CN109517045A

CN109517045A - A kind of low pH of modified is inserted into peptide

Info

Publication number: CN109517045A
Application number: CN201811459449.9A
Authority: CN
Inventors: 王颖; 魏化伟; 杨承刚
Original assignee: Beijing Zai Qin Biological Medicine Co Ltd
Current assignee: Beijing Zai Qin Biological Medicine Co Ltd
Priority date: 2017-12-19
Filing date: 2018-11-30
Publication date: 2019-03-26
Anticipated expiration: 2038-11-30
Also published as: CN109517045B; CN110790829B; CN110790829A

Abstract

The invention discloses a kind of low pH of modified to be inserted into peptide, which is that its extracellular fragment sequence is repeated one or more times acquisition in the sequence basis of low pH insertion peptide already existing in the prior art.The present invention proves that improved low pH insertion peptide targeting navigates to tumor cell surface using the tumour cell of in vitro culture；Improved low pH insertion peptide target tumor tissue is proved using tumor mouse model and is maintained for a long time in vivo.In addition the research of the invention finds that, the extracellular fragment of the low pH insertion peptide of modified can be used as antigen and prepare antibody, which can be used for oncotherapy.The low pH insertion peptide of modified of the invention is clinically with a wide range of applications.

Description

A kind of low pH of modified is inserted into peptide

Technical field

The invention belongs to field of biomedicine, it is related to a kind of low pH insertion peptide of modified, the invention further relates to by modified The composition that low pH insertion peptide is constituted.

Background technique

In past 10 years, chemotherapy has very important effect in oncotherapy, while also by people Extensive concern.But traditional anti-tumor drug still has many limitations, such as they cannot be distinguished normal tissue and swell Tumor tissue can also cause fatal adverse reaction so that therapeutic efficiency is very low what is more.Therefore, improving selectivity becomes anti- The key of tumour medicine research and development.Anti-tumor drug can be specifically transmitted to tumor tissues by targeted drug delivery system, and And can reduce intake of the normal tissue to anti-tumor drug, its adverse reaction can be reduced, improve clinical therapeutic efficacy.At present Targeted drug delivery system is many kinds of, some have been used in clinical treatment.However, also there is identical receptor in the normal tissue Expression, they can equally identify the targeting ligand, and only identification is horizontal lower, this make its target efficiency and therapeutic effect by Apparent limitation.

The maximum difference of tumor tissues and normal tissue is, the former extracellular environment slant acidity.In recent years, with tumor group The acidic micro-environment knitted is that the anti-tumor drug of target spot is comparatively fast developed.Since height of the tumour cell to glucose absorbs, Glucose forms acidic environment by glycolysis at lactic acid under oxygen free condition；On the other hand, the abnormal vascular of tumour causes tumour to supply oxygen Insufficient, tumour cell transformation growth is out of control to cause anoxic and metabolism is not normal increases anaerobic metabolism；Tumour cell self by Up-regulation hypoxia inducible factor eventually leads to tumor group to adapt to the acidic environment after low-oxygen environment and corresponding glycolysis generation lactic acid The pH value for knitting microenvironment is 5.7-7.0, hence it is evident that lower than the pH 7.4 of normal tissue.Acidic micro-environment is to improve anti-tumor drug choosing One very effective target spot of selecting property.

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.Because being easy the property that the dissolubility difference that agglutination generates is film peptide Matter, pHLIP also has the tendency that agglutination as film peptide, especially under conditions of high concentration and/or low pH, in the water-soluble of neutral pH In liquid, concentration existing for pHLIP monomer is less than 30 μ g/ml, and at low ph conditions, the pHLIP peptide of state I I and state I II are whole Exist with monomeric form.The deliquescent reduction of many studies have shown thats peptide as caused by the change in structure will lead to peptide and film Binding ability and the conformation of entire peptide change.The stability of peptide is very important property in blood, because of blood Protease in liquid can degrade the peptide of L-type Amino acid profile in several minutes.Although the polypeptide of D type Amino acid profile is relatively stable It is more, but due to they can be changed chirality, be not appropriate for in conjunction with specific receptor.Because of pHLIP and double points of lipid There is no the interactions of specificity between sublayer, so the not unexpected pHLIP being made of L-type or D type of people is with identical Biophysics and tumor-localizing characteristic, more and more evidences show pHLIP positioning do not need it is any specificity point The generation of sub- binding events.Only a significant difference is, the left hand helix of D-pHLIP formation cross-film, and L-pHLIP shape At the right-handed helix of cross-film.Compared with the peptide of penetrating cell, pHLIP is rested in cell membrane after being inserted into cell membrane, one end Into cytoplasm, the other end enters extracellular space.Therefore, peptide has dual transmission capacity: a kind of ability is that it can be by cargo To cell surface, another ability is that the cargo molecule that cannot wear film can be injected or is discharged into cytoplasm by it for molecule system.For Realize the first ability, cargo molecule can be connected to the N-terminal of pHLIP, such cargo molecule have wide in range polarity and Image probe such as is delivered to and acid organize and its stable is lain in cell membrane surface by size, the example of an application.In order to It realizes second of ability, cargo molecule can be passed through a C-terminal for being keyed to pHLIP that can be sheared in cytoplasm, Such key entry disulfide bond that can be sheared, one application example be anti-tumor drug is delivered to tumor tissues and by its It imports in tumour cell matter and plays a role, such as fluorescent dye, cyclic peptide, polarity toxin and peptide nucleic acid.

As that studies pHLIP deepens continuously, limit of the application of discovery wild type pHLIP by some key factors System, such as in vivo remove slowly, c-terminus the electrically charged influence to film insertion process.Scholars attempt by pHLIP amino acid Sequence is adjusted, and designs the pHLIP derivative with more preferable performance.PHLIP sequence regulative mode is specifically included that and is 1. cut at present Remove or invert the film insert end of wild type pHLIP sequence；2. with glutaminic acid residue, positively charged lysine residue or protonation Non-standard amino acid residue (γ-carboxylic acid, alpha-amido ethanedioic acid) replace the part or all of aspartic acid of transmembrane region.Through sequence The pHLIP derivative generated is adjusted, such as pHLIP variant 3 (excision film insert end), can reduce by pHLIP electrically charged, quickenings PHLIP enters the process that cell membrane forms transbilayer helix, improves its tumor-targeting.PHLIP variant 7 is keeping good targeting Property while accelerate release rate in blood, be advantageously implemented the internal delivering of drug.The sequence of current pHLIP can be used for reference Column adjusting method develops more advanced pHLIP derivative.

