Construction and Production of Concatameric Human TNF Receptor-Immunoglobulin Fusion Proteins

Tumor necrosis factor-$\alpha$ (TNF-$\alpha$) and lymphotoxin-$\alpha$ (LT-$\alpha$, TNF-$\beta$) can initiate and perpetuate human diseases such as multiple sclerosis (MS), rheumatoid arthritis (RA), and insulin-dependent diabetes mellitus (IDDM). TNFs can be blocked by the use of soluble TNF receptors. However, since monomeric soluble receptors generally exhibit low affinity or function as agonists, the use of monomeric soluble receptors has been limited in the case of cytokines such as TNF-$\alpha$, TNF-$\alpha$, interleukin (IL)-1, IL-4, IL-6, and IL-13, which have adapted to a multi component receptor system. For these reasons, very high-affinity inhibitors were created for the purpose of a TNFs antagonist to bind the TNFR and trigger cellular signal by using the multistep polymerase chain reaction method. First, recombinant simple TNFR-Ig fusion proteins were constructed from the cDNA sequences encoding the extracellular domain of the human p55 TNFR (CD120a) and the human p75 TNFR (CD120b), which were linked to hinge and constant regions of human $IgG_1$ heavy chain, respectively using complementary primers (CP) encoding the complementary sequences. Then, concatameric TNFR-Ig fusion proteins were constructed using recombinant PCR and a complementary primer base of recombinant simple TNFR-Ig fusion proteins. For high level expression of recombinant fusion proteins, Chinese hamster ovary (CHO) cells were used with a retroviral expression system. The transfected cells produced the simple concatameric TNFR-Ig fusion proteins capable of binding TNF and inactivating it. These soluble versions of simple concantameric TNFR-Ig fusion proteins gave rise to multiple forms such as simple dimers and concatameric homodimers. Simple TNFR-1g fusion proteins were shown to have much more reduced TNF inhibitory activity than concatameric TNFR-Ig fusion proteins. Concatameric TNFR-Ig fusion proteins showed higher affinity than simple TNFR-Ig fusion proteins in a receptor inhibitor binding assay (RIBA). Additionally, concatameric TNFR-Ig fusion proteins were shown to have a progressive effect as a TNF inhibitor compared to the simple TNFR-Ig fusion proteins and conventional TNFR-Fc in cytotoxicity assays, and showed the same results for collagen induced arthritis (CIA) in mice in vivo.