CN1201369A - Stimulation of cell-mediated immune responses by targeted particulated genetic immunization - Google Patents
Stimulation of cell-mediated immune responses by targeted particulated genetic immunization Download PDFInfo
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- CN1201369A CN1201369A CN96198106A CN96198106A CN1201369A CN 1201369 A CN1201369 A CN 1201369A CN 96198106 A CN96198106 A CN 96198106A CN 96198106 A CN96198106 A CN 96198106A CN 1201369 A CN1201369 A CN 1201369A
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Abstract
The present invention relates to various methods of genetic immunization for the purpose of providing antigen-specific immunity in a mammalian host, including a human host. The invention is based on the ability to direct particulate polynucleotides which express an antigenic protein or protein fragment to the cytoplasm of host target cells, such as antigen presenting cells. A directed delivery of such particulate polynucleotides to the cytoplasm of antigen presenting cells will stimulate antigen-specific CTL production, thus promoting destruction of affected cells such as neoplastic cells and virally infected cells.
Description
1. foreword
For carrying out genetic immunization, be core of the present disclosure place at the mammalian body internal stimulus antigen specific immune that comprises the human host.This specification discloses the target cell to the host, as the kytoplasm transmission particle polynucleotides of antigen presenting cell.These particle polynucleotide encodings can be near the antigen protein or the antigen protein fragment of target cell kytoplasm.The expression of antigen gene causes antigen specific immune reaction, and it comprises but is not only limited to the inducing action of (CTL) of the former specificity cell toxicity T lymphocyte of antagonism.Antigen is able to by endogenous MHC I approach antigen-presenting peptide on film near kytoplasm.Film by endogenous MHC I approach is presented the inducing action that can stimulate the antigen specific CTL.Derivative then antigentic specificity CTL can lead and destroy the host cell that influenced by antigen presentation, for example cell of neoplastic cell or virus infections.2. background of invention
Cytotoxic T lymphocyte (CTL) is antitumor or the key composition of effective human immunity reaction of virus infections.Cytotoxic T lymphocyte destroys neoplastic cell or virus infected cell by MHC I molecular presentation in the lip-deep antigenic peptides of infected target cell by identification.These antigenic peptides are the catabolites that are present in the exogenous proteins in the cytosol of infected cell, and are wherein processed and be and pass CTL by endogenous MHC I processing approach.
Though MHC I molecular recognition extraneous protein may be enough to discern and destroy infected target cell by CTL, also some other signal need be arranged from T lymphocyte precursor inducing antigen-specific CTL.The antigen presenting cell of specialization (APC) can provide the needed antigen induction period-MHC I part and the auxiliary signal of CTL mediation immunity.The general feature of APC comprises that MHC I and II express, to the expression of the various adhesion molecules of APC-lymphocytes interactions particular importance, and the expression of costimulatory molecules such as CD 80 and CD 86.The example of APC comprise macrophage and dendritic cell (comprise the bright Chinese Schwann Cells of epidermis of skin, the dendritic cell of skin, and lymph node and and spleen in dendritic cell).
Carry out immunity inoculation in the body with killed tumour cell, virus infected cell or protein component, effort with the inducing antigen-specific ctl response does not generally all have successfully, infers that its reason is that protein in the extracellular fluid can not enter cytosol and present approach near MHC I.
The genetic immunization method has several attractive features.Several vivo gene transfer methods that comprise retrovirus or adenovirus mediated gene transfer and directly inject naked DNA have caused transgene expression (relevantly to comment referring to Krishnaw etc., 1995, nature medical science (NatureMed.) 1:521-522 and Pardoll etc., 1995, immunity (Immunity) 3:165-169).
People such as Williams (1991, studies show that NAS's journal (Proc.Natl.Acad.Sci.USA) 88:2726-2730) causes the expression of protein luciferase in the integral skin cell after the firefly luciferase genes is sent in biology bombardment (biobalistic).Be not devoted to ctl response in these researchs, do not consider the directed cell-mediated immunoreactive specificity host cell that produces yet.
People such as Tang (1992, nature (Nature) 356:152-154) utilize biological bombardment (biobalistic) to install the humoral response that produces exogenous proteins.The hGH encoding gene that will be under the control of CMV promotor or beta-actin promotor is sent to the epidermal tissue of mouse.Detect the anti-hGH antibody that produces in the mouse reaction of body inherence to this immunologic process.People such as Tang are the openly genetic immunization of the immunization route of cell guiding mediation not.People such as Tang do not have open or the directly genetic immunization of guiding APC cell of prompting.
People such as Fynan (1993, the journal 90:11478-11482 of NAS) use the plasmid DNA construction body of encoding influenza virus hemagglutinin glycoprotein further to confirm people's such as Tang discovery.People such as Fynan will by particle gun to epidermis DNA delivery bag by the humoral response that gold grain produced and other mechanism contrast; and the discovery biological bombardment of use (biobalistic) device 1) generation is to 95% protective effect of lethal influenza viruse attack; 2) be the valid approach the most of dna immunization inoculation; prove its in fact than in mucous membrane, the muscle or intravenous administration more effective, and 3) the DNA requirement lacks 250 to 2500 times than salt solution inoculation.People such as Fynan are not open or point out the APC cell that directly leads to carry out genetic immunization.Do not mention the immunity of CTL mediation.
Liu and colleague thereof (Montgomery etc., 1993, DNA cell biology (DNACell Biol.) 12:777-783; Ulmer etc., 1993, science (Science) .259:1745-1749; Donnelly etc., 1995, natural medical science 1:583-587) but proved the antigentic specificity ctl response of nondirectional non-specific intramuscular injection naked DNA inducing anti-disease poisonous protein and the protective immunity of antiviral attack.These researchs are not disclosed as carries out genetic immunization to APC directional transmissions genetic stocks.
People such as Sun (1995, the journal 92:2889-2893 of NAS) utilize biological bombardment (biobalistic) device to produce antitumor reaction in the mouse body.The author has directly transported the plasmid construction body of expressing IL-6 to the mouse interior tumor position.The expression of IL-6 provides a kind of non-specific cytokine gene therapy that is oriented to tumour of form.There is not the antigen specific immune of proposition at tumour.People such as Sun are not open or prompt for genetic immunization and direct target APC cell.
People such as Kundig (1995, science, 268:1343-1346) proof, it is very crucial that proteantigen is positioned on the lymphoid organ for inducing antigen-specific ctl response in the body.But do not study genetic immunization.
Kovacsovics-Bankowski and Rock (1995, science 267:243-246) prove that usually the phagosome of the proteantigen that can not present by the endogenous approach of MHC I is to the cytosol approach.The author infers that can enter MHCI on the protein fact of particle form of internalization in the phagosome presents required kytoplasm approach.But this research does not indicate genetic stocks complete on the function carries out cytosol to the kytoplasm approach by phagosome ability.
People such as Falo (1995, nature medical science 1:649-653) prove that directly the delivery of particles proteantigen can produce the antineoplastic immune that antigentic specificity CTL mediates in the mouse body in animal body, thereby provide foundation for the phagosome in the support to kytoplasm approach.They prove that if with the particle form administration, protein is injected directly in the animal body, its phagosome that can enter APC specifically is to the kytoplasm approach.But this literary composition does not describe the genetic immunization method in detail.Do not point out the remains material that gives in the body by this approach functional completeness enter the ability of APC or other types cell cytoplasm.
Pardoll and Beckerleg (1995, immune 3:165-169) have commented the immunology of naked DNA vaccine recently.They point out emphatically the importance of the dna immunization inoculation mechanism of further determining present the unknown.Specifically, their conclusion is: " mechanism of illustrating the reaction of naked DNA activate immunity is very important.Have only by these research, could introduce flexibly and modify, " to make it on quality and quantity, bringing into play maximum function.
Although put down in writing many achievements in research in the above-mentioned list of references, can be but still need to develop at particular cell types in the host, with the immunity of stimulator antigen specific CTL mediation, and promote the genetic immunization method of direct destruction host's internal specific neoplastic cell or virus infected cell conversely.The present invention notes and has satisfied this needs.3. brief summary of the invention
The present invention relates to treatment or preventative genetic immunization to mammalian hosts, this method comprises the dna fragmentation of the target cell transfer encoding antigen protein in mammalian body, dna fragmentation in the host cell inner expression reorganization, and continue after by this host cell antigen-presenting peptide, thereby the immunity or the humoral immunity of irritation cell mediation, or both.
The invention further relates to treatment or preventative genetic immunization to mammalian hosts, it comprises that the specificity target cell in the mammalian hosts body transmits the dna sequence dna of coding for antigens protein or its biological active fragment.The antigenic peptide of expressing from this dna fragmentation is to infected cell-specific, and continue back stimulator antigen specific CTL generation, thereby promotes the destruction of the cell of infected cell such as neoplastic cell and virus infections.
The invention still further relates to the particle polynucleotides and carry out genetic immunization and seeded with mammalian host, in the cytosol of target cell, transmit these transgenosis polynucleotides then based on particle.In case enter within the target cell, the particle polynucleotides are promptly expressed certain protein or its biological active fragment, thereby produce suitable antigenic peptide fragment, and are presented on the target cell membrane by endogenous MHC I approach.Suitably presenting the purpose antigenic peptides by MHC I approach can stimulate CTL to produce and and then promotion neoplastic cell or the isocellular destruction of virus infected cell.
