CN103097398A - Immunogenic peptide and composition having the peptide for preventing or treating hpv-related disease - Google Patents

Immunogenic peptide and composition having the peptide for preventing or treating hpv-related disease Download PDF

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CN103097398A
CN103097398A CN2011800441028A CN201180044102A CN103097398A CN 103097398 A CN103097398 A CN 103097398A CN 2011800441028 A CN2011800441028 A CN 2011800441028A CN 201180044102 A CN201180044102 A CN 201180044102A CN 103097398 A CN103097398 A CN 103097398A
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李炅律
林钟伯
张善弼
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Biocore Co Ltd
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Abstract

The present invention relates to an immunogenic peptide and a composition having the peptide for preventing or treating a HPV-related disease, and more specifically, to an epitope of HPV type 16 E7 for inducing the generation of cytotoxic T lymphocytes specific to an HLA-A*2402 type cervical cancer patient, and a composition having the peptide for preventing or treating a HPV-related disease.

Description

Immunogenic peptide and the composition that is used for prevention or treatment HPV relative disease that comprises this peptide
Technical field
The composition that is used for prevention or treatment HPV relative disease that the present invention relates to immunogenic peptide and comprise this peptide, in more detail, relate to the composition that is used for prevention or treatment HPV relative disease of inducing the HPV Class1 6E7 epi-position that generates the specific cytotoxic T lymphocyte of HLA-A*2402 type Patients with Cervical Cancer and comprising this peptide.
Background technology
In the malignant tumour that cervical cancer occurs in the women, sickness rate is in second, and the whole world has the patient more than 350,000 to die from cervical cancer every year, and its quantity continues to increase (Pisani P etc., Int J Cancer2002; 97(1): 72-81.).Known cervical cancer mainly causes by human papillomavirus (human papillomavirus:HPV), proved and surpass in the HPV of kind more than 100 more than 40 kind and play a role in female sex organs (de Villiers EM etc., Virology 2004; 324:17-27).In polytype HPV, particularly HPV Class1 6 and HPV Class1 8 are found in Patients with Cervical Cancer at most, can predict it and tumour has important relationship, and be known, wherein the infection rate of HPV Class1 6 in Patients with Cervical Cancer the highest (Munoz N etc., N Engl J Med2003; 348(6): 518-27).
Prove, when HPV infects normal cell, viral oncogene Insertion Into Host Cell (host cell) gene, express the Various Diseases poisonous protein, wherein E6 and E7 are powerful cancer proteins, it suppresses the effect as p53 and the RB of tumor-inhibiting factor, and promotes to change and continue growth (zur Hausen H.Nat Rev Cancer 2002 to cancer cells; 2(5): 342-50).Known, it is the function of RB that E7 suppresses tumor-inhibiting factor, promotes that S phase gene is activity (zur Hausen H etc., the Nat Rev Cancer 2002 of cyclin A and cyclin E; 2(5): 342-50; Phelps WC etc., Cell 1988; 53(4): 539-47), and, be CIP1(p21 by suppressing cell cycle protein dependent kinase inhibitor) and the effect of function KIP1(p27) etc., hinder the apoptosis of cells infected, induce to tumour and change (Funk JO etc., Genes Dev1997; 11(16): 2090-100; Zerfass-Thome K etc., Oncogene1996; 13(11): 2323-30).Viral cancer protein like this is owing to not finding in normal cell, therefore in the immunotherapy take all kinds cancer as object, become very strong target (target), and become gradually in order to generate the cytotoxic T lymphocyte (cytotoxic T lymphocyte:CTL) that to treat cervical cancer and will look into a main object of the research that the HPV epi-position is such.
Up to now, for develop the research of inducing the specific E7 epi-position that generates HPV Class1 6 specific cytotoxic T lymphocytes in order to treat cervical cancer for, although the result (Kast WM etc., the J Immunol1994 that study centered by most HLA-A*0201 type patient and prove to account in human leucocyte antigen (HLA) (the human leukocyte antigen:HLA) allelotrope (allele) the mankind are arranged; 152:3904-12), but the research contents that does not almost not prove in the HLA-A*2402 type patient as another main HLA-DR alleles of the mankind, truth that Here it is.
Therefore, in the present invention, by flow cytometry assay (flow cytometry) and cytotoxicity (cytotoxicity) test, find out the HPV Class1 6E7 epi-position of inducing the founder cell toxic T lymphocyte, wherein, described cytotoxic T lymphocyte can be treated the Patients with Cervical Cancer with HLA-A*2402 type.
Summary of the invention
Technical task
The present invention proposes in view of above-mentioned necessity, the object of the invention is to, a kind of HPV Class1 6E7 peptide of inducing the founder cell toxic T lymphocyte is provided, and wherein, described cytotoxic T lymphocyte can be treated the Patients with Cervical Cancer with HLA-A*2402 type.
Another object of the present invention is, a kind of composition for preventing or treat above-mentioned HPV relative disease is provided.
Solve the problem means
To achieve these goals, the invention provides identical mutant on peptide with 9-10 aminoacid sequences that are derived from HPV Class1 6E7 protein and function thereof.
In addition, in the present invention, above-mentioned peptide sequence preferably comprises CDSTLRLCV(sequence number 16) or LCVQSTHVDI(sequence number 35) aminoacid sequence, but be not limited to this.
In the present invention, " identical on function " reaches " identical in fact biological function or activity " and refers to respectively, in the situation that measure the biologic activity of polypeptide separately by identical step, its biologic activity approximately 50% to approximately 100% or the biologic activity of its above degree with polypeptide that this polypeptide is compared in show.For example, the flow cytometry assay that utilizes of Fig. 3 is confirmed the generation of IFN-γ of HPV Class1 6E7 epitope specificity and/or the utilization of Fig. 4 51The Cr method for releasing ( 51Cr release assay) confirm HPV Class1 6E7 epi-position (9mer, 10mer) specific test-results of killing and wounding the effect of cervical cancer cell is, peptide identical on function of the present invention refers to, demonstrate the bioactive peptide identical in fact with peptide of the present invention in Fig. 6 c and Fig. 6 d by the step as shown in Fig. 6 b, preferably, peptide identical on above-mentioned functions preferably has CYQSTHVDI(sequence number 43) or CYVTLRVCL(sequence number 47) aminoacid sequence, but be not limited to this.
In addition, the invention provides a kind of nucleic acid of peptide of encode the invention described above sequence number 16 or sequence number 35.
In addition, in the present invention, the gene of the peptide of optimized encoding sequence number 16 has the base sequence of sequence number 48, and the gene of the peptide of encoding sequence numbers 35 has the base sequence of sequence number 49, but bring out on these base sequences the halmatogenesis such as more than one displacement occurs, lack, increase, all halmatogenesis genes that demonstrate the activity identical with target effect of the present invention are also included within scope of the present invention.
