CN103539840A - Epidermal growth factor receptor (EGFR) mimotope peptide and application thereof - Google Patents
Epidermal growth factor receptor (EGFR) mimotope peptide and application thereof Download PDFInfo
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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
The present invention is that application number is dividing an application of 201010023003.9 application for a patent for invention.
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 tyrosine kinase receptor that molecular weight is 170KD, and it plays considerable effect for propagation and the differentiation of different cells.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 propagation of cell, the angiogenesis of the invasion and attack of tumour, inside tumor and the transfer of tumour.EGFR cross to express in the property tumour of many epitheliums source, and crosses the level expressed and patient's prognosis has close contacting.EGFR cross to express normally the amplification by gene, also or because Urogastron sudden change causes.The mutant of modal EGFR is EGF-R ELISA III form variation body (EGFRvIII), it has mainly lacked 801 bases of encoding sequence exon 2 to the 7 exons, this has directly caused the amino acid whose disappearance in 267 of EGFR extracellular regions, and aobvious sub-connection place has formed a new glycine outside.EGFRvIII is at cerebral glioma, and mammary cancer, can be detected in nonsmall-cell lung cancer and prostate cancer, in healthy tissues, does not express.
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., Monoclonal antibody806inhibits the growth of tumor xenografts expressing either the de2-7or amplified epidermal growth factor receptor (EGFR) but not wild-type EGFR.Cancer Res.2001; 61 (14): 5355-61.) can identify more specifically EGFR and the EGFRvIII expressing, research shows the Growth of Cells that mAb806 can suppress to express EGFR or express EGFRvIII.The clinical I phase is studied and shows; the mouse-human chimeric CH806 of mAb806 monoclonal antibody specifically target tumor tissue (Scott AM etc., A phase I clinical trial with monoclonal antibody ch806targeting transitional state and mutant epidermal 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 epitope for the epidermal growth factor receptor-specific monoclonal antibody806reveals that it preferentially recognizes an untethered form of the receptor.J Biol 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 healthy tissues, if be directly used in human body, may be difficult to produce corresponding monoclonal antibody because of immunological tolerance, therefore need to find the analogue epi-peptide (mimotope) of this section of polypeptide, for the development of anti-tumor vaccine.The inventor has screened a monoclonal antibody 12H23 (international patent application no: PCT/CN2009/074090) in early-stage Study, the EGFR that it can be identified EGFRvIII and cross express, and can effectively suppress in vivo the growth of HuH7-EGFRvIII human liver cancer cell and SMMC-7721 cell in experiment in vitro.Research finds that the epitope polypeptide sequence of this antibody is also
287cGADSYEMEEDGVRKC
302.
Monoclonal antibody is applied to clinical some restrictions that still exist, and such as high medical expense, dissatisfactory curative effect still, from the side effect of mouse-anti or chimeric antibody 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, the problem cann't be solved with regard to likely solving monoclonal antibody.Analogue epi-peptide, structurally synantibody is similar in conjunction with epi-position, but aminoacid sequence is likely different, is conducive to so a large amount of antibody producing for this epi-position in human body.Therefore, the vaccine that produces 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 a kind of have immunogenic material and application thereof.
The present invention also aims to provide the pharmaceutical composition that contains described acceptor analogue epi-peptide or there is immunogenic material.
In a first aspect of the present invention, a kind of isolated polypeptide is provided, described polypeptide is the polypeptide that is selected from aminoacid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2.
In another aspect of this invention, provide a kind of immunogenic material that has, described material is connected with antigen, and described antigen contains the aminoacid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; Or described substance gives expression to antigen, described antigen contains the aminoacid sequence shown in SEQ ID NO:1 or SEQ ID NO:2.
In a preference, the described immunogenic material that has is to connect the protein macromolecule that (coupling) has antigen.
In another preference, the described immunogenic material that has having described in immunogenic material comprises: keyhole limpet hemocyanin; And
Be coupled to the antigen of described keyhole limpet hemocyanin, described antigen contains the aminoacid sequence shown in SEQ ID NO:1 or SEQ ID 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 being 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 antibody, produce pharmaceutical composition, described antibody recognition EGF-R ELISA.
In a preference, described polypeptide is for the preparation of the pharmaceutical composition of prevention or treatment tumour.
In another aspect of this invention, provide the described purposes with immunogenic material, for the preparation of induction antibody, produce pharmaceutical composition, described antibody recognition EGF-R ELISA.
