CN1480463A - Chirality peptide antigen and compon of bacterin based on core sequence of mucin 1 - Google Patents
Chirality peptide antigen and compon of bacterin based on core sequence of mucin 1 Download PDFInfo
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Abstract
A peptide vaccine able to stimulate organism to generate the antineoplastic immunoreaction, especially the chiral peptide antigen based on core sequence of mucoprotein 1, the composite vaccine containing said peptide antigen, its preparing process, and its application as immunogen in stimulating organism to generate immuno reaction are disclosed.
Description
The present invention relates to be used to stimulate body to produce the peptide vaccine of anti tumor immune response, particularly relate to chirality peptide antigen based on the core sequence of MUC-1, contain the antigenic vaccine composition of said peptide, its preparation method and stimulating body to produce application in the immune response as immunogen.
The basis of tumor immunity is the expression and the existence of tumour antigen.Recent two decades comes, and has found the multiple tumour antigen relevant with tumor development in succession, and one of them is exactly a Saliva Orthana.The Saliva Orthana high molecular that to be a class expressed by various normal or pernicious epithelial cells (〉=200KDa) transmembrane glycoprotein, the known glycoprotein that has at least nine kinds of forms.Various Saliva Orthanas all have similar structure, all are rich in Serine, Threonine and proline(Pro), and have the tandem repetitive sequence of different numbers.Wherein MUC-1 (MUC1) is first discovery, can be by the Saliva Orthana of anti-epithelial cell monoclonal antibody identification.The MUC1 polypeptide backbone by extracellular fragment, stride the film section and born of the same parents' inner segment three parts are formed.Wherein, stride film section and born of the same parents' inner segment and have conservative property between kind, in the performance of MUC1 function, play an important role so infer it.Extracellular fragment contains 20-125 successive tumor-necrosis factor glycoproteins, and each tumor-necrosis factor glycoproteins comprises 20 amino acid, i.e. PDTRPAPGSTAPPAHGVTSA.Wherein S, T, A, G and P five seed amino acids account for more than 50%, and P is to the formation of MUC1 space structure and and then the immunogenicity of molecule played a significant role.The oligosaccharides that the O-of nearly 50-90% connects is connected on the Serine and threonine residues of Saliva Orthana core area (the series connection iterons of 20 amino acid primitives), thereby the rigidity and snappiness (the Strous et al. of whole molecule have been improved, Crit.Biochem.Biol.25:57,1992).Known each series connection repeats to include 5 possible glycosylation sites, and is the immunodominance zone of containing epitope between paired Threonine and Serine.
People MUC1 gene is positioned on the karyomit(e) 1q21, and contains 7 exons.A key character of MUC1 gene is its polymorphism, and promptly its 2nd exon contains many successive tumor-necrosis factor glycoproteinss (VNTR).Each VNTR comprises 60 bases and is rich in GC.From 20 to 125 of the VNTR numbers of different people do not wait.
Present studies show that MUC1 both can bring out antitumor CTL immune response (MHC is restrictive and non-, and MHC is restrictive), can suppress immunologically competent cell killing and wounding tumour cell simultaneously again.High-caliber MUC1 expresses and the prognosis of tumour is negative correlation, and prompting MUC1 may participate in immunoreactive adjusting.Discover, see the lip-deep Saliva Orthana of tumor cell membrane and be different from Saliva Orthana on the normal epithelium cell in some aspects.For example, be added in that O-dextran on the Saliva Orthana that normal breast produces is based on core 2 and be compound with it, be added in O-dextran on the mammary cancer Saliva Orthana and then be based on core 1.This just means that some epitope in the tandem repetitive sequence is crested in normal Saliva Orthana, then expose in the relevant Saliva Orthana of tumour.Because also carry new sugar antigen decision base on the mammary cancer Saliva Orthana molecule, so whole molecule is different from the normal breast Saliva Orthana on antigenicity.Someone finds that mammary cancer, carcinoma of the pancreas, colorectal carcinoma, prostate cancer and some other secretory tissue tumours have the mucinous expression of unusual glycosylation.Evidence suggests, suffer from the patient of mammary cancer, colorectal carcinoma and the ovarian cancer of transfer at some, the orosomucoid level improve with immunotherapy after antitumor reaction and the reduction of patient's survival rate relevant (MacLean etal., J.Immunotherapy, 20:70,1997).Also find in addition, relevant MUC1 Saliva Orthana of the tumour of affinity purification and synthetic MUC1 polypeptide core tumor-necrosis factor glycoproteins can suppress the proliferative response of human T-cell to the polyclone stimulator, and the multiple number of connecting in the inhibition degree of on cell proliferation and the MUC1 polypeptide core (the Agawal et al that is directly proportional, Nature Med.4:43,1998).People such as Chen (Chen L, et al, J.Immunol.139:351-359,1997) further find that the MUC1 of tumor cell secretion can suppress the lymphocytic propagation of T, make it to be in Go/G! Phase.Jerome and colleague thereof studies show that, exist in the lymphoglandula of some tumour patient can with the cytotoxic lymphocyte (CTL) of people's Saliva Orthana reaction, and the activity of CTL (Jerome et al in the anti-MUC1 antibody MUC1 positive target cell capable of blocking, CellularImmunity and Immunotherapy of Cancer, 321-328,1990).On the other hand, the experiment of carrying out with the reactive monoclonal antibody of epithelial cell tumour and relevant healthy tissues shows, tumour cell is because of the unusual glycosylation of its Saliva Orthana molecule, and may have the epitope different with the normal cell Saliva Orthana.The Saliva Orthana molecule glycosylation of tumor cells expression is incomplete, and sugar chain is shorter relatively, thereby causes the exposure (Devine et al, CancerRes., 51:5826,1991) of Saliva Orthana molecule tandem repetitive sequence on cell surface.
