CN103539840B

CN103539840B - Epidermal growth factor receptor (EGFR) mimotope peptide and application thereof

Info

Publication number: CN103539840B
Application number: CN201310442612.1A
Authority: CN
Inventors: 李宗海; 杨麟; 王华茂; 王海; 蒋华; 石必枝
Original assignee: Shanghai Cancer Institute
Current assignee: Shanghai Cancer Institute
Priority date: 2010-01-20
Filing date: 2010-01-20
Publication date: 2015-04-01
Anticipated expiration: 2030-01-20
Also published as: CN103539840A

Abstract

The invention relates to an epidermal growth factor receptor (EGFR) mimotope peptide and an application thereof. The mimotope peptide can be competitively combined with an EGFR-resistant antibody, which shows that a mimotope is similar to a natural epitope in biology and structure. The mimotope peptide provided by the invention is beneficial to the great generation of specific antibodies in animal bodies and capable of solving the technical problems that the natural peptide epitope immunogenicity is low and the generated antibodies are insufficient.

Description

EGF-R ELISA analogue epi-peptide and application thereof

The divisional application of the present invention's to be application number be application for a patent for invention of 201010023003.9.

Technical field

The invention belongs to biological technical field; More specifically, the present invention relates to EGF-R ELISA analogue epi-peptide and application thereof.

Background technology

EGF-R ELISA (EGFR) is a molecular weight is the tyrosine kinase receptor of 170KD, and it plays considerable effect for the propagation of different cell and differentiation.The enhancing of the vicious transformation of cell and the signal path of EGFR has close contact.The activation of the signal path of EGFR can promote the transfer of the propagation of cell, the invasion and attack of tumour, the angiogenesis of inside tumor and tumour.EGFR is process LAN in many epithelial origin tumours, and the level of process LAN and the prognosis of patient have close contacting.The process LAN of EGFR normally by the amplification of gene, also or due to Urogastron sudden change causes.The mutant of modal EGFR is EGF-R ELISA type III varient (EGFRvIII), its major deletions 801 bases of encoding sequence exon 2 to the 7th exon, this directly results in the amino acid whose disappearance in 267, EGFR extracellular region, and aobvious sub-connection place defines a new glycine outside.EGFRvIII is at cerebral glioma, and mammary cancer, can be detected in nonsmall-cell lung cancer and prostate cancer, not express in the normal tissue.

Because EGFR has important impact in the generation evolution of cancer, therefore for the methods for the treatment of of EGFR also in continuous development.MAb806 monoclonal antibody (Luwor RB etc., Monoclonalantibody806inhibits the growth of tumor xenografts expressing either thede2-7or amplified epidermal growth factor receptor (EGFR) but notwild-type EGFR.Cancer Res.2001; 61 (14): 5355-61.) can identify EGFR and EGFRvIII of process LAN more specifically, research shows that mAb806 can suppress process LAN EGFR or express the Growth of Cells of EGFRvIII.The clinical I phase is studied and shows; the mouse-human chimeric CH806 of mAb806 monoclonal antibody can specifically target tumor tissue (Scott AM etc., A phase I clinical trialwith monoclonal antibody ch806targeting transitional state and mutantepidermal growth factor receptors.Proc Natl Acad Sci U S A.2007; 104 (10): 4071-6.).Peptide sequence on mAb806 monoclonal antibody identification EGFR is ²⁸⁷cGADSYEMEEDGVRKC ³⁰²(Johns TG etc., Identification of the epitopefor the epidermal growth factor receptor-specific monoclonal antibody806reveals that it preferentially recognizes an untethered form of the receptor.JBiol Chem.2004Jul16; 279 (29): 30375-84.).So this section of peptide sequence is expected to as vaccine.But because this section of polypeptide just exists in the normal tissue, if be directly used in human body, may be difficult to produce corresponding monoclonal antibody because of immunological tolerance, therefore need the analogue epi-peptide (mimotope) finding this section of polypeptide, for the development of anti-tumor vaccine.The present inventor has screened a monoclonal antibody 12H23 (international patent application no: PCT/CN2009/074090) in early-stage Study, it can identify the EGFR of EGFRvIII and process LAN, and effectively can suppress the growth of HuH7-EGFRvIII human liver cancer cell and SMMC-7721 cell in experiment in vitro in vivo.Research finds that the epitope polypeptide sequence of this antibody is also ²⁸⁷cGADSYEMEEDGVRKC ³⁰².

Monoclonal antibody is applied to and clinically still there are some restrictions, such as high medical expense, still dissatisfactory curative effect, from mouse-anti or chimeric antibody side effect and need repeatedly immunity could produce enough tiring.If carry out immune human body with the structural simulation polypeptide of antibody combining site, make human body self produce active immunity, and can continue to produce antibody, just likely solve the problem that monoclonal antibody cann't be solved.Analogue epi-peptide, structurally synantibody is similar in conjunction with epi-position, but aminoacid sequence is likely different, is conducive to a large amount of antibody produced for this epi-position in human body like this.Therefore, the vaccine producing active immunity, in the urgent need to finding the analogue epi-peptide of described epitope polypeptide, can be induced in this area.

Summary of the invention

The object of the present invention is to provide EGF-R ELISA analogue epi-peptide and application thereof.

The present invention also aims to provide one to have immunogenic material and application thereof.

The present invention also aims to provide containing described acceptor analogue epi-peptide or the pharmaceutical composition with immunogenic material.

