CN1398187A - Method and compsns. for treating liver cell cancer - Google Patents
Method and compsns. for treating liver cell cancer Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4715—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
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- A61K39/4643—Vertebrate antigens
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- A61K39/46448—Cancer antigens from embryonic or fetal origin
- A61K39/464481—Alpha-feto protein
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Abstract
A method for preventing or for treating cancer in a mammal, where the cancer cells express at least a part of an alpha fetoprotein molecule at the cell surface, the method comprising the step of creating an immune response in the mammal to at least part of the amino acid sequence of an alpha fetoprotein molecule, where the immune response comprises activating alpha fetoprotein peptide specific T lymphocytes against the cancer cells. A composition for preventing or for treating cancer comprising a peptide having at least part of the sequence of alpha fetoprotein.
Description
About being subjected to the federal research of supporting or the statement of exploitation
The present invention carries out under the support of Federal Government, Contract NO NIH/NCI ROICA 77623.Federal Government has some right to the present invention.
The cross reference of related application
The exercise question that the application requires on February 10th, 2000 to submit to is the income of the U.S. Patent application 60/181,966 of " being used to prevent or treat the method and composition of hepatocarcinoma "; And the application is the part continuation application of the exercise question of JIUYUE in 2000 submission on the 12nd for the U.S. Patent application 09/660,252 of " using the hepatocarcinoma through substituting alpha-fetoprotein to treat "; And be of the part continuation application of the exercise question of JIUYUE in 2000 submission on the 14th for the U.S. Patent application 09/662,505 of " using the hepatocarcinoma treatment of alpha-fetoprotein cDNA "; Their content intact is collected herein by reference.
Background
Primary hepatocarcinoma is to cause main causes of death by cancer in the world wide.Hepatocarcinoma (HCC) is the common type of primary hepatocarcinoma, and global sickness rate is approximately annual 1200000 examples.In some area, such as Southeast Asia and Subsahara Africa, hepatocarcinoma is one of modal malignant tumor.As if the altofrequency of this disease relevant at these geographic high incidences with viral hepatitis.
Effective treatment of hepatocarcinoma is limited to non-metastatic disease individuality at present, and relates to ocal resection, follows or do not follow liver transplantation.Yet, because the recurrence after the excision, even excision and transplanting can not be cured most of tumors.Embolic chemotherapy also is invalid to a great extent.
Therefore, still need be at effective treatment of hepatocarcinoma.It is desirable to, this Therapeutic Method is applicable to the lesser developed countries with high incidence.In addition, this Therapeutic Method should be applicable to the individuality of suffering from unresectable tumor and metastatic disease.
General introduction
In one embodiment, the present invention is used in mammal prevention or treatment method for cancer, and wherein cancerous cell is at least a portion of cell surface expression alpha-fetoprotein molecule.This method is included in the mammal step that causes at the immunne response of the partial amino-acid series at least of alpha-fetoprotein molecule, and wherein immunne response comprises the alpha-fetoprotein peptide specific T lymphocyte of activation at cancerous cell.In one embodiment, alpha-fetoprotein peptide specific T lymphocyte is a cytotoxic T lymphocyte.In a preferred embodiment, the alpha-fetoprotein molecule is SEQID NO:2.In an especially preferred embodiment, the alpha-fetoprotein molecule is selected from down group: the 137-145 position residue of SEQ ID NO:2, the 158-166 position residue of SEQ ID NO:2, the 325-334 position residue of SEQ ID NO:2 and the 542-550 position residue of SEQ ID NO:2.In one embodiment, cancer is a hepatocarcinoma.In another embodiment, mammal is the people.
In a preferred embodiment, the step of initiation immunne response comprises that to one or more compositionss of administration, wherein said composition comprises the peptide of the partial amino-acid series at least with alpha-fetoprotein.In an especially preferred embodiment, peptide is selected from down group: the 137-145 position residue of SEQ IDNO:2, the 158-166 position residue of SEQ ID NO:2, the 325-334 position residue of SEQID NO:2 and the 542-550 position residue of SEQ ID NO:2.In another preferred embodiment, this peptide is selected from down group: the 1-9 position residue of SEQ ID NO:2, the 178-186 position residue of SEQ ID NO:2, the 218-226 position residue of SEQ ID NO:2, the 235-243 position residue of SEQ ID NO:2, the 306-315 position residue of SEQ ID NO:2, the 485-493 position residue of SEQ ID NO:2, the 492-500 position residue of SEQ ID NO:2, the 507-516 position residue of SEQ ID NO:2, the 547-556 position residue of SEQ ID NO:2 and the 555-563 position residue of SEQ ID NO:2.
In another preferred embodiment, the step that causes immunne response comprises that to one or more compositionss of administration wherein said compositions comprises the dendritic cell through one or more peptide pulses of at least a portion that constitutes aminoacid sequence SEQ ID NO:2.In also having an embodiment preferred, the step that causes immunne response comprises that to one or more compositionss of administration wherein said compositions comprises the dendritic cell through the recombinant adenoviral vector transduction of coding alpha-fetoprotein.
In another embodiment, the present invention relates to be used for preventing or treating the method for hepatocarcinoma the people, wherein cancerous cell is at least a portion of cell surface expression alpha-fetoprotein molecule, thereby this method comprises by the people being used one or more compositionss and activates step at the alpha-fetoprotein cytotoxic T lymphocyte of cancerous cell at least a portion of aminoacid sequence SEQID NO:2, and wherein said compositions comprises and is selected from down the peptide of organizing: the 137-145 position residue of SEQ ID NO:2, the 158-166 position residue of SEQ ID NO:2,325-334 position residue with SEQ ID NO:2.
In another embodiment, the present invention is the method that is used in people's prevention or treatment hepatocarcinoma, wherein cancerous cell is at least a portion of cell surface expression alpha-fetoprotein molecule, thereby this method comprises by the people being used one or more compositionss and activates step at the alpha-fetoprotein cytotoxic T lymphocyte of cancerous cell at least a portion of aminoacid sequence SEQ IDNO:2, and wherein said compositions comprises and is selected from down the peptide of organizing: the 542-550 position residue of SEQ ID NO:2.
In another embodiment, the present invention is the method that is used in people's prevention or treatment hepatocarcinoma, wherein cancerous cell is at least a portion of cell surface expression alpha-fetoprotein molecule, thereby this method comprises by the people being used one or more compositionss and activates step at the alpha-fetoprotein cytotoxic T lymphocyte of cancerous cell at least a portion of aminoacid sequence SEQ IDNO:2, and wherein said compositions comprises and is selected from down the peptide of organizing: the 1-9 position residue of SEQ ID NO:2, the 178-186 position residue of SEQ ID NO:2, the 218-226 position residue of SEQ ID NO:2, the 235-243 position residue of SEQ ID NO:2, the 306-315 position residue of SEQ ID NO:2, the 485-493 position residue of SEQ ID NO:2, the 492-500 position residue of SEQ ID NO:2, the 507-516 position residue of SEQ ID NO:2, the 547-556 position residue of SEQ ID NO:2,555-563 position residue with SEQ ID NO:2.
In another embodiment, the present invention is the method that is used in people's prevention or treatment hepatocarcinoma, wherein cancerous cell is at least a portion of cell surface expression alpha-fetoprotein molecule, thereby this method comprises by the people being used one or more compositionss and activates step at the alpha-fetoprotein cytotoxic T lymphocyte of cancerous cell at least a portion of aminoacid sequence SEQ IDNO:2 that wherein said compositions comprises the dendritic cell through one or more peptide pulses of at least a portion that constitutes aminoacid sequence SEQ ID NO:2.One or more peptides that are used for the pulse dendritic cell are selected from down group: the 137-145 position residue of SEQ ID NO:2, the 158-166 position residue of SEQ ID NO:2, the 325-334 position residue of SEQ ID NO:2 and the 542-550 position residue of SEQ IDNO:2.
In another embodiment, the present invention is the method that is used in people's prevention or treatment hepatocarcinoma, wherein cancerous cell is at least a portion of cell surface expression alpha-fetoprotein molecule, thereby this method comprises by the people being used one or more compositionss and activates step at the alpha-fetoprotein cytotoxic T lymphocyte of cancerous cell at least a portion of aminoacid sequence SEQ IDNO:2 that wherein said compositions comprises the dendritic cell through the recombinant adenoviral vector transduction of coding alpha-fetoprotein.
In another embodiment, the present invention is the isolated peptides that can be used for preventing or treating cancer, and this peptide is selected from down group: the 137-145 position residue of SEQ ID NO:2, the 158-166 position residue of SEQ ID NO:2 and the 325-334 position residue of SEQ ID NO:2.In a preferred embodiment, the present invention is the compositions that is used to prevent or treat cancer, wherein said composition comprises one or more peptides that are selected from down group: the 137-145 position residue of SEQ ID NO:2, the 158-166 position residue of SEQ ID NO:2 and the 325-334 position residue of SEQ ID NO:2 present in an amount at least sufficient to cause the immunne response at alpha-fetoprotein in mammal.Also can comprise adjuvant in the compositions.In another embodiment, the present invention is used for comprising the step of the people being used one of one of these peptides or these compositionss in people's prevention or treatment method for cancer.
The present invention also comprises the means that are used to prevent or treat cancer, wherein comprises one or more peptides that are selected from down group: the 137-145 position residue of SEQ ID NO:2, the 158-166 position residue of SEQ ID NO:2, the 325-334 position residue of SEQ ID NO:2 and the 542-550 position residue of SEQ ID NO:2.
In another embodiment, the present invention is the isolated peptides that can be used for preventing or treating cancer, and this peptide has the sequence according to the 542-550 position residue of SEQ ID NO:2.In a preferred embodiment, the present invention is the compositions that is used to prevent or treat cancer, and wherein said composition comprises the peptide that has according to the sequence of the 542-550 position residue of SEQ ID NO:2.Also can comprise adjuvant in the compositions.In another embodiment, the present invention is used for comprising the step of the people being used one of this peptide or these compositionss in people's prevention or treatment method for cancer.
