Modified diphtheria toxins

Inactive Publication Date: 2009-01-08
ANGELICA THERAPEUTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0026]Provided herein is a method of enhancing activity of an anti-cancer agent (e.g., RNA transfected DCs, anti-CLTA4 antibodies, MISIIR scFvs, etc.), by administering a DT variant-IL2 fusion protein described herein. In one embodiment, a DT variant-IL2 fusion protein is administered followed by administration of the anti-cancer agent. In one non-limiting example, the DT variant-IL2 fusion protein is administered at least four (4) days prior to the anti-cancer agent.
[0027]Also provided herein is a method of treating a metastatic cancer via reduction or elimination of Tregs by administering an anti-cancer agent (e.g., RNA transfected DCs, anti-CLTA4 antibodies, MISIIR scFvs, etc.) and a DT variant-IL2 fusion protein described herein. Metastatic tumors include, for example, metastatic renal cell carcinoma, metastatic prostate cancer, metastatic ovarian cancer and metastatic lung cancer. In one embodiment, a DT variant-IL2 fusion protein is administered followed by administration of the anti-cancer agent. In one non-limiting example, the DT variant-IL2 fusion protein is administered at least four (4) days prior to the anti-cancer agent.
[0028]In another aspect, provided herein is a method of treating a prostate tumor, an ovarian tumor, a lung tumor or a melanoma via reduction or elimination of Tregs by administering an anti-cancer agent (e.g., RNA transfected DCs, anti-CLTA4 antibodies, MISIIR scFvs, etc.) and a DT variant-IL2 fusion protein described herein. In one embodiment, a DT variant-IL2 fusion protein is administered followed by administration of the anti-cancer agent. In one non-limiting example, the DT variant-IL2 fusion protein is administered at least four (4) days prior to the anti-cancer agent.
[0029]Provided herein is a method for making a composition comprising the steps of: (a) constructing a vector comprising a nucleic acid sequence which encodes a polypeptide having an amino acid sequence of any of SEQ ID NOS: 4-147 or a polypeptide having two or more modifications of SEQ ID NOS: 4-147, an

Problems solved by technology

Severe VLS can cause fluid and protein extravasation, edema, dec

Method used

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  • Modified diphtheria toxins
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  • Modified diphtheria toxins

Examples

Experimental program
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Effect test

example 1

Construction, Expression and Purification of DT Variant and DT-Fusion Proteins

[0262]Construction of DT Variant and DT-Fusion Proteins

[0263]A truncated DT-based toxophore comprising a methionine residue at the N-terminus and amino acid residues 1 through 386 (SEQ ID NO: 2) of the native DT (now residues 2-387 in the truncated toxophore) is constructed as DT387 or residues 1-382 of DT387. The DT-based toxophore can also comprise a methionine residue at the N-terminus, amino acid residues 1 through 386 (SEQ ID NO: 2) of the native DT (now residues 2-387 in the truncated toxophore), and residues 484-485 of native DT, constructed as DT389. DT387 and DT389 contains three (x)D(y) motifs at residues 7-9 (VDS), residues 29-31 (VDS), and residues 290-292 (IDS). DT382 contains residues 1-382 of DT387 or DT389. Other C-terminal truncated DT constructs as described herein can be used in the assays provided herein for testing functionality of DT variants. One would understand that modifications m...

example 2

Cell Permeabilization Assays

[0270]Human vascular endothelial cells are maintained in EGM media (obtained from Cambrex, Walkersville, Md.). Sub-confluent early passage cells are seeded at equivalent cell counts onto plastic cover slips. Purified, endotoxin free wild type DT toxophore and mutants are labeled with the fluorescent tag F-150 (Molecular Probes, Eugene, Oreg.) through chemical conjugation. HUVECs are incubated with equivalent amounts of the labeled toxophores. The media is then aspirated, and the cells are then washed, fixed and prepared for analysis. Examination of the cells on cover slips from different treatment groups permits the analysis of the number of cells labeled by the fluorescent toxophore. No targeting ligand is present on the toxophore and, consequently, the level of HUVEC interaction is proportional only to the toxophores affinity for HUVECs. Comparisons are carried out using a fluorescent microscope and comparing the number of cells labeled from at least te...

example 3

[0274]This example describes a method for testing ADP-Ribosyltransferase Activity. Ribosome inactivating protein toxins, such as diphtheria toxin, catalyze the covalent modification of translation elongation factor 2 (EF-2). Ribosylation of a modified histidine residue in EF-2 halts protein synthesis at the ribosome and results in cell death. Ribosyltransferase assays to determine catalytic activity of the DT387 mutants are performed in 50 mM Tris-Cl, pH8.0, 25 mM EDTA, 20 mM Dithiothreitol, 0.4 mg / ml purified EF-2, and 1.0 μM [32P]-NAD+(10 mCi / ml, 1000 Ci / mmol, Amersham-Pharmacia). The purified mutant proteins are tested in a final reaction volume of 40 μl. The reactions are performed in 96 well, V-bottom microtiter plates (Linbro) and incubated at room temperature for an hour. Proteins are precipitated by addition of 200 μl 10% TCA and collected on glass fiber filters, and radioactivity is determined by standard protocols. Traditional methods for measuring ADP-ribosylation use per...

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Abstract

The present application relates to compositions of modified diphtheria toxin and fusion proteins containing modified diphtheria toxin that reduce binding to vascular endothelium or vascular endothelial cells, and therefore, reduce the incidence of Vascular Leak Syndrome, as well as methods of making the compositions. The present application also relates to a polypeptide toxophore from a modified diphtheria toxin, where the modification is at least one amino acid residue at the amino acid residues 6-8, 28-30 or 289-291 of an unmodified native diphtheria toxin. Also described are fusion proteins which contain a modified diphtheria toxin and a non-diphtheria toxin fragment which contains a cell binding portion. The modified diphtheria toxins described can be used for the treatment of a malignant disease or a non-malignant disease.

Description

CROSS-REFERENCE[0001]This application claims the benefit of U.S. Provisional Application No. 60 / 945,556, filed Jun. 21, 2007 (attorney docket number 33094-702.101), U.S. Provisional Application No. 60 / 954,278, filed Aug. 6, 2007 (attorney docket number 33094-702.102), U.S. Provisional Application No. 61 / 032,888, filed Feb. 29, 2008 (attorney docket number 33094-702.103), U.S. Provisional Application No. 61 / 042,178, filed Apr. 3, 2008 (attorney docket number 33094-702.104), U.S. Provisional Application No. 60 / 945,568, filed Jun. 21, 2007 (attorney docket number 33094-703.101), U.S. Provisional Application No. 60 / 954,284, filed Aug. 6, 2007 (attorney docket number 33094-703.102), U.S. Provisional Application No. 61 / 032,910, filed Feb. 29, 2008 (attorney docket number 33094-703.103), and U.S. Provisional Application No. 61 / 042,187, filed Apr. 3, 2008 (attorney docket number 33094-703.104), each of which applications is incorporated herein in its entirety by reference.BACKGROUND OF THE ...

Claims

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Application Information

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IPC IPC(8): A61K39/05C07K14/195A61P35/04
CPCA61K38/16A61K39/0011A61K39/39A61K47/48261C07K14/34A61K2039/5156A61K2039/55533A61K2039/55544A61K47/48269A61K47/6415A61K47/642A61P35/00A61P35/02A61P35/04A61P37/00A61P43/00A61K38/164C07K14/55C07K19/00
Inventor DAVIS, CLAUDE GEOFFREYDATTA, DEEPSHIKHA
Owner ANGELICA THERAPEUTICS
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