CN106928335A - Her-2 polypeptides, composition and preparation method and application for tumour - Google Patents
Her-2 polypeptides, composition and preparation method and application for tumour Download PDFInfo
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Abstract
The present invention relates to polypeptide field, and in particular to a class is used for Her-2 polypeptides, composition comprising Her-2 polypeptides of tumour and its preparation method and application.The sequence of wherein described Her-2 polypeptides is selected from the one kind in following sequence：SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4.Especially breast cancer, lymph cancer, prostate cancer or lung cancer show good therapeutic effect, and lower toxic and side effect for tumour for Her-2 polypeptides of the invention, the composition comprising Her-2 polypeptides, show good medicinal property.
Description
Technical field
The present invention relates to polypeptide field, a specifically related to class is used for Her-2 polypeptides, composition comprising Her-2 polypeptides of tumour and its preparation method and application, is furthermore applied to prepare or treat relevant disease or the related drugs such as prostate cancer, lymph cancer, lung cancer particularly breast cancer.
Background technology
Her-2/neu genes were found by Slamon and Alex first in 1985, Slamon in 1987 etc. points out that Her-2/neu Overexpressions are relevant with breast cancer relapse and survival rate first, Her-2/neu is identified as a kind of new tumor marker (Slamon DJ, Clark GM, Wong SG, et al.Science, 1987,235 (4785)：177-82).
Her-2/neu is a kind of proto-oncogene, and expression is mainly started in embryonic development, the detectable a small amount of expression of normal structure after adult.The many tumours of the mankind, such as breast cancer of 20%-30%, 20% soft nest cancer, non-small cell lung cancer, stomach cancer, carcinoma of mouth, often P185HER2 is caused to over-express because Her-2/neu gene magnifications and (or) transcription are abnormal, this is short with the Preventive of tumour and life cycle closely related.Researchs to 26 Patients with Osteosarcoma find, though amplification and mutation without Her-2/neu, the patient expression Her-2/neu for having 42%, this expression is relevant with early stage transfer and low survival, illustrate Her-2/neu genes tumour generation with develop in play an important role.
Heat shock protein GP-96 is protein a kind of highly conserved and in monomorphism, is most one of rich in protein in endoplasmic reticulum.GP-96 has molecular chaperone function, can be with intracellular many Antigenic Peptide Non-covalent bindings.GP-96 is the key protein that antigen is offered.Offer approach there are some researches show what, GP-96 participated in MHC I class antigens.Zoopery also confirms that preparing immune mouse using the GP-96 for from tumour cell separate can produce the specific protective cellullar immunologic response of anti-corresponding tumour.Human granulocyte-macrophage colony stimulating factor (rhGM-CSF) is a kind of glycoprotein cytokine of 14kd, is that HPC survival in vitro, increment and differentiation are necessary.Experiment shows that rhGM-CSF can significantly improve effect that is antitumor and promoting marrow hemopoiesis.Her-2/neu is an important target molecules for developing mab treatment breast cancer.Have the treatment that two kinds of monoclonal antibodies for Her-2/neu have been widely used in Her-2/neu positive breast cancers in the world at present, can be used alone or with chemotherapeutic use in conjunction.
The content of the invention
Problem of the prior art solved by the invention is：The epitope that existing tumor vaccine haves the shortcomings that differs, strong and therapeutic effect is not bad for immunogenicity.
Composition it is an object of the invention to provide a class Her-2 polypeptides and containing Her-2 polypeptides, can induce immune response, attack tumour cell.Such compound can strengthen cell immune system so as to attack and prevent the growth and diffusion of tumour cell, or cancer growth and diffusion.
Specifically, the invention provides following technical scheme：
The invention provides a kind of Her-2 polypeptides for tumour cell, the sequence of the Her-2 polypeptides is selected from the one kind in following sequence：
SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4.
Preferably, described Her-2 polypeptides are natural amino acid or the amino acid modified through blocking group.
Preferably, described blocking group be selected from tertbutyloxycarbonyl, the oxygen tert-butyl group, the tert-butyl group, trityl, 2,2,4,6,7- pentamethyl Dihydrobenzofuranes -5- sulfonyls, pi-allyl in one or more more than.
Preferably, described Her-2 polypeptides include chain polypeptide or ring type polypeptide.
Preferably, described ring type polypeptide is intramolecular cyclization, and its cyclic mode is selected from one or more in S-S, CO-S, CO-NH key.
Present invention also offers a kind of preparation method of the Her-2 polypeptides for tumour cell, comprise the following steps：
(1) solid-phase synthesis are used, according to the connection order of Her-2 polypeptides in the presence of coupling agent and reaction dissolvent, the coupling of amino acid, Her-2 polypeptide of the synthesis with full guard is carried out successively；
(2) cracking obtains Her-2 polypeptides.
Preferably, the coupling agent is selected from the one kind in following several combinations：(1) N, N '-DIC and I-hydroxybenzotriazole,
(2) hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus, I-hydroxybenzotriazole and N, N '-diisopropylethylamine,
(3) 2- (7- azos BTA)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester and N, N '-diisopropylethylamine,
(4) hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus, I-hydroxybenzotriazole and N- methylmorpholines,
(5) 2- (7- azos BTA)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester and N- methylmorpholines；
The reaction dissolvent is selected from N, more than one or more in N '-dimethylformamide, dichloromethane, 1-METHYLPYRROLIDONE, dimethyl sulfoxide (DMSO).
