Antibody to CD40 Ligand Inhibits Both Humoral and Cellular Immune Responses to Adenoviral Vectors and Facilitates Repeated Administration to Mouse Airway

Gene Therapy (1997) 4, 611–617  1997 Stockton Press All rights reserved 0969-7128/97 $12.00 Antibody to CD40 ligand inhibits both humoral and cellular immune responses to adenoviral vectors and facilitates repeated administration to mouse airway

A Scaria1, JA St George1, RJ Gregory1, RJ Noelle2, SC Wadsworth1, AE Smith1 and JM Kaplan1 1Genzyme Corporation, Framingham, MA; 2Department of Microbiology, Dartmouth Medical School, Lebanon, NH, USA

Adenoviral vectors have been used successfully to transfer against murine CD40 ligand inhibits the development of the human CFTR cDNA to respiratory epithelium in animal neutralizing antibodies to adenoviral (Ad) vector. MR1 also models and to CF patients in vivo. However, studies done decreased the cellular immune response to Ad vector and primarily in mice, indicate that present vector systems have allowed an increase in persistence of transgene limitations. Among other things, transgene expression in expression. Furthermore, when administered with a the lung is transient and the production of neutralizing anti- second dose of Ad vector to mice preimmunized against bodies against adenovirus correlates with a reduced ability vector, MR1 was able to interfere with the development of to readminister a vector of the same serotype. Here we a secondary antibody response and allowed for high levels demonstrate that in mice, a transient blockade of costimu- of transgene expression upon a third administration of lation between activated T cells and B cells/antigen vector to the airway. presenting cells using a monoclonal antibody (MR1)

Keywords: adenovirus vectors; immune response; anti-CD40 ligand; repeat administration

Introduction using immunodeficient mice have shown that this process is dependent on MHC class II presentation of the E1-deleted replication-defective adenoviral (Ad) vectors input viral proteins and activation of CD4+ T cells and are attractive vehicles for gene transfer because of their can be induced by inactive as well as active viral par- ability to transduce a wide variety of dividing and non- ticles,6 (Kaplan et al, unpublished data). 1–5 dividing cells in vivo. Such vectors have been used for Several investigators have tested the use of broad gene transfer to the respiratory epithelium of experi- immunosuppressants11 and cytoablative agents16 to over- mental animals and of individuals with cystic fibrosis come the immune response of the host to first generation 2–5 (CF). Studies from several laboratories have suggested Ad vectors. Kay et al17 have shown that transient coad- that administration of high doses of first generation Ad ministration of soluble CTLA4-Ig along with an intra- vector results in only transient gene expression in the venous injection of Ad vector expressing a nonimmuno- lung due, at least in part, to destruction of vector-trans- genic transgene product (human ␣-1 anti-trypsin) leads duced cells by host cellular immune responses + to persistent transgene expression from mouse liver. (predominantly CD8 cytotoxic T cells) directed against CTLA4-Ig blocks the B7-CD28 pathway of T cell costimu- 6–9 viral proteins and/or immunogenic transgene products. lation.18 Although Ad-specific antibody levels were Reduction of this response has been reported following reduced in CTLA4-Ig-treated mice, the inhibition was not the use of second generation vectors with decreased viral sufficient to allow secondary gene transfer under the con- 10,11 gene expression and with transgenes encoding self ditions tested.17 Yang et al19 demonstrated that coadmin- 8 rather than foreign proteins. istration of interferon-␥ (INF-␥) or interleukin-12 (IL-12) The treatment of chronic diseases like CF with Ad vec- with recombinant Ad vector diminished the formation of tors will likely require repeated administrations through- neutralizing antibodies and allowed readministration of out the lifetime of the patient. However, another limi- vector to mouse airways. However, IL-12 is a potent tation of current vectors is the difficulty in obtaining mediator which affects Th1 type CD4+ T cell responses successful readministration using a vector of the same Ad and is involved in stimulating natural killer cells and pro- serotype. Several groups have now demonstrated that a moting the differentiation of cytotoxic T cells.20–22 INF-␥ strong dose-dependent humoral immune response is is known to up-regulate MHC class I on antigen induced by Ad vectors leading to the development of presenting cells.23 Thus, both INF-␥ and IL-12, while cap- 6,7,12–15 neutralizing antibodies to adenoviruses. Studies able of inhibiting humoral immunity, might enhance the elimination of Ad vector-transduced cells by CTLs. Activated T cells play a critical role in the generation of Correspondence: A Scaria, Genzyme Corporation, One Mountain Road, humoral and cellular immune responses. The interaction Framingham, MA 01701, USA between the T cell receptor (TCR) and antigen-major his- Received 30 October 1996; accepted 12 February 1997 tocompatibility complex (MHC) expressed on the surface Repeat administration of adenoviral vector A Scaria et al 612 of an antigen presenting cell (APC) is necessary but not sufficient for the optimal activation of T cells which also requires additional costimulatory signals provided by several receptor–ligand pairs including B7-CD28 and CD40-CD40 ligand (CD40L).18 CD40L is expressed transiently at high levels on activated CD4+ T cells.24,25 The costimulation provided by CD40L on T cells interacting with CD40 on B cells and other APCs seems to be essential for thymus-dependent humoral immunity25–28 and may also play an important role in the generation of cellular immune responses through the production of helper cytokines.28–30 In this article, we describe experiments aimed at blocking this costimulation pathway transiently using a monoclonal antibody against CD40L. The results of these experiments show that, in the context of administration of an Ad vector to mouse airways, anti- CD40L inhibited both humoral and cellular immune responses.

