Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1995-03-14
2003-02-04
Nolan, Patrick J. (Department: 1644)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S328000
Reexamination Certificate
active
06514942
ABSTRACT:
BACKGROUND OF THE INVENTION
A. Field of the Invention
The present invention relates generally to the field of molecular biology, and particularly to the area of natural and synthetic peptides. More particularly, the invention discloses HER-2
eu peptide, DNA segment, antibody compositions. Various methods for making and using these compositions are disclosed, such as, for example, the use of peptides and antibodies in various pharmacological and immunological applications, including the stimulation of cytotoxic T-lymphocytes and cancer therapies.
B. Description of the Related Art
1. HER-2
eu Proto-Oncogene
The HER-2
eu proto-oncogene (HER-2) encodes a transmembrane protein whose expression is enhanced in a number of breast and ovarian tumors and correlates with tumor aggressiveness. Because of its expression on normal epithelial cells, HER-2 can be defined as a tumor-associated antigen (Ag) and may be of interest as a target of a therapeutic anti-tumor T-cell response. A CD3
+
CD8
+
CD4
−
line isolated from cell cultures has been shown to lyse HLA-A2
+
, HER-s
+
ovarian tumors but not natural killer (NK) target K562 cells, and showed significantly higher lysis of HER-2
high
than of HER-2
low
ovarian tumors. Some inhibition of lysis was inhibited by HER-2 peptide-pulsed HLA-A2
+
targets, suggesting that some epitopes may be present on tumor cells associated with HLA-A2.
2. Tumor-Reactive T-Cells
Tumor reactive T-cells have been reported to mediate therapeutic responses against human cancers (Rosenberg et al., 1988). In certain instances, in human immunotherapy trials with tumor infiltrating lymphocytes (TIL) or tumor vaccines, these responses correlated either with in vitro cytotoxicity levels against autologous tumors (Aebersold et al., 1991) or with expression of certain HLA-A,B,C gene products (Marincola et al., 1992). Recent studies (Ioannides et al., 1992) have proposed that in addition to virally encoded and mutated oncogenes, overexpressed self-proteins may elicit some degree of tumor-reactive cytotoxic T-lymphocytes (CTLs) in patients with various malignancies (Ioannides et al., 1992; Ioannides et al., 1993; Brichard et al., 1993; Jerome et al., 1991). Autologous tumor reactive CTLs can be generated from lymphocytes infiltrating ovarian malignant ascites (Ioannides et al., 1991), and overexpressed proteins such as HER-2 may be targets for CTL recognition (Ioannides et al., 1992).
Information on epitopes of self-proteins recognized in the context of MHC Class I molecules remain limited, despite a few attempts to identify epitopes capable of in vitro priming and Ag-specific expansion of human CTLs. For example, peptide epitopes have been proposed which are likely candidates for binding on particular MHC Class I Ag (Falk et al., 1991), and some studies have attempted to define peptide epitopes which bind MHC Class I antigens.
Short synthetic peptides have been used either as target antigens for epitope mapping or for induction of in vitro primary and secondary CTL responses to viral and parasitic Ags (Bednarek et al., 1991; Gammon et al., 1992; Schmidt et al., 1992; Kos and Müllbacher, 1992; Hill et al., 1992). Unfortunately, these studies failed to show the ability of proto-oncogene peptide analogs to stimulate in vitro human CTLs to lyse tumors endogenously expressing these antigens.
3. Synthetic Peptides and T-Cell Epitope Mapping
Synthetic peptides have been shown to be a useful tool for T-cell epitope mapping. However in vivo and in vitro priming of specific CTLs has encountered difficulties (Alexander et al., 1991; Schild et al., 1991; Carbone et al., 1988). It is generally considered that in vitro CTL priming cannot necessarily be achieved with peptide alone, and in fact, a high antigen density is thought to be required for peptide priming (Alexander et al., 1991). Even in the limited instances when specific priming was achieved, APC or stimulators were also required at high densities (Alexander et al., 1991).