Saliva Orthana is above-mentioned these character of MUC1 particularly, the particularly exposure of Saliva Orthana core sequence on the tumour cell, and immunity system is to the response capacity of these structures of Saliva Orthana molecule, make we might with Saliva Orthana particularly MUC1 as the target molecule of immunotherapy of tumors, utilization is carried out tumour-specific immunotherapy (Devine et al based on mucinous peptide vaccine, Cancer Res., 51:2908,1991).
In recent years, some investigators design has also prepared a series ofly based on Saliva Orthana particularly synthetic or the antigen peptide of reorganization or the binding substances of its sugar or fat of MUC1, or contains the immune regulation composite of this class peptide and relevant composition.For example, United States Patent (USP) 5,744 discloses for No. 144 and to have comprised that at least 5 MUC1 series connection repeat, and is connected with 5 additional amino acid whose synthetic MUC1 sample peptides at tandem repetitive sequence N or C-terminal.Said peptide is not having to present native conformation under the glycosylated situation.United States Patent (USP) 6,177, disclose for No. 256 a kind of as the immunogenicity anti-tumor vaccine, the binding substances of forming by oxidation seminose and people's Saliva Orthana MUC1 polypeptide.United States Patent (USP) 6,168 discloses the slow-released carrier that contains based on the antigen peptide of the series connection iteron of MUC1 core peptide for No. 804, and the application in causing Th1 type anti tumor immune response.Wherein said antigen peptide comprises at least one T cell epitope of being made up of 12-25 amino acid.In addition, people such as Sameul (Sameul, J.et al, Int.J.Cancer, 75:295-302,1998) with the synthetic peptide of liposome MUC1 and with phosphatide as the adjuvant immunity mouse, the result induces and has produced T1 type cell immune response, and most of immunized animal has produced MUC1 specific T-cells, INF and IgG.People such as Reddish (Reddish, M.et al, Int.J.Cancer, 76:817-823,1998) to comprise that 16 amino acid whose MUC1 synthesize the crosslinking protein of peptide (BP16) and KLH as immunogen, and with Detox as adjuvant immunity metastatic breast cancer patient, the most of subjects of result have produced MUC1 Restricted CTL reaction.People such as Karanikas (Karanikas V, et al., J.Clin.Invest.100:2786-92,1997) use the binding substances immune milk carninomatosis people of the synthetic peptide and the Toxoid,tetanus that contain a tumor-necrosis factor glycoproteins, and the result has only brought out faint immune response.People such as Kim (Kim, SK et al., Vaccine 19:530-537,2001) use 12 kinds of different adjuvants or composite adjuvant to cooperate the conjugated protein immune mouse of synthetic MUC1 and KLH, the result shows that composite adjuvant can induce anti-MUC1 antibody response better than single adjuvant, shows to select suitable adjuvant to have vital role for the immunogenicity that improves polypeptide vaccine.
An object of the present invention is to provide immunogenic peptide based on the MUC1 tandem repetitive sequence, be characterised in that said MUC1 sequence comprises that 1-5 series connection repeats, wherein each series connection repeats to contain at least a D-type amino acid, and two ends of sequence are proline(Pro).
According to a preferred embodiment of the invention, said immunogenic peptide has the aminoacid sequence shown in the SEQ ID NO:1.
According to a preferred embodiment of the invention, wherein said immunogenic peptide based on the MUC1 tandem repetitive sequence has the special immunogenicity of MUC1 that has improved.
According to a preferred embodiment of the invention, wherein said immunogenic peptide based on the MUC1 tandem repetitive sequence prepares with the peptide synthetic method.
Another object of the present invention provides MUC1 specificity vaccine composition, and said vaccine composition is made up of the immunogenic peptide that is defined as above and one or more pharmaceutically acceptable adjuvants basically.
According to a preferred embodiment of the invention, wherein said epidemic focus peptide can be and be selected from the carrier protein chemical coupling of serum albumin, Protalbinic acid and spoon azurin.
A further object of the present invention relates to the application of vaccine composition in inducing the reaction of MUC1 specificity antineoplastic immunity that is defined as above.
According to a preferred embodiment of the invention, the reaction of wherein said MUC1 specificity antineoplastic immunity is a MUC1 SC lymphocyte reaction.
The present invention generally relates to and is used to stimulate body to produce the peptide vaccine of anti tumor immune response, particularly relate to chirality peptide antigen based on the core sequence of MUC-1, contain the antigenic vaccine composition of said peptide, its preparation method and stimulating body to produce application in the immune response as immunogen.
As everyone knows, cellular immunization is brought into play key effect in immunologic tumor rejection.The T lymphocyte can be discerned and target cell surface MHC1 class or the about 8-12 of 2 quasi-molecule bonded length amino acid whose antigen peptide.Then discern and MHC1 quasi-molecule bonded endogenous peptide as the cytotoxic lymphocyte (CTL) of the main effects cell that kills and wounds target cell.Show that on evidence the processing treatment that synthetic peptide antigen need not pass through antigen presenting cell (APC) can combine with the MHC quasi-molecule, and in the activate immunity reaction, bring into play the same effectiveness of endogenous antigen peptide.
