Target-specific, cytotoxic, recombinant pseudomonas exotoxin

• US 5,705,163 A
• Filed: 06/05/1995
• Issued: 01/06/1998
• Est. Priority Date: 01/02/1990
• Status: Expired due to Fees

• Current Assignee: The United States of America As Represented By The Secretary of Agriculture
• Original Assignee: The United States of America As Represented By The Secretary of Agriculture
• Inventors: Fitzgerald, David, Chaudhary, Vijay K., Pastan, Ira
• Primary Examiner(s): Feisee, Lila
• Assistant Examiner(s): DAVIS, MINH TAM B
• Application Number:
  US08/461,233
• Time in Patent Office: 946 Days
• Field of Search: 424/183.1, 424/236.1, 424/260.1, 935/11, 435/69.1, 435/69.7, 435/71.3, 435/875, 530/387, 530/391.7, 530/356
• US Class Current: 424/260.1
• CPC Class Codes: 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
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/21 from Pseudomonadaceae (F)
• CPC Class Codes: C07K 14/495 Transforming growth factor [TGF]
• CPC Class Codes: C07K 14/70514 CD4
• CPC Class Codes: C07K 16/2866 against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C07K 2317/77 Internalization into the cell
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: 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
• CPC Class Codes: C07K 2319/55 containing a fusion with a toxin, e.g. diphteria toxin
• CPC Class Codes: C07K 2319/75 containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
• CPC Class Codes: Y10S 435/875 Pseudomonas aeruginosa

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

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.

3. 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:

space="preserve" listing-type="equation">R.sup.1 --R.sup.2 --R.sup.3 --L--(R.sup.4).sub.n

wherein,

R¹ is a positively charged amino acid;

R² is a negatively charged amino acid;

R³ is a negatively charged amino acid;

R⁴ is a positively charged amino acid; and

n is zero or 1.

4. The method of claim 3, wherein R¹ is selected from the group consisting of R and K.

5. The method of claim 4, wherein R² is selected from the group consisting of E and D.

6. The method of claim 4, wherein R³ is selected from the group consisting of E and D.

7. The method of claim 4, wherein n is 1 and R⁵ is selected from the group consisting of K and R.

8. The method of claim 4, wherein the carboxy terminal sequence is selected from the group consisting of REDLK, KEDLK, REISLR, REDL, and KDEL.

9. The method of claim 4, wherein the carboxy terminal sequence is KDELKDELKDEL.

10. The method of claim 4, wherein the first recognition molecule is an antibody or a portion of an antibody which recognizes the target cell.

11. 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.

12. The method of claim 4, wherein the first recognition molecule is TGFα

or CD4.

13. The method of claim 4, wherein the first recognition molecule is inserted after amino acid 607 of the PE.

14. The method of claim 4, wherein a second recognition molecule is attached to the amino terminus of said Pseudomonas exotoxin.

15. The method of claim 14, wherein the second recognition molecule is different from the first recognition molecule.

16. The method of claim 14, wherein the second recognition molecule is anti-Tac(Fv).

17. The method of claim 14, wherein the recombinant PE is TGFα

-anti-Tac(Fv)-PE40.

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.

18. 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.

19. 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.

20. 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.

21. The method of claim 19, wherein the first recognition molecule is TGFα

or CD4.