Adenovirus-transduced dendritic cells stimulate cellular ... - Nature

Gene Therapy (2001) 8, 1255–1263  2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt

RESEARCH ARTICLE

Adenovirus-transduced dendritic cells stimulate cellular immunity to melanoma via a CD4+ T cell-dependent mechanism J Steitz, J Bru¨ck, J Knop and T Tu¨ting Department of Dermatology, J Gutenberg-University, Langenbeckstr 1, D-55101 Mainz, Germany

We previously showed that genetic immunization of C57BL/6 mice with recombinant adenovirus encoding human TRP2 (Ad-hTRP2) was able to circumvent tolerance and induce cellular and humoral immune responses to murine TRP2 associated with protection against metastatic growth of B16 melanoma. In the present study we compared delivery of Ad-hTRP2 with cultured dendritic cells (DC) and direct injections of Ad-hTRP2. We show that application of Ad-hTRP2 with cultured DC enhanced protective immunity to B16 melanoma cells. Most importantly, delivery of recombinant adenovirus with DC alters the character of the immune response resulting in preferential stimulation of strong cellular immunity in the absence of significant humoral immunity to the

encoded antigen. Adoptive transfer of lymphocytes from mice immunized with Ad-hTRP2-transduced DC confirmed that cellular components of the immune response were responsible for rejection of B16 melanoma. The protective efficacy of Ad-hTRP2-transduced DC clearly depended on the presence of CD4+ T helper cells. Furthermore, ADhTRP2-transduced DC, but not direct injection of Ad-hTRP2, were effective in the presence of neutralizing anti-adenoviral antibodies. These preclinical studies demonstrate the superiority of melanoma vaccines consisting of cultured DC transduced with recombinant adenoviruses encoding melanoma antigens. Gene Therapy (2001) 8, 1255–1263.

Keywords: genetic immunization; recombinant adenovirus; TRP2; dendritic cells; melanoma

Introduction Genetic immunization strategies are being developed in many laboratories for the prevention and treatment of infectious diseases and cancer.1–3 Immunization of mice with plasmid DNA or recombinant viruses expressing model antigens has been shown to stimulate strong humoral and cellular immune responses including antigen-specific CD8+ CTL and CD4+ Th cells. Antigen-specific immunity was able to protect mice from normally lethal challenges with antigen-transfected transplantable tumor cell lines.4–7 This novel method of immunization has also become attractive for the development of melanoma vaccines, because a number of antigens recognized by cellular components of the immune system have been identified at the molecular level in melanoma patients.8– 13 These melanoma antigens include normal cellular proteins such as the tyrosinase family of enzymes involved in melanin synthesis which are endogenously expressed in melanocytes and melanoma cells.10–13 Preclinical studies have been performed with plasmid DNA or recombinant viruses encoding the tyrosinase-related proteins 1 and 2 (TRP1 and TRP2) in mice.14–19 However, induction of immune responses to these self antigens could only be observed using immunogenic recombinant viruses. Pre-

Correspondence: T Tu¨ting Received 28 March 2001; accepted 23 May 2001

sumably, these strategies were impaired by mechanisms responsible for maintenance of peripheral tolerance.20 We and others have recently reported that genetic immunization of C57BL/6 mice with human but not with murine TRP2 was able to circumvent peripheral tolerance and induce coat depigmentation as a sign of autoimmune-mediated destruction of melanocytes associated with protective immunity against metastatic growth of B16 melanoma cells.17,18 Importantly, immunization with human but not with murine TRP2 cDNA was able to stimulate CD8+ T cells in vivo recognizing the H2-Kbbinding peptide SVYDFFVWL which derives from an evolutionary conserved region of both murine and human TRP2 corresponding to amino acids 180–188. This peptide has been identified as a melanoma rejection antigen in C57BL/6 mice using a Kb-restricted CTL line specific for B16 melanoma cells.21 Interestingly, this same peptide also binds to HLA-A2 and is recognized by melanoma-reactive CTL in human melanoma patients.13 Additionally, we found that mice immunized with human but not with murine TRP2 cDNA produced antibodies reactive with human TRP2 and cross-reactive with murine TRP2. The sequence of human TRP2 differs from that of murine TRP2 by about 20% at the amino acid level. These differences appear to be responsible for escaping peripheral tolerance, possibly by providing an epitope for the induction of CD4+ T helper cells. We observed that induction of protective immunity against metastatic growth of B16 melanoma cells was considerably stronger after injection of recombinant

Ad-transduced DC stimulate melanoma immunity J Steitz et al

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adenoviruses when compared with gene gun immunization using plasmid DNA. However, direct injections of recombinant adenoviruses for vaccine purposes are associated with several potential problems. Most importantly, adenoviruses will infect a variety of tissues leading to widespread expression of antigen and potential undesired toxicity, for example, in the liver. Furthermore, induction of neutralizing antibodies to viral envelope proteins can significantly impede vaccine efficacy.4,22–24 This is of considerable importance for future clinical applications since adenoviruses are common human pathogens and neutralizing antibodies are frequently found in patients. Direct transduction and activation of dendritic cells (DC) in vivo appears to be required for induction of cellular immunity following genetic immunization.6,25,26 Activated DC migrate to secondary lymphoid organs where they are uniquely capable of stimulating naive antigenspecific T cells.27 Advances in cell culture techniques allowing for the generation of large numbers of immunostimulatory DC in vitro from bone marrow precursors with the help of recombinant GM-CSF and IL-4 provide the opportunity for their application as a vaccine adjuvant.28–30 We and others have shown that cultured DC can be efficiently transduced with recombinant adenovirus expressing murine TRP2 and applied successfully as a melanoma vaccine in mice in vivo.31,32 This strategy may avoid problems associated with direct injections of recombinant adenoviruses. In the present study, we investigated how delivery of a recombinant adenovirus expressing human TRP2 (AdhTRP2) with cultured DC influences the resulting immune response in comparison with direct injections of Ad-hTRP2. We assessed the induction of protective immunity against B16 melanoma cells and analyzed the stimulation of cellular and humoral immune responses specific for TRP2. These experiments provide important information for the clinical application of adenovirustransduced DC which are currently being evaluated in first trials for the immunotherapy of patients with advanced melanoma.

