Transient Immunosuppression with 15-Deoxyspergualin Prolongs Reporter Gene Expression and Reduces Humoral Immune Response After Adenoviral Gene Transfer

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Gene Therapy (1998) 5, 85–90  1998 Stockton Press All rights reserved 0969-7128/98 $12.00 Transient immunosuppression with 15-deoxyspergualin prolongs reporter gene expression and reduces humoral immune response after adenoviral gene transfer

G Cichon and M Strauss Department of Molecular Cell Biology , Humboldt-University of Berlin, Max-Delbru¨ck House, Robert-Ro¨ssle Strasse 10, D-13122 Berlin-Buch, Germany

A strong immune response against transgenic cells is one markedly slowed the decline of reporter gene expression important limitation for long-term expression after adenovi- and a positive effect was still detectable 200 days after ral gene transfer in mammals. Continuous pharmacological gene transfer. At the same time, antibody production was immunosuppression has been shown to ameliorate reduced by 50–60%. Continuous treatment with 15-DSG immune reactions and to prolong reporter gene expression. (10 mg/kg twice weekly) led to a further small increase of In this study, we explored the effect of short-term immuno- gene expression but reduced antibody formation by 80– suppression for long-term gene expression and its impact 90%. A short course of FK 506 and cyclosporin A (CsA), on antibody formation. Immunosuppression with FK 506 had conferred a negative effect on gene expression. Both (1 mg/kg/day), cyclosporin A (20 mg/kg/day) and 15-deoxy- groups showed an even faster reduction in gene spergualin (10 mg/kg/day) was performed in NMRI mice. expression compared with the control group. The results Expression of the reporter gene human alpha-1-antitrypsin of this investigation suggest that 15-DSG could serve as (hAAT) and antibody formation was monitored for 7 an effective supplement for viral gene therapy protocols. months. A 5-day course of 15-deoxyspergualin (15-DSG)

Keywords: 15-deoxyspergualin; immunosuppression; adenoviral gene transfer; human alpha-1-antitrypsin; long-term expression

Introduction account for the main source of neutralising antibodies. Since fiber or other proteins can hardly be removed from Recombinant adenoviruses are the most efficient in vivo the capsid, antigenicity can be circumvented to some 1 gene transfer vehicles currently available. One important extent by employment of recombinant viral vectors with target organ for the therapy of inherited diseases with fiber proteins of varying serotypes.10 The second source adenoviral vectors is the liver due to its high efficacy of of antigenicity, the expression of viral genes, can be infection and a considerable liver tropism after systemic avoided by construction of recombinant viruses with 2 application. Two receptors have been found so far which extended deletions6,11 and finally to the replacement of can mediate cellular uptake of the adenovirus, the first the entire viral genome.12,13 The contribution of these new 3 one is an alpha-v-integrin receptor and the so-called viral constructs for long-term expression has not been CAR-receptor (coxsackievirus and adenovirus receptor) reported so far. The third problem, which is important in 4 which has recently been cloned. The employment of gene therapy in general, the potential antigenicity of the recombinant adenoviruses for the transfer of therapeutic neoantigen, is difficult to assess and may be different for genes has one important limitation. Long-term gene versatile gene products. For these reasons, even in the so- expression is limited due to strong cellular and humoral called gutless adenoviral vectors of the latest gener- host immune responses that inactivates or destroys trans- ation,14 immunogenicity will remain a problem and some genic cells and blocks repeated administration of viral kind of immunosuppression will become an unavoidable 5–7 vectors by formation of neutralising antibodies. There component of adenoviral gene transfer protocols. Several are three main sources of antigenicity in adenoviral vec- strategies have already been applied successfully to over- tors which contribute to the strength and character of the come these problems. The constitutive expression of an immune response: viral proteins building the viral cap- immunomodulatory gene (gp19) from the adenoviral E3 8 6 sid, viral proteins persistently expressed from the vector region has been shown to reduce the cytotoxic activity of 9 and the transgene product itself. Antifiber antibodies CD8 cells.15,16 Other genes with similar functions are the UL18 of the cytomegalovirus, ICP47 of herpes simplex virus or the EBNA1 of the Epstein–Barr virus.17–19 They Correspondence: G Cichon suppress the immune recognition and promote viral rep- Received 8 May 1997; accepted 26 August 1997 lication by interfering with mechanisms of