In view of the extensive existence of MUC-1 (MUC1) in multiple tissue and organ epithelial surface, with and unconventionality expression in corresponding tumor tissues, so might be with it as a kind of potential oncobiology mark, and use the peptide that particularly comprises the epitope of MUC1 and different number tandem repetitive sequences based on the MUC1 sequence as immunogen, carry out the specificity active immunity treatment of corresponding tumour.Yet, for the MUC1 molecule that does not contain sugar chain, particularly comprising the immunogen of less relatively number (for example) series connection multiple based on MUC1, its immunogenicity is very low undoubtedly.In order to overcome this critical problem, the present invention attempts to start with from the following aspects and improves the antienzyme degradation capability and the optical stability of molecule, to improve the stability and the immunogenicity of molecule: (1) rationally adjusts the sequence of amino acid of MUC1 molecule core area keeping and not changing under the prerequisite of MUC1 epitope (APDTR); (2) replace inherent L type amino acid in the wild sequence with one or more D type amino acid, to improve the resistance to enzymolysis ability and the receptor-binding activity of molecule; (3) terminal add or replace proline(Pro) at two of said peptide with optical stability; And (4) are coupled to the immunogenic peptide of synthetic based on MUC1 on the carrier protein macromole polypeptide chain that is selected from serum albumin, Protalbinic acid and spoon azurin.
Therefore, an object of the present invention is to provide immunogenic peptide based on the MUC1 tandem repetitive sequence, be characterised in that said MUC1 sequence comprises that 1-5 series connection repeats, wherein each series connection repeats to contain at least a D-type amino acid, and two ends of sequence are proline(Pro).
In general, in proper range, the series connection multiple number immunogenicity of many gained polypeptide more is strong more, but in order to observe because of adding the immunogenic influence of local chirality feature that D-type amino acid caused to synthetic peptide, and for the convenience on preparing, the present invention particularly preferably is the synthetic peptide that only contains a repeating unit.
According to the preferred embodiments of the invention, said MUC1 core series connection repeating unit has aminoacid sequence as follows:
ProAlaHisGlyValThrD-SerAlaProAspThrArgProAlaProGlyD-SerThrAlaPro(SEG?ID?NO：1)
Can according to the synthetic antigen peptide of the present invention of solid-phase peptide synthetic technology known in the art (for example referring to Steward, J.M.and Young J.D., Solid Phase Peptide Synthesis, 2ndEd., Pierce Chemical Company, Rockford, I11. (1984)).In the solid-phase peptide synthetic method, at first C-terminal amino acid is connected on the suitable solid phase carrier or resin.Be used for synthetic C-terminal carboxyl peptide preferred solid phase carrier be 4-hydroxymethyl phenoxy methyl-copolymerization (vinylbenzene-1% divinylbenzene).The preferred solid phase carrier that is used for the C-terminal acid amides is 4-(2 ', 4 '-dimethoxy phenyl-Fmoc-aminomethyl)-benzene acetamide oxide ethylamide resin (Apllied Biosystem Co.).Can be by N, N '-dicyclohexylcarbodiimide (DCC), N, N '-DIC (DIC) or 2-(1-hydrogen-1-benzotriazole base)-1,1,3,3-tetramethyl-urea phosphofluoric acid (HBTU) makes C-terminal amino acid be coupled to (50-100 ℃, reaction is 1 to 24 hour in methylene dichloride or DMF solvent) on the resin.When solid phase carrier is 4-(2 ', 4 '-dimethoxy phenyl-Fmoc-aminomethyl) benzene acetamide oxide ethylamide resin, should with the coupling of above-mentioned C-terminal amino acid before with secondary amine such as piperidines the cracking of Fmoc group is fallen.Can use adjacent 124 Triazole-1-base-N, N, N ', N '-tetramethyl-urea phosphofluoric acid (HBTU, 1 equivalent) in DFM with the coupling of de-protected 4-(2 ', 4 '-dimethoxy phenyl-Fmoc-aminomethyl) benzene acetamide oxide ethylamide resin.
When solid phase synthesis finishes, can use lytic reagent (for example thioanisole, water, dithioglycol and trifluoroacetic acid) process resin bonded peptide, to remove the synthetic peptide from resin and to make it protection.If the C-terminal of peptide is an alkylamide, can carries out ammonia with alkylamine and separate with cracking and go out resin.Perhaps, can carry out transesterification with ethanol and handle removing polypeptide, and then carry out that ammonia is separated or directly change amide group and handle.Can use above-mentioned several cracking agents to remove the side chain protected group.
Can use ion exchange chromatography, hydrophilic adsorption chromatography, silica gel adsorption chromatography, partition chromatography, a series of chromatography step purifying such as high performance liquid chromatography (HPLC), particularly reversed-phase HPLC de-protected synthetic peptide fully.
Can use the Applied biosystems 477A type protein sequence instrument of being furnished with the 120A analyser, analyze the aminoacid sequence of the peptide of purifying with automatic Edman chemical method.Can use laser desorption and electrospray mass spectrometry to measure the molecular mass of each peptide sequence.
Therefore, for the purposes of the present invention, can at first use automatic or semi-automatic polypeptide synthesizer (for example semi-automatic Peptide synthesizer of ACT90 type of American ACT company production), basically according to the synthetic immunogenic peptide of the present invention of the known solid phase method of peptide synthesis.Briefly, can be at first at the synthesizer reaction flask molten required solid-phase resin that fully rises, then with DIC (DIC), a hydration hydroxybenzotriazole (HoBt) and diisopropylethylamine (DIPEA) as coupling agent, in the DMF solvent, repeat the solid-phase peptide Connection Step, until finishing the synthetic of whole peptide chain by fixed routine.
