CN109467607A - A kind of acid-sensitive fusogenic peptide of target tumor and its application - Google Patents
A kind of acid-sensitive fusogenic peptide of target tumor and its application Download PDFInfo
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Abstract
The invention discloses a kind of acid-sensitive fusogenic peptide of target tumor and its application, which is that the 4th structural domain for being inserted into peptide and tumor surface antigen Her2 by low pH is formed by connecting.Fusogenic peptide of the invention can be inserted on tumor cell membrane under acidic environment and in the 4th structural domain of cell surface display Her2, and success is identified by tumour medicine Herceptin, and the fusogenic peptide can effectively treat tumour.Research achievement of the invention, which overcomes existing tumour medicine, can only treat the defect of a kind of cancer or a kind of parting that can only treat a kind of cancer, by the way that tumor surface to be marked to fusogenic peptide of the invention, Herceptin is realized for the therapeutic efficiency of all cancers, this is for clinical treatment tumour with the meaning of milestone.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to it is swollen in targeted therapy to further relate to the fusogenic peptide for a kind of fusogenic peptide
Effect in tumor.
Background technique
In recent years, as China's rapid economic development, people's material and culture level are continuously improved, life style also occurs huge
It is big to change, also changed along with our living environment, such as water quality deterioration, air quality decline etc..Due to life
Great changes, malignant tumour, cardiovascular disease has occurred in the change of mode and the decline of environmental quality, the China human mortality cause of death
The main reason for non-communicable diseases such as disease and chronic disease have become China's death, wherein brought by malignant tumour
Death occupies very big ratio, becomes the problem of we can not ignore.
Currently, the common treatment method of malignant tumour has operative treatment, chemotherapy, radiotherapy etc., but due to pernicious
Tumour has differentiation degree low, and cellular morphology differs greatly with normal tissue cell, disorganized and frequent recurrence or transfer
The features such as, there are still many still unsolved problems for the Clinics and Practices of malignant tumour.Malignant tumour early stage often without any symptom,
When there is the symptom on body, it is mainly tumor-infiltrated, compressing organ and DISTANT METASTASES IN caused by, be at this moment often in, evening
Phase, middle and advanced stage tumour is all not satisfactory using various therapeutic effects, and the overwhelming majority can not cure.It does not only shift at present
Tumour can take surgical resection therapy, late malignant tumour operation is difficult and postoperative several years still can recur or shift.Chemistry
Medicinal treatment is the important means in oncotherapy, however current anti-tumor drug that there are targetings is poor, therapeutic effect is bad,
The disadvantages of toxic side effect is big, destructible body immunity.Therefore, how current anticancer drug faces enormous challenge is avoiding
Use effective carrier by drug-rich in tumor tissues while damaging other normal tissues.
In the middle of the 19th century, Arthus Bernard first proposed the concept of " interior environment ", that is, cells survival
Microenvironment be extracellular fluid.Every physicochemical property of cell micro-environment is in metastable state under normal physiological conditions, when
After the stable state of cell micro-environment is destroyed, it will lead to the various pathology of cell appearance and sexually revise.Abnormal extracellular subacidity ring
The induction and maintenance in border, it is considered to be tumour is formed and the key link of progress.The study found that metastases are that it is most fatal
On one side, metastases are growth course of the malignant cell to the position other than primary tumo(u)r, are tumour patient death
One of main reason.Metastases are positively correlated with its cell migration ability, and it was discovered by researchers that tumour cell acid discharge
Intensity is positively correlated with its transfer ability, tumour cell external solution again micro- acidity, therefore tumour slightly acidic environment have become it is anti-
One of the hot spot of tumor area research.
