Gene Therapy (2002) 9, 537–546 2002 Nature Publishing Group All rights reserved 0969-7128/02 $25.00 www.nature.com/gt RESEARCH ARTICLE Transient blocking of both B7.1 (CD80) and B7.2 (CD86) in addition to CD40–CD40L interaction fully abrogates the immune response following systemic injection of adenovirus vector C Ziller1, F Stoeckel1, L Boon2 and H Haegel-Kronenberger1 1TRANSGENE, Strasbourg, France; and 2Tanox Pharma BV, Amsterdam, The Netherlands Blockade of the CD40–CD40L and CD80/CD86–CD28 of the immune response against Ad. Using either of these costimulatory pathways represents a strategy to inhibit the mAb pairs, a second vector could be administered 1 month immune response against Ad vectors designed for gene after the first injection but with lower efficiency than in naive therapy applications. Since most previous studies have used animals. Thus, CD86 stands as the pivotal B7 molecule a CTLA4-Ig fusion molecule binding to both CD80 and involved in the development of the immune response against CD86, the respective roles of these B7 molecules remained Ad. However, only the blockade of both CD80 and CD86 undefined. We have studied the effect of blocking mono- in addition to CD40L fully inhibited the humoral and cellular clonal Abs (mAbs) directed against the costimulatory mol- responses against the Ad vector, such that readministration ecules CD40L, CD80 and CD86, alone or in different combi- after 1 month was as efficient as in naive animals. At the nations, on the humoral and cellular immune responses time of readministration, treated animals had regained their against Ad. Groups of mice were transiently treated with ability to mount a normal immune response to the second each combination of blocking mAbs upon systemic injection Ad vector, showing that tolerance was not induced. of a first Ad vector. Combinations of anti-CD80 + anti-CD86 Gene Therapy (2002) 9, 537–546. DOI: 10.1038/sj/gt/3301684 or anti-CD40L + anti-CD86 mAbs resulted in strong inhibition Keywords: costimulatory molecules; gene therapy; adenovirus vector Introduction repeated systemic administrations do not achieve efficient re-transduction of the target tissues due to the The immune response directed against Ad vectors rep- production of circulating neutralizing antibodies resents a major limitation for their use in gene therapy (NAbs).1,6–9 In mouse models, transient depletion or applications which require repeated administrations. blocking of the CD4+ T cell population decreases the pro- Transduction of target tissues such as the liver or the duction of anti-Ad antibodies (Abs) and permits vector lung by non-replicative Ad vectors is inflammatory and readministration.10,11 However, these strategies induce induces humoral and cellular responses involving CD4+ + profound immunosuppression, potentially detrimental to and CD8 T cells which can be directed against adenovi- patients with genetic diseases. Furthermore, it may not 1–4 ral proteins and the transgene product. Cytotoxic T apply to primates as suggested by a recent study show- lymphocytes (CTLs) directed against immunogenic trans- ing that blocking of CD4+ cell responses did not decrease genes have been shown to limit the persistence of trans- the humoral response following vector instillation in the 3,4 duced cells. CTLs directed against adenoviral proteins lung of Rhesus monkeys.12 Ideally, a non-depleting treat- have also been suspected to affect the long-term persist- ment inducing only transient suppression of the immune ence of the viral genome by destroying transduced cells, response against the virus should be achieved. Such 5 due to leaky expression of late viral genes. However, approach should not lead to immunological tolerance to expression of a non-immunogenic transgene can be rela- Ad proteins, since patients must remain capable of tively stable and long-lasting, suggesting that anti-Ad responding against infection by wild-type Ad. CTLs only play a minor role in the elimination of Antigen presentation leads to productive lymphocyte 2–4 transduced cells. activation when co-stimulatory signals are delivered by The humoral response to Ad proteins constitutes the the binding of surface molecules such as CD40, main obstacle to vector readministration. Particularly, CD80/B7.1 and CD86/B7.2, present on antigen- presenting cells (APCs) and up-regulated in the context of inflammation, with their ligands on T cells.13–15 In the Correspondence: H Haegel-Kronenberger, Experimental and Clinical Immunology, TRANSGENE, 11 rue de Molsheim, 67082 Strasbourg absence of co-stimulation, antigen recognition by the TCR Cedex, France has been shown to induce T cell anergy or unresponsive- Received 3 August 2001; accepted 28 January 2002 ness.15–19 CD28 and CTLA4 present on the surface of T Costimulation blockade and Ad vector readministration C Ziller et al 538 cells can bind indifferently CD80 and CD86 molecules on ated 4 weeks later, following administration of an Ad APCs, but deliver opposite signals: the