CN1247614C - Preparation of antigen sensitized human dendron shaped cell and its use - Google Patents
Preparation of antigen sensitized human dendron shaped cell and its use Download PDFInfo
- Publication number
- CN1247614C CN1247614C CN 02136518 CN02136518A CN1247614C CN 1247614 C CN1247614 C CN 1247614C CN 02136518 CN02136518 CN 02136518 CN 02136518 A CN02136518 A CN 02136518A CN 1247614 C CN1247614 C CN 1247614C
- Authority
- CN
- China
- Prior art keywords
- cell
- tumour
- heating temperature
- antigen
- hour
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 239000000427 antigen Substances 0.000 title claims abstract description 88
- 102000036639 antigens Human genes 0.000 title claims abstract description 84
- 108091007433 antigens Proteins 0.000 title claims abstract description 84
- 238000002360 preparation method Methods 0.000 title abstract description 16
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 82
- 210000004443 dendritic cell Anatomy 0.000 claims abstract description 62
- 210000004027 cell Anatomy 0.000 claims abstract description 46
- 238000000034 method Methods 0.000 claims abstract description 36
- 206010070834 Sensitisation Diseases 0.000 claims abstract description 29
- 230000008313 sensitization Effects 0.000 claims abstract description 29
- 238000004321 preservation Methods 0.000 claims abstract description 13
- 206010009944 Colon cancer Diseases 0.000 claims abstract description 6
- 210000004881 tumor cell Anatomy 0.000 claims description 45
- 238000010438 heat treatment Methods 0.000 claims description 30
- 239000000203 mixture Substances 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 16
- 239000006228 supernatant Substances 0.000 claims description 15
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 12
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 10
- 238000005336 cracking Methods 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 10
- 244000309466 calf Species 0.000 claims description 9
- 210000002966 serum Anatomy 0.000 claims description 9
- 239000002609 medium Substances 0.000 claims description 7
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 claims description 6
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 5
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 5
- 235000011089 carbon dioxide Nutrition 0.000 claims description 5
- 201000010989 colorectal carcinoma Diseases 0.000 claims description 5
- 239000003937 drug carrier Substances 0.000 claims description 5
- 239000008194 pharmaceutical composition Substances 0.000 claims description 5
- 229920006395 saturated elastomer Polymers 0.000 claims description 5
- 101001002709 Homo sapiens Interleukin-4 Proteins 0.000 claims description 4
- 102000055229 human IL4 Human genes 0.000 claims description 4
- 201000001441 melanoma Diseases 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 238000010257 thawing Methods 0.000 claims description 4
- 206010005003 Bladder cancer Diseases 0.000 claims description 3
- 208000017897 Carcinoma of esophagus Diseases 0.000 claims description 3
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 3
- 208000032612 Glial tumor Diseases 0.000 claims description 3
- 206010018338 Glioma Diseases 0.000 claims description 3
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 3
- 206010025323 Lymphomas Diseases 0.000 claims description 3
- 208000002454 Nasopharyngeal Carcinoma Diseases 0.000 claims description 3
- 206010061306 Nasopharyngeal cancer Diseases 0.000 claims description 3
- 206010030155 Oesophageal carcinoma Diseases 0.000 claims description 3
- 206010033128 Ovarian cancer Diseases 0.000 claims description 3
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 3
- 206010060862 Prostate cancer Diseases 0.000 claims description 3
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 3
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 3
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 3
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 3
- 201000011510 cancer Diseases 0.000 claims description 3
- 230000002490 cerebral effect Effects 0.000 claims description 3
- 201000010881 cervical cancer Diseases 0.000 claims description 3
- 230000001186 cumulative effect Effects 0.000 claims description 3
- 201000005619 esophageal carcinoma Diseases 0.000 claims description 3
- 208000021045 exocrine pancreatic carcinoma Diseases 0.000 claims description 3
- 210000003734 kidney Anatomy 0.000 claims description 3
- 208000032839 leukemia Diseases 0.000 claims description 3
- 201000007270 liver cancer Diseases 0.000 claims description 3
- 208000014018 liver neoplasm Diseases 0.000 claims description 3
- 201000005202 lung cancer Diseases 0.000 claims description 3
- 208000020816 lung neoplasm Diseases 0.000 claims description 3
- 201000011216 nasopharynx carcinoma Diseases 0.000 claims description 3
- 201000000498 stomach carcinoma Diseases 0.000 claims description 3
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 3
- 230000002934 lysing effect Effects 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 abstract description 4
- 230000005809 anti-tumor immunity Effects 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 2
- 208000029742 colonic neoplasm Diseases 0.000 abstract 1
- 238000005057 refrigeration Methods 0.000 abstract 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 16
- 101710163595 Chaperone protein DnaK Proteins 0.000 description 14
- 101710178376 Heat shock 70 kDa protein Proteins 0.000 description 14
- 101710152018 Heat shock cognate 70 kDa protein Proteins 0.000 description 14
- MDDIUTVUBYEEEM-UHFFFAOYSA-N azane;pyrrolidine-1-carbodithioic acid Chemical compound N.SC(=S)N1CCCC1 MDDIUTVUBYEEEM-UHFFFAOYSA-N 0.000 description 10
- 239000003795 chemical substances by application Substances 0.000 description 10
- 210000001744 T-lymphocyte Anatomy 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 239000006285 cell suspension Substances 0.000 description 7
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 7
- 230000036039 immunity Effects 0.000 description 7
- 230000001939 inductive effect Effects 0.000 description 7
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 6
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 102000005962 receptors Human genes 0.000 description 6
- 108020003175 receptors Proteins 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- 230000001464 adherent effect Effects 0.000 description 5
- 239000002671 adjuvant Substances 0.000 description 5
- 230000000118 anti-neoplastic effect Effects 0.000 description 5
- 210000000612 antigen-presenting cell Anatomy 0.000 description 5
- 230000028993 immune response Effects 0.000 description 5
- 238000009169 immunotherapy Methods 0.000 description 5
