CA2233278A1 - Stimulation of cell-mediated immune responses by targeted particulate genetic immunization - Google Patents
Stimulation of cell-mediated immune responses by targeted particulate genetic immunization Download PDFInfo
- Publication number
- CA2233278A1 CA2233278A1 CA002233278A CA2233278A CA2233278A1 CA 2233278 A1 CA2233278 A1 CA 2233278A1 CA 002233278 A CA002233278 A CA 002233278A CA 2233278 A CA2233278 A CA 2233278A CA 2233278 A1 CA2233278 A1 CA 2233278A1
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- antigen
- tumor rejection
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- particulate
- cells
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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
STIMULATION OF CELL-MEDIATED IMMUNE RESPONSES BY TARGETED
PARTICULATE GENETIC IMMUNIZATION
1. INTRODUCTION
Genetic immnni7~tion for the purpose of stim~ ting antigen-specific i,.,...~ ily in a m~mm~ n host, in-~ln-ling a human host, is at the core of the present riicnlos---e. This crerific~tion discloses delivery of particulate polynucleotides to the cytoplasm of host 20 target cells, such as antigen ~e5~ .t;.~g cells. These particulate polynurl~ootides encode an antigenic protein or antigenic protein fr~gment which ~rce~C~s the cytoplasm of the target cell. Expression of the antigen gene results in antigen-specific immnn~ ~e~l,onses, infhl-ling but not limited to, the induction of antigen-specific cytotoxic T-lymphocytes (CTLs). Cytosolic access of the antigen allows membrane pl~se~.ti.tion of the ~ntigenic 25 peptide through the endogenous MHC class I pathway. Membrane ~ on via the endoge.,ous MHC class I pathway stimlll~t~s the induction of antigen-specific CTLs.
Tn~luced antigen-specific CTLs then target and destroy antigen-e~yl~-~sing affected host cells such as neoplastic cells or virally infected cells.
CA 02233278 l99X-03-27 WO 97/11605 PCT~US96/15728 _, 2. BACKGROUND OF THE INVENT~QN
Cytotoxic T-lymphocytes (CTLs) are a critical component of effective human imml-nP responses to tumors or viral infections. Cytotoxic T-lymphocytes destroyneoplastic cells or virus infected cells through recognition of antigenic peptides ~ s~nt~d by MHC class I mokFcule$ on the surface of the ~ffected target cells. These antigenic peptides are degra~i~tion products of foreign proteins present in the cytosol of the affected cell, which are ~ ce~5ed and p.~se.,ted to CTLs through the endogenous MHC class I
~,-ocF~;ng pathway.
Althc--gh the recognition of a foreign protein in the context of the MHC class ImoleclllF may be s~lffi~iF~nt for the recognition and destruction of affected target cells by CTLs, the induction of antigen-specific CTLs from T-lymphocyte precursors requires ~rlriition~l signals. Spe~i~li7Fcl antigen plFs~ g cells (APCs) can provide both the antigen-MHC class I ligand and the ~r~c~ccory signals required in the indllction phase of CTL-meAi~t~d immunity. General properties of APCs include MHC class I and class II
expression, ~ sion of various adhesion molecules important for APC-lymphocyte int~r~cti~n, and expression of costimlll~tory molecules such as CD80 and CD86.
FY~mrles of APCs include macrophages and dendritic cells (including c~lt~neollc ~ide.,llal Langerhans cells, dermal dendritic cells, and dendritic cells resident in Iymph nodes and spleen).
Attempts to induce antigen-specific CTL responses in vivo by immuni7~tion with killed tumor cells, killed virus-infected cells, or component proteins have generally been uncllcceccful, presumably because proteins in the extracellular fluids cannot enter the cytosol and access the MHC class I p.ese.~ ion pathway.
Genetic immuni7~tion possess several attractive features. Several in vivo gene transfer mFtho~ic result in transgene expression, including retroviral or adenoviral ...e~ t~d gene transfer, and direct injection of naked DNA (for a review, see Krishnaw, et al., 1995, Nature Med. 1: 521-522 and Pardoll, et al., 1995, Immunity 3:165-169.) Williarns, et al. (1991, Proc. Natl. Acad. Sci. USA 88: 2726-2730) showed the e~ç,_~;on of the protein luciferase in intact epidermal cells following biolistic 30 (binb~ tic) delivery of the firefly luciferase gene. CTL responses were not addressed in these studies, nor were specific host cells targeted to generate cell-mF~ tFd immllne .. .I.o.~ c W O 97/11605 PCT~US96/15728 Tang, et al. (1992, Na~ure 356: 152-154) utilized a biolistic (biob~lictic) device to produce a humoral response to a foreign protein. A gene encoding hGH under control of either the CMV promoter or the B-actin promoter was delivered to the epidermal tissue of mice. Anti-hGH antibodies were detected in mice in response to this immuni7~tionS plocedure. Tang, et al. does not disclose genetic immunization targeting the cell-mrAi~trd immnne pathway. Direct targeting of APC cells for genetic i.. ~ ;on is not Aicrlocl~d or suggested by Tang, et al.
Fynan, et al. (1993, Proc. Natl. Ac~d. Sci. USA 90: 11478-11482) confirmed the finAingc of Tang, et al. by using a plasmid DNA construct enroAing an infll~en7~ virus 10 hPm~gglntinin glycop-o~ . Fynan, et al. col,lp~d humoral responses gener~tPd by gene gun delivery of DNA coated gold beads to the epidermis with other me~h~ ...c and found that the use of a biolistic (biob~licti~) device 1) resulted in 95% protection to a lethal influen7~ ch~llrnge~ 2) was the most effiril~nt route for DNA immnni7~fign, proving to be s--bst~nti~lly more effective than mucosal, intramuscular, or intravenous ~Aminict~ion, and, 3) .~C~lUi~ 250 to 2500 times less DNA than saline inocul~tionc Direct targeting of APC cells for genetic immuni_ation is not Aicnlosed or suggested by Fynan, et al. CTL-m~Ai~tpd immunity is not addressed.
Liu and colleagues (Montgomery et al., 1993, DNA Cell Biol. 12:777-783; Ulmer et al., 1993, Science. 259:1745-1749; Donnelly et al., 1995, Nature Medicine 1:583-20 587.) have Aemonctrated that untargeted, nonq~erific intr~m--ccnl~r injection of naked DNA induces antigen-specific CTL responses to viral proteins and ~lote~;ti~re imm--nity to viral rh~llrng~. The studies do not disclose targeting of genetic m~t~ri~l to APCs for genetic im...~ ;9.,l Sun, et al. (1995, Proc. Natl. Acad. Sci. USA 92: 2889-2893) utilized a biolistic (biob~lictic) device to produce an anti-tumor response in mice. The authors delivered a plasmid con~LIu-;~ e~pressillg IL-6 directly to a tumor site in mice. E~ .sion of IL-6 afforded a form of cytokine gene therapy nons~ec;l;r~lly directed at the tumor. Antigen-specific illllllll.l;~y to tumors was not cl~imrd- Direct targeting of APC cells for genetic illlllln~ ;on is not Aicrlr$rd or suggested by Sun, et al.
Kundig et al. (1995, Science. 268: 1343-1346) clemorlct ate that protein antigenloc~li7~tion to the Iymphoid organs is critical for the induction of antigen-specific CTL
onse~ in vivo. Genetic immuni7~tir,n is not addressed.
WO 97/11605 PCT~US96/15728 Kovacsovics-Bankowski and Rock (l99S, Science 267: 243-246) demonctrate a phagosome-to-cytosol pathway for protein AntiQenc not normally plesentcd through the MHC class I endogenous pathway. The authors cpertllAte that proteins in particulate forrn internAli7e~ within phagosol"es are in fact able to enter the cytosolic pathway for MHC
S class I P1~ ,~ nAI;O" The capacity of fi-nt~tic~nAlly intact genetic mAtPrial to enter the cytosol through a ~hagosc""e-to-cytosol pathway is not add.c..scd.
Falo, et al. (l99S, Na~ure Med. 1: 649-653) offer in vivo support for ph~5,....~-to-cytosol pathway by showing that delivery of particulate protein antigen directly into animals results in antigen-specific CTL m~'liA-~d tumor immnni~y in mice. They 10 ~.omonct.s3te that proteins injected directly into animals in vivo can cpecificAlly enoer the phagos~""c cytosol pathway of APCs if A-lminictered in particulate form. No details are forwarded leg~rding genetic imm-mi7~tinn procedures. The capacity of in vivo ~cl...i";~ d genetic m~t~riAl to enter the cytosol of APCs, or other cell types,fnnctionAIly intact through this pathway is not addressed.
Pardoll and Recl~rlPg (l99S, ~mmuni~y 3:165-169) have recently reviewed the immnr~ology of naked DNA vaccines. They emphAci7~ the i,.,pc.lLance of ~irlition~l studies to define the cu.-cnlly unknown mechAnicm of DNA immnni7~iQn Specific~lly, they c~-ncll-de that "it will be i-"~oiL~nt to dissect the ".e~ hAIlicmc by which it (naked DNA) activates immune ~ 7~nSCS. It is only through these studies that int~lligt n~
20 m~xiific~tic~nc can be introduced to ~A~ ', both qualitatively and q---AnfitAtively its imAtf potency."
Despite the efforts ~o. -...c.lt~ in the above ~cÇcrc.~ce material, there remains a need to develop a genetic i~nllll..l; ~tion protocol which specificAIly targets cell types within the host to 5timnlAt.o antigen-specific CTL mloAiAt~d i...~.~m~ily and in turn ~..,."ole 25 direct dc;.~ ion of specific neoplastic or virally infected cells within the host. The present invention both addresses and meets this need.
PARTICULATE GENETIC IMMUNIZATION
1. INTRODUCTION
Genetic immnni7~tion for the purpose of stim~ ting antigen-specific i,.,...~ ily in a m~mm~ n host, in-~ln-ling a human host, is at the core of the present riicnlos---e. This crerific~tion discloses delivery of particulate polynucleotides to the cytoplasm of host 20 target cells, such as antigen ~e5~ .t;.~g cells. These particulate polynurl~ootides encode an antigenic protein or antigenic protein fr~gment which ~rce~C~s the cytoplasm of the target cell. Expression of the antigen gene results in antigen-specific immnn~ ~e~l,onses, infhl-ling but not limited to, the induction of antigen-specific cytotoxic T-lymphocytes (CTLs). Cytosolic access of the antigen allows membrane pl~se~.ti.tion of the ~ntigenic 25 peptide through the endogenous MHC class I pathway. Membrane ~ on via the endoge.,ous MHC class I pathway stimlll~t~s the induction of antigen-specific CTLs.
Tn~luced antigen-specific CTLs then target and destroy antigen-e~yl~-~sing affected host cells such as neoplastic cells or virally infected cells.
CA 02233278 l99X-03-27 WO 97/11605 PCT~US96/15728 _, 2. BACKGROUND OF THE INVENT~QN
Cytotoxic T-lymphocytes (CTLs) are a critical component of effective human imml-nP responses to tumors or viral infections. Cytotoxic T-lymphocytes destroyneoplastic cells or virus infected cells through recognition of antigenic peptides ~ s~nt~d by MHC class I mokFcule$ on the surface of the ~ffected target cells. These antigenic peptides are degra~i~tion products of foreign proteins present in the cytosol of the affected cell, which are ~ ce~5ed and p.~se.,ted to CTLs through the endogenous MHC class I
~,-ocF~;ng pathway.
Althc--gh the recognition of a foreign protein in the context of the MHC class ImoleclllF may be s~lffi~iF~nt for the recognition and destruction of affected target cells by CTLs, the induction of antigen-specific CTLs from T-lymphocyte precursors requires ~rlriition~l signals. Spe~i~li7Fcl antigen plFs~ g cells (APCs) can provide both the antigen-MHC class I ligand and the ~r~c~ccory signals required in the indllction phase of CTL-meAi~t~d immunity. General properties of APCs include MHC class I and class II
expression, ~ sion of various adhesion molecules important for APC-lymphocyte int~r~cti~n, and expression of costimlll~tory molecules such as CD80 and CD86.
FY~mrles of APCs include macrophages and dendritic cells (including c~lt~neollc ~ide.,llal Langerhans cells, dermal dendritic cells, and dendritic cells resident in Iymph nodes and spleen).
Attempts to induce antigen-specific CTL responses in vivo by immuni7~tion with killed tumor cells, killed virus-infected cells, or component proteins have generally been uncllcceccful, presumably because proteins in the extracellular fluids cannot enter the cytosol and access the MHC class I p.ese.~ ion pathway.
Genetic immuni7~tion possess several attractive features. Several in vivo gene transfer mFtho~ic result in transgene expression, including retroviral or adenoviral ...e~ t~d gene transfer, and direct injection of naked DNA (for a review, see Krishnaw, et al., 1995, Nature Med. 1: 521-522 and Pardoll, et al., 1995, Immunity 3:165-169.) Williarns, et al. (1991, Proc. Natl. Acad. Sci. USA 88: 2726-2730) showed the e~ç,_~;on of the protein luciferase in intact epidermal cells following biolistic 30 (binb~ tic) delivery of the firefly luciferase gene. CTL responses were not addressed in these studies, nor were specific host cells targeted to generate cell-mF~ tFd immllne .. .I.o.~ c W O 97/11605 PCT~US96/15728 Tang, et al. (1992, Na~ure 356: 152-154) utilized a biolistic (biob~lictic) device to produce a humoral response to a foreign protein. A gene encoding hGH under control of either the CMV promoter or the B-actin promoter was delivered to the epidermal tissue of mice. Anti-hGH antibodies were detected in mice in response to this immuni7~tionS plocedure. Tang, et al. does not disclose genetic immunization targeting the cell-mrAi~trd immnne pathway. Direct targeting of APC cells for genetic i.. ~ ;on is not Aicrlocl~d or suggested by Tang, et al.
Fynan, et al. (1993, Proc. Natl. Ac~d. Sci. USA 90: 11478-11482) confirmed the finAingc of Tang, et al. by using a plasmid DNA construct enroAing an infll~en7~ virus 10 hPm~gglntinin glycop-o~ . Fynan, et al. col,lp~d humoral responses gener~tPd by gene gun delivery of DNA coated gold beads to the epidermis with other me~h~ ...c and found that the use of a biolistic (biob~licti~) device 1) resulted in 95% protection to a lethal influen7~ ch~llrnge~ 2) was the most effiril~nt route for DNA immnni7~fign, proving to be s--bst~nti~lly more effective than mucosal, intramuscular, or intravenous ~Aminict~ion, and, 3) .~C~lUi~ 250 to 2500 times less DNA than saline inocul~tionc Direct targeting of APC cells for genetic immuni_ation is not Aicnlosed or suggested by Fynan, et al. CTL-m~Ai~tpd immunity is not addressed.
Liu and colleagues (Montgomery et al., 1993, DNA Cell Biol. 12:777-783; Ulmer et al., 1993, Science. 259:1745-1749; Donnelly et al., 1995, Nature Medicine 1:583-20 587.) have Aemonctrated that untargeted, nonq~erific intr~m--ccnl~r injection of naked DNA induces antigen-specific CTL responses to viral proteins and ~lote~;ti~re imm--nity to viral rh~llrng~. The studies do not disclose targeting of genetic m~t~ri~l to APCs for genetic im...~ ;9.,l Sun, et al. (1995, Proc. Natl. Acad. Sci. USA 92: 2889-2893) utilized a biolistic (biob~lictic) device to produce an anti-tumor response in mice. The authors delivered a plasmid con~LIu-;~ e~pressillg IL-6 directly to a tumor site in mice. E~ .sion of IL-6 afforded a form of cytokine gene therapy nons~ec;l;r~lly directed at the tumor. Antigen-specific illllllll.l;~y to tumors was not cl~imrd- Direct targeting of APC cells for genetic illlllln~ ;on is not Aicrlr$rd or suggested by Sun, et al.
Kundig et al. (1995, Science. 268: 1343-1346) clemorlct ate that protein antigenloc~li7~tion to the Iymphoid organs is critical for the induction of antigen-specific CTL
onse~ in vivo. Genetic immuni7~tir,n is not addressed.
WO 97/11605 PCT~US96/15728 Kovacsovics-Bankowski and Rock (l99S, Science 267: 243-246) demonctrate a phagosome-to-cytosol pathway for protein AntiQenc not normally plesentcd through the MHC class I endogenous pathway. The authors cpertllAte that proteins in particulate forrn internAli7e~ within phagosol"es are in fact able to enter the cytosolic pathway for MHC
S class I P1~ ,~ nAI;O" The capacity of fi-nt~tic~nAlly intact genetic mAtPrial to enter the cytosol through a ~hagosc""e-to-cytosol pathway is not add.c..scd.
Falo, et al. (l99S, Na~ure Med. 1: 649-653) offer in vivo support for ph~5,....~-to-cytosol pathway by showing that delivery of particulate protein antigen directly into animals results in antigen-specific CTL m~'liA-~d tumor immnni~y in mice. They 10 ~.omonct.s3te that proteins injected directly into animals in vivo can cpecificAlly enoer the phagos~""c cytosol pathway of APCs if A-lminictered in particulate form. No details are forwarded leg~rding genetic imm-mi7~tinn procedures. The capacity of in vivo ~cl...i";~ d genetic m~t~riAl to enter the cytosol of APCs, or other cell types,fnnctionAIly intact through this pathway is not addressed.
Pardoll and Recl~rlPg (l99S, ~mmuni~y 3:165-169) have recently reviewed the immnr~ology of naked DNA vaccines. They emphAci7~ the i,.,pc.lLance of ~irlition~l studies to define the cu.-cnlly unknown mechAnicm of DNA immnni7~iQn Specific~lly, they c~-ncll-de that "it will be i-"~oiL~nt to dissect the ".e~ hAIlicmc by which it (naked DNA) activates immune ~ 7~nSCS. It is only through these studies that int~lligt n~
20 m~xiific~tic~nc can be introduced to ~A~ ', both qualitatively and q---AnfitAtively its imAtf potency."
Despite the efforts ~o. -...c.lt~ in the above ~cÇcrc.~ce material, there remains a need to develop a genetic i~nllll..l; ~tion protocol which specificAIly targets cell types within the host to 5timnlAt.o antigen-specific CTL mloAiAt~d i...~.~m~ily and in turn ~..,."ole 25 direct dc;.~ ion of specific neoplastic or virally infected cells within the host. The present invention both addresses and meets this need.
3. SUMMARY OF THE INVENTION
The present invention relates to th~ peutic or prophylactic genetic i-, -- -- ~;,~t;o.
of a m~mm~ n host which comprises delivery of a DNA fragment which encodes an antigenic protein to a target cell within the m~mm~ n host, expression of the 5 l~o-llbilldnt DNA fr~gmPnt within the host cell, and subs~ucnt pl~,f ~t;~tion of the antigenic peptide or peptides by the host cell so as to stimul~tP- cell-mPAi~tPd immlmity, hllmor~l ;.. I.. ;~y, or both.
The present invention further relates to th~P~relltir or prophylactic genetic immuni7~tion of a ,,m~mm~ n host which comprises delivery of a DNA sequence 10 PnCoding a antigenic protein or biologically active fragment thereof to a specific target cell within the m~mm~ n host. Antigenic peptides eA~ 5sed from the DNA
are specific to an affected cell and a,lL.s~u~,ntly ctim~ tP antigen specific CTL
production, thereby promoting destruction of affected cells such as neoplastic cells and virally infectP~ cells.