In addition, the invention provides the nucleic acid of the peptide of encoding sequence respectively numbers 43 or sequence number 47.
These CYQSTHVDI(m3, No. 3, replaced peptide) and CYVTLRVCL(m7, No. 7, replaced peptide) peptide can utilize the RNA codon to infer its gene order, following multiple combination (for example, sequence number 50-107) can occur.
Table 1
Table 1 is that coding modified peptides (modified peptide) 3 is the gene order table of the peptide of sequence number 43.
Table 2
Figure BDA00002915182000032
Table 2 is that coding modified peptides 7 is the gene order table of sequence number 47.
In addition, the invention provides and comprise (a) above-mentioned peptide and (b) pharmaceutical composition of pharmaceutically useful carrier.
Above-mentioned composition infects relative disease for treatment or prevention HPV, for example bowenoid papulosis (bowenoid papulosis), anus abnormal (anal dysplasia), respiratory tract or papilloma of conjunctiva, uterine neck SARS type hyperplasia, cervical cancer, carcinoma vulvae (vulval cancer) or prostate cancer are useful, but are not limited to this.
In the present invention, above-mentioned composition is characterised in that, it is HLA-A*2402 type patient-specific.
In addition, peptide of the present invention or nucleic acid can optionally be made particulate, liposome or immunostimulating complex (ISCOM) (can only comprise saponin(e as activeconstituents) or peptide of the present invention be put into other the transportation means that is suitable for supplying to subject make preparation.In the situation that use particulate, its multipolymer that preferably has as Poly(D,L-lactide-co-glycolide (poly-lactic-co-glycolic acid, PLGA) and so on is polymeric matrix.
In mammals, immune response (for example, the cell immune response that comprises the reaction of MHC class I-mediation or class II-mediated immunity) can be by immunogenic peptide being applied to the mammals with the MHC molecule of being combined with immunogenic peptide, i.e. the mankind, man like ape, dog, cat, rabbit, ox, mouse and trigger.The peptide of the invention described above can be used with the part of particulate, liposome or ISCOM or solution.
Another method utilization of using peptide of the present invention comprises the expression vector of the nucleotide sequence of the peptide of the present invention of encoding.The signal sequence that this nucleotide sequence also can optionally be encoded and be connected with the peptide of the invention described above.When this nucleic acid encoding signal sequence, preferably its coding is from the signal sequence Met Ala Ile Ser Gly Val Pro Val Leu Gly Phe Phe Ile Ile Ala Val Leu Met Ser Ala Gln Glu Ser Trp Ala(sequence number 108 of HLA-DR.alpha).In this situation, sequence of the present invention can have sequence number 16 for example or 35 or the sequence of its halmatogenesis body.Preferred this nucleic acid does not comprise the virogene that causes that nucleic acid infects, and complete (intact) E7 protein of not encoding.
Nucleic acid of the present invention can be included in plasmid, can optionally be provided in the particulate that comprises polymeric matrix.In a preferred embodiment, polymeric matrix must be made of the multipolymer of PLGA.Preferred this particulate for example has 0.02～20 micron or the about diameter below 11 microns.Preferred a plurality of particulate has 0.02～20 micron or the about diameter below 11 microns.
The cell that comprises plasmid of the present invention also is within the scope of the present invention.This cell for example can be B cell or other antigen presenting cells (APC).This cell can cultivated or keep under its condition of plasmid expression peptide of encoding.
Nucleic acid of the present invention and plasmid for for example the above-mentioned plasmid of mentioning being applied to mammals with " naked DNA " mammals for example in the mankind method of induction of immunity reaction useful.This mammals can have danger or the disease of HPV infection, cervical atypical hyperplasia and/or cervical cancer.Nucleic acid of the present invention and plasmid also can insert in particulate, liposome, ISCOMS or other the suitable transportation means as above-mentioned record.
If not special definition in specification sheets, all scientific terminologies that use in this specification sheets and technical term have the common meaning equivalent in meaning of understanding with those skilled in the art.
The nucleic acid of the peptide of putting down in writing in the present invention and this peptide of coding can be used for causing the immune response for HPV E7 protein.
Peptide of the present invention can be connected with transportation (trafficking) sequence that this peptide is transported to organ in cell.This transportation sequence is to play the aminoacid sequence of the function of transportation in the cell of regulating the peptide be attached with this transportation sequence (guide from [Dan to [Dan or cell surface move).Such transportation sequence can be transported to this polypeptide ER, lysosome or endosome, can comprise signal peptide (translate duration is directed to protein the N-end sequence of ER), for example the ER of KDEL and so on is detained the lysosome target sequence of sequence and KFERQ or QREFK and so on.
Short aminoacid sequence can play the function with the signal of organ in protein target specific cells.For example find the hydrophobicity signal peptide in the amino-terminal end of the protein that goes to ER.
Such transportation sequence can be Met Ala Ile Ser Gly Val Pro Val Leu Gly Phe Phe Ile Ile Ala Val Leu Met Ser Ala Gln Glu Ser Trp Ala(sequence number 108 for the HLA-DR.alpha leader sequence).If this part is enough to polypeptide of the present invention is transported to ER, can only comprise the part (for example at least 9 amino-acid residues) in above-mentioned specific 25 residue sequence of this signal peptide.
In some cases, process (namely shearing) in order to utilize signal peptidase, the sequence of the HPV HPV-16 E7 peptide of the present invention of preferably encoding has variation with the part of being connected the sequence connection.Recognition sequence for signal peptide is recorded in Von Heijne, NAR14:4683,1986.
When building the DNA of coding peptide of the present invention, can Application standard technology (for example referring to the technology of putting down in writing in WO94/04171).This construct can be included in increases the appended sequence of expressing in the human cell, for example the RNA of suitable promotor, encoding sequence stablizes 5 ' and 3 ' sequence, intron (can be arranged in the sequence 5 that is encoded ' or 3 ' either side) and Poly(A) attachment site, and the replication orgin and the selective marker that make this construct to select and to copy protokaryon and/or eucaryon host.
Can have kalamycin resistance gene (519-1313 position), SV40 early promoter (131-484 position) and thymidine kinase (thymidine kinase, TK) poly-adenosine site (1314-1758 position) in plasmid of the present invention.Kalamycin resistance gene and associated adjustment sequence just are used for the purpose of selection, if do not need selection or not preferred, can remove from plasmid.