- 239000002504 physiological saline solution Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- 150000003384 small molecules Chemical class 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 230000030741 antigen processing and presentation Effects 0.000 description 4
- 230000000890 antigenic effect Effects 0.000 description 4
- 239000000969 carrier Substances 0.000 description 4
- 230000005847 immunogenicity Effects 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 230000006044 T cell activation Effects 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 101710113864 Heat shock protein 90 Proteins 0.000 description 2
- 102100034051 Heat shock protein HSP 90-alpha Human genes 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 2
- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- 238000001815 biotherapy Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000005138 cryopreservation Methods 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000012447 hatching Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000036737 immune function Effects 0.000 description 2
- 239000000568 immunological adjuvant Substances 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 230000003526 lymphopoietic effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 210000005087 mononuclear cell Anatomy 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- -1 poly(lactic acid) Polymers 0.000 description 2
- 229920000747 poly(lactic acid) Polymers 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- 102100038222 60 kDa heat shock protein, mitochondrial Human genes 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 108010058432 Chaperonin 60 Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 101000578784 Homo sapiens Melanoma antigen recognized by T-cells 1 Proteins 0.000 description 1
- 101001094545 Homo sapiens Retrotransposon-like protein 1 Proteins 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 102100028389 Melanoma antigen recognized by T-cells 1 Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 229920000954 Polyglycolide Polymers 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 230000005867 T cell response Effects 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000005975 antitumor immune response Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 229960002152 chlorhexidine acetate Drugs 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000036046 immunoreaction Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 239000002398 materia medica Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 210000003071 memory t lymphocyte Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000004719 natural immunity Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000006174 pH buffer Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000003950 pathogenic mechanism Effects 0.000 description 1
- 102000023856 peptide binding proteins Human genes 0.000 description 1
- 108091008399 peptide binding proteins Proteins 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004633 polyglycolic acid Substances 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- MCSINKKTEDDPNK-UHFFFAOYSA-N propyl propionate Chemical compound CCCOC(=O)CC MCSINKKTEDDPNK-UHFFFAOYSA-N 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 210000003370 receptor cell Anatomy 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000011287 therapeutic dose Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Images
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to a new sensitization method for human dendritic cells, preparation and preservation technology, and applications of the human dendritic cells to antitumor immunity. The new sensitization method for the dendritic cells and the preparation and preservation technology are used clinically, and have the advantages of high sensitization efficiency, few requirement of antigens, long cell preservation time, high resuscitation rate after refrigeration, adaptability to clinical applications, etc. The human dendritic cells are particularly suitable for treating cancers such as colon cancer, etc.
Description
Technical field
The present invention relates to biology and medical field, relate more specifically to a kind of preparation method of tumour antigen and the method for preparing APDC with the antigen that this method makes.
Background technology
Dendritic cell (Dendritic Cells, DC) be a kind of antigen presenting cell that exists in the normal human with specific function, be called " the natural adjuvant " of body, can directly absorb, processing, antigen-presenting, stimulating intravital primary tape T cell activation, is " the startup person " of immune response.DC can also promote the propagation and the activation of B cell by direct or indirect mode in addition, the regulation and control humoral immunoresponse(HI); Stimulate the memory T cell activation to bring out secondary immune response; , the natural immunity nonspecific with NK interaction influence replied, so DC can be described as " perpetrator " of immune response in the body.DC is in the key link of tumour immunity, can absorb and processing treatment tumour antigen and transfer initiatively specificity antineoplastic immunity reaction killing tumor cell of body.In the biotherapy of numerous tumours, enjoy attention owing to bringing into play the autonomous antineoplastic immune function of patient's body based on the immunotherapy of DC.
Utilize DC to carry out the immunotherapy of tumour, main purpose be induce patient's body produce tumour-specific, lasting immune response.In these therapies, report at most, research the most deep, most widely used general, curative effect is the most definite is to use the dendritic cell of tumour antigen sensitization to tumor treatment.The clinical observation material of having reported shows, the curative effect of this therapy surpasses the various biotherapies of in the past being reported, also be tool wish a kind of, the application prospect of this therapy is widely had an optimistic view of.
The key principle of immunotherapy of tumors is given full play to body's immunological function exactly to kill tumour cell.Because DC is in the key link of tumour immunity, can absorb with the processing treatment tumour antigen and transfer initiatively specificity antineoplastic immunity reaction of body, therefore, utilize the functional characteristics of DC, before the DC maturation, give full play to the function of its antigen uptaking, giving tumour antigen stimulates, the DC that will absorb tumour antigen again is fed back in the body, the DC that carries tumour antigen is when offering antigenic information to the T cell, activated the T cell, thus the cytotoxic T lymphocyte (CTL) of inducing body to produce to have the specific killing tumour cell, thereby the patient produced certain therapeutic action.