In a preference, described has immunogenic material for the preparation of the pharmaceutical composition of prevention or treatment tumour or oral lichen planus and hickie.
In another aspect of this invention, provide a kind of pharmaceutical composition, described pharmaceutical composition contains:
The polypeptide that one or more of significant quantity (2 kinds) are described, or one or more (2 kinds) described there is immunogenic material; With pharmaceutically acceptable carrier.
In a preference, described pharmaceutical composition is vaccine.
In another preference, described pharmaceutical composition also contains the immunological adjuvant of significant quantity.
In another aspect of this invention, provide a kind of medicine box, in described medicine box, contain: the polypeptide described in one or more; Or there is immunogenic material described in one or more; 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 has shown the binding specificity analysis of the phage clone after monoclonal antibody 12H23 is to enrichment.
Wherein, 1,2,3 is 12H23 and the displaying M13 phage clone of specific peptide sequence or the combination situation of wild-type M13 phage, and 4 is the combination situation of homotype control antibodies and described phage clone, and 5 is the combination situation of BSA and described phage clone.
Fig. 2 has shown that employing ELISA carries out phage competitive binding assay.
1 is to be combined situation in conjunction with 12H23 (left side) with the competition of G-HCL with the phage of contrast IgG (right side);
2 is to be combined situation in conjunction with 12H23 (left side) with the competition of N12-806 with the phage of contrast IgG (right side);
3 is to be combined situation in conjunction with 12H23 (left side) with the competition of S1-GFP with the phage of contrast IgG (right side);
4 is to be combined situation in conjunction with 12H23 (left side) with the competition of cc16 with the phage of contrast IgG (right side);
5 is to be combined situation in conjunction with 12H23 (left side) with the competition of control peptide with the phage of contrast IgG (right side).
Fig. 3 has shown the joint efficiency of the synthetic polypeptide of elisa assay and 12H23.
Wherein, 1 for 12H23 antibodies is to synthetic polypeptide WHTEILKSYPHE – KLH; 2 for 12H23 antibodies is to synthetic polypeptide LPAFFVTNQTQD-KLH; 3 for 12H23 antibodies is to synthetic polypeptide control peptide-KLH; 4 for 12H23 antibodies is to KLH.
Fig. 4 has shown that Western blot detects the reactivity that anti-polypeptide serum and EGFRvIII or EGFR are crossed the clone of expression.The combination situation of the antibody of monoclonal antibody 12H23 or mimic epitopes inducing peptide (in mouse serum) and Huh7-EGFRvIII and A431 lysis albumen, EGFRvIII and EGFR can detect (swimming lane 1 at 130kDa and 170kDa respectively, positive control), mice serum (swimming lane 2) by the immunity of WHTEILKSYPHE-KLH conjugate produces band with lysis albumen at 130kDa and 170kDa, mice serum (swimming lane 3) by the immunity of LPAFFVTNQTQD-KLH conjugate produces band with lysis albumen at 130kDa and 170kDa, by the mice serum (swimming lane 4) of control peptide-KLH conjugate immunity or there is no band by the mice serum (swimming lane 5) of KLH immunity separately.
Fig. 5 has shown immunofluorescence result, 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 inventor, through deep research, discloses a kind of analogue epi-peptide of EGF-R ELISA first.Described analogue epi-peptide can, competitively in conjunction with the antibody (as 12H23) of anti-EGFR, show that mimic epitopes is close with natural epi-position in biology and structure.Analogue epi-peptide of the present invention is conducive to produce in a large number in animal body specific antibody, has overcome the technical barrier that native peptides epi-position immunogenicity is low, can not produce enough antibody.
As used herein, described " analogue epi-peptide " refers to polypeptide (as table 1) or its derived peptide with aminoacid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, its in 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, " separated " refers to that material separates (if natural substance, primal environment is natural surroundings) from its primal environment.For example, polynucleotide and polypeptide under the native state in active somatic cell do not have separation and purification, if but other materials that same polynucleotide exists together with native state with polypeptide separate, for separation and purification.
As used herein, " immunocompetence " or " immunogenicity " refers to by the specificity humoral in natural, restructuring or synthetic vaccine-induced mammalian body and/or the ability of cellullar immunologic response.
As used herein, " immunne response " comprises cellularity and/or body fluid immunne response, and they are enough to produce the antibody of specificity anti-epidermal growth factor receptor; Or the disease that prevents or suppress to be 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), has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various vehicle or thinner.