binding of CD28 vector encoding a second reporter transgene (Ad-h␥IFN) contributes to T cell activation while CTLA4, which is in the absence of a costimulation-blocking treatment. The induced later during activation, down-regulates the T cell immune response was further analyzed after readminis- response and participates in the induction of peripheral tration to investigate the effect of the costimulation-block- tolerance.20–25 CD40L/CD154 is the ligand for CD40 and ing treatment on the capacity to elicit a posteriori an is transiently induced on T cells upon antigen recog- immune response against Ad. nition. Stimulation of CD40 molecules, present on the surface of B cells, by CD40L plays a crucial role in the Results development of the humoral response, including Ig iso- type switching and regulation of B cell proliferation. In Abrogation of the humoral response against Ad vector addition, activation of APCs through CD40 results in by blocking CD40L, CD80 and CD86 increased expression of CD80 and CD86 co-stimulatory We first tested the effect of all combinations of blocking molecules.13 Importantly, CD40 signalling in dendritic mAbs directed against CD40L, B7.1/CD80 and cells induces their functional maturation by up-reg- B7.2/CD86 on the anti-Ad immune response when ulating the expression of MHC and co-stimulatory mol- injected at the time of first vector administration. Figure ecules on their surface and the secretion of cytokines, 1 describes the experimental scheme comprising 10 such as IL-12.26 groups of 10 C57Bl/6 mice each. At day 0, an Ad vector In mouse models, a large body of evidence has sug- encoding human factor IX (Ad-hFIX, 109 IU/mouse) was gested that the blockade of either or both of the CD40– injected into the tail vein of animals treated with the CD40L and B7–CD28 co-stimulatory pathways can blocking mAbs that were injected intraperitoneally. We inhibit the development of an immune response against chose to use the C57Bl/6 mouse strain because it was Ad vectors.27–30 Cellular and humoral reponses against shown to be tolerant to the hFIX transgene product after Ad were partially inhibited by treatment with anti- i.v. injection of Ad-hFIX, resulting in sustained hFIX CD40L mAb, which allowed vector readministration to expression in the serum.4,37 Levels of this transgene pro- the lungs in mice and also in primates.29–31 Most encour- duct in the circulation therefore reflect the number of aging results have been obtained in the study of Kay et transduced cells in the targeted tissues. A second vector, al,28 where simultaneous blocking of CD40 and of B7 mol- encoding human ␥IFN (Ad-h␥IFN, 109 IU/mouse), was ecules using an anti-CD40L mAb and a CTLA4-Ig fusion administered intravenously 4 weeks later (day 28). Dos- molecule resulted in successful secondary Ad-mediated age of circulating h␥IFN was used as read-out to assess gene transfer to the mouse liver. These results showed the efficiency of readministration. that blockade of both co-stimulatory pathways is neces- The humoral response against Ad was measured fol- sary to efficiently inhibit the anti-Ad immune response lowing the first administration of Ad-hFIX and the following systemic administration. efficiency of immunosuppression was evaluated by com- Because the CTLA4-Ig molecule binds both CD80 and paring mouse groups that received the mAbs to the CD86, studies using this molecule do not allow the untreated group (Figure 1b, group 8, first vector injected respective contribution of B7 molecules to the immune response against Ad to be deciphered. The requirement of blocking either or both of CD80 and CD86 costimu- latory molecules in addition to CD40–CD40L interaction to achieve optimal suppression of the immune response remains to be resolved. Distinct and seemingly contradic- tory roles have been reported for CD80 and CD86 mol- ecules notably in the context of autoimmune diseases and tolerance induction.14–17,25,32–35 Transgenic mice over- expressing CD80 on mature B cells make reduced humo- ral responses to T-dependent antigens.36 Allowing CTLA4 engagement by CD80 has been shown to inhibit T cell activation in the absence of CD28.24 Since CD80 is up-regulated later than CD86 on the surface of activated APCs,15,17 it could be hypothesized that leaving CD80 molecules available could favor their interaction with CTLA4 on T cells and thereby contribute to down-regu- lation of T cell responses. CTLA4 shows around 10-fold greater affinity for B7 molecules than CD28 does, which is reflected by the capacity of the CTLA4-Ig fusion pro- Figure 1 Experimental design. (a) C57Bl/6 mice were treated or not with blocking mAbs against CD40L, CD80 and/or CD86 and injected i.v. with tein to inhibit CD28-mediated T cell responses. 109 IU of Ad-hFIX. The immune response against the Ad vector was To address these points and attempt to down-regulate evaluated and detection of the transgene product was followed in the the immune response against Ad with maximal efficacy, serum.