Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Fusion protein or fusion polypeptide
Reexamination Certificate
2000-12-13
2004-07-27
Chan, Christina (Department: 1644)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Fusion protein or fusion polypeptide
C424S190100, C424S193100
Reexamination Certificate
active
06767542
ABSTRACT:
BACKGROUND OF THE INVENTION
Stimulation of an immune response is dependent upon the presence of antigens recognized as foreign by the host immune system. Bacterial antigens such as
Salmonella enterica
and
Mycobacterium bovis
BCG remain in the phagosome and stimulate CD4 T-cells via antigen presentation through major histocompatibility class II molecules. In contrast, bacterial antigens such as
Listeria monocytogenes
exit the phagosome into the cytoplasm. The phagolysosomal escape of
L. monocytogenes
is a unique mechanism which facilitates major histocompatibility class I antigen presentation of listerial antigens. This escape is dependent upon the pore-forming sulfhydryl-activated cytolysin, listeriolysin O (LLO).
The ability of
L. monocytogenes
to break down the vacuole within a host cell and enter the cytoplasm has led to its use as a recombinant vaccine. U.S. Pat. No. 5,830,702 describes vaccines comprising attenuated mutants of Listeria spp. genetically engineered to express foreign antigens in the cytoplasm of infected macrophages and other cells. Several approaches for expressing the antigen in Listeria spp. are described including generation of a fusion protein of a selected foreign antigen and a listerial protein, preferably an enzyme involved in lysis of host vacuoles. In particular, a fusion protein encoding the hly promoter and the first 416 amino acids of LLO fused in-frame to the entire coding sequence of the NP antigen was constructed in
E.coli
and on transformation to
Listeria monocytogenes
is demonstrated to secrete a 105 kDA protein that reacts with antiserum to LLO and NP (col. 24 of '702 patent). Recombinant
L. monocytogenes
secreting a fusion protein comprising listeriolysin O and NP (LLO-NP) was demonstrated to target infected cells for lysis by NP-specific class I-restricted cytotoxic T cells. In contrast, a hemolysin-negative
L. monocytogenes
strain expressing LLO-NP presented the antigen in a class II restricted manner (Ikonimidis et al.
J. Exp. Med.
1994 180:2209-2218). Thus, from these studies it was surmised that hemolysin-dependent bacterial escape from the vacuole is necessary for class I presentation in vitro.
The escape function of
L. monocytogenes
has also been transferred to
Bacillus subtilis
and attenuated Salmonella ssp. strains (Bielecki et al.
Nature
1990 354:175-176, Gentschev et al.
Infect. Immun.
1995 63:4202-4205).
S. enteric
and
M. bovis
BCG vaccine carriers which secrete listeriolysin O have also been constructed (Kaufman, S. H. and Hess,
J. Immunol. Lett
. January 1999 65(1-2):81-4). These constructs are taught to be capable of introducing antigens into the MHC class II and MHC class I pathway, resulting in stimulation of both CD4 and CD8 T-cells. Comparison of
S. enterica
vaccines which display the same listerial antigen in secreted and somatic form showed the secreted antigen display to be superior to the somatic antigen display (Kaufman, S. H. and Hess,
J. Immunol. Lett
. January 1999 65(1-2):81-4).
WO 99/10496 discloses recombinant BCG strains secreting hemolytically active hly with an improved MHC class I-restricted immune response for use as a vaccine against tuberculosis.
Administration of purified listeriolysin O encapsulated in liposomes has also been reported to be effective in the induction of antigen-specific Th1-dependent protective immunity to various kinds of intracellular parasitic bacteria in vivo (Tanabe et al.
Infect. Immun
. February 1999 67(2):568-75).
PEST sequences in eukaryotic proteins have long been identified. It has been taught that proteins containing amino acid sequences that are rich in prolines (P), glutamic acids (E), serines (S) and threonines (T), generally, but not always, flanked by clusters containing several positively charged amino acids, have rapid intracellular half-lives (Rogers et al.