The present invention also comprises the means that are used to prevent or treat cancer, wherein comprises the peptide that has according to the sequence of the 542-550 position residue of SEQID NO:2.
Describe in detail
In one embodiment, the present invention can separately or unite the one group of peptide that is used for the treatment of hepatocarcinoma.In another embodiment, the present invention is the method for preventing or treating hepatocarcinoma by independent or co-administered one or more peptides of the present invention or the compositions that comprises one or more peptides of the present invention.In another embodiment, the present invention is the method for preventing or treating hepatocarcinoma by the dendritic cell of using recombinant adenovirus (AdV) carrier transduction that passes through one or more peptide pulses of the present invention or process coding alpha-fetoprotein.
About 80% hepatocarcinoma activates the expression of alpha-fetoprotein again.Mus and people's T cell bank can both be discerned AFP derived peptide epi-position in I type MHC environment.Therefore, although in embryo development procedure, be exposed to this oncofetal protein of high blood plasma level, be not that all AFP specific T-cells are all lost in immune individual generating process.
The present invention relates to by human a-fetoprotein is the evaluation of the deutero-peptide of SEQ ID NO:2, and it is subjected to the identification in human T-cell storehouse when being presented in the environment of HLA-A*0201.Summarize as table 1 hereinafter, identified 4 kinds of AFP derived peptide, be called " dominance " peptide.They are PLFQVPEPV, hAFP
137-145, the 137-145 position residue of SEQ ID NO:2; FMNKFIYEI, hAFP
158-166, the 158-166 position residue of SEQID NO:2; GLSPNLNRFL, hAFP
325-334, the 325-334 position residue of SEQ ID NO:2; And GVALQTMKQ, hAFP
542-550, the 542-550 position residue of SEQ ID NO:2.Each all has one or two anchor residues.
In concentration dependent I type binding assay, each dominance peptide is all stablized HLA-A*0201 on the T2 cell.They are stable in the kinetic determination method of dissociating to reach 2-6 hour.In addition, each dominance peptide is all induced peptide specific T cell by several normal HLA-A*0201 donors external.Importantly, these hAFP peptide specifics T cell can also be discerned HLA-A*0201 in cytotoxicity assay and IFN γ ELISPOT algoscopy
+/ AFP positive tumor cell.These information are summarized in hereinafter table 1.Table 1: the general introduction peptide position sequence anchor T2 of dominance peptide is in conjunction with the concentration kinetics hAFP that dissociates relatively
137-1454 hours hAFP of 137-145 PLFQVPEPV 2 2.5mM
158-1664 hours hAFP of 158-166 FMNKFIYEI 2 0.5mM
325-3342 hours hAFP of 325-334 GLSPNLNRFL 2 10mM
542-550542-550 GVALQTMKQ 1＞100mM 6 hours
As what the present invention proved, the activation of these T cells can realize by present these dominance peptides in the immunostimulation environment, comprise presenting of full-time antigen presentation dendritic cell.Process coding alpha-fetoprotein cDNA is that these 4 kinds of dominance peptide epitopes will be processed and present to the dendritic cell of recombinant adenovirus (AdV) carrier transduction of SEQ ID NO:1 in the environment of MHC, also will induce the AFP specific T-cells to activate.The HLA-A*0201/K of similar immunity
bMice is the cell of identification process AFP peptide pulse in the release of cytokines algoscopy also.In addition, people and the HLA-A*0201/K that stimulates through the AFP peptide
bMouse T cell is replied identification through the target of hAFP transformation and the human liver cell cancerous cell of (on than low degree) natural expression AFP.At last, use mass spectrography by from HLA-A*0201
+The complicated peptide mixer of HCC cell eluting has been identified at least 3 kinds of AFP epi-positions.Thereby multiple evidence shows that each of these 4 kinds of dominance peptides all has immunogenicity, and obtains natural process and present in the environment of HLA-A*0201.
Also identified to have the faint or less other 10 kinds of peptides of replying but in the algoscopy of more than one types, being positive that reproduce, be called " subdominance " peptide.These 10 kinds of peptides are MKWVESIFL, the 1-9 position residue of SEQ ID NO:2; ILLWAARYD, the 178-186 position residue of SEQ ID NO:2; LLNQHACAV, the 218-226 position residue of SEQ ID NO:2; FQAITVTKL, the 235-243 position residue of SEQ ID NO:2; TTLERGQCII, the 306-315 position residue of SEQID NO:2; CIRHEMTPV, the 485-493 position residue of SEQ ID NO:2; PVNPGVGQC, the 492-500 position residue of SEQ ID NO:2; NRRPCFSSLV, the 507-516 position residue of SEQ IDNO:2; TMKQEFLINL, the 547-556 position residue of SEQ ID NO:2; And NLVKQKPQI, the 555-563 position residue of SEQ ID NO:2.
Though compositions disclosed by the invention and method mainly are to use one or more dominance peptides hAFP
137-145Be 137-145 position residue, the hAFP of SEQ ID NO:2
158-166Be 158-166 position residue, the hAFP of SEQ ID NO:2
325-334Be 325-334 position residue and the hAFP of SEQ ID NO:2
542-550Be the 542-550 position residue of SEQ ID NO:2, but be to use 10 kinds of subdominance peptides one or more substitute in 4 kinds of dominance peptides one or more or with it associating also belong within the scope of the present invention.
The evaluation of dominance peptide and subdominance peptide will be discussed now in more detail.At first, be that SEQ ID NO:2 (Genbank numbering: J00077, J00076 and V01514) identifies potential peptide sequence in conjunction with HLA-A*0201 by hAFP.The length of these peptides is 9 or 10 aminoacid, and the 2nd is isoleucine, leucine or methionine; Perhaps, according to the length of peptide, the 9th or 10 is isoleucine, leucine or valine, or the two furthermore.Use the hereditism of University of Wisconsin computer (the University of Wisconsin of group, Genetics Computer Group) " searching pattern " (find patterns) program screening hAFP sequence is SEQ ID NO:2, has identified 74 kinds of such peptides.Use standard technique to synthesize this 74 kinds of peptides.
Stablize in the algoscopy at HLA-A*0201, each of this 74 kinds of peptide material standed fors is all tested with the concentration dependent of T2 cell combining.Evening before that day, in the expression of room temperature insulation with increase cell surface I type MHC molecule, with each peptide incubated overnight, the concentration range of peptide was 0.1-100mM then with T2 (TAP deficiency) cell.With anti-HLA-A2 antibody (BB7.2) and goat anti-mouse-FITC with cell dyeing after, measure the stability of HLA-A*0201 by flow cytometry.To be used as positive control in conjunction with Flu substrate peptide (58-66 amino acids) HLA-A*0201 (Flu) strongly.
Then, measure the stability of MHC-peptide complexes by the kinetic determination method of dissociating.PH3.2 citrate-phosphatic acid buffer with gentleness is peeled off HLA-A*0201 LCL.Immediately with the concentration of 200mM with each peptide pulse to cell, promptly when having 3 μ g/ml b2 microglobulins in room temperature insulation 1 hour.Wash excessive peptide off, and cell is incubated 0,2,4 and 6 hour in 37 ℃.When each time point finishes, clean cell, and pair cell surface HLA-A2 expresses and dye, analyze by flow cytometry then.If average fluorescent strength with respect to peeling off but does not raise 1.5 times with the cell of peptide pulse at least, think that then peptide-I type MHC complex is stable.In the kinetic determination method of dissociating, all 4 kinds of dominance stabilized peptides reach 2-6 hour.
Then 4 kinds of dominance peptides are carried out other immunology and physio-chemical study.These researchs comprise in vitro study, and wherein (1) is used to generate AFP peptide specific human T-cell culture with peptide, and these cultures had both had peptide specific, were subjected to the identification of the cell of natural expression AFP again; (2) will be used to generate identification AFP positive cell and through the dendritic cell of AdVhAFP transduction through the AFP specificity human T-cell of the AFP negative cells of overdominance peptide pulse.These researchs also comprise research in the body, and wherein (1) has the splenocyte of identification polypeptide and AFP positive cell with the transgenic mice of peptide immunity; (2) AdVhAFP/DC mice immunized identification AFP positive cell and through the AFP negative cells of overdominance peptide pulse.These studies show that, the dominance peptide has immunogenicity, and self has immunogenicity AFP, and the dominance peptide is by natural process and being presented on the AFP positive cell surface, and AFP/DC or dominance peptide can be used for generating the AFP specific T-cells, these cell cellulation factors and to kill and wound A be positive cell.In addition, mass spectrography is used to identify the AFP peptide by the positive hepatocellular carcinoma cells of AFP surface physically.
At first, originally HLA-A2*0201 human T-cell culture is carried out repeatedly peptide to stimulate, with the proof peptide in human T-cell's lab environment immunogenicity and peptide specific T cell recognition through the ability of the target of AFP transfection.Prepare large quantities of T cell cultures by PBMC, and test is discerned through the peptide pulse and is expressed the ability of two kinds of targets of AFP between the 3rd and 7 weeks of amplification through each dominance AFP derived peptide pulse (adding KLH, IL-7 and IL-2).These culture amplification peptide specifics T cell, evidence are that they can be at the JY cell of discerning the pulse of process particular peptide but not through contrast MART-1 in EL ISPOT algoscopy
7-35Secretion IFN γ behind the JY of pulse.Parental cell with respect to unmodified or the empty AdVRR5 transduction of process, AFP peptide specific T cell mass is also discerned the HLA-A2*0201 melanoma cells (M202) through negative stable transfection of AFP and process AdVhAFP transduction, and the frequency that is shown as the AFP specific T-cells that generates IFN γ increases.In order to assess the ability of identification HLA-A*0201+, be that HepG2 (is Hep3B with respect to the positive HCC of HLA-A2-/AFP) carries out cytotoxicity and ELISPOT algoscopy to the hepatocellular carcinoma cells of natural expression AFP.