Results

Anti-CD40 ligand suppresses development of Ad-specific antibodies To explore the role of the CD40–CD40L interaction in the generation of antibodies to Ad vectors, BALB/c mice were injected intraperitoneally with anti-CD40L monoclonal antibody (MR1, 200 ␮g/injection per mouse on days −2, +2, +6 and +10). Ad2/CFTR2 vector (109 IU) was instilled intranasally on day 0. Analysis of serum from these animals by ELISA showed a marked decrease in anti-Ad (IgG + IgM + IgA) titers in MR1-treated mice for up to 41 days (Figure 1a). Analysis of bronchoalveolar lavage (BAL) fluid revealed a parallel drop in Ad-specific IgA levels in MR1-treated mice (Figure 1b). On day 38 after the first administration of Ad2/CFTR2, a vector of the same serotype, Ad2/␤Gal2, was administered to the different groups of mice. Expression of ␤- galactosidase was measured by a quantitative assay on Figure 1 (a) Adenovirus-specific serum antibodies. Ad2/CFTR2 vector day 41. As might have been predicted from the reduced (109 IU) was instilled on day 0 and MR1 (200 ␮g per mouse per injection) − + + + levels of pulmonary anti-Ad IgA, ␤-galactosidase levels was given IP on days 2, 2, 6 and 10. Serial two-fold dilutions of were elevated in MR1-treated mice compared with serum collected at different time-points were analyzed by ELISA. Data are presented as the mean titer of four individual animals ± standard error control mice (Figure 2). of the mean (s.e.m.). *P Ͻ 0.001 using Student’s t test, for all time-points comparing ± MR1. (b) Adenovirus-specific IgA levels on day 40. BAL Decreased CTL response against Ad vector, increase in samples obtained from mice on day 40 following administration of vector persistence and decreased inflammation were analyzed by ELISA. Results are expressed as the mean IgA concen- ± Ͻ BALB/c mice were instilled intranasally with 109 IU of tration from three individual mice s.e.m. *P 0.05 using Student’s t test. Ad2/␤Gal-4 on day 0 and injected with MR1 on days −2, +2, +5 and +8. As shown in Figure 3a, spleen cells from MR1-treated mice showed decreased yet measurable lev- characterized by inflammatory cell infiltrates was present els of CTL activity compared with spleen cells from on day 5 and was not yet resolved at day 21. On day 5, untreated control mice, albeit using an assay that is not there were no differences noted between the lungs of strictly quantitative. mice treated with the vector either with or without MR1 Since CTLs have been implicated in the loss of trans- antibody treatment (data not shown). However, on day gene expression, we tested whether administration of 21, there were fewer inflammatory changes in all regions MR1 with an Ad vector would give rise to prolonged of the lungs from mice treated with the antibody (Figure transgene expression. As shown in Figure 3b, transgene 4). The inflammatory cell infiltrate was markedly reduced expression measured by a quantitative galactosidase in the peribronchial/peribronchiolar and perivascular assay, declined to background levels by day 21 in the regions. untreated controls. By comparison, in MR1-treated mice, transgene expression also declined but remained consist- Secondary antibody response in a preimmunized host ently higher than in untreated mice. MR1 was tested for its ability to interfere with the sec- Lung tissue from the mice was examined for evidence ondary antibody response in mice that had been preim- of histopathological changes in the peribronchial, peri- munized with Ad vector. Mice were instilled with 108 IU vascular and alveolar regions. Lung inflammation of Ad2/CFTR2 on day 0 and readministered with 108 IU Repeat administration of adenoviral vector A Scaria et al 613