In the known object that is infected or might be infected by HPV by HPV by HPV infection, suspection, can use peptide of the present invention and nucleic acid as prevention or treatment vaccine.Other suitable objects comprise the symptom that presents the HPV-relative disease or the people who looks like diseases.The vaccine of bowenoid papulosis (bowenoid papulosis), anus abnormal (anal dysplasia), respiratory tract or papilloma of conjunctiva, uterine neck SARS type hyperplasia, cervical cancer, carcinoma vulvae (vulval cancer) or prostate cancer for example be treated or be prevented to peptide of the present invention and vaccine can as the vaccine for the treatment of or prevent the infection relative disease of HPV bacterial strain 16.
HPV infects or HPV infects relative disease in order to treat, and peptide of the present invention or nucleic acid can be used separately or use together with other treatment rule as known in the art such as chemotherapeutic drug, radioactive rays and operation.In addition, peptide of the present invention and nucleic acid can adjuvant or cytokine (or nucleic acid of the Codocyte factor) be mixed and are used as well known in the art with the other treatment of finding out in order to promote immune response, example.
Peptide of the present invention or nucleic acid can be for the manufacture of prevention or the medicine for treatment things of HPV infection or HPV infection relative disease.
Delivery system of the present invention can be used for that peptide or the DNA construct of expressing this peptide are transported to wish and promote immunoreactive suitable cell to HPV.
The nucleic acid of peptide of the present invention or this peptide of encoding can the Application standard method, for example Donnelly et al., J.Imm.Methods176:145,1994 and Vitiello et al., J.Clin.Invest.95:341, the such method of method of putting down in writing in 1995 is used.
Peptide of the present invention or nucleic acid can be with forms as known in the art, for example with in intramuscular injection, intravenous injection, intra-arterial injection, intradermal injection, abdominal injection, nose, intravaginal, bowel lavage, subcutaneous injection mode use, the powder that perhaps they can be by for example comprising particulate or the suction of solution are applied in gi tract, mucous membrane or respiratory organs.Can local (for example uterine cervix or infection site) or systemic administration.
The nucleic acid of peptide of the present invention or this peptide of encoding can use the pharmaceutically useful carrier of soliquid, powder, physiological saline, lipid, liposome, microballoon (microspheres) or nanometer spherical particle and so on to carry.They can form mixture or relevant or exposed to transportation means, can carry with the delivery system as known in the art of lipid, liposome, particulate, gold, nanoparticle, polymkeric substance, condensing agent, polyose, polyamino acid, tree, saponin(e, absorption enhancing substance or lipid acid and so on.
Preferred 1 the every 1kg body weight of consumption is used approximately 0.1～100 micromolar peptide or the approximately DNA of 1～200 microgram.Certainly, as known in this area, the consumption to the patient of regulation depends on many factors, and this factor comprises patient's height, body surface area, age, the specific compound of using, sex, time of application and approach, general health state and the other drug of using simultaneously.Determine neatly suitable amount of application in the limit of power of the pharmacist with common knowledge.
Also can use the other standards transmission method of particle gun (biolistic) transmission or external (ex vivo) processing and so on.In extracorporeal treatment, can obtain for example antigen presenting cell (APCs), dendritic cell, peripheral blood lymphocytes or medullary cell from patient or suitable donor, after Activated in Vitro, be applied to this patient with this immune composition.
Can use United States Patent (USP) the 5th, 783, the particulate that the material of putting down in writing in 567 is included is transported to intracellular transportation means as the giant molecule that is used for DNA or peptide and so on.They comprise in the shell that is enclosed in polymkeric substance or enter the giant molecule of polymeric matrix.Particulate is for example brought into play the effect of keeping the such characteristic of keeping giant molecule of the DNA that entered with undecomposed state.Particulate can also be used for the giant molecule pulse is carried and is transported to specific position or is transported to specific cells or the target cell group.
Polymeric matrix can be the biodegradation type multipolymer of Poly(D,L-lactide-co-glycolide, starch, gelatin or chitin and so on.Particulate can be used in particular for making DNA molecular to maximize to the cytophagous transfer of object.Perhaps tissue can be injected or be implanted to this particulate.
Peptide of the present invention can be applied to object by lipid known in this area, tree or liposome.As everyone knows, for example carry the liposome of the nucleic acid of immune peptide or this peptide of encoding to cause in vivo ctl response (Reddy et al., J.Immunol.148:1585,1992; Collins et al., J.Immunol.148:3336-3341,1992; Fries et al., Proc.Natl.Acad.Sci.USA89:358,1992; Nabel et al., Proc.Nat.Acad.Sci.(USA) 89:5157,1992).
Peptide of the present invention and nucleic acid can use size to be 30-40nm, electronegative, immunostimulating complex (Immune Stimulating Complexes with cage structure, ISCOMS) use, this immunostimulating complex is separately or with cholesterol and Quil A(saponin(e with saponin(e) mix simultaneously.Peptide of the present invention and nucleic acid can be used simultaneously or use respectively with ISCOMS.
Protective immunity be comprise use ISCOMS as for the toxoplasmosis of the transportation means of antigen, and the multi-infection experimental model of the viral-induced tumour of Epstein-Barr in generate (Mowat et al.; Immunology Today12:383-385,1991).Find, the ISCOMS of low antigen amount that is made into the 1ug of capsule produces the ctl response (Takahashi et al., Nature344:873-875,1990) of class I mediation.
The ability that causes immunoreactive peptide of the present invention and nucleic acid can be analyzed with the immunoreactive method of mensuration known in this area.For example, the generation of cytotoxic T cell can be by using expression or the standard of MHC tetramer, the mensuration cell within a cell factor 51The Cr method for releasing shows.The standard analysis of ELISA or ELISPOT and so on also can be used for measuring the cytokine spectrum that helps the T cell activation.T cell proliferation can also use as known in the art other analyze and 3The analysis of H-Thymine deoxyriboside uptake assay and so on is measured.The B cell response can be measured with the analysis of ELISA and so on.
The additive method of digital picture, cytology vaginoscope and histological examination and so on can be used for measuring peptide of the present invention and the effect of nucleic acid for the relevant focus of papilloma virus or papilloma virus level.
Below describe the present invention in detail.
The research about the adopting property CD8+T cell therapy take HPV Class1 6E7 as object of before carrying out is that the white race (cacasian) with HLA-A*0201 type carries out as main, and has shown the several specific E7 epi-position that demonstrates anticancer effect.But the achievement in research of the basic HPV Class1 6E7 epi-position that does not obtain for the adopting property CD8+T cell therapy that accounts for the HLA-A*2402 type patient who occupies the majority in the whole world population anthous over half (Asian) actually.