Meanwhile, present invention also offers a kind of composition for tumour cell, the composition contains Her-2 polypeptides, also containing more than selected from one or more in following albumen：GP-96 albumen, rhGM-CSF albumen or the monoclonal antibody for Her-2/neu.
Preferably, the sequence of the Her-2 polypeptides is selected from the one kind in following sequence：
SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4.
Preferably, the Her-2 polypeptides and the mass ratio of the GP-96 albumen are 0.5:1~2：1.
It is furthermore preferred that the Her-2 polypeptides are 1 with the mass ratio of the GP-96 albumen：1.
Preferably, the Her-2 polypeptides and the mass ratio of the rhGM-CSF albumen are 0.5:1~2：1.
It is furthermore preferred that the Her-2 polypeptides are 1 with the mass ratio of the rhGM-CSF albumen：1.
Preferably, the Her-2 polypeptides and the monoclonal antibody mass ratio for Her-2/neu are 0.5:1~2：1.
It is furthermore preferred that the Her-2 polypeptides are 1 with the mass ratio of the monoclonal antibody for Her-2/neu：1.
Preferably, the described monoclonal antibody for Her-2/neu is selected from the one kind in Herceptin or handkerchief trastuzumab.
Preferably, the composition is vaccine combination.
Present invention also offers a kind of pharmaceutical composition, composition described in Her-2 polypeptides described in any of the above of free form of the pharmaceutical composition comprising effective dose or pharmaceutical acceptable salt or more any one as active component, also comprising one or more medicinal carrier substance and/or diluent.
Wherein, the pharmaceutically acceptable salt that the present invention is used includes hydrochloride, hydrobromate, sulfate, phosphate, nitrate and acetate, oxalates or tartrate, wherein preferred acetate.
Meanwhile, present invention also offers the Her-2 polypeptides described in any of the above, or more the purposes of epidemic disease composition or described pharmaceutical composition described in any one in for preparing prevention and/or treatment anticancer medicine.
Her-2 polypeptides described in any of the above, or more composition described in any one or described pharmaceutical composition for prevent and/or treating cancer field in purposes.
Preferably, described cancer includes breast cancer, prostate cancer, lymph cancer, lung cancer, preferably breast cancer.
What the present invention was obtained has the beneficial effect that：The present invention has found a series of polypeptides by the research of Her-2/neu proto-oncogenes family, it individually or with GP-96 albumen, rhGM-CSF albumen etc. is combined, the vaccine combination for obtaining, cytotoxic T lymphocyte (CTL) can be stimulated to destroy cancer cell, medicine as prevention and treatment tumour or cancer, is particularly well-suited to breast cancer.Her-2 polypeptides of the invention or the composition containing Her-2 polypeptides will be useful for the medicine of the preparation treating cancer that curative effect is more notable, side effect is lower and its relevant disease.
Brief description of the drawings
Fig. 1 is the external killing activity figure to Raji of cytotoxic T cell (CTL) of HER2-GP96 inductions.
Fig. 2 is the external killing activity figure to MCF-7 of cytotoxic T cell (CTL) of HER2-GP96 inductions.
Fig. 3 is the external killing activity figure to PC3 of cytotoxic T cell (CTL) of HER2-GP96 inductions.
Fig. 4 is the external killing activity figure to SBC-5 of cytotoxic T cell (CTL) of HER2-GP96 inductions.
Fig. 5 is the external killing activity figure to HEK293 of cytotoxic T cell (CTL) of HER2-GP96 inductions.
Specific embodiment
As described above, it is an object of the invention to：A kind of Her-2 polypeptides for tumour, the composition containing Her-2 polypeptides and its preparation method and application are provided.
Wherein, in the preferred embodiment of the present invention, the invention provides a kind of Her-2 polypeptides for tumour, its sequence is SEQ ID No.1.
In another preferred embodiment of the invention, the invention provides a kind of Her-2 polypeptides for tumour, its sequence is SEQ ID No.2.
In another preferred embodiment of the invention, the invention provides a kind of Her-2 polypeptides for tumour, its sequence is SEQ ID No.3.
In yet another preferred form of the invention, the invention provides a kind of Her-2 polypeptides for tumour, its sequence is SEQ ID No.4.
Meanwhile, present invention also offers a kind of composition, its Her-2 polypeptide and GP-96 albumen for containing any of the above sequence.
Above-described Her-2 polypeptides or composition can be used for treating the related disease of tumour or the medicine related for preparing same tumour, especially lymph cancer, breast cancer, prostate cancer and lung cancer.