As used herein, " significant quantity " or " immune significant quantity " refers to that with single dose or a continuous agent part, giving individual amount is effective to treatment or prevention.This consumption is determined the assessment of medical conditions and other correlative factor according to treated individual healthy state and physiological situation, the individual classification for the treatment of (as non-human primates etc.), the ability of individual immunity system synthesis antibody, required degree of protection, the preparation of vaccine, treatment doctor.Estimate that this consumption, by the scope relatively wide, can determine 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 essentially identical with external monoclonal antibody 806, can identify EGF-R ELISA III form variation body (EGFRvIII) and cross the EGF-R ELISA (EGFR) of expressing, and in experiment in vitro, can effectively suppress HuH7-EGFRvIII human liver cancer cell and the propagation of crossing the A431 human lung carcinoma cell of expressing EGFR in vivo.Yet mab treatment application clinically also exists many bottlenecks, the method that passive antibody immunity needs repeated multiple times immunity just can obtain effectively tiring and addresses these problems is exactly a kind of new vaccines of research and development, can be by the mimic epitopes immune mouse of antibody recognition by using, excite the active immunity of mouse, make himself to produce biological property and be similar to the antibody of 12H23, thereby play the effect of targeted therapy.The inventor screens monoclonal antibody 12H23 with phage library, by euzymelinked immunosorbent assay (ELISA) verify obtained 2 mimic epitopess (shown in sequence SEQ ID NO:1 or SEQ ID NO:2) all can be effectively in conjunction with monoclonal antibody 12H23.Then by immune mouse after these 2 mimic peptide coupling high molecular weight proteins, assess in the rear mice serum of immunity for high molecular weight protein, mimic epitopes and EGFRvIII and the antibody titer of crossing the EGFR expressing, and the antibody in the results show serum can be identified above-mentioned antigen effectively, and HuH7-EGFRvIII human liver cancer cell and cross the A431 human lung carcinoma cell express EGFR.The results show 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 be by antibody recognition.Meanwhile, the peptide section that these screen does not need with natural epi-position identical on space structure, only need to have nature epi-position by the function of antibody recognition.Described analogue epi-peptide can solve the problem that passive immunization brings and can become a kind of therapeutic or preventative new drug as a vaccine.
After obtaining the aminoacid sequence of cicada analogue epi-peptide of the present invention, those skilled in the art can prepare this epitope peptide easily, and it can be synthetic polypeptide, recombinant polypeptide.Epitope peptide of the present invention can be the product of chemosynthesis, or uses recombinant technology for example, to produce from protokaryon or eucaryon host (, bacterium, yeast and mammalian cell).
The present invention also comprises fragment, derivative and the analogue of described analogue epi-peptide.As used herein, term " segment ", " derivative " refer to " analogue " biological function, structure or the active polypeptide that substantially keeps analogue epi-peptide of the present invention identical.Analogue epi-peptide segment of the present invention, derivative and analogue can be: (1) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferably conservative amino acid residue), or (2) have the polypeptide of substituted radical in one or more amino-acid residues, or (3) mature polypeptide and another compound are (such as extending the compound of polypeptide transformation period, polyoxyethylene glycol for example) merge formed polypeptide, or (4) additional aminoacid sequence is blended in this peptide sequence and the polypeptide (fusion rotein) that forms.
Described analogue epi-peptide itself can be used as the antibody that vaccine immunity animal produces identification EGF-R ELISA; Or the formation fusion rotein that can be connected with other albumen, immune animal is to produce the antibody of identification EGF-R ELISA; Or can form and there is immunogenic material with the coupling of macromole phase; Or can be expressed or be illustrated on cell surface or bacteriophage coat protein surface, immune animal be to produce the antibody of identification EGF-R ELISA.
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 for example 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-stranded.Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise the epitope peptide of the present invention of encoding, and can be also the polynucleotide that also comprise 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 the fragment of polypeptide, analogue and derivative 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 conventionally.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 prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.Described recombination method is normally cloned into carrier by described polynucleotide, then proceeds to cell, then by ordinary method separation from the host cell propagation, obtains relevant sequence.Can synthesize relevant sequence by the method for synthetic in addition.
At present, can be completely by chemosynthesis, obtain the DNA sequence dna of code book invention epitope peptide (or its fragment, or derivatives thereof).Then this DNA sequence dna can be introduced in various existing DNA moleculars as known in the art (as carrier) and cell.In addition, also can will suddenly change and introduce in protein sequence of the present invention by chemosynthesis.