In the present invention, the PBMC for being supplied with from normal HLA-A*2402 type blood donation person uses the HPV-16 E7 that is synthesized to process, after cultivating 1 week or 2 weeks, measure the expression degree of IFN-γ, selected inducing to the candidate of the peptide of CD8+T cytodifferentiation used cytotoxicity experiment, namely 51The Cr method for releasing is confirmed actual anticancer effect.As shown in Figure 1, in 14 15mer peptides that synthesized, E761-75(CDSTLRLCVQSTHVD), E7 67-81(LCVQSTHVDIRTLED) peptide is compared with other HPV-16 E7s, demonstrate the generation of the IFN-γ of high value, for the E7 61-75(CDSTLRLCVQSTHVD that confirms to screen with flow cytometry assay) and E767-81(LCVQSTHVDIRTLED) epi-position whether in fact HLA-A*2402 type cervical cancer cell is demonstrated anticancer effect, utilize 51The Cr method for releasing is measured the cell killing effect, and result use E7 67-81(LCVQSTHVDIRTLED as can be known) test group processed of epi-position demonstrates and compares obvious high cell killing effect with negative control group.On the other hand, in flow cytometry assay, the expression degree of IFN-γ and CD69 demonstrates the 67-81(LCVQSTHVDIRTLED with E7) the similar E7 61-75(CDSTLRLCVQSTHVD of epi-position) epi-position and E7 67-81(LCVQSTHVDIRTLED) epi-position compares, and the cell killing effect shows low (Fig. 2) unexpectedly.The reason that such result occurs is predicted to be, and the reality that is caused by the aminoacid sequence difference of two E7 epi-positions is different to the immunity degree of bringing out of cervical cancer cell.
In fact, the 15mer peptide that utilization is as above found out for the correct epi-position that presents in the MHC class I molecule of searching cancer cells, composition length is the peptide of 9mer and 10mer respectively, the length of the epi-position that this length is combined with MHC class I molecule by known conduct, and utilize flow cytometry assay analysis, result demonstrates E761-69(CDSTLRLCV) and E7 67-76(LCVQSTHVDI) the peptide activity of inducing cytotoxic T cell effectively, high immunogenicity (immunogenic) had (Fig. 3) thereby can predict it.In addition, utilize the E7 61-69(CDSTLRLCV that filters out) and E7 67-76(LCVQSTHVDI) peptide, after cultivating PBMC, utilize 51The Cr method for releasing is measured the cervical cancer cell fragmentation effect, and result can confirm, and two kinds of peptides all demonstrate the 67-81(LCVQSTHVDIRTLED with E7) epi-position compares higher effect (Fig. 4).
Meanwhile, utilize the computer program of predictive molecule bonding force, with E7 61-69(CDSTLRLCV) and E7 67-76(LCVQSTHVDI) aminoacid sequence of peptide is replaced as the form high with HLA-A*2402 molecule bonding force, with synthetic like this CYQSTHVDI(m3, No. 3, replaced peptide) and CYVTLRVCL(m7, No. 7, replaced peptide) after peptide is processed and is cultivated PBMC, implement flow cytometry assay, result can be confirmed, compare with existing peptide, induce the activity (Fig. 6～9) of higher cytotoxic T cell.In addition, with E7 61-69(CDSTLRLCV) and E7 67-76(LCVQSTHVDI) peptide is replaced as the form high with HLA-A*2402 molecule bonding force, with synthetic like this CYQSTHVDI(m3, No. 3, replaced peptide) and CYVTLRVCL(m7, No. 7, replaced peptide) after peptide is processed and cultivated PBMC, measure the cell killing effect, result as can be known, compare with existing peptide, demonstrate higher cell killing effect (Fig. 6～9).Can confirm thus, by utilizing E761-69(CDSTLRLCV) and E767-76(LCVQSTHVDI) epi-position come the activity of inducing cytotoxic T cell, can actual effectively act on killing and wounding of cervical cancer cell, and can confirm, its the variation peptide, be CYQSTHVDI(m3, No. 3, replaced peptide) and CYVTLRVCL(m7, No. 7, replaced peptide) can be effectively applied to treatment and the vaccine development (Figure 10～12) of HLA-A*2402 type Patients with Cervical Cancer.
By former studies, confirmed anticancer effect (Patel S etc., the Curr Opin Obstet Gynecol2009 of cytotoxic T cell in the experiment that utilizes the cervical cancer animal model; 21(1): 54-9; Kim TY etc., Cancer Res2002; 62:7234-40).In the present invention, for the anticarcinogenic effect of the E7 epi-position in vivo confirming to filter out, utilizing HLA-A*2402 type cervical cancer cell pearl is SiHa, builds mouse model.It is SiHa that the BALB/c nude mice in 5 ages in week is injected the cervical cancer cell strain, after forming tumour, has used PBMC or the CD8+T cell processing and cultivate with the HPV-16 E7 that filters out, and has confirmed tumour removing or the inhibition of cytotoxic T cell.By shown in result as can be known, when using E7 67-76(LCVQSTHVDI) when the PBMC that processes and cultivate is applied to the mouse that is formed with tumour, compare with the negative control group of not carrying out any processing, the growth of tumour is suppressed (Figure 10).
In addition, to use E7 61-69(CDSTLRLCV), E7 67-76(LCVQSTHVDI), CYQSTHVDI(m3, No. 3, replaced peptide) and CYVTLRVCL(m7, No. 7, replaced peptide) after the CD8+T cell of processing and cultivating is applied to mouse, when confirming tumour big or small, can confirm the growth of comparing tumour with the negative control group of not carrying out any processing and greatly be suppressed (Figure 11 and 12).Can confirm thus, with the E7 61-69(CDSTLRLCV that filters out in the test in vivo by the experimentation on animals that utilizes the cervical cancer mouse model), E7 67-76(LCVQSTHVDI), CYQSTHVDI(m3, No. 3, replaced peptide) and CYVTLRVCL(m7, the growth inhibitory effect that No. 7, replaced peptide) peptide is processed and cultured cells toxicity T cell has cervical cancer.
Result is, by flow cytometry assay and cytotoxicity experiment HPV Class1 6 E7 67-81(LCVQSTHVDIRTLED) can be used as the epi-position that can generate the specific cytotoxic T cell of cervical cancer, measurable arriving, E7 67-81(LCVQSTHVDIRTLED) in 15 aminoacid sequences, length is the E7 61-69(CDSTLRLCV of 9mer and 10mer) and E7 67-76(LCVQSTHVDI) epi-position really has in HLA-A*2402 and express and induction of immunity originality.
In addition, E7 61-69(CDSTLRLCV) and E7 67-76(LCVQSTHVDI) CYQSTHVDI(m3 of the replaced form of the amino acid of peptide, No. 3, replaced peptide) and CYVTLRVCL(m7, No. 7, replaced peptide) peptide demonstrates higher anticancer effect, can prove that they are to generate effective epi-position for the vaccine development of HLA-A*2402 type cervical cancer and the required cytotoxic T lymphocyte of adoptive immunity cell therapy.