After peptide chain is synthetic, sloughs blocking group and downcut required peptide from solid-phase resin.Precipitation and air-dry after, with the required synthetic peptide of high back voltage liquid chromatography (RP-HPLC) purifying and use its molecular weight of mass spectrometric determination (referring to embodiment 1).
Since synthetic peptide of the present invention in vivo with experiment in vitro in equal former activity of specific immune (seeing for details hereinafter) of having had clear improvement of performance, so might be with it as antigen-like material, cooperate suitable immunological adjuvant, preparation has the vaccine composition of clinical value.
Therefore, another object of the present invention provides MUC1 specificity vaccine composition, and said vaccine composition is made up of the immunogenic peptide that is defined as above and one or more pharmaceutically acceptable adjuvants basically.
According to a preferred embodiment of the invention, wherein can be and be selected from the carrier protein chemical coupling of serum albumin, Protalbinic acid and spoon azurin as the said immunogenic peptide of the primary activity composition of vaccine composition of the present invention.For this reason, for example can use glutaraldehyde method (Shen Guanxin, Zhou Rulin chief editor, modern immunological experiment technology, the 1st edition, 8-9 page or leaf, Hubei science tech publishing house) to realize that synthetic peptide is with carrier protein such as mice serum is albuminous combines.
Vaccine composition of the present invention can contain one or more pharmaceutically acceptable adjuvants.These adjuvants include but not limited to bacille Calmette-Guerin vaccine (BCG), granulocyte-macrophage colony stimutaing factor (GM-CSF), interleukin-22 (IL-2) and Zadaxin (TP).
Discovering in recent years, with regard to immunity system, GM-CSF can act on antigen presenting cell--dendritic cell, and then performance promotes immune response, regulates the effect of immunologic function.In addition, studies show that in a large number that Zadaxin is to improve effective preparation of immunologic function, and may be by regulating the immunocompetence that the T cell function is regulated body.Therefore, the present invention selects for use cell-macrophage colony stimulating factor and Zadaxin as the composite adjuvant for preparing MUC1 specific peptide vaccine especially.
In order to study the biologic activity of the synthetic peptide vaccine composition of modifying based on the present invention; we use the mouse inbred lines that has inoculated breast cancer cell GZ.A5.3H.4 (as transfection the clone of people MUC1 overall length cDNA) as experimental subjects; observe result of treatment and the immune protective effect of vaccine composition of the present invention respectively to tumor animal; and further observed synthetic peptide vaccine to the influence of mouse lymphocyte multiplication capacity and the ability of inducing specific CTL killing activity.In the experiment, add GM-CSF or TP is an experimental group with synthetic peptide vaccine of the present invention (being designated hereinafter simply as D-MUC1), with be added with equally GM-CSF or TP based on MUC1 core area repeating unit but do not contain the positive contrast of the amino acid whose synthetic peptide vaccine of D-type (being designated hereinafter simply as L-MUC1), and be blank with physiological saline.For the influence of the non-specific antineoplastic immune activity of getting rid of GM-CSF or TP to observations, also set up two positive controls in the experiment in addition, promptly GM-CSF group and TP organize.
In the treatment experiment, each organizes mouse lymphocyte, and (10 micrograms/ml) external continued stimulus is after 4 days, and the lymphocyte proliferation activity of D-MUC1+TP treated animal is significantly higher than control group, but the L-MUC1 group is then suitable substantially with control group through synthetic peptide.On the other hand, D-MUC1+GM-CSF, D-MUC1+TP, GM-CSF and TP group mouse lymphocyte are through synthesizing peptide (after external evoked 8 days of the 5-10 microgram/ml), each group has all produced the reaction of MUC1 specific CTL, and shows the specific killing activity to the GZ.A5.3H.4 cell of expressing MUC1 protein.In the immunoprotection experiment; each organizes mouse lymphocyte, and (the external continued stimulus of 5 micrograms/ml) is after 4 days through synthetic peptide; the lymphocyte proliferation activity of D-MUC1+TP group and D-MUC1+GM-CSF treated animal is significantly higher than control group, but the L-MUC1 group is then suitable substantially with control group.On the other hand, L-MUC1, D-MUC1+GM-CSF, D-MUC1+TP, GM-CSF and TP group mouse lymphocyte are through synthesizing peptide (after external evoked 8 days of the 5-10 microgram/ml), each treated animal has all produced the reaction of MUC1 specific CTL, and show specific killing activity to the GZ.A5.3H.4 cell of expressing MUC1 protein, but wherein the reactive behavior of L-MUC1 is then low slightly.
In addition, we have also analyzed the ability of synthetic inducing peptide humoral immunization of the present invention, found that, mouse only produces faint anti-MUC1 antibody response (data not shown goes out) after twice vaccine inoculation.
