Hydrodynamic Limb Vein Delivery of a Xenogeneic DNA Cancer Vaccine Effectively Induces Antitumor Immunity

Abstract

Tumor-associated antigens (TAA) are typically poorly immunogenic “self” antigens. An effective strategy to break tolerance and induce antitumor immunity is by genetic vaccination, employing the orthologous TAA-sequence from a different species. We recently developed a clinically relevant approach for intravascular hydrodynamic limb vein (HLV) delivery of nucleic acids to skeletal muscle. Using the human gp100 xenogeneic TAA in the murine B16 melanoma model, we show that genetic vaccination of mice by HLV plasmid DNA delivery was highly effective at breaking tolerance against the homologous murine gp100 (mgp100) TAA and induced prophylactic antitumor protection. HLV vaccination resulted in an anti-hgp100 humoral and cellular response, with 4–5% of CD8+ T cells being gp10025–33-epitope-specific. Vaccinated animals demonstrated in vivo cytolytic activity against human and mgp10025–33 peptide-pulsed targets. Antitumor immunity could be adoptively transferred by splenocytes from human gp100-vaccinated animals. Furthermore, a durable antitumor memory response was established as ∼3% of CD8+ T cells were gp10025–33 antigen-specific in mice 6 months after vaccination. Following a single HLV human gp100 DNA boost, this level increased to ∼17% and protected animals from subsequent B16 tumor rechallenge. Our results warrant further consideration of HLV as a clinically relevant method for cancer gene therapy. Tumor-associated antigens (TAA) are typically poorly immunogenic “self” antigens. An effective strategy to break tolerance and induce antitumor immunity is by genetic vaccination, employing the orthologous TAA-sequence from a different species. We recently developed a clinically relevant approach for intravascular hydrodynamic limb vein (HLV) delivery of nucleic acids to skeletal muscle. Using the human gp100 xenogeneic TAA in the murine B16 melanoma model, we show that genetic vaccination of mice by HLV plasmid DNA delivery was highly effective at breaking tolerance against the homologous murine gp100 (mgp100) TAA and induced prophylactic antitumor protection. HLV vaccination resulted in an anti-hgp100 humoral and cellular response, with 4–5% of CD8+ T cells being gp10025–33-epitope-specific. Vaccinated animals demonstrated in vivo cytolytic activity against human and mgp10025–33 peptide-pulsed targets. Antitumor immunity could be adoptively transferred by splenocytes from human gp100-vaccinated animals. Furthermore, a durable antitumor memory response was established as ∼3% of CD8+ T cells were gp10025–33 antigen-specific in mice 6 months after vaccination. Following a single HLV human gp100 DNA boost, this level increased to ∼17% and protected animals from subsequent B16 tumor rechallenge. Our results warrant further consideration of HLV as a clinically relevant method for cancer gene therapy. IntroductionAlthough cancer cells are less immunogenic than foreign pathogens, the immune system is clearly capable of recognizing and eliminating tumor cells. Tumor-associated antigens (TAAs) recognized by the immune system are typically self-antigens1Houghton AN Gold JS Blachere NE Immunity against cancer: lessons learned from melanoma.Curr Opin Immunol. 2001; 13: 134-140Crossref PubMed Scopus (171) Google Scholar and are poorly immunogenic as a result of central and peripheral tolerance, a process that prevents the development of autoimmune disorders against “self” molecules. Mutated or altered self (heteroclitic)-antigens are better able to activate T-cell antitumor immunity through the formation of a more sustained major histocompatibility complex/altered self-antigen complex on the surface of professional antigen-presenting cells, such as dendritic cells.2Dyall R et al.Heteroclitic immunization induces tumor immunity.J Exp Med. 1998; 188: 1553-1561Crossref PubMed Scopus (187) Google Scholar This effectively breaks immunological tolerance to the self antigen. One form of presenting the immune system with an altered self antigen is through the expression of a closely related ortholog derived from a different species.3Srinivasan R Wolchok JD Tumor antigens for cancer immunotherapy: Therapeutic potential of xenogeneic DNA vaccines.J Transl Med. 2004; 2: 12Crossref PubMed Scopus (41) Google Scholar In the B16 murine melanoma model, vaccination with xenogeneic human gp100 (hgp100) by viral vector,4Zhai Y et al.Cloning and characterization of the genes encoding the murine homologues of the human melanoma antigens mart1 and gp100.J Immunother. 1997; 20: 15-25Crossref PubMed Scopus (94) Google Scholar direct intramuscular injection,5Tanaka M et al.Induction of a systemic immune response by a polyvalent melanoma-associated antigen DNA vaccine for prevention and treatment of malignant melanoma.Mol Ther. 2002; 5: 291-299Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar or particle-mediated (i.e., gene gun) plasmid DNA (pDNA) delivery induced antitumor protection against B16 tumor challenge.4Zhai Y et al.Cloning and characterization of the genes encoding the murine homologues of the human melanoma antigens mart1 and gp100.J Immunother. 