Dynamic equilibrium is remain (such as between normal cell and surrounding organizational environment for the maintenance normal physiological activity moment
Ion distribution, the synthesis of protein and enzyme, air pressure and pH etc.), the two collective effect is cell Proliferation, differentiation, apoptosis and thin
The secretion and expression of the various factors of cellular surface provide a stable environment.Once this dynamic equilibrium is destroyed, just there is disease
Occur.And it is excessive to differ from the former extracellular acid solution between tumor tissues and normal tissue, pH is in acidity.Human normal
The extracellular normal physiological pH value of tissue maintains 7.4, and intracellular pH (pHi) value is 7.2.This phenomenon is by polysaccharide point
Solution, Na+ are reversely cooperateed with, and Cl, bicarbonate ion pump, the number of mechanisms such as exchange of sodium ion and potassium ion cooperatively form
's.But in most of tumor tissues, the variation of intraor extracellular pH gradient is reversed, that is, pHi > pH.The study found that passing through
The methods of microelectrode, nuclear magnetic resonance spectroscopy detect the pH value inside and outside tumour cell, and tumour cell pH is between 6.15~6.8, in acid
Property, and pHi is about 7.2, is in neutrality even alkalinity.The opposite phenomenon of this intraor extracellular pH value mainly with the metabolism of tumor tissues
It is related.The growth of tumour needs a large amount of nutriment, but due to tumor neogenetic blood vessels disorder and itself non-functional capillary
Blood vessel causes nutriment supply insufficient, is unable to satisfy the oxygen demand of tumour hypermetabolism, this Partial tumors tissue is caused to lack
Oxygen.The state of anoxic hinders cell and obtains energy by mitochondrial respiratory chain, and the tumour cell for being now in anaerobic condition is logical
It crosses activation hypoxia inducible factor and thus activates the signal cascade reaction in downstream as generated glycolysis relevant enzyme, up-regulation glycolysis generation
It thanks, so that tumour cell is able to adapt to anaerobic environment and survive.Clinical discovery, most of malignant growth and development process
In all there are internal anoxic zones, and usually there is necrosis in these regions, are also easier to that metastases occur.Even if aerobic
Tumour cell still carries out glycolysis under environment, on the one hand this unique metabolic process of tumour cell maintains the steady of pH intracellular
On the other hand state and the basic physiological function of tumour cell also result in the formation of the outer slightly acidic environment of tumour cell, this is also
The result of natural selection in tumour evolutionary process.Even result of study shows to need not move through the mutant cell of glycolysis,
Extracellular matrix is still in acid.This discovery explanation, the acid phenomenon of tumor tissues are perhaps derived from tumour cell essence.Cause
This, the feature of tumour slightly acidic environment can be used as a kind of research for targeting target and being used for tumour cell medical domain, and find
Targeted probes to acid-sensitive are the key that solve the problems, such as this.
Low pH from the transbilayer helix PROTEIN C of bacteria rhodopsin is inserted into peptide (pHLIP, pH low insertion
Peptide) special nature just because of it in acidic micro-environment is at research hotspot in recent years.PHLIP is a kind of water
The polypeptide of dissolubility is inserted into cell bilayer lipid membrane and forms stable cross-film α spiral.Peptide folds and film insertion is by neutral or alkali
Property (pH > 7.4) pH drop to faintly acid (pH=7.0-6.5 or lower) driving.There are three types of Main Morphologies for pHLIP tool:
The form I of water is dissolved under neutral pH without structure, under neutral ph without structure and the state I I that is integrated on cell membrane surface, in acid
Property pH under be inserted into and be threaded through with α the state I II of cell membrane.Therefore, the binding force of peptide chain and cell membrane ratio is neutral when low pH
Under the conditions of binding force it is several times high, this targetedly targets acidity diseased tissue for pHLIP and provides advantageous basis.Research
It was found that the N-terminal of pHLIP is to be free on cell membrane surface, and C-terminal is embedded in and penetrates into cell when low pH.Therefore, small molecule
It is covalently bound to the N-terminal of pHLIP, tumour cell film surface can be transported to by pHLIP in low pH.Davies etc. is utilized
PHLIP as the image probe in blood platelet construct the gold nano grain system of rare earth element package a kind of for cell at
Picture, the experiment key be that its C-terminal can be embedded into the cell by pHLIP in pH≤6.5, thus by fluorescent molecule etc. at
As small molecule transport it is intracellular.
