Man-made superantigens: Tumor-selective agents for T-cell-based therapy

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Abstract

Superantigens (SAgs) are a collection of bacterial and viral proteins with potent immunostimulatory properties. SAgs bind to Major Histocompatibility Complex Class II (MHC II) molecules of antigen presenting cells (APCs) and activate a high frequency of T lymphocytes. To target a T-cell attack against tumor cells we genetically linked tumor-specific antibody Fab fragments to the SAg Staphylococcal enterotoxin A (SEA). Fab-SEA fusion protein efficiently targeted to solid tumors and induced a T-cell-mediated eradication of established metastases in animal models. Successful therapy was T-cell-dependent and required tumor specificity of the Fab moiety of the Fab-SEA fusion protein. Due to the high affinity of SAg for MHC II, a limitation of this approach was retention of Fab-SEA proteins in normal tissues expressing MHC II, which caused systemic immune activation and dose limiting toxicity. We recently solved the structure of SEA and applied structure-based drug design to develop a novel generation of `man-made' SAg with improved pharmacological and pharmacokinetic properties. Mutation of the major MHC II binding site of SEA substantially reduced retention in MHC II+ tissues and systemic toxicity, while local immune activation at targeted tumor sites was retained. The Fab-SEA mutants display a 10 000-fold higher affinity for tumor tissue compared to normal tissue and the therapeutic window was improved >100-fold compared to native Fab-SEA protein. Thus protein engineering can be applied to convert harmful bacterial toxins into tolerable tumor-specific agents.

Introduction

T-cells possess a multitude of cytostatic effector functions, including direct cell mediated cytotoxicity and production of growth-suppressive and pro-inflammatory cytokines. The frequency of spontaneously occurring tumor-specific T-cells in tumor-bearing individuals is generally low and insufficient for interfering with progressive tumor growth.

Expansion of tumor-reactive cytotoxic T lymphocytes (CTLs) by experimental approaches has shown that CTL can interfere with tumor growth in animal tumor models, which supports the application of T-cell-based approaches in cancer immunotherapy 1, 2. However, the clinical utility of antigen-specific CTL is limited by the heterogeneous expression of the tumor-specific peptide antigen/MHC complexes and downregulation of the complexes in human tumors [3]. In contrast, a number of tumor cell surface molecules defined by monoclonal antibodies (mAbs) seem to be abundantly and homogeneously expressed on certain tumor types [4]. In vivo biodistribution studies demonstrate that antibodies can be useful for recognition and specific targeting to tumors [5]. However, antibodies penetrate poorly in solid tumors and are generally not very potent in inducing tumor cell death in vivo.

Superantigens (SAgs) are bacterial and viral proteins characterized by their ability to induce strong T-cell-mediated immune responses 6, 7, 8. SAgs bind as unprocessed proteins to MHC II molecules on APCs and interact with T-cells bearing particular T-cell receptor Vβ chains (TCR Vβ). Conventional peptide antigens engage the hypervariable V, D, J segments of the TCRα and β chains, resulting in activation of <0.01% of all T-cells. In contrast, the binding of SAg to the Vβ region of the TCR leads to activation of a large fraction (2–30%) of all T-cells 9, 10.

We have introduced a novel binding specificity in SAg by genetic fusion to the Fab fragment of a tumor specific mAb 11, 12, 13, 14, 15. Treatment with such fusion proteins conveys superantigenicity to the tumor and induces a powerful anti-tumor T-cell response.

Section snippets

Staphylococcal enterotoxins as prototype bacterial superantigens

The family of bacterial SAgs includes toxins produced by gram positive Staphylococcal and Streptococcal strains, mycoplasma and certain gram negative bacteria. The Staphylococcal enterotoxins (SEs) are the most well characterized members of this family and may serve as prototype SAgs [6].

Antibody-targeted superantigens

The ability of SEs, at picomolar concentrations, to induce generation of tumor-suppressive cytokines (e.g. TNF and IFNγ) and activation of CTL (e.g. SDCC) suggested their use in cancer immunotherapy.

The application of SAgs for cancer therapy is, however, limited by low and heterogenous expression of MHC class II in most tumor types.

To introduce a novel binding specificity in SEA we genetically fused SEA to a Fab fragment of a tumor-reactive monoclonal antibody 11, 12, 13.

The Fab-SEA fusion

Dynamics of Fab-SEA induced intratumoral immune responses

The dynamics of Fab-SEA therapy in mice carrying syngeneic B16 melanoma pulmonary metastases was studied at the tumor site and systemically in lymphoid tissues by immunohistochemistry.

Macrophages and dendritic cells accumulate in the tumor 2 h after the first Fab-SEA injection. This influx of dendritic cells and macrophages was accompanied by induction of TNFα and the chemokines MIP1α and MIP1β [31]. A few hours later the adhesion molecule VCAM-1 was detected on pulmonary vascular endothelial

Engineering of tumor-selective superantigens

Fab-SEA therapy is efficient and well tolerated at doses of 1 mg/kg in normal C57/Bl6 mice. At these doses Fab-SEA induced high systemic levels of pro-inflammatory cytokines such as IL6, IL1β, TNF and IFNγ 25, 33. However, most mouse strains are rather insensitive to high levels of pro-inflammatory cytokines and are not useful for predicting toxicity in humans. In contrast, injection of 10–100 ng/kg Fab-SEA protein to Cynomolgus monkeys resulted in high systemic cytokine levels (e.g. TNF), and

Pharmacokinetic properties of Fab-SEA (wt) protein

The distribution of Fab-SEA was studied using 125I-labelled protein in C57/Bl6 mice carrying B16 lung tumors followed by whole body autoradiography. Tumor localization was clearly demonstrated for Fab-SEA and the corresponding Fab fragment (Fig. 3a and b). Targeting to lymphoid tissue was seen only with the SEA-containing fusion proteins (Fig. 3a and c). The specificity of the Fab fragment was crucial since SEA fused to an irrelevant Fab did not localize to the tumor (Fig. 3c). Plasma

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      Because all tumor cells do not express MHC class II, to make superantigens selective for tumor antigens, Dohlsten et al. (6–8) exploited the conjugates between WT superantigen staphylococcal enterotoxin A (SEA) from S. aureus and antibody specific for tumor antigens. Because of the high affinity of SEA for MHC class II, a limitation of this approach was a retention of Ab-SEA fusion proteins in normal tissues expressing MHC class II, which caused systemic immune activation and dose-limiting toxicity (9). Therefore to lower the systemic effect of Ab-SAg fusion proteins, the Asp-227 to Ala (D227A) substitution was introduced into the SEA, reducing binding activity to MHC class II without affecting the TCR binding (10, 11).

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