In a first aspect of the present invention, provide a kind of isolated polypeptide, described polypeptide is the polypeptide being selected from aminoacid sequence as shown in SEQ IDNO:1 or SEQ ID NO:2.

In another aspect of this invention, provide one to have immunogenic material, described material is connected with antigen, and described antigen contains SEQ ID NO:1 or the aminoacid sequence shown in SEQ ID NO:2; Or described substance gives expression to antigen, described antigen contains SEQ ID NO:1 or the aminoacid sequence shown in SEQ ID NO:2.

In a preference, the described immunogenic material that has connects the protein macromolecule that (coupling) has antigen.

In another preference, the described immunogenic material that has had described in immunogenic material comprises: keyhole limpet hemocyanin; And

Be coupled to the antigen of described keyhole limpet hemocyanin, described antigen contains SEQ ID NO:1 or the aminoacid sequence shown in SEQID NO:2.

In another preference, described antigen comprises:

The polypeptide of aminoacid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2; And

The connection peptides be connected with described polypeptide, described connection peptides has 1-20 (preferably 4-10) amino acid.

In another preference, described connection peptides is positioned at the carboxyl terminal of the polypeptide of aminoacid sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

In another preference, described connection peptides has the aminoacid sequence shown in SEQ ID NO:4.

In another aspect of this invention, provide a kind of polynucleotide of separation, the polypeptide described in described polynucleotide encoding.

In another aspect of this invention, provide the purposes of described polypeptide, for the preparation of induction of antibodies produce pharmaceutical composition, described antibody recognition EGF-R ELISA.

In a preference, described polypeptide is for the preparation of prevention or the pharmaceutical composition for the treatment of tumour.

In another aspect of this invention, provide the described purposes with immunogenic material, for the preparation of induction of antibodies produce pharmaceutical composition, described antibody recognition EGF-R ELISA.

In a preference, described has immunogenic material for the preparation of prevention or the pharmaceutical composition for the treatment of tumour or oral lichen planus and hickie.

In another aspect of this invention, provide a kind of pharmaceutical composition, described pharmaceutical composition contains:

One or more of significant quantity polypeptide described in (2 kinds), or described in one or more (2 kinds), there is immunogenic material; With pharmaceutically acceptable carrier.

In a preference, described pharmaceutical composition is vaccine.

In another preference, the immunological adjuvant of described pharmaceutical composition also containing significant quantity.

In another aspect of this invention, provide a kind of medicine box, contain in described medicine box: the polypeptide described in one or more; Or described in one or more, there is immunogenic material; Or described pharmaceutical composition.

Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.

Accompanying drawing explanation

Fig. 1 shows the binding specificity analysis of monoclonal antibody 12H23 to the phage clone after enrichment.

Wherein, 1,2,3 be 12H23 with show the M13 phage clone of specific peptide sequence or wild-type M13 phage in conjunction with situation, 4 be isotype control Ab and described phage clone in conjunction with situation, 5 be BSA and described phage clone in conjunction with situation.

Fig. 2 shows and adopts ELISA to carry out phage competitive binding assay.

1 in conjunction with the competition binding situation of 12H23 (left side) with the phage and G-HCL that contrast IgG (right side);

2 in conjunction with the competition binding situation of 12H23 (left side) with the phage and N12-806 that contrast IgG (right side);

3 in conjunction with the competition binding situation of 12H23 (left side) with the phage and S1-GFP that contrast IgG (right side);

4 in conjunction with the competition binding situation of 12H23 (left side) with the phage and cc16 that contrast IgG (right side);

5 in conjunction with the competition binding situation of 12H23 (left side) with the phage and control peptide that contrast IgG (right side).

Fig. 3 shows the joint efficiency of elisa assay improvement on synthesis and 12H23.

Wherein, 1 for 12H23 antibodies is to improvement on synthesis WHTEILKSYPHE – KLH; 2 for 12H23 antibodies is to improvement on synthesis LPAFFVTNQTQD-KLH; 3 for 12H23 antibodies is to improvement on synthesis control peptide-KLH; 4 for 12H23 antibodies is to KLH.

Fig. 4 shows the reactivity that Western blot detects the clone of anti-polypeptide serum and EGFRvIII or EGFR process LAN.The antibody (in mouse serum) of monoclonal antibody 12H23 or mimic epitopes inducing peptide and Huh7-EGFRvIII and A431 cytolytic proteins in conjunction with situation, EGFRvIII and EGFR can detect (swimming lane 1 respectively at 130kDa and 170kDa, positive control), band is produced with cytolytic proteins at 130kDa and 170kDa by the mice serum (swimming lane 2) of WHTEILKSYPHE-KLH conjugate immunity, band is produced with cytolytic proteins at 130kDa and 170kDa by the mice serum (swimming lane 3) of LPAFFVTNQTQD-KLH conjugate immunity, by the immunity of control peptide-KLH conjugate mice serum (swimming lane 4) or there is no band by the mice serum (swimming lane 5) of KLH immunity separately.

Fig. 5 shows immunofluorescence results, and serum used or antibody are respectively: the mice serum after A, use WHTEILKSYPHE-KLH immunity; Mice serum after B, use LPAFFVTNQTQD-KLH immunity; Serum after C, contrast polypeptide-K LH immunity; Mice serum after D, KLH immunity; E, 12H23 monoclonal antibody.

