CN1313484C

CN1313484C - 12 amino acid analog epi-position of human B cell specificity membrane molecule CD20 and polypeptide epi-position vaccine configurated by said analog epi-position

Info

Publication number: CN1313484C
Application number: CNB2005100963700A
Authority: CN
Inventors: 张英起; 李萌; 颜真
Original assignee: Fourth Military Medical University FMMU
Current assignee: Fourth Military Medical University FMMU
Priority date: 2005-11-17
Filing date: 2005-11-17
Publication date: 2007-05-02
Anticipated expiration: 2025-11-17
Also published as: CN1786021A

Abstract

The present invention discloses a mimic epitope of 12 amino acids of a specificity expression membrane molecule CD20 of a human B cell and a polypeptide epitope vaccine constructed by the mimic epitope. A human-mouse chimeric monoclonal antibody Rituximab which is used for treating B lymphocytic leukemia and lymphoma and is authorized for sale by FDA in US is used as a ligand to screen out the mimic epitope of the CD20 molecule from a stochastic presentation peptide library of a bacterial virus 12. Gln-Asp-Lys-Leu-Th-Gln-Try-Pro-Lys-Try-Leu-Glu which as the mimic epitope can be specifically identified by a single Mabthera antibody. The mimic epitope of the 12 amino acids is chemically synthesized and is chemically coupled with eleocellariumhaemophile hemocyanin (KLH) to obtain a vaccine successfully constructed. A protein vaccine constructed by the polypeptide mimic epitope vaccine can induce antiserum in view of natural CD20 molecules from the body of a mouse. The antiserum has the similar function with the Mabthera for killing CD20+ cells. The antiserum can be applied to an autogenous vaccine of the CD20 molecule of the human body after being further prepared. Thereby, the passive immunization treatment of the single antibody is replaced or replenished by an active immunity method, and a basis is provided for overcoming the treatment defects caused by the single antibody.

Description

12 aminoacid mimic epitopes of human B cell specificity membrane molecule CD20 and the polypeptide epitope vaccine that makes up with this mimic epitopes thereof

Technical field

The invention belongs to the medical biotechnology field, be specifically related to phage random present the affinity detection of the screening in peptide storehouse, screened phage and order-checking, polypeptide synthetic and coupled, animal immune experiment, induce technology such as sero-fast external activity detection.Further relate to 12 aminoacid mimic epitopes of a kind of human B cell specificity membrane molecule CD20 and can induce polypeptide epitope vaccine in vivo at the autoantibody of CD20 molecule with what this mimic epitopes made up.

Background technology

1.CD20 the correlative study of molecule and monoclonal antibody thereof

1.1 CD20 molecule

The CD20 molecule is a significant molecule that is expressed in more than 95% normal or the B cell surface that worsens, the desirable target antigen when being the treatment B cell lymphoma.The gene of coding CD20 is positioned at chromosomal t (11 No. 11; 14) (q13; Q32) zone, the molecular weight that it is made up of 297 amino acid are about the non-glycosylated phosphorprotein of 33kD, and the initial pre-B cell stage that is expressed in disappeared until the plasmocyte phase.Monocyte, tranquillization and activated T cell and non-lymphocyte are not all expressed the CD20 molecule.The CD20 molecule is four membrane-spanning proteins, aminoterminal and carboxyl terminal all are positioned at the plasma membrane inboard, in the third and fourth ring district that forms by 43 amino-acid residues of striding between the film district, constituted its main epitope, also be the target position of various monoclonal antibody molecularitys.Internalization is not obvious after CD20 molecule and its antibodies, also do not have tangible CD20 molecule obscission, and no free CD20 exists in human serum.

At present, the biological function of relevant CD20 molecule also imperfectly understands, and majority thinks that it is a function of exercising calcium channel with polymeric form, is the component of regulating B cell proliferation, differentiation signal approach.CD20 is positioned on the Lipid Rafts (lipid rafts) of cytolemma, and Lipid Rafts is a zone of being rich in phosphatide on the after birth, is considered to the platform of a signal conduction.The kinases member (Lyn, Fyn and Lck) and the PAG (p75/85) of the Src family on CD20 and the Lipid Rafts link to each other, and PAG raises Csk to Lipid Rafts, thereby make Lyn, Fyn and Lck keep inactivated state.The CD20 molecule is close mutually under the effect of antibody, and the function of the crosslinked even super polymer performance calcium channel that forms when crosslinked takes place, and the extracellular Ca2 ion is flowed in the cell; In addition, the tyrosine protein kinase of Src family because near and mutually activate, the initiating signal approach is mobilized the release of endogenous calcium ion.The two causes the rising of intracellular calcium concentration, thereby the operation of cell cycle exerts an influence, and regulates cell proliferation and differentiation, even causes the apoptosis of cell.

1.2 the current situation of the monoclonal antibody of CD20 molecule

In about 30 years of past, the monoclonal antibody technique that proposes by K  hler and Milstein for a series of diseases in-vitro diagnosis opportunity is provided.Yet the antigenic characteristic of the specific recognition of monoclonal antibody was just just shown up prominently in nearly 10 years in the immunotherapy of human diseases.At present, monoclonal antibody (mAbs) is used to the diagnosis and the treatment of cancer in clinical, control autoimmune disease, graft-vs-host reaction and allograft rejection, and brain (ridge) marrow inflammation, myocardial damage and the reversible drug intoxication etc. of the initiation of treatment infectation of bacteria.

The CD20 molecule after the high expression level of human malignant B cell and antibodies not the multifrequency nature of internalization etc. make it become the important target of Antybody therapy malignant B cells.Rituximab (Rituxan ^, IDEC-C2B8), the trade(brand)name Mabthera was used for the treatment of non-Hodgkin lymphoma (non-Hodgkin ' s lymphoma) in 1997 by FDA approval.It is the chimeric antibody of a people mouse, comprises the variable region of mouse source IDEC-C2B8 2B8 (Ibritumomab) and the constant region of humanized IgG 1 heavy chain and κ chain.Mabthera has similar avidity and people tissue reaction activity to its parental antibody.Be with respect to its advantage of 2B8 monoclonal antibody: 1) the humanized zone can mediate normal host's effector function; 2) its long half time is in simple murine antibody; 3) generation of the reduction anti-mosaic monoclonal antibody of host (HACA).Rituximab can kill and wound CD20 by two kinds of approach of lysis (CDC) of antibody-dependent cytotoxicity effect (ADCC) and complement-mediated ⁺Bone-marrow-derived lymphocyte.People's such as Demiden experiment in vitro has confirmed that Rituximab can induce CD20 ⁺B lymphoma cell apoptosis.At present, think that mechanism that Ritaximab removes the Malignant B cell also is to activate host's immunologically competent cell, make the sensitization etc. again of chemotherapy tolerance lymphocyte.

The side effect of Rituximab comprises shiver with cold, heating, feels sick, fatigue and weak, headache, itch, tongue and throat's enlargement (angioedema) etc.Other symptoms have vomiting, bronchospasm, asthma, moderate ypotension, rhinitis, fash, urticaria, Blushing, cancer-related pain and the irregular pulse or the angina pectoris symptom that have existed are increased the weight of etc.Rituximab dosage 375mg/m ², weekly 1 intravenous drip totally 4 times be 1 course of treatment, average 1 the course of treatment expense be about 120,000 Renminbi.Minimum treatment was 4 courses of treatment, and alleviating rule fully is 27%.Also have simultaneously the use of discovering rituximab to cause the disappearance of B cell surface CD20 molecule, perhaps this also is one of reason at the bottom of the rituximab service efficiency.Rituximab is used for the monoclonal antibody formulation of oncotherapy as first by FDA approval, and its treatment that appears as non-Hodgkin lymphoma provides new direction.

In recent years, FDA has ratified the relevant monoclonal antibody medicines (in February, 2002 and in June, 2003) of two kinds of CD20 molecules again, radioimmunoassay treatment monoclonal antibody-Zevalin and Bexxar (Tositumoma and ¹³¹I Tositumoma).Zevalin is the mouse source monoclonal antibody 2B8 (Ibritumomab) of isotropic substance 90Y mark.Bexxar is a radio isotope ¹³¹The epi-position identification of I mark more depends on the mouse resource monoclonal antibody-anti-B1 monoclonal antibody (Tositumomab, IgG2a-λ) of the polymeric existence of CD20.These two kinds put exempt from medicine by antibody-mediated radio isotope to tumor locus, bring into play radioactivity cell toxicant direct killing tumour cell, overcome the shortcoming that radiotherapy can not the whole body administration; Compare with simple immunotherapy, show stronger ability of killing and wounding contiguous tumour cell.

