Hydrodynamic Limb Vein Delivery of a Xenogeneic DNA Cancer Vaccine Effectively Induces Antitumor Immunity
Authors: Zane C Neal, Mary K Bates, Mark R Albertini, Hans Herweijer
Journal: Molecular Therapy (2007)

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 independent antitumor immunity.Surgery. 2000; 128: 273-280Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 8Rakhmilevich AL et al.Effective particle-mediated vaccination against mouse melanoma by coadministration of plasmid DNA encoding gp100 and granulocyte-macrophage colony-stimulating factor.Clin Cancer Res. 2001; 7: 952-961PubMed Google ScholarA safe and efficient gene delivery method is paramount to the successful application of promising gene therapy strategies in the clinical setting.9Glover DJ Lipps HJ Jans DA Towards safe, non-viral therapeutic gene expression in humans.Nat Rev Genet. 2005; 6: 299-310Crossref PubMed Scopus (499) Google Scholar, 10Wells DJ Opening the floodgates: clinically applicable hydrodynamic delivery of plasmid DNA to skeletal muscle.Mol Ther. 2004; 10: 207-208Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar We recently described a non-viral gene transfer method that hydrodynamically delivers DNA (or other nucleic acids) intravascularly via a limb vein to reliably and effectively transfect skeletal muscle tissue.11Hagstrom JE et al.A facile nonviral method for delivering genes and sirnas to skeletal muscle of mammalian limbs.Mol Ther. 2004; 10: 386-398Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar Hydrodynamic limb vein (HLV, trademarked as Pathway IV) delivery was shown equally effective in small and large animals for the delivery and expression of pDNA in limb myofibers, including mice, rats, dogs, and monkeys. HLV delivery is a simple and repeatable procedure that results in minimal myofiber damage or cellular infiltrate,12Toumi H et al.Rapid intravascular injection into limb skeletal muscle: a damage assessment study.Mol Ther. 2006; 13: 229-236Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar while efficiently expressing secreted and non-secreted transgene products. This method involves transient isolation of the limb vasculature by a tourniquet in combination with rapid intravenous injection of a relatively high-volume pDNA solution.13Zhang G Budker V Wolff JA High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA.Hum Gene Ther. 1999; 10: 1735-1737Crossref PubMed Scopus (813) Google Scholar Importantly, HLV gene delivery represents a clinically applicable hydrodynamic delivery technology with a potential for loco-regional as well as systemic gene therapy utility in patients.10Wells DJ Opening the floodgates: clinically applicable hydrodynamic delivery of plasmid DNA to skeletal muscle.Mol Ther. 2004; 10: 207-208Abstract Full Text Full Text PDF PubMed Scopus (23) Google ScholarEmploying HLV gene delivery, we recently showed that this method is a highly efficient process to genetically immunize research animals (mice, rats, and rabbits) for the induction of high-titer humoral responses against secreted and non-secreted transgene products.14Bates MK et al.Genetic immunization for antibody generation in research animals by intravenous delivery of plasmid DNA.Biotechniques. 2006; 40: 199-208Crossref PubMed Scopus (29) Google Scholar In this study, we evaluated HLV gene transfer technology to deliver the human gp100 xenogeneic DNA cancer vaccine in the murine B16 melanoma model. We tested whether the HLV-delivered hgp100 DNA vaccine was able to overcome immune tolerance and elicit prophylactic protection, characterize the humoral and cellular immune responses, and assess long-term antitumor memory.ResultsHLV-delivery of hgp100 xenogeneic DNA vaccine breaks tolerance and provides prophylactic antitumor protection against B16 tumor challengeWe assessed whether the HLV delivery of a hgp100 expression vector to mice