Immune Modulation

Other specific strategies include reduction of the costimulatory signaling activity using the CTLA4 immunoglobulin Fc (CTLA4-Ig) and blocking CD40-CD40L interaction (Fig. 2). Interaction of APC with processed adenovirus antigens or adenovirus transgene antigens with T cells through the MHC-T-cell receptor (TCR) ligation constitutes signal 1, which is an incomplete signal and can induce T-cell nonresponsiveness or anergy. A second signal can be provided by interaction with B7-1 or B7-2 (CD80/CD86) on the APC with CTLA4 (CD 152) or CD28 on the T cell (Fig. 2). A soluble form of CTLA4-Ig (sCTLA4-Ig) can bind to B7-1 and B7-2 and block interactions of this molecule with membrane bound CTLA4 on T cells. The anti-adenovirus response can be decreased and there is prolonged expression of the adenovirus transgene in the presence of either sCTLA4-Ig or in the presence of an adenovirus that expresses CTLA4-Ig (AdsCTLA4-Ig).

Kay et al. [17, 18] demonstrated that systemic coadministration of recombinant adenovirus with sCTLA4-Ig leads to persistent adenovirus gene expression in mice without long-term immunosuppression. Ideguchi et al. [19] utilized local administration of adenovirus that expressed ¡3-galactosidase as well as CTLA4-Ig (AdsCTLA4-Ig) in the central nervous system and showed that this combination decreased T-cell infiltration and also decreased the anti-adenovirus antibody titer. Expression of P-galactosidase at the injection site in the striatum and corpus callosum peaked at day 6 and remained until day 60 in both control and treated groups at about the same level despite suppression of the inflammatory response. Guibinga et al. [20] showed that the combination of CTLA4-Ig plus FK506 resulted in prolonged adenovirus vector-mediated production of dystrophin compared to treatment with either immunosuppressive alone. Schowalter et al. [21] demonstrated that murine CLTA4-Ig markedly prolonged adenovirus transgene expression in the liver and diminished formation of neutralizing antibodies as well as decreasing the proliferative response without causing irreversible immune suppression. Ali et al. [22] used AdsCTLA4-Ig administration in association with intraocular administration

Figure 2 Role of costimulatory molecules to induce cytotoxic or helper T-cell response and promote anti-Ad antibody production. Signal 1 consists of the processed antigen presented by the MHC expressed by APCs stimulation of T cells. These antigens are recognized by specific T-cell receptors (TCRs) on T cells. This interaction is also assisted by CD8 expressed on cytotoxic T cells or CD4 expressed on helper T cells that interact with MHC. In addition to this central, specific T-cell-APC interaction, costimulatory molecules are necessary for optimal T-cell response. These consist of CD28 and CTLA4 (CD152) expressed on T cells that interact with B7.1 and B7.2 (CD80 and CD86), expressed on APCs. This interaction can be blocked by soluble CTLA4-lg (sCTLA4-lg) that tightly binds to B7.1 and B7.2 and prevents the interaction of this molecule expressed on APCs with CD28/CTLA-4 expressed on T cells. A second costimulatory molecule pathway consists of CD40 ligand (CD154) expressed on T cells that interact with CD40 expressed on APCs. This interaction can be blocked by administration of a soluble CD40-lg (sCD40-lg) which binds with the CD40 ligand and prevents the interaction between CD40 and CD40 ligand.

Figure 2 Role of costimulatory molecules to induce cytotoxic or helper T-cell response and promote anti-Ad antibody production. Signal 1 consists of the processed antigen presented by the MHC expressed by APCs stimulation of T cells. These antigens are recognized by specific T-cell receptors (TCRs) on T cells. This interaction is also assisted by CD8 expressed on cytotoxic T cells or CD4 expressed on helper T cells that interact with MHC. In addition to this central, specific T-cell-APC interaction, costimulatory molecules are necessary for optimal T-cell response. These consist of CD28 and CTLA4 (CD152) expressed on T cells that interact with B7.1 and B7.2 (CD80 and CD86), expressed on APCs. This interaction can be blocked by soluble CTLA4-lg (sCTLA4-lg) that tightly binds to B7.1 and B7.2 and prevents the interaction of this molecule expressed on APCs with CD28/CTLA-4 expressed on T cells. A second costimulatory molecule pathway consists of CD40 ligand (CD154) expressed on T cells that interact with CD40 expressed on APCs. This interaction can be blocked by administration of a soluble CD40-lg (sCD40-lg) which binds with the CD40 ligand and prevents the interaction between CD40 and CD40 ligand.

of adenovirus encoding ß-galactosidase and demonstrated reduced immune response to adenovirus, as well as prolonged expression of the transgene in retinal cells. Kay et al. [17] showed that combined treatment with sCTLA4-Ig and anti-CD40 ligand resulted in prolonged adenovirus-mediated gene expression for up to 1 year in the liver and the ability to readminister adenovirus in 50% of mice. Following readministration, there was persistent secondary gene expression lasting 200-300 days, and diminished spleen proliferative response, tumor necrosis factor (TNF)-ot and IFNy production and decreased production of neutralizing antibodies. Chirmule et al. [23] showed that despite the absence of CD40-CD40 ligand interaction in CD40 ligand knockout mice, after administration of LacZ into the mouse lung, these mice developed a functional humoral response to the vector evidenced by germinal center formation and anti-adenovirus IgGl and IgA that resulted in effective neutralization of virus and prevented effective readministration of the virus.