The present invention also relates to genetic immuni7~tion with particulate polynucleotides and inocul~tion of a m~mm~ n host and subsequent delivery of these particulate-based transgenic polynucleotides to the cytosol of the target cell. Once within the conr;n~s of the target cell, the particulate polynucleotide t~ Gaa~s a protein or hiologic~lly active fr~gmPnt thereof whereby an a~plupliate ~ntigenic peptide fr~gmPnt is 20 r~ d and ~lGsenlGd to the target cell membrane via the Pn~iQg~nous MHC class I
pathway. Proper presentation of the ~ntigenir peptide or peptides of interest through the MHC class I pathway stimnl~t~ps CTL production and in turn promotes destruction of cells such as neoplastic cells or virally infected cells.
Thel~,iol.;, the present invention also relates to in YiW metho-ls of therapeutic or 25 prophylactic genetic imml-ni7~ric n of a m~mm~ n host which comprises generating a DNA r..t~ ..t which G~pl~ sses an antigenic protein or antigenic protein r, ~"--- -t diaLIibuling the DNA fragment on a particle surface which results in a particulate polynurleotidP7 inocnl~ting the m~mm~ n host with said particulate polynucleotide and delivering the particulate polynuc1Poti-ie to the cytoplasm of a target cell of the 30 m~mm~ n host so that the e,~ aed antigenic protein or antigenic protein fragment is ~lesenl~;d to the Illelllblane surface of said target cell through the MHC class I pathway.
- In the present invention it is preferable that the m~mm~ n host be a human.
W O 97/11605 PCT~US96/15728 It is also preferable in the various embodiments disclosed within this cpecifie~tion that the DNA fr~gmtont of interest express l) a tumor rejection antigen or an ~ntig~nin protein fr~gm.ont or 2) a viral antigen or an antigenic protein fragment. Examples of human TRAs which may be utilized in the present invention include but are not limited to 5 MAGE-l, MAGE 3, Melan-A, gplOO, p53, CEA and HER2/neu. Examples of viral ~ntigenc which may be utilized in the present invention include but are not limited to HIV
gpl20, HIV gpl60, TnfluPn7~ virus nucl~p-v~eill and Hepatitis B surface antigen The ~.~rc..~d target cell in the present invention is an APC while the l -~r~...,d 10c~1i7~tinn or migration of the APC target cell is the Iymphoid tissue of the human host 10In one embo~lim~ont of the present invention the m~mm~ host is immnni7~~
with the particulate polynucleotide by ~Iti1i7ing a ...i.~;o~..3jectile bombardment device.
Spe~ifi-~lly, a m~mm~ n host is imm--ni7~d by inocul~tion with a particulate polynuc1~oti-1e by a biolistic (biob~1icti~) procedure such that the particulatepolynucleotide enters the cytoplasm of at least an ~rvp-iate number of host cells. The 15 tr~ncgeni~ polynucleotide is e~ e,sed at biologically effective levels such that ~nti~enic peptide r.~g...~ are presented to the endogenous MHC class I pathway and displayed on the membrane surface of the host cells Endogenous host cell membrane pr~s~ont~tion of the introduced antigen promotes induction of antigen-specific CTLs, which in turn circulate throughout the m~mm~ n host, p~re~ably a human host, to destroy neop!~ctir 20 cells or virally infected cells.
In a specific embo lim~nt of the present invention a m~mm~ n host, ~ Çelably a human, is i.. ni,. ~ with a particulate polynll~1eoti~le by microprojectile bOmb~l"l~.~L
inoc~ tion such that the particulate polynucleotide enters the cytoplasm of at least an d~-v~riale number of host cells, in~ln~ing APCs, in the path of the projectiles as a direct 25 result of non-specific projectile bombardment. When the skin is bombarded, APCs of the skin which may be bombarded include, but are not limited to epidermal Langerhans cells, kPr~tin~cyte5, or dermal d~n<lritic cells. When the lymphoid tissue is bo,--ba ded, APCs of the lymphoid tissue which may be bv--.ba-ded include, but are not limited to resident tl.on~lritic cells, ...a~.vpl1ages~ stromal cells, T-lymphocytes, or B-lymphocytes.
30In another embodiment of the present invention a m~mm~ n host is immnni7~A
with a particulate polynueleoti~le by direct injection, including but not limited to S~IJC~ V~C injection, epidermal injection, dermal injection, Iymphatic injection and W O 97/11605 PCT~US96/15728 --7--intra venous injection. The particulate polynucleotide enters host cells and is eA~l.,ss~d at bio1cgi~11y effective levels such that ~nti~nic peptide fra~mPntc are pleaented to the endogenous MHC class I pathway and displayed on the membrane surface of the hostcell. Endogenous target cell membrane pr.osPnt~tion promotes the induction of antigen-S specific CTLs, which in turn circulate throughout the m~mm~ n host, plcrel~bly ahuman host, to destroy neoplastic cells or virally infected cells.
In an esre~i~11y preferred embodiment the particulate polynucleotide is delivered to a human host by s~bcut~ntqous injection and targeted to APCs through a phagosome-to-cytosol pathway and ~AI~lcased at biologically effective levels by APCs. The present invention fiic.-1oSes that direct injection of the particulate polynucleotide complex via a ~uL.~ n~Qus inoculation route results in targeted delivery to APCs and antigen ~ ion in the Iymphoid tissue.
In another especially ~ ll~ embodiment of the present invention, s~b~ .n~us injection for direct targeting to an APC involves delivery of a particulate polynucleotide 15 ~ncorling a tumor rejection antigen (TRA) or biologically active fr~gm~nt thereof.
Directed delivery of a TRA particulate polynucleotide in this manner will m~ximi7~ entry of tumor specific ~ntiglonic peptides into the class I pathway as well as avoiding s~bct~nti~1 particulate translocation within non-APC cells.
In another ecpeci~l1y pl~:f~lled embodiment of the present invention, 5nb~ n~
20 injection for direct targeting to an APC involves delivery of a particulate polynucleotide f-n~o-iing a viral antigen or biologically active fr~gm~ont thereof. Directed delivery of a viral antigen ~n~orling particulate polynucleotide in this manner will m~cimi7~ entry of viral specific ~ntig~nic peptides into the class I pathway as well as avoiding s~~bst~nti~
particulate tr~n~10c~tion within non-APC cells.
2~ It will also be known to the skilled artisan that particul~tes composed of a variety of m~t~-ri~1~ including but not limited to gold, iron, and synthetic plastics can access the phagosome-to-cytosol pathway used to target APCs in this invention.
In another embodiment of the present invention a m~mm~ n host is im.~
by injection, inf 1~iing but not limited to subcutaneous injection, epidermal injection, 30 dermal injection, lymphatic injection and intra venous injection, with syngeneic APCs that have been antigen loaded in vitro by co-inc~lb~tion with particulate polynucleotide.
- Particulate polym~e1eoti~es may, for example, enter the APC in vitro by either WO 97/11605 PC~AUS96/15728 microprojectile bombdld~ t or the pha~osonlc to-cytosol pathway. Specifically, am~mm~ n host is i,.. l.. ,;,f~ with particulate polynucleotide transfected APCs, such that the particulate polynucleotide ~ific~lly enters APCs in vitro and the APCs are injected into the host. Subsequent to uptake by APCs, the transgenic polynllcleoti-ie is c~ c;~scd S at bi~-logir~lly effective levels such that ~ntigenic peptide fragments are prucess~ and pl~-ltcd through the endogenous MHC class I pathway and displayed on the In~ 7l,~..P
surface of the APCs. After inje~tion of such APCs, Pn~og~nous APC cell ~ b~
~e~ ~ ;on of antigen plUIIIOIe~t the in-iuction of antigen-specific CTLs either at the sitc of injection, or in the lymphoid tissue. Induced antigen-spccific CTLs in turn circulate 10 throughout thc m~mm~ n host, plcfcldbly a human host, to destroy neoplastic cells or virally infected cells.
In another embodiment of the present invention a m~mm~ n host is i~.. n.~
by injection, inriuding but not limitcd to suhc~lt~neous injection, epidermal injc,_~ion, dermal injection, lymphatic injection and intra venous injection, with syngeneic APCs that 15 have been ~ n~r~ ~ in vitro with particulate polynllrleoti~ies Sperific~lly, a m~mm~ n host is i.. ;,.~i with particulate polynucleotide transfected APCs, such that the particulate polynucleotide enters APCs in vitro and the APCs are then injected into the host. Particulate polynucl~oti~les may, for example, enter the APC in vitro by either mic~u~J;ecLile bomb~d~ ,--t or the phagosome-to-cytosol pathway. APCs are tr~n~fer~d 20 in vitro with antigen enrorling polynucleotide and/or polynucleotides enCQrling a or mol~clll.o5 which increases the effiriPnry of the antigen pres~nting function of the APC. Such mol~ s include but are not limited to cytokines and costimlll~tory mole~ s F~;....l.les of cytokines include but are not limited to IL-12, IL-2, and IL4.
F~ ks of costim~ tory molecules include but are not limited to CD80 and CD86.
25 Subse IL~ellL to entry into APCs, the tr~n~genic antigen enro(ling polym~clcQ~ is CAIJl.,sS~ at biologin~lly effective levels such that antigenic peptide fragments are p-uces~ and ple~.e.-l~:d through the endogenous MHC class I pathway and displayed on the ln~.lllJl~lc surface of the APCs. After injection of such APCs, endogenous APC cell m .. b .. r p~ tirJn of antigen promotes the in~lction of antigen-specific CTLs either 30 at the site of injection, or in the lymphoid tissue. Induced antigen-specific CTLs in turn circulate throughout the m~mm~ n host, ~l~r~.dbly a human host, to destroy n~ ~p~ r cells or virally infected cells. S~ll.s~ enl to entry into APCs, the transgenic cytokine -W O 97/11605 PCT~US96/157Z8 _g and/or costimul~t(~ry PncoriinE polynucleotide is c~p-~cs~.cd at biologically effective levels such that the antigen presenting function of the APC results in the indtlction of antigen specific immun~ responses either at the site of the injection, or in the lymphoid tissue.
It will also be known to the skilled artisan that polynucleotides can be l,~c ~ .t~.
S onto partir~ t~os colllposed of a variety of m~tPri~lc inclllAin~ but not limited to gold, iron, and synthetic plastics.
It will also be known to the skilled artisan that various recombinant vectors may be used to ~ od the ~ sg~.le sequence to be applied in particulate form. The p.~f~..~l vector, due primarily to ease of h~nAling, is a DNA plasmid vector.
It will be known to the skilled artisan that APCs can be obtained from a variety of host tissue in~ g, but not limited to bone marrow and pefiphc~l blood, and that said APCs can be manipulated in YitrO and then reintroduced into said host.
It is an object of the present invention to provide th~r~reutic or prophylactic genetic im~ inn against neoplastic cells.
It is another object of the present invention to provide therapeutic or prophylactic genetic immnni7~ti~n against viral infections.
It is an object of the present invention to prc.vide for genetic immuni7~tion of a m~mm~ n host, ~cre~bly a human, by targeting particulate polynucleotides encoAin~
tumor rejection antigen genes to host imml.ne cells involved in generating a CTL.~ e It is an object of the present invention to provide for genetic immnni7~tion of a m~mm~ n host, preferably a human, by kllc,cLh~g particulate polynllc~ootiAes r.,~o~ g tumor rejection antigen genes to host antigen prP~enting cells 1O~1i7ed within or capable Of tr~ffirl~in~ to host Iymphoid tissue so as to gen~.dle a CTL response.
It is an object of the present invention to provide for genetic immuni7~tion of a m~mmz~ n host, ~ cÇc~dbly a human, by targeting particulate polymlcleotides ~ncoAing viral genes to host immnnç cells involved in generating a CTL response against the specific viral infection It is an object of the present invention to provide for genetic immnni7~tion of a m~mm~ n host, preferably a human, by targeting particulate polynucleotides çncoAing vi~al genes to host antigen ~ese~ g cells loc~li7~A within host lymphoid tissue so as to a CTL ~.~.~onse.
The present invention relates to th~ peutic or prophylactic genetic i-, -- -- ~;,~t;o.
of a m~mm~ n host which comprises delivery of a DNA fragment which encodes an antigenic protein to a target cell within the m~mm~ n host, expression of the 5 l~o-llbilldnt DNA fr~gmPnt within the host cell, and subs~ucnt pl~,f ~t;~tion of the antigenic peptide or peptides by the host cell so as to stimul~tP- cell-mPAi~tPd immlmity, hllmor~l ;.. I.. ;~y, or both.
The present invention further relates to th~P~relltir or prophylactic genetic immuni7~tion of a ,,m~mm~ n host which comprises delivery of a DNA sequence 10 PnCoding a antigenic protein or biologically active fragment thereof to a specific target cell within the m~mm~ n host. Antigenic peptides eA~ 5sed from the DNA
are specific to an affected cell and a,lL.s~u~,ntly ctim~ tP antigen specific CTL
production, thereby promoting destruction of affected cells such as neoplastic cells and virally infectP~ cells.
The present invention also relates to genetic immuni7~tion with particulate polynucleotides and inocul~tion of a m~mm~ n host and subsequent delivery of these particulate-based transgenic polynucleotides to the cytosol of the target cell. Once within the conr;n~s of the target cell, the particulate polynucleotide t~ Gaa~s a protein or hiologic~lly active fr~gmPnt thereof whereby an a~plupliate ~ntigenic peptide fr~gmPnt is 20 r~ d and ~lGsenlGd to the target cell membrane via the Pn~iQg~nous MHC class I
pathway. Proper presentation of the ~ntigenir peptide or peptides of interest through the MHC class I pathway stimnl~t~ps CTL production and in turn promotes destruction of cells such as neoplastic cells or virally infected cells.
Thel~,iol.;, the present invention also relates to in YiW metho-ls of therapeutic or 25 prophylactic genetic imml-ni7~ric n of a m~mm~ n host which comprises generating a DNA r..t~ ..t which G~pl~ sses an antigenic protein or antigenic protein r, ~"--- -t diaLIibuling the DNA fragment on a particle surface which results in a particulate polynurleotidP7 inocnl~ting the m~mm~ n host with said particulate polynucleotide and delivering the particulate polynuc1Poti-ie to the cytoplasm of a target cell of the 30 m~mm~ n host so that the e,~ aed antigenic protein or antigenic protein fragment is ~lesenl~;d to the Illelllblane surface of said target cell through the MHC class I pathway.
- In the present invention it is preferable that the m~mm~ n host be a human.
W O 97/11605 PCT~US96/15728 It is also preferable in the various embodiments disclosed within this cpecifie~tion that the DNA fr~gmtont of interest express l) a tumor rejection antigen or an ~ntig~nin protein fr~gm.ont or 2) a viral antigen or an antigenic protein fragment. Examples of human TRAs which may be utilized in the present invention include but are not limited to 5 MAGE-l, MAGE 3, Melan-A, gplOO, p53, CEA and HER2/neu. Examples of viral ~ntigenc which may be utilized in the present invention include but are not limited to HIV
gpl20, HIV gpl60, TnfluPn7~ virus nucl~p-v~eill and Hepatitis B surface antigen The ~.~rc..~d target cell in the present invention is an APC while the l -~r~...,d 10c~1i7~tinn or migration of the APC target cell is the Iymphoid tissue of the human host 10In one embo~lim~ont of the present invention the m~mm~ host is immnni7~~
with the particulate polynucleotide by ~Iti1i7ing a ...i.~;o~..3jectile bombardment device.
Spe~ifi-~lly, a m~mm~ n host is imm--ni7~d by inocul~tion with a particulate polynuc1~oti-1e by a biolistic (biob~1icti~) procedure such that the particulatepolynucleotide enters the cytoplasm of at least an ~rvp-iate number of host cells. The 15 tr~ncgeni~ polynucleotide is e~ e,sed at biologically effective levels such that ~nti~enic peptide r.~g...~ are presented to the endogenous MHC class I pathway and displayed on the membrane surface of the host cells Endogenous host cell membrane pr~s~ont~tion of the introduced antigen promotes induction of antigen-specific CTLs, which in turn circulate throughout the m~mm~ n host, p~re~ably a human host, to destroy neop!~ctir 20 cells or virally infected cells.
In a specific embo lim~nt of the present invention a m~mm~ n host, ~ Çelably a human, is i.. ni,. ~ with a particulate polynll~1eoti~le by microprojectile bOmb~l"l~.~L
inoc~ tion such that the particulate polynucleotide enters the cytoplasm of at least an d~-v~riale number of host cells, in~ln~ing APCs, in the path of the projectiles as a direct 25 result of non-specific projectile bombardment. When the skin is bombarded, APCs of the skin which may be bombarded include, but are not limited to epidermal Langerhans cells, kPr~tin~cyte5, or dermal d~n<lritic cells. When the lymphoid tissue is bo,--ba ded, APCs of the lymphoid tissue which may be bv--.ba-ded include, but are not limited to resident tl.on~lritic cells, ...a~.vpl1ages~ stromal cells, T-lymphocytes, or B-lymphocytes.
30In another embodiment of the present invention a m~mm~ n host is immnni7~A
with a particulate polynueleoti~le by direct injection, including but not limited to S~IJC~ V~C injection, epidermal injection, dermal injection, Iymphatic injection and W O 97/11605 PCT~US96/15728 --7--intra venous injection. The particulate polynucleotide enters host cells and is eA~l.,ss~d at bio1cgi~11y effective levels such that ~nti~nic peptide fra~mPntc are pleaented to the endogenous MHC class I pathway and displayed on the membrane surface of the hostcell. Endogenous target cell membrane pr.osPnt~tion promotes the induction of antigen-S specific CTLs, which in turn circulate throughout the m~mm~ n host, plcrel~bly ahuman host, to destroy neoplastic cells or virally infected cells.
In an esre~i~11y preferred embodiment the particulate polynucleotide is delivered to a human host by s~bcut~ntqous injection and targeted to APCs through a phagosome-to-cytosol pathway and ~AI~lcased at biologically effective levels by APCs. The present invention fiic.-1oSes that direct injection of the particulate polynucleotide complex via a ~uL.~ n~Qus inoculation route results in targeted delivery to APCs and antigen ~ ion in the Iymphoid tissue.
In another especially ~ ll~ embodiment of the present invention, s~b~ .n~us injection for direct targeting to an APC involves delivery of a particulate polynucleotide 15 ~ncorling a tumor rejection antigen (TRA) or biologically active fr~gm~nt thereof.
Directed delivery of a TRA particulate polynucleotide in this manner will m~ximi7~ entry of tumor specific ~ntiglonic peptides into the class I pathway as well as avoiding s~bct~nti~1 particulate translocation within non-APC cells.
In another ecpeci~l1y pl~:f~lled embodiment of the present invention, 5nb~ n~
20 injection for direct targeting to an APC involves delivery of a particulate polynucleotide f-n~o-iing a viral antigen or biologically active fr~gm~ont thereof. Directed delivery of a viral antigen ~n~orling particulate polynucleotide in this manner will m~cimi7~ entry of viral specific ~ntig~nic peptides into the class I pathway as well as avoiding s~~bst~nti~
particulate tr~n~10c~tion within non-APC cells.