Adopt the direct sensitization dendritic cell of tumour antigen, can induce body to produce the anti-specific cellullar immunologic response of tumour after feeding back in the body, but how efficiently, apace the sensitization that obtains tumour antigen and be used for dendritic cell remain the important topic of immunotherapy of tumors area research.Although for some tumour antigen certain understanding is arranged at present, but the tumour antigen of most tumours is still the unknown; Known antigenic peptide might not be induced best anti tumor immune response, as the CTL cell that from the melanoma patients peripheral blood, induces can not the present tumour of cloning of recognition antigen such as gp100, MART1 etc., prompting may exist other unknown tumour antigens effectively inducing antitumor immunity reply; At the effective inducing antitumor immunity rejection of the ctl clone of single antigen decision family, may bring into play the antitumor action of the best at the acting in conjunction of a plurality of antigens decision family.
Heat shock protein(HSP) (Heat shock protein, HSP) be that a class guards, extensively is present in the class protein in protokaryon and the eukaryote at the organic evolution camber, it is the induced product of biomass cells stress reaction, HSP is bringing into play important effect in the immunoreaction process of body, by participating in important physical processes such as antigen processing submission, T cell activation, closely related with physiology such as the antineoplastic immune of body, anti-infectious immunity, autoimmune disorder and pathomechanism, be the important immune molecule of a class.Heat-shock protein family can be divided into four classes according to molecular weight: HSP90 family, HSP70 family, HSP60 family and small molecules HSP family.Wherein HSP70 family is a most conservative and topmost class among the HSP.
The experiment of Armold shows, with transfection the HSP90 family molecule gp96 immune mouse in cell source of β-Gal, can produce specific CD8 at β-Gal
+CTL.Blachere etc. in external reconstruct the polypeptide complex of HSP70 and gp96, the result shows that HSP itself does not induce specific immunity, and HSP bonded polypeptide can induce the CTL of antigen-specific, but peptide is combined in other peptide-binding proteins such as the white protein of mouse, then non-immunogenicity.
The experiment of Srivastava shows, the HSP70 polypeptide complex in tumour cell source can be induced special antineoplastic immune, and confirm, this immunogenicity is small-molecule substance rather than the HSP that comes from the HSP polypeptide complex, through the reversed-phase HPLC analysis, the molecular weight of these small-molecule substances is 1-5kD, these small-molecule substances have been removed, HSP then loses its immunogenicity, but adjuvant sample effect and the immunogenicity of small-molecular peptides of the only immune effect that can not obtain with these small-molecule substance immune mouses, so HSP in the antigen presentation process is indispensable two integral parts of immunotherapy of HSP mediation.
Blachere etc. studies show that, HSP can become CD8
+A kind of new adjuvant of t cell responses.Moroi etc. adopt the OVA257-264T cell epitope as antigen peptide, external with HWDFAWPW is this can be crosslinked with the protein bound peptide binding domain of HSP70, form hybrid protein, the result induces the antigenic specific CTL at OVA effectively, and can obviously resist the attack of E.G7-OVA tumour cell, use OVA antigen peptide or HSP70 then can not induce ctl response separately, show that the HSP70 molecule has tangible adjuvant sample effect to the activation of T cell.
Arnold-Schild etc. have confirmed that the reaction of HSP inductive specific CTL is owing to there is the acceptor of HSP on the full-time antigen presenting cell (APC), can make APC specifically by the endocytosis of hsp receptor mediation antigen peptide, infer the existence of hsp receptor from morphology.
On the J.Exp.Med in June, 2000, Castellino has confirmed that with Singh-Jasuja etc. there is hsp receptor in the DC surface in different experiments respectively, and infer that hsp receptor is saturable, confirmed that simultaneously HSP bonded antigen is to rely on and the non-dependence approach of TAP by TAP in born of the same parents, to the MHC-I quasi-molecule, and induced subsequently ctl response by submission.This acceptor is proved to be on the APC such as being present in DC, scavenger cell and B cell, and is not present on the T cell.Therefore can think that it is the important mechanism of HSP as adjuvant that HSP interacts by hsp receptor and APC.Studies show that the efficiency ratio of this receptor-mediated antigen presentation mode is engulfed high 1000～10000 times.
Discoveries such as Asea, HSP70 molecule can be exercised similar cytokine-like effect outside born of the same parents.HSP70 can produce combining of high-affinity with monocyte, induces it to produce IL-6, IL-1 isoreactivity cytokine, and can induce the DC maturation, raises its MHC-II and CD86 developed by molecule, secretion IL-12 and TNF-α, thereby the antigen presentation ability of enhancing DC.Therefore adopt HSP to induce DC as immunological adjuvant, can the antigenic submission of target mediation, the submission efficient of enhancement antigen, thus induce stronger ctl response.
Yet, up to now, also do not have effectively preparation initiation at the method for the tumour antigen of the specific immune response of tumour.Therefore, this area presses for that exploitation is new can effectively to cause tumour antigen at the immunne response of tumour and preparation method thereof.
Summary of the invention
Purpose of the present invention just provides and a kind ofly can effectively cause tumour antigen at the immune induction of tumour and preparation method thereof.
Another object of the present invention provides with dendritic cell of tumour antigen sensitization of the present invention and preparation method thereof.
In a first aspect of the present invention, a kind of extracting method of tumour antigen is provided, comprise step:
(a) at 42 ℃ of heat treated tumour cells;
(b) at 55 ℃ of heat treated tumour cells;
(c) make the tumour cell cracking of step (b);
(d) remove cell debris, separate the supernatant liquor that obtains to contain tumour antigen.
In a preference, lysing cell is realized for 1-10 time by the freeze thawing tumour cell.
In another preference, step (a) and treatment time (b) are 0.1-100 hour.
In a second aspect of the present invention, a kind of tumour antigen is provided, it is the mixture of heat shock protein(HSP)-antigen peptide, this mixture prepares by following steps:
(a) at 42 ℃ of heat treated tumour cells;
(b) at 55 ℃ of heat treated tumour cells;
(c) make the tumour cell cracking of step (b);
(d) centrifugal removal cell debris, acquisition contains the supernatant liquor of heat shock protein(HSP)-antigen peptide.