could effectively confer protection against challenge with B16 melanoma cells. Following the HLV delivery of 25 μg of pCMV-hgp100 on days 0, 10, and 20, mice were challenged by an intradermal (i.d.) injection of 104 B16 tumor cells on day 30. We initially challenged vaccinated mice with a B16 variant tumor cell that stably expresses the hgp100 molecule (B16-hgp100), to determine whether mice were protected from tumors expressing the cognate immunogen delivered by the genetic vaccine. As shown in Figure 1a, 100% of the mice that received the hgp100 vaccine by HLV delivery lacked B16-hgp100 tumor growth. Similarly, genetic vaccination by intravascular hydrodynamic tail vein injection, a gene delivery procedure used with research animals that results in transgene expression predominantly by hepatocytes,13Zhang G Budker V Wolff JA High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA.Hum Gene Ther. 1999; 10: 1735-1737Crossref PubMed Scopus (813) Google Scholar resulted in the prophylactic protection of 75% of hgp100-vaccinated mice. Alternatively, all control mice that received vaccination with the luciferase (luc)-expressing plasmid, pCMV-Luc, by HLV or hydrodynamic tail vein delivery, exhibited demonstrable B16-hgp100 tumor development by days 9–12 and had to be sacrificed by days 22 owing to progressive tumor growth. A single HLV or hydrodynamic tail vein delivery of hgp100 pDNA was unable to induce protection against tumor challenge (not shown).Although HLV delivery of the hgp100 pDNA vaccine appears sufficient to elicit effective antitumor protection against B16-hgp100 tumor challenge (Figure 1a), overcoming tolerance and mediating antitumor protection directed against the mgp100 self-TAA, constitutively expressed by B16 tumor cells, is considered immunologically more challenging. We therefore vaccinated mice with an increased dose of 50 μg of pDNA per delivery. Control animals that were HLV luc-vaccinated exhibited progressive tumor development when challenged with B16-hgp100 or B16 tumors (Figure 1b), with only 1 of 13 B16-hgp100-challenged and two of 13 B16-challenged mice remaining tumor-free at day 21. As before, hgp100-vaccinated mice exhibited protection against B16-hgp100 tumor challenge with 13 of 13 (100%) remaining tumor-free through the duration of the experiment (day 32). Tolerance was broken against mgp100, as 13 of 13 hgp100-vaccinated mice that were challenged with B16 tumor also exhibited complete antitumor protection.Vaccination promotes expansion of gp100-specific CD8+ T cellsWe evaluated the ability of HLV-delivered hgp100 genetic vaccination to induce a gp100-specific T-cell response by determining the frequency of hgp10025−33/H2Db-tetramer+ CD8+ T cells. The gp10025−33 peptide sequence is the most well-characterized H2Db-restricted epitope within the gp100 molecule.15Overwijk WW et al.Gp100/pmel 17 is a murine tumor rejection antigen: induction of “self”-reactive, tumoricidal t cells using high-affinity, altered peptide ligand.J Exp Med. 1998; 188: 277-286Crossref PubMed Scopus (403) Google Scholar The hgp10025−33/H2Db-tetramer binds and identifies CD8+ T cells with T-cell receptor specificity to the gp10025−33 epitope.16Wang LX et al.Interleukin-7-dependent expansion and persistence of melanoma-specific T cells in lymphodepleted mice lead to tumor regression and editing.Cancer Res. 2005; 65: 10569-10577Crossref PubMed Scopus (77) Google ScholarMice were HLV vaccinated and bled 10 days after the final pDNA administration to obtain peripheral blood mononuclear cells (PBMCs) for the identification of gp100-specific T cells. Tetramer analysis of group-pooled PBMCs indicated that 2.6% of CD8+ T cells were gp10025−33 antigen-specific in animals that received two HLV deliveries of 50 μg of hgp100 pDNA on days 0 and 10 in the right hind limb (Figure 2b), versus 0.5% in unvaccinated control mice (Figure 2a). Mice