At present, three kinds of independent uses of antibody of this of listing have all obtained better curative effect.The combined chemotherapy drug use all has good coordinating effect.But the phenomenon that also exists patient's cell surface CD20 antigen after treatment, to lose, thereby influence the curative effect of monoclonal antibody.Anti-CD20 antibodies medical expense height in addition, the reaction of HAMA and HACA has also taken place in part patient, how to find more perfect methods of treatment also to need further exploration.

2. therapeutic vaccine

2.1 progress

In recent years, in the process of the acute and chronic disease of treatment, demonstrated good effect at the proteic monoclonal antibody of mankind itself.But, the costliness of cost and the inconvenience of using have limited the widespread use of monoclonal antibody, therefore, turn to and seek proteic active immunity vaccine by accepting this antibody protein passively at the mankind itself, both the therapeutic modality with active immunity substituted passive immunization, became the developing direction of protein drug.At present, the research of therapeutic vaccine has become a focus, relates to a lot of diseases, as: chronic viral infection, allergy, tumour, Alzheimer thatch disease, diabetes, hypertension, obesity and rheumatic arthritis etc.

Therapeutic vaccine makes people's immunity system help reaction.Most vaccine can be divided into two big classes: a class is to induce body generation humoral immune reaction, produces antibody; Another kind of is to induce body generation cell immune response, produces cytotoxic T cell (CTLs).Back one class therapeutic vaccine is mainly used in the treatment of tumour and disease of viral infection.

All by inducing production of antibodies to protect body, this just proves by inducing antibody to treat infectious diseases is a kind of effective methods of treatment to most preventative vaccines.Compare with preventative vaccine, the development of therapeutic vaccine will be many slowly, just see the hope of success up to therapeutic vaccine in recent years.Simultaneously, monoclonal antibody is indicating that in obtained immense success aspect the treatment disease therapeutic vaccine that can induce antibody to produce in vivo has vast potential for future development.In fact, it is possible that the endogenous specific antibody that has had animal experiment to show to induce certain level is treated disease, as: blocking-up TNF-α is with the treatment diseases associated with inflammation.

It is very effective aspect treatment rheumatoid arthritis and Crohn ' s disease (the disconnected property ileitis of joint) that humanized anti-TNF-alpha monoclonal antibodies has been proved to be.The blocker listing of several TNF-α has been arranged at present, comprise two kinds of monoclonal antibody (infliximab, adalimumab) and a kind of receptor blocking agent (etanercept), they are helping thousands of patient to palliate the agonizing sufferings, and annual income reaches 2,000,000,000 dollars.So the TNF-α of blocking-up overexpression can reach the effect of treatment disease.Verifiedly in animal experiment can specificity induce the neutrality antibody of TNF-α, and the inductive antibody titers is enough to treat the scorching model of joint of animal by active immunity.

Other animal experiment shows and can treat following disease by producing high titre antibody in the inductor: the vaccine at Angiotensin can be treated hypertension; Can treat Eosinophilia's disease that pathogenic agent causes at the vaccine of IL-9; Vaccine at IL-5 can be treated asthma; Vaccine at N-methyl-D-aspartate receptor-1 (NMDAR1) can be treated apoplexy.In addition, the immunity at some sexual hormoue such as human chorionic gonadotrophin (human chorionicgonadotro-pin HCG) can reduce the intravital hormonal readiness of women and reach contraceptive effect; Vaccine at gonadotropin-releasing hormone (GnRH) can be used for the treatment of advanced prostate cancer; Among the carcinoma of the pancreas patient, the antibody that utilizes therapeutic vaccine to induce at gastrin (gastrin) can prolong patient's life late.

So, is there which problem in the research of therapeutic vaccine? as if this problem is answered recently in triturating what happens at the therapeutic vaccine of Alzheimer thatch disease.Alzheimer thatch disease is a kind of very suitable disease for the treatment of with therapeutic vaccine that seems, the pathogenic process of this disease reaches several years even many decades.If can induce long-term antibody with therapeutic vaccine, be undoubtedly a kind of ideal methods of treatment so, especially this patient often forgets and takes medicine.

The feature of Alzheimer thatch disease is the deposition of patch in the brain, contains the A beta-peptide in this patch, and this A beta-peptide is that (amyloid precursor protein APP) derives for precursor protein from amyloid.In the gene of coding APP was undergone mutation the crowd that the generation that causes the A beta-peptide increases, Alzheimer thatch disease was just beginning to have taken place in one's early years.Expressing in the transgenic mice brain of APP of this sudden change has a large amount of plaque deposition, and the patch of finding in this patch and the sick patient's brain of Alzheimer thatch is similar.Add that with the A beta-peptide strong immunological adjuvant comes immune this transgenic mice that the patch in the mouse brain is reduced, the spiritual expression of mouse is clearly better.Subsequently, a kind of therapeutic vaccine at Alzheimer thatch disease has begun clinical trial, after it has good tolerability in clinical I phase evidence, the clinical II phase that comprises more than 300 patient has been followed by having begun, but unexpected be 6% subjects owing to produce the sterility encephalitis and be forced to stop to test.Studies confirm that subsequently it doesn't matter for the titre of this side effect and antibody, in fact, the patient that tried who does not produce antibody response also got the sterility encephalitis.In addition, in a patient's brain, find to have a large amount of lymphocytic infiltrations.These results of study are consistent with a kind of hypothesis, and that side effect of finding in patient is because A beta-peptide specific T lymphocyte causes, rather than since antibody cause.This just requires to develop the autoimmune s-generation vaccine that does not cause the T cell mediated.So, how is the effect of this therapeutic vaccine at Alzheimer thatch disease? during the clinical II phase of mentioning in front tests, in the patient who induces anti-A beta-peptide antibody some cognitive power weaken certain postponement, some patients are arranged in addition in this year of receiving treatment state of consciousness improvement has been arranged.If when vaccine design, can solve its safety issue, so in the near future, will occur at the therapeutic vaccine of Alzheimer thatch disease.

Another kind of slightly different vaccine is lower to the requirement of security, and that is exactly the vaccine at habit-forming medicine.Vaccine at Cocaine and Nicotine in animal experiment has reduced the level of these medicines in brain, has eliminated their habituation symptom, and experimental animal is being accepted immune then medicine no longer the dependence.In testing in the clinical I phase, the Cocaine vaccine has been proved to be good tolerability, and can induce good antibody response, and now, people are longing for its validity result.

2.2 induce the theoretical investigation of autoantibody

Is therapeutic vaccine at oneself protein how to induce the antibody of autoimmunization system generation at oneself protein? the specific antibody that produces sufficiently high titre is with the treatment relative disease, and therapeutic vaccine must overcome three obstacles: T cell tolerance, Blymphocyte tolerance, induce antibody under the situation that does not have adjuvant and antigen prolonged action preparation.As everyone knows, the human immune system mainly starts to attack to external invader, and body itself is not attacked, and this may be because body has the ability that can discern " nonego " and " oneself ".Immune this specific character is commonly called tolerance or anergy.Tolerance occurs in B cell and T cell levels.In general, the T cell tolerance is stricter.Concerning many antigens, when the T cell tolerance took place, normal B cell strain but existed in vivo.In fact, have three kinds of mechanism to cause immunological tolerance: cell strain is rejected, and promptly specific lymphocyte is thoroughly rejected from lymphocyte populations; Immunity is incompetent, and promptly specific lymphocyte exists, but its function can not be activated; Immunity is ignored, and the lymphocyte that promptly has immunologic function exists, but owing to do not run into the autoantigen that exists with the antigen form, so can not be activated.Concerning the T cell, inducing tolerance and incompetent major organs is thymus gland (central tolerance), but induces tolerance also can carry out in periphery.Blymphocyte tolerance is mainly induced in marrow, but also can induce in periphery.Usually, antigenic immunological tolerance is easier to be illustrated for enriching of generally expressing.