It is following research heat using the targeted system that the acidic environment taxis exploitation of pHLIP is conducive to treatment malignant tumour
Point.
Summary of the invention
The present invention is completed based on following design: tumour has heterogeneity, even if the tumour in same tumor tissues is thin
Cellular surface may express different proteantigens, and the tumour of the expression antigen can only be killed for the drug of a certain proteantigen
Cell, but do not have lethal effect to the tumour cell for not expressing the antigen, these tumour cells survive, and to form growth excellent
Gesture, so that the tumor patient produces drug resistance to the drug.If by a kind of proteantigen in all tumor cell surfaces
It all expresses, then the drug that will lead to for the proteantigen can thoroughly kill all tumour cells.For different tumours
Tissue is also such.The present invention is by the 4th structural domain of the molecule parting marker Her2 of breast cancer cell surface expression and targets
The low pH insertion peptide of tumour connects to form fusogenic peptide, which can target any solid tumor cell and in tumour cell table
Face is shown, for the drug such as Herceptin of the 4th structural domain of Her2, so that it may to any including breast cancer cell
Cancer cell carries out lethal effect, expands the scope of application of the tumour medicine.
One of the objects of the present invention is to provide a kind of acid-sensitive fusogenic peptides of target tumor.
The second object of the present invention is to provide the preparation method of above-mentioned fusogenic peptide.
The third object of the present invention is to provide application of the above-mentioned fusogenic peptide in neoplasm targeted therapy.
To achieve the goals above, present invention employs following technical solutions:
According to an aspect of the present invention, described to melt the present invention provides a kind of acid-sensitive fusogenic peptide of target tumor
Closing peptide includes low pH insertion peptide, and tumor surface antigen or its functional domain, the functional domain of the tumor surface antigen are
The structural domain that antibody is identified and combined.
Further, the fusogenic peptide includes low pH insertion peptide, the functional domain of tumor surface antigen, the tumor surface
The functional domain of antigen is the structural domain that antibody is identified and combined.
Can be used for constructing the tumor surface antigen of fusogenic peptide of the present invention example include but is 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.
Can be used for constructing fusogenic peptide of the present invention low pH insertion peptide include sequence be SEQ ID NO.1 shown in polypeptide or its
Variant.
Sequence is that abbreviation WT, the variant of WT include Var1-Var16 to polypeptide shown in SEQ ID NO.1 in the present invention.
The sequence of WT and its variant is 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 its functional domain of the invention is connected to the low pH by Linker and is inserted into peptide
N-terminal.
The Linker be it is commonly used in the art, sequence can be (GGGS) m, be also possible to (GGGGS) m, wherein m
=natural number.
In specific embodiments of the present invention, the Linker sequence is GGGGS (SEQ ID NO.19).
In specific embodiments of the present invention, the sequence of low pH insertion peptide of the invention is as shown in SEQ ID NO.1.
In specific embodiments of the present invention, tumor surface antigen is Her2.
In specific embodiments of the present invention, the functional domain of tumor surface antigen is the 4th structural domain of Her2 albumen
Or the Her2 albumen the 4th is possessed in its function similar structures domain, the function similar structures domain of the 4th structural domain of Her2 albumen
The activity of structural domain binding antibody.
Further, the 4th domain sequence of Her2 albumen for use in the present invention is as shown in SEQ ID NO.18.
The function similar structures domain of the 4th structural domain of Her2 albumen includes the Her2 albumen that sequence is SEQ ID NO.18
Four structural domains by one or several amino acid residues substitution and/or deletion and/or addition and with shown in SEQ ID NO.18
The sequence amino acid sequence with the same function as shown in SEQ ID NO.18 derived from polypeptide.
The function similar structures domain of the 4th structural domain of Her2 albumen includes that sequence is amino acid shown in SEQ ID NO.18
Sequence has at least 80% homology (also known as sequence identity), it is highly preferred that with amino acid shown in SEQ ID NO.18
The homology of sequence at least about 90% to 95%, what the amino acid sequence of 96%, 97%, 98%, 99% homology of Chang Wei was constituted
Polypeptide.