Her-2 small peptides of the present invention derive from Her-2/neu proto-oncogene man's family sequences, Her-2/neu proto-oncogene man's family sequences are analyzed by computer, carry out assisting sifting and obtain the Her-2 small peptides that length is 16 peptides, by the way that further study show that the combination of aforementioned polypeptides and GP-96 albumen, rhGM-CSF albumen or monoclonal antibody for Her-2/neu etc. has high activity on the growth of tumour cell and diffusion is suppressed, Her-2 short peptide sequences are as follows：
SEQ ID No.1：SRMARDPQRFVVIQNE；
SEQ ID No.2：CAHYKDPPFCVARCPS；
SEQ ID No.3：YGLGMEHLREVRAVTS；
SEQ ID No.4：AVENPEYLTPQGGAAP；
Some conventional abbreviations have following meanings in the present invention；
Fmoc：Fluorenylmethyloxycarbonyl
Fmoc-AA：The amino acid of fluorenylmethyloxycarbonyl protection
DIC：N, N '-DIC
DCC：N, N '-dicyclohexylcarbodiimide
PyBOP：Hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus
HATU：2- (7- azos BTA)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester
HOBt：I-hydroxybenzotriazole
tBu：The tert-butyl group
OtBu：The oxygen tert-butyl group
Trt：Trityl
OtBu：Tert-butoxy
Pbf：2,2,4,6,7- pentamethyl Dihydrobenzofuranes -5- sulfonyls
Ser：Serine
Arg：Arginine
Met：Methionine
Ala：Alanine
Asp：Aspartic acid
Pro：Proline
Gln：Glutamine
Phe：Phenylalanine
Val：Valine
Ile：Isoleucine
Asn：Asparagine
Glu：Glutamic acid
DMF：N, N '-dimethylformamide
MeOH：Methyl alcohol
DCM：Dichloromethane
NMP：1-METHYLPYRROLIDONE
DMSO：Dimethyl sulfoxide (DMSO)
TFA：Trifluoracetic acid
Piperidine：Hexahydropyridine
DMAP：DMAP
DIEA：N, N '-diisopropylethylamine
TMP：2,4,6- trimethylpyridines.
It is this invention provides the synthetic method of Her-2 small peptides, its step is as follows：
Step 1, in the presence of activator systems, Fmoc-Glu (OtBu)-resin is obtained by resin solid phase carrier and Fmoc-Glu (OtBu)-OH couplings；
Step 2, by solid-phase synthesis, the amino acid with N-terminal Fmoc protections and side chain protected is coupled according to Her-2/neu proto-oncogene man's family sequence peptide backbone peptide sequences successively；
Step 3, cracking, purifying is freezed, and obtains Her-2/neu proto-oncogene man's family sequence peptides and the like.
Wherein, resin solid supports described in step 1 use 2-CTC resins, the activator systems are selected from DIEA, TMP or NMM, Fmoc-Glu (the OtBu)-resin is Fmoc-Glu (OtBu)-CTC resins of 0.30~0.70mmol/g substitution values, can be synthesized with oneself, it is also possible to directly by being commercially available.
Wherein, resin solid supports use Wang resin described in step 1, the activator systems are made up of DIC, HOBt and DMAP, Fmoc-Glu (the OtBu)-resin is Fmoc-Glu (the OtBu)-Wang resin of 0.30~0.70mmol/g substitution values, can be synthesized with oneself, it is also possible to directly by being commercially available.
Wherein, the solid phase synthesis process described in step 2,
1) it is 1 to use by volume ratio：The deprotection liquid of 4 piperidines and DMF composition removes the Fmoc protection groups on Fmoc-Glu (OtBu)-resin, obtains H-Glu (OtBu)-resin；
2) in the presence of coupling agent system, the asparagine of H-Glu (OtBu)-resin and Fmoc protections and side chain protected is coupled and obtains Fmoc-Asn (Trt)-Glu (OtBu)-resin；
3) repeat step 1), 2), carry out the coupling of amino acid successively according to Her-2/neu proto-oncogene man's family sequence peptide backbone peptide sequences, coupling amino acid order is：
Fmoc-Gln(Trt)-OH、Fmoc-Ile-OH、Fmoc-Val-OH、Fmoc-Val-OH、
Fmoc-Phe-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Pro-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Ala-OH、Fmoc-Met-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Ser(tBu)-OH；
The coupling agent system includes condensing agent and reaction dissolvent, and the coupling agent includes condensing agent and reaction dissolvent, and the condensing agent is selected from the one kind in following several combinations：(1) N, N '-DIC and I-hydroxybenzotriazole,
(2) hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus, I-hydroxybenzotriazole and N, N '-diisopropylethylamine,
(3) 2- (7- azos BTA)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester and N, N '-diisopropylethylamine,
(4) hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus, I-hydroxybenzotriazole and N- methylmorpholines,
(5) 2- (7- azos BTA)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester and N- methylmorpholines；
The reaction dissolvent is selected from N, more than one or more in N '-dimethylformamide, dichloromethane, 1-METHYLPYRROLIDONE, dimethyl sulfoxide (DMSO).
Described vaccine combination, wherein Her-2 small peptides and GP-96 albumen, rhGM-CSF albumen or the content ratio for the monoclonal antibody of Her-2/neu are 1：1.
Make further detailed description to the present invention with specific embodiment below in conjunction with the accompanying drawings.But, these embodiments are only for the explanation present invention, without limiting the scope of the present invention.
Wherein, reagent used in embodiment in the present invention is the conventional commercial reagent in this area and manufacturer's model of instrument is as follows：
Fmoc protected amino acids raw material, 2-CTC resins and Wang resin are conventional commercial reagent, and purchased from gill biochemistry (Shanghai) Co., Ltd., chemistry is pure；
Amino-acid resin, purchased from gill biochemistry (Shanghai) Co., Ltd.；
Dimethylformamide, purchased from Jiangshan Chemical Co Ltd, Zhejiang；
DIC, purchased from Zibo paradise mountain Chemical Co., Ltd.；
HOBT, purchased from Suzhou Highfine Biotech Co., Ltd.；
N, N '-diisopropylethylamine (DIEA), purchased from Suzhou Wu Fan bio tech ltd；
Mass spectrograph, model MALDI-TOF 4700, vendor A B SCIEX；
Preparative high performance liquid chromatography instrument, model LC3000, Beijing Chuangxin Tongheng Science and Technology Co., Ltd. of manufacturer.