2~ It will also be known to the skilled artisan that particul~tes composed of a variety of m~t~-ri~1~ including but not limited to gold, iron, and synthetic plastics can access the phagosome-to-cytosol pathway used to target APCs in this invention.
In another embodiment of the present invention a m~mm~ n host is im.~
by injection, inf 1~iing but not limited to subcutaneous injection, epidermal injection, 30 dermal injection, lymphatic injection and intra venous injection, with syngeneic APCs that have been antigen loaded in vitro by co-inc~lb~tion with particulate polynucleotide.
- Particulate polym~e1eoti~es may, for example, enter the APC in vitro by either WO 97/11605 PC~AUS96/15728 microprojectile bombdld~ t or the pha~osonlc to-cytosol pathway. Specifically, am~mm~ n host is i,.. l.. ,;,f~ with particulate polynucleotide transfected APCs, such that the particulate polynucleotide ~ific~lly enters APCs in vitro and the APCs are injected into the host. Subsequent to uptake by APCs, the transgenic polynllcleoti-ie is c~ c;~scd S at bi~-logir~lly effective levels such that ~ntigenic peptide fragments are prucess~ and pl~-ltcd through the endogenous MHC class I pathway and displayed on the In~ 7l,~..P
surface of the APCs. After inje~tion of such APCs, Pn~og~nous APC cell ~ b~
~e~ ~ ;on of antigen plUIIIOIe~t the in-iuction of antigen-specific CTLs either at the sitc of injection, or in the lymphoid tissue. Induced antigen-spccific CTLs in turn circulate 10 throughout thc m~mm~ n host, plcfcldbly a human host, to destroy neoplastic cells or virally infected cells.
In another embodiment of the present invention a m~mm~ n host is i~.. n.~
by injection, inriuding but not limitcd to suhc~lt~neous injection, epidermal injc,_~ion, dermal injection, lymphatic injection and intra venous injection, with syngeneic APCs that 15 have been ~ n~r~ ~ in vitro with particulate polynllrleoti~ies Sperific~lly, a m~mm~ n host is i.. ;,.~i with particulate polynucleotide transfected APCs, such that the particulate polynucleotide enters APCs in vitro and the APCs are then injected into the host. Particulate polynucl~oti~les may, for example, enter the APC in vitro by either mic~u~J;ecLile bomb~d~ ,--t or the phagosome-to-cytosol pathway. APCs are tr~n~fer~d 20 in vitro with antigen enrorling polynucleotide and/or polynucleotides enCQrling a or mol~clll.o5 which increases the effiriPnry of the antigen pres~nting function of the APC. Such mol~ s include but are not limited to cytokines and costimlll~tory mole~ s F~;....l.les of cytokines include but are not limited to IL-12, IL-2, and IL4.
F~ ks of costim~ tory molecules include but are not limited to CD80 and CD86.
25 Subse IL~ellL to entry into APCs, the tr~n~genic antigen enro(ling polym~clcQ~ is CAIJl.,sS~ at biologin~lly effective levels such that antigenic peptide fragments are p-uces~ and ple~.e.-l~:d through the endogenous MHC class I pathway and displayed on the ln~.lllJl~lc surface of the APCs. After injection of such APCs, endogenous APC cell m .. b .. r p~ tirJn of antigen promotes the in~lction of antigen-specific CTLs either 30 at the site of injection, or in the lymphoid tissue. Induced antigen-specific CTLs in turn circulate throughout the m~mm~ n host, ~l~r~.dbly a human host, to destroy n~ ~p~ r cells or virally infected cells. S~ll.s~ enl to entry into APCs, the transgenic cytokine -W O 97/11605 PCT~US96/157Z8 _g and/or costimul~t(~ry PncoriinE polynucleotide is c~p-~cs~.cd at biologically effective levels such that the antigen presenting function of the APC results in the indtlction of antigen specific immun~ responses either at the site of the injection, or in the lymphoid tissue.
It will also be known to the skilled artisan that polynucleotides can be l,~c ~ .t~.
S onto partir~ t~os colllposed of a variety of m~tPri~lc inclllAin~ but not limited to gold, iron, and synthetic plastics.
It will also be known to the skilled artisan that various recombinant vectors may be used to ~ od the ~ sg~.le sequence to be applied in particulate form. The p.~f~..~l vector, due primarily to ease of h~nAling, is a DNA plasmid vector.
It will be known to the skilled artisan that APCs can be obtained from a variety of host tissue in~ g, but not limited to bone marrow and pefiphc~l blood, and that said APCs can be manipulated in YitrO and then reintroduced into said host.
It is an object of the present invention to provide th~r~reutic or prophylactic genetic im~ inn against neoplastic cells.
It is another object of the present invention to provide therapeutic or prophylactic genetic immnni7~ti~n against viral infections.
It is an object of the present invention to prc.vide for genetic immuni7~tion of a m~mm~ n host, ~cre~bly a human, by targeting particulate polynucleotides encoAin~
tumor rejection antigen genes to host imml.ne cells involved in generating a CTL.~ e It is an object of the present invention to provide for genetic immnni7~tion of a m~mm~ n host, preferably a human, by kllc,cLh~g particulate polynllc~ootiAes r.,~o~ g tumor rejection antigen genes to host antigen prP~enting cells 1O~1i7ed within or capable Of tr~ffirl~in~ to host Iymphoid tissue so as to gen~.dle a CTL response.
It is an object of the present invention to provide for genetic immuni7~tion of a m~mmz~ n host, ~ cÇc~dbly a human, by targeting particulate polymlcleotides ~ncoAing viral genes to host immnnç cells involved in generating a CTL response against the specific viral infection It is an object of the present invention to provide for genetic immnni7~tion of a m~mm~ n host, preferably a human, by targeting particulate polynucleotides çncoAing vi~al genes to host antigen ~ese~ g cells loc~li7~A within host lymphoid tissue so as to a CTL ~.~.~onse.
4. BRIEF DESCRIPTlON OF THE FIGURES
Figure 1 shows function~l presentation of ovalbumin by the transfected tumor cell lines MO4 and EG7. Microcultures were prepared with the T-cell hybridoma RF33.70S (anti-OVA+ Kb) and the inriie~t~d number of transfected (squares) or untransfected (circles) tumor cells in the presence (open symbols) or absence (closed symbols) of added exogenous OVA-peptide SIINFEKL (lOng/ml~ as described (Rock, et al., 1990, J.
Immunol. 45:804-811). After 18hrs incub~til~n, sl~pern~t~ntc were harvested and assayed for IL-2 using the indicator cell line HT2 (Rock, et al., 1990, J. Immunol. 145: 804-811).
(A) B16 and the OVA-~ sre~;led subrk ne MO4. (B) EL4 and the OVA tr~n~fect~l ELAsu'~clon~o EG7. OVA ~1~ C~ tion by the OVA-transfected tumors was not ~i~nifirantly enh~nred by the presence of exogenous SIINFEKL in the assay cultures.
Figure 2 shows that OVA expression by the B16 derived melanoma MO4 does not signifie~ntly effect in vivo tumor growth or host survival following tumor ~h~ilPnge. Mice were rh~llen~ed with MO4 (circles) or B16 (squares) (5 x 104/mouse, i.d., bilateral, mid-flanks). Tumor size (Figure 2A) was a~p~ced 3x/week and is reported as the average tumor area in square milTimetl~rs until the first death occurred in each group. Survival (Figure 2B) is recorded as the percentage of surviving ~nim~lc All experiments inch~
S mice/group and were repeated at least three times. Mice bf~cQmin~ monbund were~rrjfie~1 Figure 3 shows that immunization by cutaneous delivery of OVA encoding DNA
induces OVA-specific CTLs and antigen-specific, CTL mP~Ti~tPd protection from lethal ch~llen~e with the OVA expressing m~l~nom~ MO4. In vitro restimulated splenocytes from OVA-immuni7~d (g~neti~ y immnni7~i as desc,il,ed in Example Section 7) micewere assayed for cytolytic function against the OVA-tr~ncfect~Pd Iymphoma EG7 (closed squares) or the untr~n~fectPd parent EL4 (open triangles) (A). Effector populations were incubated with complement alone (open squares) or with mAbs against CD4+(closed tri~ngle5), CD8+ (open circles), or Thyl.2+ (closed circles) Iymphocytes and complement, then assayed for cytolytic activity against EG7 targets (B). In C-F, mice were gerletif~lly jmm-lni7~d with OVA (closed squares) or lacZ (open squares) and boosted 7 days later. Groups of immnni7Pd mice were challenged 7 days after the final iml.. ;,~lion (day 0) with either the B16 m~ nom~ (D), or the OVA-e,.~,~saing sub~k~ne W O 97/11605 PCT~US96/15728 MO4 (C). Alternatively, immunized mice were divided into Z groups, one of which was deFIet~cl of CD8+ Iymphocytes by i.p. injection of anti-CD8 mAb 7 and 9 days after the last immuni7~tion. Intact (E) and CD8+ dcplered (E;) mice were then ch~llen~ed 10 days after the final immnni7~tiQn (day 0) with MO4. Survival was reported as the pe.c~..t~ge S of surviving animals (C-~;). Animals surviving on day 60 had no sign of tumor growth.
All e~ ntc in-~luded S mice per group and were ~t:~eated at least 3 times. Mice that became moribund were sacrificed according to animal care guidelines.
Figure 4 shows that antigen ~ lg cells internalize and express particulate polynucleotides, and process and present the ~ essed antigen through the MHC class I
10 restricted p.ucessi~g pathway. Dendritic cells were prepared by ~epl~tin~ bone marrow cells of lymphocytes and culturing overnight in RPMI 1640 suppl~m~ontPd with 10%FCS, L-glnt~min~ antibiotics and 2-ME in 24 well plates at 106 cells/well. Cells were repleted on day 1 at 2.5 x 105 cells/well with GM-CSF (103U/ml, Sigma, St. Louis, MO) andmurine rIL-4 (103U/ml, Genzyme, Cambridge, MA) and loosely adherent cells were 15 harvested on day 8. By flow cytometric analysis, these dendritic cells e~plessed CD45, CD44, CDllb (Mac-1), CD18, CD80, cD86 and class I and class II MHC :lnti~onc Dendritic cells were pulsed 2hrs at 37~C with or without OVA peptide (20ng/ml) +B2-microglobin (B2-M, 10~1/ml, human, Sigma) in reduced serum media (Optimen, Gibco, Grand Island, NY). Cells were then washed extensively, resucrended in PBS and 20 irr~ t~d (2000 rad) before injection into naive mice. The indicated number of bone marrow derived dendritic APCs were cocultured with OvA-enco~ling particulate polynucleotides prepared as described in Example Section 7 (SO~I/ml/106 cells of 7mg/ml partirul~tlos) using either Fe beads (closed squares) or gold beads (closed circles) as the particulate substrate or soluble OVA protein (2mg/ml) (open squares) for 24hrs., washed, 25 and then the inrii~t~d number of APCs were co-cultured in microcultures with the T-cell hybridoma RF33.70 (anti-OVA+ Kb). After 18hrs incub~tion, supernatants were harvested and assayed for IL-2 using the indicator cell line HT2 (Rock, et al., 1990, J.
Immunol. 145: 804-811).
Figure S shows a co~ ~ison of particulate polynucleotide immunization 30 a~iminictered by biolistic (biob~lictic) or subcutaneous injection. Groups of 5 C57BI/6 mice were immuni7PA, boosted on day 7, and then nh~llenged by intradermal iniecti~n of - 5 x 105 MO5 tumor cells in each flank 7 days after boosting. Immuni7~tions CQI~ d W O 97/11605 PCT~US96/15728 of: (A) B-Gal encoding particulate polynuc1eotiAes prepared and delivered by biolistic (binb~licti~-) aAminictration as described in Example Section 7; (B) OVA enCQAing particulate polynucleotides p~ cd and delivered by biolistic (biob~lictic) ~dminictr~inn as des~ribe~ in Fy~mrle Section 7; (C) an excess quantity of OVA encoAing S poly--ucl~otide without parti~ul~t~os delivered by, ~b~ eo~C injection; or, (D) an equivalent amount of OVA PnroAin~ particulate polynucleotiAPs p-~el~d-~d as in F~
Section 7 but aAminict~ed by sl~hcnt~n~ous injection. Data is presented as % of animals tumor free in each group 50 days after tumor ch~ nge.
Figure 6 shows that imml-ni7~fion with APCs which had been co-incubated with 10 particulate polynucleotide enroAing OVA protects animals from ch~ nge by OVA-eA~.es~ g melanoma MOS, Groups of 5 C57Bl/6 mice were im~ ..i;~ suh~ -n~lcly dS d~ ;bed in Figure S on one occ~cion and then .-h~ onged by intlddelllldl injection of 1 x 105 MO5 tumor cells in each flank 10 days after immnni7~tion Imm ~ nc concicted of either: (open squares) particulate polynucleotide encoAing the irrelevant 15 antigen B-g~l~rtnci~i~c~ (1OO~L1 of 7mg/ml particulate solution (particulate wt./vol PBS) per hind leg bil~ttorally; (open circles) salable pAc-neo-OVA (100~1 per hind leg c~an ap~-v..;-~-trly equivalent quantity of DNA/animal); or (closed squares) 5 x 104 bone marrow derived dendritic cells/100~1 hind legs s.q. (prepared as described in FY~-F'- 7).
Survival was reported as the pelccn~ge of surviving ~nim~lc All experiments in~luded 5 20 mice per group. Mice that became ~.-o-ib.lnd were sacrificed according to animal care guidt~lin~s 5. DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term ~mAmmAliAn host" includes members of the animal kingdom, in~lu~ling but not limited to human beings.
As used herein, the term "DNA fragment" may include any nucleotide sequence, S either DNA or RNA, which contAinc apl)rol,-iate coding region and regulatory sequences to result in target cell expression of an Antig~nic protein or AntigPnic protein frAgm~nt for cell ~I-e.-~b-~le prP~PntAtion via the enllogenol~C MHC Class I pathway.
As used herein, the term "particulate polynucleotide" may refer to a particulatemade from materials in~h-fiing but not limited to gold, iron, and synthetic plastics 10 wherein the particle comprises a population of DNA frAgmPnts as defined in the ~ce ling p~. ,.g. ,.ph The present invention relates to thprAreutir or prophylactic genetic im...~ ;,,.l;nn of a m~mm~ n host which comprises delivery of a DNA fragm~nt to a target cell within the mAmmAliAn host, expression of the DNA frAgm~Pnt within the target cell, and 15 subse4uent prestontAtior~ of a recombinant antigenic peptide(s) within the target cell so as to stim--lAt~o cell-me~liAtPd immnnity~ humorAl immnnity, or both.
The present invention further relates to therApeutic or prophylactic genetic immllni7Ation of a mAmm~liAn host which comprises delivery of a DNA fragment ~ncorlin~ a protein or biologi~Ally active fragment thereof to a specific target cell within 20 the m~mmAli~n host. Antigenic peptides ~A~ sed from the DNA sequence are specific to an ~ffected cell such as a nP~pl~ric cell or virally infected cell. Antigen specific CTL
production is ~timulAtPd, thereby promoting destruction of target cells such as ne~plaslic cells and virally infected cells.
The present invention ~licclos~ps genetic immunization mPtho~ic for llcat~ent or25 pl~ ,.tion of tumors or viral infections. Cytotoxic-T-cells are a significant co...pon ~~t of the immlln~ ~nse to tumors and viral infectinn~- Cytotoxic-T-cells kill neoplastic cells or virus-infected cells through the recogniti~n of antigenic peptides ~ ,ent~d by MHC class I mol~pclllp~s on the surface of the tumor target. These peptides are derived from tumor AntigPnc that are synthP~i~pd by the affected cell and ~legrA~Ied in the cytosol.
30 Attempts to induce tumor-specific CTL responses in vivo by immnni~Ati~n with killed tumor cells or component proteins have generally been ~n~lccessful~ presumably because W O 97/11605 PCT~US96/15728 proteins in the ~xtr~re~ r fluids cannot enter the cytosol and access the MHC class I
;on pathway.
A specific embodiment of the present invention relates to genetic immnni7~tion with particles cont~ining the DNA fr~gmPnt ~ s~ing the protein of interest. Such5 particles cont~ining a DNA fr~gml~nt are referred to throughout this ~pecific~tion as 'particulate polynucleotides'. In vivo cell delivery is best accomplished by ~lmini~tering the DNA r.~l~-... nt or protein of interest in particulate form, such as coated beads or gold particles. The protein or biologically active ~ .ne..t is ~A~ sscd subsequent to cytosolic delivery within the target cell. The e~ ssed protein or protein fragment specific to the 10 affected cell provides a substrate for generating an ~ntigenic peptide(s) for prestont~tion to T-lymphocytes via the endogenous MHC class I pathway. Ap~ iate presentation of the ~nti~enic peptide or pepti~les of interest through the MHC class I pathway ~timlll~tç CTL
pro-iuction and in tum promote destruction of the affected cell.
The present invention is based on the premise that a DNA fragment ~ ssi,-g a 15 tumor rejection an~igen (TRA) or viral antigen or active fragment thereof may be targeted to a specific cell so as to promote the cascade of events cu~ in~ting in CTL-m p.ote,;Li-~e tumor immnnity To this end, the present invention discloses induction of CTL~ d immnnity by transfecting a target host cell with a DNA construct in particulate form which encodes an antigenic protein. The imm~lni7in~ protein is produced 20 intr~rellul~rly and hence has access to the MHC class I restricted p,- ~r~ ion paLllway.
Naturally processed e~iLopes result, and the transfected cells may produce the immnni7ing protein for several days, potentially f~rilit~ting more intense immunogenic 5timlll~tio~.
In a specific embodiment of the present invention a m~mm~ n host is immlmi7rd with a particulate polynucleotide by a microprojectile bombardment device such that the 2~ particulate polynucleotide enters the cytoplasm of at least an a~l .u~liate number of host cells, ~leL.~bly inrluriing but not nrcr~rily limited to APCs, in the path of the projectiles as a direct result of non-specific projectile bombardment. When the skin is bombarded, APCs of the skin which may be bombarded include, but are not limited to epidermal Langerhans cells, keratinocytes, or dermal dendritic cells. When the Iymphoid 30 tissue is bombarded, APCs of the Iymphoid tissue which may be bombarded include, but are not limited to resident dendritic cells, macrophages, stromal cells, T-lymphocytes, or B-lymphocytes. The n,~nSgrnic polynucleoti-ir is ~ "essed at biologically effective levels W O 97/11605 pcTrus96/ls728 -15-such that antigenic peptide fragments are processed and presented through the endogenous MHC class I pathway and displayed on the membrane surface of the APC cells.
Endogenous APC cell membrane presentation of antigen promotes the induction of antigen-specific CTLs either at the site of bombardment, or after trafficking of bombarded S cells to the lymphoid tissue. Incluced antigen-specific CTLs in turn circulate LhIUU~IIUU
the m~mm~ n host, preferably a human host, to destroy neoplastic cells or virally infçctod cells.
A m~mm~ n host may be immnni7~d with a particulate polynucleotide by a biolistic (biob~ tic) delivery procedure such that the particulate polynucleotide 10 cperific~lly enters host cells, including APCs, through the phagosome-to-cytosol pathway.
