# US 2022/0033463 A1 · Preparation and Application of Soluble Tim-3 Recombinant Protein and Mutant Protein Thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2022/0033463 A1 - Document ID: US20220033463A1 - Status: Published application - Kind: A1 - Application number: 17/506,676 - Filed: 2021-10-21 - Published: 2022-02-03 - Earliest claimed: 2019-10-24 - Assignees: Zhejiang University - Applicants: Zhejiang University - Inventors: Ying Yang; Zhi Chen; Haihong Zhu; Yanning Liu; Guohua Lou; Yu Shi - Web page: https://patentgrep.com/patent/US20220033463A1 ## CPC - C07K 14/70503: {Immunoglobulin superfamily} - A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract The present application belongs to the field of biotechnology, and particularly relates to preparation and use of soluble Tim-3 recombinant protein and a mutant protein thereof. The soluble Tim-3 recombinant protein is used to prepare a drug with the function of regulating monocytes or a drug with the function of enhancing tumor immune response, and the amino acid sequence of the soluble Tim-3 recombinant protein is as shown in SEQ ID NO: 1. In addition, the present application further provides the mutant proteins of the sTim-3 recombinant protein and preparation and use of the mutant protein. The mutant recombinant protein is obtained by screening through a directed evolutionary technology, with the function of regulating monocytes or the function of enhancing tumor immune response, and combines with a host codon optimization method to improve the expression efficiency. ## Claims ### Claim 1 Use of Soluble Tim-3 recombinant protein in preparing a drug with the function of regulating monocytes or a drug with the function of enhancing tumor immune response, wherein the amino acid sequence of the soluble Tim-3 recombinant protein is as shown in SEQ ID NO: 1. ### Claim 2 (depends on 1) Use according to claim 1, wherein the drug with the function of regulating monocytes can be used for inhibiting overactivity of the monocytes in patients with inflammation, and the drug with the function of enhancing tumor immune response can be used for enhancing immune response in cancer patients to avoid host's immune escape. ### Claim 3 A purification method of soluble Tim-3 recombinant protein, comprising the following steps: performing fluid exchanging on cell culture solutions of eukaryotic CHO cells, prokaryotic E. coli and insect baculovirus expression system containing recombinant human soluble Tim-3 through microfiltration clarification and ultrafiltration concentration, and then passing through cation and anion chromatographic column, molecular sieve chromatographic column, hydrophobic chromatographic column and/or affinity chromatographic column to obtain the soluble Tim-3 recombinant protein with enough purity, wherein the amino acid sequence of the soluble Tim-3 recombinant protein is as shown in SEQ ID NO: 1. ### Claim 4 A soluble Tim-3 recombinant mutant protein, wherein the mutant protein is a soluble Tim-3 recombinant mutant protein obtained in the mode that: on the basis of a gene sequence corresponding to human Tim-3 (NP_116171.3) extracellular domain amino acid (SEQ ID NO: 1), mutant genes are obtained by screening through a directed evolutionary technology, and then expressed, wherein the mutant sites of the mutant genes do not involve conserved sequences among species, but occur in non-conservative sequence positions. ### Claim 5 A soluble Tim-3 recombinant mutant protein, wherein the amino acid sequence of the soluble Tim-3 recombinant mutant protein is one of the sequences as shown in SEQ ID NOs: 2-7. ### Claim 6 (depends on 4) The soluble Tim-3 recombinant mutant protein according to claim 4, wherein the soluble Tim-3 recombinant mutant protein further comprises a His tag sequence preceded by a linker peptide. ### Claim 7 (depends on 6) The soluble Tim-3 recombinant mutant protein according to claim 6, wherein the His tag sequence is HHHHHH, and the linker peptide is (G)nS, n=1-4. ### Claim 8 (depends on 4) A preparation method of the soluble Tim-3 recombinant mutant protein according to claim 4, wherein on the basis of the gene sequence corresponding to the human Tim-3 (NP_116171.3) extracellular domain amino acid (SEQ ID NO: 