Figure 2 Efficient re-administration of Ad vector. Ad2/CFTR2 (109 IU) and MR1 administrations were as described for Figure 1. Ad2/␤Gal2 virus was administered to mice on day 38 and the lungs assayed for ␤- galactosidase levels on day 41. Data are presented as means ± s.e.m. (n = 4). *P Ͻ 0.05 using Student’s t test, comparing ± MR1. of Ad2/CFTR2 on day 50. We had previously determined that an intranasal instillation of 108 IU of Ad vector elicits both humoral and cellular immune responses to the vector in several strains of mice including BALB/c (data not shown). MR1 injections were given around the time of the second virus administration on days 44, 48, 52 and 56. Sera from the different groups of animals were analyzed both for anti-Ad ELISA titers (IgG + IgM + IgA) and Ad neutralizing titers at various time-points. The secondary antibody response measured by ELISA was only slightly decreased in the MR1-treated mice but was less sustained (Figure 5a). The antibody titers declined more rapidly and by the time of the third vector administration on day 99, both the ELISA titers and Ad neutralizing titers were clearly decreased in the mice that had received MR1 along with the second administration of Ad vector (Figure 5a and b). The ␤-galactosidase encoding Ad2/␤Gal-2 vector was given as the third administration on day 99 and ␤- galactosidase levels in the lung were measured 3 days later on day 102. Control mice that had received the first (day 0) and second dose (day 50) of Ad2/CFTR2 showed only 6% of the ␤-galactosidase activity in lung homogen- ates compared with naive mice that received a single intranasal administration of Ad2/␤Gal-2 (Figure 6). Mice that received MR1 around the time of the second administration of Ad2/CFTR2 showed 10-fold higher levels of Figure 3 (a) Decreased CTL response against Ad vector. Mice were transgene expression after the third administration com- instilled intranasally with 109 IU of Ad2/␤Gal4 vector on day 0 and pared with untreated control mice (Figure 6). The effec- injected with MR1 (250 ␮g per mouse per injection) on days −2, +2, +5 tiveness of the third vector administration in the MR1- and +8. On day 21, spleen cells were collected, restimulated in vitro with infected syngeneic fibroblasts and tested for cytolytic activity. Results treated animals was comparable to that obtained in ± naive mice. shown are mean percentage lysis s.e.m. from triplicate wells at various effector:target ratios. (b) Increased persistence of transgene expression. Lungs from parallel groups of mice were assayed for ␤-galactosidase levels Discussion at various time-points after Ad2/␤Gal4 administration. Data are presented as means ± s.e.m. (n = 4). Ad-based gene therapy for genetic diseases such as cystic fibrosis will likely require repeated administrations due to the transient nature of transgene expression from cur- a clinical reality. We and others6,7,13,14 have shown that rent vectors. While improvements in vector design administration of Ad vectors to the airways leads to the should increase persistence of expression to some degree, activation of Ad-specific T and B cells. The cellular the barrier of humoral immunity against Ad also needs immune response includes CD8+ T cells which, in many to be overcome before Ad-based gene therapy becomes animal models, appear to be involved in the rapid decline Repeat administration of adenoviral vector A Scaria et al 614

Figure 4 Decreased lung inﬂammation in MR1-treated mice. Mice were instilled with 109 IU of Ad2/CFTR2 on day 0 and given MR1 (200 ␮g per mouse per injection) on days −2, +2, +6 and +10. Lungs were ﬁxed and examined for histopathological changes. Results shown are for day 21.