antigen Transient immunosuppression with 15-DSG G Cichon and M Strauss 86 processing and MHCI-mediated antigen presentation in wild-type infections. Another very effective way of pre- venting an adaptive immune response which could even lead to long-lasting tolerance induction in rodents is the functional inhibition of naive CD4 cells during antigen exposition with monoclonal antibodies.15,20,21 Blocking the costimulatory CTLA4-B7.2 pathway with a monclonal antibody leads to similar results as the application of an anti-CD4 antibody.22 Antibody formation is entirely pre- served and long-lasting gene expression after adenoviral gene transfer has been shown in mouse models.15,20,21 Pharmacological immunosuppression with the immunosuppressants cyclosporin A or FK506 has been shown to reduce immune response and prolongs reporter gene expression.7,23,24 Formation of neutralising antibodies was not prevented, rendering the repeated application of adenoviral vectors inefficient. Since long-term immune suppression is accompanied by an increased suscepti- bility for infectious diseases, and a certain risk for the development of lymphoproliferative and hematological disorders and several organ dysfunctions, we explored the effect of a short period of immunosuppression on long-term gene expression. Beside cyclosporin A and FK 506, 15-deoxyspergualin (15-DSG), an immunosuppressant currently under investigation in clinical phase III trials for the prophylaxis of transplant rejection25 was tested. In contrast to FK 506 and cyclosporin A which both interfere with the IL-2 signal transduction pathway, 15-DSG interferes with heat shock proteins of the HSP70 Figure 1 (a) Serum levels of hAAT in mice after systemic application of family which are involved in antigen processing and 1 × 1010 p.f.u. Ad-hAAT. Effect of immunosuppression with FK506 (1 26 mg/kg/day for 5 days), cyclosporin A (20 mg/kg/day for 5 days), 15-DSG MHCI-mediated antigen presentation. 15-DSG has ++ shown a strong immunosuppressive potency in several (10 mg/kg/day for 5 days) and DSG . Mean values along the time course are shown. A second dose of 5 × 109 p.f.u. was administered on day 215 allograft transplantation models and has been employed after the initial application. (b) Serum hAAT levels of single mice 30 days once in an adenoviral gene therapy protocol for the after gene transfer (gene expression in animal 4 of the cyclosporin group reduction of antibody formation.27 Its effect on the dur- (1150 ng/ml) and animals 2, 3 and 4 of the FK506 (1975 ng/ml, 675 ation of gene expression has not been investigated so far. ng/ml, 250 ng/ml) were too low to be drawn in the limits of this graph). For the differences DSG–control, DSG++–control, control–cylosporin and control–FK506, the KS test revealed P Ͻ 0.05. Results The repeated administration of 5 × 109 p.f.u. (Ad-hAAT) Long-term expression of human alpha-1-antitrypsin did not cause any measurable increase of gene Blood samples were taken once before mice received expression. 1 × 1010 p.f.u. of Ad-hAAT vector and on days 5, 10, 20, 30, 60, 100, 150, 200 and 220 after application and ana- Antibody formation against adenoviral proteins lysed for hAAT by an ELISA. Animals of all groups The level of antibody formation directed against vector showed a strong initial hAAT expression ranging from antigens was determined on day 30, 100, 150 and 200 0.8 mg/ml to 3.2 mg/ml between individuals on day 5 after virus application (Figure 2). Continuous treatment (Figure 1). Only minor differences could be observed between the groups on day 5 when peak expression was seen. Figure 1b shows the linear proportion of hAAT expression on day 30 to demonstrate especially the DSG effect. The observed differences between both DSG groups and the control group became significant from day 30. The decline of expression was not a continuous one but showed some kind of steps. The control group lost approximately 90% of expression between days 20 and 30. Human AAT levels in the FK506-treated and the CsA-treated groups dropped earlier than in the control group. In these two groups, a strong decline was notable already between days 10 and 20. In the DSG group, Figure 2 Time course of antibody formation against adenoviral antigens × 10 expression was prolonged and the main decline was after application of 1 10 p.f.u. Ad-hAAT. Effect of short-course observed between days 30 and 60. Only a tendentious immunosuppression with 15-DSG (10 mg/kg/day), FK506 (1 mg/kg/day) and cyclosporin A (20 mg/kg/day) and continuous treatment with 15- but no significant difference could be observed between DSG (10 mg/kg twice weekly). Each column represents the average value both DSG groups. In the control group, one animal of four animals. Titers were ranging between approximately 1/4096 (15- showed gene expression comparable to the DSG group. DSG twice weekly) and 1/131 072 (control group); n = 4. Transient immunosuppression with 15-DSG G Cichon and M Strauss 87 with DSG twice weekly for 150 days reduced the anti- pression of antibody development against antigens with body formation by 80–90%. After DSG withdrawal on a smaller molecular weight is somehow more efficient day 150, a slight induction was seen during the following than suppression of antigens with a higher weight 50 days. Compared with the control group immuno- (Figure 3). However, humoral response to penton base, suppression with DSG for 5 days reduced antibody for- penton-associated and to fiber proteins could be detected mation by about 50–60% during the whole course of 200 in all mice of the DSG++ group. days. In the control group, antibody titers increased up to day 150, while titers in the FK506 and the CsA group Expression of bacterial ␤-galactosidase in mouse liver peaked on day 30 and declined over the next 6 months. The highest expression of ␤-galactosidase (␤-gal) in livers To illustrate the difference in the antibody pattern of the control group was measured 6 days after appli- between the DSG++ and the control group, we probed a cation of 1 × 1010 p.f.u. Ad-␤-gal. Expression dropped Western blot containing separated solubilized total aden- over the next 2 weeks and reached almost background ovirus protein with serum of mice taken at day 150. Fig- after 3 weeks (Figure 4). After 6 weeks none of the control ure 3 shows the immunoblot along with the Commassie- animals showed any remaining ␤-galactosidase activity stained viral extracts. Mice showed only a moderate in the liver. The daily treatment with high doses of cyclo- response to the abundantly present hexon protein and sporin A (100 mg/kg/day) or 15-DSG (5 mg/kg/day) led did not react significantly with any of the core proteins. to a sustained high level of ␤-galactosidase for the whole Penton base, penton-associated and fiber protein seemed period of immunosuppression (21 days). Differences to be strong antigens, despite being present in moderate occurred in the rate of decline after withdrawal of amounts. Within the control group, differences were immunosuppression. ␤-Gal expression in all animals of observed regarding the reactivity against individual anti- the CsA group had returned back to baseline expression gens. The first mouse did not show any humoral on day 42, while all animals in the DSG group (DSG for response to hexon protein, the second one did not 21 days) were still expressing approximately 10% of respond to protein IX and the third mouse did not show maximum values. The group which received 15-DSG for any detectable response to the hexon-associated protein. 5 days only showed a slower decline of expression com- All animals in the DSG++ group responded to immuno- pared with the control group. On day 42, the members of suppression with a strong reduction of antibody develop- this group still expressed about 3% of maximum values. ment, at the same time all of them showed hAAT ␮ expression values between 10.5 and 15 g/ml. The anti- Discussion body pattern in the DSG++ group suggests that the sup- This investigation suggests that a short treatment of mice with 15-deoxyspergualin has a long-standing positive effect on gene expression after adenoviral gene transfer while transient immunosuppression with FK506 and cyclosporin A leads to premature termination. These results were quite surprising since short-term immunosuppression with cyclosporin A and FK506 had been ben- eficial in several transplant models28–30 and after adenoviral gene transfer in skeletal muscles.23 It has been reported that even tolerance induction against the lym- phocytic choriomeningitis virus in mice occurred after transient high-dose treatment with cyclosporin A.31 We could clearly confirm a strong positive effect of high-dose cyclosporin A treatment on ␤-galactosidase expression as long as immunosuppression was continued, but after withdrawal expression declined rapidly. Both groups (15- DSG for 5 days and 21 days) showed a slower reduction rate of expression compared with the control and the CsA group. The different effects of 15-DSG compared with

Figure 3 Comparison of serum antibody pattern against adenoviral antigens of control and immunosuppressed animals 150 days after virus application. Electrophoresis of adenoviral proteins (12 ␮g each lane) was performed on a 12% acrylamide gel, one lane (left) was directly stained with Coomassie-blue to visualize viral proteins. The other lanes were blotted and each lane was probed separately with sera of three mice of the control and the DSG++ group (15-DSG 5 mg/kg twice weekly for 150 days). The Figure 4 Time course of ␤-galactosidase expression in mice after appli- graph above the Western blot shows the corresponding serum hAAT levels. cation of 1 × 109 p.f.u. Ad-␤-gal. Effect of immunosuppression with high- Gene expression in two animals (2 and 3) of the