The invention effect
by the present invention as can be known, in HPV Class1 6E7, length is the 67-81(LCVQSTHV DIRTLED of 15mer) can be used as the epi-position that can generate the specific cytotoxic T cell of cervical cancer, confirmed wherein E7 61-69(CDSTLRLCV) and E7 67-76(LCVQSTHVDI) epi-position has higher anticarcinogenic effect, E7 61-69(CDSTLRLCV) and E7 67-76(LCVQSTHVDI) CYQSTHVDI and the CYVTLRVCL peptide of the replaced form of the amino acid of peptide demonstrate higher anticancer effect, can prove that they are to generate effective epi-position for the vaccine development of HLA-A*2402 type cervical cancer and the required cytotoxic T lymphocyte of adoptive immunity cell therapy.
Description of drawings
Fig. 1 utilizes flow cytometry assay to confirm the figure of the generation of the specific IFN-γ of HPV Class1 6E7 epi-position (15mer).Donor 1:HLA-A*2402/2601.
Fig. 2 utilizes 51The Cr method for releasing is confirmed the figure of the fragmentation effect of the specific cervical cancer cell of HPV Class1 6E7 epi-position (15mer).Donor 2:HLA-A*2402/3303(E: effector cell (effector), T: target cell (target cell)).
Fig. 3 utilizes flow cytometry assay to confirm the figure of the generation of the specific IFN-γ of HPV Class1 6E7 epi-position (9mer/10mer).Donor 3:HLA-A*0203/2402.
Fig. 4 utilizes 51The Cr method for releasing is confirmed the figure of the fragmentation effect of the specific cervical cancer cell of HPV Class1 6E7 epi-position (9mer, 10mer).Donor 4:HLA-A*2402/3101.(E: effector cell, T: target cell)
Fig. 5 utilizes 51The Cr method for releasing comes relatively the figure of the cervical cancer cell fragmentation effect of the CTL that separates from PBMC and the removed PBMC of CTL.Donor 5:HLA-A*0206/2402.(E: effector cell, T: target cell)
Fig. 6～9th confirmed by replacement amino acid and the figure of the cytotoxic effect of synthetic HPV Class1 6E7 epitope specificity, (Fig. 6) be E7 61-69(9mer, peptide 16) and E7 67-76(10mer, peptide 35) with the integrated structure prognostic chart of HLA-A*2402 molecule, (Fig. 7) to calculate by amino-acid substitution and the figure in conjunction with energy of synthetic HPV Class1 6E7 peptide, being (Fig. 8) to utilize flow cytometry assay to confirm the figure of generation of the IFN-γ of the HPV Class1 6E7 epitope specificity synthetic by displacement, is (Fig. 9) to utilize 51The Cr method for releasing is confirmed the figure of fragmentation effect of the cervical cancer cell of the HPV Class1 6E7 epitope specificity synthetic by displacement, (c) donor 6:HLA-A*2402/2402(d) donor 7:HLA-A*2402/3101.(E: effector cell, T: target cell)
Figure 10～12nd, the figure of the anticancer therapy effect of confirmation cytotoxic T cell in the cervical cancer mouse model, (Figure 10) be to be applied to the mouse of implanted device Cervical Tumor with the PBMC that the HPV-16 E7 that filters out is processed and cultivated after, measure the figure of the size variation of tumour, be (Figure 11) after being applied to the mouse of implanted device Cervical Tumor with the CTL that the HPV-16 E7 that filters out is processed and cultivated, measure the size variation of tumour and the figure of weight (Figure 12).(a) donor 8:HLA-A*0206/2402(b) donor 9:HLA-A*2402/3303.
Embodiment
Below, embodiment is described in more detail the present invention by indefiniteness.But following embodiment should not be construed as circumscription of the present invention in following embodiment just for illustration the present invention puts down in writing.
Embodiment 1: peptide (peptide) synthetic
Whole aminoacid sequences for HPV Class1 6E7 protein, utilize computerized algorithm (computer algorithms), after separately synthetic in the mode of 15mer big or small overlapping (overlapping) separately, utilize high performance liquid chromatography (high performance liquid chromatography:HPLC) instrument, final selection purity is the peptide more than 95%, amounts to synthetic 14 (table 3).In order to find the epi-position that really presents in the peptide that is 15mer 14 length of being synthesized in MHC class I molecule, select 2 peptides that anticarcinogenic effect is high by testing sieve, after the mode that repeats with 9mer and the 10mer of the size of known epi-position as presenting in MHC class I molecule is separately synthetic, utilize the HPLC instrument, synthetic 25 purity are the peptide (table 4) more than 95% altogether.Filter out 2 peptides that anticarcinogenic effect is high from the peptide that 25 length of being synthesized are 9mer and 10mer, based on this, utilize computerized structural analysis software, predict in fact the highest with HLA-A*2402 molecule bonding force aminoacid sequence, displacement composite part amino acid (table 5).After synthetic peptide was dissolved in 1%DMSO PBS by this way, then freezing thawed and used in room temperature before using in-20 ℃.Peptide of the present invention is the A﹠amp that entrusts Korea S Yan Qi prefecture; Pep company synthesizes.
Table 3
Sequence number Amino acid position Aminoacid sequence
1 1-15 MHGDTPTLHEYMLDL
2 7-21 TLHEYMLDLQPETTD
3 13-27 LDLQPETTDLYCYEQ
4 19-33 TTDLYCYEQLNDSSE
5 25-39 YEQLNDSSEEEDEID
6 3l-45 SSEEEDEIDGPAGQA
7 37-51 EIDGPAGQAEPDRAH
8 43-57 GQAEPDRAHYNIVTF
9 49-63 RAHYNIVTFCCKCDS
10 55-69 VTFCCKCDSTLRLCV
11 6l-75 CDSTLRLCVQSTHVD
12 67-8l LCVQSTHVDIRTLED
13 73-87 HVDIRTLEDLLMGTL
14 79-93 LEDLLMGTLGIVCPI
15 85-98 GTLGIVCPICSQKP
The HPV Class1 6E7 peptide (15mer) that in table 3, expression is synthesized.
Table 4
Sequence number Amino acid position Aminoacid sequence
16 6l-69 CDSTLRLCV
17 62-70 DSTLRLCVQ
l8 63-7l STLRLCVQS
19 64-72 TLRLCVQST
20 65-73 LRLCVQSTH
21 66-74 RLCVQSTHV
22 67-75 LCVQSTHVD
23 68-76 CVQSTHVDI
24 69-77 VQSTHVDIH
25 70-78 QSTHVDIRT
26 7l-79 STHVDIRTL
27 72-80 THVDIRTLE
28 73-81 HVDIRTLED
29 61-70 CDSTLRLCVQ
30 62-71 DSTLRLCVQS
31 63-72 STLRLCVQST
32 64-73 TLRLCVQSTH
33 65-74 LRLCVQSTHV
34 66-75 RLCVQSTHVD
35 67-76 LCVQSTHVDI
36 68-77 CVQSTHVDIR
37 69-78 VQSTHVDIRT
38 70-78 QSTHVDIRTL
39 7l-80 STHVDIRTLE
40 72-81 THVDIRTLED
The HPV Class1 6E7 peptide (9mer and 10mer) that in table 4, expression is synthesized.