Therefore, the invention still further relates to method in treatment or the mammiferous body of preventative genetic immunization, this method comprises the dna fragmentation that produces antigen expressed protein or antigen protein fragment, this dna fragmentation is distributed on the particle surface that produces the particle polynucleotides, be sent in the kytoplasm of mammal target cell with said particle polynucleotides seeded with mammalian host and with these particle polynucleotides, thereby expressed antigen protein or antigen protein fragment be presented on the film surface of said target cell by MHC I approach.
Preferred mammalian hosts is the people among the present invention.
In disclosed each embodiment of this specification, preferably interested dna fragmentation expresses 1) tumor rejection antigen or antigen protein fragment, or 2) viral antigen or antigen protein fragment.The people TRA example that can utilize in the present invention comprises but is not only limited to MAGE-1, MAGE3, Melan-A, gp100, p53, CEA and HER2/neu.The example that can be used for viral antigen of the present invention comprises but is not only limited to HIV gp120, HIV gp160, influenza virus nucleoprotein and hepatitis B surface antibody.
Preferred target cell is APC among the present invention, and preferred orientation of APC target cell or migration position are human host's lymphatic tissues.
In one embodiment of the invention, utilize the microparticle bombardment device to use particle polynucleotides immunity mammal.Specifically, by biological bombardment method with the inoculation of particle number thuja acid with immune mammalian hosts, thereby the particle polynucleotides are entered in the kytoplasm of proper number host cell at least.Express transgenic polynucleotides on the biology level of significance are so that antigenic peptide fragment is presented to endogenous MHC I approach and represents on the film surface of host cell.The endogenous host film that is imported into antigen is presented the generation of inducing that promotes antigentic specificity CTL, and the latter enters particularly human host's circulation of mammalian hosts, to destroy the cell of neoplastic cell or virus infections.
In a particular of the present invention, use particularly people of particle polynucleotides immunity mammalian hosts through the microparticle bombardment inoculation method, be by making the particle polynucleotides enter proper number host cell at least with the emitting substance approach, comprising the kytoplasm of APC as the direct result of non-specific emitting substance bombardment.When bombardment skin, the APC of the skin that can be bombarded comprises but is not only limited to the bright Chinese Schwann Cells of epidermis, horn cell or skin dendritic cell.When the bombardment lymphatic tissue, the lymphatic tissue that can be bombarded comprises but is not only limited to resident dendritic cell, macrophage, stroma cell, T lymphocyte or bone-marrow-derived lymphocyte.
In another embodiment of the invention, with for example hypodermic injection of direct injection, epidermis injection, corium injection, lymph injection and intravenous injection, with particle polynucleotides immunity mammalian hosts.The particle polynucleotides enter host cell and with effective horizontal expression biologically, the antigenic peptide fragment be presented to endogenous MHC I approach and represent on the film surface of host cell.The endogenous target cell membrane is presented the inducing action that can promote the antigen specific CTL, and the latter is circulated in whole mammalian hosts, better is the human host, to destroy the cell of neoplastic cell or virus infections.
In a particularly preferred embodiment, through hypodermic injection particle poly thuja acid is sent in human host's body, to cytosol approach guiding APC, and express it with the biology level of significance by phagosome by APC.The invention discloses by the subcutaneous vaccination approach and directly inject, transmit the particle polynucleotides and in lymphatic tissue, carry out antigen presentation to the APC orientation.
In another particularly preferred embodiment of the present invention, send the particle polynucleotides of codes for tumor rejection antigen (TRA) or its biological active fragment through the hypodermic injection APC that directly leads.Directly send TRA particle poly thuja acid by this way and can make the tumour specific antigen peptide farthest enter the I classpath, and avoid the transposition in non-APC cell of a large amount of particles.
In another particularly preferred embodiment of the present invention, send the particle polynucleotides of coding viral antigen or its biological active fragment through the hypodermic injection APC that directly leads.The particle polynucleotides of directly sending the coding viral antigen by this way can make the virus-specific antigenic peptides enter the I classpath most possibly, and can avoid the transposition in non-APC cell of a large amount of particulate antigens.
Those skilled in the art will be appreciated that, the particle of being made up of various materials comprises but be not only limited to gold, iron and synthetic plastic, can be near the phagosome that be used for the present invention's guiding APC to the kytoplasm approach.
In another embodiment of the invention, through injection, include but not limited to hypodermic injection, epidermis injection, the injection of corium skin, lymph injection and intravenous injection, with being incubated the back altogether in the homology APC of external loading antigen immunity mammalian hosts with the particle polynucleotides.The particle polynucleotides for example can through microparticle bombardment or by phagosome to the kytoplasm approach at the external APC that enters.Specifically, with the immune mammalian hosts of the APC of particle polynucleotides transfection.The particle polynucleotides promptly in the external APC that enters specifically, can be expelled to this APC in the host then like this.After taking in, on the biology level of significance, express these transgenosis polynucleotides, thereby the antigenic peptide fragment and represents on the film surface of APC by the processing of endogenous MHC I approach with present by APC.Behind these APC of injection, the endogenous APC cell membrane of antigen is presented the inducing action that promptly promotes antigentic specificity APC in injection site or lymphatic tissue.Derivative antigentic specificity CTL itself is again in the particularly human host's body-internal-circulation of whole mammalian hosts, to destroy the cell of neoplastic cell or virus infections.
In another embodiment of the invention, through injecting the injection of for example hypodermic injection, epidermis, the injection of corium skin, lymph injection and intravenous injection, with in external by the homology APC of particle polynucleotides transfection immunity mammalian hosts.Specifically, the APC immunity mammalian hosts that transforms with particle polynucleotides so that the particle polynucleotides are in the external APC that enters, and then is expelled to this APC in the host.For example can microprojectile bombardment methods or make the particle polynucleotides in the external APC of entering through phagosome to kytoplasm approach.In the polynucleotides of external use coding for antigens and/or encode that one or more can increase the polynucleotides transfection APC of molecule of the effectiveness of APC antigen presentation function.Such molecule comprises but is not only limited to cell factor and costimulatory molecules.The example of cell factor comprises but is not only limited to IL-12, IL-2 and IL-4.The example of costimulatory molecules comprises but is not only limited to CD 80 and CD 86.After entering APC, the polynucleotides of coding transgenosis antigen are expressed on the biology level of significance, so promptly cross by endogenous MHC I approach processing antigen-presenting fragments of peptides and make it to be presented on the film surface of APC also.Behind these APC of injection, the endogenous APC cell membrane of antigen is presented the inducing action that promptly promotes antigentic specificity CTL in injection site or the lymphatic tissue.Derivative antigentic specificity CTL this in mammalian hosts human host's systemic circulation particularly, to destroy the cell of neoplastic cell or virus infections.After entering APC, the transgenic cell factor and/or costimulating factor coded polynucleotide are able to be expressed on the biology level of significance, cause the antigentic specificity reaction in the injection site or in lymphatic tissue with the antigen presentation function that causes APC.
Those skilled in the art also can know, polynucleotides are deposited on the particle of being made up of as gold, iron and synthetic plastic various materials.
Those skilled in the art also can know, can utilize various recombinant vectors to produce the transgenic sequence of using with particle form.Mainly due to easy operating, thus with the DNA plasmid vector as preferred vector.
Those skilled in the art also can know, can be from comprising but be not only limited to the various host tissues of marrow and peripheral blood and obtain APC, and can behind the said APC of external manipulation, it be imported among the said host.
An object of the present invention is to provide the treatment or the preventative genetic immunization method of anti-neoplastic cell.
Another object of the present invention provides the treatment or the preventative genetic immunization method of viral infection resisting.
A further object of the present invention provides to participating in the particle polynucleotides that the host immune cell interior orientation that produces ctl response imports the codes for tumor rejection antigen gene, and with to mammalian hosts, preferably the human host carries out the method for genetic immunization.
A further object of the present invention provides to being positioned mammal, particularly maybe can lead to the adenoid host antigen of host in human host's lymphatic tissue and present the particle polynucleotides that cell directional imports the codes for tumor rejection antigen gene, so that produce the genetic immunization method of ctl response.
A further object of the present invention provide to the mammal of participating in the ctl response that produces anti-specificity virus infections particularly human host's body interior orientation import the particle polynucleotides of the viral gene of encoding, said host is carried out the method for genetic immunization.
A further object of the present invention provide to be positioned mammal particularly the adenoid host antigen of human host be in the delivery cell particle polynucleotides that import the coding viral gene so that in said host, produce the genetic immunization method of ctl response.4. Brief Description Of Drawings
It is that MO4 and EG7 carry out the functional of ovalbumin and present that Fig. 1 shows through transfection tumor cell.Stating under external source OVA peptide SIINFEKL (10ng/ml) (Rock etc., 1990, the Journal of Immunology 45:804-811) existence condition of (open symbols) being arranged or do not have that (filled symbols) added, with T quadroma RF33.70 (anti-OVA+K
b) and transfected (square frame) that specify number or the tumour cell of not transfected (circle) prepare micro-culture.Be incubated after 18 hours, the results supernatant also uses indicating clone HT2 (Rock etc., 1990, Journal of Immunology 145:804-811) to detect IL-2.(A) the subclone MO4 of B16 and OVA transfection.(B) the EL4 subclone EG7 of EL4 and OVA transfection.There is not the OVA that obviously improves the tumour of OVA transfection to present when in the test culture, having exogenous SIINFEKL.