control (both Ͻ1.5 ng/ml) dose cyclosporin A (100 mg/kg/day) and 15-DSG (5 mg/kg/day) for 21 group were too low to be drawn in the limits of this graph. days and a short course of 15-DSG (5 mg/kg/day) for 5 days; n = 3. Transient immunosuppression with 15-DSG G Cichon and M Strauss 88 those of FK506 and cyclosporin A on the other side are fiber and penton base are strong antigens in NMRI mice. most likely caused by the different mechanisms of action. The exeptional behavior of single mice regarding the 15-DSG has been shown to exert an inhibitory effect on expression pattern and the difference in antigen- cytotoxic T lymphocyte (CTL) response.32,33 It seems to antibody pattern reflects the heterogenicity at the H2 inhibit primarily the early induction phase rather than locus in the outbred strain NMRI. the effector phase of the cell-mediated lysis.34 It inhibits The long-lasting immunosuppressive effect of 15-DSG lymphocyte proliferation in the mixed lymphocyte reac- on the background of its affinity to heatshock proteins tion,33 kappa-light chain presentation in B cells and cyto- leads to a new and interesting theory about T lymphocyte kine production in monocytes.35 The molecular basis of activation which has recently been suggested by Matz- these diverse functions seems to be binding and inhi- inger et al.37,38 This theory says that activation of naive bition of certain heat shock proteins and of the nuclear T lymphocytes during the first contact with an antigen transcription factor kappaB (NF-␬B).26 Since heat shock requires some kind of alarm reaction. The model pro- proteins are responsible for partial denaturation and poses the existence of mediators that are released or acti- renaturation of proteins during their transport through vated upon contact with antigen, assigning them a membranes, their inactivation affects the transport of dangerous quality. These mediators are still hypothetical MHC molecules into the endoplasmatic reticulum and but heat shock proteins may be good candidates for this the Golgi apparatus. function. Thus, 15-deoxyspergualin could serve as a A second immunological function related to antigen rewarding tool for the investigation and understanding processing and presentation is the denaturation of cellu- of immune functions and we have shown here that it can lar proteins or potential antigens before fragmentation at be an effective supplement for adenoviral gene therapy the proteasome and MHC-mediated presentation. In con- protocols. trast to 15-DSG, FK506 and cyclosporin A interfere with the expression of IL-2 in T lymphocytes which is required for self-activation and clonal proliferation. Both sub- Materials and methods stances inhibit the dephosphorylation of transcription factors involved in IL-2 transcription.36 Antigen fragmen- Preparation of recombinant adenovirus tation and MHC-mediated presentation is one of the Recombinant replication-deficient adenoviruses contain- early steps in the adaptive immune response while IL- ing the cDNA of the human alpha-1-antitrypsin and the 2 release and T lymphocyte activation is a downstream bacterial ␤-galactosidase gene, both driven by an RSV reaction. It has been shown that co-expression of viral promoter, were provided by M Kay (Markey-Center, genes, which interfere with MHC I presentation, had a University of Washington, Seattle, WA, USA). Both markedly positive effect on gene expression.15 This obser- viruses were related to the adenovirus strain dl30939 vation together with our findings suggest that an inter- which is still carrying a functional gp19 gene in E3.15,39 vention in the early phase of antigen recognition is of Viruses were propagated on human embryonic kidney greater relevance for long-term effects than blocking cells (293 cells) and harvested 2 days after infection. Lysis m downstream effector functions like IL-2 release. The was performed in 0.1% NP40, 1 m MgCl2, PBS and viral observed rebound reaction could be mediated by a shift particles were purified by two rounds of cesium chloride from a primary TH2 to a TH1 response. In this particular density gradient centrifugation.40 Cesium chloride was case, we would expect a reduced humoral response in removed by gel filtration on a Sephadex G-25 column the FK506 and the cyclosporin group compared with the (Pharmacia, Uppsala, Sweden). Equilibration was perfor- control group. We could indeed observe a reduced med with injection buffer containing 3 mm KCl, 1 mm humoral response (Figure 2) in both groups, but the MgCl2, PBS and sterile filtration was carried out through reduction could be related to the shorter expression of a 0.45 ␮m filter (Schleicher and Schuell FP 030/2, Dassel, viral genes as well. There are only a few and contro- Germany). Virus suspension was supplemented with versial reports as to possible rebound reactions after tran- 10% glycerol and stored in aliquots at −78°C. Titration sient immunosuppression with cyclosporin A or FK506. was carried out in an endpoint dilution assay on 293 cells. 