Table 5
Sequence number Existing aminoacid sequence Replaced aminoacid sequence
41 LCVQSTHVDI(68-76) CIQSTHVDI
42 LCVQSTHVDI(68-76) CYQSTSVDL
43 LCVQSTHVDI(68-76) CYQSTHVDI
44 LCVQSTHVDI(68-76) CYQSTHVDL
45 CDSTLRLCV(61-69) CYATLRVCL
46 CDSTLRLCV(61-69) CYSTLRACL
47 CDSTLRLCV(61-69) CYVTLRVCL
The HPV Class1 6E7 peptide (amino-acid substitution) that in table 5, expression is synthesized.
Embodiment 2: the preparation of cell
From the HLA-A*2402 type blood donation person's (table 4) that signs letter of consent in research blood, utilize centrifugation (centrifugation), separate PBMC (peripheral blood lymphocytes, peripheral blood mononuclear cell).The words of simple declaration, use Ficoll-Hypaque 1.077, after forming concentration gradient (density-gradient), flow into carefully donor's blood thereon, then implemented at normal temperatures centrifugation 15 minutes with the speed of 2000rpm, then only gather leukocytic cream (buffy coat), clean 2 times and be used for testing with PBS.
Table 6
Blood donation person (donor) Sex HLA-A HLA-B HLA-C
1 The man A*2402/2601 15，35 03，04
2 The man A*2402/3303 07，44 07，07
3 The man A*0203/2402 35，5l 07，14
4 The man A*2402/3001 13，55 01，06
5 The man A*0206/2402 07，59 01，07
6 The man A*2402/2402 40，40 03，04
7 The man A*2402/3101 35，51 03，14
8 The man A*0206/2402 35，54 01，03
9 The man A*2402/3303 40，58 03，08
Table 6 is the tables about HLA-A*2402 type blood donation person.
Embodiment 3: generate autologous dendritic cell (autologous dendritic cell) by PBMC
To put into by the PBMC that centrifugation obtains in the T75 flask that contains RPMI nutrient solution (10%FBS, 5% microbiotic), cultivate 2 hours under 37 ℃.Then, remove the cell that swims, in being attached to the monocyte of flask (monocyte), add interleukin 4 (IL-4,1000U/ml) and granulocyte-macrophage colony stimutaing factor (GM-CSF, 800U/ml).Cultivate beginning in the time of rear the 2nd day, the 4th day, add respectively IL-4(1000U/ml) and GM-CSF(1600U/ml).According to the growth of cell, exchange at any time the RPMI nutrient solution.Cultivate beginning in the time of rear the 5th day, add tumor necrosis factor-alpha (TNF-α, 1000u/ml) (Lim JB etc., Exp Hematol2006; 34(3): 296-307; Provenzano M etc., J Immunother2002; 25:342-351).
Embodiment 4: the generation of the polyclone cytotoxic T cell of peptide specific
The HLA-A*2402 type blood donation person's of superfreeze PBMC is thawed, in every hole is filled with the 24 hole culture vessels of RPMI nutrient solution of 2ml, add the cell of proper amt.Cultivate beginning in the time of rear the 1st day, peptide to be tested (10 μ g/ml) and interleukin II (IL-2,1000U/ml) are added in each hole.Between incubation period, add every three days IL-2(1000U/ml), and the exchange nutrient solution.Cultivate beginning in the time of rear the 7th day, add and use dendritic cell (cell count is minimum is more than 1/10th of cytotoxic T cell number) and the peptide (20 μ g/ml) that generates by aforesaid method.
Embodiment 5: utilize flow cytometry assay (flow cytometry) to measure cell internal interference element-γ (IFN-γ) generates
Cultivate beginning in the time of rear the 7th day, the cytotoxic T cell that is added with dendritic cell and peptide was cultivated 1 hour under 37 ℃.At this moment, in the test group one group adds phytoh(a)emagglutinin (PHA, 0.25 μ g/ml).After cultivation, add respectively the Cyanein (brefeldin A, BFA) of 10 μ g, then cultivated 5 hours under 37 ℃.From each hole collecting cell, use PBS(phosphate buffered saline buffer, phosphate-buffered saline thereafter) clean.After cleaning, add the PBS of the EDTA that contains 1mM concentration, cultivated 10 minutes at 37 ℃.When cultivating end, clean 2 times with the PBS that comprises 5%FBS, add respectively fluorescently-labeled antibody, be the mouse anti human CD3+ antibody of perdinin-chlorophyll-protein (PerCP) combination and the mouse anti human CD8+ antibody of phycoerythrin (PE) combination with the 1:100 ratio, then under 4 ℃, cultivated 15 minutes with dark (dark) state.After cultivation, use respectively lysate (lysing solution) and rupture of membranes agent (permeabilization solution) to process, add fluorescein isothiocyanate (fluorescein isothiocyanate, FITC) the mouse anti human IFN-gamma antibodies of combination is then under 4 ℃, cultivated 30 minutes with dark state.Thereafter, clean 2 times with the PBS that comprises 5%FBS, fixing with 1% formaldehyde (formaldehyde), then utilize fluorescence-activated cell sorting (fluorescence activated cell sorting, FACS) (Kern F etc., Eur J Immunol2000 are measured and analyzed to device; 30:1676-1682; Gratama JW etc., Cytometry2004; 58:79-86).
Embodiment 6: cytotoxicity experiment (cytotoxicity assay)
Take the cervical cancer cell strain as target cell (target cell), use 51The Cr method for releasing is implemented cytotoxicity experiment.Simple declaration to the HLA-A*2402 type cervical cancer cell strain of cultivating, is added the Na of 0.1mCi in the RPMI nutrient solution 2 51CrO 4, cultivated 45 minutes under 37 ℃.After cleaning 2 times with PBS, the target cell of proper amt is added in cytotoxic T cell with various ratios, and cultivated 5 hours under 37 ℃.When cultivating end, carry out centrifugation 5 clocks with 1500rpm, in pre-prepd γ-counter test tube (γ-counter tube), each moves into 100 μ l.Utilize gamma-radiation counter (γ-ray counter) to measure and analyze.The degree that the specific target cell of cytotoxic T cell is killed and wounded utilizes formula [(the spontaneous cpm of experiment cpm-)/(the spontaneous cpm of maximum cpm-)] * 100 to calculate (Bao L etc., Biol Blood Marrow Transplant2008; 14:1156-1162).
