Pseudomonas exotoxin A with less immunogenic B cell epitopes

Patent metadata

Field	Value
Publication number	US-9206240-B2
Application number	US-201214241782-A
Country	US
Kind code	B2
Filing date	Sep 13, 2012
Priority date	Sep 16, 2011
Publication date	Dec 8, 2015
Grant date	Dec 8, 2015

Abstract

The invention provides a Pseudomonas exotoxin A (PE) comprising an amino acid sequence having a substitution of one or more of amino acid residues E420, D463, Y481, L516, R563, D581, D589, and K606, wherein the amino acid residues are defined by reference to SEQ ID NO: 1. 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 selected from the group consisting of E420, D463, Y481, L516, R563, D581, and D589 as defined by reference to SEQ ID NO: 1 are, independently, substituted, with the proviso that when the amino acid residue at position 516 is substituted with alanine, at least one of the amino acid residues E420, D463, Y481, R563, D581, and D589 is substituted,
wherein the PE has a further substitution selected from the group consisting 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 E282, E285, P290, R313, N314, P319, D324, E327, E331, Q332, D403, D406, R412, R427, E431, R432, R458, D461, R467, R490, R505, R513, E522, R538, E548, R551, R576, Q592, and L597 as defined by reference to SEQ ID NO: 1, and
wherein the PE optionally has (i) a further substitution selected from the group consisting of one or more amino acid residues within one or more T-cell epitopes, (ii) 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 a combination of (i) and (ii).

2. The PE of claim 1 , wherein the substitution of one or more of amino acid residues E420, D463, Y481, L516, R563, D581, and D589 is a substitution of, independently, alanine, glycine, serine, or glutamine in place of one or more of amino acid residues E420, D463, Y481, L516, R563, D581, and D589.

3. The PE of claim 1 , wherein the substitution of one or more of amino acid residues E420, D463, Y481, L516, R563, D581, and D589 is a substitution of one or more of amino acid residues D463, Y481, and L516.

4. The PE of claim 1 , wherein the substitution of one or more of amino acid residues E420, D463, Y481, L516, R563, D581, and D589 is a substitution of one or more of amino acid residues E420, Y481, R563, D581, and D589.

5. The PE of claim 1 , wherein the substitution of one or more of amino acid residues E420, D463, Y481, L516, R563, D581, and D589 is a substitution of one or more of amino acid residues E420, D463, Y481, L516, R563, and D581.

6. 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, R458, D461, R467, R490, R505, 8513, E522, R538, E548, R551, R576, K590, Q592, and L597, as defined by reference to SEQ ID NO: 1.

7. The PE of claim 6 , wherein the further substitution of an amino acid within one or more B-cell epitopes is a substitution of, independently, alanine, glycine, or serine in place of one or more amino acid residues R427, R458, R467, R490, R505, and R538.

8. The PE of claim 7 , wherein the substitution of one or more of amino acid residues E420, D463, Y481, R563, D581, and D589 is a substitution of alanine in place of amino acid residue D463, 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 R458;
(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.

9. The PE of claim 1 , wherein the PE has a further substitution of one or more amino acid residues within one or more T-cell epitopes.

10. The PE of claim 1 , wherein the PE has a deletion of one or more continuous amino acid residues of residues 1-273 and 285-394 as defined by SEQ ID NO: 1.

11. A pharmaceutical composition comprising (a) the PE of claim 1 and (b) a pharmaceutically acceptable carrier.

12. A chimeric molecule comprising (a) a targeting agent conjugated or fused to (b) the PE of claim 1 .

13. The chimeric molecule of claim 12 , wherein the targeting agent is a monoclonal antibody.

14. A method of inhibiting the growth of a target cell, which method comprises contacting the cell with the PE of claim 1 , in an amount effective to inhibit growth of the target cell.

Assignees

• Pastan Ira H
• Onda Masanori
• Liu Wenhai
• Us Health

Inventors

• Pastan Ira H
• Onda Masanori
• Liu Wenhai

Classifications

• C07K16/1214

  Pseudomonadaceae (F) · CPC title

• C07K2317/56

  variable (Fv) region, i.e. VH and/or VL · 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

• A61K38/164 Primary

  from bacteria · CPC title

• C07K2317/622

  Single chain antibody (scFv) · CPC title