In addition, prepare CTL by dendritic cell through the AdV transduction.In brief, will reach 2 hour with AdVhAFP or AdVMART1 with infection multiplicity (moi) 1000 transductions with the dendritic cell that GM-CSF and IL-4 are incubated preparation by PBMC.Clean the dendritic cell after transduceing, irradiation, and with 1-2 * 10
5Cell/ml coating is as the stimulus object of CTL preparation.By the magnetic bead exclusive method by in the body non-adherent cell, eliminating CD4, CD14, CD19 and CD56+ cell effector lymphocyte's (common 80% is CD8+ in the colony, shows) with preparation CD8+ enrichment.With the CD8+ cell with through the transduction dendritic cell with 2 * 10
6Cell/ml is coated with in the body culture medium at 5% of interpolation 10-25ng/ml IL-7.In culture, added 10U/ml IL-2 in every 3-4 days.With 1 dendritic cell the ratio of 10-20 CD8+CTL is stimulated CD8+CTL once more from body AdV transduction dendritic cell with fresh weekly.Identify the performance of most of cultures weekly, distinguish CD4+ and CD8+ cell.Each dominance peptide has all been induced peptide specific T cell by several normal HLA-A*0201 donors external.
Because the external human T-cell who stimulates through AdVhAFP/DC in CTL and ELISPOT algoscopy all specific recognition through the target of hAFP transfection, so then study this 4 kinds of dominance peptides, measure the specific recognition whether they are subjected to the T cell of process AdVhAFP/DC stimulation.Cultivate after 7-21 days, to the cytotoxicity and the frequency of the CD8 enrichment T cell tests hAFT peptide specific IFN gamma cells factor cellulation that stimulates with AdVhAFP/DC weekly.AdVhAFP/DC T cell culture has cytotoxicity to the JY cell through the pulse of one of 4 kinds of AFP peptides, and indication can have been increased at the CTL of these peptides by the peripheral blood of normal donor.With through after the LCL of body peptide pulse or JY cell stimulate again, these bulk cultures things also comprise with respect to MART-1
27-35The IFN γ secretory cell special to the AFP peptide that frequency is much higher, indication is except hAFP
542-550In addition, the also natural dendritic cell by through the AdVhAFP transduction of other 3 kinds of dominance peptides obtain processing and present.
The T cell culture that stimulates through AdVhAFP/DC also has low-frequency cytokine cellulation, and the latter can discern A*0201
+/ AFP is positive, and hepatocarcinoma is HepG2, but nonrecognition A*0201
-/ AFP is positive, and HCC is Hep3B.Detect the T lymphocyte of synthetic Th1 cytokine IFN γ and TNF α, and do not detect Th2 cytokine IL-4.Be coated with not containing the T cell and also detect IL-10 when hepatocarcinoma is, the generation of indicating this cytokine is from tumor cell.
As described below with HLA-A*0201/K
bTransgenic mice is used to screen 74 kinds of peptides, and to determine whether these peptides have immunogenicity, whether natural obtaining in the environment of HLA-A*0201 processed and presented.HLA-A*0201/K
bThe transgenic female mice is raised by the Animal House of University of California (Los Angeles) radiation oncology system at present at first available from Harlan-SpragueDawley (Indianapolis city, Indiana State, the U.S.).
About the peptide immunity, the subcutaneous acceptance 100 μ g of mice were with 1: 1 emulsive AFP or control peptide in complete Freund's adjuvant.Be used in the complete Freund's adjuvant after emulsive each peptide immunity, the draining lymph node cell generates IFN γ in identification behind the cell of hAFP stable transfection.In addition, can in dendritic cell (process is expressed the transduction of the adenovirus (AdVhAFP) of AFP) mice immunized spleen, identify alpha-fetoprotein peptide specific T cell.Thereby 4 kinds of natural obtaining in I type environment of dominance peptide are processed and are presented, and have immunogenicity.
Then, confirm the interior immunogenicity of body of these 4 kinds of dominance peptides.With pulse each dominance peptide immunity HLA-A*0201/K to the homology dendritic cell
bMice.To at external use immunodominant peptide or MART-1 peptide or use the splenocyte that stimulates once more through Jurkat/AFP or Jurkat/MART cells transfected system to carry out IFN γ specificity ELISPOT algoscopy.The immunity of each hAFP peptide and subsequently stimulating once more of each peptide or Jurkat/AFP induced a large amount of AFP specificity IFN γ cellulations.No matter whether stimulate once more, from not showed cell toxicity and the IFN γ generation of lymphocyte of PBS injection mice.Use MART-1
27-35The peptide mice immunized generates the MART-1 specificity and replys and do not generate the AFP peptide and reply.
Then, prepare dendritic cell by the differentiation in GM-CSF and IL-4 by myeloid progenitor, and transduce with comprising the recombinant adenoviral vector that hAFP cDNA is SEQ ID NO:1 (AdVhAFP).In RPMI/2%FCS with the dendritic cell of moi 100 transduction In vitro culture.Transduce and carried out 2 hours in 37 ℃.Clean dendritic cell then and with every animal 0.2ml PBS 5 * 10
5Individual dendritic cell are resuspended, are used for injection.In all situations, survival rate surpasses 95%.In immunity 2 weeks of back, use Jurkat cell to stimulate mouse boosting cell once more through hAFP (Jurkat/AFP) or MART-1 (Jurkat/MART) stable transfection.By the frequency that ELISPOT mensuration AFP specificity and MART-1 specificity IFN γ discharge, calculate the meansigma methods of 3 groups of independent experiments, p＜0.02.At 5 hours
51Cr discharges the cytotoxicity of having measured in the algoscopy at Jurkat/AFP and Jurkat/MART.
Secondly, to being that the dominance peptide of eluting has carried out HPLC and mass spectrum is identified by the HLA-A*0201 human hepatocellular carcinoma.The general introduction of these analysis results is shown in hereinafter table 2.
For the eluting peptide, HepG2 and Hep3B cell are cleaned after 3 times with PBS, insulation is 1 minute in 5ml citrate-phosphate buffer pH3.2.With suspension centrifugal (800 * g, 5 minutes), obtain supernatant, add up to every kind of cell line to collect the acellular supernatant of 500ml.With the experiment material lyophilizing and be stored in-20 ℃.
Freeze-dried material is dissolved in 30ml water/acetonitrile/trifluoroacetic acid (W/A/TFA volume ratio 95/5/0.1) again.This solution is drawn into analytical reversed-phase HPLC post (Keystone Scientific C with the 0.2ml/min flow velocity
18Betasil, 250mm * 2mm, 5mm granular size, 100 apertures) on, chromatographic column is used the W/A/TFA balance in advance.Use the cumulative linear gradient of 0.1%TFA in acetonitrile (time/% acetonitrile=0/5,5/5,55/100,60/100) to carry out eluting.215 and 280nm place monitoring chromatographic column eluent light absorption value, and collect 1 minute fraction.On different chromatographic columns, use identical separation gradient to obtain to have time of staying corresponding to the synthetic peptide of the aminoacid sequence of immunostimulatory peptides.
Carrying out the MALDI-TOF mass spectrography, is HepG2 (HLA-A2 to analyze by AFP generation hepatocellular carcinoma cells
+) and Hep3B (HLA-A2
-) the HPLC fractionated peptide of acid eluting.Voyager-REACTION PRODUCTS (PerSeptive Biosystems, thunder Framingham city not, Massachusetts) Matrix Assisted Laser DesorptionIonization/Time-Of-Flight (MALDI-TOF) is used to obtain mass spectrum.This equipment uses the rustless steel target, and sample is deposited thereon and be dry.All spectrum carry out external calibration with insulin, cause exactness high in quality usually in ± 0.1%.Freeze dried HPLC fraction is resuspended in 70% acetonitrile that 10 μ l contain 0.1%TFA.With this material of 1 μ l and 1 μ l substrate alpha-cyano-4-hydroxycinnamic acid (Sigma is dissolved in 70%ACN/0.1%TFA with 10mg/ml) point sample together.Scanning m/z 500-7000 obtains spectrum.
The maldi analysis of HPLC fraction has determined that nearly all fraction all comprises the nearly different peptides of 20 kinds of mass range 700-1500Da, although usually there is advantage signal.In this complex mixture, identify the m/z value corresponding to hAFP
158-166Be 158-166 position residue, the hAFP of SEQ ID NO:2
325-334Be 325-334 position residue and the hAFP of SEQ ID NO:2
542-550It is the protonated molecule of calculating gained list isotropism ((M+H) of the 542-550 position residue of SEQ ID NO:2
+) the peak, hAFP
542-550, hAFP
158-166And hAFP
325-334Be present in the peptide set by HepG2 cell eluting.In a HPLC fraction, identify the peptide of m/z 975.6 from the set of HepG2 peptide.HAFP
542-550Calculating gained (M+H)
+Be 975.5, have corresponding to hAFP
542-550The time of staying of synthetic peptide of aminoacid sequence that is the 542-550 position residue of SEQ ID NO:2 is 21.2 minutes.In addition, in sample that only contains substrate and HPLC fraction 18-22, do not observe signal at m/z 975.5 ± 1 places from the Hep3B eluting.Similar, according to the prediction of standard peptide performance, found also that in suitable fraction m/z is corresponding to hAFP derived from HepG2
158-166Be 158-166 position residue and the hAFP of SEQ ID NO:2
325-334It is the calculating gained (M+H) of the 325-334 position residue of SEQ IDNO:2
+The peak.In the fraction of gathering by the peptide of Hep3B eluting, there are not these peaks.In a fraction, observe the peak of m/z 1152.2, illustrate to have hAFP
325-334It is the sodium adduct of the 325-334 position residue of SEQ ID NO:2.