Summary of the invention

One of the objects of the present invention is to provide low pH insertion peptides of a kind of modified and preparation method thereof.

The second purpose of this hair is to provide the composition comprising the low pH insertion peptide of above-mentioned modified, and the composition is used for Treatment, diagnosis or the mark of disease.

The third object of the present invention is to provide the antibody by the low pH insertion peptide of above-mentioned modified for antigen preparation.

In order to complete above-mentioned purpose, this invention takes following technical solutions:

Peptide is inserted into the present invention provides a kind of low pH of modified, and the sequence of the low pH insertion peptide of the modified contains following sequence Column: the extracellular fragment of the low pH insertion peptide of WT or its variant is repeated once, is repeated twice or is repeated twice polypeptide sequence achieved above.

Preferably, the variant of the low pH insertion peptide of WT includes Var1-Var16.

The sequence of the low pH insertion peptide of WT or its variant is as follows:

WT:ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT(SEQ ID NO.2)；

Var1:ACEDQNPYWARYADWLFTTPLLLLDLALLVDG(SEQ ID NO.3)；

Var2:ACEDQNPYWRAYADLFTPLTLLDLLALWDG(SEQ ID NO.4)；

Var3:ACDDQNPWRAYLDLLFPTDTLLLDLLW(SEQ ID NO.5)；

Var4:ACEEQNPWRAYLELLFPTETLLLELLW(SEQ ID NO.6)；

Var5:ACDDQNPWARYLDWLFPTDTLLLDL(SEQ ID NO.7)；

Var6:CDNNNPWRAYLDLLFPTDTLLLDW(SEQ ID NO.8)；

Var7:ACEEQNPWARYLEWLFPTETLLLEL(SEQ ID NO.9)；

Var8:CEEQQPWAQYLELLFPTETLLLEW(SEQ ID NO.10)；

Var9:CEEQQPWRAYLELLFPTETLLLEW(SEQ ID NO.11)；

Var10:ACEDQNPWARYADWLFPTTLLLLD(SEQ ID NO.12)；

Var11:ACEEQNPWARYAEWLFPTTLLLLE(SEQ ID NO.13)；

Var12:ACEDQNPWARYADLLFPTTLAW(SEQ ID NO.14)；

Var13:ACEEQNPWARYAELLFPTTLAW(SEQ ID NO.15)；

Var14:TEDADVLLALDLLLLPTTFLWDAYRAWYPNQECA(SEQ ID NO.16)；

Var15:CDDDDDNPNYWARYANWLFTTPLLLLNGALLVEAEET(SEQ ID NO.17)；

Var16:CDDDDDNPNYWARYAPWLFTTPLLLLPGALLVEAEET(SEQ ID NO.18)；

The part that horizontal line is drawn in above-mentioned sequence is the extracellular fragment sequence of low pH insertion peptide.Var1-Var 16 is WT Variant.

The low pH that sequence the is SEQ ID NO.2-18 extracellular fragment for being inserted into peptide is repeated once, be repeated twice or is repeated twice Polypeptide sequence achieved above includes:

(extracellular fragment)_n+ Linker+SEQ ID NO.2-18, wherein the .. of n=1,2,3,4 ... ....

Linker sequence for use in the present invention can be (GGGS) m, wherein m=1,2,3,4 ... ....

In a specific embodiment of the present invention, it is SEQ ID that the sequence of the low pH insertion peptide of the modified, which is by sequence, The extracellular fragment of the Var7 of NO.9 is repeated once the sequence of acquisition, sequence are as follows: ACEEQNPGGGSACEEQNPWARYLEWLFPTETL LLEL(SEQ ID NO.1)。

In a specific embodiment of the present invention, although by taking Var7 as an example, it was demonstrated that the extracellular fragment of low pH insertion peptide repeats The sequence obtained after primary, which compares former sequence, has a more useful effect, but those skilled in the art's research according to the present invention Achievement can directly skimble-skamble other variants being inferred to for WT, the sequence that extracellular fragment obtains after being repeated once be compared Former sequence equally can have more useful effect, because of the invention the experimental results showed that the extracellular fragment of low pH insertion peptide The general character of advantage, so the variant of the above WT and the WT including Var1-Var 16 are inserted into peptide by improved low pH It is all included in the scope of protection of the present invention.

The present invention provides a kind of composition, the composition includes the low pH insertion peptide of mentioned-above modified.

Further, the composition further includes functive, and the functive includes therapeutic agent, diagnosticum, mark molecule.

Functive pH insertion peptide low with mentioned-above modified can connect in N-terminal or connect in C-terminal.Tool For body, if therapeutic agent plays therapeutic effect by the molecule on cell surface, then the therapeutic agent need to be connected to low pH insertion peptide N-terminal, if therapeutic agent plays therapeutic effect by the molecule of cell interior, then the therapeutic agent need to be connected to the C of low pH insertion peptide End；Diagnosticum is used to show the presence of disease pathology state, and can connect diagnosticum shows that it on cell surface in N-terminal Show, also can connect shows that it in cytoplasm in C-terminal；Mark molecule is for making cell membrane surface not include the mark molecule Cell increase the expression of the mark molecule, therefore in general, mark molecule is connected to the N-terminal of low pH insertion peptide.

Further, the therapeutic agent include but is not limited to antibody drug, small-molecule drug, antibiotic, polypeptide, peptide nucleic acid, Nanoparticle, liposome.

Antibody drug can be the antibody drug for any tumor cells, as long as it can treat the tumour.Antibody Drug includes: molecular targeted monoclonal antibody medicine, targeting antibodies coupling drug, bispecific antibody drug, targeting immunity inspection point medicine Object etc..The example of such antibody drug includes but is not limited to: Rituximab, Herceptin, WAY-CMA 676, A Lundan Anti-, ibritumomab tiuxetan, tositumomab, Avastin, Cetuximab, Victibix, difficult to understand, promise monoclonal antibody, 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) list Anti-, 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, Aunar Zhu Dankang, AVM hereinafter monoclonal antibody, Di Nuosaimai, gemtuzumab, Necitumumab, Atezolizumab.