It is known that, conventionally, the modification of one or more amino acid will not influence albumen in a protein or polypeptide
The function of matter.Those skilled in the art can approve the amino acid for changing single amino acids or small percentage or to amino acid sequence
Individual additions, missing, insertion, replacement are conservative modifications, and wherein the change of protein and peptide generates the albumen with identity function
Matter or polypeptide.It is well known in the art for providing the Conservative substitution tables of intimate amino acid.
The function similar structures domain of the 4th structural domain of Her2 albumen also includes to amino acid sequence shown in SEQ ID NO.18
The non-conservative modification of column, as long as the polypeptide by modification still is able to retain the biological activity of binding antibody.
Preferably, the functional domain of tumor surface antigen is the 4th structural domain of Her2 albumen, sequence such as SEQ ID
Shown in NO.18.
In specific embodiments of the present invention, the sequence of fusogenic peptide of the invention is as shown in SEQ ID NO.20.
According to a further aspect of the invention, the present invention provides a kind of tumor marker system, the tumor marker systems
Including mentioned-above fusogenic peptide.
Further, the tumor marker system can also 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 invention, the present invention provides a kind of targeting therapy for tumor system, the target tumors
Treatment system includes mentioned-above tumor marker system.
Further, the targeting therapy for tumor system can also include tumor-killing system, the tumor-killing system packet
Include the antibody for tumor surface antigen.
Tumor-killing system of the invention can be CAR-T or TCR-T system, pass through immunocyte, such as T cell, expression
For the antibody or TCR of tumor surface antigen.Tumor-killing system is also possible to ADC (antibody drug conjugates)
System, i.e. antibody coupling toxin (Pseudomonas Exotoxin PE38, diphtheria toxin, times carcinomycin duocarmycin, golden yellow Portugal
Grape coccus enterotoxin A/E-120, shiga toxin, ricin (WA)), it is chemotherapeutics (Irinotecan, exatecan, adriamycin), small
Molecule inhibitor (benefit Statins, calicheamycin class, CHROMATOGRAPHIC FRACTIONATION AND MASS, tubulysin, antimicrobial, urase difficult to understand), liposome, nanometer
Gold particle etc..In addition, tumor-killing system is also possible to Immunocytokines, i.e., by certain immune cell factors and antibody
Link, such as IL-2, IL-12, TNF-α, IL-10, TGF-β.It also include bispecific antibody killer-system, i.e., an antibody is known
Not Rong He peptide linkage antigen or antigenic domains, another antibody identifies other antigens.
The action principle of targeting therapy for tumor system of the invention is as follows: the fusogenic peptide in tumor marker system is inserted in low pH
Enter and be inserted on cell membrane under the action of peptide, the 4th structure domain views of Her2 are in tumor cell surface, tumor-killing system
Antibody drug tumor cell surface antigen, antigen-antibody combine, thus by tumor-killing system aggregates in tumor tissues, it is thorough
The specific killing tumour cell at bottom.
According to a further aspect of the invention, the present invention provides mentioned-above fusogenic peptides to construct mentioned-above swell
Application in tumor tagging system.
According to a further aspect of the invention, the present invention provides mentioned-above fusogenic peptides to construct mentioned-above target
Application into system of tumor treatment.
According to a further aspect of the invention, the present invention provides mentioned-above tumor marker systems in building front institute
The application in targeting therapy for tumor system stated.
Specifically, targeting therapy for tumor system may include two subsystems, and a subsystem is tumor marker system,
Including the mentioned-above fusogenic peptide of the present invention, another subsystem is tumor-killing system, including for tumor surface antigen
Antibody.
Antibody for tumor surface antigen of the invention can be any antibody for tumor surface antigen.It is described anti-
Body includes monoclonal antibody, bispecific antibody.
Antibody for tumor surface antigen of the invention also includes the antigen binding for the antibody of tumor surface antigen
Part, further, the antigen-binding fragment of the antibody include Fab, Fab ', F (ab ') 2, Fv or single-chain antibody.