Therefore, in the HPLC fractionated peptide set of positive HepG2 cell but not the negative Hep3B cell of HLA-A*0201 eluting, identified 3 kinds potential quality candidate in these 4 kinds of peptides by HLA-A*0201.In 3 kinds of peptides being identified, the peak is observed in the multiple scanning to point sample.In a fraction, observe broad peak, surpassed the range of allowable error of this physicochemical analysis at m/z 1020.9 places from the set of HepG2 peptide.Therefore, can not prove on the surface of HepG2 cell and have hAFP
137-145It is the 137-145 position residue of SEQ ID NO:2.
There to be the dominance peptide in order on immunology, confirming in these fraction, in the ELISPOT algoscopy, 1ml to be used for stinging once more the mice spleen cell of menstruating on time after pregnancy AdVhAFP/DC immunity from the various HPLC fraction of HepG2 or Hep3B cell.Observe 200-250 speckle/10 by the fraction that comprises the dominance peptide
6Individual cell is observed 100-130 speckle/10 by other fraction
6Individual cell is observed maximum 50 speckle/10 by the Hep3B fraction
6Individual cell.This has further supported the mass spectrum of dominance peptide to identify.
| Table 2 | |||||||
| Peptide 1 | The HPLC time of staying (min) of peptide 2 | Calculate gained (M+H) +，3 | Observed among the HepG2 (M+H) +，4 | Observed among the Hep3B (M+H) +，5 | Identify (M+H) +HPLC fraction # 6 | Immunological response fraction (IFN γ ELISPOT) 7 | |
| ??HepG2 | ??Hep3B | ||||||
| hAFP 542-550 | ??21.2 | ??975.5 | ??975.6 | Do not have | ??21 | ??20，21 | ??0 |
| hAFP 158-166 | ??28.9 | ??1204.6 | ??1204.9 | Do not have | ??28 | ??27，28， ??29 | ??0 |
| hAFP 137-145 | ??28.1 | ??1025.6 | Do not have | Do not have | ??- | ??27，28， ??29 | ??0 |
| hAFP 325-334 | ??27.7 | ??1130.6 | ??1130.1 | Do not have | ??28 | ??27，28， ??29 | ??0 |
Footnote: 1. peptide identifies; 2. the typical HPLC time of staying of peptide is synthesized in contrast; Prospective quality/
Charge measurement; 4. observed mass measured value in from the acid eluting peptide of HepG2; 5. observed mass measured value in from the acid eluting peptide of Hep3B; 6. in acid eluting peptide, observe from HepG2 hAFP peptide mass measured value fraction (minute); 7. the fraction that contains the splenocyte that can sting the initiation of menstruating on time after pregnancy AdVhAFP/DC peptide once more by IFN γ ELISPOT.
EXAMPLE Example 1: the method for preventing or treating hepatocarcinoma by administration for peptides
According to one embodiment of the invention, provide the method that is used to prevent or treat the hepatocarcinoma patient.This method comprises selects suitable patient, and the HLA-A*0201+ patient such as suffering from the positive hepatocarcinoma of AFP uses one or more peptides of the present invention to the patient then.The patient is used the peptide of sufficient dosage, and preferred repetitive administration repeatedly, thereby cause immunne response, and cause immunne response thus at hepatocarcinoma at AFP.
In a preferred embodiment, peptide is hAFP
137-145Be 137-145 position residue, the hAFP of SEQ ID NO:2
158-166Be 158-166 position residue, the hAFP of SEQ ID NO:2
325-334Be 325-334 position residue and the hAFP of SEQ ID NO:2
542-550It is the associating of the 542-550 position residue of SEQ ID NO:2.In another preferred embodiment, described one or more peptides have been applied 2-5 time.In an especially preferred embodiment, peptide has been used 3 times.In another preferred embodiment, described one or more peptides have been used 3 times with the interval in 2 weeks.
In a preferred embodiment, peptide is an intradermal administration, and to use the path also be suitable although those skilled in the art can understand other with reference to disclosure book.
In a preferred embodiment, each of one or more peptides is all used after the emulsifying in 0.5mlMontanide ISA-51.Therefore, when four kinds of peptides of associating, they are used after the emulsifying in adding up to 2ml Montanide ISA-51.Peptide after the emulsifying is divided into quarter, and every part is applied to different loci.In a preferred embodiment, these one or more peptides are to use with the dosage of every kind of about 50-2000 μ g.In a preferred embodiment, these one or more peptides are to use with the dosage of every kind of about 100-1000 μ g.In an especially preferred embodiment, these one or more peptides are to use with the dosage of every kind of about 500 μ g.
Method and composition of the present invention is used for the treatment of several the AFP positive/A2.1+ hepatocarcinoma patients.According to this method, with four kinds of peptide hAFP
137-145Be 137-145 position residue, the hAFP of SEQ ID NO:2
158-166Be 158-166 position residue, the hAFP of SEQ ID NO:2
325-334Be 325-334 position residue and the hAFP of SEQ ID NO:2
542-550Be every patient of 542-550 position residue immunity of SEQ ID NO:2.With peptide emulsifying in 0.5ml Montanide ISA-51, the associating back adds up to 2ml.Peptide after the emulsifying is divided into quarter, and every part is applied to different loci.Measuring periphery T cell by ELISPOT and tetramer algoscopy replys.These tests show that these four kinds of AFP derived peptide have immunogenicity in vivo, even also are like this among the high patient of AFP serum levels before immunity.
First patient (being called AFP-A1) suffers from AFP positive recurrence, unresectable/A2.1+ hepatocarcinoma.Three immunity are used to him in interval with 2 weeks, and each immunity all is to contain this four kinds of peptides, every kind 100 μ g in MontanideISA.Simultaneously, set up the outer PBMC culture of parallel body by blood before this first patient's the immunity, and with every kind of peptide repetition pulse.According to the inspection of ELISPOT, all there is clearly external identification in all peptides in the 28th day culture; According to the inspection of tetramer algoscopy, find the clearly external identification of 2 kinds of existence, i.e. hAFP to 3 kinds of peptides
137-145Be 137-145 position residue and the hAFP of SEQ ID NO:2
325-334The 325-334 position residue that is SEQ ID NO:2 has, and to hAFP
158-166The 158-166 position residue that is SEQ ID NO:2 does not but have.Tetramer algoscopy can not be assessed AFP
542Whether the existence of specific T-cells, because AFP
542The equipment that peptide tetramer can not be produced these reagent folds.Reply indication in the body to hAFP
137-145Be 137-145 position residue, the hAFP of SEQ ID NO:2
158-166Be 158-166 position residue and the hAFP of SEQ ID NO:2
542-550Be the clear identification of the 542-550 position residue of SEQ ID NO:2, and trending towards discerning hAFP for the second time and after the immunity for the third time
325-334It is the 325-334 position residue of SEQ ID NO:2.
Second patient (being called AFP-A2) has 70 years old white man male that ethanol brings out the liver cirrhosis medical history, and its hepatitis B and hepatitis C all are negative.He presents swelling, and finds the 8cm lump at the liver lobus sinister.His AFP level was 10 at that time, 400ng/ml.Liver biopsy has disclosed the hepatocarcinoma of abundant differentiation at the sclerosis liver.He experimentizing property antifungal FV-462 is attempted, but PD and generation ototoxicity.Check back 7 months, use, he is used four kinds of peptide hAFP according to the solution of the present invention
137-145Be 137-145 position residue, the hAFP of SEQ ID NO:2
158-166Be 158-166 position residue, the hAFP of SEQ ID NO:2
325-334Be 325-334 position residue and the hAFP of SEQ ID NO:2
542-550It is the associating of the 542-550 position residue of SEQ ID NO:2.He has accepted twice immunity, at interval two weeks.The tetramer after twice immunity and ELISPOT data show detect the t cell response at all 4 kinds of AFP peptide epitopes.
Therefore, these result's indications, this method has caused the detected immunne response of being undertaken by the AFP specific T-cells in the hepatocarcinoma patient who is in a bad way.Embodiment 2: by using the method for preventing or treating hepatocarcinoma through the dendritic cell of peptide pulse of the present invention
According to one embodiment of the invention, provide the method that is used to prevent or treat the hepatocarcinoma patient.This method comprises selects suitable patient, such as the HLA-A*0201+ patient who suffers from the positive hepatocarcinoma of AFP, then the patient is used the dendritic cell of one or more peptide pulses of the present invention of process.The patient is used the dendritic cell of sufficient dosage, and preferred repetitive administration repeatedly, thereby cause immunne response, and cause immunne response thus at hepatocarcinoma at AFP.
In a preferred embodiment, dendritic cell are to use hAFP
137-145Be 137-145 position residue, the hAFP of SEQ ID NO:2
158-166Be 158-166 position residue, the hAFP of SEQ ID NO:2
325-334Be 325-334 position residue and the hAFP of SEQ ID NO:2
542-550It is the associating pulse of the 542-550 position residue of SEQID NO:2.In another preferred embodiment, dendritic cell have been used 2-5 time.In an especially preferred embodiment, dendritic cell have been used 3 times.In another preferred embodiment, dendritic cell were used 3 times at interval in 2 weeks.
In a preferred embodiment, dendritic cell are intradermal administration, and to use the path also be suitable although those skilled in the art can understand other with reference to disclosure book.In one embodiment, dendritic cell are with about 1 * 10
5With 1 * 10
8Between dosage use.In another preferred embodiment, dendritic cell are with about 1 * 10
6With 1 * 10
7Between dosage use.In an especially preferred embodiment, dendritic cell are with about 5 * 10
6Dosage use.
According to the technology that those skilled in the art will know that, prepare dendritic cell by in tissue culture, being exposed to the adherent autologous peripheral blood mononuclear cell that granulocyte-macrophage colony stimutaing factor (GM-CSF) and interleukin-4 (IL-4) reached for 1 week.Adopt the cell separation mononuclear cell of method (leukapheresis) by Ficoll-Hypaque is centrifugal by deriving from a leukocyte list, and frozen in liquid nitrogen until being used to produce dendritic cell.The mononuclear cell that will thaw cleans once with saline, and with 2-4 * 10
7Individual cell/25cm
2The density of Costar bottle is with 2.5-5 * 10
6The concentration coating of individual survivaling cell/ml culture medium (the hot deactivation autoserum+gentamycin of RPMI 1640+5%).Make it in 37 ℃ after adherent 2 hours, clean by saline and remove non-adherent cell.Attached cell was cultivated 7 days when having rhGM-CSF (800U/ml) and rhIL-4 (500U/ml) in complete medium.Clinical grade GM-CSF is provided by Immunex, and IL-4 is provided by Schering-Plough.
