Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-01-31
2001-08-07
Feisee, Lila (Department: 1806)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S070210, C530S387100, C530S412000, C530S418000
Reexamination Certificate
active
06270978
ABSTRACT:
BACKGROUND OF THE INVENTION
Peptides are transported in and out of human cells by several different transport mechanisms. One influx peptide transporter has been located in the brush border of the epithelial cells of the intestinal mucosa. Properties of this intestinal transporter (hereinafter “influx peptide transporter”) have been studied in situ in intestinal mucosa preparations and in vitro with brush border membrane vesicles, isolated enterocytes, and cell culture.
Many different solutes, including small peptides, antibiotics, oral angiotensin converting enzyme (ACE) inhibitors, and oral renin inhibitors are transported into the cytoplasm of the enterocyte by the influx peptide transporter. (e.g.,
Ganapathy and Leibach
, 1991
, Curr. Biol
. 3:695-701; Okano et al., 1986
, J. Biol. Chem
. 261:14130-14134; Nakashima et al., 1984
, Biochem. Pharm
. 33:3345-3352). The influx peptide transporter plays a pivotal role in the absorption of certain oral drugs, including &bgr;-lactams and ACE inhibitors. Out of 27 &bgr;-lactam antibiotics examined, the influx peptide transporter was able to distinguish between those that are orally absorbed in humans and those that are not (Tabas et al., 1991, 31st Interscience Conference on Antimicrobial Agents and Chemotherapy Abstract No. 164). Moreover, the influx peptide transporter has been demonstrated to transport a number of oral &bgr;-lactam antibiotics but not parenteral &bgr;-lactam antibiotics in studies using human intestinal Caco-2 cells and rabbit intestinal brush-border membranes (Dantzig et al., 1992
, Biochim. Biophys. Acta
1112:167-173; Dantzig et al., 1992, 32nd Interscience Conference on Antimicrobial Agents and Chemotherapy, Anaheim, Calif., Abstract No. 1460; Snyder et al., 1992, 32nd Interscience Conference on Antimicrobial Agents and Chemotherapy Abstract No. 1461; Okano et al., 1986
, J. Biol. Chem
. 261:14130-14134.) Similar studies have been conducted to examine the ability of the influx peptide transporter to predict which ACE inhibitors are orally absorbed (Friedman and Amidon, 1989
, Pharm. Res
. 6:1043-1047).
Influx peptide transporter activity has been identified as a 127,000 dalton membrane protein from rabbit intestinal mucosa by photoaffinity labeling methods employing radiolabeled penicillin or a radiolabeled cephalexin analog (Kramer, 1987
, Biochim. Biophys. Acta
905:65-74; Kramer et al., 1988
, Biochem. Pharmacol
. 37:247-2435). A purified 127,000 dalton protein from rabbit intestinal mucosa preparations reconstituted into liposomes resulted in binding and transport activities, and polyclonal antibodies to this putative transporter were prepared (Kramer et al., 1990
, Biochim. Biophys. Acta
1030:50-59). Monoclonal antibodies (MAbs) reactive with human influx peptide transporter have not been previously described.
Proteins that are expressed in discrete locations in the body can be used as markers to determine the origin of cells or tissues. For example, carcinoembryonic antigen is a protein situated only in the colon and is used as a marker for colon tumor cells (Shrively, 1985
, Crit. Rev. Oncol. Heamatol
. 2:355-399). MAbs raised to unique proteins, such as the human influx peptide transporter, can be used to identify tumors and metastases that originate from a tissue that expresses that antigen. This may be done as a diagnostic in the laboratory or may be used in vivo with an imaging agent.
SUMMARY OF THE INVENTION
The present invention provides monoclonal antibodies (MAbs), immunoreactive fragments or recombinants thereof, reactive with human influx peptide transporter, a protein of approximately 120,000 dalton molecular weight.
