# US 2015/0307587 A1 · RTRAIL Mutant and Monomethyl Auristatin E Conjugate Thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2015/0307587 A1 - Document ID: US20150307587A1 - Status: Published application - Kind: A1 - Application number: 14/437,987 - Filed: 2013-06-20 - Published: 2015-10-29 - Earliest claimed: 2012-10-25 - Assignees: Zhejiang University a university - Applicants: Zhejiang University - Inventors: Shuqing CHEN; Liqiang PAN - Web page: https://patentgrep.com/patent/US20150307587A1 ## CPC - C07K 14/70575: {NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 (NGF C07K14/48, TNF C07K14/525)} - A61K 47/64: Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent (peptidic linkers A61K47/65) - A61K 47/65: Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers - A61P 35/00: Antineoplastic agents - 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) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS ## Abstract Disclosed are an rTRAIL mutant and monomethyl auristatin E (MMAE) conjugate thereof. The amino acid sequence thereof is as represented by SEQ ID No.1. Also disclosed are a coding gene of the rTRAIL mutant and expression system, recombinant vector and expression unit containing the coding gene. Also disclosed are an rTRAIL mutant-vcMMAE conjugate and preparation and uses thereof. The conjugate of the present invention has the biological functions of both the rTRAIL mutant and the MMAE; the MMAE is directionally transferred to a tumor cell through the specific binding between the rTRAIL mutant and a death receptor on the surface of the tumor cell, and is released and takes effect in the tumor cell, thus killing the tumor cells less sensitive or even resistant to TRAIL, and reducing the toxicity generated by the separate administration of the MMAE. ## Claims ### Claim 1 An rTRAIL mutant, characterized in that the amino acid sequence is represented by SEQ ID No.1. ### Claim 2 (depends on 1) A gene encoding the rTRAIL mutant according to claim 1, characterized in that the base sequence is represented by SEQ ID NO.2. ### Claim 3 (depends on 2) An expression unit, a recombinant vector or an expression system containing the gene according to claim 2. ### Claim 4 An rTRAIL mutant-MMAE conjugate, characterized in that it is formed by conjugating monomethyl auristatin E (MMAE) with an rTRAIL mutant trimer through a linker, the amino acid sequence of the rTRAIL mutant is represented by SEQ ID No.1, and said linker is a maleimide-modified valine-citrulline dipeptide. ### Claim 5 (canceled) ### Claim 6 (depends on 4) An use of the rTRAIL mutant-MMAE conjugate according to claim 4 in preparation of anti-tumor drugs. ### Claim 7 A method of preparing an rTRAIL mutant-MMAE conjugate, comprising: (1) An rTRAIL mutant polymer is depolymerized and re-polymerized to generate an rTRAIL mutant trimer; (2) The rTRAIL mutant trimer is mixed with a MMAE having a linker to perform a coupling reaction; (3) After completion of the reaction, the rTRAIL mutant-MMAE conjugate is obtained by separation and purification; wherein, the amino acid sequence of the rTRAIL mutant is represented by SEQ ID No.1; said linker is a maleimide-modified valine-citrulline dipeptide. ### Claim 8 (depends on 7) The method according to claim 7, wherein said rTRAIL mutant trimer is prepared by a method comprising: dissolving the rTRAIL mutant in a buffer containing zinc ion, adding tris(β-chloroethyl)phosphate thereinto, and reacting under water-bath for 1-3 hours at 30° C.˜40° C. ### Claim 9 (depends on 7) The method according to claim 7, wherein said coupling reaction is performed at a temperature of no higher than 4° C. for 30˜60 min. ### Claim 10 (depends on 7) The method according to claim 7, wherein said separation and purification includes: removing the substances with molecular weights below 10 kDa from the reaction solution by ultrafiltration, removing the precipitate by centrifugation to give a supernatant, and sterilizing the supernatant by filtration to obtain the rTRAIL mutant-MMAE conjugate.