1997; 20: 15-25Crossref PubMed Scopus (94) Google Scholar, 6Gold JS et al.A single heteroclitic epitope determines cancer immunity after xenogeneic DNA immunization against a tumor differentiation antigen.J Immunol. 2003; 170: 5188-5194Crossref PubMed Scopus (91) Google Scholar, 7Hawkins WG et al.Immunization with DNA coding for gp100 results in cd4 t-cell antitumor Full Text Full Text PDF PubMed Scopus Google Scholar, et particle-mediated vaccination against melanoma by of plasmid DNA encoding gp100 and 2001; Google and gene delivery method is to the of gene in the gene expression in PubMed Scopus Google Scholar, the clinically hydrodynamic delivery of plasmid DNA to skeletal Ther. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar We recently a gene method that DNA nucleic a limb vein to and effectively skeletal et al.A method for genes and to skeletal of Ther. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar limb vein as delivery was effective in and animals for the delivery and expression of in limb and HLV delivery is a and that results in or cellular et intravascular limb skeletal a Ther. 13: Full Text Full Text PDF PubMed Scopus Google Scholar and This method of the limb by a in with of a of foreign gene expression in after vein of plasmid Ther. PubMed Scopus Google Scholar HLV gene delivery a clinically hydrodynamic delivery with a potential for as as systemic gene in the clinically hydrodynamic delivery of plasmid DNA to skeletal Ther. 2004; Full Text Full Text PDF PubMed Scopus Google HLV gene we recently that this method is a highly process to animals and for the of humoral against and et immunization for in animals by delivery of plasmid PubMed Scopus Google Scholar In this we HLV gene to the human gp100 xenogeneic DNA cancer vaccine in the murine B16 melanoma We the DNA vaccine was able to immune tolerance and prophylactic the humoral and cellular immune and antitumor of xenogeneic DNA vaccine breaks tolerance and prophylactic antitumor protection against B16 tumor the HLV delivery of a expression to mice could effectively protection against with B16 melanoma cells. Following the HLV delivery of of on and mice were by an of B16 tumor cells on We mice with a B16 tumor that the to mice were protected from the by the genetic in of the mice that the vaccine by HLV delivery tumor genetic vaccination by intravascular hydrodynamic vein a gene delivery with animals that results in expression by of foreign gene expression in after vein of plasmid Ther. PubMed Scopus Google Scholar resulted in the prophylactic protection of of mice that vaccination with the by HLV or hydrodynamic vein tumor development by and to be by to tumor single HLV or hydrodynamic vein delivery of was to induce protection against tumor HLV delivery of the vaccine to effective antitumor protection against tumor tolerance and antitumor protection against the by B16 tumor cells, is more We mice with an increased of of animals that were HLV tumor development with or B16 with of and of mice at mice protection against tumor with of through the of the was against as of mice that were with B16 tumor antitumor of CD8+ T the of genetic vaccination to induce a T-cell response by the of CD8+ T cells. is the epitope the gp100 et is a murine tumor of cells altered Exp Med. 1998; 188: PubMed Scopus Google Scholar and CD8+ T cells with T-cell to the et and of T cells in mice to tumor and PubMed Scopus Google were HLV and after the to peripheral cells for the of T cells. of that of CD8+ T cells were antigen-specific in animals that HLV of of on and in the limb in mice that were and in a of CD8+ T cells We mice in the tumor in HLV vaccine and and demonstrated prophylactic protection from tumor from animals CD8+ T cells In by vaccination with by Y et al.Cloning and characterization of the genes encoding the murine homologues of the human melanoma antigens mart1 and gp100.J Immunother. 1997; 20: 15-25Crossref PubMed Scopus (94) Google Scholar, 7Hawkins WG et al.Immunization with DNA coding for gp100 results in cd4 t-cell antitumor Full Text Full Text PDF PubMed Scopus Google Scholar or et and after of a of Exp Med. 2003; PubMed Scopus Google Scholar to break tolerance and induce antitumor protection against B16 or CD8+ T et and after of a of Exp Med. 2003; PubMed Scopus Google Scholar HLV vaccination with was to and antigen-specific CD8+ T cells, with the of cells to of CD8+ T cells. was of by were with and and by in the the of that for HLV delivery of of in the limb on and HLV delivery of of in and on and from mice that demonstrated protection from B16 tumor after are of from animals from gp100 vaccination induces cytolytic potential of T cells from mice was against tumor in a were from mice and in by with and as in the cytolytic mice that were by of particle-mediated delivery activity against with of HLV vaccination resulted in an response against cells with of In response against targets. of mice were of in the limb on and by HLV of on and by or of on and by were DNA vaccination and in the in as in the and activity of vaccination treatment is as cells. was in In vivo from mice were and peptide-pulsed and as in the in vivo as in the and Vaccinated mice HLV of in the limb months and were with a single HLV delivery of in the and from and and and after of targets. were as in the and in and mice to and to in and of to the mice antitumor activity against and we the of and peptide-pulsed an in vivo that and R in vivo by memory Immunol. 