Genetic Immunization With In Vivo Dendritic Cell-targeting Liposomal DNA Vaccine Carrier Induces Long-lasting Antitumor Immune Response

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1 originl rticle Genetic Immuniztion With In Vivo Dendritic Cell-trgeting Liposoml DNA Vccine Crrier Induces Long-lsting Antitumor Immune Response Arup Gru 1, Gopikrishn Moku 1,2, Suresh Kumr Gull 1,2 nd Arind Chudhuri 1,2 1 Biomterils Group, CSIR Indin Institute of Chemicl Technology, Hyderd, Telngn, Indi; 2 Acdemy of Scientific nd Innovtive Reserch (AcSIR), Chenni, Tmil Ndu, Indi A mjor limiting fctor retrding the clinicl success of dendritic cell (DC)-sed genetic immuniztions (DNA vccintion) is the scrcity of iologiclly sfe nd effective crrier systems for trgeting the ntigen-encoded DNA vccines to DCs under in vivo settings. Herein, we report on potent, mnnose receptor selective in vivo DC-trgeting liposomes of novel ctionic mphiphile with mnnose-mimicking shikimoyl hed-group. Flow cytometric experiments with cells isolted from drining lymph nodes of mice s.c. immunized with lipoplexes of pgfp plsmid (model DNA vccine) using nti-cdc ntiody-leled mgnetic eds reveled in vivo DC-trgeting properties of the presently descried liposoml DNA vccine crrier. Importntly, s.c. immuniztions of mice with electrosttic complex of the in vivo DC-trgeting liposome nd melnom ntigen-encoded DNA vccine (p-cmv-mart1) induced long-lsting ntimelnom immune response (1 dys post melnom tumor chllenge) with remrkle memory response (more thn 6 months fter the second tumor chllenge). The presently descried direct in vivo DC-trgeting liposoml DNA vccine crrier is expected to find future exploittions towrd designing effective vccines for vrious infectious diseses nd cncers. Received 1 April 215; ccepted 22 Novemer 215; dvnce online puliction 19 Jnury 216. doi:1.138/mt INTRODUCTION Dendritic cells (DCs), ody s most professionl ntigen-presenting cells, possess the unique ility of cpturing nd processing pthogenic ntigens in the peripherl lood nd tissues. The ntigen-loded DCs migrte through fferent lymphtics to the nery drining lymph nodes where they present the processed ntigen frgments in complextion with oth clssicl mjor histocomptiility complexes (MHC clss I nd II) nd nonclssicl (CD1 fmily) ntigen-presenting molecules to the resting T lymphocytes. 1 3 Becuse of such distinguishing ntigen-presenting ility of the DCs, DCs pulsed/trnsduced with tumor-ssocited or virl ntigens re finding incresing pplictions s vccines for cncer nd infectious diseses. DCs re often ex vivo trnsfected with tumor/virl ntigens-encoded DNA vccines. 4 9 Such ex vivo DC trnsfection-sed genetic immuniztion protocols, lthough highly efficient in comting cncer, re lor-intensive, time-consuming, nd expensive. Autologous DC precursors re pinstkingly isolted, the isolted utologous DC precursors re then ex vivo trnsfected with DNA vccines, nd the ex vivo trnsfected DCs finlly need to e reimplnted in recipient s ody for mounting immune response. To this end, oth virl nd nonvirl vectors re now eing used for direct in vivo trgeting of DNA vccines to DCs However, chieving long-lsting immunity through use of simple nd cost-effective in vivo DC-trgeting system remins formidle chllenge. Previously, we reported tht mnnose receptor selective liposomes of ctionic mphiphiles contining two liphtic n-hexdecyl nonpolr tils nd mnnose-mimicking shikimoyl- nd quinoyl- hed-groups with lysine spcer in etween re efficient DNA vccine crriers for ex vivo DC trnsfectionsed genetic immuniztion. 9 These priorly reported systems were found to e efficient in inducing long-lsting immune response ginst melnom in mice immunized with DCs ex vivo trnsfected with lipoplexes of melnom ntigen-encoded DNA vccines. 9 However, s descried elow, the system filed in mounting long-lsting immune response ginst melnom when used under direct in vivo DC-trgeting mode. We envisged tht the DC trnsfection efficiency of this new clss of mnnose receptor selective lipids contining mnnose-mimicking shikimoyl- nd quinoyl- hed-groups need to e further enhnced for mking their liposomes effective in trgeting DNA vccines to DCs under in vivo settings. With such rtionle in mind, in the present study, we chemiclly trnsformed the lysine side chin mino group into the trnsfection enhncing gunidine group. Herein, we show tht liposomes of the ctionic mphiphile contining mnnose-mimicking shikimoyl hed-group nd two n-hexdecyl hydrophoic tils cn trget DNA vccines to DCs under in vivo settings when the side chin mino group of the lysine spcer is gunidinylted (lipid 1, Figure 1). We show tht direct in vivo immuniztion (s.c.) of mice with electrosttic complex of the liposome of lipid 1 nd The first two uthors re co-first uthors nd contriuted eqully to this work. Correspondence: Arind Chudhuri, Biomterils Group, CSIR Indin Institute of Chemicl Technology, Trnk, Hyderd, Telngn 5 7, Indi. E-mil: rind@iict.res.in Moleculr Therpy vol. 24 no. 2, fe

2 In Vivo DC-trgeted Genetic Immuniztion The Americn Society of Gene & Cell Therpy chrge rtios t 4:1) were found to e within the rnge of nm (Supplementry Figure S). Lipid 2 Lipid 3 Figure 1 Structures of ctionic mphiphiles with mnnose-mimicking shikimoyl- (lipid 1) nd quinoyl- (lipid 2) hed-groups nd their mnnosyl nlog (lipid 3) used in the present study. melnom ntigen-encoded DNA vccine (p-cmv-mart1) induces long-lsting ntimelnom immune response (1 dys post melnom tumor chllenge) with remrkle memory response (more thn 6 months fter the second tumor chllenge). With the vilility of the presently descried ctionic lipid 1, overcoming the formidle chllenge of inducing longlsting immune response through direct in vivo trgeting of tumor ntigen-encoded DNA vccines to DCs will now ecome fesile. The presently descried direct in vivo DC-trgeting liposoml DNA vccine crriers re thus expected to find future pplictions in effective vccine developments for vrious infectious diseses nd cncers. RESULTS Chemistry The ctionic lipids 1 nd 2 (Figure 1) contining gunidinylted lysine spcer etween the hydrophoic tils nd mnnose-mimicking shikimoyl- nd quinoyl hed-groups s well s their mnnosyl nlog lipid 3 (Figure 1) were synthesized y conventionl peptide coupling of the cetyl protected shikimic, quinic cids nd mnnose to ppropritely derivtized lysinylted mphiphiles followed y quterniztion, deprotections, nd chloride ion exchnge (Supplementry Schemes S1 S3). The detils of synthetic schemes, procedures, nucler mgnetic resonnce (NMR), nd mss spectrl dt for lipids 1 3 s well s the highperformnce liquid chromtogrphy (HPLC) profiles of the purified lipids 1 3 in two different moile phses re provided in the Supplementry Informtion (Supplementry Figures S1 S9. Sizes of the liposomes nd lipoplexes Hydrodynmic dimeters (zet sizes) of the liposoml formultions were mesured y dynmic light scttering technique. The sizes of the liposoml formultions of lipids 1 3 were within the rnge of nm (Supplementry Figure S1) nd those for the lipoplexes of lipids 1 3 nd p-cmv-mart1 (lipid:dna In vitro DC trnsfection properties of lipids 1 3 Primry mmdcs (mouse one mrrow-derived DCs) were isolted from one mrrows in the tiis nd fiuls of mle C57BL/6J mice s descried previously. 