that were twice vaccinated (days 0 and 10) in both hind limbs exhibited a frequency of 4.4% hgp10025−33-specific CD8+ T cells (Figure 2c). We also evaluated mice used in the tumor challenge experiment shown in Figure 1b, which had received three HLV hgp100 vaccine deliveries (days 0, 10, and 20) and had demonstrated complete prophylactic protection from B16-hgp100 tumor challenge. The PBMCs from these animals had 4.6% hgp10025−33-specific CD8+ T cells (Figure 2d). In studies by others, vaccination with mgp100, by DNA4Zhai 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 independent antitumor immunity.Surgery. 2000; 128: 273-280Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar or peptide,17Overwijk WW et al.Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive cd8+ t cells.J Exp Med. 2003; 198: 569-580Crossref PubMed Scopus (754) Google Scholar failed to break tolerance and induce antitumor protection against B16 tumors or expand gp10025−33/H2Db-tetramer+ CD8+ T cells.17Overwijk WW et al.Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive cd8+ t cells.J Exp Med. 2003; 198: 569-580Crossref PubMed Scopus (754) Google Scholar HLV vaccination with pCMV-mgp100 pDNA was similarly unable to stimulate and expand gp10025−33 antigen-specific CD8+ T cells, with the frequency of tetramer+ cells similar to controls (not shown).Figure 2Detection of gp100-specific CD8+ T cells. Group-pooled (n=3) blood was depleted of erythrocytes by hypotonic shock before labeling. PBMCs were two-color labeled with anti-murine CD8-FITC and hgp10025−33/H2Db-tetramer-phycoerythrin and analyzed by flow cytometry. Values in the upper-right quadrant indicate the percentage of total viable CD8-staining PBMCs that stain positive for hgp10025−33/H2Db-tetramer. (a) Untreated control mice. (b) HLV delivery of 50 μg of pCMV-hgp100 in the right hind limb on days 0 and 10. (c) HLV delivery of 50 μg of pCMV-hgp100 in both right and left hind limbs on days 0 and 10. (d) Positive control PBMCs from hgp100 HLV-vaccinated mice that demonstrated protection from B16 tumor challenge (Figure 1b) collected 15 days after challenge. Results are representative of three experiments, two from studies not involving animals from Figure 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)HLV gp100 vaccination induces CTL activityThe cytolytic potential of splenic T cells from hgp100-vaccinated mice was tested against B16-hgp100 tumor targets in a standard 51Cr-release assay (Figure 3a). Splenocytes were harvested from vaccinated mice and activated in vitro by co-culture with low-dose IL2 and mitomycin-treated B16-hgp100 stimulators, before use as effectors in the cytolytic T-lymphocyte (CTL) assay. Control mice that were pCMV-Luc-vaccinated by two applications of particle-mediated delivery had negligible CTL activity against B16-hgp100 targets with only 275 lytic units (LU) of activity. However, HLV hgp100 pDNA vaccination resulted in an enhanced CTL response against B16-hgp100 cells with 1,350 LU of activity.Figure 3CTL activity. (a) In vitro response against B16-hgp100 targets. Groups of C57BL/6 mice (n=4) were vaccinated with: 50 μg of pCMV-hgp100 in the right hind limb on days 0, 14, and 28 by HLV (Pathway IV) delivery; 1 μg of pCMV-hgp100 on days 0 and 28 by Gene gun delivery; or 1 μg of pCMV-Luc (luc) on days 0 and 28 by Gene gun delivery. Group-pooled spleens were harvested 10 days following final DNA vaccination and used in the in vitro cytotoxicity assay as described in the Materials and Methods section. Cytolytic activity of each vaccination treatment group is expressed as lytic units per 107 effector cells. Assay was performed in quadruplicate wells. (b–d) In vivo cytoxicity assay. Splenocytes from naïve C57BL/6 mice were CFSE-labeled and peptide-pulsed and used as targets in the in vivo cytotoxicity assay as described in the Materials and Methods section. Vaccinated