Variable region that Fab refers to the variable region containing light chain and constant region and heavy chain and constant region are through disulfide bond
A part of the antibody molecule combined.
Fab ', which refers to, contains the Fab segment in part hinge area.
F (ab ') 2 refers to the dimer of Fab '.
Fv is referred to containing antibody heavy chain variable region, light chain variable region and the minimum antibody with whole antigen binding sites
Segment.
Single-chain antibody is referred to being connected directly by light chain variable region with heavy chain variable region or is formed by connecting by a peptide chain
Engineered antibody.
Antibody of the invention further includes the various variants of antibody, such as the change derived from Similar amino acids well known in the art substitution
Body, variant caused by the missing of amino acid, increase.
Antibody for tumor surface antigen of the invention can include one or more sugar in heavy chain and light chain variable region
Base site, as known in the art, the one or more glycosylation site present in variable region can lead to enhancing
Antibody immunogenicity, or change the pharmacokinetics of antibody due to changing antigen binding.
Antibody for tumor surface antigen of the invention can be designed as comprising modification in the region Fc, be usually to change
Become antibody one or more functional characteristics, as serum half-life, complement combine, Fc receptor combine, and/or antigen rely on it is thin
Cellular toxicity.In addition, antibody of the invention, which can be modified by sulphation, (e.g., one or more chemical groups can be connected to anti-
Body), or be modified to change its glycosylation, to change the one or more functions characteristic of antibody again.
Another modification that antibody for tumor surface antigen of the invention can be designed is Pegylation.Antibody
It can be by Pegylation to for example, increasing biology (such as serum) half-life period of antibody.It, should in order to make antibody Pegylation
Antibody or its segment are usually in the item for being suitable for one or more polyethylene glycol (PEG) group and being connected to the antibody or antibody fragment
It under part, is reacted with PEG, such as the active ester or aldehyde derivatives of polyethylene glycol.Preferably, the Pegylation be by with it is active
PEG molecule (or similar reactive water-soluble polymer) carries out acylation reaction or alkylated reaction and realizes.
Antibody example includes but is not limited to: molecular targeted monoclonal antibody medicine, targeting antibodies coupling drug, bispecific antibody medicine
Object, targeting immunity inspection point drug etc..Such antibody example is such as: Rituximab, Herceptin, WAY-CMA 676, A Lun
Monoclonal antibody, ibritumomab tiuxetan, tositumomab, Avastin, Cetuximab, Victibix, difficult to understand, promise list
Anti-, her wooden monoclonal antibody, this appropriate former times monoclonal antibody, handkerchief trastuzumab, ado- Herceptin, Ah's Torr pearl monoclonal antibody, Lei Molu monoclonal antibody, pyridine aldoxime methyliodide (PAM)
Monoclonal antibody, Beaune spit monoclonal antibody, receive Wu Dankang, Da Leimu monoclonal antibody, Nu Tuxi monoclonal antibody, the trastuzumab of resistance to former times, angstrom sieve trastuzumab, Ah
Special Zhu Dankang, AVM hereinafter monoclonal antibody, Di Nuosaimai, gemtuzumab, Necitumumab, Atezolizumab.
Term " CAR-T " in text, full name are Chimeric Antigen Receptor T-Cell Immunotherapy,
Chimeric antigen receptor T cell immunotherapy.According to this characteristic of tumor microenvironment, scientist optimizes a series of CART sequence
Column, make it in different pH value and the affinity of antigen have entirely different affinity, thus in different pH value situations
Lower activation.
" tumor surface antigen ", which refers to, as described herein newly occurs or mistake during tumorigenesis in cell surface
Spend the antigenic substance of expression.
Term " targeting antibodies coupling drug " or immune conjugate (immunoconjugate) in text.Immune conjugate
Molecule is made of monoclonal antibody and " bullet " drug two parts.The substance that can be used as " bullet " mainly has three classes, i.e. radionuclide, medicine
Object and toxin；It is connect with monoclonal antibody and respectively constitutes radio-immunity conjugate, chemo-immunity conjugate and immunotoxin.