in transgene expression.6–9 The humoral immune response, which is dependent on MHC class II-mediated antigen presentation to CD4+ T cells,6 promotes the generation of Ad-specific neutralizing antibodies of both IgA and IgG isotypes.14 This serotype-specific antibody response correlates with the reduced effectiveness of repeated administrations of a vector of the same serotype.15 In this article, we demonstrate that a transient blockade of costimulation between T cells and B cells and other APCs using a monoclonal antibody (MR1) against CD40 ligand suppresses the development of antibodies against Ad and allows for efficient readministration of vector. MR1 treatment partially decreased the cellular immune response to Ad vector and this correlated with an increase in persistence of transgene expression. As expected, MR1 treatment did not affect the nonspecific acute phase of inflammation induced by intranasal administration of Ad vector (day 5, not shown), but the Figure 5 (a) Effect of MR1 on secondary antibody response. Mice were level of pathology observed during the chronic T cell- instilled with 108 IU of Ad2/CFTR2 on days 0 and 50. MR1 injections dependent phase of inflammation6 (day 21) was mark- were given on days 44, 48, 52 and 56. Serial two-fold dilutions of sera edly reduced in MR1-treated mice. collected at different time-points were assayed for Ad-specific antibodies Most CF patients will have been exposed to human by ELISA. Data are presented as the mean titer of four individual animals ± s.e.m.. *P Ͻ 0.05 and **P Ͻ 0.02 using Student’s t test. (b) Neutraliz- adenoviruses at some point in their lives and probably ing antibody titers at the time of readministration. Sera from day 94 will have immunological memory against the serotype(s) samples were also analyzed for anti-Ad neutralizing titers as described in of adenovirus to which they have been exposed. It is Materials and methods. Data are presented as means ± s.e.m. (n = 4). possible that the pre-existing anti-Ad humoral immunity *P Ͻ 0.05 using Student’s t test. in these individuals might interfere with a single or repeated Ad vector administration. To test this directly, we studied gene expression in mice that were already allografts.35 In models of acute GVHD, it has been shown immunized against Ad vector. We found that coadminis- that anti-CD40L can completely block the generation of tration of MR1 inhibited the development and persistence anti-host CTL responses. It has been proposed that this of a secondary antibody response and resulted in high is due to the ability of anti-CD40L to interfere with the levels of transgene expression upon a third adminis- maturation of professional APC so that they do not tration of vector to the airway. This finding is consistent acquire appropriate costimulatory molecules optimally to with reports that CD40L is critical for formation of germi- present antigen.36 Recent studies confirm that CD40 may nal centers as well as their maintenance.31,32 play an important role in dendritic cell function37 and Recent studies in animal models have shown that treat- therefore impact on the priming of T cells.38 In this study, ment with anti-CD40L in vivo can block the development we have demonstrated the inhibitory effect of MR1 on of collagen-induced arthritis33 and graft-versus-host dis- humoral and cellular immune responses against Ad vec- ease (GVHD)34 and prevent rejection of pancreatic islet tors for several weeks after the last administration of Repeat administration of adenoviral vector A Scaria et al 615

Figure 6 Third administration of Ad vector. Ad2/CFTR2 (108 IU) and MR1 administrations were as described for Figure 5. Mice were then instilled with 2 × 109 IU of Ad2/␤Gal2 on day 99 and lungs were assayed for ␤-galactosidase levels on day 102. Data are presented as means ± s.e.m. (n = 4). *P Ͻ 0.05 using Student’s t test, comparing group 1 versus 2 and group 3 versus 4.

