CN101637608A - Mixture of tubercle bacillus HSP and tumor lysate - Google Patents
Mixture of tubercle bacillus HSP and tumor lysate Download PDFInfo
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Abstract
The invention provides a mixture prepared by mixing heat shock proteins of tubercle bacillus and tumor cell lysate. The mixture is used for preparing a preparation for treating individuals carrying the same tumor.
Description
Technical field
The present invention relates to a kind ofly be used to prevent and treat the heat shock protein of tumor and the mixture of tumor cell lysate.
Background technology
Tissiers utilized the sds gel electrophoresis technology to prove first in 1974, heat stress can be induced and be produced a histone matter in the fruit bat body, he is because of synthetic this histone matter of ambient temperature rising inducing cell, be called heat shock protein (heat shockprotein, HSP).Now prove, except that heat stress, some otherly stress also can activate the HSP gene, synthetic heat shock protein as under the conditions such as anoxia, heavy metal poisoning, oxidative stress, infection, hunger, wound, metabolic poison.HSP (heat shock protein) is the protein that extensively is present in the class high conservative in protokaryon and the eukaryotic cells, plays a significant role at aspects such as cell growth, growth, differentiation, genetic transcriptions.According to the molecular weight size, HSPs is divided into HSP90 (83～90kD) families, HSP70 (66～78kD) families, HSP60 family and little HSP family (sHSP).Also having molecular weight in addition is 100～110kD and characteristic is different from the macromole HSPs of above-mentioned family, and the common characteristic of HSPs is: 1. under the stress state, it is expressed and significantly increases; 2. in prokaryotic cell and eukaryotic cell, all express, have the aminoacid sequence and the encoding gene thereof of high conservative; As prokaryote and Eukaryotic HSP70 40%～60% homology is arranged, in different eukaryotes, its homology is 60%～78%.
The antigenicity of simple tumor antigen is one of difficult problem main in the present immunotherapy of tumors a little less than.After using the people HSP of reorganization and the tumor cell lysate supernatant mixes entirely, someone is used for antineoplastic experimentation (Feng ZH, Huang B, Zhang GM, et al.Investigation on theeffect of peptides mixture from tumor cells inducing anti-tumor specific immune response.Scince inchina, 2002 (45): 4,361-69.), obtained effect preferably.
Tulase HSP and people HSP homology are higher, and have many immunological adjuvant characteristics of people HSP, receive much attention in recent years.A large amount of studies show that: tulase heat shock protein (heat shock protein, HSP) 65 and 70 except that can having the submission of intersection antigen function, can also with cell surface CD40, TLR2/4 and CCR5 receptors bind, induce body cell immunity and humoral immunization by number of ways, be antineoplastic immune adjuvant (the A.A.A.Asea and A.De Maio (eds.) that application prospect is relatively arranged, Heat Shock Proteins:Potent Mediators of Inflammationand Immunity, 159-172..2007Springer), be expected to substitute the application that is used for anti-tumor aspect after people HSP makes up with full tumor cell lysate.
In the histiocyte of the mankind and animal and cell membrane can extract tumor cell lysate as tumor vaccine.Acquisition methods is a breaking method, and commonly used has: the method that colds and heat succeed each other, multigelation method, sonioation method, glass drench homogenate method, autolysis method and enzyme facture.Passing pure tumour-cell vaccine is exactly to pass through from body or allogeneic tumor cell or its crude extract, handle the back through physics, chemistry or biological means and keep its immunogenicity, but a little less than the immunogenicity of this vaccine, be difficult to cause immunne response, in this vaccine, add adjuvant such as bacillus calmette-guerin vaccine (BCG), short and small rod-like stem mattress or Fu Shi live agent to strengthen its immunogenicity, and effect is still undesirable.
Summary of the invention
The technical problem to be solved in the present invention is the immunocompetence that how to strengthen tumor antigen in the tumor cell lysate better, to reach the purpose of prevention and treatment tumor.
The technical scheme that adopts is the immunocompetence that strengthens tumor antigen in the tumor cell lysate with tulase HSP.Scheme is as follows:
1, the acquisition of tumor cell lysate
The method that obtains tumor cell lysate at present has a lot, summarizes and gets up to mainly contain preparation methoies such as multigelation, ultrasonic degradation, cell pyrolysis liquid cracking.What this research was adopted is that the multigelation method prepares tumor cell lysate.Method is roughly: with-70 ℃ freezing, 37 ℃ be dissolved as a circulation, carry out 5 circulations repeatedly, destroy situation with the trypan blue checking lysis of dyeing then.Cracking cell lysate completely can be used for follow-up research.
2, the acquisition of heat shock protein
Tulase HSP65 albumen is provided by the Bethune of Jilin University medical college molecular biology teaching and research room.
Among the present invention, our using gene engineering method obtains tulase HSP70 albumen.
Applied HSP65, HSP70 albumen are the tubercule bacillus sources in the research.HUMAN HEAT SHOCK PROTEINS Hhsp HSP70 with present widespread usage relatively has better antigenicity.It can bring into play better immune activation effect and angtigen presentation effect as a kind of external antigenic substance.
3, utilize tumor cell lysate associating HSP65, HSP70 albumen antitumor
1) preparation of tumor cell lysate: the Nacl of the tumor cell that growth conditions is good after with high pressure makes cell suspension, behind-70 ℃ and 37 ℃ of multigelations 5 times, (freeze 15min, melt 5min, shake mixing therebetween), dye to determine the cracking situation with platform phenol is blue.100% cell indigo plant is dyed all cracking death of explanation cell, and this cracking mixed liquor that includes whole tumor cell lysates will directly apply to subsequent experimental.
2) preparation of full tumor cell lysate and HSP65, HSP70 mixture: the full tumor cell lysate that will prepare mixes with the HSP65 or the HSP70 of synthetic, be put in after the mixture preparation 4 ℃ standby.
3) press down the tumor experiment in the experimental animals: with mixture immunity B16 melanoma or the Lewis lung cancer tumor-bearing mice of full tumor cell lysate and HSP65, HSP70, the time point of immunity is-7,1,8,15 days after the lotus tumor, the immunity position is a mice right hind groin, and establishes matched groups such as full tumor cell lysate, HSP65, HSP70, Nacl simultaneously.Judge the tumor-inhibiting action of medicine by observation drafting tumor growth curve, observed and recorded survival time of mice.
The effect that the present invention obtains:
Draw tumor growth curve and observed and recorded survival time of mice by observing, originally discover: corresponding full tumor cell lysate associating HSP65 or HSP70 can suppress the growth of B16 melanoma and Lewis lung cancer glucagonoma, the life cycle of significant prolongation tumor-bearing mice simultaneously significantly.These results prove: corresponding full tumor cell lysate associating HSP65, HSP70 have the effect of anti-B16 melanoma and Lewis lung cancer.Novelty of the present invention and creativeness:
Present a lot of anti-tumor experiments all concentrate on the emphasis of research on tumor cell lysate and the heat shock protein, but the two research thinking that combines is also rare.And the method that heat shock protein HSP65, HSP70 that the cracking mixed liquor that will include whole tumor cell lysates is originated with tubercule bacillus carry out antitumor research in external direct mixing is at home and abroad rarely reported especially.Feng Zuohua etc. find the effect that this mixture has tangible anti-H22 hepatocarcinoma with tumor-cell antigen peptide and the HSP70 that purification obtains after external the mixing.Michael W.Graner etc. also find to purify and obtain the growth that tumor antigen-heat shock protein complex in the tumor cell lysate can significantly suppress 12B1 lymphocytoma and B16 melanocytoma.
Two above-mentioned researchs are known research meanses the most approaching with this research.This research need not to carry out the complexity purification of tumor antigen peptide, just can use in external direct mixing with HSP65 by the resulting full tumor cell lysate of the method for easy multigelation.Therefore, it is more simple to compare top method.And include more tumor antigen in the full tumor cell lysate of using in our research, experimental results show that it has the effect of better activated immune system, inhibition growth of tumour cell than the antigen in the simple tumor cell cracking supernatant.We used HSP65, HSP70 are all the tubercule bacillus source in addition.Very all studies have shown that, HUMAN HEAT SHOCK PROTEINS Hhsp HSP70 with present widespread usage compares, HSP65, the HSP70 in tubercule bacillus source have stronger intersection and offer antigenic ability, MHI class angtigen presentation approach can be better exogenous antigen be inducted into, and then antineoplastic immune cell, performance antitumor immune function can be effectively activated.More than these novel part and main innovate point, guaranteed several researchs of this research compared with the front, will have better antineoplaston effect and potential applicability in clinical practice.