Results Delivery of Ad-hTRP2 with cultured DC enhanced protective immunity to B16 melanoma In initial experiments, we investigated the ability of genetic immunization with Ad-hTRP2-transduced DC to induce protective immunity against growth of B16 melanoma lung metastases. An adenovirus expressing green fluorescent protein (Ad-EGFP) was used for control purposes. Groups of C57BL/6 mice were also immunized with Ad-hTRP2 or Ad-EGFP injected directly in vivo. All mice were challenged intravenously with B16 melanoma cells 2 weeks later. Both direct injection of Ad-hTRP2 and delivery of Ad-hTRP2 with DC completely prevented metastatic growth of B16 melanoma in the lungs (Figure 1a and b). In a second set of experiments using Ad-␤gal in place of Ad-EGFP as the control adenovirus, all four groups of immunized mice were challenged subcutaneously with B16 melanoma cells. In our hands, protection against growth of solitary metastases in the skin requires the induction of a stronger immune response when compared with experimental induction of multiple Gene Therapy

Figure 1 Ad-transduced DC induce superior protective immunity to B16 melanoma. Groups of C57BL/6 mice were immunized by one injection of 5 × 108 p.f.u. of Ad-hTRP2 or Ad-␤gal (a, c). Alternatively, groups of mice received one injection of 2.5 × 105 Ad-hTRP2- or Ad-␤gal-transduced DC (b, d). The induction of protective tumor immunity was assessed 2 weeks later by injecting 4 × 105 B16 melanoma cells intravenously and counting the number of lung metastases after 14 days. Shown are the number of lung metastases in individual mice and the mean number of metastases indicated by the horizontal bar of a representative experiment (a, b) Alternatively, immunized mice were challenged with 105 B16 melanoma cells subcutaneously in the flank and tumor growth determined by palpation every other day. All animals with palpable tumors had eventually to be killed because of progressive tumor growth. Cumulative results of two separate experiments expressed as percent of mice without tumors in each group at the indicated time-points are depicted (c, d).

lung metastases, presumably because tumors in the periphery are much less accessible for T cells. In this experimental setting immunization with Ad-hTRP2 resulted in delayed growth of B16 melanomas in the majority of mice (Figure 1c). Delivery of Ad-hTRP2 with DC promoted stronger protective immunity against growth of subcutaneous B16 melanomas with the majority of mice being tumor free at day 60 after tumor challenge (Figure 1d). Adenovirus-transduced DC stimulate antigen-specific cellular immunity In subsequent experiments, we compared the induction of CD8+ T cells specific for TRP2 or the model antigen ␤galactosidase. Groups of C57BL/6 mice were immunized with Ad-hTRP2 or Ad-␤gal either injected directly or delivered with cultured DC and killed 2–4 weeks later. Splenocytes were harvested, and peptide-specific stimulation of CD8+ T cells in vivo evaluated in ELISPOT assays using synthetic peptides corresponding to the known immunogenic H2-Kb-binding epitopes TRP2aa180–188 and ␤galaa497–504. Antigen-specific release of IFN␥ could readily be observed in mice injected with Ad-hTRP2 or Ad␤gal. Splenocytes harvested from mice injected with Adtransduced DC routinely produced a high background of nonspecific IFN␥ production in ELISPOT assays, even when CD8+ T cells were positively selected and tested separately. In a number of assays we were not able to


demonstrate reproducibly significant reactivity against the TRP2aa180–188 peptide in mice immunized with AdhTRP2-transduced DC (data not shown). As an alternative, we investigated the induction of cellular immunity in mice following injection of adenovirus-transduced DC utilizing EGFP as a model antigen in BALB/c mice. In this system, we took advantage of the recently discovered CTL-defined H2-Kd-binding peptideepitope derived from aa200–208 of EGFP.33 Genetic immunization of BALB/c mice with Ad-EGFP results in strong induction of T cells reactive with the EGFP200–208 peptide in IFN␥ ELISPOT assays (Figure 2a). Mice injected with Ad-␤gal only produced background levels of reactivity. This confirmed our previous results indicating that the foreign protein EGFP is a powerful T cell antigen in BALB/c mice with a high frequency of precursor T cells specific for the EGFPaa200–208 peptide. Groups of BALB/c mice were also injected intravenously with Ad-EGFP- or Ad-␤gal-transduced DC. Importantly, significant reactivity with the H2-Kd-binding EGFPaa200–208peptide was detected in splenocytes harvested from BALB/c mice immunized with Ad-EGFP-transduced DC (Figure 2b). The large number of EGFP-specific T cells clearly exceeded the high background reactivity caused by the DC-based immunization strategy. In this experimental system, our results indicated that adenovirustransduced DC are able to stimulate peptide-specific T cells in vivo. Adenovirus-transduced DC do not stimulate significant levels of antibodies specific for the encoded antigen or adenoviral proteins Alternatively, we compared the induction of antibodies specific for TRP2 or the model antigen ␤-galactosidase. Groups of C57BL/6 mice were immunized with AdhTRP2 or Ad-␤gal either injected directly or delivered with cultured DC and sera samples harvested 3–8 weeks later. In order to detect antibodies specific for human TRP2 with cross-reactivity for murine TRP2, we performed immunoblot assays with lysates of B16 cells which endogenously express murine TRP2. Sera from mice injected with Ad-hTRP2 showed specific reactivity with the 75 kDa and 55 kDa bands representing mature glycosylated and immature unglycosylated TRP2 in B16 cells

