Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-08-19
2001-10-23
Huff, Sheela (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007100, C435S007210, C435S007230, C435S007240, C435S007250, C435S007700, C435S007920, C435S325000, C435S326000, C435S327000, C435S328000, C435S329000, C435S330000, C435S331000, C435S332000, C435S343000, C435S344000, C530S350000, C530S380000, C530S385000, C530S387100, C530S387300, C530S387700, C530S387900, C530S388100, C530S388150, C530S388200, C530S391300, C530S391500
Reexamination Certificate
active
06306615
ABSTRACT:
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not applicable.
FIELD OF THE INVENTION
This invention relates to monoclonal antibodies that recognize modified &bgr;-tubulin, methods of using such antibodies to detect modified &bgr;-tubulin, methods of using such antibodies to monitor &bgr;-tubulin modifying agents administered to a patient, methods of using such antibodies to isolate modified &bgr;-tubulin, and methods of detecting the anti-modified &bgr;-tubulin antibodies.
BACKGROUND OF THE INVENTION
Microtubules are composed of &agr;/&bgr;-tubulin heterodimers and constitute a crucial component of the cell cytoskeleton. Furthermore, microtubules play a pivotal role during cell division, in particular when the replicated chromosomes are separated during mitosis. Interference with the ability to form microtubules from &agr;/&bgr;-tubulin heterodimeric subunits generally leads to cell cycle arrest. This event can, in certain cases, induce programmed cell death. Thus, natural products and organic compounds that interfere with microtubule formation have been used successfully as chemotherapeutic agents in the treatment of various human cancers.
Pentafluorophenylsulfonamidobenzenes and related sulfhydryl and disulfide modifying agents (see, e.g., compound 1; 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene;
FIG. 1C
) prevent microtubule formation by selectively covalently modifying &bgr;-tubulin. For example, compound 1 does not covalently modify all of the five known &bgr;-tubulin isotypes. Instead, binding is restricted to those &bgr;-tubulin isotypes that have a cysteine residue at amino acid position 239 in &bgr;-tubulin. Such isotypes include &bgr;1, &bgr;2 and &bgr;4-tubulin. The other two isotypes (&bgr;3 and &bgr;5) have a serine residue at this particular position (Shan et al.,
Proc. Nat'l Acad. Sci USA
96:5686-5691 (1999)). It is notable that no other cellular proteins are modified by compound 1.
Although &bgr;-tubulin modification can be monitored using the tritiated derivative of compound 1 or other &bgr;-tubulin modifying agents, in many cases this method is either impractical or not sensitive enough. Specific, sensitive methods of detecting &bgr;-tubulin modification are needed for diagnostic applications and for dose monitoring in patients receiving chemotherapeutic agents that modify &bgr;-tubulin.
The present application is related to U.S. Pat. No. 5,880,151, issued Mar. 9, 1999; PCT 97/02926, filed Feb. 22, 1997; PCT 97/12720, filed Jul. 18, 1997; PCT 98/16781, filed Aug. 13, 1998; PCT 99/13759, filed Jun. 16, 1999; and PCT 99/16032, filed Jul. 15, 1999, herein each incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
Thus, the present invention provides monoclonal antibodies that selectively recognize modified &bgr;-tubulin and new methods of detecting &bgr;-tubulin modification using such monoclonal antibodies. These methods can be used to detect modified &bgr;-tubulin in patient samples, to monitor and adjust doses of &bgr;-tubulin modifying agents administered to patients, to isolate modified &bgr;-tubulin, and to detect anti-modified &bgr;-tubulin antibodies.
In one aspect, the present invention provides a monoclonal antibody that specifically binds to modified &bgr;-tubulin, the antibody selected from the group consisting of 1F6D8, 1B2C11, 3A1C11, 2C1H7, 3F2A4, 5F5C11, and 6D4D11. The hybridoma cell line 2C1H7, Accession number, PTA-2686, was deposited on Nov. 16, 2000 with American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110, USA.
In one embodiment, the antibody is covalently linked to a detectable moiety. In another embodiment, the antibody is covalently linked to a biotin moiety, an iodine moiety, or an enzyme moiety.