Particulate polynucleotides enter host cells through the ph~o50...c to-cytosol pathway either at the bombarded site (skin or lymphoid tissue) or after t~ffiçking to the Iymphoid tissue. Thus, particulate polynucleotides may enter the Iymphoid tissue either by direct trafficking of the particul~trs to the Iymphoid tissue with subsequent uptake by cells in the 15 lymphoid tissue, or by trafficking of host cells which have taken-up particulate polynnCleoti~iPs to the lymphoid tissue. Subsequent to uptake by host cells, the l.,.n~g"..ir polynucleotide is e~e~sed at biologically effective levels such that antigenic peptide f~gml~ntc are processed and presented through the endogenous MHC class I pathway and displayed on the l-.~,.--brane surface of the host APCs. Fn~oge~ous APC cell ...~...~..,..-~
20 pff ~ Iinn of antigen promotes the induction of antigen-specific CTLs either at the site of bombardment, or in the Iymphoid tissue. Induced antigen-specific CTLs in turncirculate throughout the m~mm~ n host, preferably a human host, to destroy r.P~ cl;r cells or virally infected cells.
In another embodiment of the present invention a m~mm~ n host is im......
25 with a particulate polynucleotide by direct injection, including but not limited to s~hcut~neouc injection, epidermal injection, dermal injection, Iymphatic injection and intra venous injection. The particulate polymlrleotide enters host cells and is W~ ,SSf~Xl at biologically effective levels such that antigenic peptide fragm~ntc are presented to the endogenous MHC class I pathway and displayed on the membrane surface of the host30 cell. Endogenous target cell membrane presentation promotes the induction of antigen-specific CTLs, which in turn circulate throughout the m~mm~ n host, plc;rc;~dl>ly a ~ human host, to destroy neo~ld:,Lic cells or virally infected cells.
-1~
A m~mm~ n host may be immllni7ed with a particulate polynucleoti~le such that the particulate polynucleotide specifir~lly enters host cells, inf ~ ing APCs, through the phagosome-to-cytosol pathway. Particulate polynucleotides enter host cells through the phagosome-to-cytosol pathway either at the injection site or after trafficking to the S Iymphoid tissue. Thus, particulate polynuc lPoti~ies may enter the lymphoid tissue either by direct trafficking of the parrir~ t~s to the lymphoid tissue with subsequent uptake by cells in the Iymphoid tissue, or by tr~ffir~ing of host cells which have taken-up particulate pOlynurl~oti~ec to the lymphoid tissue. Subsequent to uptake by host cells, the ~".~SC,~-~;r polynucleotide is e~ sed at biologically effective levels such that antigenic peptide 10 fr~gm~ntc are processed and presented through the endogenous MHC class I pathway and displayed on the m~...b,~nr surface of the host APCs. FndrJgenous APC cell rr.~ P
ion of antigen promotes the in~llction of antigen-specific CTLs either at the site of injertion, or in the Iymphoid tissue. Induced antigen-specific CTLs in turn circulate throughout the m~mm~ n host, l~lcr~.dbly a human host, to destroy neoplastic cells or 15 virally infected cells.
In another specific embodiment of the present invention a m~mm~ n host is imlllnl~ A with a particulate polynucleotide by direct injection, including but not limited to Sl l~c.~li.nPous injection, epidermal injection, dermal injection, lymphatic injection and intra venous injection. The composition of the particulate polynucleotide is d~cip~n~d to 20 include specific m~leculf~s which p-er~. .t ally target APC phagocytic pathways (inclutlin~ but not limited to, the m~nnose-receptor m.oAi~tP~I pathway, or Fc--~;c~-t~ ~ pathway) or facilit~t~ cytosolic access of the particulate (ir clur~in~ but not limited to endosomal ~llc.llbl~lc fusion proteins such as the viral HA protein or the listerolysin protein). Particulate polynuclPoti~les enter host cells through the phagosoJI.c 25 to-cytosol pathway either at the injection site or after trafficking to the lymphoid tissue.
Thus, particulate polynucleotides may enter the lymphoid tissue either by directtr~ffi~kin~ of the parti~ul~t~s to the lymphoid tissue with subsequent uptake by cells in the lymphoid tissue, or by traffi~-king of host cells which have taken-up particulate polynudeotides to the Iymphoid tissue. Subse~luent to uptake by host cells, the n,.i~sge~
30 polynu~l~ti-i~ is c~-cssed at biologically effective levels such that antigenic peptide fr~gm~nt~ are l~ucessed and presented through the endogenous MHC class I pathway and displayed on the membrane surface of the host APCs. Endogenous APC cell Ill.,..lb~ e present-qti- n of antigen promotes the indllction of antigen-specific CTLs either at the site of injection, or in the lymphoid tissue. Induced antigen-specific CTLs in turn circulate throughout the mqmm~ n host, preferably a human host, to destroy neoplastic cells or virally infected cells.
The present invention is exemplified on several fronts using a murine mehqnomq model df cl=l ;hed in detail in FYqmpllo Section 6. Briefly, the primary limitqtiorl in studying antigen specific tumor imm-lnity in a murine model is the lack of a defined tumor antigen l~CQ~ 7~ by MHC class I r~ctri.~t~ CTLs. Since TRAs are not fim~i~.,.. n~ y different from any other protein synthesized by the cell, except that the 10 host is not tolerant to them, a foreign protein syntheci7f~ci by a tumor should function as a tumor antigen. Tumor immnni7qtil n metho~c of the present invention are eYernrlifi~d with a murine tumor model with a defined, endogenously syntheci7Pd TRA by trqncfecting the ovalbumin (OVA) gene into the C57Bl/6 derived .,.elano",a B16. This system is attractive for several reasons: (1) the B16 melanoma is an extensively studied 15 murine tumor, (2) in vivo growth characteristics and m~tqctqciC of this tumor line are well cteri7od, and (3) ovalbumin has a well defined structure. The intracellular ocec~;..f~ and pl~ ~ .,t~lion of OVA in the C57Bl/6 mouse is known. In particular the structure of the processed peptide, presented in qccociqti~n with MHC class I Kb, is known. Assays for the fim~-tionql expression of ovalbumin peptide[SIINFEKL] in :~cco~ ;ol~ with H2-Kb using the T-T hybridoma 33.70.Al anti-OVA-Kb are also known (Kovacovics-Bankowski, et al., 1993, Proc. Natl. Acad. Sci. USA. 90: 4942-4946).Techniques to evaluate in vivo inductio~ of OVA specific CTLs in this system are also well ~escrihed (Moore, et al., 1988, Cell 54: 777-785).
One embodiment of the present invention is immunization with particulate polynu~ oti~ie of interest using a biolistic (biob-qlicric) device. Specific-qlly~ a mqmmqliqn host is im~ f.1 with a particulate polynucleotide by a biolistic (biCb-qlicti~-) pl~edu.., such that the particulate polymlcleoti-le delivery ~lu~ )L~ a series of biological events within the trqncfe~t~ cells so as to elicit p,~e~ e immunity to lethal tumor ~~h~llenge The present invention is exemplified severalfold by cutaneous antigen delivery using a 30 biolistic (biob-qlictic) device. First, introduction of particulate polynucleotides enco ling the foreign protein B-Gal results in the expression of B-Gal protein in both the epidermis and in the ~lr.qinin~ lymph nodes. Second, OVA immunization results in the in~lction of W O 97/11605 PCT~US96/15728 -18-OVA-specific CTLs. Third, using the OVA-B16 model, it is shown that OVA-immnni7~oA animals are protected from tumor ch~lltonge by an OVA eA~ g tumor, and that this protection is antigen specific and dep~n-lent on CTLs. Furthermore, the m-och~nicm of this protection goes beyond a generalized cutaneous delivery of S particulate polynucleotides via a biolistic (biobalistic) device. Instead, specific l~L..r,c~ g to phagocytic APCs and/or antigen expression in the Iymphoid tissue is central to in-hlcing antigen-specific CTLs from p-~u-¢o-~. It is therefore a preferred embodiment of the present invention that the target cell be a phagocytic APC, specifically an APC
which is loc~li7~d within, or can traffic to, the Iymphoid tissue.
In another embo~iiment of the present invention a m~mm~ n host is immnni7pd with a particulate polyml~leoti~le by direct injection, inclu~ling but not limited to subcut~neo--c injection, epidermal injection, dermal injection, Iymphatic injection and intra venous injection. The transgenic polynucleotide is ~A~I~ased at biologically effective levels such that antigenic peptide fragm~ltc are processed and presented through the 15 endogenous MHC class I pathway, and displayed on the membrane surface of the target cells. Endogenous APC cell membrane presentation promotes the in~luction of antigen-specific CTLs, which in turn circulate throughout the m~mm~ n host, preferably ahuman host, to destroy neoplastic cells or virally infected cells.
In a ~l~r~ d embo-lim~nt of the present invention the target cell of a m~mm~ n 20 host i~n"~,l";, d with a particulate polynucleotide by direct injection is a phagocytic APC.
The transgenic polynucleotide is eA~-~..¢.ed at biologically effective levels such that antigenic peptide fragm~ntc are pl~.enlc~d to the endogenous MHC class I pathway and displayed on the membrane surface of the APC cell. APC cell membrane ~ e.,l~t;gnpromotes the induction of antigen specific CTLs, which in turn circulate throughout the 25 m~mm~ n host, preferably a human host, to destroy neoplastic cells or virally infected cells.
In a l,.er~--.;d embodiment the particulate polynucleotide is delivered to a human host by subcut~nP~us injection. The present invention dicrlosec that direct injection of the particulate polynucleotide complex results in targeted delivery to phagocytic APCs and 30 gene expression in the Iymphoid tissue. It is this targeted delivery to phagocytic APCs and/or gene expression in the Iymphoid tissue via snbcl-t~neous ~rlminictration which results in superior inriuction of antigen-specific CTLs from naive precursors.
W O 97/11605 PCT~US96/15728 Therefore, central to the present invention are DNA fragments encoding a proteinor biologically active fragment thereof that enter and are ~A~ sed by APCs. The antigen of interest is expressed within the APC cytoplasm and enters the MHC class I
pathway, allowing the APCs to promote induction of antigen-specific CTLs. The data 5 presented in this disclosure supports the novel premise that the most effective manner in which to induce such antigen-specific CTLs is by direct transfer of genetic material to the APCs in the Iymphoid tissue or APCs capable of trafficKing to the Iymphoid tissue.
In another embo-iiment of the present invention a m~mm~ n host is illlllll~.~;7..!:
by injection, in~ln-ling but not limited to subcut~neQus injection, epidermal injection, 10 dermal injection, Iymphatic injection and intra venous injection, with syngeneic APCs that have been antigen loaded in vilro with particulate polynucleotide. Specifically, a m~mm~ n host is immunized with particulate polynucleotide transfected APCs, such that the particulate polynucleotide enters APCs in vitro and the APCs are injected into the host. Subsequent to entry into APCs, the transgenic polynucleotide is eA~ sed a 15 biologically effective levels such that ~nrigenic peptide fr~gm~ontc are processed and p.Gse.-lt;d through the endogenous MHC class I pathway and displayed on the membrane surface of the APCs. After injection of such APCs, endogenous APC cell membrane prestont~ti~n of antigen promotes the induction of antigen-specific CTLs either at the site of injection, or in the Iymphoid tissue. Induced antigen-specific CTLs in turn circulate 20 throughout the m~mm~ n host, preferably a human host, to destroy cells or virally infected cells.
In another specific emborlim~n~ of the present invention a m~mm~ n host is immnni7~d by injection, including but not limited to subcu~neQus injection, epidermal injection, dermal injection, Iymphatic injection and intra venous injection, with syngeneic 25 APCs that have been antigen loaded in vitro by co-incub~tion with particulatepolynucleotide. Specific~lly7 syngeneic APCs are transfected in vitro by co-incub~tion with particulate polynucleotides such that the particulate polynucleotide enters the APCs through a phagosome-to-cytosol pathway. A m~mm~ n host is immunized with particulate polynucleotide transfected APCs, such that the particulate polynucleotide 30 cpecific~lly enters APCs through the phagosome-to-cytosol pathway and the APCs are then injected into the host. Subsequent to uptake by APCs the transgenic polynucleotide is eA~ sed at biologically effective levels such that antigenic peptide fragments are W O 97/11605 PCT~US96/15728 ucessed and presented throughout the endogenous MHC class pathway and ~iicri~ced on the l1.c..~bl~ule surface of the APCs. After injection of such APCs, endogenous APC cell membrane presentation of antigen promotes the induction of antigen-specific CTLs either at the site of injection, or in the Iymphoid tissue. Induced antigen-specific CTLs in turn 5 circulate throughout the m~mm~ n host, preferably a human host, to destroy neoplastic cells or virally infected cells.
In another specific embodiment of the present invention a m~mm~ n host is immnni7~d by injection, including but not limited to subcutaneous injection, epi~e~rn:~l injection, dermal injection, Iymphatic injection and intra venous injection, with syngeneic lO APCs that have been antigen loaded in vitro particulate polynucleotide. Specifir~lly, syngeneic APCs are h~ncfe~~ed in vitro with microprojectile bo1"ba.~l",ent of APCs with particulate polynuc1e~tirle~ Subsequent to projectile bombardment into APCs, thetransgenic polynucleotide is eA~JleS5Cd at biologically effective levels such that :1ntigenir peptide fr~gmrntc are p loccased and presented through the endogenous MHC class I
I5 pathway and displaced on the membrane surface of the APCs. After injection of such APCs, endogenous APC cell membrane ~ se..1,.ti~n of antigen promotes the induction of antigen-specific CTLs either at the site of injection, or in the Iymphoid tissue. Induced antigen-specific CTLs in turn circulate throughout the m~mm~ n host, preferably a human host, to destroy neoplastic cells or virally infected cells.
In another embo~iimrnt of the present invention a r~mm~ n host is i.. ~ d by injection, inr~ ling but not limited to s~1hcut~neous injection, epidermal injection, dermal injection, lymphatic injection and intra venous injection, with syngeneic APCs that have been tr~n~fect~d in vitro with particulate polynucleotide. Sperific~lly~ a m~mm~ n host is i---...~ d with particulate polynucleotide tr~n~fe~ted APCs, such that the 25 particulate polynucleoticle enters APCs in vilro and the APCs are injected into the host.
Particulate polynll~leotifltos enter APCs in vitro by either microprojectile bomb~d,..~.lt or the phagoac,."e to-cytosol pathway. APCs are tr~ncfectrd in vitro with antigen enco~ling polynucleotides and/or polynuclcotides enrofiing a molecule or molecules which illc the effirienry of the antigen presrnting function of the APC, such as, but not limited to, 30 cytokine molecules such as IL-12, IL-2, and/or IL-4 and/or costirr nl~tory molecules such as CD80 and/or CD86. Subsequent to entry into APCs, the ~.d..SgC~iC antigen enco~ling poly..ucle~Lides is ~ r~,!7 ,ed at binlogic~lly effective levels such that ~ntig~nir~ peptide W O97/11605 PCT~US96/15728 rA~ are processed and presented through the endogenous MHC class I pathway and displayed on the membrane surface of the APCs. After injection of such APCs, endogenous APC cell membrane precentAtinn of antigen promotes the inductiQn of antigen-specific CTLs either at the site of injection or in the Iymphoid tissue. Induced S antigen-specific CTLs in tum circulate throughout the mAmmAliAn host, pre~.dbly a human host, to destroy neoplastic cells or virally infected cells. Subsequent to entry into APCs, the lldnsgeniC cytokine andlor costimnlAtory Pnco~ing polynucleotide is ~;A~
at biologically effective levels such that the antigen pr~C~nting function of the APC results in the inrlllcti~n of antigen specific immune responses either at the site of inj~ction, or in the lymphoid tissue.
It will be within the purview of the artisan of ordinary skill to inco",~,ldle a TRA
or viral antigen of choice into the system ~iicrlc~sed within this specification. FY~mples of presently available TRAs include, but are by no means limited to, MAGE-l, MAGE-3, BAGE, GAGE-l, GAGE-2, Tyrosinase, Melan-A(MART-l), gplOO(pmell7), gp75(TRP1), CEA (carçinoemhryonic antigen) as well as viral derived tumor -Antigenc from HPV, HBV, and EBV, as well as tumor ACcociAt~d oncogene/tumor SU~ gene mut~tinn /~nroded antigens such as P53, P16, RAS, HER2/neu, C-ABL, and polymorphic endothelial mucin antigens (as reviewed in Maeurer et. al., 1996, Cancer Vaccines in Clinical Tmmlmc)logy Principles and Practice, ed R. Rich, Mosby Publishing. Chpt.
123:1904-1918 and Van den Eynde et al., 1995, J. Ei~p. Med. 182:689-698). FY~m~
of viral antigens include, but are by no means limited to, Influenza nucleo~ eill (Donnelly et al., 1995, Nature Med. 1:583-587.), HIV gp 120, HIV gp 160, and T-T~pAtitic B surface antigen (as reviewed in Pardoll and Beckerleg, Immunity, l99S, 3: 165-169).
It is known to the artisan of ordinary skill that any eukaryotic promote} and/orenh~nr~r sequence available which is known to up-regulate expression of a ~IA..cg~..ir DNA sequence may be used in constructing a recombinant vector for combination with the particle of choice so as to generate the particulate polynucleQti(lt- of the present invention. Such promoter fragments include but are not limited to a cytomegalovirus 30 (CMV) promoter, a Rous Sarcoma virus (RSV) promoter, a Murine T~nkl~miA Virus(MLV) p.~,...otel, a B-actin promoter, as well as any cell-specific promoter or enh-Anr.or s~u~"ce known to be active in the target cell.
To this end, a preferred embodiment of the present invention is the use of particulate bound DNA to delive} the DNA rnrQ~iing a tumor or viral antigen specifir~lly to the APCs in the lymphoid tissue, or APCs capable of trafficking to the Iymphoid tissue, so as to promote MHC class I access of antigen, thus allowing APCs to Stim~ t.o 5 inrlllction of antigen-specific CTLs. These CTLs will then circulate throughout the host and destroy neoplastic cells or virally infected cells.
Tumor immunity based on the ~ d method of utilizing ~--bcut~nrous injection to ~-u.l-ult: a specific .~o.lse in Iymph node APCs is exemplified in Example Section 6, Example Section 7 and Example Section 8.
The following examples are offered by way of illustration of the present invention, and not by way of limi~tic.n, W O 97/11605 PCT~US96/15728 6. EXAMPLE: OVA/B16 GENETIC MURINE TUMOR MODEL
The OVA/B16 murine model was used to generate the data disclosed in FY~mple Section 7 and 8 which exemplify the claimed invention. The OVA/B16 murine system is attractive for several reasons: (1) the B16 melanoma is an extensively studied murine tumor, (2) in vivo growth characteristics and mft~ct~cic of this tumor line are well rh~cte~ized, and (3) ovalbumin has a well defined structure. The intr~rçll~ r procescing and presentation of OVA in the C57B1/6 mouse is known. In particular the structure of the p.ocessed peptide, presented in association with MHC class I Kb, is known. Assays for the filnction~l expression of ovalbumin peptide tSIINFEKL] in ~ccoci~tion with ~I2-Kb using the T-T hybridoma 33.70.AI anti-OVA-Kb are also known (Kovacovics-Bankowski, et al., 1993, Proc. Natl. Acad. Sci. USA. 90: 4942-4946).Techniques to evaluate in vivo in~ ction of OVA specific CTLs in this system are also well ~iesr~ihe~ (Moore, et al., 1988, CeU 54: 777-785).