On this basis, we use the mouse model of the GZ.A5.3H.4 tumour cell of having inoculated high level expression MUC1, have further observed treatment and the preventive effect of modified polypeptides vaccine of the present invention to animal-transplanted tumor.In to the treatment experiment that the tumor animal of inoculated tumour cell carries out in advance, tumor growth rate and the tumor weight before the execution of L-MUC1 group mouse are all approaching with control group, and these parameters of D-MUC1+GM-CSF and D-MUC1+TP group mouse then are starkly lower than control group.Visible D-MUC1+GM-CSF group tumor group is woven with pathologic necrosis on a large scale under the opticmicroscope.D-MUC1+TP, GM-CSF organize in the mouse tumor tissue with TP and also have similar necrotic lesion, but degree of necrosis and scope are all organized less than D-MUC1+GM-CSF.Histological examination finds that further in the presence of D-MUC1+GM-CSF adjuvant, D-MUC1 inductive ctl response in the mouse body causes target cell with apoptosis and downright bad mode death.In the immunoprotection experiment that the animal that inoculates synthetic peptide vaccine of the present invention is in advance carried out, in three weeks behind the above-mentioned tumour cell of animal inoculation pvaccination, disconnected neck is put to death animal and is measured tumor weight and body weight.The result as seen, the tumor growth rate of L-MUC1 group mouse and put to death before tumor weight all approaching with control group, these parameters that D-MUC1+GM-CSF and D-MUC1+TP organize mouse then are starkly lower than control group.Visible D-MUC1+GM-CSF and D-MUC1+TP group tumor group is woven with pathologic necrosis on a large scale under the opticmicroscope.Though GM-CSF, TP also have similar necrotic lesion with the L-MUC1 treated animal, degree of necrosis and scope are all organized less than D-MUC1+GM-CSF.
As a kind of tumor associated antigen, MUC1 is the proper constituent of cell.But in tumour cell, because of its glycosylation not exclusively causes structural changes, and so its less immunogenic.This also is the reason of most of epithelium tumours (as mammary cancer) patient MUC1 specific CTL hyporeactive.Therefore, the inventor has designed and synthesized based on MUC1 core area series connection repeating unit, with the antigen peptide of the amino acid whose modification of part or all of its natural L-type of D-type aminoacid replacement, and successfully to have prepared with said peptide and inert support combination of proteins thing on this basis be the vaccine composition of primary activity composition.Show with experiment in vitro in our body, synthetic peptide vaccine of the present invention can be following assisting of some cytokine such as granulocyte-macrophage colony stimutaing factor (GM-CSF) and Zadaxin (TP), induces the specificity antineoplastic cell immune response (ctl response) of tumor animal effectively.Though relevant mechanism is still indeterminate at present, but infer it to be because the one or more amino acid in (1) D-MUC1 molecule are replaced by corresponding D-type amino acid, amino acid whose this reverse reversing has had molecule to be different from the local chirality feature of its wild sequence, thereby improved the immunogenicity of molecule, improved the recognition capability of immunity system it; (2) the D-type is amino acid whose in the molecule mixes the rigidity that has changed amino acid whose optics conformation in restriction enzyme site place and molecule, thereby has improved the ability of peptide molecule opposing proteasome degradation; (3) adding with proteinic coupling of inert support and adjuvant (cytokine) has also improved the ability that the antitumor immunity of organism reaction was induced or stimulated to vaccine composition of the present invention to a certain extent.
The following example is intended to further illustrate of the present invention based on the synthetic peptide of MUC1 core series connection repetition and the production method and the advantage of vaccine thereof.What should spell out is that under the prerequisite of the spirit and principles in the present invention, any change and change that indivedual inessential technical characterictic of the present invention is done all will fall in the claim scope that awaits the reply of the present invention.
Embodiment 1: of the present invention based on the antigen peptide of MUC1 core series connection iteron and the preparation of relevant vaccine
Basically in accordance with known methods, use Advenced ChemTech 90 type peptide synthesizers to synthesize and have the peptide that shows aminoacid sequence down: N-ProAlaHisGlyValThrD-SerAlaProAspThrArgProAlaProGlyD-Se rThrAlaPro-C (SEG ID NO:1).For this reason, at first take by weighing solid-phase resin (H-Pro-CLTR, 0.9mmol), and with the abundant swelling of methylene dichloride (DCM).Use DIC, HOBt and DIPEA coupling agent then, in the DMF solvent, repeat following synthesis cycle step, add the amino acid shown in the above-mentioned peptide fragment successively:
1, at first with the Fmoc-amino acid in DCM washing (5 minutes) reactor that contains 20% piperidines;
2, use 20% piperidines that is dissolved among the DMF to handle once more 25 minutes, to remove the Fmoc blocking group from aminoacid functional group;
3, wash 2 times each 2 minutes with DMF;
4, use methyl alcohol (MeOH) to wash totally 2 minutes 1 time;
5, wash 2 times each 2 minutes once more with DMF;
6, in the presence of DIC, HOBt and DIPEA coupling agent, add the protected amino acid that is equivalent to the amino 3 times of amounts of resin, reaction is 1 hour under the room temperature;
7, wash 2 times each 2 minutes with DMF;
8, use methyl alcohol (MeOH) to wash totally 2 minutes 1 time;
9, wash 2 times each 2 minutes once more with DMF.
After synthetic the finishing, wash resin (5 minutes) to remove DMF with tetrahydrofuran (THF) (THF), air-drying resin under argon gas and nitrogen environment then, thus obtain the peptide of resin-bonded.
(trifluoroacetic acid: water: phenol: with disulfide: mixture thioanisole=82.5: 5: 5: 2.5: 5) continued stir about 60 minutes again in about 0 ℃ of following stir about 15 minutes under room temperature with the peptide of resin-bonded and the lytic reagent of pre-preparation (5 to-10 ℃ of preservations).Leach resin then also with purified trifluoroacetic acid drip washing.The resin that leaches and washed is added to by every part of 0.5ml in the centrifuge tube that contains the 8ml diethyl ether of having an appointment, behind the centrifugal resulting suspension, removes supernatant.Can repeatedly repeat this process comes out up to all peptides are all precipitated.Wash sedimentary filtrate with ether, air-dry then and lyophilize it.