17 Distinguishing DC surfce mrkers (including MHC II, totl MHC II, mnnose receptors, CDc, CD86, H2K, nd CD4) in the isolted mmdcs were confirmed y flow cytometry (Supplementry Figure S). First, we evluted DC trnsfection properties of the liposomes of lipids 1 3 y flow cytometry using GFP plsmid (s model DNA vccine). First we trnsfected mmdcs with GFP plsmid complexed with liposomes of lipids 1 3 nd equimolr 1,2-dioleyol-sn-glycero-3-phosphoethnolmine (DOPE; s co-lipid) t ctionic lipid:dna chrge rtio of 4:1 (initil trnsfection experiments cross rnge of lipid:dna chrge rtios reveled 4:1 to e the most optiml lipid:dna chrge rtio, dt not shown). Trnsfected cells were visulized y fluorescence microscopy (Figure 2) nd the reltive trnsfection efficiencies of lipids 1 3 were mesured y flow cytometry (Figure 2c). In such flow cytometric mesurements of trnsfection efficiency, the utofluorescence intensities of the untreted control cells re sutrcted from the fluorescence intensities of the treted cells. The lipoplexes of lipid 1 with mnnose-mimicking shikimoyl hed-group ws found to e the most efficient with ~2% DC trnsfection efficiency, followed y liposomes of lipids 2 nd 3 with ~% nd ~5% DC trnsfection efficiencies, respectively (Figure 2c). Commercilly ville LipofectAmine 2 ws found to e very poor in trnsfecting mmdcs (Figure 2,c). Mnnose receptor selective DC trnsfection properties of lipids 1 3 were significntly ffected when mmdcs were preincuted with 1 mg/ml mnnn, nturl lignd for mnnose receptor (Figure 2,d), which supports the notion tht the DC trnsfection y lipids 1 3 re medited vi mnnose receptor. Furthermore, towrd ddressing concentrtion-dependent inhiition y mnnn, we evluted the DC trnsfection efficiencies of lipids 1 3 using DCs preincuted with incresing mnnn concentrtions cross the mg/ml. The DC trnsfection efficiencies were dversely ffected with incresing concentrtions of mnnn (Supplementry Figure S13). Importntly, the DC trnsfection properties of lipids 1 3 were not significntly ffected when DCs were preincuted with lminrin, commercilly ville polyscchride of glucose (Supplementry Figure S13). Thus, the findings summrized in Figure 2 nd the Supplementry Figure S13, convincingly demonstrted mnnose receptor-medited DC trnsfection efficcies of ctionic mphiphiles with mnnose-mimicking shikimoyl nd quinoyl- hed-groups. In vivo DC trnsfection properties of lipids 1 3 After confirming the mnnose receptor-specific in vitro DC trnsfection efficiencies of lipids 1 nd 2, we mesured the efficiencies of the liposoml formultions of lipids 1 3 (contining equimolr DOPE) in trgeting DNA vccines to DCs under in vivo conditions using p-cmv-gfp s model for DNA vccine. The lipoplexes (liposome:dna complexes) of lipids vol. 24 no. 2 fe. 216

3 In Vivo DC-trgeted Genetic Immuniztion Bright Green Merge Bright Green Merge Pre-st. mnnn + Lipid 2 Pre-st. mnnn +Lipid 2 Lipid 3 Pre-st. mnnn +Lipid 3 Lipofect. 2k Pre-st. mnnn +Lipofect. 2k c Lipid 2 d Pre-st. mnnn + Lipid Pre-st. mnnn + Lipid Lipid Lipofect. 2K Pre-st. Pre-st. 33 mnnn 32 mnnn + + Lipid3 Lipofect K Figure 2 with the mnnose-mimicking shikimoyl hed-group is the most efficient mong the lipids 1 3 in trnsfecting dendritic cells (DCs) in vitro. () Fluorescence imges of DCs trnsfected with the lipoplexes of lipids 1 3. () Flourescence imges of DCs trnsfected with the lipoplexes of lipids 1 3 in the presence of mnnn (1 mg/ml). (c) Efficiencies of the lipoplexes of lipids 1 3 in trnsfecting mmdcs mesured y flow cytometry. (d) Efficiencies of the lipoplexes of lipids 1 3 in trnsfecting mmdcs presturted with mnnn (1 mg/ml) mesured y flow cytometry. In ech of these trnsfection experiments, ~5 1 5 cells were used nd the cells were trnsfected with lipoplexes of lipids 1 3 nd α5-gfp plsmids contining lipid:dna chrge rtios of 4:1. The degrees of GFP expression in trnsfected mmdcs were visulized using epifluorescence microscope nd quntified y flow cytometry. Trnsfection efficiencies in ech cse were lso mesured using GFP lipoplexes of the commercilly ville liposoml trnsfection kit, LipofectAmine 2 (extreme right pnels). Ech experiment ws repeted three times nd the trnsfection profiles were found to e similr in ech time. were injected s.c. into C57BL/6J mice. Twenty-four hours fter injection, the percentges of GFP-positive cells in the CDc + cells were isolted from the lymph nodes y using nti-cdc ntiody-leled mgnetic eds s descried previously. 18 Mice injected with only vehicles (5% glucose solution) were used s control. The findings in the flow cytometric study reveled significntly higher numer of GFP-positive cells (~15%) in the drining lymph nodes (CDc + cells) of mice injected with lipoplexes of lipid 1 when compred to the corresponding numers oserved in mice injected with lipoplexes of lipids 2 (~6%) nd 3 (~3%) (Figure 3,). We lso evluted the MART1 expression level in trnsfected DCs under in vivo conditions using flow cytometric method y injecting mice (n = 2) with lipoplexes of lipids 1 3 nd p-cmv-mart1. The findings summrized in Supplementry Figure S14 clerly showed liposomes of lipid 1 to e the most efficient mong lipids 1 3 for trgeting DNA vccines to DCs under in vivo conditions. Phenotypic profiling of DCs trnsfected with lipoplexes of lipid 1 nd p-cmv-β-gl Using nontrnsfected DCs s control, next we ssessed the reltive phenotypic chnges of DCs trnsfected with: the most promising lipoplex of lipid 1 nd p-cmv-β-gl, only liposomes of lipid 1 nd only p-cmv-β-gl. Expression levels of the DC mturtion mrkers CD83, CD4, CD8, nd CD86 were most significnt in DCs trnsfected with the lipoplex of lipid 1 nd p-cmv-β-gl induced (Figure 4). Importntly, DCs treted with only liposomes of lipid 1 or with only p-cmv-β-gl plsmid filed to show ny such significnt phenotypicl mturtion (Figure 4). Secreted cytokines from DCs trnsfected with lipoplex of lipid 1 nd p-cmv-β-gl With view to gin some insights into the nture of cytokines secreted y trnsfected DCs, we used ELISA kits for mesuring TNF-α, IL-6, nd IL-p7 cytokine levels in the superntnts Moleculr Therpy vol. 24 no. 2 fe