2003; 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PubMed Scopus Google Scholar were from the were and in the with or of in a of at of was splenocytes were for on with or were at a splenocytes were at and cells were adoptively transferred from mice were of and was by was by the of peptide-pulsed in HLV mice the cells were splenocytes with or and the cells were peptide-pulsed and mice were with of or or the of splenocytes from were mice or animals from the in that were of on and and demonstrated protection against tumor and were from mice splenocytes were in a of of by mice were by vein with splenocytes and with tumor cells. mice were by a of of human of of of and on were as et immunization for in animals by delivery of plasmid PubMed Scopus Google Scholar cells from were with to and the cells at cells in murine and of and were in a M M and were in a and transferred to and were at a of polyvalent was at a were developed the and on for results of of are as and are derived from of and are by the of the HLV gene delivery IntroductionAlthough cancer cells are less immunogenic than foreign pathogens, the immune system is clearly capable of recognizing and eliminating tumor cells. Tumor-associated antigens (TAAs) recognized by the immune system are typically self-antigens1Houghton AN Gold JS Blachere NE Immunity against cancer: lessons learned from melanoma.Curr Opin Immunol. 2001; 13: 134-140Crossref PubMed Scopus (171) Google Scholar and are poorly immunogenic as a result of central and peripheral tolerance, a process that prevents the development of autoimmune disorders against “self” molecules. Mutated or altered self (heteroclitic)-antigens are better able to activate T-cell antitumor immunity through the formation of a more sustained major histocompatibility complex/altered self-antigen complex on the surface of professional antigen-presenting cells, such as dendritic cells.2Dyall R et al.Heteroclitic immunization induces tumor immunity.J Exp Med. 1998; 188: 1553-1561Crossref PubMed Scopus (187) Google Scholar This effectively breaks immunological tolerance to the self antigen. One form of presenting the immune system with an altered self antigen is through the expression of a closely related ortholog derived from a different species.3Srinivasan R Wolchok JD Tumor antigens for cancer immunotherapy: Therapeutic potential of xenogeneic DNA vaccines.J Transl Med. 2004; 2: 12Crossref PubMed Scopus (41) Google Scholar In the B16 murine melanoma model, vaccination with xenogeneic human gp100 (hgp100) by viral vector,4Zhai Y et al.Cloning and characterization of the genes encoding the murine homologues of the human melanoma antigens mart1 and gp100.J Immunother. 1997; 20: 15-25Crossref PubMed Scopus (94) Google Scholar direct intramuscular injection,5Tanaka M et al.Induction of a systemic immune response by a polyvalent melanoma-associated antigen DNA vaccine for prevention and treatment of malignant melanoma.Mol Ther. 2002; 5: 291-299Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar or particle-mediated (i.e., gene gun) plasmid DNA (pDNA) delivery induced antitumor protection against B16 tumor challenge.4Zhai Y et al.Cloning and characterization of the genes encoding the murine homologues of the human melanoma antigens mart1 and gp100.J Immunother. 1997; 20: 15-25Crossref PubMed Scopus (94) Google Scholar, 6Gold JS et al.A single heteroclitic epitope determines cancer immunity after xenogeneic DNA immunization against a tumor differentiation antigen.J Immunol. 2003; 170: 5188-5194Crossref PubMed Scopus (91) Google Scholar, 7Hawkins WG et al.Immunization with DNA coding for gp100 results in cd4 t-cell antitumor Full Text Full Text PDF PubMed Scopus Google Scholar, et particle-mediated vaccination against melanoma by of plasmid DNA encoding gp100 and 2001; Google and gene delivery method is to the of gene in the gene expression in PubMed Scopus Google Scholar, the clinically hydrodynamic delivery of plasmid DNA to skeletal Ther. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar We recently a gene method that DNA nucleic a limb vein to and effectively skeletal et al.A method for genes and to skeletal of Ther. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar limb vein as delivery was effective in and animals for the delivery and expression of in limb and HLV delivery is a and that results in or cellular et intravascular limb skeletal a Ther. 13: Full Text Full Text PDF PubMed Scopus Google Scholar and This method of the limb by a in with of a of foreign gene expression in after vein of plasmid Ther. PubMed Scopus Google Scholar HLV gene delivery a clinically hydrodynamic delivery with a potential for as as systemic gene in the clinically hydrodynamic delivery of plasmid DNA to skeletal Ther. 2004; Full Text Full Text PDF PubMed Scopus Google HLV gene we recently that this method is a highly process to animals and for the of humoral against and et immunization for in animals by delivery of plasmid PubMed Scopus Google Scholar In this we HLV gene to the human gp100 xenogeneic DNA cancer vaccine in the murine B16 melanoma We the DNA vaccine was able to immune tolerance and prophylactic the humoral and cellular immune and antitumor