Fig. 2 shows tumor growth in vivo or the host's survival rate after the OVA expression of the melanoma MO4 that B16 derives does not influence tumor challenge significantly.With MO4 (circle) or B16 (square frame) (5 * 10
4/ mouse, both sides midriff hypodermic injection) the attack mouse.Estimate tumour size (Fig. 2 A) weekly for 3 times and provide the result, in every group, occur till first animal dead with average tumor area (square centimeter number).Represent survival rate (Fig. 2 B) with the percentage of surviving animals.All experiments are every group of 5 mouse all, and repeat at least 3 times.Put to death dying mouse.
Fig. 3 shows that transdermal delivery OVA coding DNA carries out immunity inoculation to induce the protective effect of the specific CTL of OVA mediation, stops the lethal hit due to the melanoma MO4 that expresses OVA.The molten cell function (A) of the lymphoma EG7 (closed square) of the splenocyte antagonism OVA transfection of OVA immunity (as the described inherent immunity of embodiment the 7th part) mouse of analysis stimulated in vitro or not transfected parent EL4 (empty triangle).With effector cell colony only with complement (short side frame), or with anti-CD4
+(black triangle), CD8
+(open circle) or Thyl.2
+(solid circles) lymphocytic monoclone antibody and complement are incubated together, analyze the cell lysis activity (B) of anti-EG7 target then.In C-F, mouse is with OVA (closed square) or hereditary 0 immunity of LacZ (hollow square), and after 7 days booster immunization.Back 7 days of last immunity (0 day) is with B16 melanoma (D), or the subclone MO4 (C) that expresses OVA attacks each group by mice immunized.Perhaps, will be divided into 2 groups by mice immunized, wherein 7 the and 9 days anti-CD8 mAb of lumbar injection in one group of immune the last time back are to get rid of CD8
+Lymphocyte.Attack complete (E) and CD8 in back 10 days of last immunity (0 day) with MO4
+(F) mouse that exhausts.Percentage record survival rate with surviving animals (C-F).The animal that still survived in the 60th day does not have the sign of tumor growth.All experiments all are every group of 5 mouse, and repeat at least 3 times.Show loving care for guide to kill dying mouse according to animal.
Fig. 4 shows the antigen presenting cell internalization and expresses the particle polynucleotides, and processes and present the antigen of being expressed by MHCI class restriction processing approach.Bone marrow cell in getting rid of lymphocyte also is being added with among the RPMI 1640 of 10%FCS, L-glutaminate, antibiotic and 2-ME, in 24 hole flat boards with 10
6The density overnight incubation of individual cells/well is prepared dendritic cell.In the 1st day with 2.5 * 10
5The density of cells/well is spread cell again to apply and is being contained GM-CSF (10
3U/ml, Sigma, St.Louis is MO) with mouse rIL-4 (10
3U/ml, Genzyml, Cambridge in medium MA), and gathered in the crops the cell of loose adhesion in the 8th day.Through the flow cytometry analysis, show that these dendritic cells have expressed CD45, CD44, CD11b (Mac-1), CD18, CD80, CD86 and I class and II class MHC antigen.Reduce blood serum medium (Optimen, Gibco, Grand Island, NY) in, add or do not add OVA peptide (20ng/ml)+B2M (β 2-M, 10 μ l/ml, under situation Sigma) with 37 ℃ of pulses of dendritic cell 2 hours.Thoroughly wash cell then, be resuspended among the PBS and irradiation (2000rad) it, be then injected in the mouse body that is used to first test.Use iron granules (closed square) or golden particulate (open circle) as particle matrix or use OVA albumen (2mg/ml) (black box) with the tree-shaped APC of the bone marrow derived that specifies number and particle polynucleotides (50 μ l/ml/10 by the coding OVA of the preparation of method described in embodiment the 7th part
6Cell, 7mg/ml particle) cultivated altogether 24 hours, washing, then with the APC that specifies number in micro-culture with T quadroma RF33.70 (anti-OVA+K
b) cultivate altogether.Be incubated after 18 hours, the results supernatant also uses indicating clone HT2 to detect IL-2 (Rock etc., 1990, Journal of Immunology 145:804-811).
Fig. 5 demonstration is carried out the comparison of particle polynucleotides immunity inoculation through biology bombardment (biobalistic) or hypodermic injection administration.Every group of 5 C57B1/6 mouse of immunity carried out booster immunization on the 7th day, then behind booster immunization the 7th day in every side flank portion intracutaneous injection 5 * 10
5Individual MO5 tumour cell is to attack it.The immunity material comprises: (A) by the particle polynucleotides described method preparation of embodiment the 7th joint and bombard coding β-Gal that (biobalistic) administration transports through biology; (b) press the particle polynucleotides that the described method of embodiment the 7th joint prepares and bombard through biology the coding OVA of (biobalistic) transmission; (c) the excessive no particle polynucleotides of the coding OVA that transmits through hypodermic injection; Or (D) press the described method preparation of embodiment the 7th joint but through the particle polynucleotides of the equivalent coding OVA of hypodermic injection administration.Be the percentage that tumour cell is attacked back 50 days no tumour animals in every group to data.
Fig. 6 show with the APC immunity inoculation that is incubated altogether of particle polynucleotides of coding OVA after, the attack of the melanoma MO5 that avoids being expressed OVA of can watching for animals; Every group of 5 C57B1/6 mouse are pressed once subcutaneous immunity described in Fig. 5, then after immunity the 10th day in every side flank portion intracutaneous injection 5 * 10
5Individual MO5 tumour cell is to attack animal.Immunity comprises with material: the particle polynucleotides (particle solution of 100 μ l concentration 7mg/ml (particle W/V PBS)) of the irrelevant antigen beta galactosidase of (hollow square) every both sides hind leg injection coding; (empty circles) commercially available pAc-neo-OVA (every hind leg 100 μ l contain the DNA/ animal of about equivalent); Or (closed square) every hind leg 5 * 10
4The dendritic cell of the individual bone marrow derived/100 μ l aqueous solution (pressing embodiment 7 described method preparations).Survival rate is represented with the percentage of surviving animals.All experiments all comprise every group of 5 animals.Show loving care for guide to kill dying mouse according to animal.5. Fa Ming detailed description
Term used herein " mammalian hosts " comprises each member of the animal kingdom, comprising but be not only limited to the people.
Term used herein " dna fragmentation " can comprise any nucleotide sequence, be DNA or RNA, it contains suitable code area and regulates sequence, target cell is expressed carry out antigen protein or the antigenic protein fragment that cell membrane is presented by endogenous MHC I classpath.
Term used herein " particle polynucleotides " can refer to from comprising but not be only limited to the particle that the material of gold, iron or synthetic plastic makes that wherein particle comprises a certain amount of dna fragmentation as hereinbefore defined.
The present invention relates to treatment or preventative genetic immunization to mammalian hosts, it comprises that the target cell in the mammalian hosts body transports a kind of dna fragmentation, this dna fragmentation is expressed in target cell, in target cell, present the recombinant antigen peptide then, immunity with the irritation cell mediation, humoral immunity, or both.
The present invention relates to treatment or preventative genetic immunization to mammalian hosts, it comprises that the specificity target cell in the mammalian hosts body transports the dna fragmentation of certain protein of coding or its biological active fragment.From then on for example neoplastic cell or virus infected cell are special to infected cells for the antigenic peptides expressed of dna sequence dna.The generation of stimulator antigen specific CTL, thereby the destruction of impelling target cell such as neoplastic cell and virus infected cell.
The invention discloses the genetic immunization method for the treatment of or preventing tumour or virus infections.Cytotoxic T cell is antitumor and immunoreactive important component virus infections.Cytotoxic T cell is presented in the cell that the lip-deep antigenic peptide of tumour target cell kills neoplastic cell or virus infections by identification by MHC I quasi-molecule.These peptides that are derived from tumour antigen are synthesized by infected cells and are degraded in kytoplasm.Generally all do not succeed with the effort of tumour-specific ctl response in the inductor with killed tumour cell or protein component immunity inoculation, supposition may be because the protein in the extracellular fluid can not enter kytoplasm and present approach near MHC I class.
The particle that a particular of the present invention relates to the dna fragmentation that contains coding destination protein matter carries out genetic immunization.Be called " particle polynucleotides " in the whole text in this this explanation of particle that contains dna fragmentation.Preferably the bead that for example wraps quilt through giving particle form or the dna fragmentation or the destination protein matter of gold grain form are finished transporting to cells in vivo.In target cell, carry out promptly expressing this protein or its biological active fragment after kytoplasm transports.That is expressed presents required antigenic peptides by endogenous MHC I classpath to the T lymphocyte substrate is provided for producing the protein of infected cell-specific or protein fragments.By MHC I classpath suitably present the purpose antigenic peptides can stimulate CTL generation and and then facilitate by the destruction of transfect cell.