Vocabulary of terms among the present invention:
" tumor ": tumor (tumor) and cancer (cancer) can be exchanged use in this manual, have identical implication.Cancer cell is not subjected to Growth Control, and the tissue around can attacking also forms transfer (metastasize), jeopardizes individual life.Tumor comprises the tumor of solid tumor (solidtumors/malignancies), soft tissue sarcoma (soft tissue sarcomas) and marrow sample or lymphoid system (myeloid or lymphoidsystems).These tumors are: esophageal carcinoma, carcinoma of gallbladder, bladder cancer is (as transitional, squamous cell carcinoma), breast carcinoma (as ductal and lobular adenocarcinoma), colon cancer (as epithelial adenocarcinoma), colorectal cancer, adrenal cortical tumor, renal carcinoma (as renal cell carcinoma), hepatocarcinoma is (as hepatoma, cholangiocarcinoma), pulmonary carcinoma is (as small and large cell adenocarc inomas, squamous cell carc inoma, bronchoalveolarcarc inoma, non smal lcell lung cancer), ovarian cancer is (as epithelial adenocarcinoma, ovarian follicle cancer.ovarian germcell cancer), cervical cancer, uterus carcinoma, endometrial cancer, cancer of vagina, cancer of the vulva, cancer of pancreas (as pancreatic ductal adenocarcinomas), rectal cancer, carcinoma of prostate (as prostatic adenocarcinoma), gastric cancer, skin carcinoma is (as malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma.basal cell carcinoma, hemangiopericytoma), the cerebral tumor is (as asneuroblastomas, astrocytic brain tumors, astrocytoma, gliomas, oligodendroglioma, ependymoma, neuroblastoma, medulloblastomas, metastatic tumor cell invasion of the central nervous system), mesothelioma, Kaposi ' s sarcoma, bone tumor (as osteomas), sarcoma is (as fibrosarcoma, osteosarcoma), carcinoma of penis, retinoblastoma, carcinoma of testis, thyroid carcinoma, trophoblastic neoplasms, rhabdomyosarcoma, teratocarcinoma, Wilms ' tumor.Tumor also refers to lymph sample and marrow sample hematopoietic system cancer, includes but not limited to leukemia, lymphoma and myeloma.These diseases comprise: acute lymphoblastic leukemia (acute lymphocytic leukemia), acute nonlymphocytic leukemia (acute nonlymphocytic leukemia), chronic lymphocytic leukemia (chronic lymphocytic leukemia), chronic myeloid cell leukemia (chronic myelocytic leukemia), the T chronic myeloid leukemia, hairy cell leukemia, acute myeloid leukaemia (acute myeloid leukemia), erythroleukemia, chronic myelocytic leukemia (chronic granulocytic leukemia), comprise the Huo Jiejin lymphomas, Huo Jie king's evil lymphomas, acute myelogenousleukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, lymphosarcoma cell leukemia, polymorphonuclear cell leukemia (prolymphocytic leukemia, PLL), B cell large celllymphoma, malignant lymphoma, B cell lymphoma, t cell lymphoma, B cell acute lymphoblastic leukemia (cell-acute lymphocytic leukemia, B-ALL), B cell chronic lymphocytic leukemia (B-cell chronic lymphocytic leukemia, B-CLL), small lymphocyte lymph pomegranate, B cell lymphoblast leukemia (B-cell prolymphocytic leukemia), fluffy shape cell lymphoma (Mantle cell lymphoma), folliculus shape lymphoma (Follicular lymphoma), outside lymph node marginal zone B cell lymphoma (Extranodal marginal zone B-cell lymphomaof MALT type), lymphoma nodal marginal zone B cell (Nodal marginal zone B-cell lymphoma, splenic marginal zone B lymphoma (Splenic marginal zone lymphoma), plasmocytoma/plasma cell myeloma (Plasmacytoma/plasma cell myeloma), dispersivity large B cell lymphoid tumor (Diffuse large B-cell lymphoma), Mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma (intravascular large B-cell lymphoma), lymphoma primary effusion (primary effusionlymphoma), Burkitt lymphoma (Burkitt ' s lymphoma), multiple myeloma, cutaneous B-cell lymphoma, constitutional folliculus cutaneous B-cell lymphoma, B cell Fei Huojie king's evil lymphomas, lymphoplasma cytoid lymphoma, mucosa-associatedlymphoid tissue (MALT) lymphoma, constitutional thyroid lymphoma, intravascular malignant lymphomatosis, the chrotoplast lymphoma, intragraft angiotropic large-cell lymphoma.
" tumor cell ": in this patent, tumor cell comprises autologous tumor cell, allogeneic tumor cell, allogeneic tumor cell, and these tumor cells can be the tumor cell of the primary tumo(u)r of individuality, the tumor cell of secondary tumor, the tumor cell or the tumor cell line cell of metastatic carcinoma.
" tumor cell lysate ": tumor cell lysate is meant the lysate with the broken preparation of tumor cell.The method of broken tumor cell includes but not limited to multigelation, supersound process, Mechanical Crushing etc.
" tumor cell of genetic modification ": the gene that is used for modifying tumor cell include but not limited to the to encode encoding gene of MHC molecule, cytokine, collaborative stimulation molecule and tumor antigen, these genes can be the encoding genes of total length, also can be the parts of total length encoding gene.Transgene method includes but not limited to the method for chemistry, physical method and viral vector mediation, as calcium phosphate method, liposome method, electrotransfer method (electroporation), pressure-mediated gene transfer (pressure mediated transfer).Can be used for genetically modified vector virus has: and retrovirus, adenovirus, adenovirus relevant carriers (adenovirus-associated vectors, AAV), herpesvirus and poxvirus etc.In addition, coding MHC molecule, cytokine, collaborative stimulation molecule and tumor antigen DNA and RNA also can be used to transfection tumor cell.Compare with the tumor cell of unmodified, the tumor cell of genetic modification may obtain stronger immunogenicity, more effectively excites the antineoplastic immunne response.
" MHC molecule ": the MHC molecule is main tissue compatible complex (major histocompatibiltiy complex, MHC) the protide molecule of gene code.The MHC molecule comprises MHC I quasi-molecule and MHC II quasi-molecule.Change the encoding gene of MHC molecule over to immunogenicity (immunogenity) that tumor cell may strengthen tumor cell.
" cytokine ": cytokine (Cytokine) is the protide molecule with immunoregulatory activity.The cytokine that can be used for modifying tumor cell includes but not limited to IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-28, IL-29, G-CSF, GM-CSF, interferon-ALPHA, interferon beta, interferon gamma, tumor cell necrosin and various chemotactic factor.
" collaborative stimulation molecule ": collaborative stimulation molecule (co-stimulaory molecules) is to be expressed in antigen presenting cell (antigenpresenting cells, APC) or the protide molecule of tumor cell surface, in the process of immunne response, provide and activate CD4+T lymphocyte and lymphocytic second activation signal of CD8+T.Change the encoding gene of collaborative stimulation molecule over to immunogenicity (immunogenity) that tumor cell may strengthen tumor cell.Can be used for modifying the including but not limited to of collaborative stimulation molecule of tumor cell: CD1a, CD1b, CD1c, CD2, CD9, CD10, CD11a, CD11b, CD11c, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD40, CD70, CD80, CD83, CD86, CD137, LFA-1, LFA-2, LFA-3, VLA-1, VCAM-1, ICAM-2, VCAM-2 and chemokine receptors etc.
" TOLL sample receptor stimulating agent ": CpG ODN is the agonist of Toll sample receptor (Toll like receptors) 9.Toll sample receptor is the receptor family of a microbe-derived molecular structure of identification (pathogen correlation molecule model).The immunocyte of expressing Toll sample receptor is by being activated in conjunction with this pathogen correlation molecule model.The mankind, Toll sample receptor family has 11 members.Toll sample receptor can be discerned the material in a lot of pathogenic microorganisms source, and these materials all are TOLL sample receptor stimulating agents.As, TLR4 can discern bacteria lipopolysaccharide (LPS); The binary of TLR2 and TLR6 can be discerned lipoteichoic acid and two acyl lipopeptid; The binary of TLR2 and TLR1 can be discerned the trigalloyl lipopeptid; TLR9 can discern synthetic or derive from antibacterial and virus contains CpG oligonucleotide (CpG ODN); Imiquimod also is the agonist of TLR9; TLR5 can discern bacterial flagellin; The binary of TLR2 and TLR6 can also be discerned zymosan; TLR4 can identification of breathing road syncystial virus F protein; TLR3 can discern Poly I:C (poly IC), synthetic or derive from virus double-stranded RNA; TLR7 and TLR8 can discern the strand viral RNA; TLR4 can discern the protozoon product, as the GPI anchorin; TLR4 can also discern the product of inflammation tissue, as HSP60 and the former fragment of fiber (Foo Y.Liew, et al.Negative regulation of toll like receptor mediated immune responsesNature Reviews Immunology.Vol 5, June 2005,446-458).Studies confirm that, the TLR9 agonist can activate natural immunity reaction and the acquired immune response (Arthur M.Krieg.Therapeutic potential of Toll-like receptor 9activation.Nature Reviews Drug Discovery, Vol 5.June 2006,471-484).In the mankind's immune system, B cell and plasma cell sample dendritic cell (pDCs) are expressed TLR9.The oligonucleotide (CpG ODN) that contains CpG is the agonist of TLR9 (D.M.Klinman, Immunotherapeutic uses of CpG oligodeoxynucleotides, 428 Nat.Rev., Immunol.4 (2004) 249-258).The TLR9 agonist can be activated TLR9 to intercellular substance by endocytosis.In plasma cell sample dendritic cell, TLR9 activates and can open beginning reaction of the natural immunity fast, it is characterized in that secreting a large amount of proinflammatory factors such as IL-6, tumor necrosis factor-alpha (TNF α), I type interferon (IFN-a/ β) and interferon-induced chemotactic factor.Rely on and two paths of the non-dependence of interferon by interferon, these plasma cell sample dendritic cell can secondary activate natural immunity cell such as NK cell, mononuclear cell and neutrophil cell.By the stimulation of TLR9, the B cell has strengthened the sensitivity to antigenic stimulus, can be divided into antibody secreting cell effectively.Therefore, the stimulation of TLR9 helps acquired immunity to reply, particularly humoral immunoresponse(HI).Can secrete IFN α by the activated plasma cell sample of TLR9 dendritic cell, this IFN α can make the migration of plasma cell sample dendritic cell gather lymph node and other peripheral lymphoid tissues.At these positions, these plasma cell sample dendritic cell can activate unsensitized T cell and memory T cell, intersect to the CD8+ cytotoxic T cell and offer the soluble antigen friendship, promote the CD4 and the CD8 t cell responses of TH1 type.