The present invention also provides a method for preparing a MAb that is reactive with human influx peptide transporter comprising the steps of:
(a) immunizing an immunocompetent mammal with a source of the human influx peptide transporter;
(b) fusing lymphocytes of the immunized immunocompetent mammal with myeloma cells to form hybridoma cells;
(c) screening the hybridoma cells of step (b) for influx peptide transporter reactivity;
(d) cloning human influx peptide transporter-reactive hybridomas of step (c);
(e) culturing an influx peptide transporter-reactive hybridoma in a medium to secrete said MAb; and
(f) recovering the MAb from the culture supernatant.
The present invention further provides a process for preparing a hybridoma that produces a MAb reactive with human influx peptide transporter comprising the steps of:
(a) immunizing an immunocompetent mammal with the human influx peptide transporter;
(b) obtaining lymphocytes from the immunized mammal;
(c) fusing the lymphocytes with myeloma cells to produce hybridoma cells; and
(d) cloning a hybridoma cell that produces human influx peptide transporter reactive MAbs.
In a further embodiment, the invention provides a method of determining uptake of an agent into a cell by a protein reactive with a human influx peptide transporter-reactive MAb, immunoreactive fragments or recombinants thereof, comprising:
(a) contacting a human influx peptide transporter-reactive MAb, immunoreactive fragments or recombinants thereof, with a cell that has influx peptide transporter activity, in an aqueous solution under conditions that allow the MAb to bind to the cell;
(b) adding the agent to be tested to said solution; and
(c) determining whether transport of the agent into the cell is decreased by the presence of the MAb.
In still another embodiment, the invention provides a method of isolating human influx peptide transporter comprising:
(a) immobilizing a MAb, immunoreactive fragments or recombinants thereof, reactive with human influx peptide transporter onto a surface;
(b) contacting said immobilized MAb with a mixture containing human influx peptide transporter under conditions that allow the human influx peptide transporter to bind to the immobilized MAb;
(c) separating immobilized MAbs that are bound to human influx peptide transporter from said mixture; and
(d) recovering the human influx peptide transporter by removing the human influx peptide transporter from the MAb.
The invention also provides a method of identifying human influx peptide transporter in a biological sample comprising:
(a) contacting the sample with a MAb, immunoreactive fragments or recombinants thereof, reactive with the human influx peptide transporter;
(b) determining the level of binding of said MAb, immunoreactive fragments or recombinants thereof to the sample; and
(c) comparing the amount of the MAb, immunoreactive fragments or recombinants thereof, bound to substances present in the sample to a control sample or to a predetermined base level, so that a binding greater than the control level is indicative of the presence of the human influx peptide transporter in a biological sample.
The invention further provides a method of diagnosing a human gastrointestinal or pancreatic duct carcinoma or metastases therefrom comprising:
(a) obtaining a body sample from a patient;
(b) contacting the body sample material with a MAb reactive with the human influx peptide transporter, immunoreactive fragments or recombinants thereof;
(c) determining the level of binding of said MAb, immunoreactive fragments or recombinants thereof to the body sample material; and
(d) determining the level of binding of a MAb reactive with human influx peptide transporter to a body sample known to be free of human gastrointestinal or pancreatic duct carcinoma or metastases therefrom to establish a control;
(e) comparing the amount of the MAb, immunoreactive fragments or recombinants thereof bound to substances present in the body sample in step (c) with the amount of the MAb, immunoreactive fragments or recombinants thereof bound to substances present in the control body sample of step (d), a binding level greater than the control level being indicative of the presence of human gastrointestinal carcinoma or pancreatic duct carcinoma or metastases therefrom.
In another embodiment the invention provides a method for diagnosing the presence of a human gastrointestinal or pancreatic duct
Bright Stuart W.
Dantzig Anne H.
Sportsman J. Richard
Tabas Linda B.
Eli Lilly and Company
Feisee Lila
Ungar Susan
Webster Thomas D.
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