4 In Vivo DC-trgeted Genetic Immuniztion The Americn Society of Gene & Cell Therpy Lipid Lipid % GFP + cells Lipid 2 Lipid 3 Figure 3 is the most efficient mong the lipids 1 3 in trnsfecting dendritic cells (DCs) in vivo. () In vivo DC trnsfection properties of the lipoplexes of lipids 1 3. Lipoplexes of lipids 1 3 nd α5-gfp plsmids were injected (s.c.) into C57BL/6J mice nd drining lymph nodes were hrvested fter 24 hours. Cells were leled with nti-cdc mgnetic eds nd enriched in n Midimx columns nd the popultions of GFP-positive cells were nlyzed y flow cytometry. Cells collected from lymph nodes of mice injected (s.c.) with only vehicle (5% queous glucose) were used s negtive control. Ech experiment ws repeted three times nd similr FACS profiles were oserved in ech time. () Grphicl representtion of GFP-positive CDC + cells isolted from lymph nodes (P <.5 versus lipoplex of lipid 3 nd α5-gfp). of DCs treted with: lipoplex of lipid 1 nd p-cmv-β-gl plsmid, only liposomes of lipid 1 nd only p-cmv-β-gl plsmid. Enhnced secretions of ll these three cytokines were oserved only in DCs trnsfected with lipoplexes of lipid 1 nd p-cmv-β-gl plsmid compred with lipid 1, p-cmv-β-gl, nd untreted control (Figure 4 d). Enhnced secretions of these immunostimultory cytokines re consistent with functionl mturtions of DC fter eing trnsfected with lipoplexes of lipid 1 nd p-cmv-β-gl. In vivo immuniztion studies Towrd confirming tht s.c. immuniztion with the lipoplexes of lipids 1 3 in mice is cple of inducing immune responses under in vivo settings, first we used p-cmv-β-gl plsmid s model DNA vccine. C57BL/6J mice were s.c. immunized with p-cmv-β-gl complexed with liposomes of lipid 1 3 nd equimolr DOPE. Two weeks post third immuniztion, splenocytes nd ser were collected nd IFN-γ (signture cytokine for cellulr immune response) nd nti-β-gl ntiodies (humorl immune response) were mesured y using nti-ifn-γ-coted nd β-glctosidse-coted ELISA pltes, respectively. Mice immunized with only vehicles (5% glucose) were used s control. Importntly, the mount of IFN-γ mesured from splenocytes nd the mount of nti-β-gl ntiodies mesured in ser for the mice group immunized with lipoplexes of lipid 1 were found to e significntly higher thn those for the control mice group s well s for the mice groups immunized with lipoplexes of lipids 2 nd 3 (Supplementry Figure S15,). These initil findings prompted us to conduct in vivo DC-trgeted genetic immuniztion experiment using lipoplexes of lipid 1 (contining shikimoylhed-group) nd p-cmv-mart1 DNA vccine encoding humn MelnA/MART1 which shres 68.6% mino cid sequence identity with its murine counterprt. 19 First mice (n = 5) were immunized with the lipoplexes of lipid 1 nd p-cmv-mart1 nd were susequently chllenged with lethl dose (~1 1 5 cells/mice) of ggressive melnom tumor. Since the tumor sizes for the control mice group immunized with only vehicle (5% queous glucose) ecme too lrge on dy 3 post tumor chllenge nd hd to e scrificed, tumor growth ws monitored for 3 dys. Importntly, ll the five mice s.c. immunized with lipoplexes of lipid 1 nd p-cmv-mart1 were completely tumor free while tumor sizes in ll the five mice s.c. immunized with irrelevnt control lipoplexes of lipid 1 nd p-cmv-β-gl kept on incresing with time nd died within 29 dys post tumor chllenge (Figure 5). In shrp contrst, long-lsting (1 dys post tumor chllenge) ntimelnom protective immunity ws oserved in mice immunized with lipoplexes of lipid 1 nd p-cmv-mart1. All the five immunized mice lived completely tumor-free life for 1 dys post tumor chllenge (Figure 5). Collectively, the findings summrized in Figure 5, demonstrte tht the long-lsting ntitumor immune response inducing potentil of the in vivo DC-trgeting lipoplexes of lipid 1 nd p-cmv-mart1. Furthermore, with view to evlute memory response, ll the five immunized mice which survived 1 dys fter the first B16F1 chllenge were chllenged second time with ~1 1 5 B16F1 cells. Four out of five mice (8%) second time chllenged with tumor lived once gin completely tumor-free lives for 6 months fter the second tumor chllenge (Figure 5c,d). We lso evluted the in vivo DC-trgeted immuniztion efficcy of lipoplexes of lipid 2 nd p-cmv-mart1 nd lipoplexes of lipid 3 nd p-cmv-mart1. Importntly, 6% of mice immunized with the lipoplexes of lipid 2 nd p-cmv- MART1 lived tumor-free life, wheres only 2% of mice immunized with the lipoplexes of lipid 3 nd p-cmv-mart1 lived tumor-free life during the oserved time period of 7 dys post tumor chllenge (Supplementry Figure S16,). Stted differently, the findings summrized in Figure 5c,d convincingly demonstrted the efficiency of the presently descried in vivo DC-trgeting liposoml DNA vccine crrier of lipid 1 in inducing drmtic memory response in cncer immunotherpy. Studies on the efficcies of the lipoplexes of lipids 1 3 nd p-cmv-mart1 plsmid in regressing estlished tumor The experiments descried ove showed tht immuniztion with lipoplex of lipid 1 nd p-cmv-mart1 efficiently protected mice from susequent tumor chllenge. With view to evlute the efficcies of these lipoplexes under therpeutic settings, lipoplexes of lipids 1 3 nd p-cmv-mart1 were s.c. dministered in mice ering estlished tumors. Six- to eightweek-old femle C57BL/6J mice (ech weighing 2 22 g, n = 5) were s.c. injected with ~ B16F1 cells in their right vol. 24 no. 2 fe. 216