Further, the antibiotic includes antitumor antibiotics, is generated by microbial metabolism with anti-tumor activity Chemical substance.Antitumor antibiotics for use in the present invention include C1027, mitomycin, adriamycin, CC-1065, Adozelesin, ducarmycins, gilvusmycin, Tetracyclines, cinnamamide, MMI-166, batimastat, green tea Polyphenol, salviandic acid A, C3368-A, C3368-B, rheum emodin, tricyclic pyranone, gel danamycin, 17AAG, Japanese yew acid, Epothilone A, epothilone B, Calicheamicin, lidamycin.

Further, the small-molecule drug is usually signal transduction inhibitor, it can specifically block tumour growth, Necessary signal transduction pathway in breeding, to achieve the purpose that treatment, the example of small-molecule drug includes but unlimited In: Imatinib, nilotinib, Dasatinib, everolimus, Tarceva, Sutent, according to Shandong for Buddhist nun, Sorafenib, gram Azoles replaces Buddhist nun, pazopanib, Gefitinib, Carfilzomib, tropsch imatinib, Axitinib, Rui Gefeini, Wei Luofeini, Xi Luomo Take charge of, moor that for Buddhist nun, it is happy cut down for Buddhist nun, olaparib, VEGF Trap, Ceritinib, romidepsin, Ai Le for Buddhist nun, Baily department he, it is rich It relaxes rich for Buddhist nun, pabishta, Afatinib, Pa Lifuming, Trimetinib, darafinib, Tan Ximo for Buddhist nun, Vande Thani, card Department, Lapatinib, Vorinostat, Venetoclax, Gleevec, Iressa.

Further, the example of the polypeptide include but is not limited to toxin, cyclic peptide, micro-pipe inhibiting factor, it is proteinase activated by Body.The example of toxin such as amanitin, the example of cyclic peptide such as phalloidine, micro-pipe inhibiting factor example such as monomethyl Ai Rui statin E (MMAE), proteinase activated receptors example such as P1AP.

Further, the peptide nucleic acid includes anti-miR (antisense nucleic acid) oligonucleotides peptide.

Further, nanoparticle includes chitosan targeted nanoparticle, long-circulating nanoparticles, polylactic acid nano particle, solid lipid Nanoparticle, carries Doxorubicin nanometer grain, Superparamagnetic Iron Oxide nanoparticle at Jenner's grain of rice.

Further, the liposome includes phosphatide, cholesterol.

Phosphatide of the present invention includes but is not limited to soybean lecithin (SPC), polyethylene glycol 1000 vitamin E succinic acid Ester (TPGS), two myristoyl lecithin (DMPC), dilauroyl lecithin (DLPC), distearoyl lecithin (DPPC), two Palmityl lecithin (DPPC), distearoylphosphatidylcholine (DSPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1- palm fibre Palmitic acid acyl -2- myristoyl lecithin (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1- stearoyl -2- palmityl ovum Phosphatide (SPPC), egg yolk lecithin (EPC), hydrogenated soybean lecithin (HSPC), dioleyl lecithin (DOPC), dioleoyl phospholipid acyl Ethanol amine (DOPE), dilauroylphosphatidylglycerol (DLPG), two palm phosphatidyl glycerols (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), di-myristoyl phosphatidic acid (DMPA), dipalmitophosphatidic acid (DPPA), two Myristoyl phosphatidyl-ethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), two myristoyl phosphatidylserines (DMPS), two palmityl phosphatidyls, two serine (DPPS), cephalin acyl serine (PS), cranial nerve sphingomyelins (BSP), two palm fibres Palmitic acid acyl sphingomyelin (DPSP), distearyl sphingomyelin (DSSP), in Distearoyl Phosphatidylethanolamine (DSPE) Any or several mixing, wherein being preferably: soybean lecithin (SPC), Distearoyl Phosphatidylethanolamine (DSPE) Or dioleoylphosphatidylethanolamine (DOPE).

Further, the diagnosticum includes fluorescent dye.Fluorescent dye include but is not limited to Alexa750, Alexa546, Alexa647、Cy5.5、DyLight 680、DyLight 680 4*PEG-conjugate(DyP680)、680RD (IR680)、800CW (IR800), indocyanine green ICG, PE, Percy-Cy5.5, FITC, APC, Cy7, FITC, GFP、Alexa Fluar488、Bidipy、Fluo-3、Propidium Iodide(PI)、PerCP、PE-Cy5、PE-Teses Red、7-AAD、PE-Cy7、PE-Alexa Flour750、Alexa Fluor660、Alexa Fluor700、APC-Cy7、APC- Alexa Flour750、Hoechsr33342-Blue、DAPI、Hoechsr33342-Red、arific Blue、Cascade Blue、Alexa Flour 405、Parific orange。

Further, the mark molecule includes that tumor surface antigen or its functional domain, tumor surface antigen refer to The antigenic substance for newly occurring or over-expressing in cell surface during tumorigenesis.

The example of tumor surface antigen 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、PD-L2。

The present invention also provides a kind of neoantigen, the neoantigen sequence includes the low pH insertion peptide of mentioned-above modified Extracellular fragment sequence or its variant sequence thereof.

In specific embodiments of the present invention, the neoantigen sequence is as shown in SEQ ID NO.19.

Neoantigen of the invention is with the following functions: (1) antigenic；(2) it is pierced after being connect with carrier protein as immunogene Exciting object generates the antibody of specificity.

The preparation method of neoantigen of the invention can use chemical synthesis: utilizing polypeptide automatic synthesizer, pass through solid phase method Synthetic antigen.

The present invention also provides a kind of nucleic acid molecules, the mentioned-above neoantigen of nucleic acid molecule encoding.

The present invention also provides a kind of recombinant vector, the recombinant vector is by empty carrier and the purpose base for being inserted into the empty carrier Because of composition, the target gene is mentioned-above nucleic acid molecules.

In the present invention, the known various carriers in this field can be selected in described " empty carrier " (or " carrier "), such as commercially available Various plasmids, clay, bacteriophage and retrovirus etc..The empty carrier may include a variety of common detection label (examples Such as fluorescent marker, antibiotic marker reporter gene) and restriction enzyme site.It is polyclonal that empty carrier itself can be used in construction of recombinant vector The various endonucleases in site carry out digestion and obtain linear plasmid, connect with the genetic fragment using the cutting of identical nucleic acid restriction endonuclease It connects, obtains recombinant plasmid.

The present invention also provides a kind of recombinant host cell, the recombinant host cell contains mentioned-above recombination and carries Body.