In another aspect, the present invention provides a method of detecting in a sample a modified &bgr;-tubulin isotype, the method comprising the steps of: (a) providing a sample treated with a &bgr;-tubulin modifying agent; (b) contacting the sample with an antibody that specifically binds to a modified &bgr;-tubulin isotype; and (c) determining whether the sample contains a modified &bgr;-tubulin isotype by detecting the antibody.
In another aspect, the present invention provides a method of monitoring the amount of &bgr;-tubulin isotype in a patient treated with a &bgr;-tubulin modifying agent, the method comprising the steps of: (a) providing a sample from the patient; (b) contacting the sample with an antibody that specifically binds to a modified &bgr;-tubulin isotype; and (c) determining the amount of modified &bgr;-tubulin isotype in the patient sample by detecting the antibody and comparing the amount of antibody detected in the patient sample to a standard curve, thereby monitoring the amount of modified &bgr;-tubulin isotype in the patient.
In one embodiment, the &bgr;-tubulin is modified at cysteine residue 239. In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is selected from the group consisting of 1F6D8, 1B2C11, 3A1C11, 2C1H7, 3F2A4, 5F5C11, and 6D4D11.
In one embodiment, the method further comprises the step of using a control antibody that recognizes both modified and unmodified &bgr;-tubulins. In another embodiment, the antibody is a monoclonal antibody selected from the group consisting of 3D12D1, 4B6G6, 5F1D4, 6H8E3, and 6H10C7.
In one embodiment, the method further comprises the step of using a control antibody that recognizes only unmodified &bgr;-tubulins. In another embodiment, the antibody is a monoclonal antibody selected from the group consisting of 3E10A3, 6A7F9, and 6E7G1.
In one embodiment, the step of determining whether the sample contains a modified &bgr;-tubulin isotype comprises detecting the antibody in an assay selected from the group consisting of an ELISA assay, a western blot, an immunohistochemical assay, an immunofluorescence assay, and a real time imaging assay. In another embodiment, the step of determining whether the sample contains a modified &bgr;-tubulin isotype further comprises quantitating the amount of modified &bgr;-tubulin isotype in the sample. In another embodiment, the antibody is bound to a solid substrate.
In one embodiment, the sample is selected from the group consisting of an in vitro tubulin polymerization reaction sample, a cultured cell, and a patient sample. In another embodiment, the patient sample is a blood sample. In another embodiment, the patient sample is from a cancer patient receiving chemotherapy comprising a &bgr;-tubulin modifying agent. In another embodiment, the agent is a pentafluorobenzenesulfonamide. In another embodiment, the agent is 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene. In another embodiment, the patient sample is from a human patient. In another embodiment, the method further comprises the step of adjusting the dose of &bgr;-tubulin modifying agent administered to the patient.
In another aspect, the present invention provides a method of isolating a modified &bgr;-tubulin isotype, the method comprising the steps of: (a) providing a sample treated with a &bgr;-tubulin modifying agent; (b) contacting the sample with an antibody that specifically binds to a modified &bgr;-tubulin isotype; and (c) isolating the modified &bgr;-tubulin isotype by isolating the antibody.
In one embodiment, the &bgr;-tubulin is modified at cysteine residue 239.
In another aspect, the present invention provides a method of detecting anti-modified &bgr;-tubulin antibody, the method comprising the steps of: (a) providing a sample; (b) contacting the sample with a peptide that specifically binds to anti-modified &bgr;-tubulin antibody; and (c) detecting the anti-modified &bgr;-tubulin antibody.
In one embodiment, the peptide comprises cysteine residue 239. In one embodiment, the peptide is ATMSGVTTCLRFPGQLNA (SEQ ID NO: 1), GTMECVTTCLRFPGQLNA (SEQ ID NO: 2), or KATMSGVTTCLRFPGQLNA (SEQ ID NO: 3). In another embodiment, the peptide is bound to a solid substrate. In another embodiment, the step of detecting the anti
Beckmann Holger
Santha Edit
Harris Alana M.
Huff Sheela
Townsend and Townsend / and Crew LLP
Tularik Inc.
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