Figure 2 Ad-transduced DC stimulate antigen-specific CD8+ T cells. Due to constitutive high background in ELISPOT assays after DC-based immunization, we were unable to demonstrate clearly the induction of TRP2-specific CD8+ T cells. Therefore, we used EGFP as a foreign, immunogenic CTL-defined antigen in BALB/c mice. Immunization was performed by one injection of 5 × 108 p.f.u. of Ad-EGFP or Ad-␤gal (a). Alternatively, mice received one injection of 2.5 × 105 Ad-EGFP- or Ad␤gal-transduced DC (b). Splenocytes were harvested 2–4 weeks later and reactivity of T cells with the H-2Kd-binding peptide EGFPaa200–208 tested using IFN␥ ELISPOT assays. Results of a representative experiment expressed as mean number of spot-forming cells per 106 splenocytes are shown.

(Figure 3a). These bands can also be detected with a specific rabbit antiserum raised against a synthetic peptide derived from the C-terminal domain of TRP2. In contrast, serum from adenovirus-immune mice injected with AdhTRP2 or serum from mice injected with Ad-hTRP2transduced DC did not show specific reactivity. These results were confirmed by measurement of antibodies specific for ␤-galactosidase in sera of immunized C57BL/6 mice by ELISA. ␤-Galactosidase protein-specific binding of IgG could only be observed in naive mice injected with Ad-␤gal but not in mice injected with AdhTRP2. Furthermore, immunization with Ad-␤gal-transduced DC never led to production of significant levels of ␤-galactosidase-specific antibodies. Similar results were found in BALB/c mice, where antibodies specific for EGFP were measured by ELISA. High titers of EGFP-specific antibodies were always detected in BALB/c mice following direct injection of Ad-EGFP. However, mice injected with Ad-EGFP-transduced DC never developed significant levels of EGFP-specific antibodies (Figure 3b). In some experiments, strong induction of T cells with specific reactivity for the H2-Kd-binding EGFPaa200–208 peptide was demonstrated in the absence of EGFP-specific antibodies in individual BALB/c mice injected with AdEGFP-transduced DC. Direct injection of recombinant adenovirus routinely led to strong humoral immunity against adenoviral envelope proteins. This could readily be detected in immunoblot assays with lysates of adenovirus-infected 293 cells which productively replicate recombinant adenoviruses. In contrast, injection of adenovirus-transduced DC did not induce significant amounts of antibodies reactive with adenoviral proteins (Figure 3c). These results were confirmed by ELISA using adenoviral particles as the solid phase antigen. We concluded that delivery of recombinant adenovirus with DC alters the character of the immune response resulting in stimu-

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Figure 3 Ad-transduced DC do not stimulate significant production of antigen-specific antibodies. (a) C57BL/6 mice were immunized with 5 × 108 p.f.u. Ad-hTRP2 or Ad-␤gal. Alternatively, mice received 2.5 × 105 Ad-hTRP2- or Ad-␤gal-transduced DC. Sera samples were harvested from mice 4–6 weeks after immunization, pooled, and assayed for the presence of antibodies cross-reactive with murine TRP2 in lysates of B16 melanoma cells separated by SDS-PAGE and transferred to PVDF membranes using immunoblotting techniques. Shown is a representative experiment with the indicated sera. (b) BALB/c mice were immunized with 5 × 108 p.f.u. of Ad-EGFP or Ad-␤gal. Alternatively, mice received 2.5 × 105 Ad-EGFP- or Ad-␤gal-transduced DC. Sera samples were harvested from mice 4–6 weeks after immunization, pooled, and assayed for the presence of antibodies reactive with recombinant EGFP protein as a solid phase antigen using ELISA. Representative results expressed as mean optical density (405 nm) at a serum dilution of 1:250 (± s.e.m.) are depicted. (c) Sera samples harvested from C57BL/6 mice immunized with Ad-hTRP2 or Ad-hTRP2-transduced DC were analyzed for the presence of antibodies reactive with adenoviral envelope proteins present in lysates of Ad-EGFP-infected 293 cells which productively replicate recombinant adenovirus. Gene Therapy