In a third aspect of the present invention, a kind of sensitization method of dendritic cell is provided, comprise step:
(a) at 42 ℃ of heat treated tumour cells;
(b) at 55 ℃ of heat treated tumour cells;
(c) make the tumour cell cracking of step (b);
(d) centrifugal removal cell debris, acquisition contains the supernatant liquor of tumour antigen.
(e) tumour antigen and dendritic cell in the step (d) are hatched altogether, thus the dendritic cell of acquisition sensitization.
In a preference, dendritic cell in the step (e) derives from peripheral blood lymphocytes, and described dendritic cell prepares with following method: adopt the rhGM-CSF that contains 50ng/ml, the recombinant human interleukin--4's of 10ng/ml fresh perfect medium, put 5% carbonic acid gas, 37 ℃, cultivated peripheral blood lymphocytes to the 3 days in the incubator of saturated humidity, in culturing bottle, replenish the fresh culture that 20ml contains 10% foetal calf serum, rhGM-CSF50ng/ml, rhIL-4 10ng/ml, continue to be cultured to the 6th day.
In another preference, also comprise step:
(f) dendritic cell with the sensitization that obtains is stored in the external preservation liquid, and the prescription of described preservation liquid is as follows: 42.5% RPMI-1640 substratum, and 7.5% DMSO, 50% concentration is 20% Plasbumin-25, by the cumulative volume of preserving liquid.
In another preference, in step (e), the blending ratio of tumour antigen and dendritic cell is 1 microgram antigen: 1 * 10
8Individual cell to 100 microgram antigen: 10
5Individual cell.
In another preference, described tumour cell is the tumour cell that is selected from down the group cancer: colorectal carcinoma, the esophageal carcinoma, cancer of the stomach, carcinoma of the pancreas, liver cancer, lung cancer, melanoma, kidney, bladder cancer, prostate cancer, leukemia, lymphoma, ovarian cancer, cervical cancer, nasopharyngeal carcinoma, cerebral glioma.
In a fourth aspect of the present invention, a kind of pharmaceutical composition or immune composition are provided, it contains pharmaceutically acceptable carrier, and the above-mentioned tumour antigen of the present invention or with the dendritic cell of described tumour antigen sensitization.
Description of drawings
Before and after the heating of four colorectal carcinoma patient tumors of Fig. 1 tissue, the comparison of HSP70 expression level (Westernblot), wherein heating back HSP70 expression level obviously raises
Fig. 2. induce the morphological observation of the 6th day human dendritic cell.
Fig. 3. the monocyte before inducing changes with the phenotype of inducing the back dendritic cell.
Embodiment
The inventor is through extensive and deep research, find by two step method thermal induction tumour cell, the expression of HSP in the tumour cell is obviously raise, behind multigelation, HSP carries tumour antigen and is released in the supernatant in large quantities, thereby obtain the mixture of a large amount of HSP-antigen peptide, these mixtures as tumour antigen, can be activated dendritic cell more efficiently.Finished the present invention on this basis.
At first, the extracting method of tumour antigen of the present invention may further comprise the steps:
(a) heat treated tumour cell (first heating phase) under lower high temperature
With isolating tumour cell or tissue, promptly place for some time approximately under first Heating temperature (for example about 42 ℃) in the temperature higher slightly than normal temps, for example about 0.1-100 hour, preferably 0.2-10 hour, more preferably 0.5-5 hour.
(b) heat treated tumour cell (second heating phase) under higher high temperature
With isolating tumour cell or tissue, promptly place for some time approximately under second Heating temperature (for example about 55 ℃ ℃) in the temperature higher more slightly than normal temps, for example about 0.1-100 hour, preferably about 0.2-10 hour, more preferably about 0.5-5 hour.
Usually, second Heating temperature is higher about 15-25 ℃ than first Heating temperature.
(c) make the tumour cell cracking of step (b)
Can adopt the whole bag of tricks known in the art to come the cracking tumour cell, for example mechanical lysis or chemical cracking.A kind of preferable methods is a freeze-thaw method, promptly makes lysis by multigelation.
(d) remove impurity such as cell debris, separate the supernatant liquor that obtains to contain tumour antigen.
Because HSP-antigenic peptide mixture is present in the supernatant liquor after the cracking, therefore can with Impurity removals such as cell debriss, acquisition contains the supernatant liquor of HSP-antigenic peptide mixture (being tumour antigen) with various ordinary methods (for example, centrifugal, ultrafiltration etc.).
Can be used for tumour cell of the present invention is not particularly limited, can be any tumour cell, representative example comprises that (but being not limited to) is selected from down the tumour cell of group cancer: colorectal carcinoma, the esophageal carcinoma, cancer of the stomach, carcinoma of the pancreas, liver cancer, lung cancer, melanoma, kidney, bladder cancer, prostate cancer, leukemia, lymphoma, ovarian cancer, cervical cancer, nasopharyngeal carcinoma, cerebral glioma.
With the tumour antigen of the inventive method preparation, its composition mainly is the mixture of heat shock protein(HSP)-antigen peptide.Experiment shows that after two step heat treated of the present invention, the expression of HSP in the supernatant liquor (for example HSP70) is obviously risen.
With the tumour antigen of the inventive method preparation, can be by multiple application.For example, as the original sensitization dendritic cell of immunity, thus the dendritic cell of acquisition sensitization, perhaps directly as immunogen preparing immune composition or pharmaceutical composition.