1), mutant genes are obtained by screening through a directed evolutionary technology, and then amino acids are expressed into the soluble Tim-3 recombinant mutant protein. ### Claim 9 (depends on 4) Use of the soluble Tim-3 mutant protein according to claim 4 in preparing a drug with the function of regulating monocytes or a drug with the function of enhancing tumor immune response. ### Claim 10 (depends on 9) The use according to claim 9, wherein the drug with the function of regulating monocytes can be used for inhibiting overactivity of the monocytes in patients with inflammation, and the drug with the function of enhancing tumor immune response can be used for enhancing immune response in cancer patients to avoid host's immune escape. ### Claim 11 (depends on 5) The soluble Tim-3 recombinant mutant protein according to claim 5, wherein the soluble Tim-3 recombinant mutant protein further comprises a His tag sequence preceded by a linker peptide. ### Claim 12 (depends on 11) The soluble Tim-3 recombinant mutant protein according to claim 11, wherein the His tag sequence is HHHHHH, and the linker peptide is (G)nS, n=1-4. ### Claim 13 (depends on 5) A preparation method of the soluble Tim-3 recombinant mutant protein according to claim 5, wherein on the basis of the gene sequence corresponding to the human Tim-3 (NP_116171.3) extracellular domain amino acid (SEQ ID NO: 1), mutant genes are obtained by screening through a directed evolutionary technology, and then amino acids are expressed into the soluble Tim-3 recombinant mutant protein. ### Claim 14 (depends on 5) Use of the soluble Tim-3 mutant protein according to claim 5 in preparing a drug with the function of regulating monocytes or a drug with the function of enhancing tumor immune response. ### Claim 15 (depends on 14) The use according to claim 14, wherein the drug with the function of regulating monocytes can be used for inhibiting overactivity of the monocytes in patients with inflammation, and the drug with the function of enhancing tumor immune response can be used for enhancing immune response in cancer patients to avoid host's immune escape. ### Claim 16 (depends on 5) A preparation method of the soluble Tim-3 recombinant mutant protein according to claim 5, wherein on the basis of the gene sequence corresponding to the human Tim-3 (NP_116171.3) extracellular domain amino acid (SEQ ID NO: 1), mutant genes are obtained by screening through a directed evolutionary technology, and then amino acids are expressed into the soluble Tim-3 recombinant mutant protein. ### Claim 17 (depends on 6) A preparation method of the soluble Tim-3 recombinant mutant protein according to claim 6, wherein on the basis of the gene sequence corresponding to the human Tim-3 (NP_116171.3) extracellular domain amino acid (SEQ ID NO: 1), mutant genes are obtained by screening through a directed evolutionary technology, and then amino acids are expressed into the soluble Tim-3 recombinant mutant protein. ### Claim 18 (depends on 7) A preparation method of the soluble Tim-3 recombinant mutant protein according to claim 7, wherein on the basis of the gene sequence corresponding to the human Tim-3 (NP_116171.3) extracellular domain amino acid (SEQ ID NO: 1), mutant genes are obtained by screening through a directed evolutionary technology, and then amino acids are expressed into the soluble Tim-3 recombinant mutant protein. ### Claim 19 (depends on 11) A preparation method of the soluble Tim-3 recombinant mutant protein according to claim 11, wherein on the basis of the gene sequence corresponding to the human Tim-3 (NP_116171.3) extracellular domain amino acid (SEQ ID NO: 1), mutant genes are obtained by screening through a directed evolutionary technology, and then amino acids are expressed into the soluble Tim-3 recombinant mutant protein. ### Claim 20 (depends on 12) A preparation method of the soluble Tim-3 recombinant mutant protein according to claim 12, wherein on the basis of the gene sequence corresponding to the human Tim-3 (NP_116171.3) extracellular domain amino acid (SEQ ID NO: 1), mutant genes are obtained by screening through a directed evolutionary technology, and then amino acids are expressed into the soluble Tim-3 recombinant mutant protein.