The present invention is based on a such prerequisite, and promptly the dna fragmentation of expressing tumor rejection antigen (TRA) or viral antigen or its active part may be directed to special cell, thereby facilitates the cascade reaction of the protectiveness tumour immunity process of final realization CTL mediation.For this purpose, the invention discloses with coding for antigens protein DNA construct transfection target host cell of a granular form to induce the immunity of CTL mediation.In cell, produce immunity protein, thereby have an opportunity to enter the approach of presenting of MHC I class restriction.Produce the epitope of natural process, and transfected cell immunity protein can be produced in continuous several days, stronger immunogenicity spread effect may be more helped bringing into play.
In a particular of the present invention, by microparticle bombardment device particle polynucleotides immunity mammalian hosts, so that the particle polynucleotides as the direct result of non-specific emitting substance bombardment, enter by the emitting substance path at least one member's the kytoplasm of host cell.When bombardment skin, the APC of the skin that can be bombarded comprises but is not only limited to the bright Chinese Schwann Cells of epidermis, horn cell or skin dendritic cell.When the bombardment lymphatic tissue, the adenoid APC that can be bombarded comprises but is not only limited to resident dendritic cell, macrophage, stroma cell, T lymphocyte or β lymphocyte.Express transgenic polynucleotides on the biology level of significance so that antigenic peptides is subject to processing, presented and are represented on the film surface of APC cell by endogenous MHC I classpath.The endogenous APC cell membrane of antigen is presented and can bombarded the position, or in the inducing action of after being transported to lymphatic tissue by the bombardment cell, facilitating the antigen specific CTL.Derivative antigentic specificity CTL is originally in particularly human host's body-internal-circulation of mammalian hosts, to destroy the cell of neoplastic cell or virus infections.
Can bombard (biobalistic) forwarding method by biology with particle polynucleotides immunity mammalian hosts, make the particle polynucleotides enter host cell (comprising APC) specifically by phagosome to kytoplasm approach.The particle polynucleotides are entering host cell by bombardment position (skin or lymphatic tissue) or after being transported to lymphatic tissue by phagosome to kytoplasm approach.Therefore, the particle polynucleotides can enter lymphatic tissue by the direct transportation of particle, and then are taken in by the cell in the lymphatic tissue, perhaps enter lymphatic tissue through transporting the host cell of having taken in the particle polynucleotides to lymphatic tissue.After being taken in by host cell, the transgenosis polynucleotides are promptly expressed with the biology level of significance, so that processing and by endogenous MHC I classpath antigen-presenting peptide, and it are represented on the film surface of host APC.The endogenous APC cell membrane of antigen present facilitate at the bombardment position or in lymphatic tissue to the inducing action of antigen specific CTL.Derivative antigentic specificity CTL is originally in the particularly human host's body-internal-circulation of whole mammalian hosts, to destroy the cell of neoplastic cell or virus infections.
In another embodiment of the invention, by comprising but the direct injection that is not only limited to the injection of hypodermic injection, epidermis, the injection of corium skin, lymph injection and intravenous injection with particle polynucleotides immunity mammalian hosts.The particle polynucleotides enter host cell and express it with the biology level of significance, thereby antigenic peptide fragment is presented to endogenous MHC I classpath and is presented on the film surface of host cell.Endogenous target cell membrane is presented the inducing action that can facilitate the antigen specific CTL, and the latter is in mammal human host's systemic circulation particularly, to destroy the cell of neoplastic cell or virus infections.
Available particle polynucleotides immunity mammalian hosts is so that the particle polynucleotides enter host cell specifically by phagosome to kytoplasm approach comprises in the APC.The particle polynucleotides enter host cell in the injection site or after being transported to lymphatic tissue by phagosome to kytoplasm approach.Therefore, particle can be transported directly to lymphatic tissue and be taken in by the cell in the lymphatic tissue then, the host cell that perhaps will take in particle is transported to lymphatic tissue makes the particle polynucleotides enter lymphatic tissue.After being taken in by host cell, the transgenosis polynucleotides are able to express on the biology level of significance, thereby make antigenic peptides be subject to processing and present by endogenous MHC I classpath, and represent on the film surface of host APC.The endogenous APC cell membrane of antigen is presented the inducing action of facilitating the antigentic specificity CTL in injection site or the lymphatic tissue.Derivative antigentic specificity CTL itself enters the particularly human host's circulation of whole mammal, to destroy the cell of neoplastic cell or virus infections.
In another embodiment of the invention, to comprise but the direct injecting method that is not only limited to the injection of hypodermic injection, epidermis, the injection of corium skin, lymph injection and intravenous injection with particle polynucleotides immunity mammalian hosts.Design particle polynucleotide compositions, make it comprise preferential target APC engulf approach specific molecular (comprise but be not only limited to the approach of mannose receptor mediation, or the receptor-mediated approach of Fc), or help particle near the specific molecular of kytoplasm (comprise but be not only limited to the endosome synexin, as the molten born of the same parents' fibroin of viral HA albumen or Listeria).The particle polynucleotides enter host cell in the injection site or after being transported to lymphatic tissue by phagosome to kytoplasm approach.Therefore, particle can be transported directly to lymphatic tissue and take in it by the cell in the lymphatic tissue then, the host cell that perhaps will take in the particle polynucleotides is transported to lymphatic tissue so that the particle polynucleotides enter lymphatic tissue.After being taken in by cell, the transgenosis polynucleotides are expressed with the biology level of significance, thereby make antigenic peptide fragment processed and present by endogenous MHC I classpath, and represent on the film surface of host APC.The endogenous APC cell membrane of antigen is presented and is facilitated antigen specific CTL inducing in injection site or lymphatic tissue.Derivative antigentic specificity CTL itself is circulated in particularly human host's whole body of mammal, to destroy the cell of neoplastic cell or virus infections.
Use the mouse black-in lymphoma model that describes in detail in the embodiment part 6 aspect several, to illustrate the present invention.Briefly, the major limitation with mouse model research antigentic specificity tumour immunity is the tumour antigen of determining that CTL was discerned that lacks I class MHC restriction.Because except the host does not have the tolerance TRA, the synthetic every other albumen of TRA and cell does not have basic difference, the synthetic extraneous protein of tumour all should have the function of tumour antigen.In the melanoma b16 through ovalbumin (OVA) gene transfection is derived to C57B1/6, illustrate tumour immunity inoculation method of the present invention with mouse tumor model with the synthetic TRA of definite external source.This system is attractive because of following several reasons: (1) B16 melanoma is to have passed through the mouse tumor of further investigation, and (2) have fully identified interior growth characteristics of the body of this tumour and transfer characteristic, and (3) have fully determined the structure of ovalbumin.Process in the cell of OVA in the known road C57B1/6 mouse body and present.Particularly known MHC K with the I class
bThe structure of the peptide of the process processing that is associated and presents.Also know and use the anti-OVA-K of T-T hybridoma 33.70A1
bDetect and H2-K
bThe functional expression (Kovacovics-Bankowski etc., 1993, Proc. Natl. Acad. Sci.USA 90:4942-4946) of relevant ovalbumin peptide (SIINFEKL).Also described the technology (Moore etc., 1988, cell 54:77-785) of estimating to induce in the body in this system the OVA specific CTL in detail.
One embodiment of the invention are to use biological bombardment (biobalistic) device to carry out immunity inoculation with purpose particle polynucleotides.Specifically; with biology bombardment (biobalistic) method particle polynucleotides immunity mammalian hosts; facilitate a series of biological processes of generation in transfected cell to cause the particle polynucleotides to transport, thereby cause the protective immunity of antagonism lethal tumor challenge.The present invention uses biological bombardment (biobalistic) device to carry out dermatogen and transports, so as to illustrating the present invention from several aspects.At first, the particle polynucleotides of importing coding extraneous protein β-Gal cause the β-expression of Gal protein in epidermis and draining lymph node.Secondly, the OVA immunity inoculation causes inducing the OVA specific CTL.The 3rd, that uses that the OVA-B16 model done studies show that the animal of OVA immunity is subjected to resisting the protective effect of the tumor challenge of expressing OVA, and this protective effect be antigentic specificity and depend on CTL.In addition, the mechanism of this protective effect has exceeded the scope by biological bombardment (biobalistic) device general dermal delivery.On the contrary, the antigen presentation in specificity guiding engulfing property APC and/or the lymphatic tissue is only and induces the key factor that produces antigentic specificity CTL from precursor.Therefore according to a preferred embodiment of the invention, target cell is engulfing property APC, particularly is positioned in the lymphatic tissue, maybe can be transported to adenoid APC.
In another embodiment of the invention, through comprising but the direct injection that is not only limited to the injection of hypodermic injection, epidermis, the injection of corium skin, lymph injection and intravenous injection with particle polynucleotides immunity mammal.The transgenosis polynucleotides are expressed on the biology level of significance, so that by endogenous MHC I classpath processing and antigen-presenting peptide, and make it to represent on the film surface of target cell.Endogenous APC cell membrane is presented the inducing action of facilitating the antigen specific CTL, and the latter is circulated in particularly human host's whole body of mammal again, to destroy the cell of neoplastic cell or virus infections.
