Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-10-26
2003-12-30
Wessendorf, T. D. (Department: 1639)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007200, C435S007210, C435S004000, C530S317000, C530S328000, C530S300000
Reexamination Certificate
active
06670142
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to screening methods for one-bead-one-compound combinatorial libraries and includes a screening assay that uses live cells to identify synthetic ligands that promote attachment and growth or proliferation of epithelial cells. Also included are ligands specific for epithelial cancer cells. The invention also relates to methods for isolating and capturing epithelial cells, including benign (non-cancerous) and malignant (cancerous) cells, from body fluids.
2. Description of Related Art
One-bead-one-compound combinatorial bead libraries (see Lam, Kit S. et al. “A new type of synthetic peptide library for identifying ligand-binding activity.”
Nature
354 (1991): 82-84), such as one-bead-one-compound peptide libraries, are being used to study cell adhesion properties of cancer cells. Using random peptide bead libraries and suspended cancer cells, peptide ligands that promote cell attachment have been identified for lymphoma (Park, Steven, Renil Manat, Brian Vikstrom, Nail Amro, and Kit S. Lam. “Identification of peptide ligands for &agr;4 &bgr;1 integrin receptor as potential targeting agents for non-Hodgkin's lymphoma,” abstract in
Peptides: The Wave of the Future,
2nd International Peptide Symposium in conjunction with the 17
th
American Peptide Symposium, San Diego, Calif. (Jun. 9-14, 2001)) and prostate cancer cell lines (Pennington, Michael E., Kit S. Lam and Anne E. Cress. “The use of a combinatorial library method to isolate human tumor cell adhesion peptides.”
Molecular Diversity
2 (1996): 19-28; DeRoock, Ian B., Michael E. Pennington, Thomas C. Sroka, Kit S. Lam, G. Tim Bowden, Elisabeth L. Bair, and Anne E. Cress. “Synthetic Peptides Inhibit Adhesion of Human Tumor Cells to Extracellular Matrix Proteins.”
Cancer Research
61 (Apr. 15, 2001): 3308-13).
In the existing methods, live cells in suspension are incubated for about one to four hours with a bead library, and the library is then screened for beads with peptide ligands that promote cell attachment. This is done by visual selection—the beads are examined under a dissecting microscope and those beads with attached cells are removed using a micropipet. Further steps are then performed to confirm that the removed beads are in fact capable of binding the particular type of cells tested. Then, the peptides on those beads are sequenced. (See Pennington et al., “Use of a combinatorial library method,” 19-28.)
In another existing method of testing live cells for peptide ligands that affect cell growth on culture plates, a bead library is prepared having selectively cleavable peptides such that a proportion of the peptide on each bead is attached to the bead by a cleavable linker. When the library is treated with a cleaving agent, enough of the peptides are released from the beads to cause the biological effect, and the rest of the peptides remain bound to the beads to allow for later sequencing. Suspended cells are incubated in tissue culture wells with a few beads and with peptides released from the beads. The effect of the released peptides on the cells (inhibition or stimulation of cell growth) is determined, and the corresponding beads are removed. The sequences of the attached peptides are then determined. (See U.S. Pat. No. 5,510,240, issued Apr. 23, 1996 to Lam, Kit S. et al.)
The existing methods, however, are not satisfactory in certain cases. The methods are difficult to use with epithelial cells, which include the majority of solid cancer cell cultures, such as lung cancer cells, that exist as adherent cultures rather than as suspended cells. With incubation periods of only a few hours, these cells are often only weakly attached to the beads and may easily fall off, rendering the screening method less accurate because some beads with attached cells are missed. Also, the existing methods may not detect cell surface receptors that may be altered by trypsin and/or EDTA. Trypsinization is commonly used to separate tissues or cell cultures into a single-cell suspension for testing with a combinatorial library. The treatment with trypsin may eliminate some, or alter the conformation of, cell surface receptors. In addition, the existing methods do not select for peptide ligands that promote cell growth or proliferation, but, rather, for ligands involved in cell attachment, particularly short-term attachment.