The recombinant vector can be converted, transduceed or is transfected into host cell by the method for this field routine, It is preferably electroporated such as Calcium Chloride Method chemical conversion, electroporation；The host cell can be prokaryotic cell or eukaryon Cell, preferably Escherichia coli, hay bacillus, yeast (such as Pichia pastoris) or various animal and plant cells, the more preferable host Cell is genetic engineering bacterium commonly used in the art, such as Escherichia coli, bacillus subtilis or Pichia pastoris.

Method commonly used in the art can be used, neoantigen of the invention is separated and purified from recombinant host cell.Example Such as, it is centrifugated culture medium and recombinant host cell, high-pressure homogenization smudge cells, centrifugal filtration remove cell fragment, affine layer Analysis purifying neoantigen.For isolating and purifying the product of resulting neoantigen, method commonly used in the art can be used and carry out purity Identification.For example, Coomassie Brilliant Blue, Kjeldahl's method, biuret method, lowry method, ultraviolet absorption method, affinity chromatography, antigen are anti- Body method, electrophoretic analysis (such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis), analysis by sedimentation, diffusion analysis, permanent solubility Method, protein spectrum etc..

The present invention also provides a kind of fusion protein, the fusion protein include mentioned-above neoantigen and with it is described The albumen or polypeptide of neoantigen connection.

Further, the fusion protein includes mentioned-above neoantigen and the carrier egg with neoantigen coupling It is white.

Carrier protein for use in the present invention includes but is not limited to KLH (keyhole limpet hemocyanin), bovine serum albumin(BSA) (BSA), ovalbumin OVA etc..Since KLH (keyhole limpet hemocyanin) immunogenicity is strong, binding site is more, and immune effect is preferable, And it farther out with immune animal affiliation, uses it to be not easy to cause cross reaction as carrier protein, is therefore preferred.

Fusion protein of the invention has immunogenicity and specificity, is a kind of immunogene, can be used to immune animal to Prepare the antibody for mentioned-above neoantigen of specificity.

The present invention also provides a kind of new antibodies, the new antibodies are with mentioned-above neoantigen or with mentioned-above Fusion protein is prepared.

Preferably, above-mentioned new antibodies of the invention are monoclonal antibodies.

Monoclonal antibody of the invention can be used the ordinary skill in the art and be prepared, system commonly used in the prior art Include: for the method for antibody

(1) based on mouse/rabbit hybridoma technology.

Basic step: animal immune, cell fusion, hybridoma screening and monoclonal antibody detection, hybridoma gram Longhua, the identification of monoclonal antibody and preparation etc..

(2) antibody screening techniques in library are shown based on phage antibody.

Basic step: bone-marrow-derived lymphocyte 1. is separated from the tissue such as peripheral blood or spleen, lymph node, extracts mRNA and reverse transcription For cDNA；2. applying antibody light chain and heavy chain primer, need to expand different Ig gene pieces by round pcr according to build library Section；3. constructing phage vector；4. expression vector converts bacterium, repetoire is constructed.Pass through the affine absorption-of the antigen more taken turns Elution-amplification, finishing screen select the antibody cloning of antigen-specific.

(3) based on the screening technique of clone antibody stock.

The mentioned-above low pH of the present invention is inserted into peptide, and the ordinary skill in the art can be used and be prepared, such conjunction It include: synthesis in solid state, liquid phase synthesis at technology.

The principle of synthesis in solid state is: the carboxyl terminal of amino acid is fixed on insoluble resin by appropriate connection molecule On, it is successively then condensed amino acid by sloughing amino protecting group on this resin, extends peptide chain, until obtaining required polypeptide. Finally Side chain protective group and the pyrolysis product from resin are removed with appropriate reagent.Compared with liquid phase, Solid-phase synthesis peptides advantage exists In: (1) every step reaction only need to simply filter and wash resin, can reach purifying purpose, overcome classical liquid phase synthesizing method In each step product require purifying difficulty, operate it is time saving, it is laborsaving；(2) soluble reagents can be excessive so as to react Complete and acquisition high yield, excess reagent can simply use solvent washing, and filtering is removed；(3) all reactions can be at one It is carried out in container, therefore avoids the formality and loss of reaction intermediate transfer；(4) if selecting connection molecule appropriate and splitting Solution condition, the renewable recycling of macromolecule resin.

The strategy of solid-phase synthetic peptide includes Boc solid phase method, Fmoc solid phase method.In a specific embodiment of the invention, Present invention uses Fmoc solid phase methods.

The present invention provides the low pH insertion peptides of mentioned-above modified to prepare the application in mentioned-above neoantigen.

The present invention provides the low pH insertion peptides of mentioned-above modified to prepare answering in mentioned-above fusion protein With.

The present invention provides the low pH insertion peptides of mentioned-above modified to prepare mentioned-above composition or new antibodies In application.Specifically:

The present invention provides the low pH insertion peptides of mentioned-above modified in preparing tumour medicine targeting delivery system Using.Mentioned-above tumor therapeutic agent and low pH insertion peptide connects, low pH is relied on and is inserted into peptide to the targeting of slightly sour environment, Specific killing is carried out by tumor therapeutic agent targeted delivery to tumor tissues and to it.

The present invention provides the low pH insertion peptides of mentioned-above modified to prepare the application in diagnosing tumor tool.It will use It is connect in mentioned-above diagnostic agent for tumor with low pH insertion peptide, relies on low pH insertion peptide to the targeting of slightly sour environment, make to swell Tumor diagnostic reagent targeted delivery judges it is swollen whether subject suffers to tumor tissues and the presence of marked tumor tissue, with this Tumor.

The present invention provides application of the low pH insertion peptide of mentioned-above modified in preparation tumour mark system.Will before Tumor surface antigen described in face is connect with low pH insertion peptide, is relied on low pH insertion peptide to the targeting of slightly sour environment, is made tumour Surface antigen targeted delivery simultaneously rests on tumor cell surface, identifies tumour cell by tumor surface antigen, is conducive to be directed to The tumour medicine of the specific antigen can play lethal effect to the tumour.By taking HER2 as an example, Herceptin is only to HER2 sun Property patient with breast cancer have therapeutic effect, by by low pH be inserted into peptide connection HER2 targeting be positioned at HER2 negative breast cancer suffer from The breast cancer cell surface of person expands so that Herceptin can also play therapeutic effect to HER2 Breast Cancer Patients with Negative Axillary The scope of application of Herceptin.