In a preferred embodiment of the invention, be engulfing property APC through direct injection with the mammalian hosts target cell of particle polynucleotides immunity.The transgenosis polynucleotides are expressed on the biology level of significance, so that antigenic peptide fragment is presented to endogenous MHC I classpath and represents on the film surface of APC cell.The APC cell membrane is presented the inducing action of facilitating the antigen specific CTL, and the latter carries out particularly human host's systemic circulation of mammal again, to destroy the cell of neoplastic cell or virus infections.
In a preferred embodiment, through hypodermic injection the particle polynucleotides are delivered in human host's body.The invention discloses direct injection particle polynucleotides compound causes to the targeted delivery of engulfing property APC and the gene expression in the lymphatic tissue.Thisly just come in engulfing property APC targeted delivery and/or in lymphatic tissue, carry out the advantage inducing action that gene expression just causes producing from original precursor antigentic specificity CTL by hypodermic injection.
Therefore, center of the present invention be enter APC and by the coded protein of expression or the dna fragmentation of its biological active fragment.In the APC kytoplasm, express interested antigen and enter the MHCI classpath, make APC be facilitated inducing action the antigen specific CTL.The data that provide in the disclosure are supported the argument that this is new, and the effective means of promptly inducing this antigentic specificity CTL is that genetic stocks is directly transferred in the APC in the lymphatic tissue, perhaps can be transported in the adenoid APC.
In another embodiment of the invention, through comprising but be not only limited to the injecting method of the injection of hypodermic injection, epidermis, the injection of corium skin, lymph injection and intravenous injection, with the homology APC immunity mammalian hosts of having carried out antigen load at external use particle polynucleotides.Specifically, promptly use the APC immunity mammalian hosts of particle polynucleotides transfection, cause the particle polynucleotides to be expelled in the host at the external APC of entering and with this APC.After entering APC, the transgenosis polynucleotides are expressed on the biology level of significance, so that antigenic peptide fragment is processed and present by endogenous MHC I classpath, and represent on the film surface of APC.Behind these APC of injection, the endogenous APC cell membrane of antigen is presented the inducing action of promptly facilitating the antigentic specificity CTL in injection site or the lymphocyte.Derivative antigentic specificity CTL itself is circulated in particularly human host's whole body of mammal again, to destroy the cell of tumour cell or virus infections.
In another particular of the present invention, by comprising but be not only limited to the injection of the injection of hypodermic injection, epidermis, the injection of corium skin, lymph injection and intravenous injection, with being incubated the homology APC immunity mammalian hosts of having carried out antigen load external altogether with the particle polynucleotides.Specifically, external and particle polynucleotides are incubated altogether with transfection homology APC, thereby make the particle polynucleotides enter APC by phagosome to kytoplasm approach.With the APC immunity mammalian hosts of particle polynucleotides transfection, promptly the particle polynucleotides enter APC specifically by phagosome to kytoplasm approach, and then this APC is expelled in the host.Enter after the APC, the transgenosis polynucleotides are expressed on the biology level of significance, so that antigenic peptide fragment is processed and present by endogenous MHC I classpath, and are shown on the film surface of APC.After injecting such APC, the endogenous APC cell membrane of antigen is presented the inducing action of promptly facilitating the antigentic specificity CTL in injection site or the lymphatic tissue.Derivative antigentic specificity CTL itself enters particularly human host's systemic circulation of mammal again, to destroy the cell of neoplastic cell or virus infections.
In another particular of the present invention, through comprising but be not only limited to the injecting method of the injection of hypodermic injection, epidermis, the injection of corium skin, lymph injection and intravenous injection, with the homology APC immunity mammalian hosts of having carried out antigen load at external use particle polynucleotides.Specifically, external through coming transfection homology APC with particle polynucleotides microparticle bombardment APC.After in bombarding APC, the transgenosis polynucleotides are expressed on the biology level of significance, so that antigenic peptide is processed and presented by endogenous MHC I classpath, are shown in then on the film surface of APC.After injecting such APC, endogenous APC cell membrane to antigen is presented the inducing action that can facilitate the antigentic specificity CTL in injection site or the lymphatic tissue, derivative antigentic specificity CTL itself is again by particularly human host's body-internal-circulation of mammal, to destroy the cell of neoplastic cell or virus infections.
In another embodiment of the invention, through comprising but be not only limited to the injecting method of the injection of hypodermic injection, epidermis, the injection of corium skin, lymph injection and intravenous injection, with in the homology APC of external use particle polynucleotides transfection immunity mammalian hosts.Specifically, with the APC immunity mammalian hosts of particle polynucleotides transfection, even the particle polynucleotides are expelled in the host in the external APC of entering and with this APC.Make the particle polynucleotides in the external APC of entering by microparticle bombardment or phagosome to kytoplasm approach.At the polynucleotides of external use coding for antigens and/or encode that certain can increase the polynucleotides transfection APC of molecule of the antigen presentation functional efficiency of APC, said molecule comprises but is not only limited to cell factor molecules such as IL-12, IL-2 and/or IL-4, and/or costimulatory moleculeses such as CD80 and/or CD86.After entering APC, the polynucleotides of coding transgenosis antigen are expressed on the biology level of significance, thereby make antigenic peptide processed and present by endogenous MHC I classpath, and appear on the film surface of APC.After injecting such APC, the endogenous APC cell membrane of antigen is presented the inducing action of just facilitating the antigentic specificity CTL in injection site or the lymphatic tissue.Derivative antigentic specificity CTL itself is circulated in particularly human host's whole body of mammal again, to destroy the cell of neoplastic cell or virus infections.After entering APC, the polynucleotides of the coding transgenic cell factor and/or costimulatory molecules obtain expressing on the biology level of significance, with the antigen presentation function of performance APC, thereby induce the antigen specific immune in injection site or the lymphatic tissue to react.
It is in those of ordinary skills' the skill that the TRA that selects or viral antigen are incorporated in the disclosed system of this specification.Present available TRA comprises but is not only limited to MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, tyrosinase, Melan-A (MART-1), gp100 (pme117), gp75 (TRP1), CEA (carcinomebryonic antigen), and from HPV, the tumour antigen that HBV and EBV virus are derived, tumour the be correlated with antigen such as the p53 of oncogene/tumor suppressor gene sudden change coding, p16, RAS, HER2/neu, (relevant summary is referring to Maeurer etc. for C-ABL and polymorphic endothelium mucin antigen, 1996, clinical immunology principle and the tumor vaccine part of putting into practice in the book, R.Rich edits, Mosby publishing house, Chpt.123:1904-1918; Van den Eynde etc., 1995, The Journal of Experimental Medicine (J.Exp.Med.) 182:689-698).The example of viral antigen comprises but is not only limited to nucleoprotein influenza (Donnelly etc., 1995, natural medical science 1:583-587), HIV gp120, HIV gp160 and hepatitis B surface antibody are (referring to Pardoll and Beckerleg, immunity, 1995,3:165-169).
Those of ordinary skills know, can use any eukaryotic promoter and/or the enhancer sequence of known rise transgenosis dna sequence dna expression at the construction of recombinant vector that is used for combining with the particle of selecting, so that produce particle polynucleotides of the present invention.Such promoter fragment comprises but is not only limited to cytomegalovirus (CMV) promotor, Rous sarcoma virus (RSV) promotor, murine leukemia virus (MLV) promotor, β actin promoter, and known in target cell activated any cell specificity promotor or enhancer sequence.
For this purpose, a preferred embodiment of the present invention is the application that combines the particle of DNA, be delivered to specifically among the APC in the lymphatic tissue with DNA codes for tumor or viral antigen, maybe can be transported among the APC in the lymphatic tissue, thereby promote antigen near MHC I classpath, thereby allow APC to stimulate inducing antigen-specific CTL.These CTL just are circulated in host's whole body and destroy neoplastic cell or the cell of virus infections then.
Described for example in embodiment the 6th part, embodiment the 7th part and embodiment the 8th part based on utilizing hypodermic injection to start the tumour immunity of this method for optimizing of specific reaction among the lymph node APC.
Following embodiment is intended to illustrate the present invention, rather than restriction the present invention.6. embodiment: OVA/B16 heredity mouse tumor model
Use the OVA/B16 mouse model to obtain illustrating disclosed data in embodiments of the invention the 7th and 8 parts.The attractive several reasons of OVA/B16 mouse system is: (1) B16 melanoma is the mouse tumour of having furtherd investigate, (2) fully identified growth characteristics and transfer characteristic in the body of this tumor cell line, and (3) ovalbumin has the structure of fully determining.It is known processing and present in the cell of OVA in the C57B1/6 mouse.Particularly known the structure of processed peptide, itself and MHC I class K
bBe associated and present.Also known road uses the anti-OVA-K of T-T hybridoma 33.70.A1
bDetect ovalbumin peptide [SIINFEKL] and H2-K
bRelevant functional expression (Kovacovics-Bankowski etc., 1993, Proc. Natl. Acad. Sci.USA 90:4942-4946).Also describe estimation in detail and in this system, induced the technology of OVA specific CTL (Moore etc., 1988, cell 54:777-785) in the body.
