Modified Pseudomonas exotoxin A

Patent metadata

Field	Value
Publication number	US-9388222-B2
Application number	US-201414505590-A
Country	US
Kind code	B2
Filing date	Oct 3, 2014
Priority date	Oct 6, 2013
Publication date	Jul 12, 2016
Grant date	Jul 12, 2016

Abstract

The invention provides a Pseudomonas exotoxin A (PE) comprising an amino acid sequence having a substitution of one or more B-cell and/or T-cell epitopes. The invention further provides related chimeric molecules, as well as related nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions. Methods of treating or preventing cancer in a mammal, methods of inhibiting the growth of a target cell, methods of producing the PE, and methods of producing the chimeric molecule are further provided by the invention.

First claim

The invention claimed is:

1. A Pseudomonas exotoxin A (PE) comprising a PE amino acid sequence wherein one or more of amino acid residues F443, R456, L477, R494, and L552 as defined by reference to SEQ ID NO: 1 are, independently, substituted, wherein the PE optionally has:
(i) a further substitution of one or more amino acid residues within one or more B cell epitopes, and the further substitution for an amino acid within one or more B-cell epitopes is a substitution of, independently, one or more of amino acid residues D403, D406, R412, R427, E431, R432, D461, R463, R467, R490, R505, R513, E522, R538, E548, R551, R576, Q592, and L597 as defined by reference to SEQ ID NO: 1,
(ii) a further substitution of one or more amino acid residues within one or more T-cell epitopes,
(iii) a deletion of one or more continuous amino acid residues of residues 1-273 and 285-394 as defined by SEQ ID NO: 1, or
(iv) a combination of any one, two, or three of (i)-(iii).

2. An isolated, mutated Pseudomonas exotoxin A (PE), comprising a sequence of the following formula:
R 1 n -FCS-R 2 n -R 3 n -PE functional domain III
wherein:
n=0 or 1 independently for each of R 1 , R 2 and R 3 ,
R 1 =1 to 10 amino acid residues
FCS=a furin cleavage sequence of amino acid residues, which sequence is cleavable by furin and has an amino end and a carboxyl end,
R 2 =1 to 10 amino acid residues;
R 3 =1 or more contiguous residues of residues 365-394 of SEQ ID NO: 1; and,
PE functional domain III=residues 395-613 of SEQ ID NO:1, wherein one or more of amino acid residues F443, R456, L477, R494, and L552 as defined by reference to SEQ ID NO: 1 are, independently, substituted, wherein the PE optionally has:
(i) a further substitution of one or more amino acid residues within one or more B cell epitopes, and the further substitution for an amino acid within one or more B-cell epitopes is a substitution of, independently, one or more of amino acid residues D403, D406, R412, R427, E431, R432, D461, R463, R467, R490, R505, R513, E522, R538, E548, R551, R576, Q592, and L597 as defined by reference to SEQ ID NO: 1,
(ii) a further substitution of one or more amino acid residues within one or more T-cell epitopes, or
(iii) both (i) and (ii).

3. The mutated PE of claim 2 , further wherein the FCS is represented by the formula P4-P3-P2-P1, wherein P4 is an amino acid residue at the amino end, P1 is an amino acid residue at the carboxyl end, P1 is an arginine or a lysine residue, and the sequence is cleavable at the carboxyl end of P1 by furin.

4. The mutated PE of claim 3 , further wherein the FCS (i) further comprises amino acid residues represented by P6-P5 at the amino end, (ii) further comprises amino acid residues represented by P1′-P2′ at the carboxyl end, (iii) wherein if P1 is an arginine or a lysine residue, P2′ is tryptophan, and P4 is arginine, valine or lysine, provided that if P4 is not arginine, then P6 and P2 are basic residues, and (iv) the sequence is cleavable at the carboxyl end of P1 by furin.

5. The mutated PE of claim 2 , further wherein the PE functional domain III consists of the sequence of residues 395 to 613 of SEQ ID NO: 1.

6. The mutated PE of claim 2 , wherein the mutated PE comprises one or more contiguous residues of residues 365-394 of SEQ ID NO: 1 between the FCS and the PE domain III.

7. The mutated PE of claim 2 , wherein n is 1 for R 1 and R2.

8. The mutated PE of claim 2 , wherein the FCS is SEQ ID NO: 8.

9. The mutated PE of claim 2 , wherein R 1 =a linker of the amino acid sequence of SEQ ID NO: 282, R 2 =a linker of the amino acid sequence SEQ ID NO: 284, and the FCS=SEQ ID NO: 8.

