25 30
Asp Glu Gly Thr
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<210>2
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<213> Artificial Sequence (Artificial Sequence)
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PheThr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Gly
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<210>3
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<212>PRT
<213> Artificial Sequence (Artificial Sequence)
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Ala Cys Glu Asp Gln Asn Pro Tyr Trp Arg Ala Tyr Ala Asp Leu Phe
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<211>27
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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1 510 15
Pro Thr Glu Thr Leu Leu Leu Glu Leu Leu Trp
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<210>6
<211>25
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
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<210>7
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<212>PRT
<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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<212>PRT
<213> Artificial Sequence (Artificial Sequence)
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<210>10
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<212>PRT
<213> Artificial Sequence (Artificial Sequence)
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<210>11
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<212>PRT
<213> Artificial Sequence (Artificial Sequence)
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Pro Thr Thr Leu LeuLeu 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
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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
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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
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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)
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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>4
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>18
Gly Gly Gly Ser
1
<210>19
<211>139
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>19
Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly
1 5 10 15
Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe
20 25 30
Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala
35 40 45
Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp
50 55 60
Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp
65 70 75 80
Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala
85 90 95
Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr
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Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu
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Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro
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<213> Artificial Sequence (Artificial Sequence)
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Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly
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20 25 30
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35 40 45
Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp
50 55 60
Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp
65 70 75 80
Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala
85 90 95
Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr
100 105 110
Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu
115 120 125
Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Gly Gly Gly Ser Ala
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Cys Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu Phe
145 150 155 160
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Glu Gly Thr
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Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe
20 25 30
Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala
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Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp
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Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp
65 70 75 80
Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala
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Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr
100 105 110
Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu
115 120 125
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Thr Glu Thr Leu Leu Leu Glu Leu
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<213> Artificial Sequence (Artificial Sequence)
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Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala
35 40 45
Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp
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Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp
65 70 75 80
Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala
85 90 95
Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr
100 105 110
Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu
115 120 125
Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Gly Gly Gly Ser Ala
130 135 140
Cys Asp Asp Gln Asn Pro Trp Arg Ala Tyr Leu Asp Leu Leu Phe Pro
145 150 155 160
Thr Asp Thr Leu Leu Leu Asp Leu Leu Trp
165 170
Claims (10)
1. A fusion peptide of a low pH insertion peptide, comprising a low pH insertion peptide, a tumor surface antigen or a functional domain thereof, which is a domain recognized and bound by an antibody against the tumor surface antigen;
preferably, the low pH insertion peptide comprises a polypeptide with a sequence shown as SEQ ID NO.1 or a variant thereof, and more preferably, the variant sequence of the polypeptide with the sequence shown as SEQ ID NO.1 is shown as SEQ ID NO.2-SEQ ID NO. 17.
More preferably, the tumor surface antigens include 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, PD-L2, CD 47;
most preferably, the tumor surface antigen is CD 47.
2. The fusion peptide of claim 1, wherein the tumor surface antigen or functional domain thereof is linked to the N-terminus of the low pH insertion peptide by Linker; preferably, the Linker sequence is (GGGS) m or (GGGGS) m, wherein m is a natural number; more preferably, the Linker sequence is GGGS.
3. The fusion peptide according to claim 1 or 2, characterized in that the functional domain of the tumor surface antigen CD47 is a polypeptide formed by the amino acids shown in SEQ ID No.19 or a polypeptide derived from the amino acid sequence shown in SEQ ID No.19 by substitution and/or deletion and/or addition of one or several amino acid residues of the amino acid sequence shown in SEQ ID No.19 and having the same function as the sequence shown in SEQ ID No. 19.
4. The fusion peptide of any one of claims 1-3, wherein the sequence of the fusion peptide is set forth in SEQ ID No. 20-22.
5. An anti-tumor pharmaceutical composition, comprising the fusion peptide of any one of claims 1 to 4; preferably, the pharmaceutical composition further comprises an anti-tumor drug targeting a tumor surface antigen or a functional domain thereof; more preferably, the anti-tumor drug comprises an antibody to a tumor surface antigen.
6. The pharmaceutical composition of claim 5, wherein the antibody comprises rituximab, trastuzumab, gemtuzumab, alemtuzumab, ibritumomab, tositumomab, bevacizumab, cetuximab, panitumumab, ofatumumab, dinomumab, ipilimumab, bretuzumab, pertuzumab, ado-trastuzumab, atrozumab, ramucirumab, pembrolizumab, bannatuzumab, nivolumab, dinumumab, dinumuxeumab, nixituzumab, rituximab, erlitumumab, erlotinuzumab, avizumab, denosumab, gemumab, necumumab, Necitumumab, ateuzumab.
7. A tumor marker system comprising the fusion peptide of any one of claims 1-4.
8. A targeted tumor therapy system comprising the tumor marker system of claim 7; preferably, the targeted tumor therapy system comprises the tumor marker system of claim 7, a tumor killing system comprising an antibody to a tumor surface antigen or a functional domain thereof in the fusion peptide of any one of claims 1 to 4.
9. The use of the fusion peptide of any one of claims 1 to 4, comprising the use of any one of:
(1) the application in preparing antineoplastic drugs; preferably, the anti-tumor drug comprises the pharmaceutical composition of claim 5 or 6;
(2) use in the preparation of a tumor marker system according to claim 7;
(3) use in the preparation of a targeted tumor therapy system according to claim 8.
10. Use of a tumor marker system according to claim 7 for the preparation of a targeted tumor therapy system according to claim 8.
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| 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 |
| CN201911208044.2A Pending CN110698565A (en) | 2018-11-30 | 2019-11-30 | Tumor-targeting CD38-pHLIP fusion peptides |
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Also Published As
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| CN110713548A (en) | 2020-01-21 |
| CN110698565A (en) | 2020-01-17 |
| CN110760008A (en) | 2020-02-07 |
| CN109517073A (en) | 2019-03-26 |
| CN110713547B (en) | 2022-10-04 |
| CN110760008B (en) | 2023-02-07 |
| CN110713548B (en) | 2022-11-04 |
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