The freeze-drier model and manufacturer that the vacuum drying and freeze-drying mentioned in embodiment are used are described as follows：
Freeze-drier：Freeze dryer FD-3 (Beijing Bo Yikang laboratory apparatus Co., Ltd)；
Lyophilisation condition：Lyophilized plate is put into freezer compartment of refrigerator (- 20 DEG C), pre-freeze 6h.Freeze dryer is opened, refrigeration is opened, more than precooling 30min sets freeze-drying curve as follows：
First paragraph：16h is run at -27 DEG C；Second segment：4h is run at -5 DEG C；3rd section：2h is run at 5 DEG C；4th section：16h is run at 30 DEG C.
(1) preparation of HER2 small peptides
Embodiment one
SEQ ID No.1：
The synthesis of H-Ser-Arg-Met-Ala-Arg-Asp-Pro-Gln-Arg-Phe-Val-Val-Ile-Gl n-Asn-Glu-OH
(1) weigh 0.1mmol substitution values be 0.61mmol/g Fmoc-Glu (OtBu)-CTC resins, add solid phase reaction post in, washed with DMF 1 time, with DMF swelling Fmoc-Glu (OtBu)-CTC resins 30 minutes after, use DMF：Pyridine volume ratio is 4：1 mixed solution sloughs Fmoc protections, is then washed with DMF 6 times, weighs Fmoc-Asn (Trt)-OH 0.5mmol, HOBt 0.5mmol, and it is 1 to add volume ratio：1 DCM and DMF mixed solutions, after adding 80 μ l DIC (0.5mmol) to activate under ice-water bath, in the above-mentioned reaction column equipped with resin of addition, after reacting 2 hours at room temperature, reaction end is judged with ninhydrin method detection, if resin water white transparency, then it represents that reaction is complete；Resin develops the color, then it represents that reaction not exclusively, it is necessary to react 1 hour, this criterion judges reaction end suitable for subsequent amino-acid coupling with ninhydrin method detection again.
(2) the step of repeating above-mentioned removing Fmoc protections and add corresponding amino acid couplings, is sequentially completed Fmoc-Gln (Trt)-OH, Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Gln (Trt)-OH, Fmoc-Pro-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Ala-OH, Fmoc-Met-OH, Fmoc-Arg (Pbf), the coupling of Fmoc-Ser (tBu)-OH.Coupling is finished, and peptide resin is washed 3 times with DMF, and DCM is washed 3 times, and MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, is drained and is obtained the thick peptide resins of 0.527g.
(3) crack：The peptide resin of 0.527g full guards is weighed, is added in the three neck round bottom flask of 25mL, by TFA：Methyl phenyl ethers anisole：Thioanisole=95：3：2 volume ratio configuration lysate 10mL, during lysate added into above-mentioned resin, room temperature reaction 2 hours, filtering, resin after washing cracking with a small amount of TFA 3 times, be added to liquid after concentration in ice ether and precipitate 1 hour by merging filtrate, concentration, centrifugation, absolute ether centrifuge washing 6 times, vacuum drying, obtains thick peptide 182.7mg.
(4) purify：By thick peptide with after 50ml water dissolves.Target product (16.9mg, 8.68%) then is obtained after C18 posts are purified for 2 times, turn salt, freeze-drying, MALDI-TOF assay products are used, the m/z values for finding the molecular ion peak of protonation are 1945.87 (theoretical amount is 1945).The wherein specific purification condition of C18 posts：First time purification condition：Mobile phase is：A phases：0.1%TFA；B phases：Acetonitrile, Detection wavelength 220nm collects purpose peak cut.Second purification condition：Mobile phase is：A phases：0.3% acetic acid；B phases：Acetonitrile.Detection wavelength 220nm, collects purpose peak cut.Turn salt operation：Turn salt condition：Mobile phase：A phases：20mM ammonium acetates-the aqueous solution；B phases：Acetonitrile；Detection wavelength 220nm.Purpose peak cut is collected, concentrated by rotary evaporation is lyophilized to obtain target product.
Embodiment two
SEQ ID No.2：
The synthesis of H-Cys-Ala-His-Tyr-Lys-Asp-Pro-Pro-Phe-Cys-Val-Ala-Arg-Cy s-Pro-Ser-OH
(1) weigh 0.1mmol substitution values be 0.57mmol/g Fmoc-Ser (tBu)-CTC resins, add solid phase reaction post in, washed with DMF 1 time, with DMF swelling Fmoc-Ser (tBu)-CTC resins 30 minutes after, use DMF：Pyridine volume ratio is 4：1 mixed solution sloughs Fmoc protections, is then washed with DMF 6 times, and it is 1 to weigh Fmoc-Pro-OH 0.5mmol, HOBt 0.5mmol and add volume ratio：1 DCM and DMF mixed solutions, after adding 80 μ l DIC (0.5mmol) to activate under ice-water bath, in the above-mentioned reaction column equipped with resin of addition, after reacting 2 hours at room temperature, reaction end is judged with ninhydrin method detection, if resin water white transparency, then it represents that reaction is complete；Resin develops the color, then it represents that reaction not exclusively, it is necessary to react 1 hour, this criterion judges reaction end suitable for subsequent amino-acid coupling with ninhydrin method detection again.