In the immune response at exotic antigen, T cell and B cell are worked in coordination and could be produced antibody effectively: when being subjected to the exotic antigen immunity, specific B cell conjugated antigen produces initial activation signal.In addition, B cell endocytic antigen presents the mixture of antigen peptide and mhc class ii molecule on its surface.Usually, the B cell can not activate the TH cell.Activate the TH cell, dendritic cell is absolutely necessary, the dendritic cell antigen uptaking, and, activate the TH cell at the mixture of its cell surface antigen-presenting peptide and mhc class ii molecule.The antigen peptide that TH cell recognition B cell surface after the activation presents and the mixture of mhc class ii molecule cause other conversion of B cell proliferation, production of antibodies and antibody class.If lack the synergy of TH cell because of immunological tolerance, so just can not produce antibody.In design process at the vaccine of oneself protein, if autoantigen merged with foreign protein or peptide carrier or be coupled at, just might walk around the TH cell tolerance: the specific B cell of autoantigen just can absorb this autoantigen and coupled carrier proteins, and present the mixture of carrier peptides and MHC II quasi-molecule on its surface, because the TH cell does not have immunological tolerance to carrier proteins, so can be activated, thus collaborative from the specific antibody of the B of antigen-specific cell generation at autoantigen.

Compare with the T cell tolerance, it is much loose that Blymphocyte tolerance is wanted.In fact, in many cases, autospecific B cell occurs with normal frequency in vivo, will be activated if be subjected to the acting in conjunction of antigen and TH cell.So to many solubility oneself proteins, have its specific b cells strain in the body, especially when this albumen is not very expression more than needed especially like this.For this proteinoid or polypeptide, it is linked to each other with carrier proteins, walk around the T cell tolerance, just might produce effective vaccine.In fact, utilize this strategy, derived at the antibody response of multiple self hormone.

2.3 phage surface presents peptide storehouse Study on Technology progress at random

1985, Smith reported allogenic polypeptide in the result that the single stranded phage surface presents, and the phage surface that grows up thus presents technology and developed rapidly in recent years, becomes instrument effective and important in biological study and the Application Areas.This technology is that encoding exogenous polypeptide or proteic gene segment are inserted in the gene of phage capsid protein, thereby allogenic polypeptide or albumen are presented in phage surface.Owing to constitute the permutation and combination that the deoxynucleotide of exogenous genetic fragment can be at random, so can make up and present different allogenic polypeptides or proteic phage surface presents polypeptide or protein pool, and then be applied to the screening of enzyme substrates, the screening of antibody, the screening of part, the research of albumen interphase interaction, aspects such as the diagnosis of disease.

2.3.1 phage presents system

2.3.1.1 filobactivirus presents system

Filobactivirus is the phage that a class has screw mandrel shape form, belongs to Inoviridae (Inoviride) in classification, Inovirus (Inovirus).Being used for the phage that the surface presents is the colibacillary phage of a class specific infection, comprises M13, fd, f1, If1 and Ike etc.They have similar structure.With the M13 phage is example, and its core is the single-stranded cyclic DNA of 6000bp, contains 10 genes, the 10 kinds of different albumen of encoding respectively.The gene VIII main capsid protein pVIII that encodes wherein, gene III, VI, VII, IX be coded auxiliary capsid protein pIII, pVI, pVII, pIX respectively.The M13 phage only infects the intestinal bacteria of tool F cilium with its less important capsid protein pIII.

Filobactivirus self has many characteristics that are suitable for making up peptide library.Can be received in as it and to insert allogenic polypeptide in the capsid protein, and will be presented in the virion surface behind the exogenous protein expression, be convenient to by corresponding acceptor or antibody recognition.Even the life cycle of exogenous array viral interference, also can be by dual-gene or phagemid system with expression of polypeptides in the surface, produce the chimeric phage that carries wild-type and recombinant capsid protein.Be easy to amplification, the phage after the reorganization can obtain amplification by ehec infection.For screen can specificity in conjunction with the polypeptide phage of a certain target molecule also ehec infection again, amplification back its nucleotide sequence of sequencing analysis.Phage is still very stable under various elution requirements (as low pH), can infect the very high titre of formation and (generally reach 10 ¹²), high like this titre is enough to represent clones all in the storehouse.

That be usually used at first that phage surface presents is capsid protein pIII or pVIII.PIII is an albumen maximum in the capsid protein, is positioned at the caudal end of phage, and its C-terminal is anchored on the inner membrance, outside N-terminal is free in.Its is discerned and in conjunction with colibacillary F cilium, its maximum characteristics are that the size of foreign protein or polypeptide is not had strict restriction, and the albumen to 50kD is successfully presented greatly.Because the multiple of copying of pIII has only 3-5, can screen the antigenic determinant of high-affinity, thus normal first-selected albumen for presenting, but in the research of immunology and biovaccine, be restricted.The exogenous molecules that presents with pVIII just can produce stronger immunological response by the stimulating organism body under the situation of no any adjuvant, and nearly about 2700 of its copy numbers, therefore can be used for screening the lower antibody of avidity, but the pVIII of small molecular weight can not hold more than 6 amino acid whose allogenic polypeptides, and this point has limited the fusion of allogenic polypeptide and pVIII.High copy pVIII has the potential using value in vaccine development.

2.3.1.2 other phage presents system

Except that filobactivirus, people have also been developed some other phage and have been presented system, as T4 phage and lambda particles phage.When foreign protein is presented, can merge with the C-terminal of T4 phage minor coat protein, also can by with the lambda particles phage capsid on the proteic N-terminal of D merge or merge with the C-terminal of the main tail protein pV of lambda particles phage.Present system with filobactivirus and compare, these two kinds present system all with the form release progeny virus particle of virulent phage, so the albumen that is presented can be bigger, even also can be to the virose albumen of host.

2.3.1.3 phagemid presents system

When presenting foreign protein with pIII or pVIII, foreign protein may have influence on some performances of phage.Particularly for pIII, it is essential when ehec infection, if its function is affected, then phage can't well be infected intestinal bacteria, and in order to address this problem, people have made up phagemid and presented system.In this system, foreign protein is encoded by phagemid, under the help of auxiliary type phage, a small amount of fusion rotein from phagemid had both been contained on the surface of the new phage that produces, contain a large amount of wild-type proteins again from the auxiliary type phage, even foreign protein has negative impact to phage like this, also by a large amount of wild-type protein " dilution ".Phagemid presents the appearance of system, also makes some new presentation modes become possibility.As utilize this system, and proofs such as Jespers can present foreign protein at pVI PROTEIN C end, and Gao etc. prove that the pIX protein N terminal can present antibody fragment at pVII, and proofs such as Fuh also can present foreign protein at the C-terminal of pVIII.Like this, all capsid proteins of M13 all can present system by phagemid and be used for presenting of foreign protein or polypeptide.

2.3.2 phage peptide library The Application of Technology

The appearance of phage display technology and develop into the extensive development of people and the screening new type antineoplastic medicine provides strong means.Along with the development of phage-displayed polypeptides storehouse technology, use the trend that little peptide analogue antigen antibody response has become development simultaneously.This be because: (1) contains the small peptide of the Key residues antigenic determinant on can simulated albumin matter; (2) in most cases, several Key residues and the formed non covalent bond of its binding molecule have constituted whole bonded major portions, i.e. interaction between the protein is to realize by the intersegmental interaction of partial oligopeptide.

2.3.2.1 interactional amino-acid residue analysis between the protein

Phage surface presents the research that peptide storehouse technology has been widely used in the interaction sites between the protein.For example: integrate the film surface receptor of plain (integtins) communication, by cell growth, differentiation, migration essential.Koivunen etc. screen with the random phage peptide storehouse of pentapeptide, six peptides, seven peptides, nonapeptide respectively and integrate the little peptide of plain bonded, from four peptide storehouses, all screen the little peptide that comprises the RGD sequence, otherwise, from random peptide library, screen the little peptide of bonded with it with the plain segment of the fiber adhesion that contains the RGD sequence, all have the sequence of CWDDG/LWLC.This sequence is similar to the sequence that combines little peptide MTSDDL with adenovirus penton RGD site that Hong etc. screens.Two groups confirm that all the KDDLW sequence is present in the plain subunit of integration, and participate in the crosslinked action of RGD.Present and triage techniques by the phagocytosis body surface, determine to integrate plain and RGD sequence albumen between interactional important residue, integrate plain specific inhibitor for design valuable information be provided.