Mouse and cell-line.Age in 5-8 week, female C57BL/6 mouse was available from JacksonLaboratories, Bar Harbor, ME.EL4 is the C57BL/6T lymphocyte, and EG7 is the subclone (Moore etc., 1988, cell 54:777-785) of the EL4 of chicken ovalbumin (OVA) transfection.The mouse melanoma b16 that C57BL/6 derives (Fidler etc., 1976, cancer research (Cancer Res.) 36:3160-3165) derives from American type culture collection (ATCC).MO4 makes up (Falo etc., 1995, natural medical science 1:649-653 by stating method with pAc-Nco-OVA plasmid transfection B16; Moore etc., 1988, cell 54:777-785).Monoclone antibody is (in Balb/c nu/nu mouse through lumbar injection GKf1.5 cell (3 * 10 from hybridoma GK1.5 (anti-CD4, ATCC TIB-207), 2.43
6) and IFA (0.5ml/ mouse) and produce anti-CD8 antibody) preparation.
Behind OVA transfection B16 melanoma cells, select and separate transfected B16 melanoma subclone MO4.As what detected according to OVA peptide (SIINFEKL) presenting on the RF33.70 cell, parent's melanoma b16 and OVA transfectant are expressed the functional K of similar level on cell surface
b(Fig. 1).On the contrary, MO4/5 rather than B16 can stimulate hybridoma (Fig. 1) in the presence of the peptide that does not have external source to add.This endogenous that has shown transfection antigen produces, processes and present.Importantly, do not change immunogenicity (Fig. 2) in the body of tumour significantly by the endogenous expression of MO4 OVA.B16 and the MO4 tumor growth rate (Fig. 2 A) in the mouse body that is used to for the first time test is similar, and host's survival rate also is (Fig. 2 B) like this.7. embodiment: carry out genetic immunization by biology bombardment administration
The location that extraneous protein is expressed-immunity back 24 or 48 hours results tissue specimen (skin or draining lymph node), washing and being dissolved under 4 ℃ in 2% formaldehyde-0.2% glutaraldehyde among the PBS is fixed in PBS.Thoroughly wash fixing sample with PBS, (be dissolved in the 1mg/ml X-gal of PBS, 5mM ferricyanic acid potassium, 5mM hydroferrocyanate, 2mM MgCl in 37 ℃ at the X-gal staining solution then
2) in the insulation 18 hours.The tissue of dyeing is made paraffin section also to be redyed with 0.1% Kernechrot.
Genetic immunization-50mg 0.95 μ m gold bead grain and 100 μ l0.1M spermidines are merged quilt is wrapped in also ultrasonic processing 5 seconds with preparation DNA gold grain.In turn, add 132 μ g plasmid DNA and 200 μ l CaCl in succession
2Make this mixture at room temperature precipitate 5-10 minute.Centrifugal pellet (10,000rpm, 30 seconds) is also washed in cold ethanol 3 times, is suspended in the final concentration that reaches 7mg gold/ml in the 7ml ethanol then again.With this solution application of sample in Tefzel
The pipe (Agracetus) in and static 5 minutes.Remove ethanol and particle is attached on the pipe sidewall, then at N with the 20rpm rotation
2Gas is dry down.The drying tube that is lined with particle is cut into 0.5 inch sections, and in the bottle of paraffin sealing, preserve standby with desiccant.Use Accell gene emitter, bombard twice (each 0.5mg gold grain=0.5 inch pipe range) so that animal is carried out vaccine inoculation to the abdomen area of shaving hair with 300psi emission pressure.With pAc-Neo-OVA plasmid (Moore etc., 1988, cell 54:777-785) or pIEglacZ plasmid (providing by the Nadia Jouroud) immune animal that is in the following lacZ gene of CMV promotor control is provided.For the experiment that relates to hypodermic injection particle polynucleotides (Fig. 5), prepare the particle of DNA bag quilt and the DNA of hypodermic injection equivalent as stated above.For some animal (Fig. 5), the free plasmid DNA that hypodermic injection is excessive.In of the PBS hypodermic injection administration of both sides middle rib belly with 100 μ l volumes.For in-vitro transfection APC (Fig. 4), weight Biomag ferric oxide particles such as use or gold grain similarly prepare the DNA particle as particle matrix.
For the experiment that relates to hypodermic injection particle polynucleotides (Fig. 5 and 6), prepare the particle of DNA bag quilt as stated above and inject equivalent or description of drawings in the DNA of specified amount.For some animal (Fig. 5 and 6), the free plasmid DNA that hypodermic injection is excessive.In of the PBS hypodermic injection administration of both sides back leg with 100 μ l.
For in-vitro transfection APC (Fig. 4 and 6), the Biomag ferric oxide particles (iron particle) or the gold grain of weight such as same use fully similarly prepare the DNA particle as stated above as particle matrix.Obtain tree-shaped APC by the method for describing in the Brief Description Of Drawings from marrow, different is does not add in the tissue culture medium (TCM) of IL-4 with 25 μ l concentration is 37 ℃ of insulations of particle polynucleotides solution (particle weight/PBS volume) 18 hours of 7mg/ml.By the method preparation of describing in the Brief Description Of Drawings and the dendritic cell of injections of antigens pulse.Briefly, get rid of in the lymphocyte bone marrow cell and in RPMI 1640 medium that are added with 10%FCS, L-glutaminate, antibiotic and 2-ME, in 24 hole flat boards with 10
6The density overnight incubation of cells/well.The 1st day with cell with 2.5 * 10
5The density shop of cells/well is applied and is being contained CM-CSF (10
3U/ml, Sigma, St.Louis is MO) with mouse rIL-4 (10
3U/ml, Genzyme, Cambridge gathers in the crops the cell of loose adhesion in flat board MA) and in the 8th day.Through the flow cytometry analysis, show that these dendritic cells have expressed CD45, CD44, CD11b (Mac-1), CD18, CD80, CD86 and I class and II class MHC antigen.Be with or without OVA peptide (20ng/ml)+β2Wei Qiudanbai (people β 2-M, 10 μ l/ml, minimizing blood serum medium Sigma) (Optimen, Gibco, Grand Island, NY) in 37 ℃ of pulses of dendritic cell 2 hours.Thoroughly wash cell then, be suspended among the PBS again and irradiation (2000rad), be then injected in the mouse body that is used to first test.
Cell toxicity test-by the work of describing in the past the method for very little change (Falo etc., 1995, natural medical science 1:649-653) stimulate the splenocyte of immunized animal again.Briefly, 1 week after the immunity inoculation, through with the irradiation (20,000rad) the EG7 cell (10 * 10
6) cultivate to stimulate splenocyte (30 * 10 once more altogether
6).Gather in the crops the effector cell after 5 days, and in round bottom microwell plate (200 μ l) effector cell-target cell ratio and 2 * 10 to point out
4Individual
51The target cell of Cr mark is cultivated.Using mAb to add complement in some cases, detect by stating method (Rock etc., 1993, Journal of Immunology 150:1244-1251) then to get rid of the T cell subsets among the effector cell.Cultivate after 4 hours for 37 ℃, collect 100 μ l supernatants from the micro-culture that repeats for three times, counting also calculates the percentage that specificity discharges by stating method (Falo etc., 1995, natural medical science 1:649-653).The result provides as the mean value of three parts of cultivations.Three parts of SEM that repeat culture always are less than 15% of average.
Protection test-by the method for stating is with pointed antigen gene construct immunity C57BL/6 mouse.Also estimate the tumor survival rate as stated above with the tumor challenge animal.Briefly, back seven days of last immunity (0 day), flank portion is with the 50% lethal dose (LD of tested animal in both sides
50) or the 4th part in 2 intracutaneous injection melanoma cells (2 * 10 of tumor cell number of pointing out
4), to attack OVA animal immunity or the lacZ immunity.Percentage record survival rate according to surviving animals.In PBS, the melanoma cells of injection is washed 3 times.Being injected cell visible with the blue method of exclusion inspection of trypanosome is alive more than 95%.All are tested every group and include 5 mouse and repeating at least 3 times.Show loving care for guide to kill dying mouse according to the animal of Pittsburgh University Medical Center.In some experiments, the CD8 that gets rid of animal
+Cell.The the 7th and 9 day intraperitoneal injection CD8mAb (2.43) after immunity as previously mentioned attacked (Falo etc., 1995, natural medical science 1:649-653) at the 10th day with tumour cell then for this reason.
The ability of the biological bombardment of estimation immunity inoculation inducing antigen-specific CTL.With altogether at the most 2.64 μ gOVA coding DNAs be sent in the skin of abdomen with 2 overlapping pulses, be used to the C57B1/6 mouse of testing for the first time with immunity, and after 7 days, carry out same booster immunization.The homology mouse thymus knurl EG7 cell of OVA is expressed in the splenocyte cracking of external these mouse that stimulate again of process, but parent's tumour EL4 cell (Fig. 3 A) of not cracking untransfected.Therefore, the target cell lysis is an antigentic specificity, and the OVA that depends on the tumour target expresses.The T cell subsets that uses mAb to get rid of in the effector cell colony proves that lysis depends on the Thy1 with I class MHC Restricted CTL effector cell feature
+, CD8
+Subgroup (Fig. 3 B).