5 In Vivo DC-trgeted Genetic Immuniztion flnks. On dy 15 when tumor ecme plple, mice were s.c. immunized with lipoplexes of lipids 1 3 nd p-cmv-mart1. This tretment ws repeted on dys 18 nd 21 post tumor inocultion. Findings summrized in Figure 6, showed tht the mice group immunized with lipoplex of lipid 1 nd p-cmv- MART1 were more efficient in inhiiting tumor growth compred to mice groups treted with corresponding lipoplexes of lipids 2 nd 3. The overll survivility of tumor-ering mice group immunized with lipoplexes of lipid 1 nd p-cmv- MART1 (45 dys) ws lso higher thn tht for tumor-ering mice group immunized with corresponding lipoplexes of lipids 2 nd 3 (Figure 6). Cytotoxic T lymphocyte cytokine ssys Next, towrd proing the reltive contriutions of humorl nd cellulr immune responses, we performed the conventionl cytotoxic T lymphocyte (CTL) ssy nd mesured the mounts of secreted IFN-γ nd IL-4 (signture cytokines for cellulr nd humorl immune responses, respectively) in the superntnts from the coculture of the effector cells (splenocytes isolted from immunized mice) nd the trget B16F1 melnom cells. Lysis of the trget melnom cells y the primed splenocytes ws studied cross the Effector:Trget cell rtio of 1:1 1:1. Importntly, while the effector splenocytes isolted from mice immunized with lipoplexes of lipid 1 nd p-cmv-mart1 lysed ~6% trget melnom cells, significntly reduced trget cell lysis (~2%) could e effected y splenocytes isolted from mice immunized with lipoplexes of lipid 1 nd control irrelevnt p-cmv-β-gl plsmid (Figure 7). This showed tht the mice group immunized with in vivo DC-trgeting lipoplexes of lipid 1 nd p-cmv-mart1 ws more efficient in inducing CTL response ginst trget cell (B16F1). Consistent with such trget cell selective lytic ctivity of the effector splenocytes, results in the cytokine secretion ssys lso reveled remrkly higher mounts of IFN-γ (compred to secreted IL-4) secreted y the ctivted T cells (Figure 7,c). IFN-γ eing distinguishing mrker of cellulr immune response, the results shown in Figure 7,c re consistent with the supposition tht cell-medited immunity plys crucil role ehind the presently oserved remrkle ntitumor immune response p-cmv-β gl /p-cmv-β gl CD CD CD CD4 Moleculr Therpy vol. 24 no. 2 fe

6 In Vivo DC-trgeted Genetic Immuniztion The Americn Society of Gene & Cell Therpy IL-p7 secretion (pg/ml) p-cmv-β gl c 4, /p- CMV-β gl IL-6 secretion (pg/ml) 3,5 3, 2,5 2, 1,5 1, 5 p-cmv-β gl /p- CMV-β gl d TNF-α secretion (pg/ml) 6, 5, 4, 3, 2, 1, p-cmv-β gl /p- CMV-β gl Figure 4 Trnsfection of dendritic cells (DCs) with lipoplex of lipid 1 nd p-cmv-β-gl induces phenotypic mturtion nd enhnced IL-p7, IL-, nd TNF-α cytokines. () Phenotype nlysis. DCs treted with only liposomes of lipid 1, only p-cmv-β-gl, nd lipoplexes of lipid 1 nd p-cmv-β-gl were stined fter 24 hours with FITC-leled ntiodies ginst costimultory molecules CD4, CD8, CD83, nd CD86 nd nlyzed y flow cytometry. The results re representtive of dt from three independent experiments. Secretion of () IL-p7 (P <.5 versus untrnsfected DCs), (c) IL-6 (P <.5 versus untrnsfected DCs), nd (d) TNF-α (P <.5 versus untrnsfected DCs) from DCs treted with the lipoplexes of lipid 1 nd p-cmv-β-gl. CD8 + T-cell depletion studies Two groups of C57BL/6J mice (n = 5 for ech group) were immunized thrice with the lipoplex of lipid 1 nd p-cmv-mart1 on dy, 7, nd 14. The first group received i.p. injections of nti-cd8 mas (2.43) on dys 4, 1, 2, 6, 13, 17, 21, 25, 29, nd the second immunized group did not receive ny nti-cd8 mas. A third unimmunized group ws injected with 5% queous glucose (vehicle control group). All the three groups were chllenged with B16F1 cells (on dy 28 for the first two groups) nd tumor growths were mesured y slide cliper. The mice group tht did not receive nti-cd8 mas lived tumor-free lives fter eing immunized with the lipoplex of lipid 1 nd p-cmv-mart1 while ll five memers of the unimmunized control mice group developed tumors within 15 dys (Figure 8). Importntly, four out of five mice which were immunized nd depleted of CD8 + cells developed tumors upon chllenge with tumor cells (Figure 8). These findings convincingly demonstrted tht cell-medited (CD8 + T cells) immunity is plying criticl role in inducing ntitumor immune response for the presently descried in vivo DC-trgeting liposoml DNA vccine crrier. DISCUSSION DNA vccintion, i.e., use of nked plsmid DNA s vccine to prime the immune system, offers numer of prcticl enefits which re not esily chievle with other existing forms of vccines such s ttenuted viruses, suunit or recominnt protein vccines, whole tumor cells, etc There re numer of distinct dvntges in using pdnas in genetic immuniztion. They re esy to design nd construct nd their lrgescle production is cost-effective. In contrst to ttenuted virl vccines whose storge nd glol delivery re complicted y the need of keeping the vccines cold, plsmid DNAs re firly stle t room temperture. 25 Since the ntigens encoded in the DNA vccines re expressed in situ, their posttrnsltionl modifictions hppen in similr wy s in the cse of infection with cognte pthogens. Another distinguishing feture of DNA vccines is tht the immune response induced y such genetic immuniztion re often primrily cellulr in nture which is elieved to ply crucil roles for effective vccintion ginst pthogens (e.g., viruses) nd cncers. 26,27 Despite ll these distinguishing dvntges, the low in vivo cell trnsfection efficiencies of nked plsmid DNA, difficulty in preferentilly trgeting of DNA vccines to professionl ntigen-presenting cells (DCs), nd the modest intrinsic djuvnt ctivity of DNA vccines re impeding their clinicl success. To this end, immuniztion with utologous DCs ex vivo trnsfected with ntigen-encoded DNA vccines hve shown promise in the pst. 1,4 9,28,29 Such ex vivo DC trnsfection-sed immuniztion protocols re lor-intensive, time-consuming, expensive, nd not ptient-friendly vol. 24 no. 2 fe. 216

7 In Vivo DC-trgeted Genetic Immuniztion Tumor volume (mm 3 ) 4, 3,5 3, 2,5 2, 1,5 1, 5 /p-cmv-β-gl /p-cmv-mart1 % Tumor-free mcie /p-cmv-mart1 /p-cmv-β-gl c Tumor volume (mm 3 ) 4,5 4, 3,5 3, 2,5 2, 1,5 1, Dys fter tumor implnttion /p-cmv-mart1 3 d % Tumor-free mcie Dys fter tumor implnttion /p-cmv-mart Dys fter second tumor implnttion Dys fter second tumor implnttion Figure 5 Direct in vivo immuniztion with lipoplexes of lipid 1 nd MART1 (melnom ntigen)-encoded DNA vccine (p-cmv-mart1) protects syngeneic C57BL/6J mice from lethl melnom chllenge with remrkle memory response. () Six- to eight-week-old femle syngeneic C57BL/6J mice (ech weighing 2 22 g, n = 5) were immunized (s.c) with lipoplexes of lipid 1 nd p-cmv-mart1 nd lipoplexes of lipid 1 nd p-cmv-β-gl (s negtive control) using 15 μl 5% glucose solution contining 15 μg DNA, 4:1 lipid:dna rtio, three times with 7-dy intervls. Two weeks post third immuniztion, mice were chllenged with melnom tumor y s.c. injection of ~1 1 5 B16F1 cells. Tumor volumes (V = ½ 2 where, = mximum length of the tumor nd = minimum length of the tumor mesured perpendiculr to ech other) were mesured with slide clipers for up to 3 dys. Results represent the mens ± SD for n = 5 tumors (P <.5 versus tumor sizes for lipoplexes of lipid 1 nd p-cmv- MART1; sttisticl nlysis ws performed