recipient mice had received three HLV deliveries 50 μg of pCMV-hgp100 in the right hind limb three months earlier, and were boosted with a single 50 μg HLV delivery of pCMV-hgp100 in both hind limbs 10 days before the cytotoxity assay. Flow cytometric histograms depict three viable CFSE-labeled target populations (M1, low; M2, intermediate; and M3, high) from naïve (b and d) and hgp100-vaccinated (c and e) recipient spleens 20 h after i.v. injection of targets. Target populations were peptide-pulsed, as indicated in the figure, and used in paired comparisons between naïve and vaccinated recipient mice (b compared to c, and d compared to e). Percent values in c and e represent specific cytotoxicity of target populations relative to the Flu-pulsed control population.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To assess whether hgp100-vaccinated mice exhibited antitumor activity against hgp100 and mgp100-expressing targets, we evaluated the killing of hgp10025−33 and mgp10025−33 peptide-pulsed targets using an in vivo cytotoxicity assay that compares target cell survival ratios between vaccinated and naïve animals.18Barber DL Wherry EJ Ahmed R Cutting edge: rapid in vivo killing by memory cd8 t cells.J Immunol. 2003; 171: 27-31Crossref PubMed Scopus (352) Google Scholar HLV hgp100 DNA-vaccinated mice received an additional HLV delivery (i.e., boost) of 50 μg of hgp100 pDNA in both hind limbs 10 d before the assay. Naïve C57BL/6 splenocytes were used as targets and vital dye labeled with various concentrations of carboxyfluorescein succinimidyl ester (CFSE) and peptide-pulsed with the Db-restricted epitopes from hgp10025−33 (KVPRNQDWL), mgp10025−33 (EGSRNQDWL), the influenza matrix protein (Flu: ASNENMETM), or left unpulsed before mixing and injection into naïve and hgp100-vaccinated recipient mice. Tetramer analyses of individual hgp100-vaccinated recipient mice 24 h before assay indicated that 12–16% of CD8+ T cells from PBMCs were hgp10025−33 antigen-specific (not shown). Splenocytes from recipient mice were harvested 20 h later and analyzed by flow cytometry for the frequency of each of the three (low, medium, and high) CFSE-labeled population of target cells. Flu-peptide-pulsed targets were considered the control target population. When comparing the killing of hgp10025−33 peptide-pulsed relative to Flu peptide-pulsed targets in naïve (Figure 3b) and hgp100-vaccinated/boosted (Figure 3c) mice, 67% of hgp100-pulsed targets were killed in the vaccinated/boosted recipient. Comparing hgp10025−33 and mgp10025−33 peptide-pulsed targets in naïve (Figure 3d) and vaccinated/boosted (Figure 3e) recipients showed that hgp100 and mgp100 peptide-pulsed targets were susceptible, with 88 and 56% killing observed, respectively.Splenocytes can transfer HLV vaccine-induced immunityGenetic vaccination of mice with hgp100 results in a T-cell-dependent antitumor response mediated predominantly by CD8+ T cells.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, 7Hawkins WG et al.Immunization with DNA coding for gp100 results in cd4 t-cell independent antitumor immunity.Surgery. 2000; 128: 273-280Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 19Yang S et al.Murine dendritic cells transfected with human gp100 elicit both antigen-specific cd8(+) and cd4(+) t-cell responses and are more effective than DNA vaccines at generating anti-tumor immunity.Int J Cancer. 