Wilson et al. [24, 25] used combined treatment with an adenovirus vector expressing sCTLA4-Ig to block CD28 stimulation and a monoclonal antibody against CD40 ligand to demonstrate prolonged adenovirus transgene expression after intratracheal administration. In addition, secondary administration and transgene expression after secondary administration was prolonged in the lung, but there was increased reaction from the liver. These results indicate that the mechanisms limiting transgene expression in the airways and the alveoli are different to those of the liver. Stein et al. [26] have shown that combined treatment of Ad-human factor IX (FIX) with an anti-CD40 ligand antibody MR-1 as well as depletion of macrophage liposomes resulted in prolonged expression of AdFIX as well as higher levels of plasma FIX. This persistence was accompanied by inhibition of anti-adenovirus IgG, and decreased IL-10 and IFN-y production from spleen lymphocytes following reexposure to virus particles in vitro. This treatment regimen also enabled secondary and tertiary infusions of AdFIX which was superior to treatment with CD40 ligand blockade alone. Kuzmin et al. [27] utilized macrophage depletion in combination with blockade of CD40 ligation to demonstrate the prolonged expression of transgene after administration of El-deleted adenovirus. This resulted in a decreased cellular and humoral response as well as the induction of transgene tolerance in the animals. Animals that were rendered immunologically unresponsive to vector and transgene antigens regained their ability to mount a productive immune response against the vector after recovery of immune function but remained unresponsive to the transgene product. Stein et al. [28] used an anti-CD40 ligand (anti-CD154) in combination with adenovirus-mediated low-density-lipoprotein receptor (LDLR) gene transfer in LDLR-deficient mice to demonstrate that these mice express LDLR on hepatocytes and maintain cholesterol levels below or within the normal range for at least 92 days.

It has been more difficult to eliminate B-cell responses. B-cell production of neutralizing antibodies is decreased after treatment with anti-CD40 or soluble CD40 [29] and deoxyspergualin [30-32]. Readministration of adenovirus vector has been achieved in the lungs of nonhuman primate by blocking of CD40-CD40 ligand interactions. A humanized anti-CD40 ligand antibody hu5C8 was used to treat primates in the presence of administration of adenovirus vectors [23]. These animals produced IgM but did not develop secretory IgA or neutralizing antibodies. This is significant since this is the first demonstration that anti-Ad neutralizing antibodies could be inhibited in a primate system by inhibiting the CD40 interaction with CD40 ligand.

A third approach includes modification of the adenovirus vector to reduce the immune response [33-36]. A more universal strategy for decreasing response to adenovirus vectors includes production of the "gutless" adenovirus which greatly reduces the immune response to Ad and its transgene [37-40]. It was initially demonstrated that constitutive expression of the immune modulatory gpl9 K protein in adenovirus vectors reduced the cytotoxic response. Further refinement of vectors including the removal of E4 also resulted in prolonged transgene expression [34], A gutless Ad that was depleted of all adenovirus genes, showed prolonged expression of |3-galactosidase in muscle. This prolonged expression correlated with a decrease in the infiltration of CD4+ and CD8+ lymphocytes. However, in LacZ transgenic mice, which was predicted to result in immunologic tolerance to |3-galactosidase expression, there was prolonged expression of the vector DNA, indicating that the immune response to this "gutless" Ad was primarily against p-galactosidase and that the response to the adenovirus vector lacking all genes was minimal [37], Gene therapy expression using adenovirus vectors with deletions of the El, E2A, E3, and E4 regions could be prolonged when combined with immunosuppressive drugs including cyclophosphamide and FK506.

One limitation of immunomodulating therapy has been that it is not specific for the adenovirus or transgene. The present review will focus on our attempts to reduce the immune response mediated by TNFa and other cytokines. A second strategy to prolong gene therapy expression is to ablate the immune response to cells that are the target of gene therapy. Such apoptosis-inducing factors include TNFa but also Fas ligand and TNF receptor apoptosis-inducing ligand (TRAIL). We have also developed methods to specifically reduce the T-cell response to Ad and also new methods to prevent B cell responses including blocking of the TNF receptor (TNFR) homolog transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI) signal in B cells. These strategies strongly suggest that it will be possible to develop strategies to ablate the immune response to adenovirus including the cytotoxic response that leads to the loss of cells carrying the transgene.

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