Development and characterization of recombinant human Fc:OX40L fusion protein linked via a coiled-coil trimerization domain

Abstract

OX40 (CD134) is a potent costimulatory molecule found on the surface of activated CD4⁺ and CD8⁺ T cells. Immunotherapy with OX40 agonists administered in vivo has demonstrated efficacy in several murine tumor models. A phase I clinical trial is currently underway in patients with advanced cancer using a mouse anti-CD134 monoclonal antibody. Therapy with this antibody will likely be limited to one cycle because patients develop neutralizing human anti-mouse antibody (HAMA). Therefore, we developed a humanized OX40 agonist that links the extracellular domain of human OX40L to the Fc domain of human IgG₁ via a trimerizing isoleucine zipper domain (ILZ). Physical characterization by velocity sedimentation revealed that this novel construct, hFcILZOX40L, was assembled into hexamers in which the Fc domains formed three disulfide-bonded dimers and the ILZ-OX40L domains formed two trimers. Trimerization of the ILZ domain was necessary to achieve appropriate assembly. In vitro biologic activity of the hFcILZOX40L hexamer was equivalent to the activity of agonist antibodies in plate-bound assays and was superior when the agonists were tested as soluble agents. Our ultimate goal is to use this recombinant molecule in a future clinical trial, and we feel that the OX40L hexamer will have equivalent or superior agonist activity in vivo when compared to an anti-OX40 antibody.

Introduction

Members of the TNF superfamily and their receptors play critical roles in T-cell immunity. These include initiation, expansion, differentiation, and down-regulation of immune responses (see Watts, 2005, for review). Typically, T-cell priming is initiated by the engagement of the T-cell receptor (TCR) and peptide/MHC complex accompanied by costimulation via CD28/B7.1. Reciprocal interactions between the APC and T cell can lead to additional costimulation late during priming that can substantially modulate the overall T-cell response. The TNF super family receptor, OX40, provides one such late priming costimulatory signal (Mallett et al., 1990, Paterson et al., 1987). OX40 expression peaks 24–48 h after TCR engagement and then diminishes over the following 3 days (Gramaglia et al., 1998), while OX40L is expressed on APCs during a similar time frame. The expression of both OX40 receptor and OX40L depends on early events such as the strength of TCR engagement, CD28 signaling, and CD40 ligation (Croft, 2003, Weinberg, 2002).

Maximizing signaling via OX40 using exogenous agonists has profound effects on the T-cell response to both proteins and tumor cells following immunization (Croft, 2003, Sugamura et al., 2004). Analysis of CD4⁺ T-cell responses following in vivo OX40 engagement showed an increase in antigen-specific T-cell expansion, enhanced cytokine production, and an increase in the generation and stability of a memory T cells (Evans et al., 2001, Huddleston et al., 2006, Kaleeba et al., 1998, Weinberg et al., 1998). In mouse tumor models, OX40 engagement, with an agonist antibody or soluble ligand in the absence of immunization, produced antitumor effects against breast and colon cancers, melanoma, sarcoma, and glioma (Kjaergaard et al., 2000, Kjaergaard et al., 2001, Morris et al., 2001, Pan et al., 2002, Weinberg et al., 2000). The detection of OX40⁺ T cells in a variety of human solid tumors by immunohistochemistry (Ladanyi et al., 2004, Petty et al., 2002, Ramstad et al., 2000) suggests that activated tumor-infiltrating T cells could serve as targets for activating tumor immunotherapy. In animal models for cancer and autoimmune disease, these OX40⁺ T cells were sorted and shown to recognize tumor or auto antigens, respectively (Weinberg, 2002). Strategies to exploit the OX40-OX40L system include agonists such as anti-OX40 monoclonal antibodies or recombinant soluble OX40L, and antagonists, which include monoclonal antibodies to OX40L or OX40:Ig (al-Shamkhani et al., 1996, Ali et al., 2004, Weinberg et al., 1999). We have initiated a phase I clinical trial using a mouse anti-human OX40 monoclonal antibody in patients with advanced cancer. As expected, human anti-mouse antibody (HAMA) responses developed within these patients and limited application of anti-OX40 therapy to one cycle. While a single cycle of therapy was sufficient in mouse tumor models, it is likely that repeated cycles of OX40 agonist therapy would be needed to treat patients most effectively. Therefore, we developed a recombinant soluble human OX40L protein, which upon binding its target could potentially be a more potent agonist than the antibody and/or may also have a shorter half-life in vivo compared to humanized antibody. The shorter half-life might be beneficial because it would limit the agonist signal to a narrower window and the human origin of the construct would permit multiple cycles of therapy.

Ligands in the TNF family are typically homotrimeric type II transmembrane proteins that contain a small cytoplasmic N-terminal domain, a transmembrane domain, a stalk region, and a C-terminal receptor-binding domain that contains the signature TNF homology sequence and the trimerizing interactions (Bodmer et al., 2002). When CD40L (Haswell et al., 2001, Morris et al., 1999, Shiraishi et al., 2004), FASL (Holler et al., 2003, Shiraishi et al., 2004), TRAIL (Walczak et al., 1999, Wu et al., 2001), and 4-1BBL (Rabu et al., 2005) were expressed as soluble recombinant molecules, their biological activity was enhanced by inclusion individually or, in some cases, in combination of (a) the stalk region, (b) an additional trimerizing domain, and (c) an oligomerization domain that linked trimers together. A modified version of the alpha helical coiled-coil domain of the yeast transcription factor, GCN4, has been used as a trimerization domain in this context. When isoleucine residues occupy the a and d positions of the heptad repeat in coiled-coil alpha helical sequences (isoleucine zipper, ILZ), trimer formation with high thermal stability, T_m > 100 °C, is strongly preferred (Harbury et al., 1993). Linking together two or more trimers can be achieved by crosslinking after biosynthesis (Rabu et al., 2005) or by incorporating a fusion partner like the Fc domain of IgG. The Fc:FasL fusion protein, which has a flexible linker between these two domains, was shown to assemble into a hexamer that contained two FasL trimers linked to three Fc dimers (Holler et al., 2003) (Fig. 1B). This arrangement provided adjacent FasL trimers that proved essential for FasL activity. The Fc domain fusion partners also enhance protein expression, provide stability/longevity in the circulation, and offer a convenient tool for purification (Lo et al., 1998). In an effort to optimize the structure and function of a recombinant OX40L molecule for therapeutic use, the complete extracellular domain of human OX40L was joined to the Fc domain of IgG₁ via an ILZ domain. This is the first description of a TNF-family member Ig fusion protein joined via a trimerization domain which was produced efficiently by a eukaryotic cell line, formed a hexameric structure, and exhibited potent biologic activity.