Screened And Prepared A Modified TNFα Molecule As TNFα Autovaccine To Treat Diseases Involving In TNFα Overexpression

Background: TNFα, a cytokine with complex bioactivity, is mainly secreted by macrophages and T cells. Besides its tumoricidal activity in vivo and in vitro, TNFαalso has several other activities including antiviral activity, immuno-effective cell activating, such as macrophages and polymorphonuclear leukocytes, major histocompatible complex antigen express upregulating, the function regulation of immune system, and so on. However high level of TNFαin the body is critically involved in the pathogensis of many diseases such as autoimmune disease, inflammatory disease, dyscrasia and so on. Neutralizing TNF-αin vivo is a useful approach for the treatment of these diseases. At present, anti-TNFαmoncolonal antibodies and soluble TNFαreceptors are clinically used for the treatment of these diseases. But these drugs have some flaws. They need to be used in large amounts. In addition, they are potentially immunogenic compounds that may elicit antibody responses. Vaccines can overcome above flaws. So vaccination against TNFαmay be a useful approach for the treatment of these diseases. AIM: To screen a TNFαautovaccine and investigate whether or not this autovaccine can be used as vaccine in the treatment of diseases with high level of TNFα. METHODS: In this study, we have prepared a serial of the mTNFαtruncated proteins, which fused with GST. Then these truncated proteins were used to immunize mice. We analyzed the potential antigenic sites on mTNFαsequence to determined which fragment in mTNFαsequence is replaced by the T helper epitope by anti-serum investigation and computer help design (Kolaskar and Tongaonkar). Then we constructed three recombinant mutant mTNFαmolecules (mTNF-TT, mTNF-HEL, mTNF-PADRE) by replacing the amino acids 126-140 of mTNFαwith T cell help epitopes, TT830-844(QYIKANSKFIGITEL), HEL46-61 ( NTDGSTDYGILQINSR ) and PADRE ( AKFVAAWTLKA ) respectively and developed a simple strategy to purify these recombinant proteins. To find proper T help epitope, the serum antibodies elicited by the three proteins were compared by bind with mTNFαand neutralize cytolytic activities of mTNFαin vitro.Finally, we identified therapeutic effect of screened autovaccine, mTNF-PADRE in LPS induced endotoxic shock mice, mTNFαinduced cachexia mice and collagenⅡinduced RA mice. RESULTS: By mTNFαtruncated proteins elicited anti-serum investigation and computer help design,we found that the amino acids 126-140 of mTNFαis a proper site for T cell help epitopes replacement. After purification of constructed three recombinant mutant mTNFαmolecules (mTNF-TT, mTNF-HEL, mTNF-PADRE), the purity of the three proteins is above 95%. L929 cytolytic assay showed modified mTNFαmolecules do not possess cytolytic activity of mTNFα. Although the three recombinant mutant mTNFαmolecules can all elicite mTNFαbinding and neutralizing antibodies, mTNF-PADRE has the best results. Animal experiments results suggested that by the treatment of mTNF-PADRE without adjuvant, survival times of mice with endotoxic shock giving LPS were prolonged, and the symptoms of experimental cachexia were ameliorated. CONCLUSIONS: After the screening of T helper epitope inserted site and inserted T helper epitope, we obtained an mTNFαautovaccine, mTNF-PADRE, which can effectively elicite mTNFαneutralizing antibodies and does not possess cytolytic activity of mTNFα. Animal experiments showed mTNF-PADRE without adjuvant can ameliorated symptoms of LPS induced endotoxic shock mice, mTNF induced cachexia mice and collagenⅡinduced RA mice. So the corresponding human TNF-PADRE could be used as a candidate molecular of human TNFαautovaccine.