Reagent and processes for targeting mutant epidermal growth...

Details Reagent and processes for targeting mutant epidermal growth... Reagent and processes for targeting mutant epidermal growth...

1997-05-21

2001-05-01

Ulm, John (Department: 1646)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

C424S185100, C424S277100

Reexamination Certificate

active

06224868

ABSTRACT:

BACKGROUND OF THE INVENTION
The success of any cancer therapy is based upon its ability to distinguish neoplastic cells from normal cells. Most current chemotherapy or radiotherapy regimens are based upon differential growth rates of tumor cells. In practice, such therapies have been very successful in treating some cancers, but for many other cancers current treatments are either palliative in nature or in the long term are ineffectual. Progress in brain tumor therapy has been especially poor as the survival curve has not appreciably changed in over 60 years. Some progress has been made using biologically based modalities such as harvesting a patient's immune system or therapeutics based upon recent research in molecular biology. However, the specificity of these therapeutics for cancerous cells is poor. Much of the research in biology based therapies has focused on defining tumor specific alterations.
The idea of utilizing a patient's own immune system to destroy a tumor is perhaps the oldest biologically based cancer therapy in use. The success of this approach rests upon the identification of a suitable antigen that will elicit both a humoral and cell mediated response. Ideally, immunization should employ a tumor specific antigen which is strictly expressed on tumor cells because the immune system most efficiently recognizes an antigen that has never been encountered before (Hellstrom, I. and Hellstrom K. E.,
Annals of New York Acad Sci
1993, 690, 24-33). The identification of such antigens has been difficult; however, progress has been made recently in isolating mutated or rearranged genes. Nearly all of the alterations characterized to date, such as p53, Rb, and ras genes, affect intracellular proteins. Recent data indicate that intracellular molecule may still be recognized by cytolytic T lymphocytes; however, the relative efficiency of tumor killing is unknown.
Studies with glioma xenografts, however, have shown that protein expressed from amplified epidermal growth factor (EGF) receptor gene is on the cell surface (Humphrey et al.,
Cancer Research
1988, 48, 2231-2238). The EGF receptor gene has been shown to be amplified in 40% of glioblastoma multiform tumors (Libermann et al.,
Nature
1985, 313(5998), 144-7; Wong et al.,
Proc Natl Acad Sci USA
1987 84(19), 6899-903). This receptor has been implicated in a wide variety of tumors including those of the breast, skin and bladder (Harris, A. L.
Recent Results in Cancer Research
1989, 113, 70-77). In the majority of these studies, increased levels of receptor message, protein or EGF binding were observed. It has also been shown that in tumors with amplification of the EGF receptor gene, the gene has frequently undergone deletion and/or rearrangement (Libermann et al.,
Nature
1985, 313(5998), 144-7; Wong et al.
Proc Natl Acad Sci USA
1987 84(19), 6899-903).
The cDNA sequence corresponding to normal EGF receptor has been reported by Ullrich et al., in
Nature
1984 309, 418-425. Wong et al.,
Proc Natl Acad Sci USA
1992, 89, 2965-2969 and Vogelstein and Bigner (PCT/US90/04489) characterized the genetic alterations associated with rearrangements or deletions of this gene in five malignant gliomas. They found mutant EGF receptor protein to be present in cells exhibiting three types of genetic deletion and/or rearrangement which result in a structurally altered receptor. The first class of deletions identified results in a gap in the extracytoplasmic domain near the transmembrane domain. The second class of deletions results in elimination of the distal portion of the extracytoplasmic domain of EGF receptor. The third class is characterized by a deletion of the majority of the external domain of the EGF receptor leaving substantially only the transmembrane portion and the intracytoplasmic domain. DNA sequences encoding proteins corresponding to each of these mutant classes were disclosed. Vogelstein and Bigner suggest that these DNA sequences may be introduced into a host cell by transformation or transfection and expressed using a wide variety of host/vector combinations. A number of useful expression vectors are disclosed including the lac system, the trp system, the tac system, the trc system major operator and promoter regions of phage lambda, the control region of fd coat protein, the glycolytic promoters of yeast, the promoters of yeast acid phosphatase, the promoters of the yeast a-mating factors, and promoters derived from polyoma, adenovirus, retrovirus, or simian virus, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses of combinations thereof. Also disclosed are examples of expression hosts useful in the invention which include eukaryotic and prokaryotic hosts, such as strains of
E. coli
including
E. coli
SG-936,
E. coli
HB 101,
E. coli
W3110,
E. coli
X1776,
E. coli
X2282,
E. coli
DHI, and
E. coli
MRC1, Pseudomonas, Bacillus including
Bacillus subtilis
, Streptomyces, yeasts and other fungi, animal cells such as COS cells and CHO cells and human cells and plant cells in tissue culture. Vogelstein and Bigner suggest that the peptide product of the prokaryotic or eukaryotic hosts transformed with the DNA sequences can be employed in the production of antibodies.
The in frame deletion from nucleotide 275-1075 in the EGF receptor (referred to as class I or Type I by Vogelstein and Bigner but hereinafter referred to as Type III) was demonstrated to generate a local amino acid sequence at the fusion junction of what were distant polypeptide sequences in the intact EGF receptor. (Humphrey et al.,
Proc Natl Acad Sci USA
1990, 87, 4207-4211). A 14 amino acid peptide spanning the junction was chemically synthesized, coupled to keyhole limpet hemocyanin, and used as an immunogen in rabbits. The elicited antibody reacted specifically with the fusion peptide in ELISA. The anti-fusion antibody was purified and shown to selectively bind the glioma deletion mutant. This antipeptide antibody was suggested as an ideal candidate for tumor imaging and immunotherapy.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a vaccine which inhibits tumor formation. The vaccine comprises a peptide having sufficient similarity to a fusion junction present in a mutant human EGF receptor so that an immune response to this mutant is elicited. A method of inhibiting formation of tumors bearing a naturally occurring mutant EGF receptor by administering this vaccine is also provided.
Another object of the present invention is to provide a vaccine for inducing regression of an existing tumor which comprises a peptide having sufficient similarity to a fusion junction present in a mutant human EGF receptor so that an immune response to this mutant is elicited. Administration of this vaccine provides a method of inducing regression of an existing tumor bearing a naturally occurring mutant EGF receptor.
An object of the present invention is to provide a cell line capable of overexpressing Type III mutant EGF receptors. Methods of producing these cell lines are also provided.
Another object of the present invention is to provide antibodies raised against a cell line overexpressing Type III mutant EGF receptor or a peptide or protein expressed by these cell lines.
Another object of the present invention is to provide antisense oligonucleotides targeted to a mutant EGF receptor which decrease expression of a mutant EGF receptor.


REFERENCES:
patent: 5212290 (1993-05-01), Vogelstein et al.
patent: 5894018 (1999-04-01), Davila et al.
patent: US90/04489 (1991-03-01), None
Baltz. Vaccines for the treatment of cancer. Am. J. Health-Syst. Pharm. 52:2574-2585, Nov. 1995.*
Moscatello et al. A Naturally Occurring Mutant Human Epidermal Growth Factor Receptor as a Target for Peptide Vaccine Immunotherapy of Tumors. Canc. Res. 57:1419-1424, Apr. 1997.*
Muller et al. Specific antibosy response towards predicted epitopes of the epidermal growth factor receptor indiced by a thermostable synthetic peptide adjuvant conjugate. Clin. Exp. Immunol.

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