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lation of cellular immunity in the absence of significant humoral immunity to the encoded antigen or the adenoviral proteins. Protective tumor immunity following immunization with Ad-hTRP2-transduced DC can be adoptively transferred with splenocytes Our experiments demonstrated that Ad-hTRP2-transduced DC were able to promote strong protective immunity against B16 melanoma without induction of significant humoral immune responses to TRP2. In order to demonstrate directly that rejection of B16 melanoma is mediated by cellular components of the immune system, we performed adoptive transfer assays with splenocytes from immunized mice34 according to the procedure described by Winn. Mice were injected with Ad-hTRP2, Ad-hTRP2-transduced DC, or Ad-EGFP-transduced DC. Two to 4 weeks later, mice were killed and splenocytes harvested. Groups of naive mice were subcutaneously injected with 105 B16 melanoma cells admixed with 107 splenocytes in the flank. The protective efficacy of adoptively transferred splenocytes against growth of B16 melanoma was determined by palpation and measurement of tumor size every other day. Only splenocytes derived from mice immunized with Ad-hTRP2-transduced DC provided significant protection against growth of B16 melanoma cells in these experiments (Figure 4). In contrast, splenocytes derived from mice immunized with Ad-EGFP-transduced DC or from mice immunized with Ad-hTRP2 injected directly were not effective. Our results demonstrate that delivery of Ad-hTRP2 with cultured DC enhances the induction of cellular immunity leading to protection against metastatic growth of B16 melanoma after adoptive transfer to naive mice. Ad-hTRP2-transduced DC require CD4+ T cells for the induction of protective immunity to B16 melanoma In subsequent experiments we attempted to analyze the cellular mechanisms involved in tumor immune defense in greater detail. In particular, we wanted to investigate the role of CD4+ T helper cells which are critically required for the stimulation of most CD8+ T effector cell responses using CD4 knockout mice. Groups of CD4 knockout or wild-type C57BL/6 mice were immunized

Figure 4 Protective immunity to melanoma can be adoptively transferred with immune cells. C57BL/6 mice were immunized with 5 × 108 p.f.u. of Ad-hTRP2, 2.5 × 105 Ad-hTRP2-transduced DC, or 2.5 × 105 Ad-EGFPtransduced DC. Splenocytes were harvested 2–4 weeks later and used for adoptive transfer in a Winn assay. 107 Splenocytes were admixed with 105 B16 melanoma cells and injected subcutaneously in the flank of naive C57BL/6 mice. Tumor growth was assessed by palpation every other day. Shown are the cumulative results of three separate experiments expressed as percent of mice without tumors in each group at the indicated timepoints. Gene Therapy

with Ad-hTRP2 or Ad-EGFP and challenged intravenously with B16 melanoma cells 2 weeks later. Wildtype but not CD4 knockout mice were protected against growth of B16 melanoma lung metastases after genetic immunization with Ad-hTRP2 (Figure 5a). Loss of protective efficacy in CD4 knockout mice was associated with the inability to stimulate T cells with specific reactivity for the H2-Kb-binding TRP2aa180–188 peptide in ELISPOT assays. Delivery of Ad-hTRP2 with cultured immunostimulatory DC could potentially bypass the need for CD4+ T cell help in inducing CD8+ T effector cells. However, CD4 knockout mice immunized with Ad-hTRP2-transduced DC were also unable to reject an intravenous challenge with B16 melanoma cells (Figure 5b). Taken together, these results suggest that the protective efficacy of genetic immunization with Ad-hTRP2 against growth of B16 melanoma critically depended on CD4+ T helper cells. This requirement for CD4+ T helper cells could not be circumvented by delivery of Ad-hTRP2 with cultured DC. Ad-hTRP2-transduced DC mediate protective immunity to B16 melanoma in the presence of neutralizing immunity to adenoviral proteins Immunization of mice with recombinant adenoviruses not only stimulates cellular and humoral immunity to encoded model antigens, but also generates high titers of specific and neutralizing antibodies against adenoviral envelope proteins which may inhibit the efficiency of further immunizations with another recombinant adenovirus of the same serotype. To address this issue, neutralizing anti-adenoviral immunity was generated in groups of mice by priming with one injection of recombinant adenovirus containing an irrelevant cDNA and boosting after 2 weeks. Humoral immunity to adenoviral proteins was confirmed 2 weeks later by ELISA and by immunoblot. Groups of ‘adenovirus-immune’ and naive C57BL/6 mice were subsequently immunized once with Ad-hTRP2 or Ad-EGFP and challenged intravenously with B16 melanoma cells 2 weeks later. Melanoma metastases grew in lungs of ‘adenovirus-immune’ mice injected with Ad-hTRP2 similar to control groups immu-

Figure 5 Ad-transduced DC are not effective in CD4 knockout mice. CD4 knockout mice were immunized with 5 × 108 p.f.u. of Ad-hTRP2 or AdEGFP (a). Alternatively, CD4 knockout mice received 2.5 × 105 AdhTRP2- or Ad-EGFP-transduced DC (b). The induction of protective tumor immunity was assessed by injecting 4 × 105 B16 melanoma cells on day 14. Lungs were harvested on day 28 and the number of lung metastases on their surface counted under a dissecting microscope. Shown are the number of lung metastases in individual mice and the mean number of metastases indicated by the horizontal bar of a representative experiment.


