Purified heat shock protein complexes

Details Purified heat shock protein complexes Purified heat shock protein complexes

2002-06-20

2004-03-30

Reamer, James H (Department: 1614)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C530S402000, C530S412000, C530S413000

Reexamination Certificate

active

06713608

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to methods for purifying and synthesizing heat shock protein complexes.
2. Description of the Prior Art
Heat shock proteins (HSPs) are associated in cells with a broad spectrum of peptides, polypeptides, denatured proteins and antigens with which they form complexes. Such HSP-peptide complexes have been described as being useful in vaccines against cancers and infectious diseases by Srivastava et al., “Heat shock protein-peptide complexes in cancer immunetherapy” in
Current Opinion in Immunology
(1994), 6:728-732; Srivastava, “Peptide-Binding Heat Shock Proteins in the Endoplasmic Reticulum” in
Advances in Cancer Research
(1993), 62:153-177. The HSP-peptide complexes appear to work as vaccines, because they may function as antigen carrying and presentation molecules. The development of vaccines using such antigens has been described by Baltz, “Vaccines in the treatment of Cancer” in
Am J Health
-
Syst Pharm
(1995), 52:2574-2585. The antigenicity of heat shock proteins appears to derive not from the heat shock protein itself, but from the associated peptides, see Udono et al., “Heat Shock Protein 70-associated Peptides Elicit Specific Cancer Immunity” in
J. Exp. Med
. (1993) 178 1391-1396, Srivastava et al., “Heat shock proteins transfer peptides during antigen processing and CTL priming” in
Immunogenetics
(1994), 39:93-98, Srivastava, “A Critical Contemplation on the Roles of Heat Shock Proteins in Transfer of Antigenic Peptides During Antigen Presentation” in
Behring Inst. Mitt
. (1994), 94:37-47. HSPs appear to be part of the process by which peptides are transported to the Major Histocompatibility Complex (MHC) molecules for surface presentation.
A number of different HSPs have been shown to exhibit immunogenicity including: gp96, hsp90 and hsp70, see Udono et al., supra. and Udono et al., “Comparison of Tumor-Specific Iummnogenicities of Stress-Induced Proteins gp96, hsp90, and hsp 70” in
Journal of Immunology
(1994), 5398-5403; gp96 and grp94, Li et al., “Tumor rejection antigen gp96/grp94 is an ATPase: implications for protein folding and antigen presentation” in
The EMBO Journal
, Vol. 12, No. 8 (1993), 3143-3151; and gp96, hsp90 and hsp70, Blachere et al., “Heat Shock Protein Vaccines Against Cancer” in
Journal Of Immunotherapy
(1993), 14:352-356.
Heat shock proteins have been purified using a procedure employing DE52 ion-exchange chromatography followed by affinity chromatography on ATP-agarose, see Welch et al., “Rapid Purification of Mammalian 70,000-Dalton Stress Proteins: Affinity of the Protein for Nucleotides” in
Molecular and Cellular Biology
(June 1985), 1229-1237. However, previous methods of purifying HSPs such as this one purify the heat shock proteins without the associated peptides. Other methods that do purify HSPs together with their associated peptides are complicated and expensive.
SUMMARY OF THE INVENTION
Therefore, it is an object of the invention to provide a simple and inexpensive method for purifying heat shock proteins together with their associated peptides, polypeptides, denatured proteins or antigens from cell lysates.
It is a further object of the invention to provide a method for synthesizing heat shock protein complexes that is capable of forming these complexes from heat shock proteins and peptides, polypeptides, denatured proteins or antigens from different cells and from different species.
The present invention provides a method for purifying heat shock protein complexes comprising the steps of adding solution containing heat shock protein complexes, in which heat shock proteins are associated with peptides, polypeptides, denatured proteins or antigens, to a column containing an ADP matrix to bind the heat shock proteins complexes to the ADP matrix and then adding a buffer containing ADP to the column remove the heat shock protein complexes in all elution product.
The present invention also provides a method for synthesizing heat shock protein complexes and purifying the complexes so produced by adding heat shock proteins to an ADP matrix column to bind them to the matrix, including a solution of peptides, polypeptides, denatured proteins or antigens to the column to bind them to the heat shock proteins as heat shock protein complexes and then adding a buffer containing ADP to the column to remove the complexes in an elution product.


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Srivastava, “Peptide-Binding Heat Shock Proteins in the Endoplasmic Reticulum: Role in Immune Response to Cancer and in Antigen Presentation,”Advances in Cancer Research(1993), 62:153-177.
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