After cracking is finished, the thick peptide (using the 5-100% acetonitrile gradient) that uses Symmetry Prep C18 post as above to obtain, the peptide of lyophilize purifying like this then through 60 minutes wash-outs with HPLC method purifying.With resulting peptide with after the 0.1% trichoroacetic acid(TCA) dissolving, with a some spraying mass spectrograph determining molecular weight.
Finish being connected of synthetic peptide and carrier proteins with glutaraldehyde method then.For example can take by weighing mice serum albumin 10mg, add synthetic peptide 15mg with borate buffer solution dissolving back.Concussion adds 0.35% glutaraldehyde 1.0ml down, and at room temperature reacted 2 hours then.The about 0.25ml of adding glycerine (1mol/L) seals unreacted glutaraldehyde and kept 30 minutes, uses borate buffer solution (pH8.5) dialysed overnight then, thereby obtains required binding substances.Can use the laser desorption ionisation time-of-flight mass spectrometer to measure the molecular weight of gained binding substances, and and then definite molecular ratio of synthesizing peptide and carrier proteins.Can be 0.7 according to the albuminous 280nm light absorption ratio of 1mg/ml, calculate the protein concn of antigenic synthetic peptide solution.
In order finally to make vaccine composition of the present invention, can in advance or use the preceding interim immunological adjuvant that in said binding substances, adds proper concn.
Embodiment 2: vaccine composition of the present invention is to the influence of mouse lymphocyte multiplication capacity and specific CTL killing activity
Present embodiment uses the mouse inbred lines that has inoculated breast cancer cell GZ.A5.3H.4 (as transfection the clone of people MUC1 overall length cDNA) as experimental subjects, observes the influence of vaccine composition of the present invention to mouse lymphocyte multiplication capacity and specific CTL killing activity respectively.
Briefly, in the treatment experiment, 60 make female Balb/c mouse through subcutaneous vaccination GZ.A5.3H.4 breast cancer cell (Biomira company is so kind as to give) (5 * 10 in 6-8 week
5/ 0.1ml) back 7 days, be divided into 6 groups at random, 10 every group.In the immunoprotection experiment, a subcutaneous inoculation GZ.A5.3H.4 breast cancer cell of week (Biomira company is so kind as to give) (1 * 10 after immunity for the second time
6/ 0.1ml), be divided into 6 groups at random, 10 every group.The L-MUC1 vaccine group: back part of animal subcutaneous vaccination L-MUC1 peptide 15 micrograms/only, and weekly, twice totally; D-MUC1 vaccine+GM-CSF group: back part of animal subcutaneous vaccination D-MUC1 peptide 15 micrograms/only, weekly, totally twice, abdominal injection GM-CSF is 300ng/ days simultaneously; GM-CSF group: abdominal injection GM-CSF 300ng/ days; D-MUC1 vaccine+TP group: back part of animal subcutaneous vaccination D-MUC1 peptide 15 micrograms/only, weekly, totally twice, abdominal injection TP 0.4 mg/day simultaneously; TP group: abdominal injection TP 0.4 mg/day.
(1) immune cell function detection (
3H-TdR mixes method):: for the second time a week after the immunization, select four animals at random for every group, put to death and get spleen and prepare splenocyte suspension (2.5 * 10
6/ ml).By every hole 2.5 * 10
5The concentration in/hole (0.1ml) adds cell in 96 well culture plates.Experimental port add synthetic peptide of the present invention (after final concentration 5 micrograms/ml and 10 micrograms/ml), 37 ℃ of insulations 4 days.Each hole adds H-TdR (10 microcuries, 50 microlitres) then, and 37 ℃ are continued insulation 14 hours.Collect each hole culture, measure and calculate the average cpm value and the stimulation index SI in each hole after washing and the oven dry.Shown in following tabulation 1 of result and the table 2.
Table 1: synthetic peptide vaccine is to influence (treatment experiment) n=4 of experiment mice lymphocyte proliferation activity
Control group L-MUC1 D-MUC1+GM-CSF GM-CSF D-MUC1+TP TPMUC1 1.398 ± 0.25 1.476 ± 0.33 1.311 ± 0.18 1.193 ± 0.36 1.563 ± 0.21 1.284 ± 0.32 (5 μ g/ml) MUC1 0.958 ± 0.15 1.042 ± 0.26 1.132 ± 0.15 0.872 ± 0.26 1.217 ± 0.19
*0.974 ± 0.22 (10 μ g/ml)
*Compare P＜0.05 with control group.
Table 2: synthetic peptide vaccine is to influence (immunoprotection experiment) n=4 of experiment mice lymphocyte proliferation activity
Control group L-MUC1 D-MUC1+GM-CSF GM-CSF D-MUC1+TP TPMUC1 1.976 ± 0.33 1.182 ± 0.22 1.430 ± 0.28
*0.935 ± 0.37 2.582 ± 0.25
*± 0.32 1.284 (5 μ g/ml) MUC1 0.853 ± 0.36 0.747 ± 0.16 1.108 ± 0.29 0.910 ± 0.14 2.339 ± 0.34
*1.234 ± 0.18
*(10 μ g/ml)
*Compare P＞0.01 with control group,
*Compare P＜0.05 with control group.
