Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1997-01-06
2001-10-02
Caputa, Anthony C. (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387900, C530S388100, C530S388260, C530S388800, C530S388850, C530S389100, C530S389700, C530S387100, C435S975000, C436S064000
Reexamination Certificate
active
06297361
ABSTRACT:
BACKGROUND OF THE INVENTION
Histamine has a pivotal role in a variety of in vivo reactions. Endogenous histamine plays an important role in regulating cell proliferation in normal and neoplastic cells. Increased histamine biosynthesis and content has been reported in different human and experimental neoplasias (Cricco et al.,
Agents and Actions
, 43:17 (1994); Garcia-Caballero et al.,
Agents and Actions
, 27:227 (1989); Scolnik et al.,
Trends Pharmacol. Sci
., 6:357 (1985)). Using histamine receptor antagonists, in vitro and in vivo experiments (Van der Ven et al.,
Br. J. Cancer
, 68:475 (1990); Watson et al.,
Gut
, 34:1091 (1993)) have demonstrated that histamine acts through the specific histamine membrane receptors, H1, H2 and H3, and may regulate tumor growth and development (Cricco et al.,
Agents and Actions
, 38:175 (1993)). However, the most compelling evidence supporting a central role for histamine in neoplasia are the results of clinical trials showing increased survival of gastric cancer patients after treatment with cimetidine, an H2 receptor antagonist (Tonnesen et al.,
Lancet
, ii:990 (1988); Burtin et al.,
Eur. J. Cancer Clin. Oncol
., 24:161 (1988)). In addition to promoting proliferation of tumor cells, histamine also has potent immunosuppressive effects which can favor tumor cell growth, for example by blunting NK activity (Hellstrand et al.,
Scand. J. Immunol
, 34:741 (1991)) and by activating T-suppressor cell function (Bartholeyns et al.,
Trends Pharmacol. Sci
., 7:23 (1985)).
Histamine levels in cells and tissues are regulated by histidine decarboxylase (HDC), the only enzyme that catalyzes the formation of histamine from L-histidine. Thus, HDC is both a specific marker for histamine and an early indicator of histamine-mediated proliferation and immune suppression. Increased HDC activity has been measured in human colorectal tumor specimens (Garcia-Caballero et al.,
Agents and Actions
, 23:357 (1988)). Moreover, the inhibitory effects of &agr;-fluoromethyl-histidine, a suicide inhibitor of HDC (Watanabe et al.,
Trends Pharmacol. Sci
., 11:363 (1990)), have been demonstrated in tumor models (Bartholeyns et al.,
Cancer Res
., 44:639 (1984); Brandes et al.,
Agents and Actions
, 33 (Suppl.):325 (1991)).
Although anfibodies to HDC have been developed, the first such antibody was a preparation of polyclonal antibodies of limited use due to its species specificity, i.e., the polyclonal antibodies proved useful only for rat studies (Watanabe et al.,
Neurosci. Lett
., 39:249 (1983); Taguchi et al.,
Brain Res
., 340:235 (1985)). Yatsunami and colleagues reported the generation of a HDC monoclonal antibody (mAb), using a peptide sequence conserved across human and rat HDC (
J. Biol Chem
., 270:30813 (1995)). However, this antibody recognized only denatured HDC.
Thus, a need exists for antibodies to HDC which are useful to detect HDC in tissue specimens, e.g., antibodies which recognize native human HDC in tumor biopsies.
SUMMARY OF THE INVENTION
The present invention provides an isolated, purified antibody, or a preparation of antibodies, that specifically reacts with, or binds to, at least the native form of mammalian histidine decarboxylase (HDC), a biologically active subunit thereof, or a biologically active variant thereof. A preferred antibody of the invention is a preparation of polyclonal antibodies that specifically binds to the native form of human HDC. Preferably, the antibodies of the invention are substantially free of antibodies that do not react with HDC.