Mice and Cell Lines. Female C57BL/6 mice, 5-8 weeks old were purchased from the Jackson Laboratories, Bar Harbor, ME. EL4 is a C57BL/6 T-lymphoma, and EG7 is a chicken egg ovalbumin (OVA)-transfected subclone of EL4 (Moore, et al. 1988, Cell 54: 777-785). The C57BL/6 derived murine melanoma B16 (Fidler, et al., 1976, Cancer Res. 36: 3~60-3165) was obtained from American Tissue Type Collection (ATCC). MO4 was constructed by transfection of B16 with the pAc-Nco-OVA plasmid as described.
(Falo, et al, 1995, Nature Med. 1: 649-653, Moore, et al. 1988, Cell 54: 777-785) Monorlon~l antibodies were prepared from the hybridomas GK1.5 (anti-CD4, ATCC
TIB-207), 2.43 (anti-CD8 antibodies was raised in Balb/c nu/nu mice by i.p. injection of GKfl.5 cells (3 x 106) and IFA (0.5ml/mouse).
After OVA transfection of the B16 melanoma, and selection, the transfected B16 melanoma subclone, MO4 was icol~trd The parent melanoma B16, and the OVA
tr~ncfrct~nt express similar levels of fimction~l Kb on the cell surface as measured by f~ on of OVA peptide (SIINFEKL) to RF33.70 (Figure 1). In contrast, M04/5, but not B16, is capable of hybridoma stimulation in the absence of exogenously added peptide (Figure 1). This ~tomonct-~trS endogenous pro~llction, processing, and ~ .,l;.l;nn of the lldll~re~;led antigen. Importantly, endogenous expression of OVA by MO4 did not cignifir~ntly alter in vivo imm~lnogenicity of the tumor (Figure 2). Tumor WO 97/11605 PCT~US96/15728 growth (Figure 2A) of B16 and M04 is comparable in naive mice, as is host survival (Figure 2B).
W O 97/11605 PCT~US96/15728 7. EXAMPLE: GENETIC IMMUNIZATION VIA
BIOLISTIC (BIOBALISTIC~ ADMTNISTRATION
~ ocfl1izo~ion of Foreign Protein Expression - Tissue specimens (skin or draining lymph nodes) were harvested 24 or 48 hours after immnni~ti~n~ washed in PBS and S fixed in 2% formaldehyde - 0.2% glutaraldehyde in PBS for 30 min. at 4~C. Fixed .l~e4~ nc were washed extensively with PBS and then in~lb~t~ in X-gal staining solu'ion (lmg/ml X-ga~, 5mM pvr~ rrl ferricya~ide, 5mM fer~ocya~ide, 2mM MgCl2i~PBS) for 18 hrs at 37~C. Stained tissue was paraffin sectioned and counterstained with 0.1% nuclear fast red.
Gene~ic Immunization - DNA coated gold particles were p lcpalcd by co.. ~h;.~ g 50 mg of 0.95 ~m gold beads and 100 ~1 of 0.1 M spermidine and soni~ting for 5 cel-on~ic 132 ~L~g of plasmid DNA and 200 ~Ll of CaC12 were added sequentially whiie vortexing. This mixture was allowed to l~c~ dte at room te~llpe.dture for 5-10 mimltPc The bead preparation was then centrifuged (10,000 rpm for 30 seconds) and washed 3 15 times in cold ethanol before re-sncp~ncif)n in 7ml of ethanol to give a final conc~n~ lion of 7mg gold/ml. The solution was then loaded into Tefzelal' tubing (Agracetus) and allowed to settle for S minutes. The ethanol was removed and the beads were ~tt~h~d to the sides of the tubing by rotation at 20 rpm for 30 seconds and N2 dried. The dry tubing lined with beads was then cut into 0.5 inch sections and stored for use with ~lloci~c~nt in 20 p~r~film sealed vials. Animals were v~ccin~t~ by delivery of 2 shots (each shot c.~ l of 0.5mg gold beads = 0.5 inch of tubing) to the shaved abdominal region using the Accell Gene Delivery Device at a discharge pressure of 300 psi. Animals were i.. ln~ d with either the pAc-Neo-OVA plasmid (Moore, et al., 1988, Cell 54: 777-785) or the pTF.gl~l'7. pla~smid (provided by Nadia Jouroud) which cont~inc the lacZ gene 25 under the control of the CMV promoter. For experiments involving subcu~n.ol~us injection of particulate polynucleotides (Figure S ) DNA coated beads were p~cp~h~d as above and an equivalent quantity of DNA was injected. For some animals (Figure 5), an excess quantity of free plasmid DNA was sllhc~ n~Qusly injected. Snhcllt~neQus injectinnc were ~riminict~red in 100,.~1 volume of PBS in the mid-flanks, bilaterally. For 30 in vitro tr~ncfection of APCs (Figure 4), DNA beads were identically plc~alcd using an equivalent weight of Biomag iron oxide beads or gold beads as a particulate substrate.
W O 97/1160~ PCT~US96tlS728 For experiments involving subcutaneous injection of particulate polynucleotides (Figure 5 and 6) DNA coated beads were prepared as above and an equivalent ~uantity of DNA was injected, or the quantity specified in the figure descriptions. For some animals (Figure 5 and 6), an excess quantity of free plasmid DNA was subcutaneously injected.
Suhc~lt~n~Qus injections were adminictered in 1OO~LI volume of PBS in the hind legs, bilaterally.
For in vitro tr~n~fection of APCs (Figure 4 and 6), DNA beads were id~nt~ y prepared as des~rihe~i above using an equivalent weight of Biomag iron oxide beads (Fe beads) or gold beads as particulate substrate. Dendritic APCs were obtained from bone marrow as described in the Brief Description of the Drawings except that IL-4 was not used in tissue culture media with 25,ul of 7mg/ml particulate polynucleotide solution (particulate wt/vol. PBS) for 18hrs at 37~C. Antigen-pulsed dendritic cells were p.~
and injected as described in the Brief Description of the Drawings. Briefly, dendritic cells were prepared by depleting bone marrow cells of Iymphocytes and culturing overnight in RPMI 1640 supplemented with 10%FCS, L-gh-t~mine, antibiotics and 2-ME in 24 wellplates at 106 cells/well. Cells were repleted on day 1 at 2.5 x 105 cells/well with GM-CSF (103U/ml, Sigma, St. Louis, MO) and murine rIL-4 (103U/ml, Genzyme, Cambridge, MA) and loosely adherent cells were harvested on day 8. By flow cytoml~tric analysis, these dendritic cells e~ .sed CD45, CD44, CDllb (Mac-l), CD18, CD80, cD86 and class I and class II MHC ~nti~en~ Dendritic cells were pulsed 2hrs at 37~C
with or without OVA peptide (20ng/ml) + B2-microglobin (132-M, 10~1/ml, human, Sigma) in reduced serum media (Optimen, Gibco, Grand Island, NY). Cells were then washed extensively, resuspended in PBS and irradiated (2000 rad) before injection into naive mice.
Cytotoxi~iry Assays - Splenocytes from immllni7~d animals were r~ostimnl~t~d with minor mo~iifi~tinnc of previously described protocols. (Falo, et al., 1995, N~ure Med.
1: 649-653). Briefly, 1 week after immuni7~tic~l splenocytes (30 x lo6) were restim by coculture with irr~ t~d (20,000 rad) EG7 cells (10 x 106). Effector cells were harvested five days later and cultured with 2xl0~ s'Cr labeled targets in round bottom microwells (200~1) at the indicated effector target cell ratio. In some cases the effector cells were ~eplet~d of T-cell subsets using mAB plus complement before assay as d~ ihed (Rock, et al., 1993, J. Immunol. 150: 1244-1251). After 4 hours at 37~C, WO 97/11605 PCT~US96/1~728 lOO,ul of supernatant from triplicate microcultures was collected and counted and the p~..;e.,~ge of specific release was r~ t~d as described. (Falo, et al., 1995, Nature Med 1 649-653). Results are reported as the mean of triplicate cultures. The SEM of triplicate cultures was always less than 15% of the mean.
Pro~ec~ion Assays - C57BL/6 mice were immunized as described with the in-iir~t~
antigen gene construct. Animals were ~~h~ nged with tumors and evaluated for tumor survival as described. Briefly, seven days after the final immllni7~tion (day 0), OVA-immnni7~d or lacZ-immunized animals were f~h~llenged by intradermal inj~tinn in the mid-flanks bilaterally with melanoma cells (2 x 104) at 2 times the dose lethal to 50%
of the animals tested (LD5,~ or the number of tumor cells in~ic~t~ in section 4. Survival is recorded as the p~.cen~ge of surviving ~nim~ls Melanoma cells for injection were washed three times in PBS. Injected cells were greater than 95% viable by trypan blue exclusion. All experiments included 5 mice per group and were repeated at least three times. Mice which became moribund were sacrificed according to animal care g~lifiplin~s of the University of Pittsburgh Medical Center. In some experiments, animals were ~ pletPd of CD8+ cells. This was accomplished by i.p. injection of CD8 mAb (2.43) 7 and 9 days after immnni7~tiorl as described, followed by tumor rh~llenge on day 10 (Falo, et ai., 1995, Nature Med. 1: 649-653).
The capacity of biolistic (biobalistic) immnni7~tion to induce antigen specific CTLs was evaluated. Naive C57Bl/6 mice were immnni7ed with a total of at most 2.64~g of OVA ~n~oriing DNA delivered to the abdominal skin with 2 overlapping pulses, andidentic~lly boosted 7 days later. In vitro restim~ tPd spleen cells from these mice Iysed the syngeneic OVA-expressing murine thymoma EG7, but not the untransfected parent tumor EL4 (Figure 3A). Thus, target cell Iysis was antigen specific, depending on expression of OVA by the tumor target. Depletion of T-Cell subsets from the effector populations using mABs demonstrated that lysis depended on Thy 1+, CD8+ subsets ch~r~ct~rictic of MHC class I-restricted CTL effector cells (Figure 3B).
Groups of mice that were immnni7~d and boosted as described above were ~h~llenged 7 days later by i.d. injection of the MO4 melanoma at a distant site to d~:L~ ine the ability of biolistic (biobalistic) immuni_ation to induce protective tumor immunity. OvA-immllni7t~d mice were protected from lethal tumor ch~llenge, whiletumors in control mice (immlmi7ed similarly, but with the lacZ ll::lJUII~l gene) grew W O 97/11605 PCTAUS96/1~728 ~J-Ug~ rely and were lethal in 60% of the animals by day 40 (Figure 3C). OVA-immlmi7~ mice were not protected from ~h~ nge with the untransfected parent melanoma B16 (Figure 3D), in~ ting that p.otccLive immnnity was antigen specific, depen-ling on OVA expression by the tumor target. We evaluated the contribution of 5 CD8+ effector cells to this protective tumor immnnity by depleting groups of immlmi7Od or control (lacZ immnni7~d) animals of CD8+ effector cells through repeated i.p.injection of anti-CD8mAB before tumor ch~llpnge~ (Falo, et al., 1995, Nature Med. 1:
649-653). While OVA-imml-ni7ed animals were p.ute~;lcd from MO4 rh~ n~e, survival in im~ ed~ CD8+ T-cell ~It plet~d animals was similar to that observed in control 10 ~nim~lc, with or without T-cell depletion (Figure 3E-F). Therefore, CD8-' T-cells are ~cc.onti~l for the plotcclive tumor immnnity induced by genetic immnni7~tion in this model.
Aci~liticn~l evidence ~ u~ling the ~-uposed merh~nicm of tumor immlmity includes the demonstration of 13-g~ tocirl~ce expression in the epidermis after biolistic 15 (bi--b~lictic) delivery of the lacZ construct, and, interestingly, discrete areas of specific CAIJJ.'~,.7sion within the draining Iymph nodes. As these nodes were distant from the imm--ni~tion site, it is unlikely that Iymph node expression was a result of direct physical bombardment. There was a predomin~n~e of gold particles in the epidermis and dermis of immnni7~d skin (Imct~in~d) 24hrs after DNA delivery and lacZ expression within 20 epidermal k~ratinocytes at 48hrs. There are also discrete areas of specific staining within the fir~ining lymph nodes. TdPnti-~lly treated specimenc from mice imm--ni7~d with irrelevant DNA did not show lacZ expression. This observation p-u-ll~t~,d further analysis which is ~iicrlo5ed in FY~mplP Section 8.
W O 97/11605 PCT~US96/15728 8. EXAMPLE: GENETIC IMMUNI_ATION BY
SUBCUTANEOUS ADMINISTRATION
The materials and methods for inrhlcing tumor immunity by subcutaneous - ~lminictration of a particulate polynucleotide are as described throughout FY~mrle Section 6 and Example Section 7.
This data demonstrates an ability to crecific~lly target phagocytic APCs in the lymphoid tissue, or APCs capable of tr~ffl~king to the Iymphoid tissue, by s~lbcut~neouc injection of particulate polynucleotides in vivo.
Protection from tumor ch~ onge was equivalent within groups of mice immnni with particulate polynucleotides comprising pAc-Neo-OVA either by direct sub.~ cinjection or by biolistic (biob~lictic) adminictration (Figure 5). Concictent with the finfiingc in FY~mrlf Section 7, immnni7~tion with a particulate polynucleotide encoriing and irrelevant antigen (e.g., PTFEI~r~7) did not confer protection (Figure 5).
Snbc.,l; n~ous injection of pAc-Neo-OVA without particlll~t~s did not confer protection from a relevant tumor ch~ nge (Figure 5). These data indicate that suhcut~nt~o--c injection of particulate polynucleotides is at least as effective as biolistic (biobalistic) particulate delivery. Furthermore, as subcutaneous injection does not result in direct physical projectile bombardment of polynucleotide particles into the cytoplasm of individual host cells, expression likely requires active uptake of polynucleotides by host cells capable of phagocytosis/endocytosis. The observation that subcutaneous injection of pAc-Neo-OVA without particulates did not confer protection also implicates particulate delivery/phagocytosis as the mech~nicm of tr~ncductiom Particulate tr~ncdllction by phagocytosis implies that transgene expression is preferentially targeted to cells capable of phagocytosis, in~lu-iing APCs.
In further support of this proposed mech~ni~m is the observation that APCs (bonemarrow derived dendritic cells) co-cultured in vitro with particulate polynucleotides enco~iing ovalbumin (~ JdlCd as fles~rihe~ in Example 7) (pAc-Neo-OVA) can ctim~ t~
the OVA (SIINFEKL+ Kb) specific T cell hybridoma RF33.70 to produce IL-2 (Figure 4).
These data demonctr~t~- that these APCs filn~tion~lly express the ovalbumin gene and generate the ovalbumin peptide-Kb complex. The potency of $timnl~inn is colllpdldble to that observed with APCs pulsed with 2mg/ml of soluble OVA protein. Particulate polynnrleotirl~s are effective in this assay using either gold or Fe as the particulate CA 02233278 l99X-03-27 substrate. Thus, the incubation of parliculate polynucleotides with phagocytic APCs in vi~ro results in the en(logenQus production, processing, and presentation of the transfected antigen These observations demonstrate that particulate polynucleotides can be taken up and e~ .;,ed by APCs, and that the corresponding proteins can be functionally presented 5 to induce antigen specific immune responses.
Fll-ll,.,.lnore, a shown in Figure 6, antigen presenting cells, in this case snbcut~nPously injected bone marrow derived denllritic cells, which have been incuh~rP~d with particulate polynucleotides ~ncoriing the antigen OVA in vitro as descri~e~ above, are capable of inrlll~ing p-uL~Li~e immunity to tumor cells e~ ing the antigen gene.
In the example given, a one time ~l1minictration of 105 irradiated transfected ~i~n~1rjti~
cells/animal (5 x 104 cells per hind leg, bilaterally) induced complete protection to tumor ch~llenge. Thus, in vivo ~lmini~rPred dendritic APCs, which are potent ~imlll~tnrs of antigen-specific CTLS and l,.ule~:Live tumor immunity when peptide-loaded in vitro are at least similarly immnnogenic which antigen-loaded by co-incubation with particulate polyn~cl~oOti~
Taken together, these results indicate that subcut~neouc ~timini~tration of particulate polynucleotides has the advantage of targeting delivery of antigens to phagocytic APCs, potentially increasing the efficiency of genetic immllni~tion while reducing unwanted deleterious effects, including but not limited to tolerance inductinn or 20 mutagenesis resulting from the transfection of normal, non-APC host cells.
-
Figure 1 shows function~l presentation of ovalbumin by the transfected tumor cell lines MO4 and EG7. Microcultures were prepared with the T-cell hybridoma RF33.70S (anti-OVA+ Kb) and the inriie~t~d number of transfected (squares) or untransfected (circles) tumor cells in the presence (open symbols) or absence (closed symbols) of added exogenous OVA-peptide SIINFEKL (lOng/ml~ as described (Rock, et al., 1990, J.
Immunol. 45:804-811). After 18hrs incub~til~n, sl~pern~t~ntc were harvested and assayed for IL-2 using the indicator cell line HT2 (Rock, et al., 1990, J. Immunol. 145: 804-811).
(A) B16 and the OVA-~ sre~;led subrk ne MO4. (B) EL4 and the OVA tr~n~fect~l ELAsu'~clon~o EG7. OVA ~1~ C~ tion by the OVA-transfected tumors was not ~i~nifirantly enh~nred by the presence of exogenous SIINFEKL in the assay cultures.
Figure 2 shows that OVA expression by the B16 derived melanoma MO4 does not signifie~ntly effect in vivo tumor growth or host survival following tumor ~h~ilPnge. Mice were rh~llen~ed with MO4 (circles) or B16 (squares) (5 x 104/mouse, i.d., bilateral, mid-flanks). Tumor size (Figure 2A) was a~p~ced 3x/week and is reported as the average tumor area in square milTimetl~rs until the first death occurred in each group. Survival (Figure 2B) is recorded as the percentage of surviving ~nim~lc All experiments inch~
S mice/group and were repeated at least three times. Mice bf~cQmin~ monbund were~rrjfie~1 Figure 3 shows that immunization by cutaneous delivery of OVA encoding DNA
induces OVA-specific CTLs and antigen-specific, CTL mP~Ti~tPd protection from lethal ch~llen~e with the OVA expressing m~l~nom~ MO4. In vitro restimulated splenocytes from OVA-immuni7~d (g~neti~ y immnni7~i as desc,il,ed in Example Section 7) micewere assayed for cytolytic function against the OVA-tr~ncfect~Pd Iymphoma EG7 (closed squares) or the untr~n~fectPd parent EL4 (open triangles) (A). Effector populations were incubated with complement alone (open squares) or with mAbs against CD4+(closed tri~ngle5), CD8+ (open circles), or Thyl.2+ (closed circles) Iymphocytes and complement, then assayed for cytolytic activity against EG7 targets (B). In C-F, mice were gerletif~lly jmm-lni7~d with OVA (closed squares) or lacZ (open squares) and boosted 7 days later. Groups of immnni7Pd mice were challenged 7 days after the final iml.. ;,~lion (day 0) with either the B16 m~ nom~ (D), or the OVA-e,.~,~saing sub~k~ne W O 97/11605 PCT~US96/15728 MO4 (C). Alternatively, immunized mice were divided into Z groups, one of which was deFIet~cl of CD8+ Iymphocytes by i.p. injection of anti-CD8 mAb 7 and 9 days after the last immuni7~tion. Intact (E) and CD8+ dcplered (E;) mice were then ch~llen~ed 10 days after the final immnni7~tiQn (day 0) with MO4. Survival was reported as the pe.c~..t~ge S of surviving animals (C-~;). Animals surviving on day 60 had no sign of tumor growth.