Embodiment 7: protein-MHC mixture is measured in conjunction with the computer simulation (In silico) of affinity Method
For measure peptide and MHC molecule in conjunction with affinity, build peptide-MHC mixture model, calculate energy poor of bonding state and separate stage, calculating mixture forming energy is assumed to be more stable mixture and has higher in conjunction with affinity.At first, peptide-MHC composite structure is to simulate as the basis take the structure of PDB id 2BAK, and the mixture energy minimization utilizes take CHARMM(Chemistry at HARvard Macromolecular Mechanics) field of force is Insight II (the Accelrys Software Inc) program on basis.After minimizing the energy, utilize protein design and in conjunction with the affinity computation program, be that EGAD comes contribution degree that calculating energy forms mixture and the contact between each residue (residue).Initial setting is used to the mixture forming energy and calculates (Pokala N etc., J Mol Biol2005; 347:203-27).Take the calculation result of program as the basis, for the comparative measurement of mixture forming energy, utilize several ' pseudo_DELTA_G_complex_formation ' numerical value.Measure for the contribute energy degree of measuring each point of contact (interface) residue, collect the dG numerical value of 1 point of contact residue of each level.
Embodiment 8: utilize the experimentation on animals of cervical cancer mouse model
The right side rib of BALB/c nude mice to 5 ages in week injects 5 * 10 5Individual cervical cancer cell strain, be SiHa cell (Friedl F etc., Proc Soc Exp Biol Med1970; 135(2): 543-5), induce the formation tumour.In order to confirm cytotoxic T cell to the result for the treatment of of cervical cancer, and reach certain numerical value (40mm in the size of tumour 3～50mm 3) time, the PBMC(l that will process with each peptide * 10 7Individual) or the CTL(3 that separates * 10 6) inject Peripheral blood by mouse tail vein.The size of tumour utilizes slide calliper rule (caliper) to measure every day, utilizes formula [(a * b)/2, a is major axis, b is minor axis], calculating mean value and record.
The result of above-described embodiment is as follows.
(1) the specific length of HLA-A*2402 is the probing into of HPV Class1 6E7 epi-position of 15mer
In order to induce the candidate of the E7 epi-position of founder cell toxicity T cell in 14 HPV Class1 6E7 peptides that synthesized with filtering out HLA-A*2402 type patient-specific, PBMC to the blood donation person processes with each peptide, after 1 week, with cultivate use the IL-2(negative control group), pp65328-336(CMV A24, QYDPVAALF, positive controls) (Szmania S etc., Blood2001; 98(3): 505-12) with pp65495-493(CMV A02, NLVPMVATV, negative control group) (Solache A etc., J Immunol1999; The control group of 163:5512-8) processing respectively compares.As shown in Figure 1 as can be known, compare with the control group of only processing with IL-2,14 peptides are all induced the generation of higher IFN-γ, in the peptide of the generation of comparing the IFN-γ that demonstrates higher numerical value with negative control group (negative control), use E761-75(CDSTLRLCVQSTHVD) and E7 67-81(LCVQSTHVDIRTLED) to demonstrate the generation numerical value of IFN-γ high for the test group processed of peptide, this value is almost near the level of positive controls (positive control).In order to confirm to utilize the E7 epi-position that flow cytometry assay filters out whether to demonstrate actually the effect that HLA-A*2402 type cervical cancer cell is had an effect and killed and wounded, utilize 51The Cr method for releasing is analyzed.PBMC for the blood donation person, use respectively IL-2, E7 61-75(CDSTLRLCVQSTHVD), E7 67-81(LCVQSTHVDIRTLED) and pp65 495-493(CMV A02, NLVPMVATV, negative control group) peptide is processed, after 1 week together with the same patient's who cultivates dendritic cell, again process rear 1 week of cultivation with each peptide, then with 51The HLA-A*2402 type cervical cancer cell of Cr mark mixes, and cultivates 5 hours, utilizes the gamma-radiation tester to analyze.Check analytical results, with with IL-2 or pp65 495-493(CMV A02, NLVPMVATV) etc. the negative control group of processing is compared, and uses E7 67-81(LCVQSTHVDIRTLED) test group processed of peptide demonstrates the cell killing effect of obviously high numerical value.As can be known, use E7 61-75(CDSTLRLCVQSTHVD) test group processed of peptide is not as E7 67-81(LCVQSTHVDIRTLED) effect (Fig. 2) of peptide.
(2) the specific length of HLA-A*2402 is the probing into of HPV Class1 6E7 epi-position of 9/10mer
pass through the above results, select E7 61-75(CDSTLRLCVQSTHVD) and E7 67-81(LCVQSTHVDIRTLED) peptide is as strong epi-position candidate, be after peptide that object resynthesis length is respectively 9mer and 10mer is processed and cultivated PBMC in order to this peptide, with the IL-2(negative control group), pp65328-336(CMV A24, QYDPVAALF, positive controls) and pp6591-100(CMV A33, SVNVHNPTGR, negative control group) compare, result can be confirmed, compare with positive controls, use E7 61-69(CDSTLRLCV) and E7 67-76(LCVQSTHVDI) test group processed of peptide demonstrates higher numerical value (Fig. 3).In addition, in order to E7 67-81(LCVQSTHVDIRTLED) the peptide length of synthesize for the basis be 9mer and 10mer candidate's peptide, be E7 61-69(CDSTLRLCV) and E7 67-76(LCVQSTHVDI) PBMC is processed utilization 51The Cr method for releasing, with use pp65 495-493(CMV A02, NLVPMVATV, negative control group) and before the length of screening is the E7 67-81(LCVQSTHVDIRTLED of 15mer) control group processed of peptide compares, result can be confirmed, being the E7 67-81(LCVQSTHVDIRTLED of 15mer with length) peptide compares, and new two kinds of synthetic peptides demonstrate higher or similar cell killing effect (Fig. 4).
The confirmation of the HPV 16 specific cytotoxic effects of HPV-16 E7 that (3) caused by CTL
For the effect by the CTL that comprises in PBMC of the HPV 16 specific cytotoxic effects of HPV-16 E7 confirmed before proving causes, use the microballoon (microbead) of anti--CD8+ antibodies, after blood donation person's PBMC separation of C D8+T cell, process and cultivate with the HPV-16 E7 that filters out respectively, then utilize 51The Cr method for releasing, with that cultivate with identical condition, that PBMC that removed the CD8+T cell compares result be, having removed in the PBMC of CD8+T cell not showed cell toxic effect, the CTL that comprises in PBMC as can be known participates in cytotoxic effect (Fig. 5) really.