Peptides useful in preparing the antibodies of the invention preferably include a peptide comprising an amino acid sequence corresponding to EPEEYRERGREM (SEQ ID NO:1), VKDKYKLQ (SEQ ID NO:2), subunits or variants thereof. Thus, a preferred embodiment of the invention includes a preparation of polyclonal antibodies that specifically reacts with a protein or polypeptide which comprises a peptide having an amino acid sequence corresponding to SEQ ID NO:1, SEQ ID NO:2, a subunit or variant thereof. As described hereinbelow, polyclonal antibodies generated to HDC peptides can bind to, and label, melanoma and leukemia cells. Moreover, the polyclonal antibodies differentially stain different staged melanoma biopsies.
As used herein, the term “a variant” of a peptide of the invention is defined to mean a peptide which has at least about 70%, preferably at least about 80%, and more preferably at least about 90%, identity or homology to a peptide having SEQ ID NO:1 or SEQ ID NO:2.
As used herein, “biologically active” with respect to a subunit or variant of a HDC peptide of the invention means that the subunit or variant peptide has at least about 10%, preferably at least about 50%, and most preferably at least about 90%, the activity of a peptide having the amino acid sequence corresponding to SEQ ID NO:1 or SEQ ID NO:2. The activity of a peptide of the invention can be measured by methods well known to the art including, but not limited to, the ability of the peptide to elicit a sequence-specific immunologic response when the peptide is administered to an organism, e.g., chicken, goat, sheep or mice.
The invention also provides an expression cassette comprising a first preselected DNA segment encoding at least one immunogenic HDC peptide, e.g., a peptide comprising the amino acid sequence corresponding to SEQ ID NO:1, SEQ ID NO:2, a subunit or variant thereof, which is operably linked to a promoter functional in a host cell. The expression cassette preferably comprises a promoter functional in a prokaryotic cell. Preferably, the expression cassette further comprises a second DNA segment encoding a carrier protein, wherein the first and second preselected DNA segments are linked so as to encode a fusion peptide. The carrier protein provides T helper cell activation and, preferably, has low immunoreactivity. The expression cassettes can be incorporated into expression vectors which can be employed to transform prokaryotic or eukaryotic host cells, so as to result in expression of an immunogenic HDC peptide, preferably comprising the amino acid sequence corresponding to SEQ ID NO:1 or SEQ ID NO:2. As used herein, the term “immunogenic HDC peptide” means those regions of HDC which are capable of eliciting an immune response, wherein the resulting antibodies are capable of specifically reacting with mammalian HDC.
The invention also provides an immunogenic composition or a vaccine comprising a peptide which comprises the amino acid sequence corresponding to SEQ ID NO:1, SEQ ID NO:2, a biologically active variant or subunit thereof, preferably linked directly through a peptide bond to a carrier protein, in combination with a pharmaceutically acceptable carrier. The administration of the immunogenic composition or vaccine to a mammal induces the production of antibodies to HDC.
The invention further provides methods of detecting or determining the presence or amount of histidine decarboxylase RNA or polypeptide in a mammalian physiological sample which comprises cells (e.g., fluids comprising mammalian cells or tissue samples). One embodiment of the invention comprises a diagnostic method for detecting histidine decarboxylase RNA. The method comprises contacting an amount of DNA with an amount of at least two oligonucleotide primers under conditions effective to amplify the DNA by a polymerase chain reaction so as to yield an amount of amplified histidine decarboxylase DNA. The DNA is obtained by reverse transcription of RNA from a mammalian physiological sample which comprises cells suspected of containing histidine decarboxylase RNA. At least one oligonucleotide is a histidine decarboxylase-specific oligonucleotide. Then the presence or amount of the amplified histidine decarboxylase DNA is detected or determined.
As used herein, the term “histidine decarboxylase-specific oligonucleotide or primer” means a DNA sequence that has at least about 80%, more preferably at least about 90%, and more preferably at least about 95%, sequence identity or homology to a portion of the DNA encoding human histi
Falus Andras
Haak-Frendscho Mary
Caputa Anthony C.
Holleran Anne L.
Promega Corporation
Schwegan, Lundberg, Woessner & Kluth
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