What in addition, research was the most deep is to hold the little peptide that can simulate the EPO function that screens the peptide storehouse at random from phage surface.The glycoprotein that EPO (erythropoietin) is made up of 165 amino acid, in clinical application in the pathologic anaemia.But because the sugar chain of EPO accounts for about half of its molecular weight, not only limited the application of prokaryotic expression system, and the structure of sugar chain also determined can only subcutaneous or intravenously administrable.Wringhton etc. screen from the peptide library that cpVIII presents and the belt peptide of 8 amino acid of EPO receptors bind, but lower with the EPO receptor affinity.For obtaining the acceptor of high-affinity, made up on this basis and comprised with 8 amino acid being that core, both sides are the peptide storehouse of stochastic sequence, present by cpVIII, therefrom screen the cyclic peptide with EPO acceptor high-affinity, and its sequence and natural EPO do not have similarity.In body, external a series of active proofs that detect, these cyclic peptide can be simulated the EPO function really, induce receptor dimer to form, and carry out signal transduction, stimulate cell growth, differentiation.The discovery of EPO simulating peptide is that phage surface presents the important breakthrough that technology is used, and for design protein small molecule mimetics provides information, thereby opens up a new medicinal design and treatment approach.

, obtain to combine active little peptide with monoclonal antibody from phage random peptide library as the screening molecule with the immobilization monoclonal antibody, its conservative property sequence is identical with the natural antigen sequence or similar, has promptly simulated the natural protein continuous epitope.If be different from the natural antigen sequence fully, claim that these peptides are mimic epitopes (mimotopes), discontinuous amino acid folds and the epi-position structure of formation through the space on its simulation native protein primary structure.

Feliu etc. use the monoclonal antibody mAbMGr2 of HER2/neu receptor extracellular structural domain, screen a series of phage phenotypes from phage peptide library.Sequential analysis shows that the conservative property sequence of phage display peptide and the amino acid sequence homology of neu polypeptide chain are very low, but each the phage phenotype immune animal that screens all can produce at HER2/neu antibody.The little peptide that experiment confirm screens from random peptide library can the natural HER2/neu extracellular domain of fine simulation in by discontinuous amino acids formed space epi-position.

2.3.2.2 determine disease specific antigen mimicking thing

The little peptide that screening combines with purpose screening thing from random peptide library, existing many pieces of documents report that successfully the success or not of screening depends on high quality peptide storehouse and highly purified screening thing.Bibliographical information is arranged, screening method is done modification slightly, promptly available mixture such as whole serum or other body fluid screen from the peptide storehouse and the little peptide of object bonded.

Existing a plurality of research groups confirm that it is possible utilizing polyclonal serum to filter out the single-minded analogue antigen determinant of disease from random peptide library.This method filters out the phage that presents specificity analogue antigen determinant from a big peptide storehouse under the situation of not knowing any information of antigen in advance, this specific phage can only not react with normal serum with patients serum's reaction.And whether the sequence of analogue antigen determinant similar to initial molecule can not influence it and be applied to vaccine, thereby more meaningful aspect newfound disease.Cortese experiment group carries out a series of experiments, successfully screen can with antibodies among some disease patients serum, and not with the in combination little peptide of normal control.Usefulness such as Prezzi have infected the human serum of human hepatitis C (HCV) as the screening thing, and the little peptide of the phage of acquisition can react with the patients serum, and reactionless with control serum.With this type of peptide is that the antibody that the antigen immune rabbit obtains can be discerned HCV albumen.Now commercial human hepatitis B virus vaccine comprises that the genetically engineered recombinant human Hepatitis B virus vaccine of purifying all prepares by this method.

2.3.2.3 screening cell-specific, organ specific peptide

Constantly develop along with phage surface presents technology, perfect, developed to utilize non-purifying molecule as the screening part screening method.Report such as Barry, filters out from the peptide storehouse and cell surface receptor bonded peptide as affine screening part with whole cell.This method advantage is that cell surface receptor is presented in cell surface after through cell translation, processing, modification, for native conformation state fully.And cell neither needs to carry out purifying to acceptor molecule as part, also do not need receptor structure is analyzed.In addition, tumour cell is to the parent's profit and the particular organization location at particular organization position, and may there be special acceptor in prompting at these tissue surfaces.Can utilize biological tissue directly to present screening histocyte surface specific acceptor the peptide library, find the relevant important little peptide of cancer therapy, be used for the treatment of medical diagnosis on disease or drug targeting from phage surface.

In a word, phage small peptide storehouse technology has important significance for theories and using value.In the mutual identification of research between albumen, the prediction of protein folding and space conformation, the combining of interaction between peptide and organism and enzyme-to-substrate, antibody and antigenic interaction, aspects such as hormone and combining of acceptor demonstrate huge application potential.Having a extensive future such as the fields such as exploitation of the design of vaccine, the assignment of genes gene mapping, small-molecule drug.

Summary of the invention

The objective of the invention is to, filter out 12 aminoacid mimic epitopes of human B cell specificity membrane molecule CD20, thereby make up a kind of therapeutic vaccine that can induce in vivo, for Malignant B cell related neoplasms provides a kind of new medicine at the specific antibody of CD20 molecule.

To achieve these goals, the technical solution adopted in the present invention is: utilize the people mouse chimeric mAb Rituximab that is used for bone-marrow-derived lymphocyte type leukemia and lymphoma treating that has been gone on the market by drugs approved by FDA to present the mimic epitopes that the peptide storehouse filters out the CD20 molecule as part at random in phage 12, this 12 amino acid whose mimic epitopes of chemosynthesis and with the bloodthirsty azurin of keyhole (KLH) to carry out chemistry coupled, the vaccine that obtains successfully constructing.The associating freund's adjuvant immune health BALB/c mouse of growing up induces the antiserum(antisera) at people CD20 molecule, and this antiserum(antisera) can be brought into play the complement consistent with Rituximab and kills and wounds (CDC) function.

Particular content is:

1) with people mouse chimeric mAb Rituximab as part, present in the 12 peptide storehouses screening at phage random and obtain mimic epitopes with three kinds of CD20 molecules of people mouse chimeric mAb Rituximab high-affinity, its aminoacid sequence is respectively: Gln Asp Lys Leu Thr Gln Trp Pro Lys Try Leu Glu;

His?Glu?Glu?Asn?Asp?Leu?Arg?Try?Try?Phe?Ile?His；

Asn?Met?Ser?Ile?Gln?Try?His?Arg?Asp?Phe?Asn?Lys。

The mimic epitopes of three kinds of above-mentioned CD20 molecules and the protein sequence of CD20 molecule and gene order do not have any homology, have special affinity with CD20 molecule monoclonal antibody Mabthera.

Realize the screening method of 12 aminoacid mimic epitopes of above-mentioned human B cell specificity membrane molecule CD20, it is characterized in that, finish according to the following steps:

A) phage random presents the screening in 12 peptide storehouses

With the NaHCO3 of 0.1mol/L, pH8.6, the Rituximab solution bag of 100 μ g/mL preparation, are tipped upside down on 96 orifice plates on the clean paper handkerchief after spending the night by the elisa plate hole, and light deduction is residual liquid to the greatest extent; Use the TBS of 1% bovine serum albumin then, 50mM Tris-HCl, pH is hatched for 7.5,4 ℃, and sealing 2h adopts the TBST of 0.1%Tween 20 to wash again 6 times, all 96 orifice plates will be tipped upside down on the clean paper handkerchief the most residual liquid of light deduction at every turn;

Add 200 μ l and contain 2 * 10 with what TBST diluted ¹¹The storehouse phage solution of pfu, incubated at room 1h; The liquid that inclines is removed unconjugated phage, with TBS+0.1%Tween 20 washings 10 times, all 96 orifice plates will be tipped upside down on the clean paper handkerchief more at every turn, and light deduction is residual liquid to the greatest extent; With the phage of 100 μ L elutriant elution of bound, its elutriant prescription is: 0.2mol/L Glycine-HCl, and pH 2.2,10g/LBSA, and the adding prescription is 1mol/L Tris-HCl rapidly, the neutralizer neutralization of pH 9.1; Get 1 μ L and survey titre, remaining liq adds 20mL and is in early stage ER2738 culture (the intestinal bacteria ER2738 of logarithmic growth; The 5mL LB substratum of 20mg/L tsiklomitsin: Tryptones 10g/L yeast extract 5g/L NaCl10g/L), cultivate 4.5h, the phage that obtains increasing in 37 ℃ of thermal agitations;