The carrier of the polynucleotide that the present invention also relates to comprise described analogue epi-peptide, and the host cell that produces through genetically engineered of described carrier or mimic epitopes peptide-coding sequence, and the method for producing described analogue epi-peptide through recombinant technology.
The carrier of the polynucleotide that comprise the analogue epi-peptide described in above-mentioned coding and suitably promotor or control sequence, can be for transforming suitable host cell, with can marking protein.
Described analogue epi-peptide can or be secreted into extracellular at cell inner expression.Can utilize the albumen of and purification of Recombinant separated by various separation methods with other characteristic its physics, 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 processes, with protein precipitant, process the combination of (salt analysis method), centrifugal, the broken bacterium of infiltration, super processing, 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, produce immunological tolerance, as an embodiment preferably, 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 a kind of immunogenic material that has, 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 hyperimmunization originality, has the haptenic ability that immunogenicity is passed to coupling.
Between described analogue epi-peptide and protein macromolecule, by chemical bond, be connected or coupling mutually; Described chemical bond is covalent linkage or non covalent bond.
As a kind of preferably embodiment, mutually 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, between described analogue epi-peptide and protein macromolecule, by chemical bond, be connected; 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.
Between described analogue epi-peptide and protein macromolecule, can directly be connected (or coupling), or connect by polypeptide connexon (connection peptides).Described connexon for example 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), by terminal cysteine, forms sulfydryl coupling, and analogue epi-peptide is connected in to KLH.
In an embodiment of the present invention, in the C-terminal coupling of analogue epi-peptide connection peptides, again with keyhole limpet hemocyanin (KLH) coupling (a KLH coupling has many analogue epi-peptides), with this coupling protein repeatedly after immune mouse, after getting mice serum, ELISA detection is tired, found that antibody in serum is not only for KLH, mimic epitopes, also for N1N2-806 and EGFRVIII.Immunoblot experiment has proved that the antibody in serum can associative list reaches the HuH7-EGFRvIII human liver cancer cell of EGFRVIII and crosses the A431 human lung cancer cell line of the EGFR expressing.The inventor has done again the immunofluorescence experiment of above-mentioned cell, and result is consistent with immunoblot experiment result.Although synthetic polypeptide in conjunction with experiment in, synthetic polypeptide LPAFFVTNQTQD is lower with the avidity of 12H23, but in immunoblotting and immunofluorescence experiment, it is different that the biological characteristics of the antibody of its generation of inducing and WHTEILKSYPHE there is no, the avidity of the synthetic polypeptide of prompting and monoclonal antibody 12H23 with the antibody in mice serum and EGFRvIII and the avidity of EGFR of expression there is no positive relationship.
Composition
The present invention also provides composition, the particularly pharmaceutical composition that comprises analogue epi-peptide of the present invention or have immunogenic material, and described composition also comprises vaccine.Said composition can be used for the antibody that induction produces 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 that comprises analogue epi-peptide of the present invention or have an immunogenic material can comprise by epitope peptide or have selected buffer reagent or the adjuvant of practical use of immunogenic material; Also can comprise other material that is 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 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 formulations, 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 available stablizer, wetting agent and emulsifying agent, change osmotic pressure or maintain the various buffer reagents of suitable pH value and skin penetration enhancer etc. as complementary material.
When as vaccine, described vaccine can adopt the whole bag of tricks to prepare.Conventionally, by the whole bag of tricks well known in the art, with suitable pharmaceutical carrier and/or vehicle (vehicle), prepare vaccine of the present invention or medicine.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 that vaccine those skilled in the art are known.Adjuvant class comprises (but being not restricted to) freund's adjuvant; Alum adjuvant; Saponin adjuvant; Ribi adjuvant (Ribi ImmunoChem Research In., Hamilton, MT); Montanide ISA adjuvant (Seppic, Paris, France); Hunter ' s TiterMax adjuvant (CytRx Corp., Norcross, GA); Gerbu adjuvant (Gerbu Biotechnik GmbH, Gaiberg, Germany) etc.
When the vaccine, available known method is by analogue epi-peptide of the present invention or have immunogenic material and be applied to object.Conventionally adopt the route of administration identical with conventional vaccine and/or simulation pathogenic infection path to use these vaccines.While 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, interior, the intravenously of interior, subcutaneous, the intracutaneous of intramuscular, tracheae, lung, intranasal, oral administration or other administered parenterally approach.Can combination medicine-feeding approach if needed, or regulate by antigen peptide or disease situation.Vaccine can single dose or multiple doses give, and can comprise and give booster dose to cause and/or to maintain immunizing power.