using students unpired t-test). () Percentge of tumor-free mice immunized (s.c.) with lipoplexes of lipid 1 nd p-cmv-mart1 nd lipoplexes of lipid 1 nd p-cmv-β-gl. (c) All the C57BL/6J mice (n = 5) immunized with lipoplexes of lipid 1 nd p-cmv-mart1 which lived tumor-free life for 1 dys post first tumor chllenge were chllenged second time with ~1 1 5 B16F1 cells. The short-term ntitumor memory responses fter this second tumor chllenge re shown for 3 dys (P <.5 versus tumor sizes for lipoplex of lipid 1 nd p-cmv- MART1; sttisticl nlysis ws performed using students unpired t-test). (d) The percentges of tumor-free mice remining live up to 18 dys fter the second melnom chllenge (8%). Aimed t resolving the ove-mentioned prolems ssocited with ex vivo DC trnsfection-sed protocols for genetic immuniztion, glol efforts hve egun towrd developing protocols for direct in vivo trgeting of ntigen-encoded DNA vccines to DCs. Promising recently developed strtegies for direct in vivo trgeting of DNA vccines to DCs include use of ntiody to trget DC-specific receptors including DEC-25, DNGR-1, CDc, etc. for delivering encoded ntigens or use of DC-specific promoter (e.g., CDc) for expressing cytokines required for enhncing vccine efficcies Dftrin et l. showed the use of fifth-genertion polymidomine (G5-PAMAM) dendrimers possessing DNA-loding surfce nd covlently grfted MHC clss II-trgeting peptides s Universl DNA pltform. In vivo DC-trgeting of genetic vccine encoding oth cytomeglovirus (CMV)-driven vccine Aghsp7 nd DC-specific CDc-driven ctive trnscription fctor XBP1 hs een shown to induce oth prophylctive nd therpeutic ntitumor immunity. 13 Rghuwnshi et l. recently succeeded in developing n in vivo DC-trgeted chitosn nnoprticles for nsl DNA immuniztion ginst nucleocpsid (N) protein of severe cute respirtory syndrome coronvirus (SARS-CoV) s ntigen. 14 Co et l. constructed in vivo DC-trgeting DNA vccine y fusing tumor-ssocited ntigen HER2/neu ectodomin to single chin ntiody frgment (scfv) from NLDC-145 ntiody specific for DC-restricted surfce molecule DEC-25 nd demonstrted its potent ntitumor cellulr nd humorl immune response inducing efficcies. 15 Rviv et l. demonstrted tht mnnosylted polyion complexes re sfe nd effective systems for in vivo trgeting of genes to CDc + DCs. 16 It is importnt to emphsize tht our previously reported liposomes of ctionic lipids contining the mnnose-mimicking shikimoyl- nd quinoyl- hed-groups with pure lysine spcer (i.e., without the lysine side chin mine group eing gunidinylted s is the cse for the presently descried lipids 1 3, Figure 1) were cple of mounting long-lsting immune response only under ex vivo DC trnsfection-sed DNA vccintion. 9 DCs re hrd-totrnsfect. Although the DC trnsfection efficcies of these previously reported lipids contining pure lysine spcer etween the Moleculr Therpy vol. 24 no. 2 fe

8 In Vivo DC-trgeted Genetic Immuniztion The Americn Society of Gene & Cell Therpy Tumor volume (mm 3 ) % Survivl 5, 4,5 4, 3,5 3, 2,5 2, 1,5 1, /p-cmv-mart1 Lipid 2/p-CMV-MART1 Lipid 3/p-CMV-MART Dys fter tumor implnttion Figure 6 Antitumor efficcies of the lipoplex of lipid 1 nd p-cmv-mart1 under therpeutic settings. () Six- to eight-week-old femle C57BL/6J mice (ech weighing 2 22 g) were injected with ~1 1 5 B16F1 cells s.c. in the right flnk. On dy 15, mice were rndomly sorted into four groups (n = 5) nd ech group ws immunized s.c. with one of the following: lipoplexes of lipid 1 nd p-cmv-mart1 (squre); lipid 2 nd p-cmv-mart1 (tringle); lipid 3 nd p-cmv- MART1 (circle). One group ws injected with 5% glucose (vehicle control, dimond). This tretment ws repeted on dys 18 nd 21 fter tumor cell implnttion. Tumor volumes (V = ½ 2 where, = mximum length of the tumor nd = minimum length of the tumor mesured perpendiculr to ech other) were mesured with slide cliper for up to 25 dys (P <.5 versus lipid 1/p-CMV-MART1). () Survivl study. mnnose-mimicking hed-group nd hydrophoic tils 9 were ~%, the lipids filed in mounting long-lsting immune response ginst melnom chllenge when used on the direct in vivo immuniztion mode (Supplementry Figure S17). Since prior studies demonstrted efficient gene trnsfer properties of gunidinylted ctionic mphiphiles, 36,37 we decided to convert the mine group in the lysine side chin of our previously reported lipids 9 with gunidine group using conventionl gunidinyltion regent HgCl 2 /N,N-di-Boc-thioure. A numer of distinguishing chemicl chrcteristics contriute to the enhnced gene trnsfer efficiencies of gunidinylted ctionic mphiphiles. The gunidinium groups, ecuse of their high pk vlues (~ 13), remin protonted cross much wider rnge of ph compred to other sic groups nd therey exhiit strong electrosttic inding properties with the phosphtes group of the mcromoleculr DNA molecules under the physiologicl ph. In ddition, they form chrcteristic prllel zwitterionic N-H + O hydrogen onds with the phosphte ions of the DNA molecules nd form /p-cmv-mart1 Lipid 2/p-CMV-MART1 Lipid 3/p-CMV-MART Dys fter tumor implnttion 25 hydrogen onds with nucleic cid ses. 37 In vitro nd in vivo DC trnsfection studies clerly demonstrted tht lipid 1 ws most efficient compred to lipids 2 nd 3. Hydrodynmic dimeters of the liposomes of lipids 1 3 were found to e within the rnge nm (Supplementry Figure S1) nd the corresponding rnges for the lipoplexes of lipids 1 3 were within nm (Supplementry Figure S). Such similr hydrodynmic dimeters of the lipoplexes of lipids 1 3 re consistent with the supposition tht the sizes of lipoplexes re unlikely to ply mjor role in modulting the DC trnsfection efficiencies of lipids 1 3. We do not understnd why increse in ex vivo DC trnsfection efficiency from ~% (s ws the cse for our previous reported lipid 9 ) to just ~2% (through conversion of the lysine mino side chin to gunidine in the present study) imprts in vivo DC-trgeting ility to the lipid. Perhps the minimum (threshold) ex vivo DC trnsfection efficiency for imprting in vivo DC-trgeting ility to ctionic lipid lies ner 2%. However, deciphering such threshold ex vivo DC trnsfection of ctionic lipids, if ny, would e impossile to find out without in-depth future structure ctivity studies using more numer of structurl nlogs such s use of multiple shikimoyl- nd quinoyl- hed-groups, use of multiple gunidine groups in the hed-group regions, etc. An importnt issue is worth mentioning t this point of discussion. Although in the present study we hve demonstrted the in vivo DC-trgeting ility of the mnnose