In text term " bispecific antibody drug " refer to can antibody in conjunction with two epitopes simultaneously, it is dual anti-can be with
It is divided into two kinds, i.e. T cell recruitment type, raises site comprising tumour cell target spot-T cell, this accounts for dual anti-most of ratio,
In, T cell is raised site and is referred to CD3 (T cell), CD16 target spot (NK cell), and target spot is usually located at tumour cell；In addition, double
It is anti-2 signal paths to be inhibited in conjunction with double target sites (such as VEGF-PDGF, VEGF-Ang2), to reduce drug resistance production
A possibility that raw.
The advantages of the present invention are as follows:
The 4th structural domain of tumor surface antigen Her2 is connect by the present invention with low pH insertion peptide for the first time, and foring can mark
Remember the target tumor acid-sensitive fusogenic peptide of tumour.Research achievement of the invention greatly extends existing for a kind of cancer
Or the indication of the tumour medicine for a kind of specific parting of cancer, there is very important meaning for clinical treatment tumour
Justice.
Detailed description of the invention
Fig. 1 shows the electrophoretogram of the fusogenic peptide of the invention using SDS-PAGE identification expression and purification；
Fig. 2 shows the fluorogram using Her2 protein expression situation in cofocal observation A549 cell；
Fig. 3, which is shown, utilizes the fluorogram that fusogenic peptide of the present invention positions on A549 cell in cofocal observation neutral environment；
Fig. 4, which is shown, utilizes the fluorogram that fusogenic peptide of the present invention positions on A549 cell in cofocal observation acidic environment；
Fig. 5 shows the curve graph that the 4th structural domain-pHLIP of Her2 influences tumour growth.
Specific embodiment
The present invention is further illustrated below by embodiment.It should be understood that the embodiment of the present invention is for illustrating
The present invention is rather than limiting the invention.The simple modifications that essence according to the present invention carries out the present invention belong to the present invention
Claimed range.
The synthesis of 1 fusogenic peptide of embodiment
1, prokaryotic expression
Step:
Strain building
Amino acid sequence (SEQ ID NO.20) the design dna sequence of fusogenic peptide according to the present invention is subjected to full genome conjunction
At both ends connection restriction enzyme site Nde I and XhoI sequence when, synthesis, with both enzymes difference digestion fusogenic peptide code nucleic acid and
PET28a carrier recycles endonuclease bamhi and carrier, with the connection of T4 ligase, transformation receptor bacterium BL21 (DE3), paves plate culture,
Picked clones send sequencing.
Culture induction
Will sequencing correctly clone culture (LB culture medium, 37 DEG C), OD value arrive 0.6-0.8 when induction plus IPTG induce,
Bacterium is received in the centrifugation in 0.5mM, 4-6 hours of IPTG final concentration.
2, inclusion body purification
Step:
(1) inclusion body washs
A liquid: 50mM Tris, 2mM EDTA, pH8.0 are washed 2 times；
B liquid: 50mM Tris, 2mM EDTA, 0.1%Triton, pH8.0 are washed 1 time；
C liquid: 20mM Tris, 1M urea, pH8.0 are washed 1 time.
(2) renaturation, ni-sepharose purification
Extracting: using 8M urea, and 5mM β-Me, 0.3M NaCl, 20mM Tris, pH8.0 extract inclusion body, extract ratio 1:
20。
Renaturation dialysis: destination protein adds 10mM β-Me, and 40 DEG C restore 15 minutes, is diluted to 0.2mg/ml 10mM PB,
50μM CuCl2, pH8.0 dialyse sample, change 3 dialyzates, supernatant be collected by centrifugation.
Ni column purification: using 0.3M NaCl, and 10mM PB, pH8.0 balance chromatographic column, with the balance of the imidazoles containing 40mM after loading
Buffer is washed miscellaneous, elutes destination protein with the equilibration buffer of the imidazoles containing 300mM.Destination protein purity is greater than 95%.