Phage and colibacillary mixed solution that amplification is good are poured centrifuge tube into, in 4 ℃, and the centrifugal 10min of 10000rpm; Supernatant changes new centrifuge tube over to, and is centrifugal again, gets 80% centrifugal supernatant and changes new centrifuge tube over to, adds the PEG8000/NaCl of 1/6 volume, and its prescription is: PEG-8000 200mg/mL, NaCl 2.5M, 4 ℃ of standing over night; Next day, 4 ℃, the centrifugal 15min of 10000rpm, centrifugal supernatant inclines; Once centrifugal fast with method again, exhaust residual supernatant; The resuspended precipitation of 1mLTBS in 4 ℃ of centrifugal 5min of 10000rpm, is got off residual cell precipitation; Supernatant adds the PEG/NaCl of 1/6 volume, ice bath 1h, and 4 ℃, the centrifugal 15min of 10000rpm, the supernatant that inclines contains 0.01%NaN with 200 μ L ₃The resuspended precipitation of TBS, the centrifugal 1min of 10000rpm; Supernatant is the good elutriant of amplification, and 1 μ L is used to survey titre, and remaining liquid is deposited in 4 ℃;

200 μ L Rituximab solution newly wrap by a hole, are used for next round screening, in order to improve the preciseness of screening peptide, second take turns with the third round screening in, the concentration of Rituximab is reduced to 50mg/L and 20mg/L respectively; Repeat above screening step, in second screening of taking turns with third round, the first round of adding and second takes turns the phage of sifting out and should calculate according to titre and be equivalent to 2 * 10 ¹¹The liquid volume of pfu phage; The concentration of Tween among the TBST is increased to 0.5%; The eluted product of third round need not increase, and gets 1 μ L and carries out titer determination, and residue is deposited in 4 ℃;

From titer determination, on the flat board of bacterial plaque number＜100 at random 50 of pickings separate good plaques and increase and purifying, both dip in 50 good blue plaques of separation getting and put into the ER2738 that mid-log phase is cultivated respectively with aseptic toothpick, after 37 ℃ of thermal agitations are cultivated 4.5h, centrifugal 30s of moment.New centrifuge tube, once centrifugal again with method, get 80% supernatant, promptly get the phage liquid that increases;

B) specific phage screening

NaHCO with 100mg/L ₃PH8.6, bag is set up the 1%BSA control wells to each hole, isostructural IgG1 control wells, 4 ℃ of overnight incubation simultaneously by 96 hole elisa plates; The coating buffer that inclines adds 5mg/L BSA, 0.1mol/L NaHCO3, and the confining liquid of pH8.6,4 ℃ of sealing 2h wash 6 times with the TBST in the step 1), all will eliminate residual liquid at every turn; With 1 * 10 of 50 purifying ⁹/ hole phage adds NaHCO respectively ₃Among the pH8.6, BSA and homotype IgG antibody 1 bag are by incubated at room 2h in the hole.TBST washes 6 times, and every hole adds the mouse anti M13 phage mAb100 μ L of HRP mark, its ratio 1: 5000, incubated at room 1h; TBST washes 6 times, and 2, the light absorption value at A410nm place is measured in 2-azine-two 3-ethyl benzo thiazole phenanthroline sulfonic acid colour developings back;

C) phage presents the sequencing of peptide

According to ELISA result, filtering out 20 has higher affinity with rituximab and carries out sequencing with the low clone of other contrast avidity; The amplification of phage clone is the same, gets the phage culture supernatant; The conventional phage single-chain DNA that extracts, with the single stranded DNA is template, the sequencing primer that is provided in the phage random peptide library test kit is provided, primer sequence is 5 '-CCCTCATAGTTAGCGTAACG-3 ', on ABI 310 automatic dna sequencers, carry out automatic sequencing,, derive phage and present the aminoacid sequence of peptide at random according to the dna sequencing result, the homology of aminoacid sequence between the relatively more different clones, three kinds of mimic epitopess that can obtain screening.

2) 12 aminoacid mimic epitopes of above-mentioned human B cell specificity membrane molecule CD20 make up the method for polypeptide epitope vaccine, it is characterized in that three kinds of mimic epitopes Gln Asp Lys Leu Thr Gln Trp ProLys Try Leu Glu that above-mentioned screening is obtained; His Glu Glu Asn Asp Leu Arg Try Try Phe Ile His; Asn Met SerIle Gln Try His Arg Asp Phe Asn Lys carries out chemiluminescent polypeptide synthetic (China occasion biotech company finishes by Xi'an), the employing glutaraldehyde method is chemical coupled with synthetic polypeptide and protein carrier KLH's, KLH is mixed with 1mg/ml solution, prescription is 0.1M PBS, pH7.5; The KLH solution of 5ml preparation, with 40 polypeptide: the ratio of 1 proteic mol ratio adds synthetic polypeptide, be cooled to 2～6 ℃, the glutaraldehyde that dropwise adds 5ml 2% is to the polypeptide protein mixture, and 2～6 ℃ were constantly stirred 1 hour, pH is 7.5 PBS or normal saline dialysis, 4 ℃ are spent the night, and obtain about 10ml polypeptide protein solution, packing,-70 ℃ frozen, and the concentration of its coupled thing is 0.5mg/ml; Determine coupled effect and antibody evaluation thereof through DOT Blot, be the polypeptide epitope vaccine of the 12 aminoacid mimic epitopes structure of B cell specificity membrane molecule CD20.

3) the mimic epitopes polypeptide vaccine of mimic epitopes structure of the present invention can induce the antiserum(antisera) at this epi-position in the mouse body; This antiserum(antisera) can be discerned the CD20 molecule of the natural expression of CD20+ cell; Can effectively kill and wound the CD20+ cell.The associating freund's adjuvant immune health BALB/c mouse of growing up induces the antiserum(antisera) at people CD20 molecule, and this antiserum(antisera) can be brought into play the complement consistent with Rituximab and kills and wounds (CDC) function.

The mimic epitopes polypeptide vaccine of the present invention's preparation uses the mimic epitopes of the CD20 molecule that filters out to break through the autoimmunity tolerance in conjunction with carrier proteins KLH, generation is at the neutralizing antibody of CD20 molecule, thereby the method for using active immunity substitutes the therapeutic modality of this passive immunization of Rituximab monoclonal antibody, and then reduce the monoclonal antibody side effect, alleviate the sufferer burden.

Description of drawings

Fig. 1 be filter out 20 with Rituximab higher affinity is arranged and with the ELISA result of other contrasts (wherein (a) is homotype IgG1 contrast, (b) is with the BSA contrast) phage clone that avidity is low: the affine result of No. 1 positive phage of frame and Rituximab; No. 2 frames are the affine result with the isostructural irrelevant antibody of Rituximab; No. 3 yellow frames are the affine result with confining liquid bovine serum albumin BSA.

Confirm that through Dot Blot the vaccine KLH-P1 and the Rituximab monoclonal antibody that make up have good affinity, and KLH-P2, KLH-P3 all can not successfully combine with Rituximab, promptly can not simulate the epi-position of CD20.

Fig. 2 detects KLH-P1 immune serum (1: 10000) titre with ELISA: No. 1 frame is that GST-P1 detects antigen; No. 2 frames are the GST contrast.Be 6 mouse after the immunity 1～No. 6, No. 7 is the control mice serum of KLH immunity.

Fig. 3 is that flow cytometer detects antiserum(antisera) and CD20 ⁺The CD20 molecule bonded result of the natural expression of cell surface: A contrast antiserum(antisera) and CD20 ⁺Cell (Raji cell); B contrast antiserum(antisera) and CD20-cell (Jurkat cell); C immunity antiserum(antisera) and CD20 ⁺Cell; D immunity antiserum(antisera) and CD20 ^-Cell; E, F are respectively the fluorescence photo of C, D.

Fig. 4 is that mtt assay detects antiserum(antisera) to CD20 ⁺The result of cell killing: light frame is CD20 ⁺Cell (Raji cell); Dark frame is CD20 ^-Cell (Jurkat cell); No. 1 Rituximab positive control; No. 2 is antiserum(antisera); Be the contrast antiserum(antisera) No. 3; No. 4 is the contrast of simple complement.

Embodiment

The present invention is described in further detail below in conjunction with drawings and Examples.

According to technical scheme of the present invention, preparation people CD20 molecular therapy autovaccine is at first used affine method for screening and is utilized Rituximab to present 12 peptide storehouse (BioLabs for part at phage random ^New England) screening obtains 12 polypeptide mimic epitopes Gln-Asp-Lys-Leu-Thr-Gln-Trp-Pro-Lys-Try-Leu-Glu (glutamine-aspartic acid-Methionin-leucine-Threonine-glutamine-tryptophane-proline(Pro)-Methionin-tryptophane-leucine-L-glutamic acid) of high-affinity in, after this epi-position of chemosynthesis, using glutaraldehyde method, that itself and KLH are carried out chemistry is coupled, obtains the immunity vaccine.