receptor-specific lipoplex of lipid 1, the present findings do not preclude the possiility of mcrophge nd possily other myeloid cells (mny of which express mnnose receptors on their cell surfce) eing trnsfected y the lipoplexes of lipid 1. Since DCs re the most potent ntigen-presenting cells in mounting dptive immune response, herein our primry focus is on the in vivo DC-trnsfecting ility of the presently descried lipids. We hve no clue yet s to the origin of more DC-trgeting efficcy of lipoplex of lipid 1 compred to the lipoplexes of lipids 2 nd 3. The issue remins elusive t this stge of investigtion. Clerly further mechnistic studies such s inding studies using purified mnnose receptor need to e undertken in future towrd gining insights into the reltive in vivo DC-trgeting efficcies of the lipoplexes of lipids 1 3. Despite significnt recent progresses in the emerging field of in vivo DC-trgeted genetic immuniztion, the chllenge of designing simple, sfe, nd effective system for in vivo trgeting of DNA vccines to DCs remins formidle. To this end, using syngeneic mouse melnom model, we hve shown tht immuniztion with tumor ntigen (MART1)-encoded DNA vccines in complextion with the presently descried next-genertion liposoml DNA vccine crrier is cple of mounting long-lsting immune response ginst lethl dose of melnom tumor chllenge (ll five immunized mice lived completely tumor-free lives during 1 dys post first tumor chllenge). p-cmv-mart1 DNA vccine encodes 18 kd Humn MelnA/ MART1 ntigen which shres 68.6% mino cid sequence identity with its murine counterprt. 19 MelnA/MART1 is humn melnocyte linege-specific ntigen expressed y mjority of humn mlignnt melnom. Effective ntigen-specific immunity ensues when DCs present the processed ntigen frgments of the MART1 to oth CD4 + nd CD8 + T cells. DCs present processed ntigen frgments to CD4 + cells in complextion with vol. 24 no. 2 fe. 216

9 In Vivo DC-trgeted Genetic Immuniztion /p-cmv-mart 1 /p-cmv-β gl % Lysis 1:1 8:1 6:1 4:1 E:T rtio 2:1 1:1 3, 2,5 c 1 IFN-γ (pg)/1 G cells 2, 1,5 1, IL-4 (pg)/1 G cells /p-cmv- MART 1 /p-cmv-β Gl /p-cmv- MART 1 /p-cmv-β Gl Figure 7 Coculture of splenocytes isolted from mice immunized with lipoplexes of lipid 1 nd p-cmv-mart1 leds to effective lysis of trget B1F1 cells vi melnom specific cytotoxic T lymphocyte (CTL) response, secretion of IFN-γ nd IL-4. () Six- to eight-week-old femle C57BL/6J mice (ech weighing 2 22 g, n = 4) were immunized (s.c., three times with 7-dy intervls) with lipoplexes of lipid 1 nd p-cmv-mart1 nd lipoplexes of lipid 1 nd p-cmv- β-gl (s negtive control). Splenocytes were collected 28 dys fter the third immuniztion nd the percentges of trget melnom cell lysis (CTL responses) were mesured s descried under Mterils nd Methods (P <.5 versus control). (,c) Two weeks post third immuniztion, splenocytes were collected, stimulted for 3 dys y coculturing with trget B16F1 cells. Amounts of IFN-γ (signture cytokine for cellulr immune response) nd IL-4 (signture cytokine for humorl immune response) relesed in the coculture superntnts were determined y ELISA (P <.5 versus control). MHC II molecules which cuses differentition of CD4 + T cells into T-helper 1 (Th1) nd T-helper 2 (Th2) cells (inducers of cellulr nd humorl immune responses, respectively). When DCs present the processed MART1 protein frgments to CD8 + T cells in complextion with MHC I molecules, the ctivted CD8 + T cells differentite into CTLs (the effector T cells) which kill the ffected cells with help from CD4 + T cells. 38 Importntly, in the present study, 8% of mice (which lived completely tumor-free lives for 1 dys fter the first tumor chllenge) lived gin completely tumor-free lives for 2 dys post second tumor chllenge. Precise mechnism s to how such long-lsting immune responses ginst melnom ntigen is induced y the presently descried in vivo DC-trgeting lipoplex of lipid 1 remins n open question t this stge of investigtion. CD4 Th1 cells secret IFN-γ which induces further prolifertion of CTLs. Presumly, effective in vivo DC-trgeting of the p-cmv-mart1 DNA vccine in complextion with the liposomes of lipid 1 ensures secretion of lrge quntity of IFN-γ (s ws oserved in the present cse) y CD4 Th1 cells which in turn leds to prolifertion of CTLs including strong induction of memory T cells (Supplementry Figure S18,). Another importnt issue deserves mention t this point. Prior studies showed tht lipoplexes, in generl, re effective in locking tumor growth in mice due to their cpilities in inducing cute immune responses. 39 Findings in the CTL ssy summrized in Figure 7 clerly show tht trget cell (B16F1) lysis hppens only to the extent of ~2% y splenocytes (t E:T rtio of 1:1) of mice immunized with control lipoplex of lipid 1 nd p-cmv-β-gl plsmid (control DNA vccine with no encoded tumor ntigen). Contrstingly, the degree of trget cell lysis y splenocytes of mice immunized with lipoplexes of lipid 1 nd p-cmv-mart1 DNA vccine ws found to e ~6% s shown in Figure 7. Consistent with these findings in the CTL ssy, oth the mounts of secreted IFN-γ (mrkers of cellulr immune response) nd IL-4 (mrkers of humorl immune response) for mice immunized with lipid 1:p-CMV MART1 lipoplexes were found to e out seven times higher thn the mounts of secreted IFN-γ nd IL-4 for mice immunized with control lipoplex of lipid 1 nd p-cmvβ-gl plsmid (Figure 7,c sed on ssy performed 4 weeks fter immuniztion). Thus, nonspecific immune response inducing properties of lipoplexes re unlikely to ply mjor role in the presently descried long-lsting immune response. Furthermore, the findings in the newly conducted experiments using CD8 + depleted mice (Figure 8) clerly support the notion tht CD8 + T cells ply n importnt in mounting long-lsting immune response descried herein. Stted differently, the results summrized in Supplementry Figure S13, nd Figure 7 c convincingly demonstrted tht heightened cellulr immune Moleculr Therpy vol. 24 no. 2 fe