Embodiment

The present inventor, through deep research, discloses a kind of analogue epi-peptide of EGF-R ELISA first.Described analogue epi-peptide competitively in conjunction with the antibody (as 12H23) of anti-EGFR, can show mimic epitopes close with natural epi-position on biology and structure.Analogue epi-peptide of the present invention is conducive to producing specific antibody in a large number in animal body, overcomes the technical barrier that native peptides epitope immunogenic is low, can not produce enough antibody.

As used herein, described " analogue epi-peptide " refers to the polypeptide (as table 1) or its derived peptide with aminoacid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, its on biology and structure with the natural epi-position of EGF-R ELISA ²⁸⁷cGADSYEMEEDGVRKC ³⁰²close, there is the function that induction body produces the antibody of anti-epidermal growth factor receptor.Described " analogue epi-peptide " is in the text also referred to as " epitope peptide of the present invention " or " described epitope peptide ".In the present invention, term " polypeptide ", " albumen " are used interchangeably.

Table 1

	Sequence
		SEQ ID NO:1	WHTEILKSYPHE
SEQ ID NO:2	LPAFFVTNQTQD

As used herein, " separation " refers to that material is separated from its primal environment (if natural substance, namely primal environment is natural surroundings).Such as, the polynucleotide under the native state in active somatic cell and polypeptide do not have separation and purification, if but other materials that same polynucleotide and polypeptide exist together with native state separate, then for separation and purification.

As used herein, " immunocompetence " or " immunogenicity " refers to by the specificity humoral in the vaccine-induced mammalian body of natural, restructuring or synthesis and/or the ability of cellullar immunologic response.

As used herein, " immunne response " comprises cellularity and/or humoral immune response, and they are enough to the antibody producing specificity anti-epidermal growth factor receptor; Or prevent or suppress the disease that caused by EGF-R ELISA overexpression.

As used herein, the composition of " pharmaceutically acceptable " is applicable to people and/or Mammals and without excessive bad side reaction (as toxicity, stimulation and transformation reactions), namely has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to the carrier being used for the treatment of agent administration, comprises various vehicle or thinner.

As used herein, " significant quantity " or " immune significant quantity " refers to that it is effective for giving individual amount to treatment or prevention with single dose or a continuous agent part.This consumption according to treat that the classification (as non-human primates etc.) of individuality is treated by individual healthy state and physiological situation, institute, the ability of individual immunity system synthesis antibody, required degree of protection, the preparation of vaccine, treating physician determine the assessment of medical conditions and other correlative factor.Estimate that this consumption is by relatively wide scope, determines by normal experiment.

Analogue epi-peptide of the present invention and encoding gene thereof

The epi-position that monoclonal antibody 12H23 has been proved it is substantially identical with external monoclonal antibody 806, the EGF-R ELISA (EGFR) of EGF-R ELISA type III varient (EGFRvIII) and process LAN can be identified, and effectively can suppress the propagation of the A431 human lung carcinoma cell of HuH7-EGFRvIII human liver cancer cell and process LAN EGFR in experiment in vitro in vivo.But mab treatment application clinically also also exists many bottlenecks, the method that passive antibody immunity needs repeated multiple times immunity just can effectively be tired and addresses these problems is exactly a kind of new vaccines of research and development, by with can by the mimic epitopes immune mouse of antibody recognition, excite the active immunity of mouse, make himself generation biological property be similar to the antibody of 12H23, thus play the effect of targeted therapy.The present inventor screens monoclonal antibody 12H23 with phage library, and obtaining 2 mimic epitopess (shown in sequence SEQ ID NO:1 or SEQ ID NO:2) by euzymelinked immunosorbent assay (ELISA) checking all can effectively in conjunction with monoclonal antibody 12H23.Then by immune mouse after these 2 mimic peptide coupling high molecular weight proteins, for high molecular weight protein in assessment immunized mice serum, the antibody titer of the EGFR of mimic epitopes and EGFRvIII and process LAN, and the antibody in the results show serum can identify above-mentioned antigen effectively, and the A431 human lung carcinoma cell of HuH7-EGFRvIII human liver cancer cell and process LAN EGFR.The results show the present inventor success screens mimic epitopes from phage library.

Analogue epi-peptide of the present invention has the similar biology of nature epi-position and chemical property, therefore close in sequence, and can by antibody recognition.Meanwhile, the peptide section that these screen does not need with natural epi-position identical on space structure, only needs to have nature epi-position by the function of antibody recognition.Described analogue epi-peptide can solve as a vaccine problem that passive immunization brings and can become a kind of therapeutic or preventative new drug.

After the aminoacid sequence obtaining cicada analogue epi-peptide of the present invention, those skilled in the art can prepare this epitope peptide easily, and it can be improvement on synthesis, recombinant polypeptide.Epitope peptide of the present invention can be the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (such as, bacterium, yeast and mammalian cell).

The present invention also comprises the fragment of described analogue epi-peptide, derivative and analogue.As used herein, term " segment ", " derivative " and " analogue " refer to the polypeptide substantially keeping biological function, structure or activity that analogue epi-peptide of the present invention is identical.Analogue epi-peptide segment of the present invention, derivative and analogue can be: the polypeptide that (1) has one or more conservative or non-conservative amino acid residue (preferred conservative amino acid) to be substituted, or (2) have the polypeptide of substituted radical in one or more amino-acid residue, or (3) mature polypeptide and another compound (such as extend the compound of polypeptide transformation period, such as polyoxyethylene glycol) merge the polypeptide that formed, or the polypeptide (fusion rotein) that (4) additional aminoacid sequence is blended in this peptide sequence and is formed.

