ABSTRACT
A long-lasting recombinant human albumin-linker-erythropoietin (EPO) is a human albumin gene fused to the N-terminal of EPO with a (GGSGG)(n)-repeated linker inserted between albumin and EPO. Albumin-EPO fusion genes were co-transfected with the dhfr gene. Albumin-EPO fusion protein has three kinds of sub-types (IALE, AD2LE, AD1LE). Albumin-EPO fusion protein was quantified with human EPO ELISA. The in vitro efficacy of albumin-EPO fusion protein was estimated using F-36E cell, and in vivo efficacy of albumin-EPO fusion protein was estimated using normocythemic mice (B6D2F1). We also determined the in vivo half-life in a Sprague-Dawley rat. A PLA program analysis result demonstrated that the albumin-EPO fusion protein IALE is about 7.8-fold more potent than rHuEPO in increasing the hematocrit of normal mice.
Subject(s)
Cloning, Molecular/methods , Erythropoietin/metabolism , Recombinant Fusion Proteins/biosynthesis , Serum Albumin/biosynthesis , Analysis of Variance , Animals , Blotting, Western , CHO Cells , Cell Count , Cell Line, Tumor , Chromatography, Gel , Cricetinae , Cricetulus , Erythropoietin/chemistry , Erythropoietin/genetics , Half-Life , Humans , Mice , Polymerase Chain Reaction , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Proteins , Reticulocytes , Serum Albumin/chemistry , Serum Albumin/geneticsABSTRACT
The avidity for CD80Ig/CD86Ig and the in vitro immunosuppressive effect of recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin, produced by transgenic rice cell suspension cultures (hCTLA4Ig(P)) with CHO-derived recombinant hCTLA4Ig (hCTLA4Ig(M)), were measured. Surface plasmon resonance (SPR) was used for kinetic binding analysis: hCTLA4Ig(P) and hCTLA4Ig(M) had higher avidity for CD80Ig/CD86Ig than for CD28Ig, and the avidity for CD80Ig/CD86Ig was similar. hCTLA4Ig(P) and hCTLA4Ig(M) had similar in vitro immunosuppressive activity against the expression of T cell-derived cytokines, such as IL-2, IL-4, and IFN-gamma, but did not suppress the expression of macrophage-derived cytokines, including TNF-alpha and IL-1beta, as well as NO. Thus the immunosuppressive mechanism of hCTLA4Ig(P) is also T cell-specific and it could therefore be used as an immunosuppressive agent with an equivalent potency to that of hCTLA4Ig(M).