Thus, there is a need for a screening assay that is specific and sensitive, works well with epithelial cells, can be used to detect cell surface receptors susceptible to trypsin, and selects for ligands that promote not only cell attachment, but also cell growth or proliferation.
There is also a need for an efficient method of isolating and capturing epithelial cells, including benign and malignant cells, from body fluids, such as blood, urine, pleural effusion, pericardial effusion, ascite, and cerebrospinal fluid. In particular, there is a need for a method of isolating and capturing cancer cells to assist in diagnosis.
SUMMARY OF THE INVENTION
The present invention is directed to a method for screening a combinatorial bead library for ligands that promote the attachment and growth or proliferation of epithelial cells. The method satisfies the need for an assay that is specific and sensitive, that can be used to detect cell surface receptors susceptible to trypsin, and that can identify ligands that promote cell growth and proliferation. The method comprises introducing a suspension of live cells to a combinatorial library of small molecules, peptides, or other types of molecules, incubating the cells with the library for about 24 to 72 hours, identifying a solid phase support of the library with cells growing on the support, isolating the solid phase support, and determining the chemical structure of the compound attached to that solid phase support.
The invention also includes ligands specific for cell attachment and growth or proliferation of epithelial cancer cells, having the chemical structure of cXGXGXXc, in which “c” is D-cysteine; “X” is any L-, D-, unnatural, or modified amino acid; and “G” is glycine.
The present invention is also directed to a method for isolating and capturing cells from body fluids and satisfies the need for an efficient method of isolating and capturing epithelial cells from body fluids. The method comprises introducing a sample of body fluid to a multiplicity of beads with one or more known ligands specific for one or more particular types of cell, incubating the body fluid with the beads for about 24 to 72 hours, identifying a bead with cells growing on the bead, isolating the bead, and recovering the cells growing on the bead.
REFERENCES:
patent: 5510240 (1996-04-01), Lam et al.
patent: 5635598 (1997-06-01), Lebl et al.
patent: 5650489 (1997-07-01), Lam et al.
patent: 5651943 (1997-07-01), Lam et al.
patent: 5840485 (1998-11-01), Lebl et al.
patent: 5858670 (1999-01-01), Lam et al.
patent: 5888497 (1999-03-01), Jain et al.
patent: 6242211 (2001-06-01), Peterson et al.
patent: 2001/0005578 (2001-06-01), Prusiner
patent: WO 9827231 (1997-12-01), None
Journal of the American Chemical Soceity (2002), 124(26), 7678-80, Liu et al.*
Liu, Gang, Yemei Fan, James R. Carlson, Zhan-Gong Zhao, and Kit S. Lam. “Solution-Phase Synthesis of a 1,5-Dialkylamino-2,4-dinitrobenzene Library and the Identification of Novel Antibacterial Compounds from This Library.”J. Comb. Chem2, No. 5 (Aug. 3, 2000): 467-474.
Lam, Kit S. “Application of combinatorial library methods in cancer research and drug discovery.”Anti-Cancer Drug Design12 (1997): 145-167.
Cabilly, S., ed.Methods in Molecular Biology,vol. 87:Combinatorial Peptide Library Protocols,chaps. 1 and 2. Totowa, New Jersey: Humana Press, Inc.
Lebl, Michal, Viktor Krch{haeck over (n)}ák, Nikolai F. Sepetov, Bruce Seligmann, Peter Strop, Stephen Felder, and Kit S. Lam. “One-Bead-One-Structure Combinatorial Libraries.”Biopolymers(Peptide Science) 37 (1995): 177-198.
Liu, Gang, and Kit S. Lam. One-bead one-compound combinatorial library method. InCombinatorial Chemis
Lam Kit S.
Lau Derick H.
Millemann Audrey A.
The Regents of the University of California
Wessendorf T. D.
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