" materia medica acceptable carrier ": materia medica acceptable carrier (pharmaceutically acceptable carrier) is meant filler, diluent or the encapsulating substance of one or more solids or liquid, and this carrier can be organic, inorganic, natural or synthetic.This carrier comprises reagent of various solution, diluent, solvent, dispersant, liposome, emulsifying agent, antibacterial, antifungal, isoosmotic and delayed absorption etc.The carrier that injectable is used comprises water, normal saline, PBS buffer (phosphate buffered saline solution), balanced salt solution, glucose solution, glycerol and emulsifying agent and wetting agent etc.Emulsifying agent can comprise oil-water emulsifiers (oil/water emulsion) and water-in-oil emulsifier (o water/oil emulsion).
" treatment effective dose ": this treatment effective dose is meant after application, can stimulate anti-tumor immune response behind individuality, produces the tulase heat shock protein of oncotherapy effect and the dosage of tumor cell lysate.This " dosage " how much be decided by the standard technique that those skilled in the art should be known, also to comprise being not limited to individual size, the order of severity of health condition and disease etc. with reference to other factors.Dosage range from 1 μ g to the 100mg/ pin.In order to reach ideal therapeutic effect, those skilled in the art can adjust this application dose, and its dosage range can be 10 times to 1000 times of aforementioned range.The dosage of the tumor cell lysate in the mixture of tulase heat shock protein and tumor lysate is from containing from 1 * 10
6Individual tumor cell to 1 * 10
10The tumor cell lysate of preparation in the scope of individual tumor cell.In order to reach ideal therapeutic effect, those skilled in the art can adjust the application dose of this tumor cell lysate, and its dosage range can be 10 times to 1000 times of aforementioned range.
" pharmaceutical composition ": the mixture of acceptable carrier composition on the mixture that pharmaceutical composition (pharmaceutical composition) refers among the present invention the tulase heat shock protein of treatment effective dose and tumor cell lysate and the materia medica.Pharmaceutical composition can but to be not limited to be aqueous solution, saline solution and powder.The application mode of pharmaceutical composition can be to inject outside intestinal.Injection medicine compositions of the present invention comprises pharmaceutically acceptable aqueous solution, non-aqueous solution, dispersant, suspension, Emulsion.The mixture that acceptable carrier is formed on the mixture of tulase heat shock protein and tumor lysate and the materia medica also can be prepared to powder (Powder).The powder of the mixture of tulase heat shock protein and tumor lysate can be through lyophilizing or the preparation of vacuum drying method.Before injection, learn the acceptable carrier dissolving with aseptic water for injection or other medicines.In some cases, in order to prolong the action effect of the mixture of tulase heat shock protein and tumor lysate among the present invention, pharmaceutical composition is used after can being processed over suitable sustained release system.The mixture of tulase heat shock protein provided by the invention and tumor lysate also can with other anti-tumor agent use in conjunction, its mode is that the mixture of (1) tulase heat shock protein and tumor lysate and other anti-tumor agent mix before application; (2) mixture of tulase heat shock protein and tumor lysate and other anti-tumor agent were used in the different time; (3) mixture of tulase heat shock protein and tumor lysate and other anti-tumor agent mix before application by different route of administration application; (4) mixture of tulase heat shock protein and tumor lysate and other anti-tumor agent are formed pharmaceutical composition (pharmaceuticalcomposition) application.
" anti-tumor agent ": anti-tumor agent (anti-tumor agent) can be treated tumor and/or be excited the preparation of anti-tumor immune response, include but not limited to chemotherapeutics, cytokine, anti-tumour antibody, epithelical cell growth factor receptor (EGFR)-tyrosine kinase (TK) activation inhibitor, tumor cell mutain tyrosine kinase inhibitor, tumor vessel inhibitor, the RNA interferences, TLR agonist and nonspecific immunity strengthening agent and etc.
" chemotherapeutics ": chemotherapeutics is meant the chemicals that is used for oncotherapy.Chemotherapeutics includes but not limited to chlormethine, cyclophosphamide, nitrocaphanum, mustine hydrochlcride, carmustine, ring second Nitrosourea, thiophene is for group, Busulfan, cisplatin, nitrosourea, 5-fluorouracil, cytosine arabinoside, methotrexate, purinethol, Ismipur, hydroxyurea, amycin, epirubicin, daunorubicin, mitoxantrone, mitomycin is widely collected mycin, mitomycin C, actinomycin D, vincristine, vincaleucoblastine, vindesine, tricuspid sugi gum alkali, camptothecine, etoposide, maytansine, Elemenum Emulsion, tamoxifen, adrenocortical hormone, procarbazine, hydroxymethyl benzyl hydrazine, L-asparaginase, platinum class (cisplatin, carboplatin, JM-216), dacarbazine, hexamethylmelamine, taxotere or train beautiful Qu Sai (Alimta), nvelbine, xeloda, irinotecan, topotecan, JM-216, Docetaxel (Docetaxel), the chemotherapeutics of use in conjunction such as taxol (paclitaxel)+cisplatin, taxol+carboplatin, strong (the gemcitabine hydrochloride)+cisplatin of selecting, taxotere (Docetaxel)+cisplatin, taxol D, be good for and select+cisplatin cisplatin (Cisplatin)+vinorelbine (vinorelbine), gemcitabine (gemcitabine)+carboplatin (Carboplatin), paclitaxel (paclitaxel)+carboplatin.
" anti-tumour antibody ": the mixture of tulase heat shock protein provided by the invention and tumor lysate can with the anti-tumour antibody use in conjunction, these antibody include but not limited to HERCEPTIN (Clin Cancer Res.2006 Jul 15; 12 (14 Pt 2): 4436s-4440s), Rituximab (Rituxan.RTM.) (Blood.69:584-591,1987), anti--CTLA-4 antibody, anti-CD 40 antibodies, anti-EGF receptor antibody and anti-vegf receptor antibody etc.
" epithelical cell growth factor receptor (EGFR)-tyrosine kinase (TK) activation inhibitor ": the epithelical cell growth factor that tumor cell such as lung carcinoma cell are expressed is a receptor tyrosine kinase, and the preparation that suppresses this kinase activity can suppress to comprise the growth of lung carcinoma cell.This type of preparation include but not limited to gefitinib or Iressa (Gefitinib, Iressa) and erlotinib (Erlotinib, Tarceva) (Bezjak, A., et al.J Clin Oncol 24:3831-3837 2006).
" tumor cell mutain tyrosine kinase inhibitor " is meant the preparation that suppresses tumor cell mutain tyrosine kinase activity.This type of preparation includes but not limited to imatinib mesylate (Gleevec or STI-571 or Imatinib mesylate).Imatinib mesylate is mutein (Bcr-Abl) tyrosine kinase phosphorylation inhibitor (Rubin BP, Duensing A.Mechanisms of resistance to smallmolecule kinase inhibition in the treatment of solid tumors.Lab Invest.2006 Aug 21).
" tumor vessel inhibitor " is meant by suppressing tumor vessel formation and brings into play anti-tumor preparation.This class preparation includes but not limited to Avastin (Avastin) and grace degree (ENDOSTAR).Avastin is in conjunction with the gene engineering monoclonal antibody of VEGF (VEGF) (Gridelli C, et al.New antiangiogenetic agents and non-small cell lung cancer.Crit Rev OncolHematol.2006 Jul 12).The grace degree is a recombinant human vascular endothelial inhibin.
It is to adopt single stranded RNA to disturb the phenomenon of cognate rna function that " RNA interferences ": RNA disturbs (RNA interference is called for short RNAi).This single stranded RNA with RNA interference effect is also referred to as the RNA interferences.The various RNA interferences of keeping the tumor cell malignant phenotype of can disturbing all can produce antineoplastic action.
