Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Fusion protein or fusion polypeptide
Reexamination Certificate
1999-11-16
2004-05-04
Bugaisky, Gabriele (Department: 1653)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Fusion protein or fusion polypeptide
C424S193100, C424S195110, C512S002000, C512S012000
Reexamination Certificate
active
06730302
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the intracellular delivery, preferably the intranuclear delivery, of compounds using the heat shock protein Hsp70.
BACKGROUND OF THE INVENTION
Heat shock proteins (“Hsps”) are a family of molecular chaperone proteins which have long been known to play essential roles in a multitude of intra- and intercellular processes, including protein synthesis and folding, vesicular trafficking, and antigen processing and presentation. Hsps are among the most highly conserved proteins known, and carry out many of their regulatory activities via protein—protein interactions. Hsp70 is one member of the heat shock protein family. (Milner, C. M. and Campbell, R. D.
Immunogenetics
32:242-251 (1990), Genbank Accession No. M59828). One of the most well characterized functions of Hsp70 is to assist in the translocation of proteins across intracellular membranes into different compartments of the cell.
Intracellular transport activity has been reported for viral proteins such as the HSV-1 structural protein VP22 (Elliott, et. al., (1997)
Cell
88:223-233) and the HIV Tat protein (Vives, et. al., (1997)
J. Biol. Chem.
272:16010-16017), as well as peptide sequences derived from Antennapedia homeodomain, fibroblast growth factor (Hawiger, (1997)
Curr. Opin. Immun.
9:189-194), and most recently the neuropeptide galanin (Pooga, et. al., (1998)
FASEB J.
12:67-77). Delivery of protein substrates has been demonstrated by some of these transport peptides as well (Phelan, et. al., (1998)
Nature Biotechnology
16:440-443, Fawell, et. al., (1994)
Proc. Natl. Acad. Sci.
91:664-668, Rojas, et. al., (1998)
Nature Biotechnology
16:370-375).
Hsps also serve a number of key functions in the immune response, and over the past few years there has been increasing interest in characterizing the nature of Hsps in generating protective immunity. A series of recent studies (Román, et. al., (1996)
Immunology
88:487-492, Suzue, et. al., (1996)
J. Immunol.
156:873-879) demonstrated that Hsp70 could act as a carrier protein to enable a bound peptide or protein substrate to enter the endosomal compartment and subsequently access the MHC class II processing pathway for exogenous antigens. Such treatment with Hsp70-peptide complexes or Hsp70 fusion proteins could elicit cargo-specific proliferative T cell responses. However, other experiments in which cancer cell-derived Hsp70 used to immunize mice resulted in specific antitumor CTL responses (Udono, et al., (1993)
J. Exp. Med.
178:1391-1396) suggested that the Hsps were using the endogenous MHC class I processing pathway. These data implied that Hsp70 was able to cross the plasma membrane and enter the cytoplasmic compartment of intact cells. Earlier studies (Hightower, et al. (1989)
J. Cell Physiol.
138:257-266) reporting the release of Hsps from axonal cells by a non-heat shock dependent mechanism, support the observation that some Hsp family members can cross the plasma membranes of certain cells.
Although the above studies imply a plasma membrane translocation capacity for Hsp70, such an activity has not been directly demonstrated. It has not been shown whether or not Hsp70 could be utilized to deliver proteins across the plasma and nuclear membranes. There exists a need to deliver compounds, such as proteins or DNA, into the cell nucleus to modulate cellular activity.
Applicants have shown that the human 70 kD heat shock protein can translocate across cell membranes to rapidly gain cytoplasmic and nuclear entry. Furthermore, chimeric proteins composed of Hsp70 peptides fused to amino acids 37-409 of the p50 subunit of NF-&kgr;B (Meyer, R., et. al., PNAS 88:966-970 (1991); Genbank Accession No. M58603) also exhibit this translocation property. Though cellular import activity has been reported for various diverse peptides, intranuclear transport generally requires the presence of specific nuclear localization sequences (“NLS”). While heat shock is known to induce increased synthesis and nuclear translocation of endogenous heat shock protein following heat shock factor activation, Applicants show that nuclear localization of exogenous heat shock protein can result without prior heat shock. Applicants have also shown that the transport and nuclear localization properties of Hsp70 are retained within a 90 amino acid C-terminal fragment; successful intranuclear transport has been demonstrated utilizing a C-terminal fragment as well as larger fragments encompassing more of the peptide binding domain. Herein is the first evidence establishing the ability of exogenous Hsp70 fusion proteins to cross the cell membrane, gain nuclear entry and exert a biological function.
Therefore, an object of the present invention is to provide a carrier for delivery of molecules with biological function into both cellular and nuclear compartments. A preferred embodiment of the present invention utilizes Hsp70, or a fragment of Hsp70 as described herein, as a vehicle for directed, noninvasive delivery of molecules, such as proteins or DNA, that may modulate gene expression.
SUMMARY OF THE INVENTION
The 70 kD heat shock protein (“Hsp70”) is a highly conserved, ubiquitous protein involved in chaperoning proteins to various cellular organelles. Applicants herein show that when added exogenously to cells, Hsp70 is readily imported into both cytoplasmic and nuclear compartments. Applicants have demonstrated that Hsp70 can be used to chaperone compounds into a cell or cells. Hsp70 was used to deliver NF-&kgr;B, a key transcriptional regulator of inflammatory responses, into the nuclear compartment. Applicants herein show that a fusion protein composed of a C-terminal Hsp70 peptide and amino acids 37-409 of the p50 subunit of NF-&kgr;B was directed into the nucleus of cells, could bind DNA specifically, and activated kappa Ig expression and TNF&agr; production. Applicants' invention encompasses the use of Hsp70 as a vehicle for intracytoplasmic and intranuclear delivery of proteins or DNA to modulate gene expression and thereby control immune responses.
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Hawiger (1997) Curr. Opin. Immun. 9:189-194.
Pooga et al. (1998) FASEB J. 12:67-77.
Phelan et al. (1998) Nature Biotechnology 16:440-443.
Fawell et al. (1994) Proc.Natl.Acad.Sci 91:664-668.
Rojas et al. (1998) Nature Biotechnology 16:370-375.
Romá{acute over ( )}n et al. (1996) Immunology 88:487-492.
Suzue et al. (1996) J.Immunol 156:873-879.
Udono et al. (1993) J.Exp.Med. 178:1391-1396.
Hightower et al. (1989) J.Cell.Physiol. 138:257-266.
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Fix
Fujihara Sheri M.
Nadler Steven G.
Bristol--Myers Squibb Company
Bugaisky Gabriele
Klein Christopher A.
Sher Audrey F.
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