Should give analogue epi-peptide of the present invention or there is immunogenic material with " significant quantity "; be analogue epi-peptide or there is immunogenic amount of substance be enough to cause immunne response in selected administration path, can effectively impel protection host to resist the complication that EGF-R ELISA overexpression causes.
Selected analogue epi-peptide or have immunogenic amount of substance in each vaccine dose part is to determine without the amount of significantly side effect by causing protective immune response.Conventionally, give approximately 0.01 μ g-10mg analogue epi-peptide or there is immunogenic material/kg body weight, preferred 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.Using adjuvant and/or immunostimulant just can improve analogue epi-peptide of the present invention or have the immunne response of immunogenic material.
Medicine box
The present invention also provides a kind of medicine box of preventing and treating the disease that Urogastron overexpression causes, wherein contains analogue epi-peptide of the present invention or has immunogenic material or contain described analogue epi-peptide or have the composition of immunogenic material.In addition, in order to facilitate administration, in described medicine box, also can contain the pin of injection, immunological adjuvant, and/or pharmaceutically acceptable carrier, and/or working instructions.
Major advantage of the present invention is:
(1) the present invention has obtained two analogue epi-peptides of EGF-R ELISA, and the antibody that these two analogue epi-peptides produce as vaccine immunity animal of usining can be identified EGF-R ELISA.Described analogue epi-peptide is conducive to produce in a large number in animal body specific antibody, has overcome the technical barrier that native peptides epi-position immunogenicity is low, can not produce enough antibody.
(2) in passive immunization topmost problem be exactly transformation period of antibody short, need immunity repeatedly, the present invention has overcome this difficult problem, has also solved the immunogenic problem of mono-clonal mouse-anti simultaneously.In cancer therapy in the future, for EGFRVIII, will be a new selection with the active immunity of crossing the EGFR antigen-specific of expressing.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as people such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
The cultivation of embodiment 1, clone and protein extracting
Cross the human lung cancer cell line A431 (purchased from the Chinese Academy of Sciences) that expresses EGFR and the Bel7402 HuH7-EGFRvIII that expresses EGFRvIII after transfection (referring to Wang H etc., Epidermal growth factor receptor vIII enhances tumorigenicity and resistance to5-fluorouracil in human hepatocellular carcinoma.Cancer Lett.2009; 279 (1): 30-8.) all with the DMEM substratum (GIBCO, Grand Island, USA) that has added 10% foetal calf serum (v/v) and 1% mycillin (v/v), cultivate.After extracting cell protein, BCA protein detection kit (BCA Protein Assay Kit for protein concentration; Pierce, Rockford, IL) to measure, the cell protein packing after extracting is also kept at-80 ℃.
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 concise and to the point process is as follows: with coated damping fluid 0.1M NaHCO
3(pH8.6) dilution monoclonal antibody 12H23, and with coated 96 orifice plates of final concentration 100 μ g/ml, 4 ℃ of overnight incubation.Second day, washs after 10 times with TBST (pH7.5 comprises 0.1%Tween-20 (v/v) for 50mM Tris, 150mM NaCl), adds 37 ℃ of 300 μ l confining liquids (0.1M NaHCO3pH8.6,5mg/ml BSA) to hatch 1 hour.Hypsokinesis in 1 hour goes out confining liquid, and every hole adds 10 μ l phage stostes (with TBST, to be diluted to 100 μ l, approximately 1.5 * 10
11individual phage), hatch 1 hour for 37 ℃.Hypsokinesis in 1 hour goes out, with TBST washing 10 times, in conjunction with 0.2M Glycine-HCl (G-HCL for upper phage; 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, second day is observed clone's number.Remaining phage-infect 50ml OD is about 0.5 ER2738 bacterial strain (purchased from NEB) and increases, and 37 ℃ acutely jolt and spend the night.Second day reclaims standby by PEG/NaCl precipitation.Phage rescue efficiency is calculated as follows: rescue efficiency=(wash-out bacteriophage/input phage) * 100%.
In the elutriation process of the second third round, the concentration of coated monoclonal antibody 12H23 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: through 3 screenings of taking turns, the phage that can be combined with monoclonal antibody 12H23 is by successfully enrichment (table 2).