Realize the screening of peptide of the present invention storehouse, vaccine construction, and immune animal, the antibody calibration operation, finish according to the following steps:

3.1. phage random presents the screening in 12 peptide storehouses

Phage random dodecapeptide storehouse (Ph.D.-12 ^TMPhage display peptide library): available from U.S. New England Biolabs company, its titre is 1.5 * 10 ¹³Plaque forming unit (pfu)/mL; Diversity 2.7 * 10 ⁹Transformants.The host bacterium is intestinal bacteria E.coli ER2738, carries to be had tetracyclin resistance by the F ' factor of phage-infect, can form the α complementation with the phage that has presented 12 peptides.Sequencing primer (96gIIIsequencing primer) sequence is: ccctcatagttagcgtaacg.

3.1.1 the mensuration of phage titre

(1) cultivation of E.coli ER2738 is with aseptic technique picking one transfering loop from the frozen thing of the colibacillary glycerine of ER2738, be inoculated in LB flat board (the Tryptones 10g/L of tetracyclin resistance, yeast extract 5g/L, agar 15g/L, NaCl 10g/L) on, cultivate 24h for 37 ℃, treat dull and stereotyped the bright bacterium colony of visible white and form after, basic culture plate is placed 4 ℃ of preservations.

(2) the single colony inoculation of picking E.coli ER2738 is in the 5mL LB that contains the 20mg/L tsiklomitsin (Tryptones 10g/L yeast extract 5g/L NaCl10g/L) nutrient solution on the flat board, and 37 ℃ of shaking culture are to logarithmic growth mid-term (OD600 is about 0.5).

(3) phage is carried out 10 times of continuous gradient dilutions with LB.For the phage culture supernatant after the amplification, dilution 10 ⁸-10 ¹¹Doubly; For the screening elutriant that does not increase, dilution 10 ¹-10 ⁴Doubly.

(4) the phage sample of every part of dilution is got 10 μ l, adds respectively (200 μ l) in the cultured bacterium of step (2), quick mixing, incubated at room 1-5min.

(5) each bacterium that infects moves into 45 ℃ of pre-temperature respectively, contains top-agar 3-5ml (Tryptones 10g/L, yeast extract 5g/L, NaCl10g/L, MgCl ₂6H ₂O1g/L, agarose 7g/L) in the culture tube, add the X-gal of 40 μ l 20mg/mL and the IPTG of 16 μ l 50mg/mL, abundant mixing is poured the LB flat board of pre-temperature then rapidly into, tilts to make the top-agar uniform distribution.

(6) flat board is placed 5min, temperature is reduced, counter-rotating is dull and stereotyped then, 37 ℃ of overnight incubation.

Observe dull and stereotypedly, count blue plaque and multiply by this flat board pnagus medius dilution of sample multiple, obtain the titre of per 10 μ l phages, with blue plaque forming unit represent (plaque forming units, pfu).

3.1.2. biological the elutriation

(1) in 96 orifice plates, adds the Rituximab solution (100 μ g/mL) of 200 μ L with NaHCO3 (pH8.6) dilution of 0.1mol/L.Place the box of airtight humidity to shake gently, 4 ℃ of overnight incubation.

(2) inoculate ER2738 in advance in the LB that contains tsiklomitsin of 10mL (surveying titre uses) and 20mL (phage is used in amplification), 37 ℃ of cultivations.

(3) 96 orifice plates in (1) are taken out, the liquid that inclines tips upside down on 96 orifice plates on the clean paper handkerchief, and light deduction is residual liquid to the greatest extent.

(4) (50mM Tris-HCl pH7.5), is hatched 2h for 4 ℃ to the TBS of adding 200 μ L confining liquid 1% bovine serum albumins (BSA).

(5) TBST (TBS+0.1%Tween20) washing is 6 times, all 96 orifice plates will be tipped upside down on the clean paper handkerchief at every turn, and light deduction is residual liquid to the greatest extent.

(6) add 200 μ l and contain 2 * 10 with what TBST diluted ¹¹The storehouse phage solution of pfu, incubated at room 1h.

(7) liquid that inclines is removed unconjugated phage, and TBST washing 10 times all will tip upside down on 96 orifice plates on the clean paper handkerchief at every turn, and light deduction is residual liquid to the greatest extent.

(8), and add neutralizer (1mol/L Tris-HCl pH 9.1) neutralization rapidly with the phage of 100 μ L elutriants (0.2mol/L Glycine-HCl, pH 2.2,10g/L BSA) elution of bound; Get 1 μ L and survey titre, remaining liq adds 20mL and is in the early stage R2738 culture of logarithmic growth, cultivates 4.5h in 37 ℃ of thermal agitations, increases.

(9) will increase good phage and colibacillary mixed solution poured centrifuge tube into, in 4 ℃, and the centrifugal 10min of 10000rpm.Change centrifugal supernatant over to new centrifuge tube, once centrifugal again with method, get 80% centrifugal supernatant and go into new centrifuge tube.

(10) in centrifuge tube, add 1/6 volume PEG8000/NaCl (PEG-8000 200mg/mL, NaCl2.5M), in 4 ℃ of standing over night.

(11) take out PEG8000/NaCl next day and precipitate good liquid, in 4 ℃, the centrifugal 15min of 10000rpm, the careful centrifugal supernatant that inclines; Once centrifugal fast with method again, exhaust residual supernatant with micropipet.

(12) with 1mLTBS resuspended centrifugation, resuspended liquid is changed in the little centrifuge tube, in 4 ℃ of centrifugal 5min of 10000rpm so that residual cell precipitation is got off.

(13) centrifugal supernatant is moved in the new little centrifuge tube, add the PEG/NaCl ice bath 1h of 1/6 volume.4 ℃, the centrifugal 15min of 10000rpm, the careful centrifugal supernatant that inclines; Once centrifugal fast with method again, exhaust residual supernatant with micropipet.

(14) contain 0.01%NaN with 200 μ L ₃The resuspended precipitation of TBS, the centrifugal 1min of 10000rpm is to precipitate those undissolvable materials.Supernatant is moved into new centrifuge tube, be the good elutriant of amplification, 1 μ L is used to survey titre, and remaining liquid is deposited under 4 ℃ of environment.

(15) newly wrap with 200 μ L target protein solution, be used for next round screening by 96 orifice plates, one hole, in order to improve the preciseness of screening peptide, second take turns with the third round screening in, the concentration of target protein is reduced to 50mg/L and 20mg/L respectively.

(16) observe the result who surveys titre in the step 14, the quantity of counting locus coeruleus is to determine the titre of phage, and this phage is used for second screening of taking turns.In second screening of taking turns, the phage that the first round of adding sifts out should be equivalent to 2 * 10 according to this titre calculating ¹¹The liquid volume of pfu phage.

(17) carry out second and take turns screening, repeating step 2-15, and the concentration of Tween among the TBST is increased to 0.5%.

(18) newly wrap with 200 μ L target protein solution, be used for the third round screening by 96 orifice plates, one hole.Repeating step 2-8, the concentration of Tween is 0.5% among the TBST, and the eluted product of third round need not increase, and gets 1 μ L and carries out titer determination, and residue is deposited in 4 ℃.From titer determination, on the flat board of bacterial plaque number＜100 at random 50 of pickings separate good plaques and increase and purifying by following method, should notice that the time that the plaque of picking is cultivated can not surpass 18h this moment.(these 50 plaques are the positive bacteriophage spot of selecting)

Through the three-wheel screening, specific phage is further by enrichment, and the phage titre that obtains progressively raises.

	Titre
	Titre	The first round	The?average： 5.6×10 ⁹pfu
Second takes turns	The?average： 7.8×10 ⁹pfu	The first round	The?average： 5.6×10 ⁹pfu
Second takes turns	The?average： 7.8×10 ⁹pfu	Third round	The?average： 2.1×10 ¹⁰pfu

(19). the amplification of positive bacteriophage, the overnight culture of ER2738 is carried out dilution in 1: 100 with LB, the 1mL/ pipe is sub-packed in the culture tube of 50mL.

(20) will dip in 50 good blue plaques of separation getting with aseptic toothpick and put into above-mentioned culture tube respectively, 37 ℃ of thermal agitations are cultivated 4.5h, increase.