Described analogue epi-peptide itself can be used as vaccine immunity animal and produces the antibody identifying EGF-R ELISA; Or can be connected with other albumen formation fusion rotein, immune animal identifies the antibody of EGF-R ELISA to produce; Or can form with the coupling of macromole phase and there is immunogenic material; Or can be expressed or be illustrated in cell surface or bacteriophage coat protein on the surface, immune animal identifies the antibody of EGF-R ELISA to produce.

A kind of purposes of analogue epi-peptide of the present invention is: as chemoprophylaxis or treatment EGF-R ELISA overexpression relative disease.Described disease is such as tumour.

The polynucleotide of epitope peptide of the present invention of encoding can be DNA form or rna form.DNA can be strand or double-strand.Term " polynucleotide of coded polypeptide " can be the polynucleotide comprising epitope peptide of the present invention of encoding, and also can be the polynucleotide also comprising additional code and/or non-coding sequence.

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, the sum analogous to general Dedekind sum of polypeptide with analogue epi-peptide of the present invention.

Polynucleotide of the present invention can obtain by the method for pcr amplification method, recombination method or synthetic usually.For pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the cDNA storehouse prepared by ordinary method well known by persons skilled in the art as template, amplification and relevant sequence.Described polynucleotide are normally cloned into carrier by described recombination method, then proceed to cell, are then separated from the host cell after propagation by ordinary method and obtain relevant sequence.Relevant sequence can be synthesized in addition by the method for synthetic.

At present, the DNA sequence dna of code book invention epitope peptide (or its fragment, or derivatives thereof) can be obtained completely by chemosynthesis.Then this DNA sequence dna can be introduced in various existing DNA molecular (as carrier) as known in the art and cell.In addition, also by chemosynthesis, sudden change is introduced in protein sequence of the present invention.

The present invention also relates to the carrier of the polynucleotide comprising described analogue epi-peptide, and the host cell that described carrier or mimic epitopes peptide-coding sequence produce through genetically engineered, and the method for described analogue epi-peptide is produced through recombinant technology.

Comprise the polynucleotide of the analogue epi-peptide described in above-mentioned coding and the carrier of suitably promotor or control sequence, may be used for transforming suitable host cell, with can marking protein.

Described analogue epi-peptide at cell inner expression or can be secreted into extracellular.Can utilize its physics, the albumen of being recombinated by various separation method abstraction and purification with other characteristic of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation process, combination by protein precipitant process (salting-out method), centrifugal, the broken bacterium of infiltration, super process, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

Immunogenic substance

Because micromolecular polypeptide easily causes body to produce immunological tolerance, as a preferably embodiment, described analogue epi-peptide is connected with macromole.Described macromole can be (but being not limited to): albumen, organism.

As a kind of optimal way, the invention provides one and have immunogenic material, this material comprises described analogue epi-peptide and protein macromolecule.Preferably, described protein macromolecule is keyhole limpet hemocyanin (KLH, its sequence is as SEQ ID NO:3).As a kind of immune carrier, KLH is the macromolecular substance with high degree of immunogenicity, has haptenic ability immunogenicity being passed to coupling.

Between described analogue epi-peptide and protein macromolecule, be connected or coupling mutually by chemical bond; Described chemical bond is covalent linkage or non covalent bond.

As one preferably embodiment, mutual coupling between described analogue epi-peptide and protein macromolecule, protein macromolecule can with at least one analogue epi-peptide phase coupling.

As a kind of embodiment, described analogue epi-peptide is connected by chemical bond with between protein macromolecule; Better, described chemical bond is peptide bond.

As optimal way of the present invention, the described immunogenic material that has comprises: keyhole limpet hemocyanin (KLH) and one or more analogue epi-peptide.On space structure, coupling analogue epi-peptide on the surface of described keyhole limpet hemocyanin molecule.

Directly can be connected between described analogue epi-peptide and protein macromolecule (or coupling), or connected by polypeptide linker (connection peptides).Described connexon such as comprises 1-20 amino acid; Be preferably 4-10 amino acid.The setting of connection peptides does not affect the described immunogenicity with immunogenic material substantially.Preferably, described connection peptides is GGGGSC (SEQ ID NO:4), forms sulfydryl coupling, analogue epi-peptide is connected to KLH by terminal cysteine.

In an embodiment of the present invention, in the C-terminal coupling connection peptides of analogue epi-peptide, again with keyhole limpet hemocyanin (KLH) coupling (a KLH coupling has many analogue epi-peptides), after this coupling protein repeatedly immune mouse, after getting mice serum, ELISA detects and tires, found that antibody in serum is not only for KLH, mimic epitopes, also for N1N2-806 and EGFRVIII.Immunoblot experiment demonstrates antibody in serum can reach the A431 human lung cancer cell line of the HuH7-EGFRvIII human liver cancer cell of EGFRVIII and the EGFR of process LAN by associative list.The present inventor has done again the immunofluorescence experiment of above-mentioned cell, and result is consistent with immunoblot experiment result.Although in improvement on synthesis Binding experiment, the polypeptide LPAFFVTNQTQD of synthesis is lower with the avidity of 12H23, but in immunoblotting and immunofluorescence experiment, it to induce the biological characteristics of the antibody of generation and WHTEILKSYPHE there is no different, the avidity of prompting improvement on synthesis and monoclonal antibody 12H23 there is no positive relationship with the avidity of the EGFR of the antibody in mice serum and EGFRvIII and process LAN.