(2) the step of repeating above-mentioned removing Fmoc protections and add corresponding amino acid couplings, is sequentially completed Fmoc-Cys (Trt)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Cys (Trt)-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Tyr (tBu)-OH, Fmoc-His (Trt)-OH, the coupling of Fmoc-Ala-OH, Fmoc-Cys (Trt)-OH.Coupling is finished, and peptide resin is washed 3 times with DMF, and DCM is washed 3 times, and MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, is drained and is obtained the thick peptide resins of 0.578g.
(3) peptide resin of 0.578g full guards is weighed, is added in the three neck round bottom flask of 25mL, by TFA：Methyl phenyl ethers anisole：Thioanisole=95：3：2 volume ratio configuration lysate 10mL, during lysate added into above-mentioned resin, room temperature reaction 2 hours, filtering, resin after washing cracking with a small amount of TFA 3 times, be added to liquid after concentration in ice ether and precipitate 1 hour by merging filtrate, concentration, centrifugation, absolute ether centrifuge washing 6 times, vacuum drying, obtains thick peptide 164.7mg.
(4) by thick peptide with after 50ml water dissolves.Target product (17.8mg, 9.92%) then is obtained after C8 posts are purified for 2 times, turn salt, freeze-drying, MALDI-TOF assay products are used, the m/z values for finding the molecular ion peak of protonation are 1793.95 (theoretical amount is 1793).
Embodiment three
SEQ ID No.3：
The synthesis of H-Tyr-Gly-Leu-Gly-Met-Glu-His-Leu-Arg-Glu-Val-Arg-Ala-Va l-Thr-Ser-OH
(1) weigh 0.1mmol substitution values be 0.65mmol/g Fmoc-Ser (tBu)-CTC resins, add solid phase reaction post in, washed with DMF 1 time, with DMF swelling Fmoc-Ser (tBu)-CTC resins 30 minutes after, use DMF：Pyridine volume ratio is 4：1 mixed solution sloughs Fmoc protections, is then washed with DMF 6 times, and it is 1 to weigh Fmoc-Thr (tBu)-OH 0.5mmol, HOBt 0.5mmol and add volume ratio：1 DCM and DMF mixed solutions, after adding 80 μ l DIC (0.5mmol) to activate under ice-water bath, in the above-mentioned reaction column equipped with resin of addition, after reacting 2 hours at room temperature, reaction end is judged with ninhydrin method detection, if resin water white transparency, then it represents that reaction is complete；Resin develops the color, then it represents that reaction not exclusively, it is necessary to react 1 hour, this criterion judges reaction end suitable for subsequent amino-acid coupling with ninhydrin method detection again.
(2) the step of repeating above-mentioned removing Fmoc protections and add corresponding amino acid couplings, is sequentially completed Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Val-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Leu-OH, Fmoc-His (Trt)-OH, Fmoc-Glu (OtBu)-OH, the coupling of Fmoc-Met-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Tyr (tBu)-OH.Coupling is finished, and peptide resin is washed 3 times with DMF, and DCM is washed 3 times, and MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, is drained and is obtained the thick peptide resins of 0.578g.
(3) peptide resin of 0.578g full guards is weighed, is added in the three neck round bottom flask of 25mL, by TFA：Methyl phenyl ethers anisole：Thioanisole=95：3：2 volume ratio configuration lysate 10mL, during lysate added into above-mentioned resin, room temperature reaction 2 hours, filtering, resin after washing cracking with a small amount of TFA 3 times, be added to liquid after concentration in ice ether and precipitate 1 hour by merging filtrate, concentration, centrifugation, absolute ether centrifuge washing 6 times, vacuum drying, obtains thick peptide 164.7mg.
(4) by thick peptide with after 50ml water dissolves.Target product (17.8mg, 9.92%) then is obtained after C18 or C8 posts are purified for 2 times, turn salt, freeze-drying, MALDI-TOF assay products are used, the m/z values for finding the molecular ion peak of protonation are 1793.95 (theoretical amount is 1793).
Example IV
SEQ ID No.4：
The synthesis of H-Ala-Val-Glu-Asn-Pro-Glu-Tyr-Leu-Thr-Pro-Gln-Gly-Gly-Al a-Ala-Pro-OH
(1) weigh 0.1mmol substitution values be 0.63mmol/g Fmoc-Pro-CTC resins, add solid phase reaction post in, washed with DMF 1 time, with the swelling Fmoc-Pro-CTC resins of DMF 30 minutes after, use DMF：Pyridine volume ratio is 4：1 mixed solution sloughs Fmoc protections, is then washed with DMF 6 times, and it is 1 to weigh Fmoc-Ala-OH 0.5mmol, HOBt 0.5mmol and add volume ratio：1 DCM and DMF mixed solutions, after adding 80 μ l DIC (0.5mmol) to activate under ice-water bath, in the above-mentioned reaction column equipped with resin of addition, after reacting 2 hours at room temperature, reaction end is judged with ninhydrin method detection, if resin water white transparency, then it represents that reaction is complete；Resin develops the color, then it represents that reaction not exclusively, it is necessary to react 1 hour, this criterion judges reaction end suitable for subsequent amino-acid coupling with ninhydrin method detection again.
(2) the step of repeating above-mentioned removing Fmoc protections and add corresponding amino acid couplings, is sequentially completed Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gln (Trt)-OH, Fmoc-Pro-OH, Fmoc-Thr (tBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Pro-OH, Fmoc-Asn (Trt)-OH, Fmoc-Glu (OtBu)-OH, the coupling of Fmoc-Val-OH, Fmoc-Ala-OH.Coupling is finished, and peptide resin is washed 3 times with DMF, and DCM is washed 3 times, and MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, is drained and is obtained the thick peptide resins of 0.596g.
