Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Ricin-like toxin variants for treatment of cancer, viral or parasitic infections

a technology of ricin-like toxin and variants, which is applied in the field of ricin-like toxin variants for cancer, viral or parasitic infections, can solve the problems of limiting the usefulness of ricin-like toxin variants as therapeutics for cancer and infectious diseases, and the preparation of suitable specific cell binding components may be problematic, so as to enhance the retrograde translocation of the host major histocompatibility complex (mhc), enhance the transcytos

Inactive Publication Date: 2011-06-16
TWINSTRAND HLDG
View PDF7 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0023]Toxin targeting using the recombinant toxic proteins of the invention takes advantage of the fact that many DNA viruses exploit host cellular transport mechanisms to escape immunological destruction. This is achieved by enhancing the retrograde translocation of host major histocompatibility complex (MHC) type I molecules from the endoplasmic reticulum into the cytoplasm (Bonifacino, J. S., Nature 384: 405-406 (1996); Wiertz, E. J. et al., Nature 384: 432-438 (1996)). The facilitation of retrograde transport in diseased cells by the virus can enhance the transcytosis and cytotoxicity of a recombinant toxic protein of the present invention thereby further reducing non-specific cytotoxicity and improving the overall safety of the product.

Problems solved by technology

The ribosomes of the castor bean plant are themselves susceptible to inactivation by ricin A chain; however, as there is no cell surface galactose to permit B chain recognition the A chain cannot re-enter the cell.
The high toxicity of these toxins for mammalian cells, combined with a lack of specificity of action poses a major problem to the development of pharmaceuticals incorporating the toxins, such as immunotoxins.
A major problem with the use of such immunotoxins is that the antibody component is its only targeting mechanism and the target antigen is often found on non-target cells (Vitetta et al., Immunology Today 14:252-259 (1993)).
Also, the preparation of a suitable specific cell binding component may be problematic.
In view of the extreme toxicity of proteins such as ricin, the lack of specificity of the immunotoxins may severely limit their usefulness as therapeutics for the treatment of cancer and infectious diseases.
In view of the ubiquitous nature of the extracellular proteases utilized in these approaches, such artificial activation of the toxin precursor or immunotoxin does not confer a mechanism for intracellular toxin activation and the problems of target specificity and adverse immunological reactions to the cell-binding component of the immunotoxin remain.
It does not confer a mechanism for intracellular toxin activation and presents a problem of ensuring sufficient quantities of toxin for internalization in target cells.
Such widespread activation of the toxin does not confer target specificity and limits the usefulness of said α-hemolysin toxin as therapeutics due to systemic toxicity.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Ricin-like toxin variants for treatment of cancer, viral or parasitic infections
  • Ricin-like toxin variants for treatment of cancer, viral or parasitic infections
  • Ricin-like toxin variants for treatment of cancer, viral or parasitic infections

Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning and Expression of Proricin Variants Activated by Disease-Specific Proteases

Isolation of Total RNA

[0415]The preproricin gene was cloned from new foliage of the castor bean plant. Total messenger RNA was isolated according to established procedures (Sambrook et al., Molecular Cloning: A Lab Manual (Cold Spring Harbour Press, Cold Spring Harbour, (1989)) and cDNA generated using reverse transcriptase.

cDNA Synthesis:

[0416]Oligonucleotides, corresponding to the extreme 5′ and 3′ ends of the preproricin gene were synthesized and used to polymerase chain reaction (PCR) amplify the gene. Using the cDNA sequence for preproricin (Lamb et al., Eur. J. Biochem., 145:266-270, 1985), several oligonucleotide primers were designed to flank the start and stop codons of the preproricin open reading frame. The oligonucleotides were synthesized using an Applied Biosystems Model 392 DNA / RNA Synthesizer. First strand cDNA synthesis was primed using the oligonucleotide Ricin1729C (Table 1). Three ...