The present invention also provides a kind of pharmaceutical composition or immune composition.In described composition, contain pharmaceutically acceptable carrier (comprising thinner, vehicle etc.) and the tumour antigen of the present invention of significant quantity or the dendritic cell of sensitization.The quantity of tumour antigen is generally 10 micrograms-100 milligram/agent, preferably is 100-1000 microgram/agent.The quantity of the dendritic cell of sensitization is generally about 10
5-10
8Individual cell/agent.
Term used herein " significant quantity " refers to the therapeutical agent treatment, alleviates or prevent the amount of target disease or situation, or shows the amount of detectable treatment or preventive effect.Depend on the nature and extent of the build of this object and healthy state, illness and the therapeutical agent selecting to give and/or the combination of therapeutical agent for the accurate significant quantity of a certain object.Therefore, specifying accurately in advance, significant quantity is useless.Yet, for certain given situation, can determine this significant quantity with normal experiment, the clinicist can judge.
For the purposes of the present invention, effective dosage is for giving individual about 0.01 mg/kg to 50 mg/kg, the preferably tumour antigen of 0.05 mg/kg to 10 mg/kg.For the dendritic cell of sensitization, significant quantity is generally about 10
5-10
8An individual cell/object.
Pharmaceutical composition also can contain pharmaceutically acceptable carrier.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent (for example tumour antigen) administration.This term refers to like this some medicament carriers: they itself do not induce generation to accepting the individual deleterious antibody of said composition, and do not have undue toxicity after the administration.Suitable carriers may be big, a metabolism macromole slowly, as the virion of protein, polysaccharide, poly(lactic acid) (polylactic acid), polyglycolic acid, aminoacid polymers, amino acid copolymer and non-activity.These carriers are well known to those of ordinary skill in the art.(Mack Pub.Co. can find discussing fully about pharmaceutically acceptable vehicle in N.J.1991) at Remington ' s Pharmaceutical Sciences.
Acceptable carrier can contain liquid on the therapeutic composition Chinese materia medica, as water, salt solution, glycerine and ethanol.In addition, also may there be complementary material in these carriers, as wetting agent or emulsifying agent, pH buffer substance etc.In addition, can also contain immunological adjuvant in the immune composition.
Usually, therapeutic composition can be made injectable agent, for example liquor or suspension; Also can be made into before injection, be fit to allocate in solution or the suspension, the solid form of liquid vehicle.
In case be made into composition of the present invention, can directly give object with it.The object of waiting to prevent or treating can be an animal; Especially people.
Directly carrying said composition can or be delivered to tissue space by subcutaneous, intraperitoneal, intravenously or intramuscularly usually realizes.Composition also can be delivered to focal zone.Other administering mode comprises oral and transdermal administration etc.The therapeutic dose scheme can be single agent scheme or multi-agent scheme.
The present invention also provides the method for coming the sensitization dendritic cell with tumour antigen of the present invention, and be about to tumour antigen and dendritic cell and hatch altogether, thus the dendritic cell of acquisition sensitization.When hatching, the blending ratio of tumour antigen and dendritic cell is 1 microgram antigen: 1 * 10
8Individual cell to 100 microgram antigen: 10
5Individual cell, i.e. 1-100 microgram antigen: 1 * 10
5-1 * 10
8Individual cell.Preferably blending ratio is 1 microgram antigen: 1 * 10
7Individual cell to 100 microgram antigen: 10
6Individual cell
Incubation temperature and time are not particularly limited, and are generally under 35-37.5 ℃ the temperature and hatch 0.1-100 hour (preferably 1-48 hour).
Can be used for dendritic cell of the present invention and be not particularly limited, can adopt the dendritic cell in various sources.Especially people's dendritic cell.A kind of preferred dendritic cell derives from peripheral blood lymphocytes.
The present invention also provides a kind of method preparation for preparing dendritic cell: adopt the rhGM-CSF that contains 25-100ng/ml, the recombinant human interleukin--4's of 5-20ng/ml fresh perfect medium, put 5% carbonic acid gas, 37 ℃, cultivated peripheral blood lymphocytes to the 3 days in the incubator of saturated humidity, in culturing bottle, replenish the fresh culture that contains 10% foetal calf serum, rhGM-CSF 25-100ng/ml, rhIL-4 5-20ng/ml, continue to be cultured to the 6th day.
The dendritic cell of sensitization can ordinary method be preserved.A kind of preferable methods is that the dendritic cell of the sensitization that will obtain is stored in the external preservation liquid, the prescription of described preservation liquid is as follows: 42.5% RPMI-1640 substratum, 7.5% DMSO, 50% concentration is 20% Plasbumin-25, by the cumulative volume of preserving liquid.This prescription has recovery back dendritic cell survival rate height, in the prescription composition toxicity low, be applicable to the preservation and the recovery that are applied to clinical dendritic cell.In addition, preservation should be in liquid nitrogen or Ultralow Temperature Freezer cryopreservation.
Major advantage of the present invention is:
(1) from tumour cell, directly separates tumour antigen.Zhi Bei tumour antigen has comprised all antigens that exist in the tumour like this.Present theory thinks that the antigen in the tumour is the form existence with " antigen fingerprint ", rather than certain independent antigen, according to this theory, all there is individual difference in everyone tumour antigen, and therefore, isolating tumour antigen should be an ideal approach comparatively from tumour cell.
(2) technology is easy, and sensitization efficient height is safe in utilization.Be purified into tumour antigen the tumour cell freeze thawing supernatant after thermal induction, be used for the sensitization dendritic cell, have sensitization efficient height, required antigen amount is few, safe in utilization, prevents effects such as microbial contamination.