The present invention also provides the low pH insertion peptides of mentioned-above modified to prepare the application in CAR-T sequence.With this The low pH insertion peptide of the modified of invention can be used as completely new Scfv sequence design CAR-T sequence as the antibody that antigen selection obtains Column.

The present invention also provides mentioned-above neoantigens to prepare mentioned-above fusion protein or mentioned-above new Application in antibody.

The present invention also provides mentioned-above new antibodies to prepare mentioned-above composition, or preparation CAR-T sequence In application.

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.

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.

Term " peptide nucleic acid " (peptidenucleicacid, PNA) is that a kind of artificial synthesized DNA or RNA are similar in text Object, skeleton is made of N-2 (aminoethyl)-glycine (N (2-aminoethyl) glycine) unit of repeated arrangement, alkali It is connected between base and skeleton with methylene carbonyl key.Since it is without the phosphate group on such as DNA or RNA, electroneutral is shown, with DNA Without electric exclusion between RNA, base pairing high specificity can form stable compound, thermostabilization when combining DNA and RNA Property it is high, PNA is also not easy by protease or nuclease hydrolysis in addition, and therefore, PNA has in biological study and clinical medicine domain Wide application prospect.

In text term " monoclonal antibody " monoclonal antibody be by single B cell clone generate height it is uniform, only for The antibody of a certain specific antigen epitope, referred to as monoclonal antibody.

Polypeptide sequence in the present invention is listed according to the sequence from N-terminal to C-terminal.

The advantages of the present invention:

The present invention has carried out the improvement in sequence on the basis of known low pH insertion peptide, and improved polypeptide is in acidity Tumor tissues microenvironment in there is stronger selectivity, and it is longer to hold time in vivo.

Detailed description of the invention

Fig. 1 shows the fluorogram that low pH insertion peptide var7 is positioned on cell；

Fig. 2 shows the fluorogram that the low pH insertion peptide p-var7 of modified is positioned on cell；

Fig. 3, which is shown, studies the figure that low pH insertion peptide positions in animal body using bioluminescence imaging technology, wherein A:24h；B: 48h；C:72h；D:72h, the tumor tissues that dissection mouse obtains；

Fig. 4 shows the curve graph that 1G12 influences tumour growth；

Fig. 5 shows the statistical chart that 1G12 influences tumor weight；

Fig. 6 shows the statistical chart that 1G12 influences mouse weight；

Fig. 7 shows hepatic pathology colored graph；

Fig. 8 shows Pathological colored graph；

Fig. 9 shows lungs pathological staining figures；

Figure 10 shows large intestine pathological staining figures；

Figure 11 shows spleen pathological staining figures.

Specific embodiment

By can more easily understand the contents of the present invention refering to following embodiments, these embodiments are only further Illustrate the present invention, is not meant to limit the scope of the invention.

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

The synthesis of the low pH insertion peptide of 1 modified of embodiment

It is successively synthesized according to the sequence of SEQ ID NO.9 and SEQ ID NO.1 from c-terminus to aminoterminal.

The connection of (1) first amino acid and resin

2-Chlorotrtyl Chloride Resin resin l g is placed in the peptide synthesis column of dried and clean, 8ml is added DCM is swollen 5min, and vacuum, which is inhaled, abandons solvent.2mmol Fmoc amino acid and 5mmol DIEA are taken respectively, are dissolved in 8ml DCM, and add Enter in resin, ambient temperature with gentle shakes reaction 60min.Vacuum, which is inhaled, abandons solvent.Resin 2 times are washed with 10ml DMF, each 2min. 10ml DCM/ methanol/DIEA (80:15:5) is added, gently shakes reaction 10min, vacuum, which is inhaled, abandons solvent.It is repeated once.With 10ml DMF is washed resin 3 times, each 2min.Vacuum, which is inhaled, abandons solvent, N₂Drying.

The measurement of the Conjugate ratio of (2) first amino acid and resin

It accurately weighs dry one resin of Fmoc amino acid of 2mg to be placed in cuvette, 20% piperidines of 3ml/DMF is added, gently The dynamic reaction 10min of jog uses 20% piperidines/DMF as empty from control zeroing, and ultraviolet specrophotometer measures the 290nm of sample Absorbance value.It replication 2 times, is averaged.Conjugate ratio is calculated by the following formula:

Conjugate ratio (mmol/g)=(Abs sample)/(example weight mg*1.75)

(3) deprotection of Fmoc group

L 0ml is added into resin and is deprotected (DEBLOCK) reagent, mixes, ambient temperature with gentle shakes reaction 5min.It abandons molten Agent washs resin 3 times with 10ml DMF, each 2min.Resin 3 times are washed with 6ml isopropanol, each 5min.It is washed with 6ml hexane Resin 3 times are washed, each 5min.Vacuum, which is inhaled, abandons solvent.A small amount of resin sample is taken, is quickly surveyed with ninhydrin (Kaiser method) Determine the free amine group content on resin: by resin 2ml with ethanol washing 3 times, be separately added into 2 drop, 5% ninhydrin, 80% phenol and KCN (2ml 0.001M KCN:98ml piperidines), mixes well, 120 DEG C of 4~6min of heating.Judge that the deprotection of Fmoc group is anti- Answer degree.

The coupling reaction of (4) second amino acid

The connection of second amino acid use in-situ activation method, take the Fmoc amino acid of 2mmol, the TBTU of 4.0mmol and The HOBT of 4.0mmol is added the DIEA of addition 5mmol after minimal amount of DMF dissolution, after mixing well, is added on de- Fmoc group Resin in.Ambient temperature with gentle shakes reaction 60min.Vacuum, which is inhaled, abandons solvent.Resin 3 times are washed with 5ml methanol, each 5min.With 10ml DMF is washed resin 3 times, each 2min.Vacuum, which is inhaled, abandons solvent.A small amount of resin sample is taken to carry out ninhydrin chromogenic assay.It surveys Determine Conjugate ratio.

(5) extension of peptide chain

The Fmoc blocking group at a upper amino acid N end is sloughed with 10ml DEBLOCK reagent, 10ml DMF washs resin 3 Secondary, vacuum, which is inhaled, abandons solvent.A small amount of resin sample is taken to carry out ninhydrin chromogenic assay.Next amino acid is coupled according to (3) method. Repeat the deprotection of Fmoc blocking group and amino acid couplings reaction, until coupling obtains required polypeptide chain.

