Monoclonal antibody-superantigen fusion proteins: tumor-specific agents for T-cell-based tumor therapy.

Abstract

The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on major histocompatibility complex (MHC) The therapeutic use of naked monoclonal antibodies (mAbs) in human epithelial cancer has met with limited success (1). The amount of mAb found to accumulate in the tumor is generally low, tumor cells display extensive heterogeneity in antigen expression, and the effector mechanisms responsible for tumor-cell destruction are insufficient (1, 2). Recent progress in the molecular understanding of T-cell recognition has provided insight into the role of T cells in the response against allogeneic and neoplastic tissues. The vigorous and destructive response against an allograft involves a high frequency of T lymphocytes that, via the T-cell receptor (TCR), recognizes peptide antigens presented in the context of major histocompatibility complex (MHC) molecules. In contrast, the frequency of T cells responding to a tumor is generally low and insufficient to interfere with tumor growth. This suggests that an attractive approach for immunotherapy would be to target a high frequency of T cells to the tumor area. We have therefore developed an antibody-based therapy that provides tumor cells with superantigenicity. Superantigens (SAgs) are a family of bacterial and viral proteins that activate a high frequency of T cells to cytokine release and cell-mediated cytotoxicity (3)(4)(5). Bacterial SAgs bind with high affinity to MHC class II molecules and subsequently interact with T cells expressing particular sequences in the variable (V) region of the TCR , / chain (TCR V(3) (3)(4)(5) MATERIALS AND METHODSProtein Reagents. Recombinant SEA was expressed in E. coli and purified to homogeneity. The hybridomas secreting the C215 (IgG2a) and C242 (IgGl) mAbs, reacting with human colon cancer, were produced at Pharmacia (8, 9). E. coli HB101 was used as host during the DNA construction work (10).

Therapy of human non-small-cell lung carcinoma using antibody targeting of a modified superantigen

“…In accordance with previous findings (Dohlsten et al, 1994), Fab-SEA was a much weaker competitor for binding to MHC class II molecules than SEA. SEA and Fab-SEA showed IC 50 values of approximately 21 and 360 nM respectively in a competitive assay with biotinylated SEA to coated Raji cell plasma membranes.…”

“…The fusion proteins were produced at Pharmacia & Upjohn (Stockholm, Sweden) essentially as described (Dohlsten et al, 1994;Abrahmsén et al, 1995;Forsberg et al, 1997). The Fvencoding portions of 5T4 were cloned from the 5T4 hybridoma and fused to sequences coding for the constant regions of the murine IgG1/k antibody C242 lacking the interchain disulphide bond.…”

“…To study cytotoxicity, K562, Calu-1 or Raji cells, labelled with (Na) 2 51 CrO 4 (Amersham Solna, Sweden) were used in a standard 4 h chromium release assay (Dohlsten et al, 1994). As effector cells, an SEA reactive T cell line, prepared as described above, was used.…”

“…British Journal of Cancer (2000) The product consists of a modified variant of the variable regions from 5T4 antibody (Forsberg et al, 1997) connected to the CL and CH1 domains of the antibody C242 (Dohlsten et al, 1994). The superantigen SEA D227A is fused to the C-terminus of CH1.…”

“…To potentiate the effects of monoclonal antibodies, the use of fusions with superantigens, which are of bacterial or viral origin and activate T cells by linking the T cell receptor to MHC class II on antigen presenting cells (reviewed by Johnson et al, 1992), have previously been described by us (Dohlsten et al, 1994;Brodin et al, 1998). Thereby, large amounts of cytotoxic and cytokine producing T-cells can be targeted to destroy and initiate a powerful T cell attack against tumour cells in vivo (Dohlsten et al, 1994(Dohlsten et al, , 1995a. Most studies of antibody targeted superantigens, e.g.…”