10 In Vivo DC-trgeted Genetic Immuniztion The Americn Society of Gene & Cell Therpy responses ply crucil roles ehind the remrkly sustined protective immunity ginst melnom tumor descried herein. We hve shown tht liposomes of ctionic mphiphiles contining mnnose-mimicking shikimoyl- hed-group, two n-hexdecyl hydrophoic tils, nd gunidinylted lysine spcer in etween the mnnose-mimicking hed-groups nd nonpolr tils hold gret promise for direct in vivo trgeting of tumor ntigen-encoded DNA vccines to DCs in designing DC-sed cncer vccines. With the vilility of the presently descried simple liposoml in vivo DC-trgeting system, voiding potentilly unsfe virl vectors for trgeting DCs under in vivo settings in genetic immuniztion will now e fesile. The drmticlly long-lsting primry immune responses ginst melnom in ll immunized mice nd the remrkle memory responses delineted herein support the future systemic promises of the presently descried direct in vivo DC-trgeting liposoml DNA vccine crrier in comting cncer nd potentilly mny other chllenging infectious diseses with known pthogenic ntigens through direct DNA vccintion in niml nd humn ody. MATERIALS AND METHODS Generl procedures, mterils, nd regents. LCQ ion trp mss spectrometer (ThermoFinnign, Sn Jose, CA) equipped with n ESI source or Micromss Quttro LC triple qudrpole mss spectrometer ws used for ESI mss spectrl nlysis of the trget lipids 1 3. Vrin FT 3, 4 MHz, nd 6 MHz NMR Spectrometers were used in running 1H NMR spectr of trget lipids 1 3 nd their synthetic intermedites. The quinic nd shikimic cids used for synthesis of lipids 1 nd 2 were from Fluk (Switzerlnd) nd the d-mnnose used for synthesis of lipid 3 ws from SD Fine Chemicls, Hyderd, Indi. The EDCI nd HOBT used for coupling qunic nd shikimic cids to tertiry mine intermedite were procured from Sigm-Aldrich (St. Louis, MO), nd the silic gel (6 mesh) used for performing column chromtogrphy ws purchsed from Acme Synthetic Chemicls (Mumi, Indi). Reversed phse nlyticl HPLC nlysis using two different moile phses (A: pure methnol nd B: 95:5, v/v, methnol/wter) were used in conforming more thn 95% purities of the trget lipids 1 3. p-cmv-mart1 plsmid DNA used in the present study ws kind gift from Dr. Vn den Eynde, Ludwig Institute for Cncer Reserch, Brussels, Belgium. Dr. Nlm Mdhusudhn Ro kindly provided us with smple of p-cmv-sport-β-gl plsmid. The CTL ssy kit nd the ELISA kits for cytokine ssys were procured from Thermo Scientific (Wlthm, MA), nd Promeg (Mdison, CA). Mouse FITC-conjugted CDc, H2K, CD4, CD86, CD83, CD8, nd mnnose receptor ntiodies were purchsed from Bio-Legend (Sn Diego, CA). Mouse phycoerythrin-conjugted CD4 + nd CDc nd FITC-conjugted CD8 + ntiodies were purchsed from Cell Signling (Dnvers, MA). Mouse nti-mhc II monoclonl ntiody used ws from Chemicon (Billeric, MA). Anti-MART1 ma ws purchsed from Pierce Biotechnology (Rockford, MA). IL-p7, IL-6, nd TNF-α ELISA kits were procured from R&D Systems (Minnepolis, MN). Anti-CDc ntiody-leled mgnetic eds nd Midimx columns were purchsed from Miltenyi Biotec (Bergisch Gldch, Germny). Cholesterol, cell culture medi, fetl ovine serum, nd the FITC-conjugted rt nti-mouse secondry ntiody were purchsed from Sigm-Aldrich. NP-4, ntiiotics, nd grose were otined from Hi-medi, Indi. B16F1 (murine melnom cells) ws procured from the Ntionl Centre for Cell Sciences (NCCS), Pune, Indi. Dulecco s modified Egle s medium with 1% fetl ovine serum ws used in culturing B16F1 cells t 37 C in humidified tmosphere contining 5% CO 2 /95% ir. C57BL/6 Tumor volume (mm 3 ) % Tumor-free mice 3,5 3, 2,5 2, 1,5 1, CD 8 + T-cell depleted treted mice Treted mice Dys fter tumor implnttion CD 8 + T-cell depleted treted mice Treted mice Dys fter tumor implnttion Figure 8 CD8 + T-cell depletion results into significntly compromised ntitumor immune response. () Two groups of C57BL/6J mice (n = 5 for ech group) were immunized thrice with the lipoplex of lipid 1 nd p-cmv-mart1 on dy, 7, nd 14. The first group received i.p. injections of nti-cd8 mas (2.43) on dys 4, 1, 2, 6, 13, 17, 21, 25, nd 29 nd the second immunized group did not receive ny nti-cd8 mas. A third unimmunized group ws injected with 5% queous glucose (vehicle control group). All the three groups were chllenged with B16F1 cells (on dy 28) nd tumor growths were mesured y slide cliper. (P <.5 versus treted mice). () Percentges of tumor-free mice. mice (ech weighing 2 22 g nd 6 8 weeks old) were otined from Ntionl Institute of Nutrition, Hyderd, Indi nd ll the in vivo experiments were done following Institutionl Bio-Sfety nd Ethicl Committee Guidelines using pproved niml protocols. Preprtion of liposomes. The co-lipid DOPE nd lipids 1 3 (t mole rtio 1:1) were dissolved in chloroform nd the solvent from the resulting homogeneous solution ws removed with thin strem of nitrogen gs. The residue ws dried under high vcuum for 8 hours nd the vcuum dried residue upon hydrtion in deionized wter for overnight fforded the in vivo DC-trgeting liposomes. The pproprite weights of lipids 1 3 nd DOPE ws used such tht the resulting liposomes contined 1 mmol/l lipids 1 3 nd 1 mmol/l DOPE for ll in vitro experiments nd the corresponding concentrtions were 5 mmol/l for ll in vivo experiments. The hydrted lipid film ws then riefly vortexed (3 seconds) nd therefter sonicted to clrity using Brnson 45 sonifier t 1% duty cycle nd 25 W output power. The resulting cler queous liposomes were then complexed with plsmid DNAs for prepring lipoplexes. Hydrodynmic size mesurements y dynmic light scttering. The hydrodynmic dimeters of the liposomes nd the lipoplexes of lipids 1 3 (using p-cmv-mart1 s DNA) were nlyzed y dynmic light scttering performed on DynPro Nno dynmic light scttering system (Wytt vol. 24 no. 2 fe. 216

11 In Vivo DC-trgeted Genetic Immuniztion Technologies, Snt Brr, CA). All mesurements were performed t fixed ngle of 9 t room temperture (25 C). Isoltion of primry mmdcs. mmdcs were isolted from the tiis nd fiuls of C57/BL6J mice s descried previously. 17 Briefly, one mrrow ws collected from tiis nd fiuls of mle C57BL/6J mice. Bone nd deris from the collected one mrrow ws removed y pssing through nylon mesh nd the pss through cells were resuspended in complete DC medium (RPMI-164) contining 1% fetl ovine serum, 5 μmol/l β-mercptoethnol, 2 mmol/l glutmine, 1% nonessentil mino cids, 2 ng/ml grnulocyte-mcrophge colony-stimulting fctor, 1 ng/ml IL-4, nd 1% ntiiotic solution. Cell medi were chnged every 2 dys with fresh DC medium. The ggregted cells were dislodged y gently pipetting with RPMI fter 6 dys, the dislodged cells pulled together, nd centrifuged t 28g for 1 minutes t room temperture. The superntnt ws discrded nd the pellets resuspended in complete DC medium t cells/ml. The resuspended cells were then plced in 1-mm cell culture Petri dishes (t ~1 1 7 cells/dish) using 1 ml medium per dish. The nondherent cells were collected fter 24 hours through gently swirling the dish nd were used for oth trnsfection nd flow cytometry experiments. Cultures of the