The patient is carried out a leukocyte list adopt method (leukapheresis) to obtain at least 2 * 10
9Individual PBL, and cold preservation is in 70%RPMI 1640,20% autoserum and 10%DMSO.Respectively getting the five equilibrium lyolysis at the-7,7 and 21 days that study freezes.In immunity for the first time that day, when adopting method (leukapheresis), the leukocyte list takes in preparation autoserous blood (60ml) carrying out, and this enough is used for all cell culture.According to the technology that those skilled in the art will know that, preparation and the purification AFP of the present invention immunodominant peptide of deriving.
Following immune patients.In immunity that day, the results dendritic cell clean once with Sterile Saline, and with debita spissitudo (such as 10
6) be resuspended in each of 1ml serum-free RPMI 1640 and four kinds of immunodominant peptides of 50mg/ml.The shortest insulation is after 1 hour, precipitate A FP peptide/DC, and clean 3 times with Sterile Saline.In trypan blue, carry out cell counting, and cell is resuspended in the 0.1ml Sterile Saline, be used for intradermal injection.
Before capacity is used, the experimenter will accept skin test, promptly contain 1% dosage in the 0.1ml saline.After 30 minute observation period, they will accept the dendritic cell through the pulse of AFP peptide of capacity in the 0.1ml saline, i.e. both sides or groin intradermal injection up and down under axillary fossa.Immunity back was to patient monitoring 2 hours.Preferably, the patient accepts the pretreatment of 50mg diphenhydramine (diphenhydramine) and 650mg acetaminophen (Tylenol), and the two is all oral.Embodiment 3: by using the method for preventing or treating hepatocarcinoma through the dendritic cell of remarkable AFP adenovirus transduction
According to one embodiment of the invention, provide the method that is used to prevent or treat the hepatocarcinoma patient.This method comprises selects suitable patient, such as the HLA-A*0201+ patient who suffers from the positive hepatocarcinoma of AFP, then the patient is used the dendritic cell of transduceing through remarkable AFP adenovirus.The patient is used the dendritic cell of sufficient dosage, and preferred repetitive administration repeatedly, thereby cause immunne response, and cause immunne response thus at hepatocarcinoma at AFP.
In another preferred embodiment, dendritic cell have been used 2-5 time.In an especially preferred embodiment, dendritic cell have been used 3 times.In another preferred embodiment, dendritic cell have been used 3 times with the interval in 2 weeks.
In a preferred embodiment, the dendritic cell intradermal administration in axillary fossa down or the groin both sides, to use the path also be suitable although those skilled in the art can understand other with reference to disclosure book.In one embodiment, dendritic cell are with about 1 * 10
5With 1 * 10
8Between dosage use.In another preferred embodiment, dendritic cell are with about 1 * 10
6With 1 * 10
7Between dosage use.In an especially preferred embodiment, dendritic cell are with about 5 * 10
6Dosage use.
Adopt method (leukapheresis) product separating monocytic cell by Ficoll-Hypaque is centrifugal by the leukocyte list, and be stored in the 10%DMSO/20% autoserum.Inoculate the last week at DC,, clean once with PBS with cell thawing, and with 2-4 * 10
7Individual cell/25cm
2The density of Costar bottle is with 2.5-5 * 10
6The concentration coating of individual survivaling cell/ml culture medium (the hot deactivation autoserum of RPMI 1640+5%).Make it to clean gentleness by PBS and remove non-adherent cell in 37 ℃ after adherent 2 hours.Attached cell was cultivated 7 days when having rhGM-CSF (800U/ml) and rhIL-4 (500U/ml) in complete medium.Clinical grade GM-CSF and IL-4 are provided by Schering-Plough.
AdVhAFP is the replication defect type 5 type adenovirus vectors of E1 disappearance, and wherein people AFP cDNA is by cmv enhancer/promoters driven.The virus titer of every batch of viral end-product according to genomic DNA quantitatively and infection titer provide.The virion product is lower than 100: 1 to the ratio of biologic activity virus and is considered to acceptable.
Following immune patients.In immune that day, the results dendritic cell clean once with Sterile Saline, and with 10
6-10
7Concentration be resuspended in 1ml 2% autoserum-RPMI 1640 and 10
9-10
10Pfu/ml AdVhAFP (infection multiplicity=1000: 1).After 2 hours, AdVhAFP/DC is resuspended in the adenovirus vector that RPMI 1640-5% autoserum is not adsorbed with deactivation in 37 ℃ of insulations, then precipitation and clean 3 times with Sterile Saline.In trypan blue, carry out cell counting, and with proper number (according to patient group's difference, 10
5With 10
8Between) cell be resuspended in Sterile Saline, be used for intradermal injection.
The patient is used DC through AdVhAFP transduction, promptly under axillary fossa or the common saline of inguinal both sides intradermal injection 0.1ml.Immunity back was to patient monitoring 2 hours.Preferably, the patient accepts the pretreatment of 50mg diphenhydramine (diphenhydramine) and 650mg acetaminophen (Tylenol), and the two is all oral.
Though passed through with reference to the quite detailed discussion of some preferred embodiment the present invention, other embodiment also is possible.Therefore, the scope of claims should not be limited to the description of the contained preferred embodiment of disclosure book.
Sequence table＜110〉The Regents of the University of California
Economou，James
Butterfield，Lisa
Ribas Bruguera, Antoni＜120〉be used for the treatment of method and composition＜130 of hepatocarcinoma〉11969-5PCT＜140〉to be assigned＜141〉2001-02-12＜150〉US 60/181,966＜151〉2000-02-10＜150〉US 09/660,252＜151〉2000-09-12＜150〉US 09/662,505＜151〉2000-09-14＜160〉2＜170〉PatentIn version 3.0＜210〉1＜211〉2032＜212〉DNA＜213〉people (Homo sapiens)＜220〉＜221〉CDS＜222〉(48) .. (1874)＜400〉1tccatattgt gcttccacca ctgccaataa caaaataact agcaacc atg aag tgg 56
Met?Lys?Trp
1gtg?gaa?tca?att?ttt?tta?att?ttc?cta?cta?aat?ttt?act?gaa?tcc?aga????104Val?Glu?Ser?Ile?Phe?Leu?Ile?Phe?Leu?Leu?Asn?Phe?Thr?Glu?Ser?Arg
5???????????????????10??????????????????15aca?ctg?cat?aga?aat?gaa?tat?gga?ata?gct?tcc?ata?ttg?gat?tct?tac???152Thr?Leu?His?Arg?Asn?Glu?Tyr?Gly?Ile?Ala?Ser?Ile?Leu?Asp?Ser?Tyr20??????????????????25??????????????????30??????????????????35caa?tgt?act?gca?gag?ata?agt?tta?gct?gac?ctg?gct?acc?ata?ttt?ttt???200Gln?Cys?Thr?Ala?Glu?Ile?Ser?Leu?Ala?Asp?Leu?Ala?Thr?Ile?Phe?Phe
40??????????????????45??????????????????50gcc?cag?ttt?gtt?caa?gaa?gcc?act?tac?aag?gaa?gta?agc?aaa?atg?gtg???248Ala?Gln?Phe?Val?Gln?Glu?Ala?Thr?Tyr?Lys?Glu?Val?Ser?Lys?Met?Val
55??????????????????60??????????????????65aaa?gat?gca?ttg?act?gca?att?gag?aaa?ccc?act?gga?gat?gaa?cag?tct???296Lys?Asp?Ala?Leu?Thr?Ala?Ile?Glu?Lys?Pro?Thr?Gly?Asp?Glu?Gln?Ser
70??????????????????75??????????????????80tca?ggg?tgt?tta?gaa?aac?cag?cta?cct?gcc?ttt?ctg?gaa?gaa?ctt?tgc???344Ser?Gly?Cys?Leu?Glu?Asn?Gln?Leu?Pro?Ala?Phe?Leu?Glu?Glu?Leu?Cys