(6) peptide chain N-terminal marks Alexa647

The resin of synthetic whole amino acid sequence, sloughs the Fmoc blocking group at amino acid N end, is washed with 10ml isopropanol Resin 3 times are washed, each 5min.1.38g Alexa647,1.6g TBTU53 and 0.76ml DIEA is taken to be added on one tree of peptide after mixing In rouge, ambient temperature with gentle shakes reaction 60min.Vacuum, which is inhaled, abandons solvent.Resin 3 times are washed with 5ml methanol, each 5min.Use 10ml DMF is washed resin 3 times, each 2min.Vacuum, which is inhaled, abandons solvent.

(7) the side chain deprotection of peptide chain and from the cutting on resin

By the resin of synthetic whole amino acid sequences, resin 3 is washed with 6ml isopropanol again after being washed with 10ml DMF It is secondary, each 5min.Resin 3 times are washed with 6ml hexane, each 5min.Vacuum inhales N after abandoning solvent₂Drying, is put into cracking container In.25ml cutting reagent is added in 1g resin, and room temperature cleavage reaction 2h shakes frequently and mixes, and the mixed liquor after reaction is filtered through glass Device filters resin, collects cleavage reaction mixed liquor, is washed resin 3 times with TFA.Reaction mixture is shifted in a round-bottomed flask In, it is washed 4 times with the ether being pre-chilled in equal volume, collects precipitating.Synthesis polypeptide crude product is obtained after drying.

(8) desalination of synthesis polypeptide

Distilled water is added to dissolve polypeptide crude product.Amersham G-25 gel 15g is weighed, fills column after swelling, the column after installing Son first distills water balance with 50ml, and after balance is good, each loading 3-5ml is eluted with distilled water, UV detector inspection UV absorption at 220nm is surveyed, collects polypeptide by peak.

(9) HPLC purified polypeptide

It is isolated and purified using the 4000 preparative HPLC high performance chromatograph of Waters Delta Prep of Waters company more Peptide.Chromatographic column is radial pressurization chromatographic column (25 × 100,15 μm, DELTA PAKC18 filler), elution system are as follows: A liquid: 5% second Nitrile solution (contains 0.1%TFA)；B liquid: 95% acetonitrile solution (contains 0.08%TFA).Hand sampling, each sample introduction 1ml, flow velocity 4ml/min, linear gradient, in 45min, B liquid is raised to 50% from 5%, and 95%B liquid is then raised in 5min and does last elution. UV absorption is detected at 215nrn, is collected component by peak, is used for Mass Spectrometer Method.By the correct Fraction collection of molecular weight detection, very Sky freeze-drying, becomes the sterling of needs, spare.

The positioning on tumour cell that the low pH of embodiment 2 insertion peptide is cultivated in vitro

1, cell line

Human colorectal cancer cells system SW480 (purchase is in ADCC).

2, reagent

1640 culture medium of RPMI (solarbio), fetal calf serum (Yuan Heng JinMa Co., Ltd), PBS (pH=7.4) (Gibco), Hydrochloric acid, (var7 is standard var7, sequence are as follows: Ala-Cys-Glu-Glu-Gln-Asn-Pro- to the var7 of alexa647 label Trp-Ala-Arg-Tyr-Leu-Glu-Trp-Leu-Phe-Pro-Thr-Glu-Thr-Leu-Leu-Leu-Glu-Leu(SEQ ID NO.9)) and alexa647 label p-var7 (the extended version var7 that is repeated once of extracellular fragment that p-var7 is var7, sequence Are as follows: Ala-Cys-Glu-Glu-Gln-Asn-Pro-Gly-Gly-Gly-Ser-Ala-Cys-Glu- Glu-Gln-Asn-Pro- Trp-Ala-Arg-Tyr-Leu-Glu-Trp-Leu-Phe-Pro-Thr-Glu-Thr-Leu-Leu-Leu-Glu-Leu(SEQ ID NO.1)), alexa647 is connected on N-terminal the 2nd Cys of above-mentioned two polypeptide.

3, instrument

Super-clean bench (RONGFENG), carbon dioxide incubator (Thermo), centrifuge (Thermo), the training of laser co-focusing cell It supports ware (20mm) (Corning), electronics pH meter (Sai Duolisi), optical microscopy (Olympus), laser confocal microscope (Nikon)。

4, experimental method

(1) the SW480 cell for being in logarithmic phase is collected, culture solution is discarded, twice with physiology salt washing, is added appropriate 0.25% pancreatin digests not adherent to cell, and appropriate culture solution is added and terminates digestion, moves into 10ml test tube, 1000rpm centrifugation 5min sucks supernatant, RPMI 1640 culture medium of the 1ml containing 10% fetal calf serum is added, mixing cell is resuspended.Therefrom draw 10 μ l Cell suspension is added in cell counting board after counting, and a certain amount of cell suspension is taken to be added in laser co-focusing Tissue Culture Dish, It is adjusted afterwards with complete medium to 5*10⁵, the system of 1ml cell is put into cell incubator overnight incubation.

(2) culture solution is prepared using the PBS buffer solution of the hydrochloric acid of 1mol/L and pH=7.4.PBS is added dropwise in hydrochloric acid to delay In fliud flushing, the pH value of final Titration Buffer to 6.3.By the peptide of synthesis ((p-var7 and var7) be separately added into pH be 6.3 and It is mixed well in 7.4 PBS buffer solution, is diluted to the final concentration of 2.5 μm of ol/L of peptide, as PBS containing peptide culture solution in proportion.

(3) the SW480 cell after overnight incubation is adherent, sucks culture medium supernatant, is washed with the PBS buffer solution of pH=7.4 It washs twice.

(4) PBS buffer solution in Tissue Culture Dish is sucked, 1ml previously configured pH is separately added into two wares =6.3 and 7.4 PBS and the mixed culture buffer of peptide, and pH of the 1ml without peptide is added in other control group culture dish =7.4 PBS culture medium is put into 37 DEG C of cell incubators and is incubated for 1 hour.

(5) suck the culture solution supernatant of PBS containing peptide after being incubated for, with respectively organize different pH value without peptide PBS buffer solution It rinses three times, the rear PBS buffer solution that pH=7.4 is added.

(6) Tissue Culture Dish prepared is placed under laser confocal microscope (647mm excitation wavelength) observation carefully After birth surface fluorescence expression.

3, experimental result:

As a result as depicted in figs. 1 and 2, it is thin to be effectively inserted into human colon carcinoma in acid solution environment by var7 and p-var7 The surface born of the same parents SW480, but the film of p-var7 is inserted into Disability (Fig. 2) in neutral solution environment, and var7 remains the ability (Fig. 2), display p-var7 have stronger selectivity in acid tumor tissues microenvironment.