nized with Ad-EGFP (Figure 6a). In contrast, naive mice injected with Ad-hTRP2 were always resistant to metastatic growth of melanoma in the lungs. To investigate the effect of neutralizing anti-adenoviral antibodies on the efficacy of genetic immunization with recombinant adenoviruses, we analyzed the induction of cellular and humoral immunity to TRP2 or the control antigens EGFP and ␤-galactosidase. Using ELISPOT, ELISA and immunoblot tests, we found that stimulation of peptide-specific T cells, as well as antigen-specific antibodies, was abrogated in ‘adenovirus-immune’ mice (data not shown). Thus, immunity to adenoviral vector components abrogated the ability to stimulate protective tumor immunity with this clinically relevant melanoma antigen. In theory, administration of adenovirus with cultured DC could circumvent neutralizing anti-adenoviral immunity because the adenovirus can infect immunostimulatory cells ex vivo. To test this hypothesis, groups of ‘adenovirus-immune’ mice were generated by two injections of recombinant adenovirus containing an irrelevant cDNA as described above and subsequently immunized with Ad-hTRP2- or Ad-EGFP-transduced DC. ‘Adenovirus-immune’ mice injected with Ad-hTRP2-transduced DC did not develop any melanoma metastases in their lungs following intravenous challenge with B16 melanoma cells. In contrast, ‘adenovirus-immune’ mice injected with Ad-EGFP-transduced DC were not protected (Figure 6b). These experiments indicated that delivery of Ad-hTRP2 with DC can restore the induction of effective melanoma immunity in mice in the presence of neutralizing anti-adenoviral antibodies.

Discussion Our investigations were undertaken to assess the efficacy of a melanoma vaccine consisting of cultured, Ad-hTRP2transduced DC in comparison to direct injection of AdhTRP2. We observed that application of cultured DC as

Figure 6 Ad-transduced DC circumvent neutralizing anti-adenoviral immunity. Neutralizing antibodies to the adenoviral vector were generated in groups of C57BL/6 mice by two immunizations with 5 × 108 p.f.u. recombinant adenovirus encoding an irrelevant insert on days 0 and 14. On day 28, groups of adenovirus-immune mice were injected directly with 5 × 108 p.f.u. Ad-hTRP2 or Ad-EGFP (a). Alternatively, mice received 2.5 × 105 Ad-hTRP2- or Ad-EGFP-transduced DC (b). The induction of protective tumor immunity was assessed by injecting 4 × 105 B16 melanoma cells intravenously on day 42. Lungs were harvested on day 56 and the number of lung metastases on their surface counted under a dissecting microscope. Results of one out of three independent experiments are presented. Shown are the number of metastases in individual mice (dots) and the mean number of metastases in each group (horizontal bar).

a biological adjuvant was able to enhance the protective efficacy of Ad-hTRP2 against subcutaneous growth of B16 melanoma. This result agrees with our previous observations using a recombinant adenovirus encoding murine TRP2.32 The superior efficacy of cultured immunostimulatory DC may be in part due to their ability to break tolerance to peripherally expressed self-antigens such as TRP2. It has become clear that the immune repertoire in humans and mice contains autoreactive B cells, CTL, and Th cells recognizing tyrosinase family antigens. Normally, activation of autoreactive T cells is prevented by several mechanisms which may include ignorance, anergy, deletion, and the presence of regulatory T cells. The precise mechanisms leading to sensitization and activation of potentially self-reactive T and B cells are not known. The nature and the activation state of DC appears to be of key importance in regulating tolerance or immunity. Induction of autoimmunity is believed to be triggered when DC are activated in a proinflammatory environment, for example, in the context of an active infection with a foreign pathogen, and present self-antigens to potentially autoreactive T cells. The ability of cultured, antigen-loaded DC to induce autoimmunity and concomitant tumor immunity has been unequivocally demonstrated in transgenic mouse models.35–37 The superior efficacy of adenovirus-transduced DC in our experiments may be related to the different quality of the resulting immune response. While direct injection of Ad-hTRP2 induced both cellular and humoral immunity, we observed that Ad-hTRP2-transduced DC did not lead to significant production of antibodies either to TRP2 or to adenoviral envelope proteins. Immune defense of B16 melanoma was therefore most likely mediated by cellular components of the immune system. This was supported by adoptive transfer of tumor protection with immune cells. We believe that the superior efficacy of lymphocytes derived from mice immunized with Ad-transduced DC in the adoptive transfer assays is due to stimulation of a stronger (autoreactive) CD4+ T cell response that may also be directed against ubiquitously expressed self-antigens. However, we have no experimental evidence for this hypothesis so far. The different character of the resulting immune responses following injection of adenovirus-transduced DC versus recombinant adenovirus is a result of the different distribution of antigen expression in vivo, which critically determines the outcome of an immune response.38 Direct injection of recombinant adenovirus in naive mice leads to a large amount of antigen expressed in vivo, primarily in non-immune cells such as liver and endothelial cells. In contrast, when recombinant adenovirus is delivered with DC, a comparably small amount of antigen is expressed in vivo only in antigen-transduced, activated DC, which largely home to secondary lymphoid tissues, in particular the lymph nodes and spleen.39 Presentation of antigen by activated DC in the context of costimulatory signals and immunostimulatory cytokines leads to potent priming of CD4+ and CD8+ T cells, while presentation of antigen by non-immune cells favors T cell tolerance. This may explain why delivery of adenovirus with DC induces stronger cellular immunity when compared with direct injection. A similar induction of a predominantly cellular immune response has been reported when cultured DC pulsed with inactivated LCMV in vitro were used as a