(3), have clinical safe in utilization, characteristics that recovery efficient is high, thereby solved the critical problem of dendritic cell clinical treatment in conjunction with the special-purpose frozen storing liquid of dendritic cell of the present invention.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The preparation of tumour antigen
Get the aseptic colorectal carcinoma tumor tissues (4 cases) of excision, soaked 30 minutes with 0.05% chlorhexidine acetate solution, stroke-physiological saline solution flushing 3 times, cut with aseptic tissue tumor tissues is shredded, adding 5ml RPMI1640 substratum grinds, after 200 order steel meshes filter, collect single cell suspension, use RPMI1640 substratum re-suspended cell (1 * 10 then
7/ ml), and the aseptic frozen pipe of the 5ml that packs into, after 1 hour, 60 ℃ of water-baths continue heating 1 hour, and frozen pipe is immersed liquid nitrogen flash freezer rapidly, take out after 10 minutes through 42 ℃ of heating in water bath, put into room temperature and melt; So multigelation is 5 times, and tumor cell lysate is added 15ml Falcon centrifuge tube, and centrifugal 20 minutes of 400 * g collects supernatant.Tumour cell freeze thawing supernatant is tumour antigen through the disposable sterilized filter filtration sterilization of 0.2 μ m.
Adopt the HSP70 monoclonal antibody to resist doing the Western-blot analysis after heating and for isolating tumour antigen in the tumor tissues of heating as one, the result heats the expression of HSP70 in the tumour antigen of back and obviously increases as shown in Figure 1.
Embodiment 2
The preparation of dendritic cell
The mononuclearcell of collecting (about 50-100ml) is slowly added several 50ml Falcon centrifuge tubes that adds the 20ml lymphocyte separation medium in advance (cell suspension volume equates with the parting liquid volume), centrifugal 30 minutes of centrifuge tube 400 * g room temperature after strict trim, the interface cell is drawn with flat mouthful of Pasteur's dropper in centrifugal back, join in the 30mlRPMI1640 substratum, centrifugal 10 minutes of 200 * g, wash 3 times, collect cell, will be made into 4 * 10 with the RPMI1640 substratum that contains 10% foetal calf serum through the mononuclearcell of centrifuge washing
6The cell suspension of/ml, plant the aseptic Falcon culturing bottle of 750ml, inoculation volume 25ml, the culturing bottle that fills cell is put 37 ℃, 5% carbonic acid gas, cultivated adherent 2 hours in the incubator of saturated humidity, culturing bottle is taken out, jiggle ten for several times, non-adherent cell is hanged, discard suspension cell, slowly in bottle, add the 10mlRPMI1640 substratum again, rock the residual non-adherent cell of flush away gently, add 30ml in the culturing bottle that keeps attached cell and contain 10% foetal calf serum, rhGM-CSF 50ng/ml, the fresh perfect medium of recombinant human interleukin--4 (rhIL-4) 10ng/ml is put 5% carbonic acid gas, 37 ℃, continue in the incubator of saturated humidity to cultivate, be cultured to the 3rd day, replenish 20ml in the culturing bottle and contain 10% foetal calf serum, rhGM-CSF 50ng/ml, the fresh culture of rhIL-4 10ng/ml continues to cultivate 2-3 days, in the culturing process every day 2 observation of cell form, at the 5th, 6 day that cultivates, visible cell became the burr shape, and assemble agglomeratingly, stop to cultivate.Blow and beat the culturing bottle bottom gently with the 10ml suction pipe, part half adherent dendritic cell is hanged, nutrient solution is sucked the 50ml centrifuge tube, centrifugal 10 minutes of 200 * g collects dendritic cell.
The form and the phenotype that detect the human dendritic cell of inducing the 6th day with flow cytometer change, and Fig. 2 and Fig. 3 have shown that the form of dendritic cell and phenotype change.Simultaneously, adopt mixed lymphocyte reacion to detect its biological function.The result has confirmed that the cell that obtains is a dendritic cell.
Embodiment 3
The sensitization of dendritic cell
The dendritic cell of collecting is made into 2 * 10 with the fresh perfect medium that contains 10% foetal calf serum, rhGM-CSF 50ng/ml, rhIL-410ng/ml
6The cell suspension of/ml, the tumour antigen that adds preparation among the embodiment 1 be to final concentration 50 μ g/ml, incubated overnight, with the 50ml centrifuge tube collect through with the dendritic cell suspension of tumour antigen overnight incubation, centrifugal 10 minutes of 200 * g collects cell; Add the 40ml stroke-physiological saline solution again, 200 * g washing in centrifugal 10 minutes, continuous 3 times.Collecting cell is APDC (APDC).
Embodiment 4
Frozen and the recovery of dendritic cell
APDC (APDC) work in-process are made into 1 * 10 with the frozen storing liquid for preparing in advance (RPMI-1640 cultivates fiduciary point 42.5%, and DMSO accounts for 7.5%, 20% Plasbumin-25 and accounts for 50%)
7The suspension of/ml divides in the frozen pipe of 2ml of packing into, and the 1ml/ pipe is put into special-purpose freezing storing box (1 ℃ of slowly cooling of per minute), puts-80 ℃ of refrigerator and cooled and freezes preservation, in infusion recovery on same day cell.Plastic cup with cleaning adds 40 ℃ of pure water of 200ml, putting into warm water rapidly after frozen cell taken out from Ultralow Temperature Freezer thaws fully, then the cell of recovering is joined in the 50ml centrifuge tube and (preset the stroke-physiological saline solution 40ml of 37 ℃ of preheatings in the centrifuge tube), centrifugal 10 minutes of 200 * g washs 3 times; The stroke-physiological saline solution that contains 1% Plasbumin-25 with 10ml is made into cell suspension, and 1 * 10
6Individual/ml, divide 10 pipes, 2-8 ℃ of preservation.