85??????????????????90??????????????????95cat?gag?aaa?gaa?att?ttg?gag?aag?tac?gga?cat?tca?gac?tgc?tgc?agc???392His?Glu?Lys?Glu?Ile?Leu?Glu?Lys?Tyr?Gly?His?Ser?Asp?Cys?Cys?Ser100?????????????????105?????????????????110?????????????????115caa?agt?gaa?gag?gga?aga?cat?aac?tgt?ttt?ctt?gca?cac?aaa?aag?ccc???440Gln?Ser?Glu?Glu?Gly?Arg?His?Asn?Cys?Phe?Leu?Ala?His?Lys?Lys?Pro
120?????????????????125?????????????????130act?cca?gca?tcg?atc?cca?ctt?ttc?caa?gtt?cca?gaa?cct?gtc?aca?agc???488Thr?Pro?Ala?Ser?Ile?Pro?Leu?Phe?Gln?Val?Pro?Glu?Pro?Val?Thr?Ser
135?????????????????140?????????????????145tgt?gaa?gca?tat?gaa?gaa?gac?agg?gag?aca?ttc?atg?aac?aaa?ttc?att???536Cys?Glu?Ala?Tyr?Glu?Glu?Asp?Arg?Glu?Thr?Phe?Met?Asn?Lys?Phe?Ile
150?????????????????155?????????????????160tat?gag?ata?gca?aga?agg?cat?ccc?ttc?ctg?tat?gca?cct?aca?att?ctt???584Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Phe?Leu?Tyr?Ala?Pro?Thr?Ile?Leu
165?????????????????170?????????????????175ctt?tgg?gct?gct?cgc?tat?gac?aaa?ata?att?cca?tct?tgc?tgc?aaa?gct???632Leu?Trp?Ala?Ala?Arg?Tyr?Asp?Lys?Ile?Ile?Pro?Ser?Cys?Cys?Lys?Ala180?????????????????185?????????????????190?????????????????195gaa?aat?gca?gtt?gaa?tgc?ttc?caa?aca?aag?gca?gca?aca?gtt?aca?aaa????680Glu?Asn?Ala?Val?Glu?Cys?Phe?Gln?Thr?Lys?Ala?Ala?Thr?Val?Thr?Lys
200?????????????????205?????????????????210gaa?tta?aga?gaa?agc?agc?ttg?tta?aat?caa?cat?gca?tgt?gca?gta?atg????728Glu?Leu?Arg?Glu?Ser?Ser?Leu?Leu?Asn?Gln?His?Ala?Cys?Ala?Val?Met
215?????????????????220?????????????????225aaa?aat?ttt?ggg?acc?cga?act?ttc?caa?gcc?ata?act?gtt?act?aaa?ctg????776Lys?Asn?Phe?Gly?Thr?Arg?Thr?Phe?Gln?Ala?Ile?Thr?Val?Thr?Lys?Leu
230?????????????????235?????????????????240agt?cag?aag?ttt?acc?aaa?gtt?aat?ttt?act?gaa?atc?cag?aaa?cta?gtc????824Ser?Gln?Lys?Phe?Thr?Lys?Val?Asn?Phe?Thr?Glu?Ile?Gln?Lys?Leu?Val
245?????????????????250?????????????????255ctg?gat?gtg?gcc?cat?gta?cat?gag?cac?tgt?tgc?aga?gga?gat?gtg?ctg????872Leu?Asp?Val?Ala?His?Val?His?Glu?His?Cys?Cys?Arg?Gly?Asp?Val?Leu260?????????????????265?????????????????270?????????????????275gat?tgt?ctg?cag?gat?ggg?gaa?aaa?atc?atg?tcc?tac?ata?tgt?tct?caa????920Asp?Cys?Leu?Gln?Asp?Gly?Glu?Lys?Ile?Met?Ser?Tyr?Ile?Cys?Ser?Gln
280?????????????????285?????????????????290caa?gac?act?ctg?tca?aac?aaa?ata?aca?gaa?tgc?tgc?aaa?ctg?acc?acg????968Gln?Asp?Thr?Leu?Ser?Asn?Lys?Ile?Thr?Glu?Cys?Cys?Lys?Leu?Thr?Thr
295?????????????????300?????????????????305ctg?gaa?cgt?ggt?caa?tgt?ata?att?cat?gca?gaa?aat?gat?gaa?aaa?cct????1016Leu?Glu?Arg?Gly?Gln?Cys?Ile?Ile?His?Ala?Glu?Asn?Asp?Glu?Lys?Pro
310?????????????????315?????????????????320gaa?ggt?cta?tct?cca?aat?cta?aac?agg?ttt?tta?gga?gat?aga?gat?ttt????1064Glu?Gly?Leu?Ser?Pro?Asn?Leu?Asn?Arg?Phe?Leu?Gly?Asp?Arg?Asp?Phe
325?????????????????330?????????????????335aac?caa?ttt?tct?tca?ggg?gaa?aaa?aat?atc?ttc?ttg?gca?agt?ttt?gtt????1112Asn?Gln?Phe?Ser?Ser?Gly?Glu?Lys?Asn?Ile?Phe?Lau?Ala?Ser?Phe?Val340?????????????????345?????????????????350?????????????????355cat?gaa?tat?tca?aga?aga?cat?cct?cag?ctt?gct?gtc?tca?gta?att?cta???1160His?Glu?Tyr?Ser?Arg?Arg?His?Pro?Gln?Leu?Ala?Val?Ser?Val?Ile?Leu
360?????????????????365?????????????????370aga?gtt?gct?aaa?gga?tac?cag?gag?tta?ttg?gag?aag?tgt?ttc?cag?act???1208Arg?Val?Ala?Lys?Gly?Tyr?Gln?Glu?Leu?Leu?Glu?Lys?Cys?Phe?Gln?Thr
375?????????????????380?????????????????385gaa?aac?cct?ctt?gaa?tgc?caa?gat?aaa?gga?gaa?gaa?gaa?tta?cag?aaa???1256Glu?Asn?Pro?Leu?Glu?Cys?Gln?Asp?Lys?Gly?Glu?Glu?Glu?Leu?Gln?Lys
390?????????????????395?????????????????400tac?atc?cag?gag?agc?caa?gca?ttg?gca?aag?cga?agc?tgc?ggc?ctc?ttc???1304Tyr?Ile?Gln?Glu?Ser?Gln?Ala?Leu?Ala?Lys?Arg?Ser?Cys?Gly?Leu?Phe
405?????????????????410?????????????????415cag?aaa?cta?gga?gaa?tat?tac?tta?caa?aat?gcg?ttt?ctc?gtt?gct?tac???1352Gln?Lys?Leu?Gly?Glu?Tyr?Tyr?Leu?Gln?Asn?Ala?Phe?Leu?Val?Ala?Tyr420?????????????????425?????????????????430?????????????????435aca?aag?aaa?gcc?ccc?cag?ctg?acc?tcg?tcg?gag?ctg?atg?gcc?atc?acc???1400Thr?Lys?Lys?Ala?Pro?Gln?Leu?Thr?Ser?Ser?Glu?Leu?Met?Ala?Ile?Thr
440?????????????????445?????????????????450aga?aaa?atg?gca?gcc?aca?gca?gcc?act?tgt?tgc?caa?ctc?agt?gag?gac???1448Arg?Lys?Met?Ala?Ala?Thr?Ala?Ala?Thr?Cys?Cys?Gln?Leu?Ser?Glu?Asp
455?????????????????460?????????????????465aaa?cta?ttg?gcc?tgt?ggc?gag?gga?gcg?gct?gac?att?att?atc?gga?cac???1496Lys?Leu?Leu?Ala?Cys?Gly?Glu?Gly?Ala?Ala?Asp?Ile?Ile?Ile?Gly?His
470?????????????????475?????????????????480tta?tgt?atc?aga?cat?gaa?atg?act?cca?gta?aac?cct?ggt?gtt?ggc?cag???1544Leu?Cys?Ile?Arg?His?Glu?Met?Thr?Pro?Val?Asn?Pro?Gly?Val?Gly?Gln
485?????????????????490?????????????????495tgc?tgc?act?tct?tca?tat?gcc?aac?agg?agg?cca?tgc?ttc?agc?agc?ttg???1592Cys?Cys?Thr?Ser?Ser?Tyr?Ala?Asn?Arg?Arg?Pro?Cys?Phe?Ser?Ser?Leu500?????????????????505?????????????????510?????????????????515gtg?gtg?gat?gaa?aca?tat?gtc?cct?cct?gca?ttc?tct?gat?gac?aag?ttc???1640Val?Val?Asp?Glu?Thr?Tyr?Val?Pro?Pro?Ala?Phe?Ser?Asp?Asp?Lys?Phe
520?????????????????525?????????????????530att?ttc?cat?aag?gat?ctg?tgc?caa?gct?cag?ggt?gta?gcg?ctg?caa?acg???1688Ile?Phe?His?Lys?Asp?Leu?Cys?Gln?Ala?Gln?Gly?Val?Ala?Leu?Gln?Thr
535?????????????????540?????????????????545atg?aag?caa?gag?ttt?ctc?att?aac?ctt?gtg?aag?caa?aag?cca?caa?ata???1736Met?Lys?Gln?Glu?Phe?Leu?Ile?Asn?Leu?Val?Lys?Gln?Lys?Pro?Gln?Ile
550?????????????????555?????????????????560aca?gag?gaa?caa?ctt?gag?gct?gtc?att?gca?gat?ttc?tca?ggc?ctg?ttg???1784Thr?Glu?Glu?Gln?Leu?Glu?Ala?Val?Ile?Ala?Asp?Phe?Ser?Gly?Leu?Leu
565?????????????????570?????????????????575gag?aaa?tgc?tgc?caa?ggc?cag?gaa?cag?gaa?gtc?tgc?ttt?gct?gaa?gag???1832Glu?Lys?Cys?Cys?Gln?Gly?Gln?Glu?Gln?Glu?Val?Cys?Phe?Ala?Glu?Glu580?????????????????585?????????????????590?????????????????595gga?caa?aaa?ctg?att?tca?aaa?act?cgt?gct?gct?ttg?gga?gtt???????????1874Gly?Gln?Lys?Leu?Ile?Ser?Lys?Thr?Arg?Ala?Ala?Leu?Gly?Val
600 605taaattactt caggggaaga gaagacaaaa cgagtctttc attcggtgtg aacttttctc 1934tttaatttta actgatttaa cactttttgt gaattaatga aatgataaag acttttatgt 1994gagatttcct tatcacagaa ataaaatatc tccaaatg, 2032＜210〉2＜211〉609＜212〉PRT＜213〉people (Homo sapiens)＜400〉2Met Lys Trp Val Glu Ser Ile Phe Leu Ile Phe Leu Leu Asn Phe Thr1,5 10 15Glu Ser Arg Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu
20??????????????????25??????????????????30Asp?Ser?Tyr?Gln?Cys?Thr?Ala?Glu?Ile?Ser?Leu?Ala?Asp?Leu?Ala?Thr
35??????????????????40??????????????????45Ile?Phe?Phe?Ala?Gln?Phe?Val?Gln?Glu?Ala?Thr?Tyr?Lys?Glu?Val?Ser
50??????????????????55??????????????????60Lys?Met?Val?Lys?Asp?Ala?Leu?Thr?Ala?Ile?Glu?Lys?Pro?Thr?Gly?Asp65??????????????????70??????????????????75??????????????????80Glu?Gln?Ser?Ser?Gly?Cys?Leu?Glu?Asn?Gln?Leu?Pro?Ala?Phe?Leu?Glu
85??????????????????90??????????????????95Glu?Leu?Cys?His?Glu?Lys?Glu?Ile?Leu?Glu?Lys?Tyr?Gly?His?Ser?Asp
100?????????????????105?????????????????110Cys?Cys?Ser?Gln?Ser?Glu?Glu?Gly?Arg?His?Asn?Cys?Phe?Leu?Ala?His
115?????????????????120?????????????????125Lys?Lys?Pro?Thr?Pro?Ala?Ser?Ile?Pro?Leu?Phe?Gln?Val?Pro?Glu?Pro
130?????????????????135?????????????????140Val?Thr?Ser?Cys?Glu?Ala?Tyr?Glu?Glu?Asp?Arg?Glu?Thr?Phe?Met?Asn145?????????????????150?????????????????155?????????????????160Lys?Phe?Ile?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Phe?Leu?Tyr?Ala?Pro