(3) peptide resin of 0.596g full guards is weighed, is added in the three neck round bottom flask of 25mL, by TFA：Methyl phenyl ethers anisole：Thioanisole=95：3：2 volume ratio configuration lysate 10mL, during lysate added into above-mentioned resin, room temperature reaction 2 hours, filtering, resin after washing cracking with a small amount of TFA 3 times, be added to liquid after concentration in ice ether and precipitate 1 hour by merging filtrate, concentration, centrifugation, absolute ether centrifuge washing 6 times, vacuum drying, obtains thick peptide 172.3mg.
(4) by thick peptide with after 50ml water dissolves.Target product (16.1mg, 9.98%) then is obtained after C18 or C8 posts are purified for 2 times, turn salt, freeze-drying, MALDI-TOF assay products are used, the m/z values for finding the molecular ion peak of protonation are 1613.75 (theoretical amount is 1613).
Embodiment five
The worst polypeptide of scoring is filtered out by computer simulation, as reference substance, its sequence is SEQ ID No.5, wherein SEQ ID No.5：
The synthesis of H-Asn-Pro-Leu-Val-Lys-Ser-Pro-Asn-Gly-Val-Lys-Ile-Ser-As p-Phe-Trp-OH (reference substance)
(1) weigh 0.1mmol substitution values be 0.60mmol/g Fmoc-Trp (Boc)-CTC resins, add solid phase reaction post in, washed with DMF 1 time, with DMF swelling Fmoc-Trp (Boc)-CTC resins 30 minutes after, use DMF：Pyridine volume ratio is 4：1 mixed solution sloughs Fmoc protections, is then washed with DMF 6 times, and it is 1 to weigh Fmoc-Phe-OH 0.5mmol, HOBt 0.5mmol and add volume ratio：1 DCM and DMF mixed solutions, after adding 80 μ l DIC (0.5mmol) to activate under ice-water bath, in the above-mentioned reaction column equipped with resin of addition, after reacting 2 hours at room temperature, reaction end is judged with ninhydrin method detection, if resin water white transparency, then it represents that reaction is complete；Resin develops the color, then it represents that reaction not exclusively, it is necessary to react 1 hour, this criterion judges reaction end suitable for subsequent amino-acid coupling with ninhydrin method detection again.
(2) the step of repeating above-mentioned removing Fmoc protections and add corresponding amino acid couplings, is sequentially completed Fmoc-Asp (OtBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Ile-OH, Fmoc-Lys (Boc)-OH, Fmoc-Val-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt)-OH, Fmoc-Pro-OH, Fmoc-Ser (tBu)-OH, Fmoc-Lys (Boc)-OH, the coupling of Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Asn (Trt)-OH.Coupling is finished, and peptide resin is washed 3 times with DMF, and DCM is washed 3 times, and MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, is drained and is obtained the thick peptide resins of 0.527g.
(3) peptide resin of 0.527g full guards is weighed, is added in the three neck round bottom flask of 25mL, by TFA：Methyl phenyl ethers anisole：Thioanisole=95：3：2 volume ratio configuration lysate 10mL, during lysate added into above-mentioned resin, room temperature reaction 2 hours, filtering, resin after washing cracking with a small amount of TFA 3 times, be added to liquid after concentration in ice ether and precipitate 1 hour by merging filtrate, concentration, centrifugation, absolute ether centrifuge washing 6 times, vacuum drying, obtains thick peptide 194.3mg.
(4) by thick peptide with after 50ml water dissolves.Target product (17.0mg, 9.44%) then is obtained after C18 or C8 posts are purified for 2 times, turn salt, freeze-drying, MALDI-TOF assay products are used, the m/z values for finding the molecular ion peak of protonation are 1800.95 (theoretical amount is 1800).
(2) Activity determination of polypeptide
The polypeptide that embodiment is prepared detects its killing activity to tumour cell respectively, while they are combined with GP-96 albumen respectively, detects its killing activity to tumour cell.Wherein, used reagent is this area by conventional i.e. commercially available reagent, and used detection method is also the ripe detection method in this area.
Wherein, reagent and instrument and producer are as follows：
Tumour cell, is purchased from：ATCC
ConA-Sepharose and close chromatographic stuffing, are purchased from：Nuo Bo Riders, Beijing Science and Technology Ltd.
G25 filtration chromatography fillers, are purchased from：Shanghai Ren Kai Bioisystech Co., Ltd
DEAE-Sepharose ion-exchange chromatography fillers, are purchased from：Am Pharmacia companies of Sweden
GM-CSF, is purchased from：It is purchased from：Shanghai Pu Zhen bio tech ltd
IL-4, is purchased from：It is purchased from：Shanghai Pu Zhen bio tech ltd
ELIASA, is purchased from：Thermo
Raji (lymthoma), MCF-7 (breast cancer), PC-3 (prostate cancer), SBC-5 (ED-SCLC), HEK-293 (human embryonic kidney epithelial cells) is purchased from ATCC.