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vaccine.40 A combined cellular and humoral immune response was only elicited when DC were pulsed with live LCMV capable of replication in vivo. In agreement with observations in this viral antigen system, we could show that the inability to stimulate the production of antibodies was not due to lack of CD4+ T cell help, since the protective efficacy of Ad-hTRP2-transduced DC against growth of B16 melanoma was abrogated in CD4−/− mice. This result clearly indicated that Ad-hTRP2transduced DC stimulate CD4+ T helper cells, which are critically required for the efficacy of the vaccine. In our opinion, immunization with cultured DC, which are transduced with antigen in vitro, does not lead to significant access of intact protein to antigen-specific B cells in vivo unless the antigen is expressed in a secreted form.41 In our studies we attempted to analyze the mechanism by which immunization with Ad-hTRP2-transduced DC mediated rejection of melanoma. We were unable to demonstrate clearly the induction of T cells with specific reactivity for the H2-Kb-binding TRP2aa180–188 peptide due to consistent high background in our IFN␥ ELISPOT assays following DC-based immunization. Since TRP2aa180–188 peptide-specific T cells can only be detected at rather low frequencies after direct injection of AdhTRP2, it may well be possible that background numbers of spots masked the specific reactivity in these experiments. We alternatively performed standard cytotoxicity assays. However, restimulation of splenocytes with TRP2-peptide in vitro strongly stimulated proliferation and peptide-specific cytotoxicity of T cells even in naive or control immunized mice making it difficult to determine the efficacy of immunization. This is presumably due to the large repertoire of TRP2-specific T cells with low affinity.42 In vitro restimulation with irradiated B16 melanoma cells naturally presenting the peptide unfortunately also did not reveal significant differences in our hands and was inferior to the IFN␥ ELISPOT technique. Preliminary results of experiments with CD8 knockout mice show that protective immunity to melanoma is abrogated following immunization with Ad-hTRP2 or significantly reduced following immunization with AdhTRP2-transduced DC. This also supports a critical role for CD8+ T cells (data not shown). To verify that adenovirus-transduced DC in principle are able to stimulate MHC class I-restricted T cells in vivo, we turned to EGFP as a strong foreign model antigen in BALB/c mice. In this antigen system, induction of T cells with specific reactivity for the H2-Kd-binding EGFPaa200– 208 peptide well above background reactivity could be clearly demonstrated following injection of Ad-EGFPtransduced DC. The preferential induction of cellular immunity following immunization with Ad-EGFP-transduced DC could be confirmed in individual mice where strong reactivity of splenocytes with the EGFPaa200–208 peptide was observed in the absence of significant levels of antibodies specific for EGFP or adenoviral envelope proteins in a simultaneously harvested serum sample. In contrast, direct injection of Ad-EGFP induced strong reactivity with the EGFPaa200–208 peptide along with high titers of antibodies specific for EGFP and adenoviral envelope proteins. Our experiments touch upon the role of CD4+ T cells for the induction of protective tumor immunity. Rejection of B16 melanoma following genetic immunization with Ad-hTRP2 was critically dependent on CD4+ T cells.

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These results agree with published observations.17 Most likely, CD4+ T helper cells are necessary for optimal induction of CD8+ T effector cells.43 Antigen-transduced DC could potentially bypass the need for CD4+ T help.44 However, immunization with Ad-hTRP2-transduced immunostimulatory DC was unable to provide protection against metastatic growth of B16 melanoma in CD4−/− mice. The CD4+ T cell dependence of vaccines with antigen-transduced DC has also been reported in other tumor models.45,46 We believe that in our experimental system, the enhanced antitumor efficacy of adenovirus-transduced DC is due to the superior ability of DC in inducing a strong CD4+ T helper response with effector functions. This may also explain why cultured DC pulsed with the synthetic H2-Kb-binding TRP2aa180–188 peptide alone were unable to induce protective immunity against B16 melanoma in our hands. The potential mechanisms whereby tumor-specific CD4+ T helper cells mediate rejection of tumors is an active area of current research.43,47–49 Importantly, genetic immunization with Ad-hTRP2transduced DC but not with Ad-hTRP2 alone was able to promote protective immunity against B16 melanoma in the presence of neutralizing anti-adenoviral antibodies. This clinically important issue has been investigated before using various antigens in other tumor models. We confirmed published reports showing that induction of protective tumor immunity following direct injection of recombinant adenovirus is abrogated in mice previously exposed to the same adenoviral vector.4,23,24 Our detailed investigations show that loss of vaccine efficacy is associated with the inability to induce cellular and humoral immunity to TRP2 in adenovirus-immune mice. It has been shown that neutralizing antibodies to adenoviral envelope proteins prevents transduction of DC in vitro.23 Presumably, induction of antigen-specific cellular immunity following injection of recombinant adenovirus is abrogated in adenovirus-immune mice because resident DC are not transduced in vivo. In agreement with other investigators, we found that delivery of recombinant adenovirus with cultured DC which are transduced in vitro can overcome pre-existing anti-adenoviral immunity and restore protective vaccine efficacy.23,24,31 A variety of strategies have been proposed to circumvent the limitations imposed by neutralizing anti-adenoviral antibodies. One possibility is the sequential use of different recombinant viruses.50 An interesting alternative strategy to circumvent neutralizing anti-adenoviral immunity has recently been reported.51 Direct injection of recombinant adenoviruses in a collagen-based matrix enhanced the vaccine efficacy and was unaffected by neutralizing anti-adenoviral antibodies. In conclusion, we show that genetic immunization of C57BL/6 mice with Ad-hTRP2-transduced DC stimulated strong cellular immunity to B16 melanoma via a CD4+ T cell-dependent mechanism. Furthermore, we found that immunization with Ad-hTRP2-transduced DC but not with Ad-hTRP2 injected directly was effective in the presence of neutralizing anti-adenoviral immunity. The results of our preclinical studies provide important information for the future clinical implementation of melanoma vaccine strategies using recombinant adenoviruses expressing melanoma antigens.


