T cell growth control using hapten-specific antibody/interleukin-2 receptor chimera

Abstract

IL-2 is a cytokine that is essential for the expansion and survival of activated T cells. Although adoptive transfer of tumor-specific T cells with IL-2 is one of strategies for cancer immunotherapy, it is essential to replace IL-2 that exerts severe side effects in vivo. To solve this problem, we propose to use an antibody/IL-2R chimera, which can transduce a growth signal in response to a cognate antigen. We constructed two chimeras, in which ScFv of anti-fluorescein antibody was tethered to extracellular D2 domain of erythropoietin receptor and transmembrane/cytoplasmic domains of IL-2Rβ or γ chain. When the chimeras were co-expressed in IL-3-dependent pro-B cell line Ba/F3 and IL-2-dependent T cell line CTLL-2, gene-modified cells were selectively expanded in the absence of IL-3 and IL-2, respectively, by adding fluorescein-conjugated BSA (BSA-FL) as a cognate antigen. Growth assay revealed that the cells with the chimeras transduced a growth signal in a BSA-FL dose-dependent manner. Furthermore, STAT3, STAT5, ERK1/2 and Akt, which are hallmarks for IL-2R signaling, were all activated by the chimeras in CTLL-2 transfectant. We also demonstrated that the chimeras were functional in murine primary T cells. These results demonstrate that the antibody/IL-2R chimeras could substantially mimic the wild-type IL-2R and could specifically expand gene-modified T cells in the presence of the cognate antigen.

Introduction

Interleukin-2 (IL-2) is an important immunomodulatory cytokine that promotes proliferation, activation and differentiation of T cells, and is also necessary for B cell and natural killer (NK) cell function. IL-2 binds to IL-2 receptor (IL-2R) consisting of three subunits, i.e. IL-2Rα, IL-2Rβ and IL-2Rγ chains, and induces heterotrimerization of them, followed by signal transduction. IL-2Rα alone or IL-2Rβ alone has low affinity (K_d ≈ 10 nM or ≈100 nM) to IL-2, while IL-2Rγ alone has no detectable affinity to IL-2. A high-affinity receptor (K_d ≈ 10 pM) is composed of all three chains, whereas in the absence of IL-2Rα expression, the other two chains have intermediate affinity (K_d ≈ 1 nM) to IL-2 [1], [2]. Naïve T cells express IL-2Rγ and low levels of IL-2Rβ, but do not express IL-2Rα, resulting in less sensitivity to IL-2. The expression of IL-2Rα is restricted to activated T cells through activation of T cell antigen receptor-mediated signaling, and allows the cells to respond to IL-2 for proliferation. Although IL-2Rα is required for the high-affinity receptor complex, IL-2Rα is dispensable for signaling, since ectopic expression of either c-kit/IL-2Rβ and c-kit/IL-2Rγ chimeras or GM-CSFRα/IL-2Rγ and GM-CSFRβ/IL-2Rβ chimeras induced ligand-dependent cell proliferation of CTLL-2 T cell line [3]. These results demonstrated that a heterodimerization of IL-2Rβ and γ chains may be sufficient for transducing growth signal in T cells.

Genetic modification of T cells is an effective approach to improve anti-cancer immunotherapy and to study T cell functions. Many investigators have tried to utilize T cells for cancer immunotherapy. One promising approach is an adoptive transfer therapy, in which tumor-specific autologous T cells are isolated, expanded in vitro and reinfused with IL-2 for further expansion in vivo [4], [5], [6], [7], [8]. The problem is that because most tumor antigens have very low antigenicity and their expression levels are low, rapid expansion and long-lasting maintenance of a large number of tumor-specific T cells are difficult in vivo, leading to insufficient therapeutic effect. In fact, the targets of previous reports and clinical trials have been restricted to the tumors having high antigenicity like melanoma cells and viral-antigen-expressing tumors [9], [10], [11]. Therefore, expansion of transferred T cells in vivo is critical for an efficient immunotherapy. However, T cell expansion by administrating high-dose IL-2 causes undesirable side effects such as vascular leak syndrome, and cardiac and pulmonary dysfunction, because IL-2 also directly or indirectly activates several other immune cells including NK cells, B cells, neutrophils and macrophages [12], [13]. One possible clue to overcome this problem is to genetically modify autologous T cells with an engineered IL-2R that could respond to non-toxic substance.

We previously developed an antigen-mediated genetically modified cell amplification (AMEGA) system using an antibody/receptor chimera that triggers a growth signal in response to a specific antigen [14], [15], [16]. An anti-fluorescein single-chain Fv (ScFv) was fused to extracellular D2 domain of erythropoietin receptor (EpoR) and transmembrane and cytoplasmic domains of gp130 to create an antibody/receptor chimera (ScFvg). When IL-3-dependent murine pro-B cell line Ba/F3 was transduced with the antibody/receptor chimera, fluorescein-conjugated BSA (BSA-FL) induced oligomerization of ScFvg chains, enabling cell growth in the medium containing BSA-FL but without IL-3 [15]. In this study, we replaced the cytoplasmic domain of ScFvg with that of IL-2Rβ and γ chains to construct antibody/IL-2R chimeras that can mimic an IL-2-mediated growth signal with BSA-FL-mediated one (Fig. 1A). We investigated whether these chimeras could be functional in Ba/F3 cells, an IL-2 dependent T cell line CTLL-2 and murine primary T cells.