15-DSG reduces antibody production even after short- term treatment by about 50% despite an increased Animal procedures and immunosuppression reporter gene expression throughout the whole period of Six-week-old female NMRI mice were purchased from this trial. Permanent treatment (twice weekly 10 mg/kg) Bomholtgaard (Ry, Denmark). For investigation of hAAT reduces antibody production by on average 80–90%. A long-term expression, five groups were set up, four mice second virus dose given about 7 months after the first in each group. On day 0 each mouse received a dose of application did not lead to any measurable gene 1 × 1010 p.f.u. in 200 ␮l injection buffer (see above) via tail expression. The lack of gene expression after the boost is vein injection and a second dose of 5 × 109 p.f.u. of the not surprising since the initial humoral response is very same virus was applied on day 215 after the initial appli- strong and leads to anti-Ad titers of up to 1/131 072 cation. Immunosuppression was started 1 day before (control group). A reduction of antibody formation by virus application. Short-course suppression was applied continuous immunosuppression to titers of about 1/4096 for 5 days after gene transfer. The immunosuppressants (DSG++ group) is remarkable but still high. Since 10–20% were diluted in PBS and applied in a volume of 200 ␮l of antibodies have neutralizing qualities the remaining by daily intraperitoneal injection. The FK506 group titers are sufficient to neutralize circulating viruses after received 1 mg/kg/day FK506, the cylosporin A group the second application. The Western blot analysis of anti- received 20 mg/kg/day cyclosporin A, the DSG group gen patterns visualizes the reduction of antibody pro- received 10 mg/kg/day 15-DSG. After the initial 5-day duction, but shows at the same time that the proteins course, one group, referred to as the DSG++ group, leading to formation of neutralizing antibodies, especially received 15-DSG in a dose of 10 mg/kg twice weekly for Transient immunosuppression with 15-DSG G Cichon and M Strauss 89 150 days. The control group received intraperitoneal was removed by centrifugation (12 000 g for 10 min) and injections of PBS only. 15-Deoxyspergualin was kindly protein content was determined by Bradford assay. ␤- provided from Behring-Werke (Marburg, Germany), Galactosidase activity was measured in 4 ␮g total protein FK506 from Fujisawa (Mu¨ nchen, Germany) and cyclo- with ␤-galactosidase kit (Tropix, Bedford, MA, USA) sporin A (Sandimmun) was purchased from Sandoz according to the manufacturer’s instructions in a Berthold (Nu¨ rnberg, Germany). luminometer (Wildbad, Germany). Background For the investigation of ␤-galactosidase expression, expression in mouse liver ranged between 800–1500 rela- four groups were set up, each group containing nine tive light units (r.l.u.). NMRI mice. On day 0, 1 × 1010 p.f.u. Ad-␤-gal were applied. Immunosuppression with 15-DSG (5 mg/ Statistical analysis kg/day) was started 1 day before virus application and In addition to calculating mean values and standard continued for 5 days and in a second group for 21 days. deviation, the significance of observed differences The third group received cyclosporin A (100 mg/kg/day) between groups or single mice was determined in the for 21 days. The control group received PBS injections non-parametric Kolmogoroff–Smirnoff test (KS test). only. Three mice of each group were killed on days 6, 21 Observed differences were stated to be significant when and 42 after virus application and ␤-galactosidase P was smaller than 0.05. expression in the liver was quantified as explained below. Acknowledgements Assay for human alpha-1-antitrypsin Serum levels of human alpha-1-antitrypsin were quant- This investigation was supported particularly by the ified by sandwich-ELISA. Antibodies: goat anti-human Max-Planck-Gesellschaft, by the Bundesministerium fu¨r alpha-1-antitrypsin (Incstar, Stillwater, OK, USA), sheep Forschung und Technologie and by the Fond der Chem- anti-human alpha-1-antitrypsin-peroxidase (The Binding ischen Industrie. We thank B Goldbrich for excellent tech- Site, Birmingham, UK), ELISA plates were purchased nical support and Dr F Schnieders for helpful discussion from Nunc (F16 plates) (Nunc, Wiesbaden-Biebrich, and for careful reading of the manuscript. Germany), o-phenylenediamine (OPD) (Sigma-Aldrich, Deisenhofen, Germany) was used as substrate and meas- References ured at 490 nm in a Dynatech MR 5000 photometer (Biermann, Bad Nauheim, Germany). Mouse alpha-1- 1 Haddada H et al. Gene therapy using adenovirus vectors. Curr antitrypsin did not cross-react with the antibodies used. Top Microbiol Immunol 1995; 199: 297–306. 2 Kass Eisler A et al. 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