T lymphocytes, are purchased from：Neat (Shanghai) the bioengineering Co., Ltd of match
RPMI-1640 culture mediums, are purchased from：Beijing Baeyer enlightening Bioisystech Co., Ltd
1st, the preparation of HER2 polypeptides
HER2 polypeptides：The target polypeptides that embodiment one to five is prepared number as follows, 1334 (embodiments five), 1335 (embodiments one) respectively, 1336 (embodiments two), 1337 (embodiments three), 1338 (example IVs), purity is up to more than 95%.Wherein peptide 1334 is as reference substance.
2nd, the preparation of GP-96 albumen
Tumour cell (being purchased from ATCC) is placed in and 25min is cultivated in 42 DEG C of environment, the concentration of GP-96 is detected.Tumor cell lysis are made to discharge GP-96 albumen using multigelation, collected using ConA-Sepharose and parent's chromatography, G25 filtration chromatographies and DEAE-Sepharose ion-exchange chromatographies, NaCl salting liquids are eluted, and desalination isolates and purifies GP-96 albumen, and is identified through SDS-PAGE and Western blot.
3rd, the culture of BMDC (DC)
Human peripheral 100ml is taken, hypotonic to abolish density gradient centrifugation separation tunica albuginea after red blood cell, with the RPMI-1640 medium cultures containing 10% calf serum, cell is adjusted to 1x10 by the adherent mononuclearcell of acquisition6After/ml, the nutrient solution containing GM-CSF100ng/ml, IL-420ng/ml is added, morphological observation and S-100 dyeing are done after being cultivated 3 days in culture plate, obtain BMDC.
4th, the induction of dendritic cells (DC)
The peptide 1334 of purifying is added in BMDC nutrient solution and is cultivated 3 days to final concentration of 500 μ g/ml, continuation, be ripe BMDC by immature Dendritic Cells Induced.
1334+GP-96 is added in BMDC nutrient solution and is cultivated 3 days to final concentration of 500 μ g/ml, continuation, be ripe BMDC by immature Dendritic Cells Induced.
The GP-96 of purifying is added in BMDC nutrient solution and is cultivated 3 days to final concentration of 500 μ g/ml, continuation, be ripe BMDC by immature Dendritic Cells Induced.
HER2 small peptides (peptide 1335, peptide 1336, peptide 1337, peptide 1338) are respectively added to be cultivated 3 days to final concentration of 500 μ g/ml, continuation in BMDC nutrient solution, are ripe BMDC by immature Dendritic Cells Induced.
HER2 small peptides (peptide 1335, peptide 1336, peptide 1337, peptide 1338) are mixed with GP-96 respectively, mixed proportion is 1：1 (mass ratio), is then added to extremely final concentration of 500 μ g/ml in BMDC nutrient solution respectively, continues to cultivate 3 days, is ripe BMDC by immature Dendritic Cells Induced.
5th, GVT detection
(1) target cell prepares
Raji (lymthoma), MCF-7 (breast cancer), PC-3 (prostate cancer), SBC-5 (ED-SCLC), HEK-293 (human embryonic kidney epithelial cells) is purchased from ATCC.
(2) the preparation culture of effector cell
After BMDC is combined with T lymphocytes, can be bred with activated t cell, the generation of inducing specific cytotoxicity T lymphocytes.It is 1 by number of cells by each group maturation DC cells of above-mentioned preparation and T lymphocytes：After 5 ratio co-incubations 3 days, suspension cell is collected as cytotoxic T cell (CTL).
(3) detections of the CTL to tumor cytotoxicity activity
Respectively collect exponential phase Raji lymthomas, MCF-7 breast cancer cells, PC-3 prostate gland cancer cells, SBC-5 small cell lung cancer cells, HEK-293 cells, then respectively adjustment cell concentration be 1 × 105/ ml, is inoculated in 96 porocyte culture plates, as target cell with 100 μ l/ holes.
By each group effector cell of above-mentioned preparation according to effect target ratio (E/T) be 20：1 mixes with five groups of target cells, at the same set only add T lymphocytes as experimental comparison group, 37 DEG C, 5%CO2Under the conditions of cultivate 6h, suck supernatant, after adding PBS rinsings to remove suspension cell, 200 μ l fresh cultures are rejoined, add MTT solution (5mg/ml), after continuing to cultivate 4h, supernatant is sucked, DMSO terminating reactions are added, absorbance (OD values) is surveyed at 570nm wavelength, killing activity is calculated, every group is respectively provided with 5 multiple holes.
6th, measurement result
The external killing activity (%) to different cells of cytotoxic T cell of the HER2-GP96 of table 1 inductions
Note：Data represent with average value, n=5.
Wherein, the data in table 1 are depicted as block diagram, as shown in Fig. 1 to Fig. 5.
7th, conclusion
The BMDC of control group polypeptide 1334, GP-96 and peptide 1334+GP-96 induced maturations is can be seen that to various target cells without obvious lethal effect from the data and the block diagram of Fig. 1 to Fig. 5 in table 1；Test group HER2 small peptide (peptides 1335, peptide 1336, peptide 1337, peptide 1338) and HER2 small peptides (peptide 1335, peptide 1336, peptide 1337, peptide 1338) the ripe BMDC of+GP-96 combined inductions shows killing tumor cell (Raji, MCF-7, PC-3, SBC-5 effect), and HER2 small peptides (peptide 1335, peptide 1336, peptide 1337, peptide 1338)+GP-96 group lethal effect be far above HER2 small peptide groups, while the BMDC of each test group induced maturation to non-tumor cell HEK-293 without obvious lethal effect.Each test group is relative to control group, p<0.05, show that HER2-GP96 has the effect of induction CTL killing tumor cells in vitro, and lethal effect is not shown to non-tumor cell, with tumour-specific, and toxic and side effect is not shown.