Table 2
After from flat board, 20 phage clones of picking jolt, with AxyPrep a small amount of plasmid extraction test kit (Axygen, uion city, USA) extracting single stranded DNA.Quantitatively, deliver to Ying Jun company (Shanghai, China) order-checking.As a result, in 20 clones, have 7 different insertion sequences, in Table 3.
The sequencing result of the phage clone after table 3, enrichment (peptide sequence of phage display)
| Clone number | The peptide sequence of coding | SEQ?ID?NO: | |
| C001、C005、C007、C010、C012、 | DHARYPWLRPPA | 5 | |
| C006、C008、C018、C013、 | WHTEILKSYPHE | 1 | |
| C002、C011、 | LPAFFVTNQTQD | 2 | |
| C009、C017、 | SHVDDLGLRPLT | 6 | |
| C016 | LLADTTHHRPWT | 7 | |
| C014 | NSPRLVHTNTHN | 8 | |
| C003 | YWNASPSASGVI | 9 |
The monoclonal antibody 12H23 of 10 μ g/mL and mouse IgG 1 homotype contrast (sub-purchased from section, Hangzhou, China).By coated 96 hole microplates after coated damping fluid dilution, 4 ℃ of overnight incubation.Second day washs after 6 times with 0.5% TBST, with the TBST that contains 5% milk powder, seals, and hatches 1 hour for 37 ℃.Throw in 7 phage clones after amplification, the dilution of phage concentration gradient, hatches 1 hour for 37 ℃.Anti-(Pharmacia, New Jersey, the USA) 37 ℃ of anti-M13 phage two of 1:1000 dilution HRP mark hatched 1 hour.Then use ABTS (SIGMA, St.Louise, USA) colour developing, by microplate reader (BioRad module680, Hercules, USA), read the absorption value at 405nm place.This experiment repeats 3 times.
Result: sequence WHTEILKSYPHE and LPAFFVTNQTQD can, successfully in conjunction with monoclonal antibody 12H23, be shown in Fig. 1.
The monoclonal antibody 12H23 of 10 μ g/mL and mouse IgG 1 homotype contrast are diluted rear coated 96 hole microplates, 4 ℃ of overnight incubation with coated damping fluid.Second day washs after 6 times with 0.5% TBST, with the TBST that contains 5% milk powder, seals, and hatches 1 hour for 37 ℃.Sequence (WHTEILKSYPHE, LPAFFVTNQTQD) throws in 10 with control peptide EQKLISEEDL (MYC label, SEQ ID NO:10) every hole after ELISA identifies
11individual phage, hatches 1 hour for 37 ℃.Then use 5 kinds of different eluents (0.1M Glycine-Hcl pH2.2, N1N2-806, S1-GFP, the Immunoglobulin IgG1 of CC16 or mouse) competitive elution.Wherein N1N2-806 is the product (its sequence is AETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVCTGDETQCYGT WVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPP GTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQY TPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPVNAEFGGGSG GGSKLGVPFYSHSVRACGADSYEMEEDGVRKCKK, SEQ ID NO:11) of the combination epi-position of monoclonal antibody 806 and the N1N2 structural domain amalgamation and expression of phage pIII albumen; S1-GFP is S1 structural domain and GFP protein fusion expression (the sequence MGSSHHHHHHSSGLVPRGSHMMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGE GDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPE GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYN SHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLS TQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKNCEVVLGNLEITYVQRNYDL SFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGL KELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIV of EGFR, SEQ ID NO:12), CC16 is the epi-position (CGADSYEMEEDGVRKC, SEQ ID NO:13) of the monoclonal antibody 806 of synthetic.The phage quantity that titration method during then by elutriation is observed competitive elution, is shown in Fig. 2.
