Costimulation by CD28 sFv expressed on the tumor cell surface or as a soluble bispecific molecule targeted to the L6 carcinoma antigen

Abstract

Interaction of the CD80 (B7-1) and CD86 (B7-2) molecules on antigen presenting cells with the receptors CD28 and CTLA-4 on T cells generates signals important in the regulation of immune responses. Because this receptor system involves multiple receptor-ligand interactions, determining the function for individual receptors has been difficult. One approach is the use of antibodies and their derivatives with singular specificity as substitute ligands to explore the activities of these molecules. We have constructed recombinant mono- and bi-specific sFv molecules specific for the CD28 receptor that are capable of binding and generating costimulatory signals to activate T cells. We demonstrate that these soluble molecules are capable of higher levels of costimulation than soluble CD80Ig at equivalent concentrations. We also constructed artificial adhesion receptors on the cell surface using two different CD28-specific sFvIgs fused to the CD80 cytoplasmic and transmembrane domains. In this report, we compared costimulation by a soluble bispecific (alpha CD28-alpha L6) single chain sFvIg fusion protein to that generated by L6 antigen positive (L6+) H3347 tumor cells transduced with cell surface expressed forms of alpha CD28 sFv's. We show that the bispecific protein can target potent CD28 costimulatory activity to L6+ tumor cells in vitro. We also show that transfection of the cell surface forms of the two different CD28 sFvIgs into H3347 tumor cells allows them to generate significant costimulatory signals to activated T cells. Finally, we demonstrate that tumor cell presentation of either the soluble bispecific or transduced cell surface sFv generate similar costimulatory effects resulting in T cell activation.

Activation of lymphocytes by anti-CD3 single-chain antibody dimers expressed on the plasma membrane of tumor cells

“…These results suggest that expression of anti-CD3 scFv on tumor cells may form the basis of a novel tumor therapy strategy especially suited to tumors with defects in antigen processing or presentation. Expression of scFv with specificity for other molecules 37,38 may be useful to activate a range of effector cells at the tumor site.…”

CD83 Is a Sialic Acid-Binding Ig-Like Lectin (Siglec) Adhesion Receptor that Binds Monocytes and a Subset of Activated CD8+ T Cells

“…After 3 days, the presence of soluble protein in cell supernatant was checked by Western blot analysis and the fusion protein was purified by protein A-Sepharose 4B affinity chromatography (Zymed, South San Francisco, CA). Stable transfectants were generated in Chinese hamster ovaries cells by using CD83Ig cDNA cloned into pD18 (14).…”

“…To detect apoptosis, we used an annexin V kit (Beckman Coulter) according to the manufacturer's instructions. CD83Ig, CD80Ig (14), and CD40Ig (18) fusion proteins were biotinylated with EZ-Link N-hydroxy-succinimi-biotin (Pierce, Rockford, IL) kit according to the manufacturer's procedure. In some experiments cells, the HPB-ALL line were incubated with neuraminidase (Sigma) for 15 min at 37°C (1 U/5 ϫ 10 6 cells) or with neuraminidase together with 2.5 mg/ml of a sialidase inhibitor (2,3-dehydro-2-deoxy-Nacetylneuraminic acid, Sigma).…”

Design of transgenes for efficient expression of active chimeric proteins on mammalian cells

“…Surface expression of chimeric proteins has ranged from almost undetectable levels (Chesnut et al, 1996;de Ines et al, 1999;Moritz and Groner, 1995;Rode et al, 1996) to high levels (Brocker et al, 1993;Hayden et al. 1996;Liao et al, 2000;Roberts et al, 1994), but the factors resulting in efficient surface expression have not been characterized.…”