# US 5,705,163 A · Target-specific, cytotoxic, recombinant pseudomonas exotoxin > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,705,163 A - Document ID: US5705163A - Status: Granted - Kind: A - Application number: 08/461,233 - Filed: 1995-06-05 - Granted: 1998-01-06 - Earliest claimed: 1990-01-02 - Assignees: The United States of America as represented by the Department of Health and Human Services, National Institutes of Health - Inventors: Ira Pastan; Vijay K. Chaudhary; David Fitzgerald - Web page: https://patentgrep.com/patent/US5705163A ## CPC - C07K 14/70514: {CD4} - A61P 35/00: Antineoplastic agents - C07K 14/21: from Pseudomonadaceae (F) - C07K 14/495: Transforming growth factor [TGF] - C07K 16/2866: {against receptors for cytokines, lymphokines, interferons} - 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 2317/77: Internalization into the cell - C07K 2319/00: Fusion polypeptide - C07K 2319/02: containing a signal sequence - C07K 2319/034: containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved - C07K 2319/55: containing a fusion with a toxin, e.g. diphteria toxin - C07K 2319/75: containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones - Y10S 435/875: Pseudomonas aeruginosa - 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) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - 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) - Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS ## Abstract A target-specific, cytotoxic, recombinant Pseudomonas exotoxin is described. Such toxins are made by inserting specific recognition molecules at specific cloning sites in at least domain III near the carboxyl terminus of the PE molecule. Various modifications of the carboxyl terminus of the PE molecule to increase cytotoxicity are set forth. Multifunctional, recombinant, cytotoxic fusion proteins containing at least two different recognition molecules are provided for killing cells expressing receptors to which the recognition molecules bind with specificity. Methods for producing novel recombinant PE molecules with specific properties are described. ## Claims ### Claim 1 A method for killing a target cell, said method comprising contacting said target cell with a cytotoxic amount of a composition comprising a recombinant Pseudomonas exotoxin (PE) having a first recognition molecule for binding said target cell and a carboxyl terminal sequence of 4 to 16 amino acids which permits translocation of the PE molecule into a cytosol of said target cell, the first recognition molecule being inserted in domain III after and no acid 600 and before amino acid 613 of the PE. ### Claim 2 A method of killing targeted cells, said method comprising the step of contacting cells targeted to be killed, with a cytotoxic amount of a recombinant Pseudomonas exotoxin fusion protein containing at least two different recognition molecules for killing cells expressing receptors to which said recognition molecules specifically bind. ### Claim 3 (depends on 1) The method of claim 1, wherein said carboxyl terminal sequence comprises, in a direction from the amino terminus to the carboxyl terminus, the following amino acids: R.sup.1 --R.sup.2 --R.sup.3 --L--(R.sup.4).sub.n wherein, R.sup.1 is a positively charged amino acid; R.sup.2 is a negatively charged amino acid; R.sup.3 is a negatively charged amino acid; R.sup.4 is a positively charged amino acid; and n is zero or 1. ### Claim 4 (depends on 3) The method of claim 3, wherein R.sup.1 is selected from the group consisting of R and K. ### Claim 5 (depends on 4) The method of claim 4, wherein R.sup.2 is selected from the group consisting of E and D. ### Claim 6 (depends on 4) The method of claim 4, wherein R.sup.3 is selected from the group consisting of E and D. ### Claim 7 (depends on 4) The method of claim 4, wherein n is 1 and R.sup.5 is selected from the group consisting of K and R. ### Claim 8 (depends on 4) The method of claim 4, wherein the carboxy terminal sequence is selected from the group consisting of REDLK, KEDLK, REISLR, REDL, and KDEL. ### Claim 9 (depends on 4) The method of claim 4, wherein the carboxy terminal sequence is KDELKDELKDEL. ### Claim 10 (depends on 4) The method of claim 4, wherein the first recognition molecule is an antibody or a portion of an antibody which recognizes the target cell. ### Claim 11 (depends on 4) The method of claim 4, wherein the first recognition molecule is selected from the group consisting of a growth factor, lymphokine, cytokine, and a hormone. ### Claim 12 (depends on 4) The method of claim 4, wherein the first recognition molecule is TGF.alpha. or CD4. ### Claim 13 (depends on 4) The method of claim 4, wherein the first recognition molecule is inserted after amino acid 607 of the PE. ### Claim 14 (depends on 4) The method of claim 4, wherein a second recognition molecule is attached to the amino terminus of said Pseudomonas exotoxin. ### Claim 15 (depends on 14) The method of claim 14, wherein the second recognition molecule is different from the first recognition molecule. ### Claim 16 (depends on 14) The method of claim 14, wherein the second recognition molecule is anti-Tac(Fv). ### Claim 17 (depends on 14) The method of claim 14, wherein the recombinant PE is TGF.alpha.-anti-Tac(Fv)-PE40. ### Claim 18 (depends on 2) The method of claim 2, wherein said two different recognition molecules comprise a first recognition molecule inserted in the carboxyl terminus of said Pseudomonas exotoxin, and a second recognition molecule attached to the amino terminus of said Pseudomonas exotoxin. ### Claim 19 (depends on 18) The method of claim 18, wherein said first recognition molecule is inserted in domain III after amino acid 600 and before amino acid 613 of said Pseudomonas exotoxin. ### Claim 20 (depends on 19) The method of claim 19, wherein said first recognition molecule is inserted in domain III after amino acid 607 of said of said Pseudomonas exotoxin molecule. ### Claim 21 (depends on 19) The method of claim 19, wherein the first recognition molecule is TGF.alpha. or CD4.