Embodiment 5
APDC stimulates allosome T proliferation of lymphocytes
Separate normal people's peripheral blood mononuclear cell, put 37 ℃ of cultivations and remove attached cell after 2 hours, non-adherent cell suspends with the RPMI1640 complete culture solution of 5% foetal calf serum, crosses nylon hair post (hatching 1 hour for 37 ℃), and the T lymphocyte of preparation adds 96 orifice plates (2 * 10
5/ hole); APDC after 1 hour, is suspended from the RPMI1640 perfect medium that contains 10% foetal calf serum in 37 ℃ of processing through mitomycin (25 μ g/ml), by various dose (1 * 10
4～1 * 10
5/ hole) mix with the Allogeneic T lymphocyte in adding 96 orifice plates, establish 3 multiple holes for every group, final volume is 200 μ l; Negative control group is to add equal-volume RPMI1640 substratum in the T lymphocyte, and positive controls is the PHA of 5 μ g/ml for add final concentration in the T lymphocyte.Put 37 ℃, 5%CO
2Hatched in the incubator 96 hours, stop preceding 4 hours in cultivating, every hole adds the MTT10 μ l of 5mg/ml, under 37 ℃ of conditions, continued to hatch 4 hours, with centrifugal 10 minutes of culture plate 200 * g, every hole sucking-off 100ul supernatant, every then hole adds 10%SDS solution 100 μ l, after the crystallization of 8 hours Dai Jia Za of 37 ℃ of placements is dissolved fully, measure optical density value (570nm) with microplate reader.Adopt Student t check compare group and test group OD value.
The result is as shown in table 1, has the lymphopoietic characteristic of obvious stimulation allosome T (p＜0.05) through APDC.
Table 1. APDC stimulates the lymphopoietic ability of allosome T (p＜0.05)
| T：DC | OD | ||
| 1 | 2 | 3 | |
| 4:1 20:1 40:1 200:1 positive control negative control | 0.324 0.388 0.370 0.342 0.447 0.269 | 0.326 0.376 0.367 0.345 0.389 0.262 | 0.346 0.377 0.365 0.318 0.434 0.268 |
Embodiment 6
The application of dendritic cell
Through the cell of embodiment 4 cryopreservation resuscitations, after relevant calibrating, be injected in the 50ml injection physiological saline, with preceding cell suspension is shaken up, intravenous drip was rocked infusion bag in per 5 minutes gently in the instillation process, avoid cell settlement to pile up, and directly caused to drip off.Perhaps adopt subcutaneous injection, per injection 1ml injects weekly 1 time, and continuous 3 times is a course of treatment.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (13)
1. the extracting method of a tumour antigen is characterized in that, comprises step:
(a) under first Heating temperature, heat treated tumour cell 0.2-10 hour, described first Heating temperature is 42 ℃;
(b) under second Heating temperature, heat treated tumour cell 0.2-10 hour, described second Heating temperature is than the high 15-25 of first Heating temperature ℃;
(c) make the tumour cell cracking of step (b);
(d) remove cell debris, separate the supernatant liquor that obtains to contain tumour antigen.
2. the method for claim 1 is characterized in that, lysing cell is realized for 1-10 time by the freeze thawing tumour cell.
3. the method for claim 1 is characterized in that, step (a) and treatment time (b) are 0.5-5 hour.
4. the method for claim 1 is characterized in that, described step (a) and treatment time (b) are 1 hour, and second Heating temperature is 60 ℃.
5. a tumour antigen is characterized in that, but its sensitization dendritic cell and then stimulate allosome T lymphopoiesis, and prepare by following steps:
(a) under first Heating temperature, heat treated tumour cell 0.2-10 hour, described first Heating temperature is 42 ℃;
(b) under second Heating temperature, heat treated tumour cell 0.2-10 hour, described second Heating temperature is than the high 15-25 of first Heating temperature ℃;
(c) make the tumour cell cracking of step (b);
(d) centrifugal removal cell debris obtains supernatant liquor.
6. tumour antigen as claimed in claim 5 is characterized in that, described step (a) and treatment time (b) are 1 hour, and second Heating temperature is 60 ℃.
7. the sensitization method of a dendritic cell is characterized in that, comprises step:
(a) under first Heating temperature, heat treated tumour cell 0.2-10 hour, described first Heating temperature is 42 ℃;
(b) under second Heating temperature, heat treated tumour cell 0.2-10 hour, described second Heating temperature is than the high 15-25 of first Heating temperature ℃;
(c) make the tumour cell cracking of step (b);
(d) centrifugal removal cell debris, acquisition contains the supernatant liquor of tumour antigen;
(e) tumour antigen and dendritic cell in the step (d) are hatched altogether, thus the dendritic cell of acquisition sensitization.
8. method as claimed in claim 7 is characterized in that, described step (a) and treatment time (b) are 1 hour, and second Heating temperature is 60 ℃.