Composition

Present invention also offers the composition comprising analogue epi-peptide of the present invention or have immunogenic material, particularly pharmaceutical composition, described composition also comprises vaccine.Said composition can be used for inducing the antibody producing anti-epidermal growth factor receptor.Said composition can be used for prevention or treatment EGF-R ELISA overexpression relative disease, and described disease includes, but is not limited to: tumour, oral lichen planus and hickie.

The composition comprising analogue epi-peptide of the present invention or there is immunogenic material can comprise by epitope peptide or there is immunogenic material practical use selected by buffer reagent or adjuvant; Also can comprise other material being applicable to intended purpose.Those skilled in the art are good at selecting suitable buffer reagent, and known in the art have numerous buffers to be applicable to intended purpose.In some example, said composition can contain pharmaceutically acceptable vehicle, known in the art have multiple and without the need to discussing in detail at this.Pharmaceutically acceptable various vehicle is at the existing detailed description of multiple publication, comprise as " Remington ' s Pharmaceutical Sciences " (" Lei Mingdun pharmaceutical science ", the 19th edition (1995) Mack Publishing Co.).

Composition of the present invention can be prepared into various formulation, as injection, granula, tablet, pill, capsule, transdermal drug etc.Thinner known in the art comprises aqueous medium, vegetalitas and animality oil & fat.Also the salt of useful stabilizers, wetting agent and emulsifying agent, change osmotic pressure or the various buffer reagent of maintenance suitable ph and skin penetration enhancer etc. are as auxiliary agents.

When being used as vaccine, described vaccine can adopt various method to prepare.Usually, by various method well known in the art, prepare vaccine of the present invention or medicine with suitable pharmaceutical carrier and/or vehicle (vehicle).Suitable carrier is Sterile Saline.Also can use other water-based and non-aqueous isotonic sterile injection liquid and water-based and non-aqueous sterile suspensions (known is all pharmaceutically acceptable carrier well-known to those skilled in the art) for this reason.

In addition, the preparation of vaccine of the present invention also can contain other composition, comprises as adjuvant, stablizer, pH adjusting agent, sanitas etc.These compositions are known by vaccines arts technician.Adjuvant class comprises (but being not restricted to) freund's adjuvant; Alum adjuvant; Saponin adjuvant; Ribi adjuvant (Ribi ImmunoChemResearch In., Hamilton, MT); Montanide ISA adjuvant (Seppic, Paris, France); Hunter ' s TiterMax adjuvant (CytRx Corp., Norcross, GA); Gerbu adjuvant (GerbuBiotechnik GmbH, Gaiberg, Germany) etc.

When being used as vaccine, available known method is by analogue epi-peptide of the present invention or have immunogenic material and be applied to object.These vaccines are used in the route of administration that usual employing is identical with conventional vaccine and/or simulation pathogenic infection path.When adopting the form of vaccine composition, also can comprise pharmaceutically acceptable carrier.In addition, this composition also can comprise adjuvant, correctives or stablizer etc.

The routine and the pharmaceutically acceptable approach that give the present composition comprise: in nose, intramuscular, tracheal strips, in subcutaneous, intracutaneous, lung, intravenously, intranasal, oral administration or other parenteral route of administration.Can combination medicine-feeding approach if needed, or regulate by antigen peptide or disease event.Vaccine can single dose or multiple doses give, and can comprise and give booster dose to cause and/or to maintain immunizing power.

Should analogue epi-peptide of the present invention be given with " significant quantity " or there is immunogenic material; i.e. analogue epi-peptide or have immunogenic amount of substance and be enough to cause immunne response in selected administration routes, can effectively impel protection host to resist the complication that EGF-R ELISA overexpression causes.

Analogue epi-peptide selected in each vaccine dose part or have immunogenic amount of substance determines without the amount of obvious side effect by causing protective immune response.Usually, give about 0.01 μ g-10mg analogue epi-peptide or there is immunogenic material/kg body weight, preferably 0.1 μ g-1mg analogue epi-peptide or there is immunogenic material/kg body weight, more preferably 1 μ g-1mg analogue epi-peptide or there is immunogenic material/kg body weight.Use adjuvant and/or immunostimulant just can improve analogue epi-peptide of the present invention or the immunne response with immunogenic material.

Medicine box

Present invention also offers a kind of medicine box preventing and treating the disease that Urogastron overexpression causes, wherein containing analogue epi-peptide of the present invention or there is immunogenic material or containing described analogue epi-peptide or have the composition of immunogenic material.In addition, conveniently administration, also can contain the pin of injection, immunological adjuvant, and/or pharmaceutically acceptable carrier, and/or working instructions in described medicine box.

Major advantage of the present invention is:

(1) present invention obtains two analogue epi-peptides of EGF-R ELISA, can EGF-R ELISA be identified using the antibody that these two analogue epi-peptides produce as vaccine immunity animal.Described analogue epi-peptide is conducive to producing specific antibody in a large number in animal body, overcomes the technical barrier that native peptides epitope immunogenic is low, can not produce enough antibody.