" nonspecific immunity strengthening agent ": the preparation that is meant the individual anti tumor immune response of the non-special enhancing of energy, these preparations comprise list but are not limited to bacillus calmette-guerin vaccine (Bacillis Calmette-Gruen, BCG), coryne bacterium parvum (Corynabacterium parvum, staphylococcus aureus, the CD40 part, bacterial ribosome extract (Ribosomal fractions), bacterial membrane extract (membranar bacterial fractions), bacterial membrane proteoglycanes, polyarginine, HSP-60, HSP-70, HSP-90 and CTLA-4 inhibitor etc.
Description of drawings
The full Lewis tumor cell lysate associating of Fig. 1 HSP65 albumen anti-Lewis pulmonary carcinoma presses down the tumor curve
Abscissa is the natural law after the injection tumor, and ordinate is a tumor size.The result shows: full Lewis tumor cell lysate associating HSP65 protein groups can obviously suppress tumor growth (P＜0.01).
The full Lewis tumor cell lysate associating of Fig. 2 HSP65 albumen anti-Lewis pulmonary carcinoma survival curve
Abscissa is the natural law after the injection tumor, and ordinate is tumor-bearing mice survival rate (percentage rate).The result shows: full Lewis tumor cell lysate associating HSP65 protein groups can obviously prolong tumor-bearing mice life cycle (P＜0.01).
The anti-B16 melanoma of the full B16 tumor cell lysate associating HSP65 albumen of Fig. 3 presses down the tumor curve
Abscissa is the natural law after the injection tumor, and ordinate is a tumor size.The result shows: full B16 tumor cell lysate associating HSP65 protein groups can obviously suppress tumor growth (P＜0.05).
The anti-B16 melanoma of the full tumor cell lysate associating HSP65 albumen of Fig. 4 survival curve
Abscissa is the natural law after the injection tumor, and ordinate is the tumor-bearing mice survival rate.The result shows: full B16 tumor cell lysate associating HSP65 protein groups can obviously prolong tumor-bearing mice life cycle (P＜0.05).
The melanomatous effective ingredient of the full tumor cell lysate associating anti-B16 of HSP65 albumen of Fig. 5 gropes to press down the tumor curve
Abscissa is the natural law after the injection tumor, and ordinate is a tumor size.The result shows: full B16 tumor cell lysate associating HSP65 protein groups can obviously suppress tumor growth (P＜0.01), and its tumor killing effect is better than B16 tumor cell lysate supernatant associating HSP65 protein groups (P＜0.05).Wherein, " Seedling " represents the tumor lysate.
The melanomatous effective ingredient of the full tumor cell lysate associating anti-B16 of HSP65 albumen of Fig. 6 is groped survival curve
Abscissa is the natural law after the injection tumor, and ordinate is the tumor-bearing mice survival rate.The result shows: full B16 tumor cell lysate associating HSP65 protein groups can obviously prolong tumor-bearing mice life cycle (P＜0.05), and its effect is better than B16 tumor cell lysate supernatant associating HSP65 protein groups (P＜0.05).Wherein, " Seedling " represents the tumor lysate.
Fig. 7: the agarose gel electrophoresis of pET28a-HSP70 plasmid enzyme restriction product
Fig. 8: 12%SDS-PAGE behind the HSP70 purification
Preclinical influence takes place to mice B16 subcutaneous transplantation tumor in the full tumor cell lysate associating of Fig. 9 HSP70 albumen
Abscissa is the natural law after the injection tumor, and ordinate is that tumor takes place by incubation period.The result shows: full B16 cell lysate associating HSP70 can prolong tumor significantly take place incubation period (P＜0.05).
The anti-mice B16 of the full tumor cell lysate associating HSP70 albumen of Figure 10 subcutaneous transplantation tumor presses down the tumor curve
Abscissa is the natural law after the injection tumor, and ordinate is a tumor size.The result shows: full B16 cell lysate associating HSP70 protein groups can obviously suppress tumor growth (P＜0.05).
The anti-mice B16 of the full tumor cell lysate associating HSP70 albumen of Figure 11 subcutaneous transplantation tumor survival curve
Abscissa is the natural law after the injection tumor, and ordinate is the mice ratio for survival.The result shows: full B16 cell lysate associating HSP70 protein groups can obviously prolong tumor-bearing mice life cycle (P＜0.05).
The full tumor cell lysate associating of Figure 12 HSP70 albumen anti-mice B16 intraperitoneal transplantation tumor survival curve (preventative)
Abscissa is the natural law after the injection tumor, and ordinate is an existence mice ratio in the tumor-bearing mice.The result shows: full B16 cell lysate associating HSP70 protein groups can obviously prolong tumor-bearing mice life cycle (P＜0.05).
The full tumor cell lysate associating of Figure 13 HSP70 albumen anti-mice B16 intraperitoneal transplantation tumor survival curve (therapeutic)
Abscissa is the natural law after the injection tumor, and ordinate is an existence mice ratio in the tumor-bearing mice.The result shows: full B16 cell lysate associating HSP70 protein groups can obviously prolong tumor-bearing mice life cycle (P＜0.05).
The specific embodiment
Material:
The Lewis cell, tulase HSP65 albumen (all deriving from the Bethune of Jilin University medical college molecular biology teaching and research room), Nacl solution (production of Inner Mongol Tian Yuan Pharma Inc.), the 1ml syringe, ice chest, dried Cotton Gossypii, the IMDM culture fluid, aseptic plate, the 50ml centrifuge tube, glue head dropper, filter screen, the tumor tissues dismembyator, gauze, balance, C57BL/6 mice (purchasing laboratory animal company limited) in Beijing dimension tonneau China, mouse cage, label, feedstuff (purchasing basis animal housing) in Jilin University, bedding and padding (purchasing basis animal housing) in Jilin University, slide gauge
Method:
1) acquisition of full tumor cell lysate: with 2.2 * 10
7The Nacl of Lewis lung cancer cell after with high pressure make 1.1ml cell suspension, behind-70 ℃ and 37 ℃ of multigelations 5 times (freeze 15min, melt 5min, shake mixing therebetween), with the blue dyeing of platform phenol to determine the cracking situation.Then whole tumor cell cracking mixed liquors are put in 4 ℃ of refrigerators, are used for immunity behind the 17-18h.
2) preparation of tumor cell: with tumor tissues aseptic taking-up in super-clean bench of tumor-bearing mice, the 2g tumor tissues weighs with scale.The tumor tissues that takes out is ground with the tumor tissues dismembyator, behind gauze and strainer filtering, make single cell suspension with 12ml serum-free IMDM suspension and be used for inoculation, the quantity of inoculation is 0.5g/15, and inoculation position is that right hind is subcutaneous.
3) full tumor cell lysate is united the interior experiment of body that HSP65 albumen suppresses Lewis lung cancer: at the close subcutaneous injection 0.033g/0.2mL IMDM Lewis of about 0.7cm place of the root of the tail portion tumor tissues in C57BL/6 right side of mice back of the body bottom, the skin mound of diameter 6～8mm appears in injection site, is considered as inoculating successfully.Experiment grouping: 40 mices are divided into 4 groups at random, 10 every group mices (1) Nacl solvent matched group: the-7, injected 150 μ L Nacl solution in 1,8,15 day in the subcutaneous groin of mice right hind place; (2) full tumor cell lysate associating HSP65 protein for treatment group: the-7,1,8,15 day in the subcutaneous groin of mice right hind place injection 1 * 10
6Full tumor cell lysate+10 μ g HSP65/150 μ LNacl solution; (3) full tumor cell lysate treatment group: the-7,1,8,15 day in the subcutaneous groin of mice right hind place injection 1 * 10
6Full tumor cell lysate/150 μ L Nacl solution; (4) HSP65 protein for treatment group: the-7, injected 10 μ g HSP65/150 μ L Nacl solution in 1,8,15 day in the subcutaneous groin of mice right hind place.Measure gross tumor volume every 2d after the administration.Gross tumor volume (V) by formula V=1/2ab2 (the a=tumor is long, and the b=tumor is wide) calculates, and draws tumor growth curve, observes survival time of mice.
The result: full Lewis tumor cell lysate associating HSP65 protein groups gross tumor volume has significant difference (P＜0.01), no difference of science of statistics between other 3 groups since 12 days with other three groups; Compared significant difference (P＜0.01) with other 3 groups the life cycle of full Lewis tumor cell lysate associating HSP65 protein groups mice.Prompting tumor cell lysate associating HSP65 can suppress the growth (Fig. 1) of Lewis lung cancer glucagonoma, the life cycle (Fig. 2) of significant prolongation tumor-bearing mice simultaneously significantly.
Conclusion: the effect that full Lewis tumor cell lysate associating HSP65 has anti-Lewis pulmonary carcinoma.
