Indirect homogeneous mobility shift assays for the detection of biologics in patient samples
Inventors
Salbato, Jared • Westin, Stefan • Ling, Nicholas Chi-Kwan • Jain, Anjali • Singh, Sharat
Assignees
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Publication Number
US-11846642-B2
Publication Date
2023-12-19
Expiration Date
Abstract
The present invention provides a sensitive and specific indirect homogeneous mobility shift assay using size exclusion chromatography to measure biologics such as vedolizumab and ustekinumab in a patient sample. The assays of the present invention are particularly advantageous for detecting the presence or level of biologics that target complex or large antigens including cell surface proteins, transmembrane proteins, heavily glycosylated proteins, and multimeric proteins, as well as antigens that cannot be purified, impure antigens, and partially or substantially purified antigens. The present invention also provides isolated soluble α4β7 integrin heterodimers and isolated soluble IL-12p40 monomers that are suitable for use in the indirect assays described herein.
Core Innovation
The invention provides a detection method in which a sample from a subject administered a first biologic is contacted with an unlabeled, isolated, and soluble polypeptide, and the polypeptide is contacted in vitro with a second biologic. Binding of the polypeptide to the first biologic present in the sample is indirectly detected by measuring a form of the second biologic produced during the in vitro contacting. The detected binding determines a presence or an amount of the first biologic in the sample.
The method further includes detecting a presence or an amount of an autoantibody to the first biologic in the sample. In the described assay implementations, the indirect homogeneous mobility shift assay is carried out using size exclusion chromatography and size exclusion-high performance liquid chromatography to quantify biologics in patient samples by observing the peak area of free labeled biologic relative to a standard curve.
The assay is described as being suitable for biologics that bind complex, large, heavily glycosylated, multimeric, or unpurifiable antigens, and it provides specific soluble antigen reagents such as isolated soluble α4β7 integrin heterodimers and isolated soluble IL-12p40 monomers. The soluble antigen reagents include defined sequence and structural constraints, including identity thresholds and design features that prevent dimerization or oligomerization for IL-12p40, and may include optional covalent linking elements, linkers, affinity or epitope tags, and protease cleavage sites.
Claims Coverage
The independent claim covers two inventive features: indirect detection of a first biologic in a subject sample using an unlabeled soluble polypeptide and a measured form of a second biologic, and detection of an autoantibody to the first biologic in the same sample. Dependent refinements further constrain polypeptide identity and structure and the biologics and labels used.
Indirect detection of first biologic via unlabeled soluble polypeptide and measured form of second biologic
A detection method comprising providing a sample from a subject administered a first biologic, contacting the sample with an unlabeled, isolated, soluble polypeptide, contacting the polypeptide in vitro with a second biologic, indirectly detecting binding of the polypeptide to the first biologic in the sample by measuring a form of the second biologic, wherein the detected binding determines a presence or an amount of the first biologic in the sample.
Autoantibody detection to the first biologic
The detection method further includes detecting a presence or an amount of an autoantibody to the first biologic in the sample.
Across the provided independent claim, the core inventive coverage centers on indirect binding detection of a first biologic using an unlabeled soluble polypeptide and a measured form of a second biologic, with determination of first biologic presence or amount and detection of autoantibodies to that first biologic.
Stated Advantages
Provides a detection method that determines a presence or an amount of a first biologic in a sample by indirectly detecting binding through a measured form of a second biologic.
Enables detection of a presence or an amount of an autoantibody to the first biologic in the sample.
Described as suitable for drugs targeting complex, large, heavily glycosylated, multimeric, or unpurifiable antigens.
Provides assay validation metrics and stated ability to tolerate common serum interferents.
Documented Applications
Quantification of biologics in patient samples using an indirect homogeneous mobility shift assay implemented with size exclusion chromatography and size exclusion-high performance liquid chromatography to measure free labeled biologic peak area against a standard curve.
Assay formats for vedolizumab and ustekinumab, including anti-drug antibody assays.
Assay use for detection of autoantibodies to biologics administered to a subject.
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