(21) the good phage liquid that will increase is poured centrifuge tube into, centrifugal 30s of moment.Change centrifugal supernatant over to new centrifuge tube, once centrifugal again with method.Get 80% supernatant, be the phage liquid of amplification.Can deposit several weeks under 4 ℃, if will preserve for a long time, can add final concentration and be 50% aseptic glycerine and deposit in-20 ℃.

3.1.3. phage specificity screening (phage E LISA)

In 50 clones that this picks out, utilize phage E LISA further to select clone with the Rituximab high-affinity.

(1) with 100mg/L Rituximab (NaHCO ₃PH8.6) bag is by 96 hole elisa plates, every hole 200 μ L, set up the bag of 1%BSA to be contrasted to each Rituximab hole simultaneously, and each hole is wrapped the negative contrast by an isostructural IgG1 (from The Fourth Military Medical University cell engineering center), 4 ℃ of overnight incubation again by the hole.

(2) coating buffer that inclines adds confining liquid (pH 8.6 for 5mg/L BSA, 0.1mol/L NaHCO3), 4 ℃ of sealing 2h.

(3) the deblocking liquid that inclines, TBST washes 6 times, all 96 orifice plates will be tipped upside down on the clean paper handkerchief at every turn, and light deduction is residual liquid to the greatest extent.

(4) with the phage (1 * 10 of 50 purifying ⁹/ hole) add Rituximab respectively, BSA and homotype IgG antibody 1 bag are by incubated at room 2h in the hole.

(5) TBST washes 6 times, and every hole adds mouse anti M13 phage mAb (1: 5000) the 100 μ L of HRP mark, incubated at room 1h.

(6) TBST washes 6 times, and the light absorption value at A410nm place is measured in ABTS (2,2-azine-two 3-ethyl benzo thiazole phenanthroline sulfonic acid) colour developing back.

3.1.4. phage presents the sequencing of peptide

According to 50 clones' ELISA result, filtering out 20 has higher affinity with rituximab and carries out sequencing with the low clone's (the results are shown in Figure 1) of other contrast avidity.

(1) extraction of phage single-chain DNA

The amplification of phage clone is the same, gets the phage culture supernatant.The single stranded DNA extraction test kit of producing with Hua Shun company carries out the extraction of phage single-chain DNA, operate by process specifications: in containing the LB nutrient solution of single phage clone, add precipitated liquid, obtain the phage particle precipitation behind the high speed centrifugation, add lysate lytic phage coat protein, through resin purification, wash-out obtains phage single-chain DNA, and quantitative its concentration of ultraviolet is greater than 100ng/ μ L.

(2) Sanger double deoxidating chain termination measuring dna sequence dna

With the single stranded DNA is template, and the sequencing primer that is provided in the phage random peptide library test kit is provided, and primer sequence is 5 '-CCCTCATAGTTAGCGTAACG-3 ', carries out automatic sequencing on ABI 310 automatic dna sequencers.

(3) present the derivation and the amino acid identity analysis of peptide ammino acid sequence at random

According to the dna sequencing result, derive phage and present the aminoacid sequence of peptide at random, draw three kinds of sequences (P1, P2, P3) as follows.The homology of aminoacid sequence between the relatively more different clones.

P1：Gln?Asp?Lys?Leu?Thr?Gln?Trp?Pro?Lys?Try?Leu?Glu

P2：His?Glu?Glu?Asn?Asp?Leu?Arg?Try?Try?Phe?Ile?His

P3：Asn?Met?Ser?Ile?Gln?Try?His?Arg?Asp?Phe?Asn?Lys

3.2. the structure of vaccine

According to above sequencing result, the mimic epitopes that summary obtains three kinds of CD20 molecules is: Gln Asp Lys Leu ThrGln Trp Pro Lys Try Leu Glu, His Glu Glu Asn Asp Leu Arg Try Try Phe Ile His, Asn Met Ser Ile Gln Try His Arg Asp Phe Asn Lys.

3.2.1. the chemosynthesis of epi-position

Adopt synthetic above three peptide species of solid-phase synthesis, synthetic work is finished by Xi'an China occasion biotechnology company, and purity is more than 98%.

3.2.2. polypeptide is chemical coupled

Adopt glutaraldehyde method to carry out the chemical coupled of polypeptide and protein carrier KLH.

(1) KLH (available from Xi'an Hua Chen) is mixed with 1mg/ml (0.1M PBS, pH7.5) solution.

(2) get the above-mentioned protein solution of 5ml, with 40 polypeptide: 1 proteic ratio (mol ratio) adds synthetic polypeptide, is cooled to 2～6 ℃, and middling speed is stirred.

(3) dropwise add the glutaraldehyde of 5ml 2% to the polypeptide protein mixture.

(4) 2～6 ℃ were constantly stirred 1 hour.

(5) with above-mentioned solution with PBS (pH7.5) or normal saline dialysis, 4 ℃ are spent the night.

(6) after dialysis is finished, obtain about 10ml polypeptide protein solution, packing ,-70 ℃ are frozen.

Its coupled thing gets concentration and should be 0.5mg/ml.

3.2.3. make up the evaluation of vaccine

Determine coupled effect with DOT Blot.

(1) with coupled polypeptide KLH-Gln Asp Lys Leu Thr Gln Trp Pro Lys Try Leu Glu (KLH-P1), KLH-His Glu Glu Asn Asp Leu Arg Try Try Phe Ile His (KLH-P2), KLH-Asn Met SerIle Gln Try His Arg Asp Phe Asn Lys (KLH-P3), and KLH puts on nitrocellulose filter (NC film) so that same amount (about 3 μ g) is careful.

(2) with 2%BSA TBS (pH7.5) closing membrane more than 2 hours.

(3) TBST (0.5%Tween20) washing film is 3 times, each 10 minutes.

(4) add the rituximab (4 μ g/ml) that TBST dilutes, the room temperature jog was hatched 1 hour.

(5) the unconjugated rituximab of flush away in kind.

(6) the anti-people two of AP (alkaline phosphatase) mark of adding TBST dilution is anti-, incubated at room 30 minutes.

(7) TBST flush away two is anti-, again with TBS flush away tween 20, develops the color with alkaline phosphatase substrate developer (sigma) lucifuge.

3.3. animal immune

3.3.1 laboratory animal

BALB/c mouse, 4 ages in week are available from The Fourth Military Medical University's Experimental Animal Center.Be divided into 4 groups at random, 6 every group.

3.3.3 experiment material

Freund's complete adjuvant, Freund's incomplete adjuvant are all available from Beijing ancient cooking vessel state biological products company.The candidate vaccine KLH-P1 of above-mentioned preparation and reference protein KLH.

3.3.4 immune programme for children

(1) mouse is divided into 2 groups at random, 6 every group.

(2), and make it complete emulsification respectively with vaccine protein and carrier proteins contrast and freund's adjuvant mixing.

(3) the subcutaneous multi-point injection immune mouse in per two all backs once.Each every immunizing dose is 30～50 μ g albumen.

(4) being total to immunity four times, is Freund's complete adjuvant emulsification first, and the back is the emulsification of Freund twice, does not add adjuvant for the last time, carries out abdominal injection.

(5) the last immunity is back 10 days, and eyeball is got blood, and separation of serum.

3.4. antibody is identified

3.4.1.ELISA

3.4.1.1. material and reagent

96 hole elisa plates (Costar), NaHCO ₃PH8.6, GST-P1 (merges the mimic epitopes P1 of GST Thiadiazolidine isomerase prokaryotic expression, by these section office construction expression voluntarily), GST (Thiadiazolidine isomerase, these section office express voluntarily), the anti-people of HRP (horseradish peroxidase) mark, and anti-mouse two anti-(Wuhan Zhong Shan biological products company).ABTS(sigma)。

3.4.1.2. experimental technique

(1) GST-P1, GST 100mg/L (NaHCO ₃, pH8.6) wrap respectively, and multiple hole be set by 96 orifice plates (100 μ l/ hole).

(2) seal with TBS (pH7.5) 1%BSA.TBST (0.5%Tween 20) washing 6 times.

(3) add the serum (1: 100,1: 1000,1: 10000, TBST dilution) of every mouse respectively, and with the positive contrast of Rituximab, incubated at room 1 hour.

(4) the TBST washing is 6 times, each 3 minutes.

(5) the anti-mouse two of adding HRP mark is anti-, and positive control adds anti-people two and resists incubated at room 30 minutes.

(6) the TBST washing is 6 times, each 3 minutes.