All e~ ntc in-~luded S mice per group and were ~t:~eated at least 3 times. Mice that became moribund were sacrificed according to animal care guidelines.
Figure 4 shows that antigen ~ lg cells internalize and express particulate polynucleotides, and process and present the ~ essed antigen through the MHC class I
10 restricted p.ucessi~g pathway. Dendritic cells were prepared by ~epl~tin~ bone marrow cells of lymphocytes and culturing overnight in RPMI 1640 suppl~m~ontPd with 10%FCS, L-glnt~min~ antibiotics and 2-ME in 24 well plates at 106 cells/well. Cells were repleted on day 1 at 2.5 x 105 cells/well with GM-CSF (103U/ml, Sigma, St. Louis, MO) andmurine rIL-4 (103U/ml, Genzyme, Cambridge, MA) and loosely adherent cells were 15 harvested on day 8. By flow cytometric analysis, these dendritic cells e~plessed CD45, CD44, CDllb (Mac-1), CD18, CD80, cD86 and class I and class II MHC :lnti~onc Dendritic cells were pulsed 2hrs at 37~C with or without OVA peptide (20ng/ml) +B2-microglobin (B2-M, 10~1/ml, human, Sigma) in reduced serum media (Optimen, Gibco, Grand Island, NY). Cells were then washed extensively, resucrended in PBS and 20 irr~ t~d (2000 rad) before injection into naive mice. The indicated number of bone marrow derived dendritic APCs were cocultured with OvA-enco~ling particulate polynucleotides prepared as described in Example Section 7 (SO~I/ml/106 cells of 7mg/ml partirul~tlos) using either Fe beads (closed squares) or gold beads (closed circles) as the particulate substrate or soluble OVA protein (2mg/ml) (open squares) for 24hrs., washed, 25 and then the inrii~t~d number of APCs were co-cultured in microcultures with the T-cell hybridoma RF33.70 (anti-OVA+ Kb). After 18hrs incub~tion, supernatants were harvested and assayed for IL-2 using the indicator cell line HT2 (Rock, et al., 1990, J.
Immunol. 145: 804-811).
Figure S shows a co~ ~ison of particulate polynucleotide immunization 30 a~iminictered by biolistic (biob~lictic) or subcutaneous injection. Groups of 5 C57BI/6 mice were immuni7PA, boosted on day 7, and then nh~llenged by intradermal iniecti~n of - 5 x 105 MO5 tumor cells in each flank 7 days after boosting. Immuni7~tions CQI~ d W O 97/11605 PCT~US96/15728 of: (A) B-Gal encoding particulate polynuc1eotiAes prepared and delivered by biolistic (binb~licti~-) aAminictration as described in Example Section 7; (B) OVA enCQAing particulate polynucleotides p~ cd and delivered by biolistic (biob~lictic) ~dminictr~inn as des~ribe~ in Fy~mrle Section 7; (C) an excess quantity of OVA encoAing S poly--ucl~otide without parti~ul~t~os delivered by, ~b~ eo~C injection; or, (D) an equivalent amount of OVA PnroAin~ particulate polynucleotiAPs p-~el~d-~d as in F~
Section 7 but aAminict~ed by sl~hcnt~n~ous injection. Data is presented as % of animals tumor free in each group 50 days after tumor ch~ nge.
Figure 6 shows that imml-ni7~fion with APCs which had been co-incubated with 10 particulate polynucleotide enroAing OVA protects animals from ch~ nge by OVA-eA~.es~ g melanoma MOS, Groups of 5 C57Bl/6 mice were im~ ..i;~ suh~ -n~lcly dS d~ ;bed in Figure S on one occ~cion and then .-h~ onged by intlddelllldl injection of 1 x 105 MO5 tumor cells in each flank 10 days after immnni7~tion Imm ~ nc concicted of either: (open squares) particulate polynucleotide encoAing the irrelevant 15 antigen B-g~l~rtnci~i~c~ (1OO~L1 of 7mg/ml particulate solution (particulate wt./vol PBS) per hind leg bil~ttorally; (open circles) salable pAc-neo-OVA (100~1 per hind leg c~an ap~-v..;-~-trly equivalent quantity of DNA/animal); or (closed squares) 5 x 104 bone marrow derived dendritic cells/100~1 hind legs s.q. (prepared as described in FY~-F'- 7).
Survival was reported as the pelccn~ge of surviving ~nim~lc All experiments in~luded 5 20 mice per group. Mice that became ~.-o-ib.lnd were sacrificed according to animal care guidt~lin~s 5. DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term ~mAmmAliAn host" includes members of the animal kingdom, in~lu~ling but not limited to human beings.
As used herein, the term "DNA fragment" may include any nucleotide sequence, S either DNA or RNA, which contAinc apl)rol,-iate coding region and regulatory sequences to result in target cell expression of an Antig~nic protein or AntigPnic protein frAgm~nt for cell ~I-e.-~b-~le prP~PntAtion via the enllogenol~C MHC Class I pathway.
As used herein, the term "particulate polynucleotide" may refer to a particulatemade from materials in~h-fiing but not limited to gold, iron, and synthetic plastics 10 wherein the particle comprises a population of DNA frAgmPnts as defined in the ~ce ling p~. ,.g. ,.ph The present invention relates to thprAreutir or prophylactic genetic im...~ ;,,.l;nn of a m~mm~ n host which comprises delivery of a DNA fragm~nt to a target cell within the mAmmAliAn host, expression of the DNA frAgm~Pnt within the target cell, and 15 subse4uent prestontAtior~ of a recombinant antigenic peptide(s) within the target cell so as to stim--lAt~o cell-me~liAtPd immnnity~ humorAl immnnity, or both.
The present invention further relates to therApeutic or prophylactic genetic immllni7Ation of a mAmm~liAn host which comprises delivery of a DNA fragment ~ncorlin~ a protein or biologi~Ally active fragment thereof to a specific target cell within 20 the m~mmAli~n host. Antigenic peptides ~A~ sed from the DNA sequence are specific to an ~ffected cell such as a nP~pl~ric cell or virally infected cell. Antigen specific CTL
production is ~timulAtPd, thereby promoting destruction of target cells such as ne~plaslic cells and virally infected cells.
The present invention ~licclos~ps genetic immunization mPtho~ic for llcat~ent or25 pl~ ,.tion of tumors or viral infections. Cytotoxic-T-cells are a significant co...pon ~~t of the immlln~ ~nse to tumors and viral infectinn~- Cytotoxic-T-cells kill neoplastic cells or virus-infected cells through the recogniti~n of antigenic peptides ~ ,ent~d by MHC class I mol~pclllp~s on the surface of the tumor target. These peptides are derived from tumor AntigPnc that are synthP~i~pd by the affected cell and ~legrA~Ied in the cytosol.
30 Attempts to induce tumor-specific CTL responses in vivo by immnni~Ati~n with killed tumor cells or component proteins have generally been ~n~lccessful~ presumably because W O 97/11605 PCT~US96/15728 proteins in the ~xtr~re~ r fluids cannot enter the cytosol and access the MHC class I
;on pathway.
A specific embodiment of the present invention relates to genetic immnni7~tion with particles cont~ining the DNA fr~gmPnt ~ s~ing the protein of interest. Such5 particles cont~ining a DNA fr~gml~nt are referred to throughout this ~pecific~tion as 'particulate polynucleotides'. In vivo cell delivery is best accomplished by ~lmini~tering the DNA r.~l~-... nt or protein of interest in particulate form, such as coated beads or gold particles. The protein or biologically active ~ .ne..t is ~A~ sscd subsequent to cytosolic delivery within the target cell. The e~ ssed protein or protein fragment specific to the 10 affected cell provides a substrate for generating an ~ntigenic peptide(s) for prestont~tion to T-lymphocytes via the endogenous MHC class I pathway. Ap~ iate presentation of the ~nti~enic peptide or pepti~les of interest through the MHC class I pathway ~timlll~tç CTL
pro-iuction and in tum promote destruction of the affected cell.
The present invention is based on the premise that a DNA fragment ~ ssi,-g a 15 tumor rejection an~igen (TRA) or viral antigen or active fragment thereof may be targeted to a specific cell so as to promote the cascade of events cu~ in~ting in CTL-m p.ote,;Li-~e tumor immnnity To this end, the present invention discloses induction of CTL~ d immnnity by transfecting a target host cell with a DNA construct in particulate form which encodes an antigenic protein. The imm~lni7in~ protein is produced 20 intr~rellul~rly and hence has access to the MHC class I restricted p,- ~r~ ion paLllway.
Naturally processed e~iLopes result, and the transfected cells may produce the immnni7ing protein for several days, potentially f~rilit~ting more intense immunogenic 5timlll~tio~.
In a specific embodiment of the present invention a m~mm~ n host is immlmi7rd with a particulate polynucleotide by a microprojectile bombardment device such that the 2~ particulate polynucleotide enters the cytoplasm of at least an a~l .u~liate number of host cells, ~leL.~bly inrluriing but not nrcr~rily limited to APCs, in the path of the projectiles as a direct result of non-specific projectile bombardment. When the skin is bombarded, APCs of the skin which may be bombarded include, but are not limited to epidermal Langerhans cells, keratinocytes, or dermal dendritic cells. When the Iymphoid 30 tissue is bombarded, APCs of the Iymphoid tissue which may be bombarded include, but are not limited to resident dendritic cells, macrophages, stromal cells, T-lymphocytes, or B-lymphocytes. The n,~nSgrnic polynucleoti-ir is ~ "essed at biologically effective levels W O 97/11605 pcTrus96/ls728 -15-such that antigenic peptide fragments are processed and presented through the endogenous MHC class I pathway and displayed on the membrane surface of the APC cells.
Endogenous APC cell membrane presentation of antigen promotes the induction of antigen-specific CTLs either at the site of bombardment, or after trafficking of bombarded S cells to the lymphoid tissue. Incluced antigen-specific CTLs in turn circulate LhIUU~IIUU
the m~mm~ n host, preferably a human host, to destroy neoplastic cells or virally infçctod cells.
A m~mm~ n host may be immnni7~d with a particulate polynucleotide by a biolistic (biob~ tic) delivery procedure such that the particulate polynucleotide 10 cperific~lly enters host cells, including APCs, through the phagosome-to-cytosol pathway.
Particulate polynucleotides enter host cells through the ph~o50...c to-cytosol pathway either at the bombarded site (skin or lymphoid tissue) or after t~ffiçking to the Iymphoid tissue. Thus, particulate polynucleotides may enter the Iymphoid tissue either by direct trafficking of the particul~trs to the Iymphoid tissue with subsequent uptake by cells in the 15 lymphoid tissue, or by trafficking of host cells which have taken-up particulate polynnCleoti~iPs to the lymphoid tissue. Subsequent to uptake by host cells, the l.,.n~g"..ir polynucleotide is e~e~sed at biologically effective levels such that antigenic peptide f~gml~ntc are processed and presented through the endogenous MHC class I pathway and displayed on the l-.~,.--brane surface of the host APCs. Fn~oge~ous APC cell ...~...~..,..-~
20 pff ~ Iinn of antigen promotes the induction of antigen-specific CTLs either at the site of bombardment, or in the Iymphoid tissue. Induced antigen-specific CTLs in turncirculate throughout the m~mm~ n host, preferably a human host, to destroy r.P~ cl;r cells or virally infected cells.
In another embodiment of the present invention a m~mm~ n host is im......
25 with a particulate polynucleotide by direct injection, including but not limited to s~hcut~neouc injection, epidermal injection, dermal injection, Iymphatic injection and intra venous injection. The particulate polymlrleotide enters host cells and is W~ ,SSf~Xl at biologically effective levels such that antigenic peptide fragm~ntc are presented to the endogenous MHC class I pathway and displayed on the membrane surface of the host30 cell. Endogenous target cell membrane presentation promotes the induction of antigen-specific CTLs, which in turn circulate throughout the m~mm~ n host, plc;rc;~dl>ly a ~ human host, to destroy neo~ld:,Lic cells or virally infected cells.
-1~
A m~mm~ n host may be immllni7ed with a particulate polynucleoti~le such that the particulate polynucleotide specifir~lly enters host cells, inf ~ ing APCs, through the phagosome-to-cytosol pathway. Particulate polynucleotides enter host cells through the phagosome-to-cytosol pathway either at the injection site or after trafficking to the S Iymphoid tissue. Thus, particulate polynuc lPoti~ies may enter the lymphoid tissue either by direct trafficking of the parrir~ t~s to the lymphoid tissue with subsequent uptake by cells in the Iymphoid tissue, or by tr~ffir~ing of host cells which have taken-up particulate pOlynurl~oti~ec to the lymphoid tissue. Subsequent to uptake by host cells, the ~".~SC,~-~;r polynucleotide is e~ sed at biologically effective levels such that antigenic peptide 10 fr~gm~ntc are processed and presented through the endogenous MHC class I pathway and displayed on the m~...b,~nr surface of the host APCs. FndrJgenous APC cell rr.~ P
ion of antigen promotes the in~llction of antigen-specific CTLs either at the site of injertion, or in the Iymphoid tissue. Induced antigen-specific CTLs in turn circulate throughout the m~mm~ n host, l~lcr~.dbly a human host, to destroy neoplastic cells or 15 virally infected cells.
In another specific embodiment of the present invention a m~mm~ n host is imlllnl~ A with a particulate polynucleotide by direct injection, including but not limited to Sl l~c.~li.nPous injection, epidermal injection, dermal injection, lymphatic injection and intra venous injection. The composition of the particulate polynucleotide is d~cip~n~d to 20 include specific m~leculf~s which p-er~. .t ally target APC phagocytic pathways (inclutlin~ but not limited to, the m~nnose-receptor m.oAi~tP~I pathway, or Fc--~;c~-t~ ~ pathway) or facilit~t~ cytosolic access of the particulate (ir clur~in~ but not limited to endosomal ~llc.llbl~lc fusion proteins such as the viral HA protein or the listerolysin protein). Particulate polynuclPoti~les enter host cells through the phagosoJI.c 25 to-cytosol pathway either at the injection site or after trafficking to the lymphoid tissue.
Thus, particulate polynucleotides may enter the lymphoid tissue either by directtr~ffi~kin~ of the parti~ul~t~s to the lymphoid tissue with subsequent uptake by cells in the lymphoid tissue, or by traffi~-king of host cells which have taken-up particulate polynudeotides to the Iymphoid tissue. Subse~luent to uptake by host cells, the n,.i~sge~
30 polynu~l~ti-i~ is c~-cssed at biologically effective levels such that antigenic peptide fr~gm~nt~ are l~ucessed and presented through the endogenous MHC class I pathway and displayed on the membrane surface of the host APCs. Endogenous APC cell Ill.,..lb~ e present-qti- n of antigen promotes the indllction of antigen-specific CTLs either at the site of injection, or in the lymphoid tissue. Induced antigen-specific CTLs in turn circulate throughout the mqmm~ n host, preferably a human host, to destroy neoplastic cells or virally infected cells.
The present invention is exemplified on several fronts using a murine mehqnomq model df cl=l ;hed in detail in FYqmpllo Section 6. Briefly, the primary limitqtiorl in studying antigen specific tumor imm-lnity in a murine model is the lack of a defined tumor antigen l~CQ~ 7~ by MHC class I r~ctri.~t~ CTLs. Since TRAs are not fim~i~.,.. n~ y different from any other protein synthesized by the cell, except that the 10 host is not tolerant to them, a foreign protein syntheci7f~ci by a tumor should function as a tumor antigen. Tumor immnni7qtil n metho~c of the present invention are eYernrlifi~d with a murine tumor model with a defined, endogenously syntheci7Pd TRA by trqncfecting the ovalbumin (OVA) gene into the C57Bl/6 derived .,.elano",a B16. This system is attractive for several reasons: (1) the B16 melanoma is an extensively studied 15 murine tumor, (2) in vivo growth characteristics and m~tqctqciC of this tumor line are well cteri7od, and (3) ovalbumin has a well defined structure. The intracellular ocec~;..f~ and pl~ ~ .,t~lion of OVA in the C57Bl/6 mouse is known. In particular the structure of the processed peptide, presented in qccociqti~n with MHC class I Kb, is known. Assays for the fim~-tionql expression of ovalbumin peptide[SIINFEKL] in :~cco~ ;ol~ with H2-Kb using the T-T hybridoma 33.70.Al anti-OVA-Kb are also known (Kovacovics-Bankowski, et al., 1993, Proc. Natl. Acad. Sci. USA. 90: 4942-4946).Techniques to evaluate in vivo inductio~ of OVA specific CTLs in this system are also well ~escrihed (Moore, et al., 1988, Cell 54: 777-785).
One embodiment of the present invention is immunization with particulate polynu~ oti~ie of interest using a biolistic (biob-qlicric) device. Specific-qlly~ a mqmmqliqn host is im~ f.1 with a particulate polynucleotide by a biolistic (biCb-qlicti~-) pl~edu.., such that the particulate polymlcleoti-le delivery ~lu~ )L~ a series of biological events within the trqncfe~t~ cells so as to elicit p,~e~ e immunity to lethal tumor ~~h~llenge The present invention is exemplified severalfold by cutaneous antigen delivery using a 30 biolistic (biob-qlictic) device. First, introduction of particulate polynucleotides enco ling the foreign protein B-Gal results in the expression of B-Gal protein in both the epidermis and in the ~lr.qinin~ lymph nodes. Second, OVA immunization results in the in~lction of W O 97/11605 PCT~US96/15728 -18-OVA-specific CTLs. Third, using the OVA-B16 model, it is shown that OVA-immnni7~oA animals are protected from tumor ch~lltonge by an OVA eA~ g tumor, and that this protection is antigen specific and dep~n-lent on CTLs. Furthermore, the m-och~nicm of this protection goes beyond a generalized cutaneous delivery of S particulate polynucleotides via a biolistic (biobalistic) device. Instead, specific l~L..r,c~ g to phagocytic APCs and/or antigen expression in the Iymphoid tissue is central to in-hlcing antigen-specific CTLs from p-~u-¢o-~. It is therefore a preferred embodiment of the present invention that the target cell be a phagocytic APC, specifically an APC
which is loc~li7~d within, or can traffic to, the Iymphoid tissue.