What is finally illustrated is, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although being described in detail to the present invention by above preferred embodiment, but it should be understood by those skilled in the art that, various changes can be made to it in the form and details, without departing from claims of the present invention limited range.
Claims (18)
1. a kind of Her-2 polypeptides for tumour cell, it is characterised in that the sequence of the Her-2 polypeptides
Selected from the one kind in following sequence：
SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4.
2. Her-2 polypeptides according to claim 1, it is characterised in that described Her-2 polypeptides are
Natural amino acid or the amino acid modified through blocking group.
3. Her-2 polypeptides according to claim 2, it is characterised in that described blocking group is selected from
Tertbutyloxycarbonyl, the oxygen tert-butyl group, the tert-butyl group, trityl, 2,2,4,6,7- pentamethyl Dihydrobenzofuranes -5-
It is more than one or more in sulfonyl, pi-allyl.
4. Her-2 polypeptides according to claim any one of 1-3, it is characterised in that described Her-2
Polypeptide includes chain polypeptide or ring type polypeptide.
5. Her-2 polypeptides according to claim 4, it is characterised in that described ring type polypeptide for point
Cyclization in sub, its cyclic mode is selected from one or more in S-S, CO-S, CO-NH key.
6. the preparation method of a kind of Her-2 polypeptides for tumour cell, it is characterised in that including following step
Suddenly：
(1) solid-phase synthesis are used, according to the connection order of Her-2 polypeptides in coupling agent and reaction dissolvent
In the presence of, the coupling of amino acid, Her-2 polypeptide of the synthesis with full guard are carried out successively；
(2) cracking obtains Her-2 polypeptides.
7. preparation method according to claim 6, it is characterised in that the coupling agent is selected from following
One kind in several combinations：(1) N, N '-DIC and I-hydroxybenzotriazole,
(2) hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus, I-hydroxybenzotriazole and N,
N '-diisopropylethylamine,
(3) 2- (7- azos BTA)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester and N, N '-two is different
Propylethylamine,
(4) hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus, I-hydroxybenzotriazole and N-
Methylmorpholine,
(5) 2- (7- azos BTA)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester and N- methylmorphines
Quinoline；
The reaction dissolvent be selected from N, N '-dimethylformamide, dichloromethane, 1-METHYLPYRROLIDONE,
It is more than one or more in dimethyl sulfoxide (DMSO).
8. a kind of composition for tumour cell, it is characterised in that it is many that the composition contains Her-2
Peptide, also containing more than selected from one or more in following albumen：GP-96 albumen, rhGM-CSF albumen
Or for the monoclonal antibody of Her-2/neu.
9. composition according to claim 8, it is characterised in that the sequence choosing of the Her-2 polypeptides
One kind from following sequence：
SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4.
10. composition according to claim 8 or claim 9, it is characterised in that the Her-2 polypeptides with
The mass ratio of the GP-96 albumen is 0.5:1~2：1, preferred mass ratio is 1：1.
11. composition according to claim 8 or claim 9, it is characterised in that the Her-2 polypeptides with
The mass ratio of the rhGM-CSF albumen is 0.5:1~2：1, preferred mass ratio is 1：1.
12. composition according to claim 8 or claim 9, it is characterised in that the Her-2 polypeptides with
The monoclonal antibody mass ratio for Her-2/neu is 0.5:1~2：1, preferred mass ratio is 1：1.
13. composition according to claim 8 or 9 or 12, it is characterised in that described is directed to
The monoclonal antibody of Her-2/neu is selected from the one kind in Herceptin or handkerchief trastuzumab.
14. composition according to claim any one of 8-13, it is characterised in that the composition is
Vaccine combination.
A kind of 15. pharmaceutical compositions, free form of the pharmaceutical composition comprising effective dose or officinal salt shape
The composition described in Her-2 polypeptides or claim 8-14 described in the claim 1-5 of formula as activity into
Point, also comprising one or more medicinal carrier substance and/or diluent.
Described in Her-2 polypeptides or claim any one of 8-14 described in 16. claim any one of 1-5
Composition or claim 15 described in pharmaceutical composition for prepare prevention and/or treatment anticancer
Purposes in medicine.
Described in Her-2 polypeptides or claim any one of 8-14 described in 17. claim any one of 1-5
Composition or claim 15 described in pharmaceutical composition for prevent and/or treating cancer field in
Purposes.
Cancer described in 18. claims 16 or 17 includes breast cancer, prostate cancer, lymph cancer, lung cancer,
Preferably breast cancer.
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| CN112779311A (en) * | 2020-11-17 | 2021-05-11 | 杭州阿诺生物医药科技有限公司 | System and method for screening and evaluating tumor immunotherapy drugs |
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| CN100381460C (en) * | 2004-11-30 | 2008-04-16 | 北京市肿瘤防治研究所 | HER-2 analogue antigen epitope and peptide containing said epitope |
| CN1908015B (en) * | 2005-10-18 | 2010-09-08 | 中国人民解放军军事医学科学院基础医学研究所 | Stabilized small molecule polypeptide capable of inhibiting HER2 high expression tumour cell proliferation |
| CN104130315B (en) * | 2014-07-25 | 2017-04-05 | 国家纳米科学中心 | A kind of polypeptide of special target HER2 albumen |
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