The low pH of embodiment 3 is inserted into the positioning of peptide in animal body

1, experimental procedure:

Mouse colonic cell CT26 inoculates Balb/c mouse, and tumour grows to about 1cm, and vein injects physiology salt respectively Water (N.S.), the p-var7 of alexa647 label, the var7 of alexa647 label, dosage 60 μ of μ Μ/100 LN.S., 24,48,72 Hour living imaging (being excited with the wavelength of Cy5.5), dorsal position photograph.It puts to death mouse and takes tumor imaging.

2, experimental result

As a result such as Fig. 3 is the results show that var7 and p-var7 can mark tumour, but the marked capacity of p-var7 is more By force, and as time goes by, the fluorescence intensity decay of var7 is more significant, when by 72 hours, tumor tissues only visible a small amount of label, On the contrary, p-var7 still visible stronger fluorescent marker at 72 hours.It should be the experiment proves that p-var7 can be targeted in vivo Tumor tissues, and maintain the long period.What black arrow represented in A figure is tumor tissues.

The tumor killing effect for the antibody that embodiment 4 is prepared using p-var7 extracellular fragment as antigen is evaluated

Experimental material: MC38 cell is purchased from ATCC；P-var7 polypeptide (molecular weight 4095Da) entrusts Beijing Hua Da egg The synthesis of Co., Ltd, white matter research and development centre, is dissolved in PBS, and concentration is 40 μM；Extracellular fragment (the Ala-Cys-Glu-Glu- of p-var7 Gln-Asn-Pro-Gly-Gly-Gly-Ser-Ala-Cys-Glu-Glu-Gln-Asn-Pro, SEQ ID NO.19) connection KLH (synthesis of Beijing Jin Sirui Biotechnology Co., Ltd)；6-8 week old female C57/BL6 mouse is purchased from dimension tonneau China.

1, Antibody preparation:

(1) step: being immunized Balb/c mouse with the extracellular fragment of the p-var7 of KLH connection, prepare hybridoma, obtain 2 plants of monoclonal antibodies, The specific bond and antibody subtype of ELISA detection antibody and antigen.

(2) result:

The results show that the monoclonal antibody for being named as 1G12 and 1G1 can be in conjunction with antigen-specific, wherein 1G12 affinity is higher, 1G1 Affinity is lower, and OD value is respectively 1.4423,0.4924, and this 2 plants are IgG1 hypotype, and the antibody concentration of preparation is about 0.7mg/ml。

2, antibody tumor killing effect is evaluated

(1) establish mouse junction cancer MC38 Transplanted tumor model: MC38 is inoculated in C57/BL6 mouse, long to 1cm to diameter of tumor When, sterile removing tumour shreds, and single cell suspension is made in homogenate, strainer, and amplification is cultivated in 1640 complete mediums, will be thin Born of the same parents are injected in C57/BL6 mouse side subcutaneous abdomen, 2x10⁶A cell/only, it is long to 0.8-1cm to diameter of tumor, reject excessive and mistake Small tumour, by the almost the same mice group of tumor size.It is divided into 4 groups: p-var7 independent injection group, 10；P-var7 connection Conjunction 1G12 injection group, 10；P-var7 joint 1G1 injection group, 10；Physiological saline N.S injection group, 10.It was measured every 3 days Tumor size.

(2) medication: p-var7 administration: every 40 μM/100 μ l of intravenous injection (about 16 μ g, with reference to living before every time The experimental result of body imaging, the injection of same dose are able to observe that fluorescence in the aggregation of tumor locus, but to third day Shi Ji This recession), it was injected the same day after the completion of grouping, injection in one day is primary, primary every injection in 2 days；Antibody administration: abdominal cavity note It penetrates, dosage 5mg/kg (molar ratio of antibody and p-var7 are about 1:5), frequency of injection is identical as p-var7, and injection time is in p- 6-12 hours after var7 administration, until terminating.

(3) result:

As a result as shown in Figure 4 and Figure 5,1G12 can significantly inhibit tumour growth, and inhibiting rate does not have for 50%, 1G1 at 2 weeks Apparent tumor killing effect；Mouse physical condition is good in therapeutic process, does not occur the symptoms such as movable decline, diarrhea, weight loss, Fig. 6 shows that mouse weight is constant；Pathological examination shows that the organ for the treatment of group's Mouse Liver kidney lung spleen intestines does not occur organic change (Fig. 7-11).

The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that Specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, exist Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to of the invention Protection scope.

Sequence table

<110>Beijing Ze Qin biological medicine Co., Ltd

<120>a kind of low pH of modified is inserted into peptide

<150> 2017113770766

<151> 2017-12-19

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Ala Cys Glu Glu Gln Asn Pro Gly Gly Gly Ser Ala Cys Glu Glu Gln

1 5 10 15

Asn Pro Trp Ala Arg Tyr Leu Glu Trp Leu Phe Pro Thr Glu Thr Leu

20 25 30

Leu Leu Glu Leu

35

<210> 2

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<213>artificial sequence (Artificial Sequence)

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Ala Cys Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu

1 5 10 15

Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala

20 25 30

Asp Glu Gly Thr

35

<210> 3

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Ala Cys Glu Asp Gln Asn Pro Tyr Trp Ala Arg Tyr Ala Asp Trp Leu

1 5 10 15

Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Gly

20 25 30

<210> 4

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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

1 5 10 15

Thr Pro Leu Thr Leu Leu Asp Leu Leu Ala Leu Trp Asp Gly

20 25 30

<210> 5

<211> 27

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<213>artificial sequence (Artificial Sequence)

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Ala Cys Asp Asp Gln Asn Pro Trp Arg Ala Tyr Leu Asp Leu Leu Phe

1 5 10 15

Pro Thr Asp Thr Leu Leu Leu Asp Leu Leu Trp

20 25

<210> 6

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<213>artificial sequence (Artificial Sequence)

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Ala Cys Glu Glu Gln Asn Pro Trp Arg Ala Tyr Leu Glu Leu Leu Phe

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Pro Thr Glu Thr Leu Leu Leu Glu Leu Leu Trp

20 25

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Ala Cys Asp Asp Gln Asn Pro Trp Ala Arg Tyr Leu Asp Trp Leu Phe