(2) in passive immunization topmost problem be exactly transformation period of antibody short, need immunity repeatedly, instant invention overcomes this difficult problem, also solve the immunogenic problem of mono-clonal mouse-anti simultaneously.In cancer therapy in the future, the active immunity for the EGFR antigen-specific of EGFRVIII and process LAN will be a new selection.

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.

The cultivation of embodiment 1, clone and protein extracting

The human lung cancer cell line A431 (purchased from the Chinese Academy of Sciences) of process LAN EGFR and after transfection, express EGFRvIII Bel7402 HuH7-EGFRvIII (see Wang H etc., Epidermal growthfactor receptor vIII enhances tumorigenicity and resistance to5-fluorouracil inhuman hepatocellular carcinoma.Cancer Lett.2009; 279 (1): 30-8.) all cultivate with the DMEM substratum (GIBCO, Grand Island, USA) that with the addition of 10% foetal calf serum (v/v) and 1% mycillin (v/v).After extracting cell protein, protein concentration BCA protein detection kit (BCAProtein Assay Kit; Pierce, Rockford, IL) measure, the cell protein packing after extracting is also kept at-80 DEG C.

Embodiment 2, use monoclonal antibody 12H23 elutriation PH.D-12 phage library

PH.D-12 phage library is purchased from NEB biotech firm.The operational manual that elutriation provides according to test kit substantially carries out, and simplified process is as follows: be buffered liquid 0.1M NaHCO with bag ₃(pH8.6) dilute monoclonal antibody 12H23, and wrap by 96 orifice plates with final concentration 100 μ g/ml, 4 DEG C of overnight incubation.Second day, after TBST (50mM Tris, 150mM NaCl, pH7.5 comprise 0.1%Tween-20 (v/v)) washing 10 times, add 300 μ l confining liquids (0.1M NaHCO3pH8.6,5mg/ml BSA) 37 DEG C and hatch 1 hour.Hypsokinesis in 1 hour goes out confining liquid, and every hole adds 10 μ l phage stostes and (is diluted to 100 μ l with TBST, about 1.5 × 10 ¹¹individual phage), hatch 1 hour for 37 DEG C.Hypsokinesis in 1 hour goes out, and washs 10 times with TBST, in conjunction with on phage 0.2M Glycine-HCl (G-HCL; PH2.2), wash-out, and at once with 1MTris-HCl (pH9.1) neutralization, the phage of gradient dilution wash-out is carrying out titration containing on the LB flat board of tsiklomitsin 20 μ g/ml, within second day, observes clone's number.The ER2738 bacterial strain (purchased from NEB) that remaining phage-infect 50ml OD is about 0.5 increases, 37 DEG C of violent shaking overnight.Within second day, reclaim for subsequent use by PEG/NaCl precipitation.Phage rescue efficiency calculates as follows: rescue efficiency=(wash-out bacteriophage/input phage) × 100%.

In the panning process of the second third round, the concentration of the monoclonal antibody 12H23 of bag quilt is respectively 10 μ g/ml and 1 μ g/ml, and TBST concentration used is 0.2% and 0.5%, and all the other steps are the same.

Result: the screening taken turns through 3, the phage that can be combined with monoclonal antibody 12H23 is by successfully enrichment (table 2).

Table 2

Embodiment 3, DNA sequencing

After from flat board, picking 20 phage clones jolt, with AxyPrep mini-scale plasmid extraction agent box (Axygen, uion city, USA) extracting single stranded DNA.Ying Jun company (Shanghai, China) order-checking is delivered to quantitatively.As a result, 7 different insertion sequences are had, in table 3 in 20 clones.

The sequencing result (peptide sequence of phage display) of the phage clone after table 3, enrichment

Clone number	The peptide sequence of coding	SEQ ID NO:
			C001、C005、C007、C010、C012、C004	DHARYPWLRPPA	5
C006、C008、C018、C013、C015	WHTEILKSYPHE	1
			C002、C011、C020	LPAFFVTNQTQD	2
C009、C017、C019	SHVDDLGLRPLT	6
			C016	LLADTTHHRPWT	7
C014	NSPRLVHTNTHN	8
			C003	YWNASPSASGVI	9

Embodiment 4, specificity enzyme crosslinking immuning adsorpting test

The monoclonal antibody 12H23 of 10 μ g/mL and mouse IgG 1 Isotype control (, Hangzhou, China sub-purchased from section).After being buffered liquid dilution with bag, bag is by 96 hole microplates, 4 DEG C of overnight incubation.Second day with 0.5% TBST wash 6 times after, close with containing the TBST of 5% milk powder, hatch 1 hour for 37 DEG C.Throw in 7 phage clones after amplification, phage concentration gradient is diluted, and hatches 1 hour for 37 DEG C.Anti-(Pharmacia, New Jersey, the USA) 37 DEG C of anti-M13 phage two that 1:1000 dilutes HRP mark hatches 1 hour.Then use ABTS (SIGMA, St.Louise, USA) to develop the color, read the absorption value at 405nm place by microplate reader (BioRad module680, Hercules, USA).This experiment repetition 3 times.

Result: sequence WHTEILKSYPHE and LPAFFVTNQTQD successfully in conjunction with monoclonal antibody 12H23, can be shown in Fig. 1.