example 2

Harvesting and Affinity Column Purification of Pro-Ricin Variants

[0422]Protein samples were harvested three days post transfection. The cells were removed by centrifuging the media at 8288 g for ten minutesusing a GS3 (Sorvall) centrifuge rotor. The supernatant was further clarified by centrifuging at 25400 g using a SLA-1500 rotor (Sorvall) for 45 minutes. Protease inhibitor phenylmethylsulfonyl fluoride (Sigma) was slowly added to a final concentration of 1 mM. The samples were further prepared by adding lactose to a concentration of 20 mM (not including the previous lactose contained in the expression medium). The samples were concentrated to 700 mL using a Prep / Scale-TFF Cartridge (2.5 ft, 10K regenerated cellulose (Millipore)) and a Masterflex pump. The samples were then dialysed for 2 days in 1× Column Buffer (50 mM Tris, 100 mM NaCl, 0.02% NaN3, pH 7.5) using dialysis tubing (10 K MWCO, 32 mm flat width (Spectra / Por)). Subsequently, the samples were clarified by centrifuging ...

example 3

In Vitro Protease Digestion of Proricin Variants

[0441]Affinity-purified proricin variant is treated with individual disease-specific proteases to confirm specific cleavage in the linker region. Ricin-like toxin variants are eluted from the lactose-agarose matrix in protease digestion buffer (50 mM NaCl, 50 mM Na-acetate, pH 5.5, 1 mM dithiothreitol) containing 100 mM lactose. Proricin substrate is then incubated at 37° C. for 60 minutes with a disease-specific protease. The cleavage products consisting ricin A and B chains are identified using SDS / PAGE (Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd. ed., Cold Spring Harbor Press, 1989), followed by Western blot analysis using anti-ricin antibodies (Sigma).

[0442]Cathepsin B may be obtained from Medcor or Calbiochem. Matrix metalloproteinases may be prepared substantially as described by Lark, M. W. et al. (Proceedings of the 4th International Conference of the Inflammation Research Association Abstract 145 (1988)) and ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
temperatureaaaaaaaaaa
temperatureaaaaaaaaaa
flat widthaaaaaaaaaa
Login to View More

Abstract

The present invention provides a protein having an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence, linking the A and B chains. The linker sequence contains a cleavage recognition site for a disease specific protease such as a cancer, fungal, viral or parasitic protease. The invention also relates to a nucleic acid molecule encoding the protein and to expression vectors incorporating the nucleic acid molecule. Also provided is a method of inhibiting or destroying mammalian cancer cells, cells infected with a virus, a fungus, or parasite, or parasites utilizing the nucleic acid molecules and proteins of the invention and pharmaceutical compositions for treating human cancer, viral infection, fungal infection, or parasitic infection.

Description

[0001]This application is a divisional of U.S. patent application Ser. No. 10 / 394,511 that was filed Mar. 24, 2003 (now U.S. Pat. No. 7,375,186), which is a divisional of U.S. patent application Ser. No. 09 / 403,752 that was filed on Oct. 29, 1999 (now U.S. Pat. No. 6,593,132) which is a national phase entry application of PCT / CA98 / 00394 filed Apr. 30, 1998, which claims benefit from U.S. provisional application Ser. No. 60 / 045,148 filed on Apr. 30, 1997 (now abandoned) and U.S. provisional application Ser. No. 60 / 063,715 filed on Oct. 29, 1997 (now abandoned), all of which are incorporated herein by reference.FIELD OF THE INVENTION[0002]The invention relates to proteins useful as therapeutics against cancer, viral infections, parasitic and fungal infections. The proteins contain A and B chains of a ricin-like toxin linked by a linker sequence that is specifically cleaved and activated by proteases specific to disease-associated pathogens or cells.BACKGROUND OF THE INVENTION[0003]Bac...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/16A61K31/7088A61P31/10A61P35/00C12N15/09A61K35/76A61K36/47A61K38/00A61K48/00A61P31/04A61P31/12A61P33/00C07K14/00C07K14/415C12N1/21C12N15/29
CPCA61K38/00A61K48/00C12N2799/021C07K2319/00C07K14/415A61P31/04A61P31/10A61P31/12A61P33/00A61P35/00Y02A50/30
Inventor BORGFORD, THOR
Owner TWINSTRAND HLDG
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com