Methods of assessing protein interactions between cells
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Publication Number
US-12411130-B2
Publication Date
2025-09-09
Expiration Date
Abstract
Provided are methods of detecting and quantitatively measuring protein-protein interactions between proteins present on the surface of different cells using a sensitive antibody-based assay. Antibodies specific for each target protein are either directly labeled, or are detected by labeled secondary antibodies. The antibodies are labeled with detectable tags that are releasable when the target proteins are within proximity to each other. Also provided are methods for facilitating diagnosis, prognosis, and treatment of cancer using this assay to detect protein-protein interactions between target proteins of interest. Methods of screening test agents that impact the protein-protein interaction are also provided.
Core Innovation
The invention provides methods of quantifying a protein-protein interaction between two cells in a sample by using antibody binding compositions that bind cell-surface proteins on the respective cells. A first antibody binding composition includes a molecular tag attached via a cleavable linkage to a first protein, while a third antibody composition includes a cleavage inducing moiety that binds a second antibody binding composition. After washing, cleavage occurs when the tagged composition is within an effective proximity range of the cleavage inducing moiety, thereby releasing the molecular tag.
The released molecular tag amount is detected as a measure of the amount of protein-protein interaction between the first protein and the second protein. The method includes providing a second antibody binding composition that specifically binds the second protein and contacting the sample with the first, second, and third antibody binding compositions. The assay readout is the detected amount of released molecular tag as a proxy for the protein-protein interaction level.
A second approach reverses which component carries the cleavage inducing moiety and which carries the molecular tag: the first antibody binding composition comprises a cleavage inducing moiety, and the third antibody composition comprises the molecular tag attached via a cleavable linkage. In both approaches, inducing cleavage releases the molecular tag when the tagged antibody and the cleavage inducing moiety are within an effective proximity range.
Claims Coverage
The document includes two independent claims. Across them, the core inventive features are proximity-dependent release of a molecular tag from an antibody binding composition, followed by detection of released molecular tag as the interaction readout.
Proximity-triggered cleavage releases a tagged antibody signal
A sample comprises a first cell expressing a first cell-surface protein and a second cell expressing a second cell-surface protein; an antibody binding composition includes a molecular tag attached via a cleavable linkage; after washing, cleavage is induced when the tagged composition is within an effective proximity range of a cleavage inducing moiety, releasing the molecular tag.
Detection of released molecular tag quantifies the protein-protein interaction
The amount of released molecular tag is detected as a measure of the amount of protein-protein interaction between the first protein and the second protein.
Third antibody binding enables cleavage-inducing proximity
A second antibody binding composition specifically binds the second protein, and a third antibody composition specifically binds the second antibody binding composition and comprises a cleavage inducing moiety, enabling proximity-dependent cleavage of the molecular tag.
Cleavage-inducing moiety and molecular tag positions are interchanged
The first antibody binding composition carries the cleavage inducing moiety, and the third antibody composition carries the molecular tag attached via a cleavable linkage; after washing, cleavage of the molecular tag is induced when the third antibody binding composition is within an effective proximity range of the cleavage inducing moiety.
Both independent claims define a proximity-dependent antibody-based assay in which a molecular tag attached via a cleavable linkage is released only when a cleavage inducing moiety is positioned within an effective proximity range relative to the tagged antibody. The amount of released molecular tag is detected and used as the measure of protein-protein interaction between two cell-surface proteins, with two alternative configurations of which antibody component carries the tag versus the cleavage-inducing moiety.
Stated Advantages
Improved prediction of responsiveness to PD-1/PD-L1 acting agents by measuring PD-1–PD-L1 complex rather than individual proteins, using a threshold level comparison.
Documented Applications
Screening test agents by measuring a protein-protein interaction level in a test cell culture and comparing it to an uncontacted control cell culture to determine whether the agent disrupts or promotes interaction formation.
Predicting a cancer subject’s responsiveness to a PD-1 or PD-L1 acting agent by measuring PD-1–PD-L1 complex in a biological sample and comparing the measured level to a threshold level.
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