The anti-B16 melanoma of embodiment 2 full tumor cell lysate associating tulase HSP65 albumen
Material:
B16 cell, HSP65 albumen (all deriving from the Bethune of Jilin University medical college molecular biology teaching and research room), Nacl solution (production of Inner Mongol Tian Yuan Pharma Inc.), 1ml syringe, ice chest, dried Cotton Gossypii, IMDM culture fluid, 50ml centrifuge tube, glue head dropper, balance, C57BL/6 mice (purchasing laboratory animal company limited), mouse cage, label, feedstuff (purchasing basis animal housing), bedding and padding (purchasing basis animal housing), slide gauge in Jilin University in Jilin University in Beijing dimension tonneau China
Method:
1) acquisition of full B16 tumor cell lysate: with 2.2 * 10
7The Nacl of B16 melanoma cell after with high pressure make 1.1ml cell suspension, behind-70 ℃ and 37 ℃ of multigelations 5 times (freeze 15min, melt 5min, shake mixing therebetween), with the blue dyeing of platform phenol to determine the cracking situation.Then whole tumor cell cracking mixed liquors are put in 4 ℃ of refrigerators, are used for immunity behind the 17-18h.
2) full B16 tumor cell lysate is united HSP65 albumen and is suppressed experiment in the melanomatous body of B16: at the close root of the tail portion in C57BL/6 right side of mice back of the body bottom about 0.7cm place subcutaneous injection 3 * 10
5/ 0.2mL IMDM B16 melanoma cell, the skin mound of diameter 6～8mm appears in injection site, is considered as inoculating successfully.Experiment grouping: 40 mices are divided into 4 groups at random, 10 every group mices (1) Nacl solvent matched group: the-7, injected 150 μ LNacl solution in the subcutaneous groin of mice right hind place in 1,8,15 day; (2) full B16 tumor cell lysate associating HSP65 protein for treatment group: the-7,1,8,15 day in the subcutaneous groin of mice right hind place injection 1 * 10
6Full B16 tumor cell lysate+10 μ gHSP65/150 μ L Nacl solution; (3) full B16 tumor cell lysate treatment group: the-7,1,8,15 day in the subcutaneous groin of mice right hind place injection 1 * 10
6Full B16 tumor cell lysate/150 μ L Nacl solution; (4) HSP65 protein for treatment group: the-7, injected 10 μ g HSP65/150 μ L Nacl solution in 1,8,15 day in the subcutaneous groin of mice right hind place.Measure gross tumor volume every 2d after the administration.Gross tumor volume (V) by formula V=1/2ab2 (the a=tumor is long, and the b=tumor is wide) calculates, and draws tumor growth curve, observes survival time of mice.
The result:
Conclusion: full B16 tumor cell lysate associating HSP65 has the melanomatous effect of anti-B16.
The melanomatous effective ingredient of the embodiment 3 full tumor cell lysate associating anti-B16 of HSP65 albumen is groped
Material:
B16 cell, HSP65 albumen (all deriving from the Bethune of Jilin University medical college molecular biology teaching and research room), Nacl solution (production of Inner Mongol Tian Yuan Pharma Inc.), 1ml syringe, ice chest, dried Cotton Gossypii, IMDM culture fluid, 50ml centrifuge tube, glue head dropper, balance, C57BL/6 mice (purchasing laboratory animal company limited), mouse cage, label, feedstuff (purchasing basis animal housing), bedding and padding (purchasing basis animal housing), slide gauge in Jilin University in Jilin University in Beijing dimension tonneau China
Method:
1) acquisition of full B16 tumor cell lysate: with 4.4 * 10
7The Nacl of B16 melanoma cell after with high pressure make 2.2ml cell suspension, behind-70 ℃ and 37 ℃ of multigelations 5 times (freeze 15min, melt 5min, shake mixing therebetween), with the blue dyeing of platform phenol to determine the cracking situation.Then whole tumor cell cracking mixed liquors are put in 4 ℃ of refrigerators, are used for immunity behind the 17-18h.
2) full B16 tumor cell lysate is united HSP65 albumen and is suppressed experiment in the melanomatous body of B16: at the close root of the tail portion in C57BL/6 right side of mice back of the body bottom about 0.7cm place subcutaneous injection 3 * 10
5/ 0.2mL IMDM B16 melanoma cell, the skin mound of diameter 6～8mm appears in injection site, is considered as inoculating successfully.Experiment grouping: 40 mices are divided into 4 groups at random, 10 every group mices (1) Nacl solvent matched group: the-7, injected 150 μ L Nacl solution in 1,8,15 day in the subcutaneous groin of mice right hind place; (2) full B16 tumor cell lysate associating HSP65 protein for treatment group: the-7,1,8,15 day in the subcutaneous groin of mice right hind place injection 1 * 10
6Full B16 tumor cell lysate+10 μ gHSP65/150 μ L Nacl solution; (3) full B16 tumor cell lysate treatment group: the-7,1,8,15 day in the subcutaneous groin of mice right hind place injection 1 * 10
6Full B16 tumor cell lysate/150 μ L Nacl solution; (4) HSP65 protein for treatment group: the-7, injected 10 μ g HSP65/150 μ L Nacl solution in 1,8,15 day in the subcutaneous groin of mice right hind place.(5) B16 tumor cell lysate supernatant associating HSP65 protein for treatment group: the-7,1,8,15 day in the subcutaneous groin of mice right hind place injection 1 * 10
6B16 tumor cell cracking supernatant+10 μ gHSP65/150 μ L Nacl solution; (6) B16 tumor cell lysate supernatant treatment group: the-7,1,8,15 day in the subcutaneous groin of mice right hind place injection 1 * 10
6Tumor cell lysate supernatant/150 μ L Nacl solution.Measure gross tumor volume every 2d after the administration.Gross tumor volume (V) by formula V=1/2ab2 (the a=tumor is long, and the b=tumor is wide) calculates, and draws tumor growth curve, observes survival time of mice.
The result:
Full B16 tumor cell lysate associating HSP65 protein groups gross tumor volume has been compared significant difference since 10 days with Nacl solvent matched group (P＜0.01), full B16 tumor cell lysate group (P＜0.01), B16 tumor cell lysate supernatant associating HSP65 protein groups (P＜0.05); Full B16 tumor cell lysate associating HSP65 protein groups survival time of mice has been compared significant difference (P＜0.05) with Nacl solvent matched group, full B16 tumor cell lysate group, B16 tumor cell lysate supernatant associating HSP65 protein groups, HSP65 protein groups, compares no difference of science of statistics with B16 tumor cell lysate supernatant group.Point out full B16 tumor cell lysate associating HSP65 can suppress the melanomatous growth of B16 (Fig. 5) significantly, unite HSP65 and unite the effect (Fig. 5) that HSP65 has the melanomatous growth of more significant inhibition B16 than tumor cell lysate supernatant the life cycle (Fig. 6) of significant prolongation tumor-bearing mice, and full B16 tumor cell lysate simultaneously.
Conclusion:
Full B16 tumor cell lysate associating HSP65 has the melanomatous effect of anti-B16; B16 tumor cell lysate supernatant associating HSP65 also has anti-B16 melanoma effect; Full B16 tumor cell lysate associating HSP65 has stronger anti-B16 melanoma effect than B16 tumor cell lysate supernatant associating HSP65.
1. separate the HSP70 encoding gene: contain the recombiant plasmid pET28a-HSP70-P16 of HSP70 structural gene, teaching and research room provides by the Bethune of Jilin University medical college molecular biosciences.Adopt PCR method from pET28a-HSP70-P16 amplification HSP70 encoding gene.5 ' end the primer sequence that adopts is 5 ' ATAATTACCATGGCTCGTGCGGTCGGGATCG 3 ', and 3 ' end primer sequence is 5 ' ATATTATAAGCTTGGCCTCCCGGCCGTC 3 ', reaction condition: 940C, 45 "; 620C, 1 '; 740C, 2 ', behind 28 cycle periods, 720C extends 10min.5 of PCR product ' end has Nco I restriction enzyme site, and 3 ' end has Hind III restriction enzyme site.The sequence of bacillus calmette-guerin vaccine (BCG) the HSP70 gene that obtains is SEQ ID NO:1.
2. clone PCR products: adopt the TA cloning process, the PCR product cloning of step 1 is gone into the TA carrier, obtain recombiant plasmid pMD-18T-HSP70.Method sees that TA connects test kit (Promega), recombinant plasmid transformed host bacterium JM109 (available from Invitrogen company), getting positive colony cultivates, (Joseph.Sambrook according to a conventional method, David W.Russell.SDS alkaline lysis prepares plasmid DNA, Molecularcloning:A Laboratory Manual, 3rd ed.27-30, Cold Spring Harbor Laboratory Press, 2001) extract plasmid, adopt the terminal cessation method of two deoxidations, to insert fragment carry out sequencing (ABIPrism310TM, USA).After sequencing is correct, utilize NcoI and HindIII point of contact with its sub-clone to pET28a (+) carrier (Novagen), obtain pET28a (+)-HSP70 plasmid.Transform host bacterium JM109, get positive transformed bacteria and cultivate, extract plasmid, adopt the terminal cessation method of two deoxidations, to insert fragment carry out sequencing (ABIPrism310TM, USA).