From the result shown in table 1 and 2 as can be seen, in the treatment experiment, mouse lymphocyte is external to synthesize the peptide continued stimulus after 4 days through 10 micrograms/ml, and the lymphocyte proliferation activity of D-MUC1+TP group is apparently higher than control group, and the lymphocyte proliferation activity and the control group of L-MUC1 group are roughly suitable.Equally; in the immunoprotection experiment; mouse lymphocyte is external to synthesize the peptide continued stimulus after 4 days through 5 micrograms/ml; the lymphocyte proliferation activity of D-MUC1+TP group is apparently higher than control group; the lymphocyte proliferation activity and the control group of L-MUC1 group are roughly suitable, but D-MUC1+TP and D-MUC1+GM-CSF group lymphocyte proliferation activity obviously raise.After 4 days, the lymphocyte proliferation activity and the control group of L-MUC1 group are roughly suitable through the synthetic peptide continued stimulus of 10 micrograms/ml, but D-MUC1+TP and TP group are then apparently higher than control group.
(2) specific CTL detection (
3H-TdR mixes method): in one week of back of immunity for the second time, put to death the animal separating spleen and prepare splenocyte suspension (20 * 10
7/ ml), get the 1ml suspension and in culture plate, add synthetic peptide (37 ℃ of insulations of 5 micrograms/ml) 8 days promptly obtain the effector cell.Get the GZ.A5.3H.4 breast cancer cell suspension 1ml (1 * 10 that is in logarithmic phase then
6/ ml), add
3The reflecrtive mark rate that each cell is calculated in 50 microlitres oven dry back is washed and is got in 37 ℃ of water-baths of H-TdR (50 microcurie) 4 hours.Get radiolabeled target cell suspension 50 microlitres (40,000 cells) and add in the 96 control culture plates and in different effects/target ratio and add above-mentioned effector cell's 100 microlitres, 37 ℃ of insulations are 14 hours then.Control wells only adds target cell.After 30 minutes, collecting cell and oven dry are measured radiation counting (cpm) then and are calculated the killing activity of effector cell to target cell with tryptic digestion.Shown in following tabulation 3 of result and the table 4.
Table 3: synthetic peptide vaccine is imitated influence (treatment experiment) n=4 of experiment mice specific CTL killing activity: target control group L-MUC1 D-MUC1+GM-CSF GM-CSF D-MUC1+TP TP100: 1 22.4 ± 4.9 23.0 ± 6.3 48.0 ± 6.7
*35.6 ± 6.7
*40.5 ± 5.7
*28.6 ± 6.4
*50: 1 14.6 ± 4.4 20.3 ± 7.1 39.9 ± 8.0
*30.0 ± 3.5
*35.4 ± 3.9
*22.5 ± 5.0
*10: 1 7.0 ± 3.1 9.9 ± 3.1 28.4 ± 8.5
*27.7 ± 5.1
*22.0 ± 4.3
*11.8 ± 5.2
*
*Compare P＞0.01 with control group,
*Compare P＜0.05 with control group.
Table 4: synthetic peptide vaccine is imitated influence (immunoprotection experiment) n=4 of experiment mice specific CTL killing activity: target control group L-MUC1 D-MUC1+GM-CSF GM-CSF D-MUC1+TP TP100: 1 35.5 ± 4.5 42.2 ± 5.4
*61.0 ± 7.4 37.0 ± 6.3 55.0 ± 6.3
*38.5 ± 5.350: 1 31.6 ± 3.4 37.5 ± 5.7
*53.9 ± 5.2 35.0 ± 3.1
*43.4 ± 5.9
*35.9 ± 4.5
*10: 1 23.0 ± 2.8 29.4 ± 4.9
*38.0 ± 6.9 31.7 ± 4.1
*32.9 ± 4.2
*27.0 ± 2.9
*
*Compare P＞0.01 with control group,
*Compare P＜0.05 with control group.
From the result shown in table 3 and 4 as can be seen, in the treatment experiment, mouse lymphocyte is external to synthesize the peptide continued stimulus after 4 days through 10 micrograms/ml, the lymphocyte of D-MUC1+GM-CSF, GM-CSF, D-MUC1+TP and TP treated animal is external through synthetic inducing peptide of the present invention after 8 days, all produced the reaction of MUC1 specific CTL, and shown MUC1 specific killing activity the GZ.A5.3H.4 tumour cell.Equally; in the immunoprotection experiment; the lymphocyte of L-MUC1, D-MUC1+GM-CSF, GM-CSF, D-MUC1+TP and TP treated animal is external through synthetic inducing peptide of the present invention after 8 days; all produced the reaction of MUC1 specific CTL, and shown MUC1 specific killing activity the GZ.A5.3H.4 tumour cell.But the killing activity of D-MUC1+TP and D-MUC1+GM-CSF group is organized apparently higher than L-MUC1.
Embodiment 3: vaccine composition of the present invention is to the influence of tumor-bearing mice tumor growth
Present embodiment uses the mouse inbred lines that has inoculated GZ.A5.3H.4 breast cancer cell (as transfection the clone of people MUC1 overall length cDNA) as experimental subjects, observe vaccine composition of the present invention to the tumor-bearing mice tumor growth in vivo influence.
Basically the method according to embodiment 2 prepares animal model for tumour and grouping.In the treatment experiment, 60 make female Balb/c mouse through subcutaneous vaccination GZ.A5.3H.4 breast cancer cell (Biomira company is so kind as to give) (after 500,000/0.1ml) 7 days in 6-8 week, as above be divided into 6 groups (10 every group) at random, after immunity for the second time, put to death animal and measure gross tumor volume and weight.In the immunoprotection experiment; a subcutaneous inoculation GZ.A5.3H.4 breast cancer cell of week (Biomira company is so kind as to give) (500,000/0.1ml) after immunity for the second time; as above be divided into 6 groups (10 every group) at random, behind the inoculated tumour cell, put to death animal in 21 days and measure gross tumor volume and weight.The result is shown in table 5 and table 6.In addition, go back the tumor resection tissue in the experiment and carried out histopathologic examination's (result is not shown).