165?????????????????170?????????????????175Thr?Ile?Leu?Leu?Trp?Ala?Ala?Arg?Tyr?Asp?Lys?Ile?Ile?Pro?Ser?Cys
180?????????????????185?????????????????190Cys?Lys?Ala?Glu?Asn?Ala?Val?Glu?Cys?Phe?Gln?Thr?Lys?Ala?Ala?Thr
195?????????????????200?????????????????205Val?Thr?Lys?Glu?Leu?Arg?Glu?Ser?Ser?Leu?Leu?Asn?Gln?His?Ala?Cys
210?????????????????215?????????????????220Ala?Val?Met?Lys?Asn?Phe?Gly?Thr?Arg?Thr?Phe?Gln?Ala?Ile?Thr?Val225?????????????????230?????????????????235?????????????????240Thr?Lys?Leu?Ser?Gln?Lys?Phe?Thr?Lys?Val?Asn?Phe?Thr?Glu?Ile?Gln
245?????????????????250?????????????????255Lys?Leu?Val?Leu?Asp?Val?Ala?His?Val?His?Glu?His?Cys?Cys?Arg?Gly
260?????????????????265?????????????????270Asp?Val?Leu?Asp?Cys?Leu?Gln?Asp?Gly?Glu?Lys?Ile?Met?Ser?Tyr?Ile
275?????????????????280?????????????????285Cys?Ser?Gln?Gln?Asp?Thr?Leu?Ser?Asn?Lys?Ile?Thr?Glu?Cys?Cys?Lys
290?????????????????295?????????????????300Leu?Thr?Thr?Leu?Glu?Arg?Gly?Gln?Cys?Ile?Ile?His?Ala?Glu?ASn?Asp305?????????????????310?????????????????315?????????????????320Glu?Lys?Pro?Glu?Gly?Leu?Ser?Pro?Asn?Leu?Asn?Arg?Phe?Leu?Gly?Asp
325?????????????????330?????????????????335Arg?Asp?Phe?Asn?Gln?Phe?Ser?Ser?Gly?Glu?Lys?Asn?Ile?Phe?Leu?Ala
340?????????????????345?????????????????350Ser?Phe?Val?His?Glu?Tyr?Ser?Arg?Arg?His?Pro?Gln?Leu?Ala?Val?Ser
355?????????????????360?????????????????365Val?Ile?Leu?Arg?Val?Ala?Lys?Gly?Tyr?Gln?Glu?Leu?Leu?Glu?Lys?Cys
370?????????????????375?????????????????380Phe?Gln?Thr?Glu?Asn?Pro?Leu?Glu?Cys?Gln?Asp?Lys?Gly?Glu?Glu?Glu385?????????????????390?????????????????395?????????????????400Leu?Gln?Lys?Tyr?Ile?Gln?Glu?Ser?Gln?Ala?Leu?Ala?Lys?Arg?Ser?Cys
405?????????????????410?????????????????415Gly?Leu?Phe?Gln?Lys?Leu?Gly?Glu?Tyr?Tyr?Leu?Gln?Asn?Ala?Phe?Leu
420?????????????????425?????????????????430Val?Ala?Tyr?Thr?Lys?Lys?Ala?Pro?Gln?Leu?Thr?Ser?Ser?Glu?Leu?Met
435?????????????????440?????????????????445Ala?Ile?Thr?Arg?Lys?Met?Ala?Ala?Thr?Ala?Ala?Thr?Cys?Cys?Gln?Leu
450?????????????????455?????????????????460Ser?Glu?Asp?Lys?Leu?Leu?Ala?Cys?Gly?Glu?Gly?Ala?Ala?Asp?Ile?Ile465?????????????????470?????????????????475?????????????????480Ile?Gly?His?Leu?Cys?Ile?Arg?His?Glu?Met?Thr?Pro?Val?Asn?Pro?Gly
485?????????????????490?????????????????495Val?Gly?Gln?Cys?Cys?Thr?Ser?Ser?Tyr?Ala?Asn?Arg?Arg?Pro?Cys?Phe
500?????????????????505?????????????????510Ser?Ser?Leu?Val?Val?Asp?Glu?Thr?Tyr?Val?Pro?Pro?Ala?Phe?Ser?Asp
515?????????????????520?????????????????525Asp?Lys?Phe?Ile?Phe?His?Lys?Asp?Leu?Cys?Gln?Ala?Gln?Gly?Val?Ala
530?????????????????535?????????????????540Leu?Gln?Thr?Met?Lys?Gln?Glu?Phe?Leu?Ile?Asn?Leu?Val?Lys?Gln?Lys545?????????????????550?????????????????555?????????????????560Pro?Gln?Ile?Thr?Glu?Glu?Gln?Leu?Glu?Ala?Val?Ile?Ala?Asp?Phe?Ser
565?????????????????570?????????????????575Gly?Leu?Leu?Glu?Lys?Cys?Cys?Gln?Gly?Gln?Glu?Gln?Glu?Val?Cys?Phe
580?????????????????585?????????????????590Ala?Glu?Glu?Gly?Gln?Lys?Leu?Ile?Ser?Lys?Thr?Arg?Ala?Ala?Leu?Gly
595?????????????????600?????????????????605Val
Claims (38)
1. be used in mammal prevention or treatment method for cancer, wherein cancerous cell is at least a portion of cell surface expression alpha-fetoprotein molecule, this method is included in the mammal step that causes at the immunne response of the partial amino-acid series at least of alpha-fetoprotein molecule, and wherein immunne response comprises the alpha-fetoprotein peptide specific T lymphocyte of activation at cancerous cell.
2. the process of claim 1 wherein that alpha-fetoprotein peptide specific T lymphocyte is a cytotoxic T lymphocyte.
3. the process of claim 1 wherein that the alpha-fetoprotein molecule is SEQ ID NO:2.
4. the process of claim 1 wherein that the part of described alpha-fetoprotein molecule is selected from down group: the 325-334 position residue of the 137-145 position residue of SEQ ID NO:2, the 158-166 position residue of SEQ ID NO:2, SEQ ID NO:2, the 542-550 position residue of SEQ ID NO:2, and above-mentioned every associating.
5. the process of claim 1 wherein that cancer is a hepatocarcinoma.
6. the process of claim 1 wherein that mammal is the people.
7. the process of claim 1 wherein that the step that causes immunne response comprises that to one or more compositionss of administration, wherein said compositions comprises the peptide of the partial amino-acid series at least with alpha-fetoprotein molecule.
8. the method for claim 7, wherein peptide is selected from down group: the 325-334 position residue of the 137-145 position residue of SEQ ID NO:2, the 158-166 position residue of SEQ ID NO:2, SEQ ID NO:2, the 542-550 position residue of SEQ ID NO:2, and above-mentioned every associating.
9. the method for claim 7, wherein peptide is selected from down group: the 1-9 position residue of SEQ ID NO:2, the 178-186 position residue of SEQ ID NO:2, the 218-226 position residue of SEQ ID NO:2, the 235-243 position residue of SEQ ID NO:2, the 306-315 position residue of SEQ ID NO:2, the 485-493 position residue of SEQ ID NO:2, the 492-500 position residue of SEQ ID NO:2, the 507-516 position residue of SEQ ID NO:2, the 547-556 position residue of SEQ ID NO:2, the 555-563 position residue of SEQ ID NO:2, and above-mentioned every associating.
10. the method for claim 1, the step that wherein causes immunne response comprises that to one or more compositionss of administration wherein said compositions comprises the dendritic cell through one or more peptide pulses of at least a portion that constitutes aminoacid sequence SEQID NO:2.
11. the process of claim 1 wherein that the step that causes immunne response comprises one or more compositionss of administration, wherein said compositions comprises the dendritic cell through the recombinant adenoviral vector transduction of coding alpha-fetoprotein.
12. be used for method in people's prevention or treatment hepatocarcinoma, wherein cancerous cell is at least a portion of cell surface expression alpha-fetoprotein molecule, thereby this method comprises by the people being used one or more peptides of being selected from down group and activates step at the alpha-fetoprotein cytotoxic T lymphocyte of cancerous cell at least a portion of aminoacid sequence SEQ ID NO:2: the 137-145 position residue of SEQ ID NO:2, the 325-334 position residue of SEQ ID NO:2, and the 137-145 position residue of SEQ IDNO:2, the 158-166 position residue of SEQ ID NO:2, the 325-334 position residue of SEQID NO:2, with at least two associating in the 542-550 position residue of SEQ ID NO:2.
13. be used for method in people's prevention or treatment hepatocarcinoma, wherein cancerous cell is at least a portion of cell surface expression alpha-fetoprotein molecule, thereby this method comprises by the people being used one or more peptides of being selected from down group and activates step at the alpha-fetoprotein cytotoxic T lymphocyte of cancerous cell at least a portion of aminoacid sequence SEQ ID NO:2: the 158-166 position residue of SEQ ID NO:2 and the 542-550 position residue of SEQ ID NO:2.