In another embo~iiment of the present invention a m~mm~ n host is immnni7pd with a particulate polyml~leoti~le by direct injection, inclu~ling but not limited to subcut~neo--c injection, epidermal injection, dermal injection, Iymphatic injection and intra venous injection. The transgenic polynucleotide is ~A~I~ased at biologically effective levels such that antigenic peptide fragm~ltc are processed and presented through the 15 endogenous MHC class I pathway, and displayed on the membrane surface of the target cells. Endogenous APC cell membrane presentation promotes the in~luction of antigen-specific CTLs, which in turn circulate throughout the m~mm~ n host, preferably ahuman host, to destroy neoplastic cells or virally infected cells.
In a ~l~r~ d embo-lim~nt of the present invention the target cell of a m~mm~ n 20 host i~n"~,l";, d with a particulate polynucleotide by direct injection is a phagocytic APC.
The transgenic polynucleotide is eA~-~..¢.ed at biologically effective levels such that antigenic peptide fragm~ntc are pl~.enlc~d to the endogenous MHC class I pathway and displayed on the membrane surface of the APC cell. APC cell membrane ~ e.,l~t;gnpromotes the induction of antigen specific CTLs, which in turn circulate throughout the 25 m~mm~ n host, preferably a human host, to destroy neoplastic cells or virally infected cells.
In a l,.er~--.;d embodiment the particulate polynucleotide is delivered to a human host by subcut~nP~us injection. The present invention dicrlosec that direct injection of the particulate polynucleotide complex results in targeted delivery to phagocytic APCs and 30 gene expression in the Iymphoid tissue. It is this targeted delivery to phagocytic APCs and/or gene expression in the Iymphoid tissue via snbcl-t~neous ~rlminictration which results in superior inriuction of antigen-specific CTLs from naive precursors.
W O 97/11605 PCT~US96/15728 Therefore, central to the present invention are DNA fragments encoding a proteinor biologically active fragment thereof that enter and are ~A~ sed by APCs. The antigen of interest is expressed within the APC cytoplasm and enters the MHC class I
pathway, allowing the APCs to promote induction of antigen-specific CTLs. The data 5 presented in this disclosure supports the novel premise that the most effective manner in which to induce such antigen-specific CTLs is by direct transfer of genetic material to the APCs in the Iymphoid tissue or APCs capable of trafficKing to the Iymphoid tissue.
In another embo-iiment of the present invention a m~mm~ n host is illlllll~.~;7..!:
by injection, in~ln-ling but not limited to subcut~neQus injection, epidermal injection, 10 dermal injection, Iymphatic injection and intra venous injection, with syngeneic APCs that have been antigen loaded in vilro with particulate polynucleotide. Specifically, a m~mm~ n host is immunized with particulate polynucleotide transfected APCs, such that the particulate polynucleotide enters APCs in vitro and the APCs are injected into the host. Subsequent to entry into APCs, the transgenic polynucleotide is eA~ sed a 15 biologically effective levels such that ~nrigenic peptide fr~gm~ontc are processed and p.Gse.-lt;d through the endogenous MHC class I pathway and displayed on the membrane surface of the APCs. After injection of such APCs, endogenous APC cell membrane prestont~ti~n of antigen promotes the induction of antigen-specific CTLs either at the site of injection, or in the Iymphoid tissue. Induced antigen-specific CTLs in turn circulate 20 throughout the m~mm~ n host, preferably a human host, to destroy cells or virally infected cells.
In another specific emborlim~n~ of the present invention a m~mm~ n host is immnni7~d by injection, including but not limited to subcu~neQus injection, epidermal injection, dermal injection, Iymphatic injection and intra venous injection, with syngeneic 25 APCs that have been antigen loaded in vitro by co-incub~tion with particulatepolynucleotide. Specific~lly7 syngeneic APCs are transfected in vitro by co-incub~tion with particulate polynucleotides such that the particulate polynucleotide enters the APCs through a phagosome-to-cytosol pathway. A m~mm~ n host is immunized with particulate polynucleotide transfected APCs, such that the particulate polynucleotide 30 cpecific~lly enters APCs through the phagosome-to-cytosol pathway and the APCs are then injected into the host. Subsequent to uptake by APCs the transgenic polynucleotide is eA~ sed at biologically effective levels such that antigenic peptide fragments are W O 97/11605 PCT~US96/15728 ucessed and presented throughout the endogenous MHC class pathway and ~iicri~ced on the l1.c..~bl~ule surface of the APCs. After injection of such APCs, endogenous APC cell membrane presentation of antigen promotes the induction of antigen-specific CTLs either at the site of injection, or in the Iymphoid tissue. Induced antigen-specific CTLs in turn 5 circulate throughout the m~mm~ n host, preferably a human host, to destroy neoplastic cells or virally infected cells.
In another specific embodiment of the present invention a m~mm~ n host is immnni7~d by injection, including but not limited to subcutaneous injection, epi~e~rn:~l injection, dermal injection, Iymphatic injection and intra venous injection, with syngeneic lO APCs that have been antigen loaded in vitro particulate polynucleotide. Specifir~lly, syngeneic APCs are h~ncfe~~ed in vitro with microprojectile bo1"ba.~l",ent of APCs with particulate polynuc1e~tirle~ Subsequent to projectile bombardment into APCs, thetransgenic polynucleotide is eA~JleS5Cd at biologically effective levels such that :1ntigenir peptide fr~gmrntc are p loccased and presented through the endogenous MHC class I
I5 pathway and displaced on the membrane surface of the APCs. After injection of such APCs, endogenous APC cell membrane ~ se..1,.ti~n of antigen promotes the induction of antigen-specific CTLs either at the site of injection, or in the Iymphoid tissue. Induced antigen-specific CTLs in turn circulate throughout the m~mm~ n host, preferably a human host, to destroy neoplastic cells or virally infected cells.
In another embo~iimrnt of the present invention a r~mm~ n host is i.. ~ d by injection, inr~ ling but not limited to s~1hcut~neous injection, epidermal injection, dermal injection, lymphatic injection and intra venous injection, with syngeneic APCs that have been tr~n~fect~d in vitro with particulate polynucleotide. Sperific~lly~ a m~mm~ n host is i---...~ d with particulate polynucleotide tr~n~fe~ted APCs, such that the 25 particulate polynucleoticle enters APCs in vilro and the APCs are injected into the host.
Particulate polynll~leotifltos enter APCs in vitro by either microprojectile bomb~d,..~.lt or the phagoac,."e to-cytosol pathway. APCs are tr~ncfectrd in vitro with antigen enco~ling polynucleotides and/or polynuclcotides enrofiing a molecule or molecules which illc the effirienry of the antigen presrnting function of the APC, such as, but not limited to, 30 cytokine molecules such as IL-12, IL-2, and/or IL-4 and/or costirr nl~tory molecules such as CD80 and/or CD86. Subsequent to entry into APCs, the ~.d..SgC~iC antigen enco~ling poly..ucle~Lides is ~ r~,!7 ,ed at binlogic~lly effective levels such that ~ntig~nir~ peptide W O97/11605 PCT~US96/15728 rA~ are processed and presented through the endogenous MHC class I pathway and displayed on the membrane surface of the APCs. After injection of such APCs, endogenous APC cell membrane precentAtinn of antigen promotes the inductiQn of antigen-specific CTLs either at the site of injection or in the Iymphoid tissue. Induced S antigen-specific CTLs in tum circulate throughout the mAmmAliAn host, pre~.dbly a human host, to destroy neoplastic cells or virally infected cells. Subsequent to entry into APCs, the lldnsgeniC cytokine andlor costimnlAtory Pnco~ing polynucleotide is ~;A~
at biologically effective levels such that the antigen pr~C~nting function of the APC results in the inrlllcti~n of antigen specific immune responses either at the site of inj~ction, or in the lymphoid tissue.
It will be within the purview of the artisan of ordinary skill to inco",~,ldle a TRA
or viral antigen of choice into the system ~iicrlc~sed within this specification. FY~mples of presently available TRAs include, but are by no means limited to, MAGE-l, MAGE-3, BAGE, GAGE-l, GAGE-2, Tyrosinase, Melan-A(MART-l), gplOO(pmell7), gp75(TRP1), CEA (carçinoemhryonic antigen) as well as viral derived tumor -Antigenc from HPV, HBV, and EBV, as well as tumor ACcociAt~d oncogene/tumor SU~ gene mut~tinn /~nroded antigens such as P53, P16, RAS, HER2/neu, C-ABL, and polymorphic endothelial mucin antigens (as reviewed in Maeurer et. al., 1996, Cancer Vaccines in Clinical Tmmlmc)logy Principles and Practice, ed R. Rich, Mosby Publishing. Chpt.
123:1904-1918 and Van den Eynde et al., 1995, J. Ei~p. Med. 182:689-698). FY~m~
of viral antigens include, but are by no means limited to, Influenza nucleo~ eill (Donnelly et al., 1995, Nature Med. 1:583-587.), HIV gp 120, HIV gp 160, and T-T~pAtitic B surface antigen (as reviewed in Pardoll and Beckerleg, Immunity, l99S, 3: 165-169).
It is known to the artisan of ordinary skill that any eukaryotic promote} and/orenh~nr~r sequence available which is known to up-regulate expression of a ~IA..cg~..ir DNA sequence may be used in constructing a recombinant vector for combination with the particle of choice so as to generate the particulate polynucleQti(lt- of the present invention. Such promoter fragments include but are not limited to a cytomegalovirus 30 (CMV) promoter, a Rous Sarcoma virus (RSV) promoter, a Murine T~nkl~miA Virus(MLV) p.~,...otel, a B-actin promoter, as well as any cell-specific promoter or enh-Anr.or s~u~"ce known to be active in the target cell.
To this end, a preferred embodiment of the present invention is the use of particulate bound DNA to delive} the DNA rnrQ~iing a tumor or viral antigen specifir~lly to the APCs in the lymphoid tissue, or APCs capable of trafficking to the Iymphoid tissue, so as to promote MHC class I access of antigen, thus allowing APCs to Stim~ t.o 5 inrlllction of antigen-specific CTLs. These CTLs will then circulate throughout the host and destroy neoplastic cells or virally infected cells.
Tumor immunity based on the ~ d method of utilizing ~--bcut~nrous injection to ~-u.l-ult: a specific .~o.lse in Iymph node APCs is exemplified in Example Section 6, Example Section 7 and Example Section 8.
The following examples are offered by way of illustration of the present invention, and not by way of limi~tic.n, W O 97/11605 PCT~US96/15728 6. EXAMPLE: OVA/B16 GENETIC MURINE TUMOR MODEL
The OVA/B16 murine model was used to generate the data disclosed in FY~mple Section 7 and 8 which exemplify the claimed invention. The OVA/B16 murine system is attractive for several reasons: (1) the B16 melanoma is an extensively studied murine tumor, (2) in vivo growth characteristics and mft~ct~cic of this tumor line are well rh~cte~ized, and (3) ovalbumin has a well defined structure. The intr~rçll~ r procescing and presentation of OVA in the C57B1/6 mouse is known. In particular the structure of the p.ocessed peptide, presented in association with MHC class I Kb, is known. Assays for the filnction~l expression of ovalbumin peptide tSIINFEKL] in ~ccoci~tion with ~I2-Kb using the T-T hybridoma 33.70.AI anti-OVA-Kb are also known (Kovacovics-Bankowski, et al., 1993, Proc. Natl. Acad. Sci. USA. 90: 4942-4946).Techniques to evaluate in vivo in~ ction of OVA specific CTLs in this system are also well ~iesr~ihe~ (Moore, et al., 1988, CeU 54: 777-785).
Mice and Cell Lines. Female C57BL/6 mice, 5-8 weeks old were purchased from the Jackson Laboratories, Bar Harbor, ME. EL4 is a C57BL/6 T-lymphoma, and EG7 is a chicken egg ovalbumin (OVA)-transfected subclone of EL4 (Moore, et al. 1988, Cell 54: 777-785). The C57BL/6 derived murine melanoma B16 (Fidler, et al., 1976, Cancer Res. 36: 3~60-3165) was obtained from American Tissue Type Collection (ATCC). MO4 was constructed by transfection of B16 with the pAc-Nco-OVA plasmid as described.
(Falo, et al, 1995, Nature Med. 1: 649-653, Moore, et al. 1988, Cell 54: 777-785) Monorlon~l antibodies were prepared from the hybridomas GK1.5 (anti-CD4, ATCC
TIB-207), 2.43 (anti-CD8 antibodies was raised in Balb/c nu/nu mice by i.p. injection of GKfl.5 cells (3 x 106) and IFA (0.5ml/mouse).
After OVA transfection of the B16 melanoma, and selection, the transfected B16 melanoma subclone, MO4 was icol~trd The parent melanoma B16, and the OVA
tr~ncfrct~nt express similar levels of fimction~l Kb on the cell surface as measured by f~ on of OVA peptide (SIINFEKL) to RF33.70 (Figure 1). In contrast, M04/5, but not B16, is capable of hybridoma stimulation in the absence of exogenously added peptide (Figure 1). This ~tomonct-~trS endogenous pro~llction, processing, and ~ .,l;.l;nn of the lldll~re~;led antigen. Importantly, endogenous expression of OVA by MO4 did not cignifir~ntly alter in vivo imm~lnogenicity of the tumor (Figure 2). Tumor WO 97/11605 PCT~US96/15728 growth (Figure 2A) of B16 and M04 is comparable in naive mice, as is host survival (Figure 2B).
W O 97/11605 PCT~US96/15728 7. EXAMPLE: GENETIC IMMUNIZATION VIA
BIOLISTIC (BIOBALISTIC~ ADMTNISTRATION
~ ocfl1izo~ion of Foreign Protein Expression - Tissue specimens (skin or draining lymph nodes) were harvested 24 or 48 hours after immnni~ti~n~ washed in PBS and S fixed in 2% formaldehyde - 0.2% glutaraldehyde in PBS for 30 min. at 4~C. Fixed .l~e4~ nc were washed extensively with PBS and then in~lb~t~ in X-gal staining solu'ion (lmg/ml X-ga~, 5mM pvr~ rrl ferricya~ide, 5mM fer~ocya~ide, 2mM MgCl2i~PBS) for 18 hrs at 37~C. Stained tissue was paraffin sectioned and counterstained with 0.1% nuclear fast red.
Gene~ic Immunization - DNA coated gold particles were p lcpalcd by co.. ~h;.~ g 50 mg of 0.95 ~m gold beads and 100 ~1 of 0.1 M spermidine and soni~ting for 5 cel-on~ic 132 ~L~g of plasmid DNA and 200 ~Ll of CaC12 were added sequentially whiie vortexing. This mixture was allowed to l~c~ dte at room te~llpe.dture for 5-10 mimltPc The bead preparation was then centrifuged (10,000 rpm for 30 seconds) and washed 3 15 times in cold ethanol before re-sncp~ncif)n in 7ml of ethanol to give a final conc~n~ lion of 7mg gold/ml. The solution was then loaded into Tefzelal' tubing (Agracetus) and allowed to settle for S minutes. The ethanol was removed and the beads were ~tt~h~d to the sides of the tubing by rotation at 20 rpm for 30 seconds and N2 dried. The dry tubing lined with beads was then cut into 0.5 inch sections and stored for use with ~lloci~c~nt in 20 p~r~film sealed vials. Animals were v~ccin~t~ by delivery of 2 shots (each shot c.~ l of 0.5mg gold beads = 0.5 inch of tubing) to the shaved abdominal region using the Accell Gene Delivery Device at a discharge pressure of 300 psi. Animals were i.. ln~ d with either the pAc-Neo-OVA plasmid (Moore, et al., 1988, Cell 54: 777-785) or the pTF.gl~l'7. pla~smid (provided by Nadia Jouroud) which cont~inc the lacZ gene 25 under the control of the CMV promoter. For experiments involving subcu~n.ol~us injection of particulate polynucleotides (Figure S ) DNA coated beads were p~cp~h~d as above and an equivalent quantity of DNA was injected. For some animals (Figure 5), an excess quantity of free plasmid DNA was sllhc~ n~Qusly injected. Snhcllt~neQus injectinnc were ~riminict~red in 100,.~1 volume of PBS in the mid-flanks, bilaterally. For 30 in vitro tr~ncfection of APCs (Figure 4), DNA beads were identically plc~alcd using an equivalent weight of Biomag iron oxide beads or gold beads as a particulate substrate.
W O 97/1160~ PCT~US96tlS728 For experiments involving subcutaneous injection of particulate polynucleotides (Figure 5 and 6) DNA coated beads were prepared as above and an equivalent ~uantity of DNA was injected, or the quantity specified in the figure descriptions. For some animals (Figure 5 and 6), an excess quantity of free plasmid DNA was subcutaneously injected.
Suhc~lt~n~Qus injections were adminictered in 1OO~LI volume of PBS in the hind legs, bilaterally.
For in vitro tr~n~fection of APCs (Figure 4 and 6), DNA beads were id~nt~ y prepared as des~rihe~i above using an equivalent weight of Biomag iron oxide beads (Fe beads) or gold beads as particulate substrate. Dendritic APCs were obtained from bone marrow as described in the Brief Description of the Drawings except that IL-4 was not used in tissue culture media with 25,ul of 7mg/ml particulate polynucleotide solution (particulate wt/vol. PBS) for 18hrs at 37~C. Antigen-pulsed dendritic cells were p.~
and injected as described in the Brief Description of the Drawings. Briefly, dendritic cells were prepared by depleting bone marrow cells of Iymphocytes and culturing overnight in RPMI 1640 supplemented with 10%FCS, L-gh-t~mine, antibiotics and 2-ME in 24 wellplates at 106 cells/well. Cells were repleted on day 1 at 2.5 x 105 cells/well with GM-CSF (103U/ml, Sigma, St. Louis, MO) and murine rIL-4 (103U/ml, Genzyme, Cambridge, MA) and loosely adherent cells were harvested on day 8. By flow cytoml~tric analysis, these dendritic cells e~ .sed CD45, CD44, CDllb (Mac-l), CD18, CD80, cD86 and class I and class II MHC ~nti~en~ Dendritic cells were pulsed 2hrs at 37~C
with or without OVA peptide (20ng/ml) + B2-microglobin (132-M, 10~1/ml, human, Sigma) in reduced serum media (Optimen, Gibco, Grand Island, NY). Cells were then washed extensively, resuspended in PBS and irradiated (2000 rad) before injection into naive mice.
Cytotoxi~iry Assays - Splenocytes from immllni7~d animals were r~ostimnl~t~d with minor mo~iifi~tinnc of previously described protocols. (Falo, et al., 1995, N~ure Med.
1: 649-653). Briefly, 1 week after immuni7~tic~l splenocytes (30 x lo6) were restim by coculture with irr~ t~d (20,000 rad) EG7 cells (10 x 106). Effector cells were harvested five days later and cultured with 2xl0~ s'Cr labeled targets in round bottom microwells (200~1) at the indicated effector target cell ratio. In some cases the effector cells were ~eplet~d of T-cell subsets using mAB plus complement before assay as d~ ihed (Rock, et al., 1993, J. Immunol. 150: 1244-1251). After 4 hours at 37~C, WO 97/11605 PCT~US96/1~728 lOO,ul of supernatant from triplicate microcultures was collected and counted and the p~..;e.,~ge of specific release was r~ t~d as described. (Falo, et al., 1995, Nature Med 1 649-653). Results are reported as the mean of triplicate cultures. The SEM of triplicate cultures was always less than 15% of the mean.