Expression and the purification of embodiment 5 reorganization HSP70
Expression plasmid pET28a (+)-HSP70 conversion that builds is entered e. coli bl21 (DE3) competent cell that has prepared, coating contains the LB flat board of 100 μ g/ml kanamycin, transforming picking monoclonal on the flat board then, be inoculated in the 3ml LB fluid medium, the 200rpm shaken cultivation is spent the night on 37 ℃ of constant temperature shaking tables, with the alkaline lysis extracting and detect plasmid (Fig. 7).The monoclonal point compound plate on the LB flat board that contains 100 μ g/ml kanamycin that conclusive evidence is contained expression plasmid pET28a (+)-HSP70 is preserved.
Select the expression engineering bacteria that a strain is proved conclusively, adopt 10 liters fermentation tank, cultivate and use the iptg abduction delivering with the 1b culture medium.Adopt height to crush the bacterium method, under 500～600bar pressure, the broken bacterium of circulation 4～5 times, centrifugal receipts supernatant, supernatant is by the nickel affinity chromatography, and Q-Sepharose FF chromatography is passed through in SuperdexG-25 ultrafiltration desalination at last, the HSP70 albumen (molecular weight is 70KD) that obtains recombinating, the proteic purity of reorganization HSP70 is about 96.5%.(Fig. 8)
The anti-mice B16 of embodiment 6 full tumor cell lysate associating HSP70 albumen subcutaneous transplantation tumor
Material:
B16 cell, HSP70 albumen, PBS solution (this chamber preparation), ADP2Na (purchasing company), MgCl2 (purchasing), 1ml syringe, ice chest, dried Cotton Gossypii, IMDM culture fluid, 50ml centrifuge tube, glue head dropper, balance, C57BL/6 mice (18-22g purchases the laboratory animal company limited in Beijing dimension tonneau China), mouse cage, label, feedstuff (purchasing basis animal housing), bedding and padding (purchasing basis animal housing), slide gauge in Jilin University in Jilin University in the Beijing Chemical Plant in sigma
Method:
1) acquisition of full B16 cell lysate: with 2 * 10
7The PBS of B16 melanoma cell after with high pressure make 1ml cell suspension, behind-70 ℃ and 37 ℃ of multigelations 5 times (freeze 15min, melt 5min, shake mixing therebetween), with the blue dyeing of platform phenol to determine being stored in-70 ℃ after the cracking situation.
2) respectively organize the acquisition of vaccine: following formulation is respectively organized vaccine, is used for immunity behind the back 37 ℃ of water-bath 1h of mixing before immunity.
3) full B16 cell lysate associating HSP70 albumen suppresses experiment in the melanomatous body of B16: experiment grouping: 48 C57BL/6 mices are divided into 4 groups at random: (1) PBS solvent matched group (PBS, 16): 1st, 15 days in the subcutaneous groin of mice right hind place injection 100ul PBS solvent (the PBS solution that contains 1mM ADP2Na and 1mM MgCl2); (2) full B16 cell lysate associating HSP70 protein groups (HSP70-tumor lysate, 8): 1st, 15 days in the subcutaneous groin of mice right hind place injection 1 * 10
6Full B16 tumor cell lysate+100 μ gHSP70/100 μ l PBS solvents; (3) full B16 cell lysate group (Tumor lysate, 16): 1st, 15 days in the subcutaneous groin of mice right hind place injection 1 * 10
6Full B16 tumor cell lysate/100 μ l PBS solvents; (4) HSP70 protein groups (HSP70,8): 1st, injected 100 μ g HSP70/100 μ l PBS solvents in 15 days in the subcutaneous groin of mice right hind place.The 18th day at each the close root of the tail portion in group right side of mice back of the body bottom about 0.7cm place subcutaneous injection 2 * 10
5/ 0.2mL IMDM B16 cell, the skin mound of diameter 6～8mm appears in injection site, is considered as inoculating successfully.After the big I of tumor is touched, measure gross tumor volume every 2d.Gross tumor volume (V) by formula V=1/2ab2 (the a=tumor is long, and the b=tumor is wide) calculates, and draws tumor growth curve, observes survival time of mice.
The result: HSP70-tumor lysate group tumor takes place incubation period and PBS group and tumor lysate group significant difference (P＜0.05) is arranged (Fig. 9), HSP70-tumorlysate group tumor size had significant difference (P＜0.05), no difference of science of statistics (Figure 10) between other each group with PBS group and tumorlysate group in 11 days behind tumor inoculation; HSP70-tumor lysate group tumor-bearing mice life cycle and PBS group and tumor lysate group have significant difference (P＜0.05), no difference of science of statistics (Figure 11) between other each group.Point out full B16 cell lysate associating HSP70 can prolong the B16 tumor significantly take place incubation period, significantly suppress growth of tumor, simultaneously the life cycle of significant prolongation tumor-bearing mice.
Conclusion: the effect that full B16 tumor cell lysate associating HSP70 has preventative anti-mice B 16 subcutaneous transplantation tumors.
The anti-mice B16 of embodiment 7 full tumor cell lysate associating HSP70 albumen intraperitoneal transplantation tumor
Material:
B16 cell, HSP70 albumen, PBS solution (this chamber preparation), ADP2Na (purchasing company), MgCl2 (purchasing), 1ml syringe, ice chest, dried Cotton Gossypii, IMDM culture fluid, 50ml centrifuge tube, glue head dropper, balance, C57BL/6 mice (18-22g purchases the laboratory animal company limited in Beijing dimension tonneau China), mouse cage, label, feedstuff (purchasing basis animal housing), bedding and padding (purchasing basis animal housing) in Jilin University in Jilin University in the Beijing Chemical Plant in sigma
Method:
1) full tumor cell lysate reaches and respectively organizes the acquisition of vaccine with embodiment 6.
2) full tumor cell lysate associating HSP70 albumen suppresses experiment in the melanomatous prevention gonosome of B16:
Experiment grouping: 32 C57BL/6 mices are divided into 4 groups at random, 8 every group: (1) PBS solvent matched group (PBS): 1st, 15 days in the subcutaneous groin of mice left hind place injection 100ul PBS solvent (the PBS solution that contains 1mM ADP2Na and 1mM MgCl2); (2) full B16 cell lysate associating HSP70 protein groups (HSP70-tumor lysate): 1st, 15 days in the subcutaneous groin of mice left hind place injection 1 * 10
6Full B16 tumor cell lysate+100 μ g HSP70/100 μ l PBS solvents; (3) full B16 tumor cell lysate group (Tumor lysate): 1st, 15 days in the subcutaneous groin of mice left hind place injection 1 * 10
6Full B16 tumor cell lysate/100 μ l PBS solvents; (4) HSP70 protein groups (HSP70): 1st, injected 100 μ g HSP70/100 μ l PBS solvents in 15 days in the subcutaneous groin of mice left hind place.Under each group mice left lower abdomen peritoneum, injected 7.5 * 10 on the 18th day
4/ 0.2ml IMDM B16 melanoma cell is observed survival time of mice.
Result: HSP70-tumorlysate group tumor takes place incubation period and PBS group and tumorlysate group significant difference (P＜0.05) is arranged, the HSP70 group has significant difference (P＜0.05) with PBS group, tumor lysate and HSP70-tumor lysate, no difference of science of statistics (Figure 12) between other each group.Point out full B16 tumor cell lysate associating HSP70 can prolong the life cycle of tumor-bearing mice significantly.
Conclusion: the effect that full B16 cell lysate associating HSP70 has preventative anti-mice B16 intraperitoneal transplantation tumor.
3) full B16 cell lysate associating HSP70 albumen suppresses experiment in the melanomatous treatment gonosome of B16:
Experiment grouping: 40 C57BL/6 mices are divided into 4 groups at random, 10 every group.Test in the prevention gonosome together for every group group and each assembly side.(1) under each group mice left lower abdomen peritoneum, injected 7.5 * 10 in the 0th day
4/ 0.2ml IMDM B16 cell; (2) each group was injected corresponding prescription reagent at the 1st, 4,8 day in the subcutaneous groin of mice left hind place respectively, observed survival time of mice.
The result: HSP70-tumor lysate group survival time of mice and PBS group have significant difference (P＜0.05), no difference of science of statistics between other each group.Point out full B16 cell lysate associating HSP70 can prolong the life cycle (Figure 13) of tumor-bearing mice significantly.
Conclusion: the effect that full B16 cell lysate associating HSP70 has curative anti-mice B16 intraperitoneal transplantation tumor.