(7) add the ABTS developer, 550 type ELISA readout instruments (U.S. Bio-Rad company) 410nm reads plate.

3.4.2. flow cytometer detects (FCM)

3.4.2.1. material and reagent

(1) cell and cell cultures

People's Malignant B leukemia cell Raji cell (CD20+), Jurkat cell (CD20-) is all available from immunology teaching and research room of The Fourth Military Medical University.All cells is all cultivated in RPMI1640 substratum (GIBCO Co).The penicillin and streptomycin that contains 10% calf serum (Hangzhou folium ilicis chinensis biological factory) and 100U*ml-1 in the substratum.The cell of taking the logarithm vegetative period, counting is diluted to 1 * 10 with single cell suspension with the RPMI1640 substratum ⁷Individual cell/ml is standby.

(2) antiserum(antisera) and antibody:

Antiserum(antisera) is the antiserum(antisera) of collecting in the animal immune experiment, the negative contrast antiserum(antisera) of KLH immunity; The positive control antibodies of rituximab (0.01 μ g/ μ L); Second antibody is respectively the goat-anti mouse of FITC mark, anti-human IgG1 (Wuhan doctor's moral).The deactivation normal rabbit serum is from The Fourth Military Medical University's Experimental Animal Center.

(3) reagent:

PBS (0.01M, pH7.2); Washings (5% foetal calf serum, 4%NaN ₃, PBS); Stationary liquid (2% glucose, 1% formaldehyde, 0.02%NaN ₃, PBS).

3.4.2.2. method

(1) get 40 cell suspensions add antiserum(antisera) arranged in advance (KLH-P1, KLH), the plastic centrifuge tube of Rituximab (30 μ L), the normal rabbit serum of (PBS dilution) deactivation in 1: 20 that adds 50 μ L was again hatched 30 minutes for 4 ℃.

(2) the washings washing is 3 times, and about each washings adding 1.5ml (1000rpm * 5min).

(3) abandon supernatant, add two of 50 μ L working concentrations and resist, fully shake up, 4 ℃ of lucifuges are hatched 30min.

(4) the washings washing is 3 times, and about each washings adding 1.5ml (1000rpm * 5min).

(5) add 600 μ L stationary liquids.

(6) going up machine (model is FACS calibur, U.S. BECTON DICKINSON company) detects.

3.4.3. the cytolysis of the complement-mediated of antibody induction (CDC)

3.4.3.1. material and reagent

(1) cell and cell cultures

The same.

(2) antibody and complement

Antiserum(antisera) is the same; The positive contrast of Rituximab (0.01 μ g/ μ L); The complement source is cavy (The Fourth Military Medical University's Experimental Animal Center).

(3) reagent

5mg/ml MTT phenylbenzene tetrazole smelling salts (sigma); PBS (0.01M, pH 7.4); Dimethyl sulfoxide (DMSO) DMSO (Shanghai chemical reagent factory).

3.4.3.1. method and step

(1) 3 guinea pig heart is got blood, and separation of serum mixes the back and originates as complement.

(2) serum-free RPMI1640 substratum is adjusted Raji, and the Jurkat cell concn is 1 * 10 ⁶Cells/ml, the every pipe packing of 50 μ L adds antiserum(antisera) (KLH-P1) respectively again, contrast antiserum(antisera) (KLH), each 30 μ L of rituximab.

(3) each pipe adds 100 μ L guinea pig serum, 37 ℃, CO respectively ₂Incubator was hatched about 1 hour.

(4) centrifugal (1000rpm * 5min), abandon supernatant.

(5) each pipe adds the RPMI1640 substratum 200 μ L of 1% calf serum, MTT solution 20 μ L, 37 ℃, CO respectively ₂Incubator continued to hatch about 4 hours.

It is (6) centrifugal that (behind the 1000rpm * 5min), discard culture supernatant, every pipe adds the DMSO of 150 μ L, and concussion 10min moves into elisa plate.

(7) wavelength 490nm, microplate reader (U.S. Bio-Rad company) is read plate.

3.5 effect of the present invention:

As follows through experiment confirm effect of the present invention:

1) the monoclonal antibody Mabthera (Rituximab) that utilizes FDA approval listing has screened the mimic epitopes that the human B cell surface C D20 molecule that Mabthera discerns for part presents peptide Kucheng merit at phage random 12: Gln AspLys Leu Thr Gln Trp Pro LYs Try Leu Glu; His Glu Glu Asn Asp Leu Arg Try Try Phe IleHis; Asn Met Ser Ile Gln Try His Asp Phe Asn Lys.Has special affinity (see figure 1) through phage E LISA detection with the Mabthera monoclonal antibody.Through sequence alignment, do not have any homology with the CD20 molecule.

2), confirm that through Dot Blot KLH-Gln Asp Lys Leu Thr Gln Trp Pro Lys Try Leu Glu (KLH-P1) and Mabthera monoclonal antibody have good affinity (see figure 2) through chemosynthesis and the coupled protein vaccine that constitutes by mimic epitopes and protein carrier KLH that made up of chemistry.The simulation of this polypeptide success discontinuous amino acid and the epi-position structure that form folding on the natural CD20 molecule primary structure through the space.

3) with KLH-Gln Asp Lys Leu Thr Gln Trp Pro Lys Try Leu Glu immunity BALB/c mouse, obtain antiserum(antisera), detect and can combine with the polypeptide Gln Asp Lys Leu Thr Gln TrpPro Lys Try Leu Glu specificity of GST amalgamation and expression through ELISA, titre can reach 1: 10000 (see figure 3).

4) the flow cytometer detected result shows: the antiserum(antisera) of acquisition can combine (see figure 4) with the CD20 molecule of natural expression on the Raji cell.The complement killing experiments shows (see figure 5), and this antiserum(antisera) also can be induced and be produced the cytolysis that complement relies on, and produces the activity similar to monoclonal antibody Rituximab.

In sum, obtain and the protein vaccine of this epi-position can be applied to the autovaccine of the CD20 molecule of human body, thereby substitutes or additional monoclonal antibody passive immunotherapy in the active immunity mode, overcomes monoclonal antibody treatment defective and lays the first stone.

Claims

1. 12 aminoacid mimic epitopes of human B cell specificity membrane molecule CD20, it is characterized in that, with people mouse chimeric mAb Rituximab as part, present in the 12 peptide storehouses screening at phage random and obtain mimic epitopes with the CD20 molecule of people mouse chimeric mAb Rituximab high-affinity, its aminoacid sequence is:

Gln?Asp?Lys?Leu?Thr?Gln?Trp?Pro?Lys?Try?Leu?Glu。

2. 12 aminoacid mimic epitopes of human B cell specificity membrane molecule CD20 as claimed in claim 1, it is characterized in that, the protein sequence of the mimic epitopes of described CD20 molecule and CD20 molecule and gene order do not have any homology, have special affinity with CD20 molecule monoclonal antibody Mabthera.

3. realize that 12 aminoacid mimic epitopes of the described human B cell specificity membrane molecule CD20 of claim 1 make up the method for polypeptide epitope vaccine, it is characterized in that, mimic epitopes Gln Asp Lys Leu Thr Gln Trp Pro LysTry Leu Glu is carried out chemosynthesis, adopt glutaraldehyde method to synthesize the chemical coupled of polypeptide epitope and protein carrier KLH; With 0.1M PBS, pH7.5 is mixed with 1mg/ml solution with KLH, gets this solution of 5ml, synthesize polypeptide epitopes with 40: the ratio of 1 proteic mol ratio adds synthetic polypeptide, be cooled to 2～6 ℃, the glutaraldehyde that dropwise adds 5ml 2% is to the polypeptide protein mixture, and 2～6 ℃ were constantly stirred 1 hour, pH is 7.5 PBS or normal saline dialysis, 4 ℃ are spent the night, and obtain about 10ml polypeptide protein solution, packing,-70 ℃ frozen, and the concentration of its coupled thing is 0.5mg/ml; Determine coupled effect and antibody evaluation thereof through DOT Blot, be the polypeptide epitope vaccine of the 12 aminoacid mimic epitopes structure of B cell specificity membrane molecule CD20.

4. the method for structure polypeptide epitope vaccine as claimed in claim 3 is characterized in that, the epitope polypeptide vaccine that described mimic epitopes makes up can induce the antiserum(antisera) at this epi-position in the mouse body; This antiserum(antisera) also can be discerned CD20 ⁺The CD20 molecule of the natural expression of cell; Can effectively kill and wound CD20 ⁺Cell.