14. be used for method in people's prevention or treatment hepatocarcinoma, wherein cancerous cell is at least a portion of cell surface expression alpha-fetoprotein molecule, thereby this method comprises by the people being used one or more peptides of being selected from down group and activates step at the alpha-fetoprotein cytotoxic T lymphocyte of cancerous cell at least a portion of aminoacid sequence SEQ ID NO:2: the 1-9 position residue of SEQ ID NO:2, the 178-186 position residue of SEQ ID NO:2, the 218-226 position residue of SEQ ID NO:2, the 235-243 position residue of SEQ ID NO:2, the 306-315 position residue of SEQ ID NO:2, the 485-493 position residue of SEQ ID NO:2, the 492-500 position residue of SEQ ID NO:2, the 507-516 position residue of SEQ ID NO:2, the 547-556 position residue of SEQ ID NO:2,555-563 position residue with SEQ ID NO:2.
15. be used for method in people's prevention or treatment hepatocarcinoma, wherein cancerous cell is at least a portion of cell surface expression alpha-fetoprotein molecule, thereby this method comprises by the people being used one or more compositionss and activates step at the alpha-fetoprotein cytotoxic T lymphocyte of cancerous cell at least a portion of aminoacid sequence SEQ ID NO:2 that wherein said compositions comprises the dendritic cell through one or more peptide pulses of at least a portion that constitutes aminoacid sequence SEQ ID NO:2.
16. the method for claim 15, one or more peptides that wherein are used for the pulse dendritic cell are selected from down group: the 137-145 position residue of SEQ ID NO:2, the 158-166 position residue of SEQ ID NO:2, the 325-334 position residue of SEQ ID NO:2 and the 542-550 position residue of SEQ ID NO:2.
17. be used for method in people's prevention or treatment hepatocarcinoma, wherein cancerous cell is at least a portion of cell surface expression alpha-fetoprotein molecule, thereby this method comprises by the people being used one or more compositionss and activates step at the alpha-fetoprotein cytotoxic T lymphocyte of cancerous cell at least a portion of aminoacid sequence SEQ ID NO:2 that wherein said compositions comprises the dendritic cell through the recombinant adenoviral vector transduction of coding alpha-fetoprotein.
18. can be used for preventing or treating the isolated peptides of cancer, it is selected from down group: the 137-145 position residue of SEQ ID NO:2 and the 325-334 position residue of SEQ ID NO:2.
19. be used to prevent or treat the compositions of cancer, it comprises one or more peptides of claim 18, their amount is enough to cause the immunne response at alpha-fetoprotein in mammal.
20. the compositions of claim 19 wherein also comprises adjuvant.
21. be used for comprising the step of the people being used the peptide of claim 18 in people's prevention or treatment method for cancer.
22. be used for comprising the step of the people being used the compositions of claim 19 in people's prevention or treatment method for cancer.
23. be used for comprising the step of the people being used the compositions of claim 20 in people's prevention or treatment method for cancer.
24. be used to prevent or treat the means of cancer, wherein comprise one or more peptides that are selected from down group: the 137-145 position residue of SEQ ID NO:2, the 158-166 position residue of SEQ ID NO:2, the 325-334 position residue of SEQ ID NO:2 and the 542-550 position residue of SEQ ID NO:2.
25. can be used for preventing or treating the isolated peptides of cancer, wherein have sequence according to the 542-550 position residue of the 158-166 position residue of SEQ ID NO:2 or SEQ ID NO:2.
26. be used to prevent or treat the compositions of cancer, wherein comprise one or more peptides of claim 25, their amount is enough to cause the immunne response at alpha-fetoprotein in mammal.
27. the compositions of claim 26 wherein also comprises adjuvant.
28. be used for comprising the step of the people being used the peptide of claim 25 in people's prevention or treatment method for cancer.
29. be used for comprising the step of the people being used the compositions of claim 26 in people's prevention or treatment method for cancer.
30. be used for comprising the step of the people being used the compositions of claim 27 in people's prevention or treatment method for cancer.
31. be used to prevent or treat the means of cancer, wherein comprise the compositions of claim 19.
32. the means of claim 31 wherein also comprise adjuvant.
33. be used for comprising the step of the people being used the means of claim 31 in people's prevention or treatment method for cancer.
34. be used for comprising the step of the people being used the means of claim 32 in people's prevention or treatment method for cancer.
35. be used to prevent or treat the means of cancer, wherein comprise the compositions of claim 26.
36. the means of claim 35 wherein also comprise adjuvant.
37. be used for comprising the step of the people being used the means of claim 35 in people's prevention or treatment method for cancer.
38. be used for comprising the step of the people being used the means of claim 36 in people's prevention or treatment method for cancer.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US18196600P | 2000-02-10 | 2000-02-10 | |
| US60/181,966 | 2000-02-10 | ||
| US66025200A | 2000-09-12 | 2000-09-12 | |
| US09/660,252 | 2000-09-12 | ||
| US66250500A | 2000-09-14 | 2000-09-14 | |
| US09/662,505 | 2000-09-14 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1398187A true CN1398187A (en) | 2003-02-19 |
| CN1255427C CN1255427C (en) | 2006-05-10 |
Family
ID=27391498
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB018047580A Expired - Fee Related CN1255427C (en) | 2000-02-10 | 2001-02-12 | Method and compsns. for treating liver cell cancer |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP1253930A4 (en) |
| JP (1) | JP3876162B2 (en) |
| KR (1) | KR100482920B1 (en) |
| CN (1) | CN1255427C (en) |
| AU (1) | AU2001238179A1 (en) |
| HK (1) | HK1046853A1 (en) |
| WO (1) | WO2001058922A2 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101918433B (en) * | 2007-11-20 | 2013-06-26 | 中央研究院 | Peptides specific for hepatocellular carcinoma cells and applications thereof |
| CN113072636A (en) * | 2020-01-06 | 2021-07-06 | 香雪生命科学技术（广东）有限公司 | T cell receptor for identifying AFP and its code sequence |
| CN113321725A (en) * | 2020-02-28 | 2021-08-31 | 香雪生命科学技术（广东）有限公司 | T cell receptor for identifying AFP |
| CN114478791A (en) * | 2015-04-03 | 2022-05-13 | 优瑞科生物技术公司 | Constructs targeting AFP peptide/MHC complexes and uses thereof |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100985914B1 (en) * | 2006-12-13 | 2010-10-08 | 주식회사 바이오리더스 | Cell Surface Expression Vector for liver Cancer Specific Antigen Alpha-fetoprotein and Microorganism Transformed by Thereof |
| WO2008113970A2 (en) * | 2007-03-16 | 2008-09-25 | Ucl Business Plc | Peptides |
| KR100900742B1 (en) * | 2007-05-17 | 2009-06-08 | 크레아젠 주식회사 | Animal Models Carrying Tumors Expressing Human Liver Cancer-Specific Antigen and Method for Analyzing Prevention and Treatment Efficacy of Dendritic Cells-Derived Immunotherapeutics Using the Above |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IT1226551B (en) * | 1988-07-29 | 1991-01-24 | Sclavo Spa | IMMUNOLOGICALLY ACTIVE SYNTHETIC PEPTIDE CAPABLE OF INDUCING THE PRODUCTION OF ANTIBODIES WITH HIGH SPECIFICITY TOWARDS ALPHA-PHETOPROTEIN AND THEIR USE IN THE DIAGNOSTIC FIELD |
| CN1150030C (en) * | 1995-01-24 | 2004-05-19 | 马蒂尼克斯研究与发展公司 | Recombinant human alpha-fetoprotein and uses thereof |
| EP0979239B1 (en) * | 1997-02-13 | 2006-10-18 | The Regents Of The University Of California | Prevention and treatment of hepatocellular cancer |
-
2001
- 2001-02-12 JP JP2001558069A patent/JP3876162B2/en not_active Expired - Fee Related
- 2001-02-12 AU AU2001238179A patent/AU2001238179A1/en not_active Abandoned
- 2001-02-12 KR KR10-2002-7010174A patent/KR100482920B1/en not_active IP Right Cessation
- 2001-02-12 CN CNB018047580A patent/CN1255427C/en not_active Expired - Fee Related
- 2001-02-12 WO PCT/US2001/004539 patent/WO2001058922A2/en not_active Application Discontinuation
- 2001-02-12 EP EP01910587A patent/EP1253930A4/en not_active Ceased
-
2002
- 2002-11-18 HK HK02108331.9A patent/HK1046853A1/en unknown
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101918433B (en) * | 2007-11-20 | 2013-06-26 | 中央研究院 | Peptides specific for hepatocellular carcinoma cells and applications thereof |
| CN114478791A (en) * | 2015-04-03 | 2022-05-13 | 优瑞科生物技术公司 | Constructs targeting AFP peptide/MHC complexes and uses thereof |
| CN113072636A (en) * | 2020-01-06 | 2021-07-06 | 香雪生命科学技术（广东）有限公司 | T cell receptor for identifying AFP and its code sequence |
| CN113072636B (en) * | 2020-01-06 | 2024-05-28 | 香雪生命科学技术（广东）有限公司 | T cell receptor for identifying AFP and coding sequence thereof |
| CN113321725A (en) * | 2020-02-28 | 2021-08-31 | 香雪生命科学技术（广东）有限公司 | T cell receptor for identifying AFP |
| CN113321725B (en) * | 2020-02-28 | 2024-06-11 | 香雪生命科学技术（广东）有限公司 | T cell receptor for identifying AFP |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1255427C (en) | 2006-05-10 |
| KR20020073203A (en) | 2002-09-19 |
| WO2001058922A9 (en) | 2002-10-17 |
| EP1253930A4 (en) | 2003-05-21 |
| WO2001058922A2 (en) | 2001-08-16 |
| EP1253930A2 (en) | 2002-11-06 |
| WO2001058922A3 (en) | 2002-02-14 |
| HK1046853A1 (en) | 2003-01-30 |
| JP2003522195A (en) | 2003-07-22 |
| JP3876162B2 (en) | 2007-01-31 |
| AU2001238179A1 (en) | 2001-08-20 |
| KR100482920B1 (en) | 2005-04-14 |
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