Embodiment 5, phage competion experiment

After the monoclonal antibody 12H23 of 10 μ g/mL and mouse IgG 1 Isotype control bag are buffered liquid dilution, bag is by 96 hole microplates, 4 DEG C of overnight incubation.Second day with 0.5% TBST wash 6 times after, close with containing the TBST of 5% milk powder, hatch 1 hour for 37 DEG C.Sequence (WHTEILKSYPHE, LPAFFVTNQTQD) and control peptide EQKLISEEDL (MYC label, SEQ ID NO:10) every hole after ELISA identifies throw in 10 ¹¹individual phage, hatches 1 hour for 37 DEG C.Then eluent (0.1M Glycine-Hcl pH2.2, the Immunoglobulin IgG1 of N1N2-806, S1-GFP, CC16 or the mouse) competitive elution that 5 kinds different is used.Wherein N1N2-806 is the product (its sequence is AETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVCTGDETQCYGT WVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPP GTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQY TPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPVNAEFGGGSG GGSKLGVPFYSHSVRACGADSYEMEEDGVRKCKK, SEQ IDNO:11) of expressing in conjunction with the N1N2 domain fusion of epi-position and phage pIII albumen of monoclonal antibody 806; S1-GFP is S1 structural domain and the GFP protein fusion expression (sequence MGSSHHHHHHSSGLVPRGSHMMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGE GDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPE GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYN SHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLS TQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKNCEVVLGNLEITYVQRNYDL SFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGL KELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIV, SEQ ID NO:12) of EGFR; CC16 is the epi-position (CGADSYEMEEDGVRKC, SEQ ID NO:13) of the monoclonal antibody 806 of synthetic.Then observe the phage quantity of competitive elution by titration method during elutriation, see Fig. 2.

The synthesis of embodiment 6, polypeptide and crosslinked

Polypeptide WHTEILKSYPHE and LPAFFVTNQTQD and control peptide: EQKLISEEDL (MYC label SEQ ID NO:14) is synthesized by gill biochemical corp (Shanghai, China).A Linker (GGGGSC, SEQ ID NO:4) and keyhole limpet hemocyanin (KLH, SIGMA, USA is passed through at the C-terminal of polypeptide after synthesis, sequence D FGHSKKIRKNVHSLTAEEQNSLRRAMDDLQDDKTRGGFQQIAAFHGEPKWCPRPEA EKKFACCVHGMAVFPHWHRLLTVQGENALRKHGFTGGLPYWDWTRPMSALPHFVAD PTYDDSVSSLEEDNPYSHGHIDSVGHDTTRAVRDDLYQSPGFGHYTDIAKQVLLAL EQDDFCDFEVQFEIAHNSIHALVGGNEPYGMSTLEYFLYDPIFFLHHSNTDRLWAI WQALQKYRGKPYNTANCAIVRHDTYRKPLQPFGLDSVINPDDETREHSVPRDVFNY KDDFNYEYESLNFNGLSIAQLDRELQRIKSHDRVFAGFLLHEIGQSALVKFYVCKH HVSDCDHYAGEFYILGDEAEMPFAYDRVYKYEISQALHDLDLHVGDNFHLKYEAFN LNGGSLGGVDLSQPSVIFEPAAGSHTA, SEQ ID NO:3) phase coupling, obtain improvement on synthesis WHTEILKSYPHE-KLH, LPAFFVTNQTQD-KLH, control peptide-KLH.

Embodiment 7, improvement on synthesis Binding experiment

After the improvement on synthesis WHTEILKSYPHE-KLH of 10 μ g/mL, LPAFFVTNQTQD-KLH and negative control (MYC-KLH and KLH) are buffered liquid dilution with bag, bag is by 96 hole microplates, 4 DEG C of overnight incubation.Second day with 0.5% TBST wash 6 times after, close with containing the TBST of 5% milk powder, hatch 1 hour for 37 DEG C.Monoclonal antibody 12H23 joins in every hole with different dilution gradients, hatches 1 hour for 37 DEG C.1:1000 dilutes the sheep anti-mouse antibody (section is sub-, Hangzhou, China) of HRP mark, hatches 1 hour for 37 DEG C.Then use ABTS (SIGMA, St.Louise, USA) to develop the color, read the absorption value at 405nm place by microplate reader (BioRadmodule680, USA).This experiment repetition 3 times.

As a result, to demonstrate sequence WHTEILKSYPHE similar with the natural epi-position of monoclonal antibody 12H23 with LPAFFVTNQTQD for phage competitive assay.N1N2-806, S1-GFP, CC16 all can with sequence WHTEILKSYPHE and LPAFFVTNQTQD competition binding monoclonal antibody 12H23, see Fig. 3.

Embodiment 8, polypeptide immune

BALB/c mouse (Shanghai Inst. of Tumor Animal Lab., Shanghai, China) is divided into 4 groups, and (WHTEILKSYPHE – KLH, LPAFFVTNQTQD – KLH, control peptide (MYC – KLH) and KLH often organize 6.The above-mentioned each polypeptide of BALB/c mouse peritonaeum hemostasis immunity, immunity 3 times altogether, three times immune dosage is every mouse 100 μ g, and adjuvant used is complete Freund's adjuvant (first time immunity) and incomplete Freund's adjuvant (second and third immunity).Each immunity gets mouse tail vein blood in latter 7 days.All serum sample mixing ,-20 DEG C of preservations will be often organized after three immunity.