Material:
G422 Mus neuroglial cytoma (purchasing willing strong Instr Ltd.) in Shanghai, tulase HSP65 albumen (deriving from the Bethune of Jilin University medical college molecular biology teaching and research room), Nacl solution (production of Inner Mongol Tian Yuan Pharma Inc.), the 1ml syringe, ice chest, dried Cotton Gossypii, the IMDM culture fluid, aseptic plate, the 50ml centrifuge tube, glue head dropper, filter screen, the tumor tissues dismembyator, gauze, balance, Balb/c mice (purchasing laboratory animal company limited) in Beijing dimension tonneau China, mouse cage, label, feedstuff (purchasing basis animal housing) in Jilin University, bedding and padding (purchasing basis animal housing) in Jilin University, slide gauge
Method:
1) acquisition of full tumor cell lysate: with 2.2 * 10
7The Nacl of G422 Mus neuroglial cytoma after with high pressure make 1.1ml cell suspension, behind-70 ℃ and 37 ℃ of multigelations 5 times (freeze 15min, melt 5min, shake mixing therebetween), with the blue dyeing of platform phenol to determine the cracking situation.Then whole tumor cell cracking mixed liquors are put in 4 ℃ of refrigerators, are used for immunity behind the 17-18h.
2) preparation of tumor cell: with tumor tissues aseptic taking-up in super-clean bench of tumor-bearing mice, the 2g tumor tissues weighs with scale.The tumor tissues that takes out is ground with the tumor tissues dismembyator, behind gauze and strainer filtering, make single cell suspension with 12ml serum-free IMDM suspension and be used for inoculation, the quantity of inoculation is 0.5g/15, and inoculation position is that right oxter is subcutaneous.
3) full tumor cell lysate associating HSP65 albumen suppresses the interior experiment of body of G422 Mus neuroglial cytoma: at the right oxter injection of BALB/C mice 0.033g/0.2mL IMDM G422 Mus neuroglial cytoma.
Experiment grouping: 40 mices are divided into 4 groups at random, 10 every group mices (1) Nacl solvent matched group: the-7, injected 150 μ L Nacl solution in 1,8,15 day in the subcutaneous groin of mice right hind place; (2) full tumor cell lysate associating HSP65 protein for treatment group: the-7,1,8,15 day in the subcutaneous groin of mice right hind place injection 1 * 10
6Full tumor cell lysate+10 μ gHSP65/150 μ LNacl solution; (3) full tumor cell lysate treatment group: the-7,1,8,15 day in the subcutaneous groin of mice right hind place injection 1 * 10
6Full tumor cell lysate/150 μ LNacl solution; (4) HSP65 protein for treatment group: the-7, injected 10 μ gHSP65/150 μ LNacl solution in 1,8,15 day in the subcutaneous groin of mice right hind place.
The result: compared significant difference (P＜0.01) with other 3 groups the life cycle of G422 tumor cell lysate associating HSP65 protein groups mice.Prompting tumor cell lysate associating HSP65 can the significant prolongation tumor-bearing mice life cycle (table 1).
Conclusion: corresponding full tumor cell lysate associating HSP65 has the effect of anti-G422 Mus neuroglial cytoma growth.
Table 1G422 tumor cell lysate associating HSP65 albumen is to the influence of G422 tumor mice life span
| Group | Number of animals (n) | Life cycle (my god) |
| Full tumor cell lysate associating HSP65 protein for treatment group | ??10 | ??85.1±10.2 |
| Full tumor cell lysate treatment group | ??10 | ??58.6±7.7 |
| HSP65 protein for treatment group | ??10 | ??55.5±7.8 |
| Nacl solvent matched group | ??10 | ??30.4±6.3 |
1, the preparation of tumor cell lysate
EL4 cell (C57BL/6 mouse lymphoma cell) is derived from American Type Culture Collection (ATCC).Employing contains 10% deactivation FCS, 20 μ g/ml gentamycins, and the IMDM culture medium of 1mM HEPES (Gibco company), at 37 ℃, 5%CO
2Condition under cultivate.With EL4 cell (1 * 10
7/ ml) be suspended among the 1ml PBS (phosphate-buffered saline), do 4 and take turns freeze thawing treatment.Method is, liquid nitrogen freezing is freezing, and 37 ℃ melt.With cell lysate through supersound process, further cracking.320g, 4 ℃, centrifugal 10 minutes.Receive supernatant ,-70 ℃ of preservations.Contain from 1 * 10 among the 100 μ l PBS
6The tumor cell lysate of EL4 cell preparation.
2, the anti-EL4 cytosis of tumor cell EL4 cell lysate associating HSP65
Adopt the C57BL/6 male mice (Beijing Vital River Experimental Animals Technology Co., Ltd.) in 6～8 ages in week to carry out the antineoplastic experiment.In EL4 tumor cell lysate associating HSP65 group mice group, in the 0th day, the 7th day and the 14th day at back, right side subcutaneous injection 100 μ l EL4 cell lysates and HSP65 mixture (containing 10 μ g HSP65).If EL4 cell lysate matched group, HSP65 albumen matched group and PBS matched group.Tumor cell (EL4 cell) lysate control group mice: inject 100 μ l EL4 cells.HSP65 albumen control group mice: inject the PBS that 100 μ l contain 10 μ g HSP.PBS control group mice: inject 100 μ l PBS.In the 18th day, at C57BL/6 right side of mice back subcutaneous injection 0.1ml EL4 cell suspension.Behind inoculated tumour, begin to observe and write down the life cycle of mice.
The influence of table 2EL4 cell lysate associating HSP65 to EL4 cell tumor formation rate
| Group | Number of animals (n) | Life cycle (my god) |
| EL4 tumor cell lysate associating HSP65 group | ??20 | ??45.5±4.9 |
| EL4 cell lysate matched group | ??20 | ??28.1±4.4 |
| HSP65 albumen matched group | ??20 | ??27.4±3.7 |
| ??PBS | ??20 | ??21.2±5.5 |
The result shows that tumor cell lysate associating HSP65 group is compared significant prolongation life cycle, P＜0.05 with matched group.The resistive connection intestinal cancer effect of embodiment 10 colon cancer cell lysates associating HSP65
1, the preparation of tumor cell lysate
The CMT93 cell is the colon cancer cell (mouse rectal carcinoma cell line) (purchasing in ATCC) in C57BL/6 mice source.With the hyclone that contains 10% (v/v) deactivation and antibiotic (the IMDM culture fluid of the streptomycin of 100IU penicillin/ml and 100IU/ml), at 37 ℃, 5%CO
2Condition under cultivate.With CMT93 cell (1 * 10
7/ ml) be suspended among the 1ml PBS (phosphate-buffered saline), do 4 and take turns freeze thawing treatment.Method is, methanol dry ice is freezing, and 37 ℃ melt.Also can adopt liquid nitrogen freezing.The cell lysate that generates is dyeed with platform phenol is blue, detect no complete cell at microscopically.320g, 4 ℃, centrifugal 10 minutes.Receive supernatant ,-70 ℃ of preservations.Contain from 1 * 10 among the 100 μ l PBS
6The tumor cell lysate of CMT93 cell preparation.
2, the resistive connection intestinal cancer effect of colon cancer cell lysate associating HSP65
Adopt C57BL/6 female mice (Beijing Vital River Experimental Animals Technology Co., Ltd.) to carry out the antineoplastic experiment.Tumor cell CMT93 cell lysate associating HSP65 group mice, in the 0th day and the 7th day at back, right side subcutaneous injection 0.1ml CMT93 cell lysate and HSP65 mixture (containing 10 μ g HSP65 albumen), if CMT93 cell lysate matched group, HSP65 matched group and PBS matched group.Tumor cell (CMT93 cell) lysate control group mice: inject 100 μ l tumor cell (CMT93 cell) lysates.HSP65 control group mice: inject 100 μ l and contain the proteic PBS of 10 μ g HSP65.PBS control group mice: inject 100 μ l PBS.In the 14th day, with 2.5 * 10
6The CMT93 cell inoculation is subcutaneous to mice (6～8 ages in week, female) back, right side.From the major diameter (long diameter) and the minor axis (short diameter) that 10th day begin with slide gauge (0.002cm) measure tumor piece of injection after the tumor.Tumor size (cm
2)=major diameter (cm) * minor axis (cm).Table 3 expression be the 20th day measurement result behind the inoculated tumour.
Table 3 colon cancer cell lysate associating HSP65 is to the inhibitory action of colon cancer cell growth
| Group | Number of animals (n) | Tumor size (cm 2，x±s) |
| CMT93 cell lysate associating HSP65 | ??18 | ?0.316±0.158 |
| HSP65 albumen | ??18 | ?0.789±0.403 |
| The CMT93 cell lysate | ??20 | ?0.689±0.303 |
| ??PBS | ??20 | ?0.954±0.309 |
The result shows that HSP65 is after being applied to mice for the associating of colon cancer cell lysate, can produce the effect of significant resistive connection intestinal cancer.Colon cancer cell lysate associating HSP65 compares P＜0.05 with matched group.
