Antibody-like peptides for quantifying therapeutic antibodies
Inventors
Lebert, Dorothée • Picard, Guillaume
Assignees
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Publication Number
US-11053303-B2
Publication Date
2021-07-06
Expiration Date
Abstract
The present invention relates to a labeled chimeric non-therapeutic antibodylike protein comprising, in a hypervariable region thereof, an enzyme cleavable peptide sequence derived from a hypervariable region of a reference therapeutic antibody.
Core Innovation
The invention relates to antibody-like internal standards for mass-spectrometric quantification of therapeutic antibodies in a sample of an individual. A known amount of one or more labeled forms of a labeled chimeric non-therapeutic antibody-like protein is added to a test sample containing therapeutic antibodies, thereby providing a pre-proteolysis sample. The labeled chimeric non-therapeutic antibody-like protein is structurally similar to reference therapeutic antibodies and comprises an enzyme cleavable peptide sequence of a hypervariable region derived from those reference therapeutic antibodies.
After enzyme proteolysis, a proteolysis sample is provided comprising proteolysis labeled peptides derived from the labeled antibody-like proteins and proteolysis peptides derived from the therapeutic antibody contained in the test sample. By mass spectrometric analysis, a ratio is determined between one or more selected proteolysis labeled peptides and one or more corresponding proteolysis peptides derived from the therapeutic antibody. From the determined ratio, the amount of the therapeutic antibody in the test sample is calculated.
The described enzyme cleavable peptide sequence is derived from a hypervariable region of each of a plurality of reference therapeutic antibodies, so that corresponding peptides are produced from both the internal standard and the therapeutic antibody. The approach supports quantifying one or more therapeutic antibodies, including uniplex and multiplex quantification, while using an internal standard that is not itself a therapeutic antibody.
Claims Coverage
The document contains two independent process claims for quantifying one or more therapeutic antibodies by spiking a labeled chimeric non-therapeutic antibody-like protein, proteolyzing, determining a labeled-to-corresponding peptide ratio by mass spectrometric analysis, and calculating the therapeutic antibody amount. Both independent claims share the same structural definition of the internal standard, and one claim differs mainly in that step a) uses a composition comprising the labeled chimeric antibody-like protein.
Labeled chimeric non-therapeutic antibody-like protein with reference-derived hypervariable enzyme-cleavable peptide sequence
The labeled chimeric non-therapeutic antibody-like protein is structurally similar to a plurality of reference therapeutic antibodies and comprises an enzyme cleavable peptide sequence of a hypervariable region derived from each one of said plurality of reference therapeutic antibodies.
Spiking a known amount of labeled chimeric non-therapeutic antibody-like protein composition for ratio-based MS quantification
A test sample containing therapeutic antibodies is spiked with a known amount of one or more labeled forms, or a composition comprising, the labeled chimeric non-therapeutic antibody-like protein to provide a pre-proteolysis sample, then subjected to enzyme proteolysis to provide proteolysis labeled peptides and proteolysis peptides derived from the therapeutic antibody, followed by mass spectrometric determination of the ratio between selected proteolysis labeled peptides and corresponding proteolysis peptides derived from the therapeutic antibody and calculation of the amount of said therapeutic antibody in the test sample.
Across both independent claims, the key inventive concept is the use of a labeled chimeric non-therapeutic antibody-like protein that is structurally similar to a plurality of reference therapeutic antibodies through enzyme cleavable peptide sequences derived from hypervariable regions, enabling calculation of therapeutic antibody amount from a mass-spectrometric ratio of corresponding proteolysis labeled and therapeutic-derived peptides.
Stated Advantages
Improved accuracy in proteolysis/MS workflows, with less than ~5% difference demonstrated for Cetuximab and Bevacizumab in human serum.
Modularity for multiplexing quantification.
Stability and generic compatibility without using the therapeutic antibody as the internal standard.
Documented Applications
Quantification of one or more therapeutic antibodies in a sample of an individual by mass spectrometric analysis, including uniplex and multiplex quantification.
Use with therapeutic antibodies including Cetuximab and Bevacizumab in human serum.
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