Modified interleukin 12 and use thereof in preparing drugs for treating tumours

Abstract

The present invention discloses a modified interleukin 12 (nsIL-12) and its gene, recombinant vector and use in manufacture of a medicament for treatment of tumors. When the oncolytic adenovirus vector carrying the modified interleukin 12 gene targets tumor tissue, the modified interleukin 12 is continuously expressed at a low level and mainly distributed in the local tumor tissue, which improves the specificity to tumor cells and reduces the systemic toxicity of interleukin 12; the modified interleukin 12 shows stronger inhibitory effect on tumor growth in intraperitoneally disseminated tumors and orthotopic tumors, and has low toxicity. The modified interleukin 12 armed oncolytic viruses show excellent antitumor effects, with a significant regression of tumors and lower toxicity compared with the existing IL-12 armed virus.

What is claimed is: 
     
         1 . A method for treatment of a disease associated with IL-12 or for reducing the toxicity of IL-12, which comprises administering to a subject an oncolytic virus which comprises the nucleotide sequence encoding a modified IL-12, which is non-secretory, or is capable of expressing the modified IL-12 which is non-secretory, wherein the non-secretory IL-12 has the following structure: p40-linker-p35 or p35-linker-p40; and wherein the non-secretory IL-12 does not contain a secretory signal peptide. 
     
     
         2 . The method according to  claim 1 , wherein the disease is a cancer. 
     
     
         3 . The method according to  claim 1 , wherein the disease is pancreatic cancer, head and neck cancer, lung cancer, esophageal cancer, ovarian cancer, colorectal cancer, colon cancer or gastric cancer or an intraperitoneally disseminated tumor. 
     
     
         4 . The method according to  claim 1 , wherein p35 and p40 are natural p35 and p40 or active variants thereof. 
     
     
         5 . The method according to  claim 4 , wherein the variant is an amino acid sequence having an identity of about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more to its parent or natural amino acid sequence, and having the activity of its parent or natural amino acid sequence. 
     
     
         6 . The method according to  claim 4 , wherein the variant comprises or consists of an amino acid sequence having substitution, deletion, insertion or addition of one or several amino acids on the basis of its natural amino acid sequence or parental sequence and retaining the activity before the substitution, deletion, insertion or addition. 
     
     
         7 . The method according to  claim 1 , wherein the linker has or consists of the amino acids of SEQ ID NO:7. 
     
     
         8 . The method according to  claim 1 , wherein the non-secretory IL-12 has or consists of the amino acids of SEQ ID NO:2 or a variant thereof. 
     
     
         9 . The method according to  claim 4 , wherein the variant is an amino acid sequence having an identity of at least about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more to the amino acid sequence of SEQ ID NO:2, and having the activity of its parental or natural amino acid sequence. 
     
     
         10 . The method according to  claim 4 , wherein the variant comprises or consists of an amino acid sequence having substitution, deletion, insertion or addition of one or several amino acids on the basis of SEQ ID NO: 2 and retaining the activity before the substitution, deletion, insertion or addition. 
     
     
         11 . The method according to  claim 1 , wherein the nucleotide sequence comprises or consists of the following sequence:
 a) the nucleotide sequence shown in SEQ ID NO: 1; b) a polynucleotide capable of hybridizing under stringent conditions to the nucleotide sequence of SEQ ID NO: 1 and encoding an amino acid sequence having non-secretory IL-12 activity; or c) a complementary sequence of the above a) or b).   
     
     
         12 . The method according to  claim 1 , which is adenovirus serotype 5 vector, vaccinia virus vector or adenovirus serotype 11 vector. 
     
     
         13 . The method according to  claim 4 , wherein p35 and p40 are natural human p35 and human p40 or active variants thereof. 
     
     
         14 . The method according to  claim 4 , wherein p35 has or consists of the sequence of SEQ ID NO: 5; and p40 has or consists of the sequence of SEQ ID NO: 6. 
     
     
         15 . The method according to  claim 1 , wherein neither p40 nor p35 in the structure contains a signal peptide. 
     
     
         16 . An oncolytic virus, which comprises the nucleotide sequence encoding a modified IL-12, which is non-secretory, or is capable of expressing the modified IL-12 which is non-secretory, wherein the non-secretory IL-12 has the following structure: p40-linker-p35 or p35-linker-p40; and wherein the non-secretory IL-12 does not contain a secretory signal peptide. 
     
     
         17 . A medicine which comprises the oncolytic virus according to  claim 16 . 
     
     
         18 . A method for preparing a recombinant vector, comprises inserting the nucleotide sequence according to  claim 11  into a vector to obtain the recombinant vector capable of expressing a non-secretory IL-12.