Polypeptide WHTEILKSYPHE and LPAFFVTNQTQD and control peptide: EQKLISEEDL (MYC label SEQ ID NO:14) is synthesized by gill biochemical corp (Shanghai, China).After synthesis of the C-terminus via a peptide Linker (GGGGSC, SEQ, ID, NO: 4) and keyhole limpet hemocyanin (KLH, SIGMA, USA; sequenceDFGHSKKIRKNVHSLTAEEQNSLRRAMDDLQDDKTRGGFQQIAAFHGEPKWCPRPEAEKKFACCVHGMAVFPHWHRLLTVQGENALRKHGFTGGLPYWDWTRPMSALPHFVADPTYDDSVSSLEEDNPYSHGHIDSVGHDTTRAVRDDLYQSPGFGHYTDIAKQVLLALEQDDFCDFEVQFEIAHNSIHALVGGNEPYGMSTLEYFLYDPIFFLHHSNTDRLWAIWQALQKYRGKPYNTANCAIVRHDTYRKPLQPFGLDSVINPDDETREHSVPRDVFNYKDDFNYEYESLNFNGLSIAQLDRELQRIKSHDRVFAGFLLHEIGQSALVKFYVCKHHVSDCDHYAGEFYILGDEAEMPFAYDRVYKYEISQALHDLDLHVGDNFHLKYEAFNLNGGSLGGVDLSQPSVIFEPAAGSHTA, SEQ ID NO:3) phase coupling, obtain synthetic polypeptide WHTEILKSYPHE-KLH, LPAFFVTNQTQD-KLH, control peptide-KLH.
Embodiment 7, synthetic polypeptide are in conjunction with experiment
The synthetic polypeptide WHTEILKSYPHE-KLH of 10 μ g/mL, coated 96 hole microplates after coated damping fluid dilution for LPAFFVTNQTQD-KLH and negative control (MYC-KLH and KLH), 4 ℃ of overnight incubation.Second day washs after 6 times with 0.5% TBST, with the TBST that contains 5% milk powder, seals, and hatches 1 hour for 37 ℃.Monoclonal antibody 12H23 joins in every hole with different dilution gradients, hatches 1 hour for 37 ℃.The sheep anti-mouse antibody (section is sub-, Hangzhou, China) of 1:1000 dilution HRP mark, hatches 1 hour for 37 ℃.Then use ABTS (SIGMA, St.Louise, USA) colour developing, by microplate reader (BioRad module680, USA), read the absorption value at 405nm place.This experiment repeats 3 times.
As a result, phage competitive assay has proved that sequence WHTEILKSYPHE is similar with the natural epi-position of monoclonal antibody 12H23 with LPAFFVTNQTQD.N1N2-806, S1-GFP, CC16 all can compete in conjunction with monoclonal antibody 12H23 with sequence WHTEILKSYPHE and LPAFFVTNQTQD, sees Fig. 3.
Embodiment 8, polypeptide immune
BALB/c mouse (Shanghai Inst. of Tumor Animal Lab., Shanghai, China) is divided into 4 groups of (WHTEILKSYPHE – KLH, LPAFFVTNQTQD – KLH, control peptide (MYC – KLH) and KLH, 6 every group.Above-mentioned each polypeptide of peritonaeum hemostasis immunity for BALB/c mouse, immunity is 3 times altogether, three times immune dosage is every mouse 100 μ g, and adjuvant used is complete Freund's adjuvant (immunity for the first time) and incomplete Freund's adjuvant (second and third immunity).Mouse tail vein blood is got in each immunity for latter 7 days.After three immunity, every group of all serum sample mixed to-20 ℃ of preservations.
Embodiment 9, antibody titer detect and immunoblot experiment
ELISA method detects the serum titer of BALB/c mouse after three immunity, and serum is pressed 1:500,1:1500,1:4500,1:13500,1:40500,1:121500,1:364500 gradient dilution, observes and whether produces tiring for KLH, synthetic polypeptide and N1N2-806 and EGFRvIII.
A431 and HuH7-EGFRvIII lysis albumen forward on nitrocellulose filter after 10% SDS-polyacrylate hydrogel electrophoresis.Nitrocellulose filter is used PBS (the enlightening Shen containing 5% milk powder, Shanghai, China) 37 ℃ of sealings are after 1 hour, with monoclonal antibody 12H23 and BALB/c mouse serum (through WHTEILKSYPHE – KLH, LPAFFVTNQTQD – KLH, after control peptide (MYC – KLH and KLH) immunity, obtain) respectively 37 ℃ hatch 2 hours, the PBS1:100000 dilution containing 5% milk powder for monoclonal antibody 12H23, the PBS1:1000 dilution containing 5% milk powder for mice serum.With 37 ℃ of the sheep anti-mouse antibodies (section is sub-, Hangzhou, China) of 1:1000 dilution HRP mark, hatch 1 hour afterwards.Then with the exposure of Kodak film room temperature darkroom.