Desalination: desalination to 20mM PB, in 0.1M NaCl.
(3)SDS-PAGE
The sample of step (2) processing is subjected to SDS-PAGE.As a result such as Fig. 1 is shown, this experiment can isolate and purify out this
The fusogenic peptide of invention, note: in Fig. 1,1 swimming lane: GJ renaturing inclusion bodies；2 swimming lanes: loading is fled；3 and 4 swimming lanes: caching liquid balance；5
Swimming lane: 40mM imidazoles elution；6 swimming lanes: 300mM imidazoles elution；7,Marker.
The positioning on tumour cell that 2 fusogenic peptide of embodiment is cultivated in vitro
1, cell culture
A549 cell, which uses, contains 10% calf serum, 1,600,000 unit gentamicins/ml DMEM culture medium, at 37 DEG C,
5%CO2Cell incubator in cultivate.Cell is passed on after covering with by 1:10.
2, confocal observation positioning
A549 cell (5x105/ hole) overnight incubation on coverslip culture dish, it discards culture supernatant and is separately added into
The fusogenic peptide (60 μ g/ml) that embodiment 1 is expressed is added in the PBS of pH6.3 and 7.4, and 37 DEG C are incubated for 1 hour, abandons the corresponding pH of supernatant
PBS wash 3 times, be added pH6.3 and 7.4 PBS, be added Herceptin-FITC or TGLA-FITC (control antibodies) it is (dense
Degree is 1:400 dilution) 37 DEG C be incubated for 30 minutes, abandon supernatant and washed 3 times with the PBS of corresponding pH, the PBS of pH7.4 is added,
Confocal observation.Grouping: (1) pH6.3 untreated fish group；(2) pH6.3 fusogenic peptide (the 4th structural domain-pHLIP of her2)；(3)
PH6.3 fusogenic peptide (the 4th structural domain-pHLIP of her2)+Herceptin-FITC；(4) pH6.3 fusogenic peptide (the 4th structure of her2
Domain-pHLIP)+TGLA-FITC；(5) pH7.4 fusogenic peptide (the 4th structural domain-pHLIP of her2)+Herceptin-FITC；(6)
PH7.4 Herceptin-FITC.
3, result
Fig. 2 shows that human lung cancer cell line A549 does not express Her2.
Fig. 3 is shown, in neutral solution environment, fusogenic peptide of the invention cannot be inserted into the cell membrane of A549, cannot be thin
The 4th structural domain of Her2 is shown on after birth.
Fig. 4 is shown, in acid solution environment, fusogenic peptide of the invention is inserted into the cell membrane of A549, and shows
The 4th structural domain of Her2 on cell membrane can be identified by Herceptin.
The above results show that low pH insertion peptide connection the 4th structural domain of Her2 does not influence its low pH insertion peptide insertion cell membrane
Characteristic, while the 4th structural domain of Her2 and low pH insertion peptide connect and nor affect on its conformation.
The effect assessment of 3 Her2 of embodiment the 4th structural domain-pHLIP and herceptin treatment tumour
Experimental material: A549 cell is purchased from ATCC；Her2 the 4th structural domain-pHLIP (Her2 D4-pHLIP) protokaryon table
It reaches；Herceptin is purchased from Roche Group；6-8 week old male nude mouse is purchased from dimension tonneau China.
Experimental procedure:
A549 is inoculated in nude mice, when diameter of tumor it is long to 1cm when, sterile removing tumour shreds, and list is made in homogenate, strainer
Cell suspension cultivates amplification, by cell infusion in nude mice side subcutaneous abdomen, 1x10 in 1640 complete mediums6A cell/
Only, long to 0.5-1cm to diameter of tumor, excessive and too small tumour is rejected, by the almost the same mice group of tumor size.Altogether
The independent injection group of point 4 groups: Her2 D4-pHLIP, 10；Her2 D4-pHLIP joint herceptin injection group, 10；Her2
D4-pHLIP joint IgG1 injection group, 10；Physiological saline N.S injection group, 10.Every 3 days measurement tumor sizes.
