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).

The bacterial superantigen staphylococcal enterotoxin (SE) A (SEA) directs cytotoxic T lymphocytes (CTLs) expressing particular sequences of the T-cell receptor (TCR)

Summary Superantigens activate T-cells by linking the T-cell receptor to MHC class II on antigen-presenting cells, and novel reactivity can be introduced by fusing the superantigen to a targeting molecule. Thus, an antibody-targeted superantigen, which activates T cells to destroy tumour cells, might be used as cancer therapy. A suitable target is the 5T4 oncofetal antigen, which is expressed on many carcinomas. We constructed a fusion protein from a Fab of a monoclonal antibody recognizing the 5T4 antigen, and an engineered superantigen. The recombinant product 5T4FabV13-SEA D227A bound the 5T4 antigen expressed on the human non-small-cell lung cancer cell line Calu-1 with a K d of 1.2 nM while the substitution of Asp227 to Ala in the superantigen moiety reduced binding activity to MHC class II. 5T4FabV13-SEA D227A tumour reactivity was demonstrated in 7/7 NSCLC samples by immunohistochemistry, while normal tissue reactivity was low to moderate. 5T4FabV13-SEA D227A induced significant T-cell-dependent in vitro killing of sensitive 5T4 bearing Calu-1 cells, with maximum lysis at 10 -10 M, while the capacity to lyse MHC class II expressing cells was approximately 1000 times less effective. Immunotherapy of 5T4FabV13-SEA D227A against human NSCLC was investigated in SCID mice reconstituted with human peripheral blood mononuclear cells. Mice carrying intreperitoneally growing Calu-1 cells showed significant reduction in tumour mass and number after intravenous therapy with 5T4FabV13-SEA D227A . Thus, 5T4FabV13-SEA D227A has highly attractive properties for therapy of human NSCLC.

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