Materials and methods Animals and cell lines C57BL/6 (H-2b), BALB/c 6 (H-2d), and CD4 knock out C57BL/6 mice, 6–12 weeks old, were bred at the Central Animal Facility of the University of Mainz. CD4 knockout C57BL/6 mice were kindly provided by Dr Hengartner (Institute of Experimental Immunology, University Hospital, Zurich, Switzerland). B16 (H-2b) is a spontaneously arising murine melanoma (a kind gift of Dr S Rosenberg, National Cancer Institute, Washington, DC, USA) and was maintained in DMEM supplemented with 10% heat-inactivated FCS, 2 mm l-glutamine, 50 ␮m 2-mercaptoethanol, 100 IU/ml penicillin, and 100 ␮g/ml streptomycin (all reagents were from Life Technologies, Eggenstein, Germany). Peptides, recombinant protein and recombinant adenoviruses The H-2Kb-binding peptide SVYDFFVWL (TRP2aa180–188) derived from the murine melanosomal protein TRP2,21 the H-2Kb-binding peptide ICPMYARV (␤galaa497–504) derived from E. coli ␤-galactosidase,23 and the H-2Kdbinding peptide HYLSTQSAL (EGFPaa200–208) derived from EGFP33 were synthesized by standard FMOC chemistry and purified by HPLC in the Peptide Synthesis Facility of the University of Mainz. Peptides were dissolved at 10 ␮g/ml in PBS containing 10% DMSO and stored at −20°C. Recombinant EGFP was purchased from Clontech and recombinant ␤-galactosidase was purchased from Sigma. Proteins were stored at −20°C. The construction of E1- and E3-deleted adenoviral vectors expressing EGFP or human TRP2 has been described.7,18 The recombinant adenovirus Ad-␤gal was kindly provided by Dr Andrea Gambotto (University of Pittsburgh, PA, USA). All recombinant adenoviruses are based on Ad5 and contain the transgene under the control of the CMV immediate–early promoter. They were propagated on CRE8 or 293 cells, purified by cesium chloride density gradient centrifugation and subsequent dialysis according to standard protocols and stored at −70°C. Culture and adenoviral transduction of murine DC Murine DC were generated from bone marrow precursors as described previously.28,32 Briefly, bone marrow cells were harvested from femurs and tibias, depleted of red blood cells, and washed twice in PBS. Cells were resuspended in DC medium consisting of RPMI 1640 supplemented with 10% heat-inactivated FCS, 1000 U/ml each of recombinant murine GM-CSF and IL-4 (a kind gift of Schering-Plough Research Institute, Kenilworth, NJ, USA), 2 mm l-glutamine, 1 mm sodium pyruvate, 0.1 mm nonessential amino acids, 0.1 mm Hepes, 50 ␮m 2-ME, 100 IU/ml penicillin, and 100 ␮g/ml streptomycin and cultured (37°C, 5% CO2) in six-well plates at 1 × 106 cells/3 ml per well. All cell culture reagents were purchased from Life Technologies (Eggenstein, Germany). Cells were fed with fresh medium containing GM-CSF and IL-4 after 2 days. Loosely adherent DC were harvested and replated after 5 days for transduction with recombinant adenovirus. DC were washed twice with PBS to remove FCS and distributed at 106 cells/300 ␮l per well of a 24-well plate in DC medium without serum. Transduction with recombinant adenovirus was performed at an MOI of 250 (ie 2.5 × 108 p.f.u. per well). Cells

were incubated for 4 h at 37°C and 5% CO2. After this time, 1.5 ml of DC medium with 10% FCS was added. Cells were routinely used for immunization 48 h after transduction.

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Immunization of mice Immunization with recombinant adenovirus was performed by direct i.p. injection of 5 × 108 p.f.u. recombinant adenovirus resuspended in 0.2 ml PBS or by i.v. injection of 2.5 × 105 bone marrow-derived DC transduced in culture. Neutralizing anti-adenoviral immunity was generated by immunizing mice twice with recombinant adenoviruses expressing a different insert on days 0 and 14. Humoral immunity to adenoviral proteins was assessed on day 28 as described below. ELISPOT assays The induction of peptide-specific CD8+ T cells was measured using the ELISPOT technique. Briefly, splenocytes were harvested 2 to 4 weeks after immunization and red blood cells depleted. 106 Splenocytes per well were restimulated in 200 ␮l of CM containing 1 ␮g/ml synthetic peptide and 25 IU/ml rhIL-2 in Millipore HA plates which were coated overnight with 10 ␮g/ml (50 ␮l per well) of anti-mIFN␥ mAb (R4-6A2; Pharmingen, Heidelberg, Germany) in PBS. After 22 h cells were washed out of the plates and bound cytokines visualized by incubation with 2.5 ␮g/ml (50 ␮l per well) of biotinylated anti-mIFN␥ mAb (XMG1.2; Pharmingen) for 1.5 h at 37°C, followed by 100 ␮l per well streptavidin–peroxidase (Molecular Biochemicals Roche, Mannheim, Germany; 1:1500 dilution in PBS containing 1% BSA, and 0.05% Tween20) for 30 min at RT, and premixed peroxidase substrate kit DAB (Vector Laboratories, Heidelberg, Germany). The number of spots was counted under a dissecting microscope and expressed as mean number of spots ± s.e.m. of repeated duplicate or triplicate determinations. All experiments were performed three to five times. Antibody measurements Antibodies against ␤-galactosidase, EGFP, or adenoviral proteins present in serum samples obtained from mice after genetic immunization were measured by ELISA using the respective recombinant proteins or heatdenatured adenoviral particles as a solid-phase antigen. Briefly, microtiter plates were coated with 5 ␮g/ml protein in carbonate buffer (pH 9.6) for 16 h at 4°C and blocked with PBS containing 3% BSA and 0.05% Tween. Serum samples were serially diluted in PBS containing 1% BSA and 0.05% Tween 20 (assay buffer) and added for 2 h. Bound antibodies were detected using peroxidaseconjugated donkey anti-mouse IgG (Jackson Immunoresearch, Westgrove, PA, USA) at a 1:5000 dilution in assay buffer followed by color development with ABTS substrate system (Sigma). Plates were read in an ELISA reader at 405 nm. Alternatively, serum samples were tested for the presence of specific antibodies by Western blot. 293 Cells infected with recombinant adenovirus were solubilized in lysis buffer (20 mm Tris, 150 mm NaCl, 2 mm EDTA, 0.5% NP40, pH 7.5) containing protease inhibitors (Complete Protease Inhibitor Cocktail; Roche) 30 h after infection. Cell extracts were subjected to 10% SDS-PAGE, and electroblotted on to PVDF membranes. Membranes Gene Therapy