Medication: Her2 D4-pHLIP administration: each every 40 μM/100 μ l of intravenous injection work as after the completion of grouping
It starts to inject, and injection in one day is primary, primary every injection in 2 days；Antibody administration: intraperitoneal injection, dosage 10mg/kg, injection frequency
Rate is identical as Her2 D4-pHLIP, and injection time is 6-12 hours after Her2 D4-pHLIP administration, until terminating.
Interpretation of result:
Fig. 5 is the results show that Herceptin significantly inhibits the tumour growth of lung cancer in mice.
Although those skilled in the art should manage above only describes a specific embodiment of the invention example
Solution, these are merely examples, and protection scope of the present invention is defined by the appended claims.Those skilled in the art
Without departing from the principle and essence of the present invention, many changes and modifications may be made, but this
A little changes or modification each fall within protection scope of the present invention.
Sequence table
<110>Beijing Ze Qin biological medicine Co., Ltd
<120>a kind of acid-sensitive fusogenic peptide of target tumor and its application
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Glu Gly
Claims (10)
1. a kind of acid-sensitive fusogenic peptide of target tumor, which is characterized in that the fusogenic peptide includes low pH insertion peptide, tumour table
Face antigen or its functional domain, the functional domain of the tumor surface antigen are the antibody for the tumor surface antigen
The structural domain for identifying and combining；
Preferably, it is polypeptide or its variant shown in SEQ ID NO.1 that the low pH insertion peptide, which includes sequence, it is highly preferred that institute
Stating sequence is the variant sequence thereof of polypeptide shown in SEQ ID NO.1 as shown in SEQ ID NO.2-SEQ ID NO.17.
2. fusogenic peptide according to claim 1, which is characterized in that the tumor surface antigen 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。
3. fusogenic peptide according to claim 1 or 2, which is characterized in that the tumor surface antigen or its functional domain
The N-terminal that the low pH is inserted into peptide is connected to by Linker；Preferably, the Linker sequence is (GGGS) m or (GGGGS) m,
Wherein m=natural number；It is highly preferred that the Linker sequence is GGGS.
4. fusogenic peptide according to claim 2 or 3, which is characterized in that the tumor surface antigen is Her2.
5. fusogenic peptide according to claim 4, which is characterized in that the functional domain of the tumor surface antigen Her2 is
The 4th structural domain of Her2 albumen or its function similar structures domain, the sequence such as SEQ ID of the 4th structural domain of Her2 albumen
Shown in NO.18, the function similar structures domain of the 4th structural domain of Her2 albumen is the amino acid as shown in SEQ ID NO.18
Derive and possesses the 4th active polypeptide of structural domain binding antibody of Her2 albumen；
Preferably, the functional domain of Her2 is the 4th structural domain of Her2 albumen.
6. fusogenic peptide according to any one of claims 1-5, which is characterized in that the sequence of the fusogenic peptide such as SEQ ID
Shown in NO.20.
7. a kind of tumor marker system, the tumor marker system includes fusogenic peptide of any of claims 1-6.
8. a kind of targeting therapy for tumor system, the targeting therapy for tumor system includes tumor marker system as claimed in claim 7
System；Preferably, the targeting therapy for tumor system includes tumor marker system as claimed in claim 7, tumor-killing system, institute
Stating tumor-killing system includes the tumor surface antigen or its functional structure in fusogenic peptide of any of claims 1-6
The antibody in domain.
9. fusogenic peptide of any of claims 1-6 is preparing tumor marker systematic difference as claimed in claim 7.
10. fusogenic peptide of any of claims 1-6 or tumor marker system as claimed in claim 7 are in preparation right
It is required that the application in targeting therapy for tumor system described in 8.
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| CN111454371A (en) * | 2020-04-18 | 2020-07-28 | 北京泽勤生物医药有限公司 | PD L1-pH L IP, preparation method and application thereof in treatment of autoimmune disease |
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| 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 |
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