Embodiment 9, antibody titer detect and immunoblot experiment

ELISA method detects the serum titer of BALB/c mouse after three immunity, and 1:500 pressed by serum, 1:1500,1:4500,1:13500,1:40500,1:121500,1:364500 gradient dilution, observes whether produce tiring for KLH, improvement on synthesis and N1N2-806 and EGFRvIII.

A431 and HuH7-EGFRvIII cytolytic proteins forwards on nitrocellulose filter after the SDS-polyacrylate hydrogel electrophoresis of 10%.PBS (the enlightening Shen of nitrocellulose filter containing 5% milk powder, Shanghai, China) 37 DEG C close after 1 hour, with monoclonal antibody 12H23 and BALB/c mouse serum (through WHTEILKSYPHE – KLH, LPAFFVTNQTQD – KLH, obtain after control peptide (MYC – KLH and KLH) immunity) respectively 37 DEG C hatch 2 hours, monoclonal antibody 12H23 dilutes with containing the PBS1:100000 of 5% milk powder, and mice serum uses the PBS1:1000 containing 5% milk powder to dilute.1 hour is hatched afterwards with the sheep anti-mouse antibody (section is sub-, Hangzhou, China) 37 DEG C of 1:1000 dilution HRP mark.Then with the exposure of Kodak film room temperature darkroom.

As a result, mouse, after immunity, creates the antibody for KLH, improvement on synthesis and N1N2-806 and EGFRVIII in serum, it is tired and is respectively 1:364500, and 1:364500,1:1500,1:4500 are in table 4.Western blot experiment demonstrates antibody in serum can reach the huh7-EGFRvIII human liver cancer cell of EGFRvIII by associative list, and the A431 human lung carcinoma cell of process LAN EGFR, prove that the present inventor successfully have found the mimic epitopes of 2 12H23 monoclonal antibodies, see Fig. 4.

The antibody titer of serum after table 4, polypeptide immune

Embodiment 10, immunofluorescence experiment

By A431 and HuH7-EGFRvIII cell with every hole 1 × 10 ⁵the density of cell plants on porous plate (sea blue, Haimen, China), 37 DEG C, 5%CO ₂incubated overnight.Second day, after cleaning 3 times with PBS, fix 30 minutes by the paraformaldehyde room temperature of 4% (v/v), after cleaning 3 times with PBS, with the sheep blood serum 37 of 10% (v/v), DEG C close 1 hour.With monoclonal antibody 12H23 and BALB/c mouse serum (WHTEILKSYPHE – KLH, LPAFFVTNQTQD – KLH, control peptide (MYC – KLH and KLH) difference incubated at room 2 hours, monoclonal antibody 12H23 and mice serum all dilute with the sheep blood serum of 10%.Afterwards with sheep anti-mouse antibody (the health one-tenth that the fluorescein isothiocyanate (FITC) of 1:50 dilution marks, Shanghai, China) and 1:2000 dilute 4,6-bis-narrows base-2-phenylindone (DAPI) (Roche, Shanghai, China) incubated at room 45 minutes, finally use fluorescent microscope (Olympus, Shanghai, China) observe fluorescence.

Result: immunofluorescence experiment demonstrates antibody in serum can reach the Huh7-EGFRvIII human liver cancer cell of EGFRvIII by associative list, and the A431 human lung carcinoma cell of process LAN EGFR, prove that the present inventor successfully have found the mimic epitopes of 2 12H23 monoclonal antibodies, see Fig. 5.

The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims

1. an isolated polypeptide, is characterized in that, described polypeptide is the polypeptide of aminoacid sequence as shown in SEQ ID NO:2.

2. have an immunogenic material, it is characterized in that, described material is connected with antigen, and the aminoacid sequence of described antigen is as shown in SEQ ID NO:2; Or described substance gives expression to antigen, the aminoacid sequence of described antigen is as shown in SEQ ID NO:2.

3. material as claimed in claim 2, it is characterized in that, the described immunogenic material that has comprises:

Keyhole limpet hemocyanin; And

Be coupled to the antigen of described keyhole limpet hemocyanin, the aminoacid sequence of described antigen is as shown in SEQ ID NO:2.

4. material as claimed in claim 3, is characterized in that, described antigen is by the polypeptide of aminoacid sequence as shown in SEQ ID NO:2; And the connection peptides to be connected with described polypeptide, described connection peptides has 1-20 Amino acid profile.

5. the polynucleotide be separated, is characterized in that, described polynucleotide encoding polypeptide according to claim 1.

6. the purposes of polypeptide according to claim 1, is characterized in that, for the preparation of induction of antibodies produce pharmaceutical composition, described antibody recognition EGF-R ELISA.

7. the arbitrary described purposes with immunogenic material of claim 2-4, is characterized in that, for the preparation of induction of antibodies produce pharmaceutical composition, described antibody recognition EGF-R ELISA.

8. a pharmaceutical composition, is characterized in that, described pharmaceutical composition contains:

Polypeptide according to claim 1, or claim 2-4 is arbitrary described has immunogenic material; With

Pharmaceutically acceptable carrier.

9. pharmaceutical composition as claimed in claim 8, is characterized in that, the immunological adjuvant of described pharmaceutical composition also containing significant quantity.

10. a medicine box, is characterized in that, contains in described medicine box: polypeptide according to claim 1; Or claim 2-4 is arbitrary described has immunogenic material; Or the pharmaceutical composition described in claim 8 or 9.