1999; 83: 532-540Crossref PubMed Scopus (50) Google Scholar Adoptive transfer of splenocytes20Schreurs MW de Boer AJ Figdor CG Adema GJ Genetic vaccination against the melanocyte lineage-specific antigen gp100 induces cytotoxic t lymphocyte-mediated tumor protection.Cancer Res. 1998; 58: 2509-2514PubMed Google Scholar or T cells15Overwijk WW et al.Gp100/pmel 17 is a murine tumor rejection antigen: induction of “self”-reactive, tumoricidal t cells using high-affinity, altered peptide ligand.J Exp Med. 1998; 188: 277-286Crossref PubMed Scopus (403) Google Scholar from hgp100 DNA-vaccinated mice can confer antitumor immunity to naïve recipient mice. To determine the relative contributions of cell-mediated immunity and humoral immunity to the antitumor response observed in HLV hgp100-vaccinated mice, we performed adoptive and passive transfer studies.Naïve recipient mice were B16-hgp100 tumor-challenged 24 h following an adoptive or passive transfer procedure. B16-hgp100, and not B16, was chosen as the challenging tumor agent to ensure possible detection of suboptimal cellular or humoral antitumor immunity that may exist in recipient animals, which might otherwise be missed by B16 tumor challenge. Recipients received splenocytes, sera, or both splenocytes and sera from HLV hgp100-vaccinated donor mice that had demonstrated protection against B16-hgp100 tumor challenge. Control animals received splenocytes and sera from non-vaccinated donors, and exhibited progressive B16-hgp100 tumor growth (Figure 4a). Likewise, passive transfer of sera from hgp100-vaccinated mice did not protect against challenge (Figure 4d). Conversely, adoptive transfer of immune splenocytes resulted in a significant (P=0.0142, day 22) delay in tumor progression compared to mice that received non-immune splenocytes and sera (Figure 4c). Interestingly, mice that received cells and sera from vaccinated mice had the highest level of protection (Figure 4b), with two of four mice being completely protected from challenge and another animal exhibiting a very protracted delay in tumor progression.Figure 4Transfer of immunity. Groups (n=4) of naïve C57BL/6 recipient mice were injected with 500 μl undiluted sera or 125 × 106 splenocytes, or the combination of sera and splenocytes, from naïve or hgp100 HLV-vaccinated donor mice. Vaccinated donor animals are from the study in Figure 1b that demonstrated protection against B16-hgp100 challenge. Group (a) received both sera and splenocytes from non-vaccinated donors; group (b) received both sera and splenocytes from hgp100-vaccinated donors; group (c) received splenocytes only from hgp100-vaccinated donors; and group (d) received sera only from hgp100-vaccinated donors. Mice were challenged with 5 × 104 B16-hgp100 tumor cells 24 h following transfer procedure. All recipient mice were treated by a daily i.p. injection of 3 × 105 IU of recombinant human IL2 on days 6–9 (arrows). Data indicate B16-hgp100 i.d. tumor growth on individual animals. The vertical lines between a and c, and b and d at day 20 aids in visual comparison of tumor growth between treatment groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Vaccination facilitates development of an anti-gp100 humoral responseAs the HLV delivery procedure is highly effective at eliciting an anti-transgene humoral response,14Bates MK et al.Genetic immunization for antibody generation in research animals by intravenous delivery of plasmid DNA.Biotechniques. 2006; 40: 199-208Crossref PubMed Scopus (29) Google Scholar we assessed whether this was also the case for HLV-delivered hgp100 vaccine. Mice that were HLV hgp100-vaccinated and tested for protection in tumor challenge studies with B16 (Figure 1b) were bled 28 days following the final vaccination procedure. The sera were tested for binding to B16-hgp100, B16, and NXS2 murine neuroblastoma cells in Western blot and flow cytometric analyses (Figure 5a, b, and c, respectively). The gp100 molecule is a ∼100 kDa glycosylated protein that is cleaved into a small (∼26 kDa) carboxy-terminal fragment and a larger amino-terminal section (60–64 kDa), which is subsequently cleaved to generate ∼26 kDa and ∼34–38 kDa fragments. The latter fragment is recognized by the HMB45 monoclonal Ab (mAb).21Yasumoto K et al.Epitope mapping of the melanosomal matrix protein gp100 (pmel17): rapid processing in the endoplasmic