It is not clear when CTL induction by HER-2 peptides in vitro was observed whether this reflects secondary activation of CTL specific for, or cross-reacting with, the Ag of interest. Whether or not this cross-reactivity can constitute the foundation for development of an in vitro CTL response to tumor remains to be determined.
Therefore, what is lacking in the prior art are universal epitopes which are both immunodominant and CTL-stimulating. Moreover, methods for the use of such CTL-stimulating peptides would be most desirable in the treatment of human cancers, particularly of breast and ovarian etiology, and the development of cancer vaccines. Identifying universal oncoprotein epitopes would permit not only an increased understanding of tumor immunity and autoimmunity in humans, but would also open the door to the design of novel therapeutic strategies for proliferative cell disorders such as human cancers, and particularly breast and ovarian cancers.
SUMMARY OF THE INVENTION
The present invention seeks to overcome these and other inherent deficiencies in the prior art by providing the identification of native and synthetic proteins or peptides derived from the HER-2
eu proto-oncogene gene product, and methods for their use in stimulating cytotoxic T-lymphocytes. These selected “universal” immunodominant epitopic peptides, and their synthetically-optimized derivatives are envisioned to be useful in the development of tumor vaccines, and anti-cancer therapeutics. Pharmaceutical reagents resulting from these novel peptides and the DNA segments which encode them will also likely prove useful as test reagents for the detection of HER-2
eu-related polypeptides, facilitate the production of anti-peptide antibodies specific to a range of HER-2
eu-related polypeptides, and result in the stimulation and production of cytotoxic T-lymphocytes specific for a variety of proliferative disorders including human cancer.
Synthetic peptide analogs can be used to define CTL epitopes recognized by tumor reactive T-cells and to stimulate in vitro peptide-specific CTLs. Such CTLs can be further evaluated for recognition of targets endogenously expressing the particular antigen (Ag) and for Ag-specific adoptive therapy.
Disclosed herein are compositions and methods for their making and use in development of anti-cancer vaccines. The generation in vitro of HLA-A2-restricted CTLs using HER-2 synthetic peptide analogs as immunogens, and peripheral blood mononuclear cells (PBMC) from healthy volunteers as responder cells is also described. Lysis with isolated CD8
+
T-cells from these CTL cultures was observed using both HER-2 peptide-pulsed HLA-A2 from these CTL cultures was observed using both HER-2 peptide-pulsed HLA-A2 transfectants and HLA-A2
+
ovarian tumors expressing high levels of HER-2 as targets.
Another aspect of the invention is the development and maintenance in long-term culture a CD3
+
CD8
+
CD4
−
line by restimulation with HER-2 peptide-pulsed autologous PBMC. This line lysed HLA-A2
+
, HER-2
high
ovarian tumors, but not HLA-A2
+
, HER-2
low
ovarian tumors. Tumor lysis was inhibited by HER-2 peptide-pulsed HLA-A2
+
transfectants, demonstrating that epitopes either similar or cross-reactive with the ones recognized by CTLs on the peptide used as immunogen in vitro are present on the tumor cells. These CTL showed lower lysis of targets pulsed with unrelated peptides (analogs of Muc-1 core peptide where HLA-A2 anchors were introduced).
A novel approach to developing tumor reactive CTLs is disclosed which focuses on a target Ag expressed on the tumor of interest and identifying CTLs induced in vivo or developed in vitro that recognize this target Ag. In tumor cells the level of expression of a particular protein may be 10
2
-10
3
fold higher than in normal tissue.
The inventors expect that a number of target T-cell Ags on human tumors may be derived from proteins that are expressed at low levels in normal cells, and at significantly higher concentration in tumor cells, such as overexp
Fisk Bryan A.
Ioannides Constantin G.
Ioannides Maria G.
Fulbright & Jaworski L.L.P.
Nolan Patrick J.
The Board of Regents The University of Texas System
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