Expression and characterization of glycolipid-anchored B7-1 (CD80) from baculovirus-infected insect cells: protein transfer onto tumor cells.
Tumor cells can be modified to express immunostimulatory molecules such as B7-1 by protein transfer using purified glycosylphosphatidylinositol-anchored B7-1 (GPI-B7-1). In this study recombinant baculovirus encoding GPI-B7-1 (vBacB7-1(GPI)) was established to obtain large quantities of purified GPI-B7-1 to modify tumor cells by protein transfer. vBacB7-1(GPI)-infected insect cells showed high-level cell surface expression of GPI-B7-1 that was susceptible to PIPLC treatment. GPI-B7-1 expressed in insect cells (Bac-GPI-B7-1) mediated T cell proliferation, indicating that the GPI-B7-1 retains costimulatory activity. Moreover, Bac-GPI-B7-1 was completely solubilized in Triton X-100 at 4 degrees C compared to 22% solubilization of GPI-B7-1 expressed in CHOK1 cells, suggesting that GPI-anchored proteins expressed in insect cells may not be clustered into the detergent-insoluble fraction. SDS-PAGE analysis of Bac-GPI-B7-1 showed faster mobility (45 kDa) compared to GPI-B7-1 from CHOK1 (68 kDa) and this difference may be due to a difference in glycosylation. Cell binding assays showed that immunoaffinity-purified Bac-GPI-B7-1 retained its functional ability to bind CD28(+) cells. Moreover, when human tumor cells were incubated with this functionally active purified GPI-B7-1, an efficient transfer of B7-1 onto tumor cells was observed. These results demonstrate that GPI-B7-1 can be expressed in insect cells in a functionally active form and can be used to modify tumor cells for immunotherapeutic applications.
Nagarajan
S
S
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, 30322, USA.
Selvaraj
P
P
D000818:Animals; D018122:B7-1 Antigen / Q000096:biosynthesis* / Q000378:metabolism* / Q000494:pharmacology; D016367:Baculoviridae / Q000737:chemistry / Q000235:genetics; D018106:CD28 Antigens / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D015870:Gene Expression; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D007313:Insecta / Q000737:chemistry / Q000235:genetics / Q000821:virology; D008213:Lymphocyte Activation / Q000187:drug effects; D008562:Membrane Glycoproteins / Q000032:analysis; D017830:Octoxynol; D011485:Protein Binding; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D012995:Solubility; D014407:Tumor Cells, Cultured / Q000821:virology
D018122:B7-1 Antigen; D018106:CD28 Antigens; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D017830:Octoxynol