1 5 10 15

Pro Thr Asp Thr Leu Leu Leu Asp Leu

20 25

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<213>artificial sequence (Artificial Sequence)

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Cys Asp Asn Asn Asn Pro Trp Arg Ala Tyr Leu Asp Leu Leu Phe Pro

1 5 10 15

Thr Asp Thr Leu Leu Leu Asp Trp

20

<210> 9

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<213>artificial sequence (Artificial Sequence)

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Ala Cys Glu Glu Gln Asn Pro Trp Ala Arg Tyr Leu Glu Trp Leu Phe

1 5 10 15

Pro Thr Glu Thr Leu Leu Leu Glu Leu

20 25

<210> 10

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<213>artificial sequence (Artificial Sequence)

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Cys Glu Glu Gln Gln Pro Trp Ala Gln Tyr Leu Glu Leu Leu Phe Pro

1 5 10 15

Thr Glu Thr Leu Leu Leu Glu Trp

20

<210> 11

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Cys Glu Glu Gln Gln Pro Trp Arg Ala Tyr Leu Glu Leu Leu Phe Pro

1 5 10 15

Thr Glu Thr Leu Leu Leu Glu Trp

20

<210> 12

<211> 24

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<213>artificial sequence (Artificial Sequence)

<400> 12

Ala Cys Glu Asp Gln Asn Pro Trp Ala Arg Tyr Ala Asp Trp Leu Phe

1 5 10 15

Pro Thr Thr Leu Leu Leu Leu Asp

20

<210> 13

<211> 24

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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> 14

<211> 22

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<213>artificial sequence (Artificial Sequence)

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Ala Cys Glu Asp Gln Asn Pro Trp Ala Arg Tyr Ala Asp Leu Leu Phe

1 5 10 15

Pro Thr Thr Leu Ala Trp

20

<210> 15

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<213>artificial sequence (Artificial Sequence)

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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> 16

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<213>artificial sequence (Artificial Sequence)

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Thr Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu Pro

1 5 10 15

Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Pro Asn Gln Glu

20 25 30

Cys Ala

<210> 17

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Cys Asp Asp Asp Asp Asp Asn Pro Asn Tyr Trp Ala Arg Tyr Ala Asn

1 5 10 15

Trp Leu Phe Thr Thr Pro Leu Leu Leu Leu Asn Gly Ala Leu Leu Val

20 25 30

Glu Ala Glu Glu Thr

35

<210> 18

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<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> 19

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<213>artificial sequence (Artificial Sequence)

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Ala Cys Glu Glu Gln Asn Pro Gly Gly Gly Ser Ala Cys Glu Glu Gln

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Asn Pro

Claims

1. a kind of low pH of modified is inserted into peptide, which is characterized in that the low pH insertion peptide of the modified contains following sequence: sequence is The low pH insertion peptide of SEQ ID NO.2 or the extracellular fragment of its variant are repeated once, are repeated twice or are repeated twice achieved above Polypeptide sequence；Preferably,

It includes sequence such as SEQ ID NO.3-SEQID NO.18 that the low pH that the sequence is SEQ ID NO.2, which is inserted into the variant of peptide, Shown in polypeptide.

2. the low pH of modified according to claim 1 is inserted into peptide, which is characterized in that the low pH of the modified is inserted into peptide from N Hold the sequence of C-terminal to be expressed as follows: sequence described in (extracellular fragment) n+Linker+ be SEQ ID NO.2 low pH be inserted into peptide or its Variant, wherein the .. of n=1,2,3,4 ... ...；Preferably, the Linker sequence is (GGGS) m, wherein m=1,2,3, 4…………；It is highly preferred that the Linker sequence is GGGS.

3. the low pH of modified according to claim 1 or 2 is inserted into peptide, which is characterized in that the low pH of the modified is inserted into peptide Sequence as shown in SEQ ID NO.1.

4. a kind of composition, which is characterized in that the composition includes the low pH of modified of any of claims 1-3 It is inserted into peptide or the low pH of any of claims 1-3 for SEQ ID NO.2 is inserted into peptide or its variant；Preferably,

The composition further includes functive, and the functive includes therapeutic agent, diagnosticum or mark molecule；The functive The N-terminal of pH insertion peptide low with the modified is connect or C-terminal connection.

5. composition according to claim 4, is characterized in that, the therapeutic agent includes antibody drug, small-molecule drug, resists Raw element, polypeptide, peptide nucleic acid, nanoparticle or liposome；Preferably, the polypeptide include toxin, cyclic peptide, micro-pipe inhibiting factor or Proteinase activated receptors；Preferably, the peptide nucleic acid includes antisense nucleic acid oligonucleotide peptide；

The diagnosticum includes fluorescent dye；

The mark molecule includes tumor surface antigen or its functional domain；Preferably, 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。

6. a kind of neoantigen, which is characterized in that the neoantigen sequence includes modified of any of claims 1-3 The extracellular fragment sequence or its variant sequence thereof of low pH insertion peptide, it is preferable that the neoantigen sequence is as shown in SEQ ID NO.19.

7. a kind of nucleic acid molecules, which is characterized in that the nucleic acid molecule encoding neoantigen as claimed in claim 6.

8. a kind of recombinant vector, which is characterized in that the recombinant vector is by empty carrier and the target genome for being inserted into the empty carrier At the target gene is nucleic acid molecules as claimed in claim 7.

9. a kind of recombinant host cell, which is characterized in that the recombinant host cell contains recombination according to any one of claims 8 and carries Body.

10. a kind of fusion protein, which is characterized in that the fusion protein include neoantigen as claimed in claim 6 and with institute State the albumen or polypeptide of neoantigen connection；Preferably, the fusion protein include neoantigen as claimed in claim 6 and with institute State the carrier protein of neoantigen coupling；It is highly preferred that the carrier protein includes KLH, BSA or OVA.

11. a kind of new antibodies, which is characterized in that the new antibodies are by neoantigen as claimed in claim 6 or claim 10 The fusion protein is prepared.

12. the low pH insertion peptide of modified of any of claims 1-3 is preparing combination described in claim 4 or 5 Object prepares neoantigen as claimed in claim 6, prepares described in fusion protein described in any one of claim 10, preparation claim 11 New antibodies, or prepare the application in CAR-T sequence.

13. neoantigen as claimed in claim 6 is being prepared described in fusion protein described in any one of claim 10 or claim 11 Application in new antibodies.

14. the composition described in preparation claim 5 of new antibodies described in claim 11, or prepare in CAR-T sequence Using.