reticulum and glycosylation in the early golgi compartment.J Biol Chem. 2004; 279: 28330-28338Crossref PubMed Scopus (60) Google Scholar Analysis using HMB45 mAb against B16-hgp100 (Figure 5a, lane 5) shows the intense labeling of bands ∼38–64 kDa that represent the processed hgp100 fragments. Two bands are noted with B16 cells at the ∼60+ kDa region (lane 4), which represent fragments of the mgp100 molecule. hgp100-Transfected COS-7 cells (lane 2) exhibit two bands: the ∼100 kDa full-length hgp100 molecule and a ∼80 kDa cleavage product. Sera from hgp100-vaccinated mice also identified a distinct band at the ∼100 kDa position with B16-hgp100 cells (Figure 5b, lane 5). Additional bands were also recognized at the ∼38–64 kDa positions, as similarly identified with HMB45 (Figure 5a, lane 5). Likewise, a faint ∼100 kDa band is identified with B16 cells (lane 4), suggesting cross-reactivity of the hgp100 vaccine-induced antibodies (Abs) with the mgp100 full-length molecule expressed by B16 cells. The identification of numerous bands by post-hgp100-vaccinated sera against hgp100-transfected COS-7 cells (Figure 5b, lane 2) demonstrates that HLV hgp100 vaccination elicits a polyclonal anti-hgp100 Ab response against additional B-cell determinants not recognized by HMB45 Ab. As expected, the NXS2 murine neuroblastoma cells showed no detection of gp100 by HMB45 (Figure 5a, lane 3) or post-immune sera (Figure 5b, lane 3).Figure 5Detection of anti-gp100 Ab. Western blot detecting gp100 protein in cell extracts probed with (a) anti-hgp100 HMB45 mAb or (b) pooled sera from hgp100 HLV-vaccinated mice. Blots contain lysates from: COS-7 (lane 1); pCMV-hgp100-transfected COS-7 (lane 2); NXS2 murine neuroblastoma (lane 3); B16 (lane 4); and B16-hgp100 (lane 5) cells. Both blots were probed with a secondary horseradish peroxidase-conjugated polyvalent anti-mouse immunoglobulin Ab. (c) Detection of gp100 on the surface of B16-hgp100 cells. (Upper panel) B16-hgp100 cells were incubated with commercially available anti-hgp100 HMB45 mAb and stained with goat-anti-mouse immunoglobulin G-FITC-conjugated Ab (GAM-FITC) (light gray histogram), or stained with GAM-FITC alone (open histogram). (Middle panel) B16-hgp100 cells were incubated with pooled sera obtained from hgp100 HLV-vaccinated mice 28 days after final vaccination. Sera were diluted in phosphate-buffered saline-2% fetal calf sera and tested at 1:10 (light histogram), 1:100 (medium histogram), and 1:1,000 (dark histogram) dilution, and stained with GAM-FITC. (Lower panel) B16-hp100 incubated with pooled non-vaccinated mouse sera at 1:10 (light histogram) and 1:100 (medium histogram) dilution. Data presented as flow cytometric histograms gated on viable B16-hgp100 cells. MFI values are indicated for each histogram.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The same post-hgp100-vaccinated sera were also analyzed by flow cytometry. B16-hgp100 cells demonstrated cell-surface binding of the control anti-hgp100 mAb, HMB45, with a specific mean fluorescence intensity (MFI) ratio of 7.2 (Figure 5c, top panel). Post-hgp100-vaccinated sera was tested for binding to B16-hgp100 cells at 1:10, 1:100, and 1:1,000 dilutions and exhibited specific MFI ratio values of 26.1, 4.0, and 1.9, respectively (Figure 5c, middle panel). Control analysis with sera from non-vaccinated mice indicated background binding to B16-hgp100 cells with specific MFI ratio of 3.1 at the 1:10 sera dilution (Figure 5c, lower panel).Vaccinated mice exhibit gp100-specific CD8+ T-cell memoryThe development of long-lived CD8+ T-cell memory is a major goal of cancer immunotherapy, as it is a pivotal component in providing continuous protection from further tumor dissemination and recurrence.22Finn OJ Cancer vaccines: between the idea and the reality.Nat Rev Immunol. 2003; 3: 630-641Crossref PubMed Scopus (508) Google Scholar Successfully