Methods for blocking TNF-alpha activity in mammals with trimeric soluble TNF receptors
This invention relates to a general methodology for efficient creation of trimeric soluble receptors for therapeutic applications. The process involves gene fusion between a soluble receptor with a ligand binding domain and a trimerization tag from the C-propeptide domain of pro-collagen, which is capable of self-assembly into a covalently linked trimer. Using both in vitro bioassays and an in vivo mouse model for collagen-induced arthritis (CIA), we show that the homotrimeric soluble TNF receptor produced with such method is a more potent blocker than dimeric TNF receptor decoys in inhibiting TNF-α mediated inflammatory diseases.
1. A method for reducing TNF-alpha activity in a mammal having a TNF-alpha-mediated disorder which comprises administering to said mammal a therapeutically effective amount of a disulfide bond-linked trimeric fusion protein comprising three fused protein subunits, wherein each fused protein subunit comprises a soluble TNF-alpha receptor or functional portion thereof containing a TNF binding domain, which in turn is fused to a C-terminal portion of collagen capable of self-trimerizing said fused protein subunit to form said disulfide bond-linked trimeric fusion protein containing three TNF ligand binding domains, wherein said trimeric fusion protein has a greater binding affinity to a TNF ligand as compared to a monomeric TNF ligand binding domain.
2. The method of claim 1 , wherein the disulfide bond-linked trimeric fusion protein is administered by subcutaneous injection.
3. The method of claim 1 , wherein the disulfide bond-linked trimeric fusion protein is administered by intravenous injection.
4. The method of claim 1 , wherein the disulfide bond-linked trimeric fusion protein is administered in a series of doses separated by intervals of days or weeks.
5. The method of claim 1 , wherein the TNF-alpha-mediated disorder is an arthritic condition.
6. The method of claim 1 , wherein the TNF-alpha-mediated disorder is Crohn's disease.
7. The method of claim 1 , wherein the TNF-alpha-mediated disorder is psoriasis.
8. The method of claim 1 , wherein the TNF-alpha-mediated disorder is an inflammatory disorder.
9. The method of claim 1 , wherein the TNF-alpha-mediated disorder is sepsis.
10. The method of claim 1 , wherein the C-terminal portion of collagen is selected from the group consisting of pro.alpha.1(I), pro.alpha 2(I), pro.alpha.1(II), pro.alpha.1(III), pro.alpha.1(V), pro.alpha.2(V), pro.alpha.1(XI), pro.alpha.2(XI) and pro.alpha.3(XI)
11. The method of claim 1 , wherein the C-terminal portion of collagen comprises a mutated or deleted BMP-1 protease recognition sequence.
12. The method of claim 1 , wherein the soluble TNF-alpha receptor is soluble human TNF-alpha receptor.
13. The method of claim 1 , wherein the soluble TNF-alpha receptor is selected from the group consisting of soluble p55 TNF-alpha receptor and p75 TNF-alpha receptor.