As a result, mouse, after immunity, has produced the antibody for KLH, synthetic polypeptide and N1N2-806 and EGFRVIII in serum, and it is tired and is respectively 1:364500,1:364500, and 1:1500,1:4500 is in Table 4.Western blot experimental results show that the antibody in serum can associative list reaches the huh7-EGFRvIII human liver cancer cell of EGFRvIII, and the A431 human lung carcinoma cell of excessively expressing EGFR, the proof inventor has successfully found the mimic epitopes of 2 12H23 monoclonal antibodies, sees 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 is planted on porous plate (sea blue, Haimen, China), 37 ℃, 5%CO
2incubated overnight.Second day, cleans after 3 times with PBS, with the paraformaldehyde room temperature of 4% (v/v), fixes 30 minutes, cleans after 3 times, with the sheep blood serum 37 of 10% (v/v), ℃ seal 1 hour with PBS.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 10% sheep blood serum.(health becomes the sheep anti-mouse antibody of fluorescein isothiocyanate (FITC) mark diluting with 1:50 afterwards, Shanghai, China) and 1:2000 dilute 4,6-bis-narrows base-2-phenylindone (DAPI) (Roche, Shanghai, China) incubated at room is 45 minutes, finally uses fluorescent microscope (Olympus, Shanghai, China) observation fluorescence.
Result: immunofluorescence experiment has proved that the antibody in serum can associative list reaches the Huh7-EGFRvIII human liver cancer cell of EGFRvIII, and the A431 human lung carcinoma cell of excessively expressing EGFR, the proof inventor has successfully found the mimic epitopes of 2 12H23 monoclonal antibodies, sees Fig. 5.
All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document, are quoted as a reference separately.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 (10)
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 described antigen contains the aminoacid sequence shown in SEQ ID NO:2; Or described substance gives expression to antigen, described antigen contains the aminoacid sequence shown in SEQ ID NO:2.
3. material as claimed in claim 2, 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, described antigen contains the aminoacid sequence shown in SEQ ID NO:2.
4. material as claimed in claim 3, is characterized in that, described antigen comprises:
The polypeptide of aminoacid sequence as shown in SEQ ID NO:2; And
The connection peptides being connected with described polypeptide, described connection peptides has 1-20 amino acid.
5. separated polynucleotide, is characterized in that, described polynucleotide encoding polypeptide claimed in claim 1.
6. the purposes of polypeptide claimed in claim 1, is characterized in that, for the preparation of induction antibody, 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 antibody, produce pharmaceutical composition, described antibody recognition EGF-R ELISA.
8. a pharmaceutical composition, is characterized in that, described pharmaceutical composition contains:
Polypeptide claimed in claim 1, or the arbitrary described immunogenic material that has of claim 2-4; With
Pharmaceutically acceptable carrier.
9. pharmaceutical composition as claimed in claim 8, is characterized in that, described pharmaceutical composition also contains the immunological adjuvant of significant quantity.
10. a medicine box, is characterized in that, in described medicine box, contains: polypeptide claimed in claim 1; Or the arbitrary described immunogenic material that has of claim 2-4; Or the pharmaceutical composition described in claim 8 or 9.
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Citations (3)
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|---|---|---|---|---|
| WO2005081854A2 (en) * | 2004-02-20 | 2005-09-09 | Ludwig Institute For Cancer Research | Egf receptor epitope peptides and uses thereof |
| WO2008145400A2 (en) * | 2007-05-31 | 2008-12-04 | Medigene Ag | Mutated structural protein of a parvovirus |
| CN101602808A (en) * | 2008-06-12 | 2009-12-16 | 上海市肿瘤研究所 | Binding proteins specific and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005081854A2 (en) * | 2004-02-20 | 2005-09-09 | Ludwig Institute For Cancer Research | Egf receptor epitope peptides and uses thereof |
| WO2008145400A2 (en) * | 2007-05-31 | 2008-12-04 | Medigene Ag | Mutated structural protein of a parvovirus |
| CN101602808A (en) * | 2008-06-12 | 2009-12-16 | 上海市肿瘤研究所 | Binding proteins specific and use thereof |
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| ANGELIKA B. RIEMER ET AL.: "Mimotope vaccines: Epitope mimics induce anti-cancer antibodies", 《IMMUNOLOGY LETTERS》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106046174A (en) * | 2016-06-17 | 2016-10-26 | 杨麟 | Tocilizumab/CH12 coupled simulated epitope |
| CN106046174B (en) * | 2016-06-17 | 2019-06-04 | 杨麟 | Tocilizumab/CH12 is coupled mimic epitope peptide |
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