9. method as claimed in claim 7, it is characterized in that, dendritic cell in the step (e) derives from peripheral blood lymphocytes, and described dendritic cell prepares with following method: adopt the rhGM-CSF that contains 50ng/ml, the recombinant human interleukin--4's of 10ng/ml fresh perfect medium, put 5% carbonic acid gas, 37 ℃, cultivated peripheral blood lymphocytes to the 3 days in the incubator of saturated humidity, replenish 20ml in the culturing bottle and contain 10% foetal calf serum, rhGM-CSF 50ng/ml, the fresh culture of rhIL-4 10ng/ml continues to be cultured to the 6th day.
10. method as claimed in claim 7 is characterized in that, also comprises step:
(f) dendritic cell with the sensitization that obtains is stored in the external preservation liquid, and the prescription of described preservation liquid is as follows: 42.5% RPMI-1640 substratum, and 7.5% DMSO, 50% concentration is 20% Plasbumin-25, by the cumulative volume of preserving liquid.
11. method as claimed in claim 7 is characterized in that, in the step (e), the blending ratio of tumour antigen and dendritic cell is 1 microgram antigen: 1 * 10
8Individual cell to 100 microgram antigen: 10
5Individual cell.
12. method as claimed in claim 7, described tumour cell are the tumour cells that is selected from down the group cancer: colorectal carcinoma, the esophageal carcinoma, cancer of the stomach, carcinoma of the pancreas, liver cancer, lung cancer, melanoma, kidney, bladder cancer, prostate cancer, leukemia, lymphoma, ovarian cancer, cervical cancer, nasopharyngeal carcinoma, cerebral glioma.
13. pharmaceutical composition or immune composition is characterized in that it contains pharmaceutically acceptable carrier, and the described tumour antigen of claim 5 or with the dendritic cell of described tumour antigen sensitization.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02136518 CN1247614C (en) | 2002-08-16 | 2002-08-16 | Preparation of antigen sensitized human dendron shaped cell and its use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02136518 CN1247614C (en) | 2002-08-16 | 2002-08-16 | Preparation of antigen sensitized human dendron shaped cell and its use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1475498A CN1475498A (en) | 2004-02-18 |
| CN1247614C true CN1247614C (en) | 2006-03-29 |
Family
ID=34146511
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02136518 Expired - Lifetime CN1247614C (en) | 2002-08-16 | 2002-08-16 | Preparation of antigen sensitized human dendron shaped cell and its use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1247614C (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979507A (en) * | 2010-09-16 | 2011-02-23 | 宋鑫 | Sterilization method for bacterial tumor tissue holoantigen |
| CN108004213B (en) * | 2018-01-30 | 2020-09-22 | 北京汇智驰康生物科技有限公司 | Method and kit for rapid amplification of CIK cells |
| CN119055758B (en) * | 2024-11-05 | 2025-02-14 | 上海惠盾因泰生物科技有限公司 | Dendritic cell therapeutic vaccine preparation and preparation method and application thereof |
-
2002
- 2002-08-16 CN CN 02136518 patent/CN1247614C/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| CN1475498A (en) | 2004-02-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101837561B1 (en) | Methods and compositions for liquidation of tumors | |
| Lin et al. | A novel adjuvant Ling Zhi-8 enhances the efficacy of DNA cancer vaccine by activating dendritic cells | |
| WO2014075631A1 (en) | Method for preparing autologous tumor vaccine and use thereof | |
| Wooldridge et al. | CpG DNA and cancer immunotherapy: orchestrating the antitumor immune response | |
| Peng et al. | Treatment of subcutaneous tumor with adoptively transferred T cells | |
| Du et al. | Peptidic microarchitecture-trapped tumor vaccine combined with immune checkpoint inhibitor or PI3Kγ inhibitor can enhance immunogenicity and eradicate tumors | |
| CN105873639A (en) | Autologous tumor vaccines and methods | |
| CN1247614C (en) | Preparation of antigen sensitized human dendron shaped cell and its use | |
| CN103687602A (en) | Induction of IL-12 using immunotherapy | |
| CN109153974A (en) | Composition for enhancing lethality to abnormal cells and application thereof | |
| CN1313151C (en) | Preparation of high-performance noncellular vaccine and usage | |
| CN1607247A (en) | Dendritic cell tumor vaccine and its preparation and use | |
| CN1282485C (en) | Dendritic cell tumor vaccine loaded with apoptotic heat shock tumor cells and its preparation method and application | |
| JP6871432B2 (en) | HLA-A2 subtype-specific antigenic determinant derived from HLA-A2 that induces an antigen-specific T cell immune response to the PLK1 protein | |
| EA015510B1 (en) | A METHOD OF INCREASING THE AMOUNT OF MONONUCLEAR CELLS IN A SUBJECT SUFFICIENT OF CANCER AND USED FOR THIS PHARMACEUTICAL COMBINATION | |
| CN1239431A (en) | Immunogenic TLP compsn. | |
| CN1305528C (en) | immune adjuvant | |
| CN103710308B (en) | Muramyl dipeptide is utilized to induce the method for DC-CIK | |
| CN105622731B (en) | CTL epitope of hepatitis B core antigen and related application of CTL epitope | |
| CN105505873A (en) | Preparation method of HLA-A0201 restriction anti-Her2-neu antigen-specific CTL (cytotoxic lymphocyte) | |
| CN1321189A (en) | Induction method of cellular immunity and cells induced by cellular immunity | |
| CN101327317B (en) | A kind of injection for treating tumor and preparation method thereof | |
| CN102838679A (en) | Preparation and application of Her2-neu antigen positive tumor therapeutic vaccine | |
| GR1009868B (en) | Personalized implant against cancer | |
| CN1288166C (en) | Modified tumor antigen peptide and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CX01 | Expiry of patent term |
Granted publication date: 20060329 |
|
| CX01 | Expiry of patent term |