After respectively organizing mouse 7 days,, induce the ability of protectiveness tumour immunity to determine biological bombardment immunity inoculation method in attacking animal away from immune position intracutaneous injection MO4 melanoma cells by above-mentioned immunity and booster immunization.The OVA mice immunized can stop the lethal tumor challenge, and then the carrying out property growth of tumour of control group mice (with the similarity method immunity, but use the lacZ reporter gene), and 60% animal cause death (Fig. 3 C) was arranged in the time of the 60th day.The OVA mice immunized does not avoid suffering the changing of parent's melanoma b16 of untransfected to hit (Fig. 3 D), shows that protective immunity is an antigentic specificity, and the OVA that depends on the tumour target expresses.We are by repeating the CD8 that the anti-CD8mAb of intraperitoneal injection gets rid of immune animal or control group (the lacZ immunity) animal before tumor challenge
+The effector cell is with estimation CD8
+The effector cell is to the contribution (Falo etc., 1995, natural medical science 1:649-653) of this protectiveness tumour immunity.Though the animal of OVA immunity can avoid suffering the attack of MO4, through the immunity eliminating CD8
+The survival rate of the animal of T cell is but similar in appearance to the survival rate (Fig. 3 E-F) of getting rid of or do not get rid of the control animals of T cell.Therefore, CD8
+The T cell is absolutely necessary to the protectiveness tumour immunity of inducing through genetic immunization in this model.
Support other evidences of the tumour immunity mechanism proposed to comprise, prove that transmitting behind the lacZ construct beta galactosidase in the epidermis in the biology bombardment expresses, and interesting be that generation is specific expressed in the separate areas in draining lymph node.Because these lymph nodes away from the immunity inoculation position, are the results of direct physical bombardment so lymph node is expressed unlikely.DNA transmit back 24 hours in by the epidermis of immune skin and cortical cell (undyed) gold grain preponderate, and in epidermal keratinocyte, had lacZ express at 48 hours.The specific stain that separate areas is also arranged in draining lymph node.Mouse sample through the same irrelevant dna immunization of handling does not show that then lacZ expresses.This observed result impels us further to analyze, described in the 8th one of embodiment.8. embodiment: carry out genetic immunization through subcutaneous administration
Give described in the material and method such as embodiment part 6 and embodiment part 7 of particle polynucleotides with the induced tumor immunity through subcutaneous.
Lead specifically engulfing property APC in the lymphatic tissue of this digital proof hypodermic injection particle polynucleotides in body maybe can be transported to the ability of adenoid APC.
Use the particle polynucleotides that comprise pAc-Neo-OVA and immune respectively organize mouse by direct hypodermic injection or biological bombardment, the ability (Fig. 5) of the antitumor attack that is equal to all must be arranged.Conform to the discovery described in the embodiment part 7, particle polynucleotides (for example pIEglacZ) immunity of the antigen that has nothing to do with encoding can not provide protective effect (Fig. 5).Hypodermic injection does not have the pAc-Neo-OVA of particle that the protective effect (Fig. 5) of the relevant tumor challenge of antagonism can not be provided.These data show that hypodermic injection particle polynucleotides are effective as biological bombardment particle transports at least.In addition because hypodermic injection does not cause polynucleotides particle direct physical micropellet bombardment in the kytoplasm of indivedual host cells, so the expression of polynucleotides need engulf by having probably/host cell of endocytosis ability initiatively takes in said polynucleotides.Hypodermic injection does not have the pAc-Neo-OVA of particle not provide protective effect this observed result yet, means the Transduction Mechanism that particle transports/engulfs yet.Carry out the cell that the preferential target of particle transduction hint transgene expression has phagocytic activity by engulfing, comprise APC.
Other evidences of this mechanism that support is proposed are, observe APC (dendritic cell of bone marrow derived) in (pAc-Neo-OVA) cultivation altogether of particle polynucleotides (press described in the embodiment 7 preparation) external and the coding ovalbumin, can stimulate OVA (SIINFEKL
+K
b) specific T-cells hybridoma RF33.70 produces IL-2 (Fig. 4).These digital proofs, these APC energy functional expression ovalbumin genes also produce ovalbumin peptide-K
bCompound.The effectiveness that stimulates is with similar with the observed effectiveness of APC of 2mg/ml solubility OVA protein pulse.Use gold or iron as particle matrix, the particle polynucleotides are effective in this detection method.Therefore, the particle polynucleotides are incubated the endogenous that causes transfected antigen with engulfing property APC and produce, process and present external.These observe proof, and the particle polynucleotides can be taken in and express by APC, and corresponding proteins matter can be able to functional presenting, with the inducing antigen-specific immune response.
Moreover; as shown in Figure 6; with the antigen presenting cell of the external insulation of particle polynucleotides of coding for antigens OVA, be the dendritic cell of hypodermic bone marrow derived in this example as stated above, can induce the protective immunity of the tumour cell of anti-antigen expressed gene.In the example that provides, once come into operation 10
5Dendritic cell/the animal of individual irradiated transfection (every hind leg (bilateral) 5 * 10
4Individual cell) induced the protective effect of antitumor attack completely.Therefore, the dendritic cell APC-that comes into operation in the body its in external load be the strong stimulus-have at least of antigentic specificity CTL and protectiveness tumour immunity during peptide to through being incubated altogether to the particle polynucleotides load similar immunogenicity during antigen.
With these results consideration of putting together, always show that the subcutaneous particle polynucleotides that give have the advantage of transporting antigen to engulfing property APC orientation, potentiality ground increases the efficient of genetic immunization and reduces and do not need ill-effect, comprising but be not only limited to inducing tolerance or because of the sudden change due to the normal non-APC host cell of transfection.
Claims (67)
1. method in the treatment or the body of preventative genetic immunization mammalian hosts, this method comprises:
(a) dna fragmentation of generation antigen expressed protein or antigen protein fragment;
(b) said dna fragmentation is distributed on the particle surface, produces the particle polynucleotides;
(c) inoculate said mammalian hosts with said particle polynucleotides, and,
(d) said particle polynucleotides are transported in the kytoplasm of target cell in the said mammalian body, so that said antigen protein of being expressed or antigen protein fragment are presented on the film surface of said target cell by I class MHC approach.
2. the process of claim 1 wherein that said mammalian hosts is the people.
3. the method for claim 2, wherein said dna fragmentation expressing tumor rejection antigen, viral antigen or its antigen protein fragment.
4. the method for claim 3, wherein said target cell is an antigen presenting cell.
5. the method for claim 4, wherein said antigen presenting cell is settled down and maybe can moved in said human host's the lymphatic tissue.
6. the method for claim 5, wherein said tumor rejection antigen is selected from MAGE-1 and MAGE3.
7. the method for claim 5, wherein said tumor rejection antigen is Melan-A.
8. the method for claim 5, wherein said tumor rejection antigen is gp100.
9. the method for claim 5, wherein said tumor rejection antigen is p53.
10. the method for claim 5, wherein said tumor rejection antigen is CEA.
11. the method for claim 5, wherein said tumor rejection antigen is HER2/neu.
12. the method for claim 5, wherein said viral antigen is HIVgp120, HIVgp160.
13. the method for claim 5, wherein said viral antigen is an influenza virus nucleoprotein.
14. the method for claim 5, wherein said viral antigen is a hepatitis B surface antibody.
15. method in the body of treatment or preventative genetic immunization mammalian hosts, this method comprises:
(a) dna fragmentation of generation antigen expressed protein or antigen protein fragment;
(b) said dna fragmentation is distributed on the particle surface, produces the particle polynucleotides;
(c) use biological bombardment device to inoculate said mammalian hosts with said particle polynucleotides; And,
(d) said particle polynucleotides are transported in the kytoplasm of target cell in the said mammalian body, so that said antigen protein of being expressed or antigen protein fragment are presented on the film surface of said target cell by I class MHC approach.
16. the method for claim 15, wherein said mammalian hosts is the people.
17. the method for claim 16, wherein said dna fragmentation expressing tumor rejection antigen, viral antigen or its antigen protein fragment.
18. the method for claim 17, wherein said target cell is an antigen presenting cell.
19. the method for claim 18, wherein said antigen presenting cell are settled down and maybe can moved in said human host's the lymphatic tissue.
20. the method for claim 19, wherein said tumor rejection antigen is selected from MAGE-1 and MAGE3.
21. the method for claim 19, wherein said rejection antigen is Melan-A.
22. the method for claim 19, wherein said tumor rejection antigen is gp100.
23. the method for claim 19, wherein said tumor rejection antigen is p53.
24. the method for claim 19, wherein said tumor rejection antigen is CEA.
25. the method for claim 19, wherein said tumor rejection antigen is HER2/neu.
26. the method for claim 19, wherein said viral antigen is HIVgp120, HIVgp160.
27. the method for claim 19, wherein said viral antigen is an influenza virus nucleoprotein.
28. the method for claim 19, wherein said viral antigen is a hepatitis B surface antibody.
29. method in the body of treatment or preventative genetic immunization mammalian hosts, this method comprises:
(a) dna fragmentation of generation antigen expressed protein or antigen protein fragment;
(b) said dna fragmentation is distributed on the particle surface, produces the particle polynucleotides;
(c) inoculate said mammalian hosts through the direct said particle polynucleotides of injection; And,
(d) said particle polynucleotides are transported in the kytoplasm of target cell in the said mammalian body, so that said antigen protein of being expressed or antigen protein fragment are presented on the film surface of said target cell by I class MHC approach.