(4) cytotoxic effect of the replaced HPV-16 E7 of amino acid is confirmed
For the result that occurs before supporting, utilize the EGAD program, calculate E7 epi-position and MHC mixture in conjunction with the forming energy result, the E7 61-69(CDSTLRLCV that calculates) and E7 67-76(LCVQSTHVDI) peptide and HLA-A*2402 mixture low in conjunction with the forming energy value, be negative value, can confirm that it is stable combination (Fig. 6).Measurable thus, two kinds of peptides can be combined with the HLA-A*2402 molecule and produce immunocompetence.
be the peptide of 9mer/10mer and the bonding force between the HLA-A*2402 molecule for two kinds of length that filter out take such result as the basis, utilize Insight II program to calculate, dope by to E7 61-69(CDSTLRLCV) and E7 67-76(LCVQSTHVDI) partial amino-acid series of peptide replace the peptide of calculating higher bonding force numerical value and synthetic after (Fig. 7), PBMC was processed for 1 week and cultivates, then with pp65 328-336(CMV A24, QYDPVAALF, positive controls), pp65 495-493(CMV A02, NLVPMVATV, negative control group) and existing E7 61-69(CDSTLRLCV) and E7 67-76(LCVQSTHVDI) peptide compares, result can be confirmed, compare with control group and existing peptide, replaced PEPC YQSTHVDI(m-peptide 3, No. 3, replaced peptide) and CYVTLRVCL(m-peptide 7, No. 7, replaced peptide) generation of induced strong IFN-γ.(Fig. 8).In addition, for the CYQSTHVDI(m-peptide 3 of confirming to filter out by flow cytometry assay, No. 3, replaced peptide) and CYVTLRVCL(m-peptide 7 cell killing effect, No. 7, replaced peptide) is utilized 51The Cr method for releasing is with two kinds of peptides and existing E7 61-69(CDSTLRLCV) and E7 67-76(LCVQSTHVDI) peptide compares, result can be confirmed, compares with existing peptide, the replaced peptide of amino acid demonstrates higher cell killing effect (Fig. 9).Thus, can be with E7 61-69(CDSTLRLCV) and E7 67-76(LCVQSTHVDI) peptide is predicted as strong specificity E7 epi-position, and as can be known, if for two kinds of peptides with the form replacement amino acid the highest with HLA-A*2402 molecule bonding force, the activity of stronger inducing cytotoxic T cell.
(5) anticancer test that utilizes cytotoxic T cell to carry out in the cervical cancer mouse model
To the BALB/c nude mice, injecting the cervical cancer cell strain is SiHa, after forming tumour, utilizes the peptide that filters out by in vitro tests, confirms the anticancer effect of cytotoxic T cell.As can be known, (G1) compares with the negative control group of not carrying out any processing as shown in Figure 10～12, will use E7 67-76(LCVQSTHVDI) when the PBMC that processes and cultivate was applied to mouse, the growth of tumour was suppressed (Figure 10).Can confirm in addition, (G1) compares with the negative control group of not carrying out any processing, to use E7 61-69(CDSTLRLCV), E7 67-76(LCVQSTHVDI), CYQSTHVDI(m3, No. 3, replaced peptide) and CYVTLRVCL(m7, No. 7, replaced peptide) after the CD8+T cell of processing and cultivating is applied to mouse, when confirming tumour big or small, the growth of tumour is suppressed (Figure 11 and 12) greatly.Can confirm thus, the E7 61-69(CDSTLRLCV that filters out with the experimentation on animals by utilizing the cervical cancer mouse model, by flow cytometry assay and cytotoxicity experiment), E7 67-76(LCVQSTHVDI) CYQSTHVDI(m3, No. 3, replaced peptide) and CYVTLRVCL(m7, No. 7, replaced peptide) peptide is processed and the growth of cultured cells toxicity T cell cervical cancer inhibiting and have anticancer effect.
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Figure IDA00002915182800201

Claims (11)

1. identical mutant on an immunogenic peptide and function thereof, it has CDSTLRLCV is that sequence number 16 or LCVQSTHVDI are the aminoacid sequence of sequence number 35.
2. identical mutant on immunogenic peptide according to claim 1 and function thereof, is characterized in that, on described function, to have CYQSTHVDI be that sequence number 43 or CYVTLRVCL are the aminoacid sequence of sequence number 47 to identical mutant.
3. nucleic acid, the peptide of its coding sequence number 16 claimed in claim 1 or sequence number 35.
4. nucleic acid according to claim 3, is characterized in that, described nucleic acid has respectively the base sequence of sequence number 48 and sequence number 49.
5. nucleic acid, the peptide of its coding sequence number 43 claimed in claim 2 or sequence number 47.
6. nucleic acid according to claim 5, is characterized in that, described nucleic acid has the arbitrary base sequence of sequence number 50 to the sequence number 107.
7. one kind is used for the composition that prevention or treatment HPV infect relative disease, and it comprises (a) claim 1 or 2 described peptide and (b) pharmaceutically useful carrier.
8. composition according to claim 7, is characterized in that, described HPV infects that relative disease is that bowenoid papulosis, anus are abnormal, respiratory tract or papilloma of conjunctiva, cervical atypical hyperplasia, cervical cancer, carcinoma vulvae or prostate cancer.
9. a particulate, liposome or immunostimulating complex, it comprises peptide claimed in claim 1 as effective constituent.
10. a particulate, liposome or immunostimulating complex, it comprises nucleic acid claimed in claim 3 as effective constituent.
11. composition according to claim 7 is characterized in that, described composition is HLA-A*2402 type patient-specific.
CN201180044102.8A 2010-09-13 2011-09-09 Immunogenic peptide and comprise the composition for preventing or treat HPV relative disease of this peptide Expired - Fee Related CN103097398B (en)

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WO2019195486A1 (en) 2018-04-05 2019-10-10 Juno Therapeutics, Inc. T cell receptors and engineered cells expressing same
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MESHKAT, Z ET AL.: "CTL response to a DNA vaccine encoding E7 gene of human papillomaviurs type 16 from an Iranian isolate", 《IRANIAN JOURNAL OF IMMUNOLOGY》 *
RIEMER A.B. ET AL.: "A concerved E7-derived cytotoxic T lymphocyte epitope expressed on human papillomavirus 16-transformed HLA-A2+ epithelial cancers", 《JOURNAL OF BIOLOGICAL CHEMISTRY》 *
VITHAGNA KHAMMANIVONG ET AL.: "Paucity of functional CTL epitopes in the E7 oncoprotein of cervical cancer associated human papillomavirus type 16", 《IMMUNOLOGY AND CELL BIOLOGY》 *

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