Sequence table
＜110〉Beijing DiWeiHuaYu Biological Technology Co., Ltd
＜120〉mixture of a kind of tulase HSP and tumor lysate
<160>1
<170>Patent?In?version?3.1
<210>1
<211>1880
<212>DNA
<213>Artificial
<400>1
ccatggctcg?tgcggtcggg?atcgacctcg?ggaccaccaa?ctccgtcgtc?tcggttctgg????60
aaggtggcga?cccggtcgtc?gtcgccaact?ccgagggctc?caggaccacc?ccgtcaattg????120
tcgcgttcgc?ccgcaacggt?gaggtgctgg?tcggccagcc?cgccaagaac?caggcggtga????180
ccaacgtcga?tcgcaccgtg?cgctcggtca?agcgacacat?gggcagcgac?tggtccatag????240
agattgacgg?caagaaatac?accgcgccgg?agatcagcgc?ccgcattctg?atgaagctga????300
agcgcgacgc?cgaggcctac?ctcggtgagg?acattaccga?cgcggttatc?acgacgcccg????360
cctacttcaa?tgacgcccag?cgtcaggcca?ccaaggacgc?cggccagatc?gccggcctca????420
acgtgctgcg?gatcgtcaac?gagccgaccg?cggccgcgct?ggcctacggc?ctcgacaagg????480
gcgagaagga?gcagcgaatc?ctggtcttcg?acttgggtgg?tggcactttc?gacgtttccc????540
tgctggagat?cggcgagggt?gtggttgagg?tccgtgccac?ttcgggtgac?aaccacctcg????600
gcggcgacga?ctgggaccag?cgggtcgtcg?attggctggt?ggacaagttc?aagggcacca????660
gcggcatcga?tctgaccaag?gacaagatgg?cgatgcagcg?gctgcgggaa?gccgccgaga????720
aggcaaagat?cgagctgagt?tcgagtcagt?ccacctcgat?caacctgccc?tacatcaccg????780
tcgacgccga?caagaacccg?ttgttcttag?acgagcagct?gacccgcgcg?gagttccaac????840
ggatcactca?ggacctgctg?gaccgcactc?gcaagccgtt?ccagtcggtg?atcgctgaca????900
ccggcatttc?ggtgtcggag?atcgatcacg?ttgtgctcgt?gggtggttcg?acccggatgc????960
ccgcggtgac?cgatctggtc?aaggaactca?ccggcggcaa?ggaacccaac?aagggcgtca????1020
accccgatga?ggttgtcgcg?gtgggagccg?ctctgcaggc?cggcgtcctc?aagggcgagg????1080
tgaaagacgt?tctgctgctt?gatgttaccc?cgctgagcct?gggtatcgag?accaagggcg????1140
gggtgatgac?caggctcatc?gagcgcaaca?ccacgatccc?caccaagcgg?tcggagactt????1200
tcaccaccgc?cgacgacaac?caaccgtcgg?tgcagatcca?ggtctatcag?ggggagcgtg????1260
agatcgccgc?gcacaacaag?ttgctcgggt?ccttcgagct?gaccggcatc?ccgccggcgc????1320
cgcgggggat?tccgcagatc?gaggtcactt?tcgacatcga?cgccaacggc?attgtgcacg????1380
tcaccgccaa?ggacaagggc?accggcaagg?agaacacgat?ccgaatccag?gaaggctcgg????1440
gcctgtccaa?ggaagacatt?gaccgcatga?tcaaggacgc?cgaagcgcac?gccgaggagg????1500
atcgcaagcg?tcgcgaggag?gccgatgttc?gtaatcaagc?cgagacattg?gtctaccaga????1560
cggagaagtt?cgtcaaagaa?cagcgtgagg?ccgagggtgg?ttcgaaggta?cctgaagaca????1620
cgctgaacaa?ggttgatgcc?gcggtggcgg?aagcgaaggc?ggcacttggc?ggatcggata????1680
tttcggccat?caagtcggcg?atggagaagc?tgggccagga?gtcgcaggct?ctggggcaag????1740
cgatctacga?agcagctcag?gctgcgtcac?aggccactgg?cgctgcccac?cccggcggcg????1800
agccgggcgg?tgcccacccc?ggctcggctg?atgacgttgt?ggacgcggag?gtggtcgacg????1860
acggccggga?ggccaagctt????????????????????????????????????????????????1880
Claims (20)
1, a kind of preparation method that is used for the treatment of the preparation of tumor, it is characterized in that tulase heat shock protein and tumor cell lysate are mixed and made into mixture, be used for the treatment of suffer from tumor cell lysate on pathology for the individuality of of a sort tumor, here " being same class source tumor on the pathology with tumor cell lysate " is meant that the tumor that is used to prepare tumor cell lysate or tumor cell line tumor and the individual tumor of suffering from are to belong to of a sort tumor in the pathology classification, and " individuality " described here is people or vertebrates.
2, the method for claim 1, wherein " being same class source tumor with tumor cell lysate on pathology " comprises that autologous tumor, allogeneic tumor, xenograft tumor, the cell of these tumors can be the tumor cell of the primary tumo(u)r of individuality, the tumor cell of secondary tumor, the tumor cell or the tumor cell line cell of metastatic carcinoma.
3, the method for claim 1, wherein said tulase heat shock protein includes but not limited to tulase heat shock protein HSP65 or tulase heat shock protein HSP70.
4, the method for claim 1, wherein said tulase heat shock protein is one or more mixing.
5, the method for claim 1, wherein said tulase heat shock protein be the tulase heat shock protein in whole or in part.
6, the method for claim 1, wherein said tumor is a melanoma.
7, the method for claim 1, wherein said tumor is a lung tumors.
8, the method for claim 1, wherein said tumor is a glioma.
9, the method for claim 1, wherein said tumor cell lysate is whole compositions of tumor cell lysate or through the active ingredient of separation and purification, and the effective ingredient here is made up of in the following composition one or more: protein, nucleic acid, lipid, saccharide.
10, the method for claim 1, wherein said tumor cell lysate derive from genetic modification from body or allogeneic tumor cell, the used gene of the tumor cell of genetic modification comprises the encoding gene of MHC molecule, cytokine, collaborative stimulation molecule, tumor antigen.
11, the method for claim 1, the mixture that wherein said tulase heat shock protein and tumor cell lysate are mixed and made into are to use separately or and other one or more anti-tumor agent use in conjunction performance antitumor actions in being used for the treatment of the process of tumor.
12, the anti-tumor agent described in the claim 11 includes but not limited to chemotherapeutics, cytokine, anti-tumour antibody, epithelical cell growth factor receptor (EGFR)-tyrosine kinase (TK) activation inhibitor, tumor cell mutain tyrosine kinase inhibitor, tumor vessel inhibitor, RNA interferences, Toll sample receptor stimulating agent, nonspecific immunity strengthening agent.
13, the method for claim 1, during one or more anti-tumor agent use in conjunction performance antitumor actions of the mixture that wherein said tulase heat shock protein and tumor cell lysate are mixed and made into and other, can also and following tumor therapeuticing method in one or more use in conjunction: surgical operation, radiotherapy, interventional therapy, physical therapy.
14, in the described method of claim 1, the application dose of the mixture that tulase heat shock protein wherein and tumor cell lysate are mixed and made into is the treatment effective dose.
15, in the described method of claim 1, mixture that tulase heat shock protein wherein and tumor cell lysate are mixed and made into and the carrying of materia medica acceptable carrier are used.
16, among claim 1, the 10-15 in arbitrary described method, the mixture that tulase heat shock protein and tumor cell lysate are mixed and made into is used through intestinal external administration approach when giving individuality, that these approach comprise is subcutaneous, in the muscle, abdominal cavity, sheath, in the Intradermal, tumor, injection by the tumor, in the lymph node, the lymph node sidenote is penetrated or suck through respiratory tract.
17, among claim 1, the 10-15 in arbitrary described method, the mixture that tulase heat shock protein and tumor cell lysate are mixed and made into is to use as the active ingredient in pharmaceutical composition.
18, in claim 1, the arbitrary described method of 10-15, the mixture that tulase heat shock protein and tumor cell lysate are mixed and made into is the mixture of one or more tulase heat shock proteins and a kind of tumor cell lysate or several tumor cell lysates.
19, as in claim 1, the arbitrary described method of 10-15, when wherein said mixture gives individuality again, can with the adjuvant use in conjunction.
20, the method for claim 1, the wherein said tumor cell that is used to prepare tumor cell lysate includes but not limited to lymphoma, leukemia, hepatocarcinoma, colorectal cancer.
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| WO2024192034A3 (en) * | 2023-03-13 | 2024-12-05 | Corner Therapeutics, Inc. | Bystander antigen enhancement of tumor cell lysate immunogenicity |
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| CN101161288A (en) * | 2006-10-13 | 2008-04-16 | 长春华普生物技术有限公司 | Enganced tumour cell cracking article of oligonucleotide and method for treating knub |
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| CN101161288A (en) * | 2006-10-13 | 2008-04-16 | 长春华普生物技术有限公司 | Enganced tumour cell cracking article of oligonucleotide and method for treating knub |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2024192034A3 (en) * | 2023-03-13 | 2024-12-05 | Corner Therapeutics, Inc. | Bystander antigen enhancement of tumor cell lysate immunogenicity |
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