Anti-pY1235-MET immunological binding reagent
Inventors
Srivastava, Apurva K. • Pfister, Thomas D. • Navas, Tony • Parchment, Ralph E. • Doroshow, James E.
Assignees
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Publication Number
US-11340219-B2
Publication Date
2022-05-24
Expiration Date
2037-03-16
Abstract
Methods and compositions are described herein for assaying the presence or absence of pY1235-MET or a fragment thereof.
Core Innovation
The invention provides methods and compositions for assaying the presence or absence of pY1235-MET or a fragment thereof. Specifically, it offers an antibody that specifically binds to pY1235-MET and fragments containing pY1235, characterized by defined complementary determining regions (CDRs) in both the heavy and light chain variable regions. This antibody is capable of distinguishing pY1235 phosphorylation independently of other phosphorylation sites, such as Y1234 or Y1230.
The problem addressed by the invention lies in the difficulty of assessing MET activation and/or signaling due to the lack of specific binding reagents that detect phosphorylation at residue Y1235 of MET without cross-reactivity to other phosphorylation sites. Unlike previous antibodies that detect multiple phosphorylated tyrosines or have cross-reactivity with MET mutants or other epitopes, the disclosed antibody provides highly specific detection of pY1235-MET.
The invention further provides recombinant polynucleotides encoding the antibody, expression cassettes with promoters operably linked to these polynucleotides, host cells comprising the expression cassettes, and methods of producing the antibody. Methods of specifically detecting pY1235-MET in a sample using the antibody, identifying MET inhibitors by their ability to reduce pY1235-MET levels or ratios relative to total MET, and monitoring MET inhibitor treatments in subjects by measuring pY1235-MET before and after treatment are disclosed.
Claims Coverage
The patent includes eleven inventive features primarily directed to the anti-pY1235-MET antibody, related polynucleotides, expression systems, production methods, and diagnostic and therapeutic applications.
Antibody specifically binding pY1235-MET with defined CDRs
An antibody that specifically binds to pY1235-MET or a fragment containing pY1235, comprising heavy and light chain variable regions with CDRs having the amino acid sequences of SEQ ID NOs:3, 4, 5 (heavy chain) and SEQ ID NOs:6, 7, 8 (light chain).
Antibody with heavy chain variable segment sequence identity
An antibody comprising a heavy chain variable segment having at least 95% sequence identity to SEQ ID NO:1.
Antibody with light chain variable segment sequence identity
An antibody comprising a light chain variable segment having at least 95% sequence identity to SEQ ID NO:2.
Polynucleotide encoding the specific antibody
A polynucleotide encoding the antibody that specifically binds pY1235-MET with the defined CDRs.
Expression cassette comprising the polynucleotide linked to promoter
An expression cassette comprising the polynucleotide encoding the antibody operably linked to a promoter.
Heterologous promoter in the expression cassette
The expression cassette wherein the promoter is heterologous to the polynucleotide.
Host cell comprising expression cassette
A host cell comprising the expression cassette encoding the antibody.
Method of producing anti-pY1235-MET antibody
A method of making the specific antibody by culturing the host cell containing the expression cassette and purifying the antibody from culture media or lysate.
Method of detecting pY1235-MET in a sample
A method of specifically detecting pY1235-MET or fragments containing pY1235 by contacting a sample with the specific antibody to form an immunoconjugate and detecting its presence or absence.
Method to identify MET inhibitors by reduction of pY1235-MET
A method for identifying a test compound as a MET inhibitor by contacting cells or extracts with the compound, detecting pY1235-MET levels using the antibody, and identifying the compound as an inhibitor if pY1235-MET or the ratio of pY1235-MET to total MET is reduced relative to a control not contacted with the compound.
Method for monitoring MET inhibitor treatment by detecting pY1235-MET
A method of monitoring MET inhibitor treatment by administering a first dose to a subject, detecting pY1235-MET in a first sample, administering a second dose, providing a second sample, and again detecting pY1235-MET using the specific antibody.
The claims cover a highly specific anti-pY1235-MET antibody defined by particular CDR sequences, polynucleotides and expression systems encoding it, methods for producing the antibody, and diagnostic and therapeutic methods employing its specificity for detecting pY1235-MET and assessing MET inhibition.
Stated Advantages
Provides a highly specific antibody for detecting phosphorylation at Y1235 of MET without cross-reactivity to other phosphorylated tyrosines or MET mutants.
Enables quantitative and specific measurement of key phosphorylated MET species involved in signaling.
Facilitates sensitive pharmacodynamic assessment of MET activation and inhibition in preclinical models and clinical samples.
Allows identification and monitoring of MET inhibitors and subject stratification for MET inhibitor therapy.
Assay and antibody robustly detect pY1235 even in fragments of MET, aiding in understanding MET signaling and drug responses.
Documented Applications
Specifically detecting pY1235-MET or fragments containing pY1235 in biological samples using the antibody.
Identifying test compounds as MET inhibitors by measuring reductions in pY1235-MET or its ratio to total MET.
Monitoring MET inhibitor treatment efficacy in subjects by comparing pY1235-MET levels before and after treatment.
Selection and stratification of subjects for treatment with MET inhibitors based on pY1235-MET detection.
Use in immunoassays to assess MET phosphorylation status in cancer research and pharmacodynamic studies.
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