collected nondherent DCs were mintined in humidified tmosphere with 5% CO 2 t 37 C. FACS protocol. Cells (~4 1 6 ) were fixed in 2% formldehyde solution for 2 hours, centrifuged, nd the superntnt ws discrded. The pelleted cells were then suspended in 3 ml wsh uffer (phosphte-uffered sline (PBS) contining 2% fetl ovine serum nd.1% sodium zide) nd incuted for 3 min t 37 C. The cell suspension ws divided eqully into Eppendorf tues contining ~5 1 5 cells in ech tue, centrifuged, the superntnts discrded, nd finlly, to the cell pellets in ech tue 1 μl commercilly ville phycoerythrin-leled mouse monoclonl ntiodies t dilution of 1:1 (in wsh uffer) ws dded. Aout 1 μl wsh uffer ws dded to one tue s control. The cell suspensions were then cultured t room temperture for 2.5 hours, centrifuged, nd the superntnts were discrded. The cell pellets were wshed with wsh uffer (2 1 ml). The wshed cells were then incuted for 1 hour fter sequentil dditions of commercilly ville unleled nti- MHC II monoclonl ntiody t dilution of 1:1 nd 1 μl of FITCconjugted secondry ntiody t 1:2 dilution (for mesuring MHC II mrker profiles). The sequence of such centrifugtion nd wshing were repeted for three times nd the finl cell pellets were suspended in 1 ml wsh uffer. The flow cytometry histogrms were then recorded in FACS-clier instrument (Becton Dickinson, Frnklin Lkes, NJ). Cells were permeilized with 9% methnol for 3 minutes efore incuting with ntiodies for mesuring totl MHC II expression in DCs. The GFP expression in trnsfected mmdcs were mesured y flow cytometry fter hrvesting nd wshing the trnsfected cells with wsh uffer (2 5 μl) nd the wshed pellets were suspended in 1 ml wsh uffer. The flow cytometry histogrms were then recorded using untreted mmdcs s controls. Phenotype nlysis of trnsfected DCs were performed using FITC-leled monoclonl ntiodies s descried ove. Flow cytometric method for studying in vivo DC-trgeting. Liposomes of lipids 1 3 were complexed with α5-gfp plsmid nd the resulting lipoplexes were injected (s.c.) into C57BL/6J mice. Two drining lymph nodes were hrvested fter 24 hours nd digested in complete RPMI medium contining 4 U/ml of collgense t 37 C for 3 minutes. The resulting single-cell suspension ws wshed with PBS contining 1% ovine serum lumin. Cells were leled with nti-cdc mgnetic eds (Miltenyi Biotec), incuted t 4 C for 15 minutes nd enriched in Midimx columns (Miltenyi Biotec) following the mnufcturer s instructions. The purity of isolted enriched DCs fter ffinity purifiction over the nti- CDc mgnetic eds ws mesured y flow cytometry using FITCleled CDc ntiody. The enriched CDc + cells DCs were found to e ~98% pure y flow cytometry. Finlly the percentge of GFP + DCs in this enriched DCs were mesured y flow cytometry. DCs collected from lymph nodes of mice immunized with only vehicle were used s control. Cytokine secretion. DC culture superntnts were hrvested 24 hours fter trnsfection nd stored t 2 C until use. Levels of IL- p7, IL-6, nd TNF-α concentrtions were ssessed using commercilly ville ELISA kits (R&D Systems) y following Mnufcturer s instructions. ELISA for nti-β-gl ntiody. Anti-β-gl ntiodies were mesured using ELISA s descried previously. 4 Briefly, 96-well ELISA pltes were coted with β-gl protein (.3 μg/well). After overnight incution t 4 C, the pltes were wshed with PBS (3 2 μl) nd locked with 1% ovine serum lumin in PBS for 2 hours t room temperture. The pltes were then wshed with PBS contining.5% Tween-2 (3 2 μl), incuted with serilly diluted mouse ser (1 μl) t room temperture for 2 hours, nd wshed gin with PBS contining.5% Tween-2 (3 2 μl). Aout 1 μl of prediluted (1:1,) horse rdish peroxidse-conjugted nti-mouse secondry ntiody ws dded to ech well of the plte, incuted t room temperture for 2 hours, nd the unound secondry ntiody ws removed y wshing with PBS contining.5% Tween-2 (3 2 μl). Aout 1 μl of ABTS (Cliochem, St. Louis, MO) ws dded to ech well nd the mixture incuted in drk t room temperture for 1 minutes. The sornce ws finlly mesured t 45 nm y ELISA reder (Bio-Tek instruments, UK). In vivo immuniztion. Six- to eight-week-old femle syngeneic C57BL/6J mice (ech weighing 2 22 g, n = 5) were immunized (s.c.) thrice (with 7-dy intervls) seprtely with lipoplexes (lipoplexes of lipid 1 nd p-cmv-mart1 in one group nd the lipoplexes of lipids 1 3 nd p-cmv-β-gl in other groups) in 15 μl 5% glucose solution contining 15 μg plsmid DNA with lipid:dna chrge rtio of 4:1. The group immunized with lipoplexes of lipids 1 3 nd p-cmv-β-gl were used to mesure humorl (β-gl ntiody) nd cellulr (IFN-gmm) immune responses. The group immunized with lipoplexes of lipid 1 nd p-cmv-mart1 s well s lipoplexes of lipid 1 nd p-cmv-β-gl (s control) were used in CTL s well s cytokine (IL-4 nd IFN-gmm) ssys. Tumor chllenge experiment. Aout 1 ml cell dissocition solution (Sigm-Aldrich) ws used to hrvest B16F1 melnom cells from T25 culture flsks nd the hrvested cells were wshed with PBS (2 5 μl) nd suspended in Hnks lnced slt solution (5 1 5 cells/ml). Two weeks fter the third immuniztion, ech of the 6 8-week-old femle C57BL/6J mice (n = 5) were chllenged (s.c.) with B16F1 melnom cells in 1 μl Hnks lnced slt solution. Plple tumors were detected 2 weeks post tumor inocultion. Therefter, dily mesurements of perpendiculr tumor dimeters were tken nd whenever ny mesurements exceeded 14 mm, mice were euthnized. The second s.c. tumor chllenge (with ~1 1 5 B16F1 cells in 1 μl Hnks lnced slt solution) were performed in ll the five mice which lived tumor-free live 1 dys fter the first tumor chllenge. Survivility studies under therpeutic settings. Six- to eight-week-old femle C57BL/6J mice (ech weighing 2 22 g) were s.c. injected in the right flnks with ~1 1 5 B16F1 cells. On dy 15, mice were rndomly sorted into four groups (n = 5 in ech group). On dy 15, 18, nd 21 post tumor inocultion, mice were s.c. immunized with lipoplex of lipid 1 nd p-cmv-mart1 (Group 1); lipoplex of lipid 2 nd p-cmv-mart1 (Group 2); lipoplex of lipid 3 nd p-cmv-mart1 (Group 3); nd 5% queous glucose lone (Group 4, the vehicle control group). Tumor volumes (V = ½ 2, where = mximum length of the tumor nd = minimum length of the tumor mesured perpendiculr to ech other) were mesured with slide cliper for up to 25 dys. ELISA for in situ IFN-γ nd IL-4. The ssys for mesuring these cytokines were performed s descried previously. 41 Two weeks fter the third immuniztion, mice were scrificed nd their spleens were isolted y Moleculr Therpy vol. 24 no. 2 fe