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Pseudomonas exotoxin a with less immunogenic b cell epitopes

a technology of pseudomonas exotoxin and b cell epitope, which is applied in the direction of peptides, drug compositions, peptides, etc., can solve the problems of reducing the effectiveness of pe for treating disease, e.g., cancer, and being highly immunogenic to pe, and achieve the effect of reducing the level of antibodies

Active Publication Date: 2014-07-31
UNITED STATES OF AMERICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text describes a method for treating or preventing cancer in mammals by administering a specific molecule called PE. The patent describes various ways to use the PE molecule, including a chimeric molecule, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, and a pharmaceutical composition. The goal is to provide an effective treatment or prevention method for cancer in mammals.

Problems solved by technology

However, PE may be highly immunogenic.
Such immunogenicity may reduce the amount of PE that can be given to the patient which may, in turn, reduce the effectiveness of the PE for treating the disease, e.g., cancer.

Method used

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  • Pseudomonas exotoxin a with less immunogenic b cell epitopes
  • Pseudomonas exotoxin a with less immunogenic b cell epitopes
  • Pseudomonas exotoxin a with less immunogenic b cell epitopes

Examples

Experimental program
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Effect test

example 1

[0141]This example demonstrates the isolation and sequencing of human ScFv specific for PE38.

[0142]Blood samples were obtained from 6 patients who were treated with different recombinant immunotoxins (RITs) containing PE38 (Table 1). RNA was isolated from blood samples using PAXGENE Blood RNA Kits (PreAnalytiX GmbH, Hombrechtikon, Switzerland). First strand cDNA was synthesized from RNA using primers with the appropriate constant region (Table 2). Single bands of the correct size for VH and Vκ cDNA were obtained by using first strand cDNA as template. VH and VL fragments were amplified individually in three steps. Restriction enzyme site and linker were added into the fragment. 100 ng of the VH and VL fragments were combined in a Splicing by Overlapping Extension Polymerase Chain Reaction (SOE-PCR) for scFv formation. The scFv fragment was digested with Nco I and Not I, and subcloned into pCANTAB 5E digested with the same enzymes to construct a scFv library.

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example 2

[0145]This example demonstrates the location of human B cell epitopes.

[0146]LMB-9 contains both domains II and III of PE. To identify the phage which only binds to domain III, the binding of each clone to HA22-LR, which only had domain III and lacks domain II, was measured. Fifteen of the 56 phage clones could not bind to HA22-LR, indicating that the epitopes recognized by these 15 phage clones were located on domain II. The remaining 41 phage clones were used to identify the residues that make up the B-cell epitopes in domain III by measuring their binding to 36 substituted proteins in which individual amino acids on the surface of domain III of the protein were changed from a large bulky amino acid to alanine or glycine. These substitutions eliminated the large bulky side chains that are involved in antibody recognition and binding. The data are shown in FIG. 1 where clones with poor binding (<10%) are shown in black cells, and substituted proteins with normal reactivity are shown...

example 3

[0149]This example demonstrates the production of a low antigenic recombinant immunotoxin (RIT) for humans.

[0150]The identification of individual residues that were involved in binding to human antisera was used to design and construct immunotoxins with substitutions that eliminated reactivity with the human anti-sera yet retained cytotoxic activity and could be produced in sufficient amounts to be useful. In most cases, residues were replaced with alanine, because its small side chain reacts poorly with antibodies and it usually does not affect protein folding. Serine was also used to substantially avoid an especially hydrophobic surface.

[0151]Based on the information in the epitope mapping studies, substitutions selected from the different amino acids that destroyed the binding of the human Fvs to domain III of HA22-LR were combined. The substitutions are shown in Table 4 below. LRO5 had all the substitutions present in HA22-LR-8M (406A, 432G, 467A, 490A, 513A, 548S, 590S, 592A) a...

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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.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application claims priority to U.S. Application No. 61 / 535,668, filed on Sep. 16, 2011, which is incorporated by reference herein in its entirety.INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY[0002]Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 53,101 Byte ASCII (Text) file named “710974 ST25.txt,” dated Sep. 11, 2012.BACKGROUND OF THE INVENTION[0003]Pseudomonas exotoxin A (PE) is a bacterial toxin with cytotoxic activity that may be effective for destroying or inhibiting the growth of undesireable cells, e.g., cancer cells. Accordingly, PE may be useful for treating or preventing diseases such as, e.g., cancer. However, PE may be highly immunogenic. Accordingly, PE administration may stimulate an anti-PE immune response including, for example, the production of anti-PE antibodies, B-cells and...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/21A61K38/16A61K47/48
CPCC07K14/21A61K38/164A61K47/48484C07K16/2896C07K16/1214C07K2317/622A61K38/00C07K2317/34A61P35/00C07K16/2851C07K16/30C07K2317/56C07K2319/01C07K2319/55A61K47/6829
Inventor PASTAN, IRA H.ONDA, MASANORILIU, WENHAI
Owner UNITED STATES OF AMERICA
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