30. the method for claim 29, wherein said mammalian hosts is the people.
31. the method for claim 30, wherein directly injection is the subcutaneous route injection.
32. the method for claim 31, wherein said recombinant DNA carrier fragment expression tumor rejection antigen, viral antigen or its antigen protein fragment.
33. the method for claim 32, wherein said target cell is an antigen presenting cell.
34. the method for claim 33, wherein said antigen presenting cell are settled down and maybe can moved in said human host's the lymphatic tissue.
35. the method for claim 34, wherein said tumor rejection antigen is selected from MAGE-1 and MAGE3.
36. the method for claim 34, wherein said rejection antigen is Melan-A.
37. the method for claim 34, wherein said tumor rejection antigen is gp100.
38. the method for claim 34, wherein said tumor rejection antigen is p53.
39. the method for claim 34, wherein said tumor rejection antigen is CEA.
40. the method for claim 34, wherein said tumor rejection antigen is HER2/neu.
41. the method for claim 34, wherein said viral antigen is HIVgp120, HIVgp160.
42. the method for claim 34, wherein said viral antigen is an influenza virus nucleoprotein.
43. the method for claim 34, wherein said viral antigen is a hepatitis B surface antibody.
44. the stripped method of treatment or preventative genetic immunization mammalian hosts, this method comprises:
(a) dna fragmentation of generation antigen expressed protein or antigen protein fragment;
(b) said dna fragmentation is distributed on the particle surface, produces the particle polynucleotides;
(c) with in the external kytoplasm that is transported to the mammalian hosts target cell of said particle polynucleotides, so that said antigen protein of being expressed or antigen protein fragment are presented on the film surface of said target cell by I class MHC approach; With
(d) inoculate said mammalian hosts through the direct said target cell of injection.
45. the method for claim 44, wherein said mammalian hosts is the people.
46. the method for claim 45, wherein directly injection is through hypodermic injection.
47. the method for claim 46, wherein said recombinant DNA carrier fragment expression tumor rejection antigen, viral antigen or its antigen protein fragment.
48. the method for claim 47, wherein said target cell is an antigen presenting cell.
49. the method for claim 48, wherein said antigen presenting cell are settled down and maybe can moved in said human host's the lymphatic tissue.
50. the method for claim 49, wherein said tumor rejection antigen is selected from MAGE-1 and MAGE3.
51. the method for claim 49, wherein said rejection antigen is Melan-A.
52. the method for claim 49, wherein said tumor rejection antigen is gp100.
53. the method for claim 49, wherein said tumor rejection antigen is p53.
54. the method for claim 49, wherein said tumor rejection antigen is CEA.
55. the method for claim 49, wherein said tumor rejection antigen is HER2/neu.
56. the method for claim 49, wherein said viral antigen is HIVgp120, HIVgp160.
57. the method for claim 49, wherein said viral antigen is an influenza virus nucleoprotein.
58. the method for claim 49, wherein said viral antigen is a hepatitis B surface antibody.
59. treatment or the mammiferous stripped method of preventative genetic immunization, this method comprises:
(a) produce the dna fragmentation that is used to express the molecule that can improve APC antigen presentation function;
(b) said dna fragmentation is distributed on the particle surface, to produce the particle polynucleotides;
(c) with in the external kytoplasm of delivering to mammal place target cell of said particle polynucleotides fortune, strengthen property protein so that express said antigen presentation with the meaningful form of biology and on the meaningful level of biology;
(d) through the direct said mammalian hosts of the said place of injection cell inoculation.
60. the method for claim 59, wherein said mammalian hosts is the people.
61. the method for claim 60, wherein directly injection is to inject through subcutaneous route.
62. the method for claim 61, wherein said target cell is an antigen presenting cell.
63. the method for claim 62, wherein said antigen presenting cell are settled down and maybe can moved in said human host's the lymphatic tissue.
64. the method for claim 63, wherein said dna vector fragment expression costimulatory molecules.
65. the method for claim 64, wherein said costimulatory molecules is selected from CD80 and CD86.
66. the method for claim 63, wherein said dna vector fragment expression cell factor molecule.
67. the method for claim 66, wherein said cell factor molecule is selected from IL-12, IL-4 and IL-2.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US53555695A | 1995-09-28 | 1995-09-28 | |
| US08/535,556 | 1995-09-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1201369A true CN1201369A (en) | 1998-12-09 |
Family
ID=24134735
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN96198106A Pending CN1201369A (en) | 1995-09-28 | 1996-09-27 | Stimulation of cell-mediated immune responses by targeted particulated genetic immunization |
Country Status (15)
| Country | Link |
|---|---|
| EP (1) | EP0863704A4 (en) |
| JP (1) | JPH11512724A (en) |
| KR (1) | KR19990063672A (en) |
| CN (1) | CN1201369A (en) |
| AU (1) | AU716497B2 (en) |
| BG (1) | BG102355A (en) |
| CA (1) | CA2233278A1 (en) |
| CZ (1) | CZ92998A3 (en) |
| HU (1) | HUP9802651A3 (en) |
| NO (1) | NO981386L (en) |
| NZ (1) | NZ319891A (en) |
| PL (1) | PL325953A1 (en) |
| SK (1) | SK40198A3 (en) |
| TR (1) | TR199800573T2 (en) |
| WO (1) | WO1997011605A1 (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU3210997A (en) * | 1996-05-24 | 1997-12-09 | University Of Maryland At Baltimore | Dna vaccines for eliciting a mucosal immune response |
| TW570803B (en) * | 1997-04-09 | 2004-01-11 | Duphar Int Res | Influenza vaccine |
| US6287569B1 (en) * | 1997-04-10 | 2001-09-11 | The Regents Of The University Of California | Vaccines with enhanced intracellular processing |
| AU2674299A (en) * | 1998-02-11 | 1999-08-30 | Maxygen, Inc. | Targeting of genetic vaccine vectors |
| AU2487300A (en) | 1998-12-31 | 2000-07-31 | Chiron Corporation | Polynucleotides encoding antigenic hiv type c polypeptides, polypeptides and uses thereof |
| NZ515041A (en) * | 1999-04-21 | 2004-03-26 | Powderject Vaccines Inc | Nucleic acid immunization |
| CN1512894B (en) | 2001-03-30 | 2010-06-16 | 格林维尔医院系统公司 | Monocyte specific particulate delivery carrier |
| US7211659B2 (en) | 2001-07-05 | 2007-05-01 | Chiron Corporation | Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof |
| US11529414B2 (en) | 2020-06-23 | 2022-12-20 | Orbis Health Solutions, Llc | Viral vaccines for in vivo expression of a nucleic acid encoding an immunogenic peptide and methods of using the same |
-
1996
- 1996-09-27 KR KR1019980702134A patent/KR19990063672A/en not_active Application Discontinuation
- 1996-09-27 PL PL96325953A patent/PL325953A1/en unknown
- 1996-09-27 CA CA002233278A patent/CA2233278A1/en not_active Abandoned
- 1996-09-27 AU AU72515/96A patent/AU716497B2/en not_active Ceased
- 1996-09-27 JP JP9513762A patent/JPH11512724A/en active Pending
- 1996-09-27 CZ CZ98929A patent/CZ92998A3/en unknown
- 1996-09-27 SK SK401-98A patent/SK40198A3/en unknown
- 1996-09-27 TR TR1998/00573T patent/TR199800573T2/en unknown
- 1996-09-27 CN CN96198106A patent/CN1201369A/en active Pending
- 1996-09-27 WO PCT/US1996/015728 patent/WO1997011605A1/en not_active Application Discontinuation
- 1996-09-27 EP EP96933987A patent/EP0863704A4/en not_active Withdrawn
- 1996-09-27 NZ NZ319891A patent/NZ319891A/en unknown
- 1996-09-27 HU HU9802651A patent/HUP9802651A3/en unknown
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1998
- 1998-03-26 BG BG102355A patent/BG102355A/en unknown
- 1998-03-26 NO NO981386A patent/NO981386L/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| BG102355A (en) | 1999-04-30 |
| NZ319891A (en) | 1999-01-28 |
| TR199800573T2 (en) | 1998-07-21 |
| NO981386D0 (en) | 1998-03-26 |
| WO1997011605A1 (en) | 1997-04-03 |
| PL325953A1 (en) | 1998-08-17 |
| AU716497B2 (en) | 2000-02-24 |
| SK40198A3 (en) | 1998-11-04 |
| EP0863704A1 (en) | 1998-09-16 |
| CZ92998A3 (en) | 1998-09-16 |
| NO981386L (en) | 1998-05-28 |
| HUP9802651A2 (en) | 1999-02-01 |
| HUP9802651A3 (en) | 2001-08-28 |
| EP0863704A4 (en) | 2003-09-10 |
| JPH11512724A (en) | 1999-11-02 |
| AU7251596A (en) | 1997-04-17 |
| KR19990063672A (en) | 1999-07-26 |
| CA2233278A1 (en) | 1997-04-03 |
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