Table 5: synthetic peptide vaccine is to influence (treatment experiment) n=4 to the tumor-bearing mice tumor growth
Control group L-MUC1 D-MUC1+GM-CSF GM-CSF D-MUC1+TP TP tumor weight 34.2 ± 13.2 34.8 ± 11.3 18.8 ± 5.0
*27.0 ± 4.5 26.8 ± 7.1 31.2 ± 10.0 (mg)
*Compare P＞0.01 with control group.
Table 6: synthetic peptide vaccine is to influence (immunoprotection experiment) n=4 to the tumor-bearing mice tumor growth
Control group L-MUC1 D-MUC1+GM-CSF GM-CSF D-MUC1+TP TP tumor weight 52.7 ± 13.4 54.8 ± 21.0 31.8 ± 11.4
*49.0 ± 18.5 30.8 ± 11.7
*50.4 ± 14.5 (mg)
*Compare P＞0.01 with control group.
By these results as can be seen: in to the treatment experiment that the tumor animal of inoculated tumour cell carries out in advance, tumor growth rate and the tumor weight before the execution of L-MUC1 group mouse are all approaching with control group, and these parameters of D-MUC1+GM-CSF and D-MUC1+TP group mouse then are starkly lower than control group.Visible D-MUC1+GM-CSF group tumor group is woven with pathologic necrosis on a large scale under the opticmicroscope.D-MUC1+TP, GM-CSF organize in the mouse tumor tissue with TP and also have similar necrotic lesion, but degree of necrosis and scope are all organized less than D-MUC1+GM-CSF.Histological examination finds that further in the presence of D-MUC1+GM-CSF adjuvant, D-MUC1 inductive ctl response in the mouse body causes target cell with apoptosis and downright bad mode death.In the immunoprotection experiment that the animal that inoculates synthetic peptide vaccine of the present invention is in advance carried out, in three weeks behind the above-mentioned tumour cell of animal inoculation pvaccination, disconnected neck is put to death animal and is measured tumor weight and body weight.The result as seen, the tumor growth rate of L-MUC1 group mouse and put to death before tumor weight all approaching with control group, these parameters that D-MUC1+GM-CSF and D-MUC1+TP organize mouse then are starkly lower than control group.Visible D-MUC1+GM-CSF and D-MUC1+TP group tumor group is woven with pathologic necrosis on a large scale under the opticmicroscope.Though GM-CSF, TP also have similar necrotic lesion with the L-MUC1 treated animal, degree of necrosis and scope are all organized less than D-MUC1+GM-CSF.
Claims (8)
1, based on the immunogenic peptide of MUC1 tandem repetitive sequence, be characterised in that said MUC1 sequence comprises that 15 series connection repeat, wherein each series connection repeats to contain at least a D-type amino acid, and two ends of sequence are proline(Pro).
2, according to the immunogenic peptide of claim 1, wherein said immunogenic peptide has the aminoacid sequence shown in the SEQ IDNO:1.
3, according to the immunogenic peptide of claim 1, wherein said immunogenic peptide based on the MUC1 tandem repetitive sequence has the special immunogenicity of MUC1 that has improved.
4, according to the immunogenic peptide of claim 1, wherein said immunogenic peptide based on the MUC1 tandem repetitive sequence prepares with the peptide synthetic method.
5, MUC1 specificity vaccine composition, said vaccine composition are basically by forming as the immunogenic peptide that limits in the claim 1 and one or more pharmaceutically acceptable adjuvants.
6, according to the vaccine composition of claim 5, wherein said immunogenic peptide can be and the carrier protein chemical coupling that is selected from serum albumin, Protalbinic acid and spoon azurin.
7, according to the application in inducing the reaction of MUC1 specificity antineoplastic immunity of the vaccine composition of claim 5.
8, according to the application of claim 7, wherein said MUC1 specificity antineoplastic immunity reaction is a MUC1 SC lymphocyte reaction.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1301267C (en) * | 2005-06-21 | 2007-02-21 | 中国人民解放军军事医学科学院附属医院 | A simulated epitope peptide of MUC1 mucoprotein and its encoding DNA and use thereof |
| CN103570818A (en) * | 2012-07-27 | 2014-02-12 | 北京智飞绿竹生物制药有限公司 | Tumor antigenic polypeptide and application thereof as tumor vaccine |
| CN103570821A (en) * | 2012-07-27 | 2014-02-12 | 北京智飞绿竹生物制药有限公司 | Mucin-1 antigenic polypeptide and application thereof as tumor vaccine |
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2002
- 2002-09-03 CN CN 02129084 patent/CN1240714C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1301267C (en) * | 2005-06-21 | 2007-02-21 | 中国人民解放军军事医学科学院附属医院 | A simulated epitope peptide of MUC1 mucoprotein and its encoding DNA and use thereof |
| CN103570818A (en) * | 2012-07-27 | 2014-02-12 | 北京智飞绿竹生物制药有限公司 | Tumor antigenic polypeptide and application thereof as tumor vaccine |
| CN103570821A (en) * | 2012-07-27 | 2014-02-12 | 北京智飞绿竹生物制药有限公司 | Mucin-1 antigenic polypeptide and application thereof as tumor vaccine |
| CN103570818B (en) * | 2012-07-27 | 2016-06-29 | 北京智飞绿竹生物制药有限公司 | Tumor antigenic polypeptide and the purposes as tumor vaccine thereof |
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