MR1, at which time less than 2% of biologically active Adenoviral vectors MR1 should still be present in the serum.25 It therefore The construction of Ad2/CFTR2 and Ad2/␤Gal-2 has appears possible to readminister an Ad vector using been described previously.7 They are both Ad2-based a transient blockade of T cell costimulation without vectors with most of the E1 region deleted and replaced complete immunosuppression of the host for prolonged with the transgene and the E4 region replaced with the periods of time. open reading frame 6 (ORF6) of E4. Ad2/␤Gal-4 is ident- Since it is known that several interactions including ical to Ad2/␤Gal-2 except that it contains the complete both B7–CD28 and CD40–CD40L are essential for optimal wild-type E4 region. 18,28,39 costimulation between T cells and APC, a combined Histopathology therapy with CTLA4Ig and MR1 might be even more On the day of death, mice were killed with an IP injection effective. Although the practicability of coadministration of Somlethal (Euthasol; King Pharmaceuticals, Bristol, of viral vectors and humanized recombinant antibodies TN, USA). The lungs were cleared of blood by vascular to CF patients is questionable, such studies have and will perfusion with phosphate-buffered saline (PBS). The tra- reveal more details about the process of immune recog- chea was cannulated and the lungs and trachea removed. nition of Ad vectors and may suggest simpler strategies The lungs were fixed by inflation with 2% paraformal- to block that process. dehyde with 0.2% glutaraldehyde in PBS, pH 7.4, at a pressure of 30 cm of H2O. Following overnight fixation, Materials and methods portions of the left lung were embedded in glycometh- acrylate, sectioned and stained with hematoxylin and Antibody injections eosin. These sections were evaluated by light microscopy MR1, a hamster anti-mouse CD40 ligand monoclonal for the presence and distribution of lung inflammation antibody was produced in ascites and purified by ion without previous knowledge of treatment.40 The lung exchange HPLC as previously described.24 BALB/c mice sections were subjectively assessed for morphologic alter- were purchased from Taconic (Germantown, NY, USA) ations on a scale of 0–4: 0 = no lesion, 1 = minimal, 2 = and were typically injected intraperitoneally (IP) with a mild, 3 = moderate, 4 = severe. total of four injections of 200 or 250 ␮g of MR1/mouse Adenovirus-specific antibodies starting on day −2 relative to the time of administration Titers of Ad-specific serum antibodies were evaluated by of the Ad vector. ELISA. Serial two-fold dilutions of sample were added to Repeat administration of adenoviral vector A Scaria et al 616 the wells of a 96-well plate coated with photochemically The protein concentration in an individual sample was inactivated Ad2 (Lee Biomolecular Research, San Diego, determined using the BioRad DC reagent (BioRad, Her- CA, USA). Bound virus-specific antibodies were detected cules, CA, USA) and the results are expressed as relative by the addition of horseradish peroxidase (HRP)-conju- light units (RLU)/␮g protein. gated goat anti-mouse Ig (IgG, IgM, IgA-specific; Jackson Immunoresearch Laboratories, West Grove, PA, USA). Neutralizing antibody titers The titer was defined as the reciprocal of the highest To determine neutralizing antibody titers, serial two-fold dilution of sample which produced an OD greater dilutions of sample were incubated with live Ad2/CFTR- 490 ° than 0.1. 2 for 1 h at 37 C/5% CO2 in the wells of flat-bottom 96- To evaluate levels of Ad-specific IgA in BAL, samples well plates. At the end of the incubation period, permiss- were diluted two-fold and added to Ad2-coated plates ive 293 cells were added to the wells and the plates were ° followed by the addition of HRP-conjugated goat anti- incubated at 37 C/5% CO2 for 72–96 h. The assay was mouse IgA (␣ chain-specific; Cappel, Durham, NC, USA). read when control 293 cells incubated alone reached у For quantification, a standard curve was constructed 90% confluency. The neutralizing antibody titer was using a monoclonal antibody against mouse IgA (Harlan defined as the reciprocal of the highest dilution of sample Sera-Lab, Sussex, UK) to coat ELISA plates and capture that showed any detectable protection of 293 cells from known amounts of purified mouse IgA (Cappel). The cytopathic effects when compared with cells incubated with untreated virus or virus incubated with sero- OD490 values obtained following the addition of HRP- conjugated goat anti-mouse IgA were plotted against the negative serum. amounts of IgA standard (ng/ml) added to the wells. The concentrations of Ad-specific IgA present in BAL samples Acknowledgements were then derived from the standard curve by linear We wish to thank the Virus Production Unit at Genzyme regression analysis. Corporation for preparation of the Ad vectors used in these studies, Wiley Smith and Carol Sacks for technical Cytotoxic T cell assay help with the animal studies, Sarah Pennington, Kristen To evaluate cytotoxic T lymphocyte (CTL) activity, spleen Couture and Margaret Nichols for their participation in cells from animals in the same group were pooled and the immunological and ␤-galactosidase assays. stimulated in vitro with mitomycin-C-inactivated, syngeneic fibroblasts infected with Ad2/␤Gal-4 at a multi- References plicity of infection (MOI) of 50 for 24 h. Cells were cul- 1 Stratford-Perricaudet L et al. Evaluation of the transfer and tured in 24-well plates containing 5 × 106 spleen cells and expression in mice of an enzyme-encoding gene using a human × 4 6 10 stimulator fibroblasts per well in a 2 ml volume. adenovirus vector. Hum Gene Ther 1990; 1: 241–256. The culture medium consisted of RPMI-1640 medium 2 Rosenfeld MA et al. 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