Science
1986 234:364-369). Further, it has been shown that these sequences target the protein to the ubiquitin-proteosome pathway for degradation (Rechsteiner and Rogers TIBS 1996 21:267-271). This pathway is also used by eukaryotic cells to generate immunogenic peptides that bind to MHC class I and it has been hypothesized that PEST sequences are abundant among eukaryotic proteins that give rise to immunogenic peptides (Realini et al. FEBS Lett. 1994 348:109-113). Prokaryotic proteins do not normally contain PEST sequences because they do not have this enzymatic pathway.
However, a PEST-like sequence rich in the amino acids proline (P), glutamic acid (E), serine (S) and threonine (T) was recently identified at the amino terminus of LLO and demonstrated to be essential for
L. monocytogenes
pathogenicity (Decatur, A. L. and Portnoy, D. A. Science 2000 290:992-995). Decatur and Portnoy teach that the presence of this PEST-like sequence in LLO targets the protein for destruction by proteolytic machinery of the host cell so that once the LLO has served its function and facilitated the escape of
L. monocytogenes
from the phagolysosomal vacuole, it is destroyed before it can damage the cells.
It has now been found that the immune response to an antigen can be enhanced by fusion of the antigen to a non-hemolytic truncated form of listeriolysin O (&Dgr;LLO). It is believed that the observed enhanced cell mediated immunity and anti-tumor immunity of the fusion protein results from the PEST-like sequence present in LLO which targets the antigen for processing.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method for enhancing the immunogenicity of an antigen which comprises fusing to the antigen a non-hemolytic truncated form of listeriolysin O (&Dgr;LLO). In a preferred embodiment, the antigen is fused to a PEST-like amino acid sequence derived from
L. monocytogenes.
Another object of the present invention is to provide compositions with enhanced cell mediated immunity and anti-tumor immunity which comprise an antigen fused to a PEST-like amino acid sequence derived from a prokaryotic organism.
Yet another object of the present invention is to provide a method for invoking an enhanced cell mediated or anti-tumor immunogenic response to an antigen in an animal comprising administering to the animal a composition comprising an antigen fused to a PEST-like amino acid sequence derived from a prokaryotic organism.
REFERENCES:
patent: 5830702 (1998-11-01), Portnoy et al.
patent: 6051237 (2000-04-01), Paterson et al.
patent: WO 99/10496 (1999-04-01), None
J Immunology 146(10): 3604-3616; May 1991.*
Bielecki et al., “Bacillus subtilisexpressing a haemolysin gene fromListeria monocytogenescan grow in mammalian cells”,Nature1990 354:175-176.
Decatur A.L. et al., “A PEST-Like Sequence in Listeriolysin O Essential forListeria monocytogenesPathogenicity”,Science2000 290:992-995.
Gentschev et al., “Salmonella Strain Secreting Active Listeriolysin Changes Its Intracellular Localization”,Infect. Immun. 1995 63:4202-4205.
Ikonimidis et al., “Delivery of a Viral Antigen to the Class I Processing and Presentation Pathway byListeria monocytogenes”, J. Exp. Med. 1994 180:2209-2218.
Kaufman S.H. et al., “Impact of intracellular location of and antigen display by intracellular bacteria:implications for vaccine development”,J. Immunol. Lett. 1999 65(1-2):81-84.
Lin et al., “Treatment of Established Tumors with a Novel Vaccine That Enhances Major Histocompatibility Class II Presentation of Tumor Antigen1”,Cancer Res. 1996 56:21-26.
Pan et al., “Rsgression of Established Tumors in Mice Mediated by the Oral Administration of a RecombinantListeria monocytogenesVaccine1”,Cancer Res. 1995 55:4776-4779.
Realini et al., “Proposed roles in protein-protein association and presentation of peptides by MHC Class I receptors”,FEBS Lett. 1994 348:109-113.
Rechsteiner and Rogers, “PEST sequences and regulation by proteolysis”,TIBS1996 21:267-271.
Tanabe et al., “Induction of Protective T Cells againstListeria monocytogenesin Mice by Immunization with a Listeriolysin O-Negative Avirulent Strain of Bacteria and Liposome-Encapsulated Listeriolysin O”,Infect. Immun. 1999 67(2):568
Gunn, III George Raymond
Paterson Yvonne
Peters Christian
Chan Christina
Huynh Phuong N
Morgan & Lewis & Bockius, LLP
The Trustees of the University of Pennsylvania
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