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were blocked with 5% nonfat dry milk in T-PBS (0.1% Tween 20 in PBS) for 1 h and reacted with pooled sera from groups of immunized mice at a 1:200 dilution in TPBS + 2% nonfat dry milk for 1 h. Bound antibodies were detected with 1:5000 dilutions of peroxidase-conjugated goat anti-mouse IgG (Jackson Immunoresearch) in TPBS + 2% nonfat dry milk for 1 h and visualized using chemiluminescence on X-ray film (Amersham). Tumor protection assay and adoptive transfer of lymphocytes The induction of antitumor immunity was assessed 2 to 4 weeks after immunization. Mice were challenged by intravenous injection of 4 × 105 B16 melanoma cells which experimentally induces heavily pigmented lung metastases. The number of macroscopically visible metastases on the surface of the lungs was counted with the help of a dissecting microscope 14 days after challenge. Alternatively, mice were challenged by subcutaneous injection of 105 B16 melanoma cells in the flank. Tumor development was assessed every other day by palpation and perpendicular diameters measured using a vernier caliper. To investigate whether cellular components of the immune response were responsible for tumor rejection, splenocytes were harvested from mice 2 to 4 weeks after immunization and red blood cells depleted. 107 Splenocytes were admixed with 105 B16 melanoma cells and injected subcutaneously in the flank of naive mice. Tumor development was assessed every other day by palpation and perpendicular diameters measured using a vernier caliper. Results are expressed as percent of mice without tumor at a given time-point. All experiments included four to six mice per group and were performed three to five times. Statistical analyses Fisher’s exact method or the Student’s t test were performed to interpret the significance of differences between experimental groups. The differences were considered statistically significant when the P value was lower than 0.05.

Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (SFB432, project A12). We thank Mrs Ga¨ rtner and Mrs Alt for excellent technical assistance. We also thank Dr von Stebut and Dr Steinbrink for critical reading of the manuscript.

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41 Timares L, Takashima A, Johnston SA. Quantitative analysis of the immunopotency of genetically transfected dendritic cells. Proc Natl Acad Sci USA 1998; 95: 13147–13152. 42 Zeh HJ et al. High avidity CTLs for two self-antigens demonstrate superior in vitro and in vivo antitumor efficacy. J Immunol 1999; 162: 989–994. 43 Ossendorp F et al. Specific T helper cell requirement for optimal induction of cytotoxic T lymphocytes against major histocompatibility complex class II negative tumors. J Exp Med 1998; 187: 693–702. 44 Ridge JP, DiRosa F, Matzinger P. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and T-killer cell. Nature 1998; 393: 474–477. 45 Schnell S, Young JW, Houghton AN, Sadelain M. Retrovirally transduced mouse dendritic cells require CD4+ T cell help to elicit antitumor immunity: implications for the clinical use of dendritic cells. J Immunol 2000; 164: 1243–1250. 46 De Veerman M et al. Retrovirally transduced bone marrowderived dendritic cells require CD4+ T cell help to elicit protective and therapeutic antitumor immunity. J Immunol 1999; 162: 144–151. 47 Hung K et al. The central role of CD4+ T cells in the antitumor immune response. J Exp Med 1998; 188: 2357–2368. 48 Mumberg D et al. CD4+ T cells eliminate MHC class II-negative cancer cells in vivo by indirect effects of IFN-gamma. Proc Natl Acad Sci USA 1999; 96: 8633–8638. 49 Qin Z, Blankenstein T. CD4+ T cell-mediated tumor rejection involves inhibition of angiogenesis that is dependent on IFN gamma receptor expression by nonhematopoietic cells. Immunity 2000; 12: 677–686. 50 Irvine KR et al. Enhancing efficacy of recombinant anticancer vaccines with prime/boost regimens that use two different vectors. J Natl Cancer Inst 1997; 90: 1894–1899. 51 Siemens DR et al. Restoration of the ability to generate CTL in mice immune to adenovirus by delivery of virus in a collagenbased matrx. J Immunol 2001; 166: 731–735.

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