10. The mutated PE of claim 2 , wherein n is 0 for R 3 .

11. The mutated PE of claim 2 , wherein PE functional domain III comprises the amino acid sequence of SEQ ID NO: 37.

12. The mutated PE of claim 2 , wherein R 1 n -FCS-R 2 n =SEQ ID NO: 36.

13. The PE of claim 1 , wherein the substitution of one or more of amino acid residues F443, R456, L477, R494, and L552 is a substitution of, independently, alanine, glutamic acid, histidine, or asparagine in place of one or more of amino acid residues F443, R456, L477, R494, and L552.

14. The PE of claim 1 , wherein the substitution of L552 is a substitution of glutamic acid or asparagine in place of L552 and the substitution of L477 is a substitution of histidine in place of L477.

15. The PE of claim 1 , wherein the further substitution of an amino acid within one or more B-cell epitopes is a substitution of, independently, alanine, glycine, serine, or glutamine in place of one or more of amino acid residues E282, E285, P290, R313, N314, P319, D324, E327, E331, Q332, D403, D406, R412, R427, E431, R432, D461, R463, 8467, R490, R505, R513, E522, R538, E548, R551, R576, K590, Q592, and L597, as defined by reference to SEQ ID NO: 1.

16. The PE of claim 1 , wherein the PE has the further substitution of an amino acid within one or more T-cell epitopes, and the further substitution of an amino acid within one or more T-cell epitopes is a substitution of, independently, alanine, glycine, serine, or glutamine in place of one or more of amino acid residues R421, L422, L423, A425, R427, L429, Y439, H440, F443, L444, A446, A447, I450, 463-519, R551, L552, T554, I555, L556, and W558 as defined by reference to SEQ ID NO: 1.

17. The PE of claim 1 , wherein the substitution of one or more of amino acid residues F443, R456, L477, R494, and L552 is a substitution of alanine in place of amino acid residue F443; a substitution of alanine in place of amino acid residue R456; a substitution of histidine in place of amino acid residue L477; a substitution of alanine in place of amino acid residue R494; and a substitution of glutamic acid in place of amino acid residue L552, the PE has an arginine residue at position 458, and the further substitution of an amino acid within one or more B-cell epitopes is:
(a) a substitution of alanine for amino acid residue R427;
(b) a substitution of alanine for amino acid residue R463;
(c) a substitution of alanine for amino acid residue R467;
(d) a substitution of alanine for amino acid residue R490;
(e) a substitution of alanine for amino acid residue R505; and
(f) a substitution of alanine for amino acid residue R538;
as defined by reference to SEQ ID NO: 1.

18. The PE of claim 1 , wherein the substitution of one or more of amino acid residues F443, R456, L477, R494, and L552 is a substitution of alanine in place of amino acid residue R456, the PE has an arginine residue at position 458, and the further substitution of an amino acid within one or more B-cell epitopes is:
(a) a substitution of alanine for amino acid residue R427;
(b) a substitution of alanine for amino acid residue R463;
(c) a substitution of alanine for amino acid residue R467;
(d) a substitution of alanine for amino acid residue R490;
(e) a substitution of alanine for amino acid residue R505; and
(f) a substitution of alanine for amino acid residue R538;
as defined by reference to SEQ ID NO: 1.

19. The PE of claim 1 , wherein the substitution of one or more of amino acid residues F443, R456, L477, R494, and L552 is a substitution of alanine in place of amino acid residue F443; a substitution of alanine in place of amino acid residue R456; a substitution of histidine in place of amino acid residue L477; a substitution of alanine in place of amino acid residue R494; and a substitution of asparagine in place of amino acid residue L552, the PE has an arginine residue at position 458, and the furt

Assignees

• Us Health
• Hoffmann La Roche
• Hoffmann La Roche

Inventors

• Pastan Ira H
• Mazor Ronit
• Onda Masanori
• Lee Byungkook
• Niederfellner Gerhard
• Imhof-Jung Sabine
• Brinkmann Ulrich
• Georges Guy

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• A61K31/337 Primary

  having four-membered rings, e.g. taxol · CPC title

• A61K38/00

  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 title

• C07K2317/24

  containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered · CPC title

• C07K16/30 Primary

  from tumour cells · CPC title