Pro~ec~ion Assays - C57BL/6 mice were immunized as described with the in-iir~t~
antigen gene construct. Animals were ~~h~ nged with tumors and evaluated for tumor survival as described. Briefly, seven days after the final immllni7~tion (day 0), OVA-immnni7~d or lacZ-immunized animals were f~h~llenged by intradermal inj~tinn in the mid-flanks bilaterally with melanoma cells (2 x 104) at 2 times the dose lethal to 50%
of the animals tested (LD5,~ or the number of tumor cells in~ic~t~ in section 4. Survival is recorded as the p~.cen~ge of surviving ~nim~ls Melanoma cells for injection were washed three times in PBS. Injected cells were greater than 95% viable by trypan blue exclusion. All experiments included 5 mice per group and were repeated at least three times. Mice which became moribund were sacrificed according to animal care g~lifiplin~s of the University of Pittsburgh Medical Center. In some experiments, animals were ~ pletPd of CD8+ cells. This was accomplished by i.p. injection of CD8 mAb (2.43) 7 and 9 days after immnni7~tiorl as described, followed by tumor rh~llenge on day 10 (Falo, et ai., 1995, Nature Med. 1: 649-653).
The capacity of biolistic (biobalistic) immnni7~tion to induce antigen specific CTLs was evaluated. Naive C57Bl/6 mice were immnni7ed with a total of at most 2.64~g of OVA ~n~oriing DNA delivered to the abdominal skin with 2 overlapping pulses, andidentic~lly boosted 7 days later. In vitro restim~ tPd spleen cells from these mice Iysed the syngeneic OVA-expressing murine thymoma EG7, but not the untransfected parent tumor EL4 (Figure 3A). Thus, target cell Iysis was antigen specific, depending on expression of OVA by the tumor target. Depletion of T-Cell subsets from the effector populations using mABs demonstrated that lysis depended on Thy 1+, CD8+ subsets ch~r~ct~rictic of MHC class I-restricted CTL effector cells (Figure 3B).
Groups of mice that were immnni7~d and boosted as described above were ~h~llenged 7 days later by i.d. injection of the MO4 melanoma at a distant site to d~:L~ ine the ability of biolistic (biobalistic) immuni_ation to induce protective tumor immunity. OvA-immllni7t~d mice were protected from lethal tumor ch~llenge, whiletumors in control mice (immlmi7ed similarly, but with the lacZ ll::lJUII~l gene) grew W O 97/11605 PCTAUS96/1~728 ~J-Ug~ rely and were lethal in 60% of the animals by day 40 (Figure 3C). OVA-immlmi7~ mice were not protected from ~h~ nge with the untransfected parent melanoma B16 (Figure 3D), in~ ting that p.otccLive immnnity was antigen specific, depen-ling on OVA expression by the tumor target. We evaluated the contribution of 5 CD8+ effector cells to this protective tumor immnnity by depleting groups of immlmi7Od or control (lacZ immnni7~d) animals of CD8+ effector cells through repeated i.p.injection of anti-CD8mAB before tumor ch~llpnge~ (Falo, et al., 1995, Nature Med. 1:
649-653). While OVA-imml-ni7ed animals were p.ute~;lcd from MO4 rh~ n~e, survival in im~ ed~ CD8+ T-cell ~It plet~d animals was similar to that observed in control 10 ~nim~lc, with or without T-cell depletion (Figure 3E-F). Therefore, CD8-' T-cells are ~cc.onti~l for the plotcclive tumor immnnity induced by genetic immnni7~tion in this model.
Aci~liticn~l evidence ~ u~ling the ~-uposed merh~nicm of tumor immlmity includes the demonstration of 13-g~ tocirl~ce expression in the epidermis after biolistic 15 (bi--b~lictic) delivery of the lacZ construct, and, interestingly, discrete areas of specific CAIJJ.'~,.7sion within the draining Iymph nodes. As these nodes were distant from the imm--ni~tion site, it is unlikely that Iymph node expression was a result of direct physical bombardment. There was a predomin~n~e of gold particles in the epidermis and dermis of immnni7~d skin (Imct~in~d) 24hrs after DNA delivery and lacZ expression within 20 epidermal k~ratinocytes at 48hrs. There are also discrete areas of specific staining within the fir~ining lymph nodes. TdPnti-~lly treated specimenc from mice imm--ni7~d with irrelevant DNA did not show lacZ expression. This observation p-u-ll~t~,d further analysis which is ~iicrlo5ed in FY~mplP Section 8.
W O 97/11605 PCT~US96/15728 8. EXAMPLE: GENETIC IMMUNI_ATION BY
SUBCUTANEOUS ADMINISTRATION
The materials and methods for inrhlcing tumor immunity by subcutaneous - ~lminictration of a particulate polynucleotide are as described throughout FY~mrle Section 6 and Example Section 7.
This data demonstrates an ability to crecific~lly target phagocytic APCs in the lymphoid tissue, or APCs capable of tr~ffl~king to the Iymphoid tissue, by s~lbcut~neouc injection of particulate polynucleotides in vivo.
Protection from tumor ch~ onge was equivalent within groups of mice immnni with particulate polynucleotides comprising pAc-Neo-OVA either by direct sub.~ cinjection or by biolistic (biob~lictic) adminictration (Figure 5). Concictent with the finfiingc in FY~mrlf Section 7, immnni7~tion with a particulate polynucleotide encoriing and irrelevant antigen (e.g., PTFEI~r~7) did not confer protection (Figure 5).
Snbc.,l; n~ous injection of pAc-Neo-OVA without particlll~t~s did not confer protection from a relevant tumor ch~ nge (Figure 5). These data indicate that suhcut~nt~o--c injection of particulate polynucleotides is at least as effective as biolistic (biobalistic) particulate delivery. Furthermore, as subcutaneous injection does not result in direct physical projectile bombardment of polynucleotide particles into the cytoplasm of individual host cells, expression likely requires active uptake of polynucleotides by host cells capable of phagocytosis/endocytosis. The observation that subcutaneous injection of pAc-Neo-OVA without particulates did not confer protection also implicates particulate delivery/phagocytosis as the mech~nicm of tr~ncductiom Particulate tr~ncdllction by phagocytosis implies that transgene expression is preferentially targeted to cells capable of phagocytosis, in~lu-iing APCs.
In further support of this proposed mech~ni~m is the observation that APCs (bonemarrow derived dendritic cells) co-cultured in vitro with particulate polynucleotides enco~iing ovalbumin (~ JdlCd as fles~rihe~ in Example 7) (pAc-Neo-OVA) can ctim~ t~
the OVA (SIINFEKL+ Kb) specific T cell hybridoma RF33.70 to produce IL-2 (Figure 4).
These data demonctr~t~- that these APCs filn~tion~lly express the ovalbumin gene and generate the ovalbumin peptide-Kb complex. The potency of $timnl~inn is colllpdldble to that observed with APCs pulsed with 2mg/ml of soluble OVA protein. Particulate polynnrleotirl~s are effective in this assay using either gold or Fe as the particulate CA 02233278 l99X-03-27 substrate. Thus, the incubation of parliculate polynucleotides with phagocytic APCs in vi~ro results in the en(logenQus production, processing, and presentation of the transfected antigen These observations demonstrate that particulate polynucleotides can be taken up and e~ .;,ed by APCs, and that the corresponding proteins can be functionally presented 5 to induce antigen specific immune responses.
Fll-ll,.,.lnore, a shown in Figure 6, antigen presenting cells, in this case snbcut~nPously injected bone marrow derived denllritic cells, which have been incuh~rP~d with particulate polynucleotides ~ncoriing the antigen OVA in vitro as descri~e~ above, are capable of inrlll~ing p-uL~Li~e immunity to tumor cells e~ ing the antigen gene.
In the example given, a one time ~l1minictration of 105 irradiated transfected ~i~n~1rjti~
cells/animal (5 x 104 cells per hind leg, bilaterally) induced complete protection to tumor ch~llenge. Thus, in vivo ~lmini~rPred dendritic APCs, which are potent ~imlll~tnrs of antigen-specific CTLS and l,.ule~:Live tumor immunity when peptide-loaded in vitro are at least similarly immnnogenic which antigen-loaded by co-incubation with particulate polyn~cl~oOti~
Taken together, these results indicate that subcut~neouc ~timini~tration of particulate polynucleotides has the advantage of targeting delivery of antigens to phagocytic APCs, potentially increasing the efficiency of genetic immllni~tion while reducing unwanted deleterious effects, including but not limited to tolerance inductinn or 20 mutagenesis resulting from the transfection of normal, non-APC host cells.
-
Claims (67)
1. An in vivo method of therapeutic or prophylactic genetic immunization of a mammalian host, which comprises:
(a) generating a DNA fragment which expresses an antigenic protein or antigenic protein fragment;
(b) distributing said DNA fragment on a particle surface, resulting in a particulate polynucleotide;
(c) inoculating said mammalian host with said particulate polynucleotide; and, (d) delivering said particulate polynucleotide to the cytoplasm of a target cellwithin said mammalian host, such that said expressed antigenic protein or antigenic protein fragment is presented to the membrane surface of said target cell through the MHC class I pathway.
(a) generating a DNA fragment which expresses an antigenic protein or antigenic protein fragment;
(b) distributing said DNA fragment on a particle surface, resulting in a particulate polynucleotide;
(c) inoculating said mammalian host with said particulate polynucleotide; and, (d) delivering said particulate polynucleotide to the cytoplasm of a target cellwithin said mammalian host, such that said expressed antigenic protein or antigenic protein fragment is presented to the membrane surface of said target cell through the MHC class I pathway.
2. The method of claim 1 wherein said mammalian host is a human.
3. The method of claim 2 wherein said DNA fragment expresses a tumor rejection antigen, viral antigen or antigenic protein fragment thereof.
4. The method of claim 3 wherein said target cell is an antigen presenting cell.
5. The method of claim 4 wherein said antigen presenting cell resides within or migrates to the lymphoid tissue of said human host.
6. The method of claim 5 wherein said tumor rejection antigen is selected from the group consisting of MAGE-1 and MAGE 3.
7. The method of claim 5 wherein said tumor rejection antigen is Melan-A.
8. The method of claim 5 wherein said tumor rejection antigen is gp100.
9. The method of claim 5 wherein said tumor rejection antigen is p53.
10. The method of claim 5 wherein said tumor rejection antigen is CEA.
11. The method of claim 5 wherein said tumor rejection antigen is HER2/neu.
12. The method of claim 5 wherein said viral antigen is HIV gp120, HIV
gp160.
gp160.
13. The method of claim 5 wherein said viral antigen is Influenza virus nucleoprotein.
14. The method of claim 5 wherein said viral antigen is Hepatitis B surface antigen.
15. An in vivo method of therapeutic or prophylactic genetic immunization of a mammalian host, which comprises:
(a) generating a DNA fragment which expresses an antigenic protein or antigenic protein fragment;
(b) distributing said DNA fragment on a particle surface, resulting in a particulate polynucleotide;
(c) inoculating said mammalian host with said particulate polynucleotide using abiolistic device; and, (d) delivering said particulate polynucleotide to the cytoplasm of a target cellwithin said mammalian host, such that said expressed antigenic protein or antigenic protein fragment is presented to the membrane surface of said target cell through the MHC class I pathway.
(a) generating a DNA fragment which expresses an antigenic protein or antigenic protein fragment;
(b) distributing said DNA fragment on a particle surface, resulting in a particulate polynucleotide;
(c) inoculating said mammalian host with said particulate polynucleotide using abiolistic device; and, (d) delivering said particulate polynucleotide to the cytoplasm of a target cellwithin said mammalian host, such that said expressed antigenic protein or antigenic protein fragment is presented to the membrane surface of said target cell through the MHC class I pathway.
16. The method of claim 15 wherein said mammalian host is a human.
17. The method of claim 16 wherein said DNA fragment expresses a tumor rejection antigen, viral antigen or antigenic protein fragment thereof.
18. The method of claim 17 wherein said target cell is an antigen presenting cell.
19. The method of claim 18 wherein said antigen presenting cell resides within or migrates to the lymphoid tissue of said human host.
20. The method of claim 19 wherein said tumor rejection antigen is selected from the group consisting of MAGE-1 and MAGE 3.
21. The method of claim 19 wherein said tumor rejection antigen is Melan-A.
22. The method of claim 19 wherein said tumor rejection antigen is gp100.
23. The method of claim 19 wherein said tumor rejection antigen is p53.
24. The method of claim 19 wherein said tumor rejection antigen is CEA.
25. The method of claim 19 wherein said tumor rejection antigen is HER2/neu.
26. The method of claim 19 wherein said viral antigen is HIV gp120, HIV
gp160.
gp160.
27. The method of claim 19 wherein said viral antigen is Influenza virus nucleoprotein.
28. The method of claim 19 wherein said viral antigen is Hepatitis B surface antigen.
29. An in vivo method of therapeutic or prophylactic genetic immunization of a mammalian host, which comprises:
(a) generating a DNA fragment which expresses an antigenic protein or antigenic protein fragment;
(b) distributing said DNA fragment on a particle surface, resulting in a particulate polynucleotide;
(c) inoculating said mammalian host with said particulate polynucleotide by direct injection; and, (d) delivering said particulate polynucleotide to the cytoplasm of a target cellwithin said mammalian host, such that said expressed antigenic protein or antigenic protein fragment is presented to the membrane surface of said target cell through the MHC class I pathway.
(a) generating a DNA fragment which expresses an antigenic protein or antigenic protein fragment;
(b) distributing said DNA fragment on a particle surface, resulting in a particulate polynucleotide;
(c) inoculating said mammalian host with said particulate polynucleotide by direct injection; and, (d) delivering said particulate polynucleotide to the cytoplasm of a target cellwithin said mammalian host, such that said expressed antigenic protein or antigenic protein fragment is presented to the membrane surface of said target cell through the MHC class I pathway.
30. The method of claim 29 wherein said mammalian host is a human.
31. The method of claim 30 wherein direct injection is by subcutaneous injection.
32. The method of claim 31 wherein said recombinant DNA vector fragment expresses a tumor rejection antigen, viral antigen or antigenic protein fragment thereof.
33. The method of claim 32 wherein said target cell is an antigen presenting cell.
34. The method of claim 33 wherein said antigen presenting cell resides within or migrates to the lymphoid tissue of said human host.
35. The method of claim 34 wherein said tumor rejection antigen is selected from the group consisting of MAGE-1 and MAGE 3.
36. The method of claim 34 wherein said tumor rejection antigen is Melan-A.
37. The method of claim 34 wherein said tumor rejection antigen is gp100.
38. The method of claim 34 wherein said tumor rejection antigen is p53.
39. The method of claim 34 wherein said tumor rejection antigen is CEA.
40. The method of claim 34 wherein said tumor rejection antigen is HER2/neu.
41. The method of claim 34 wherein said viral antigen is HIV gp120, HIV
gp160.
gp160.
42. The method of claim 34 wherein said viral antigen is Influenza virus nucleoprotein.
43. The method of claim 34 wherein said viral antigen is Hepatitis B surface antigen.
44. An ex vivo method of therapeutic or prophylactic genetic immunization of a mammalian host, which comprises:
(a) generating a DNA fragment which expresses an antigenic protein or antigenic protein fragment;
(b) distributing said DNA fragment on a particle surface, resulting in a particulate polynucleotide;
(c) delivering said particulate polynucleotide to the cytoplasm of a target cell of a mammalian host in vitro, such that said expressed antigenic protein or antigenic protein fragment is presented on the membrane surface of said target cell through the MHC class I pathway; and, (d) inoculating said mammalian host with said target cell by direct injection.
(a) generating a DNA fragment which expresses an antigenic protein or antigenic protein fragment;
(b) distributing said DNA fragment on a particle surface, resulting in a particulate polynucleotide;
(c) delivering said particulate polynucleotide to the cytoplasm of a target cell of a mammalian host in vitro, such that said expressed antigenic protein or antigenic protein fragment is presented on the membrane surface of said target cell through the MHC class I pathway; and, (d) inoculating said mammalian host with said target cell by direct injection.
45. The method of claim 44 wherein said mammalian host is a human.
46. The method of claim 45 wherein direct injection is by subcutaneous injection.
47. The method of claim 46 wherein said recombinant DNA vector fragment expresses a tumor rejection antigen, viral antigen or antigenic protein fragment thereof.
48. The method of claim 47 wherein said target cell is an antigen presenting cell.
49. The method of claim 48 wherein said antigen presenting cells resides within or migrates to the lymphoid tissue of said human host.
50. The method of claim 49 wherein said tumor rejection antigen selected from the group consisting of MAGE-1 and MAGE 3.
51. The method of claim 49 wherein said tumor rejection antigen is Melan-A.
52. The method of claim 49 wherein said tumor rejection antigen is gp100.
53. The method of claim 49 wherein said tumor rejection antigen is p53.
54. The method of claim 49 wherein said tumor rejection antigen is CEA.
55. The method of claim 49 wherein said tumor rejection antigen is HER2/nue.
56. The method of claim 49 wherein said viral antigen is HIV gp120, HIV
gp160.
gp160.
57. The method of claim 49 wherein said viral antigen is Influenza virus nucleoprotein.
58. The method of claim 49 wherein said viral antigen is Hepatitis B surface antigen.
59. An ex vivo method of therapeutic or prophylactic genetic immunization of a mammalian host, which comprises:
(a) generating a DNA fragment(s) which express a molecule which enhances the antigen presentation function of an APC;
(b) distributing said DNA fragment(s) on a particle surface, resulting in a particulate polynucleotide;
(c) delivering said particulate polynucleotide to the cytoplasm of a target cell of a mammalian host in vitro, such that said expressed antigen presentation enhancing protein or proteins is expressed in a biologically significant form and at biologically significant levels;
(d) inoculating said mammalian host with said target cell by direct injection.
(a) generating a DNA fragment(s) which express a molecule which enhances the antigen presentation function of an APC;
(b) distributing said DNA fragment(s) on a particle surface, resulting in a particulate polynucleotide;
(c) delivering said particulate polynucleotide to the cytoplasm of a target cell of a mammalian host in vitro, such that said expressed antigen presentation enhancing protein or proteins is expressed in a biologically significant form and at biologically significant levels;
(d) inoculating said mammalian host with said target cell by direct injection.
60. The method of claim 59 wherein said mammalian host is a human.
61. The method of claim 60 wherein direct injection is by subcutaneous injection.
62. The method of claim 61 wherein said target cell is an antigen presenting cell.
63. The method of claim 62 wherein said antigen presenting cell resides within or migrates to the lymphoid tissue of said human host.
64. The method of claim 63 wherein said DNA vector fragment expresses a costimulatory molecule.
65. The method of claim 64 wherein said costimulatory molecule is selected from the group consisting of CD80 and CD86.
66. The method of claim 63 wherein said DNA vector fragment expresses a cytokine molecule
67. The method of claim 66 wherein said cytokine molecule is selected from the group consisting of IL-12, IL-4 and IL-2.
Applications Claiming Priority (2)
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US53555695A | 1995-09-28 | 1995-09-28 | |
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CA002233278A Abandoned CA2233278A1 (en) | 1995-09-28 | 1996-09-27 | Stimulation of cell-mediated immune responses by targeted particulate genetic immunization |
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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) |
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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 |
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1996
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- 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
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