Compositions and methods for in vitro assays of fibrin activity
Inventors
Akassoglou, Katerina • Ryu, Jae Kyu • Meyer-Franke, Anke
Assignees
Publication Number
US-11573222-B2
Publication Date
2023-02-07
Expiration Date
2035-01-09
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Abstract
The present invention provides compositions exhibiting in vivo activity of fibrin in an in vitro setting, in vitro assays comprising such compositions, methods of producing such compositions, and methods of using such compositions and assays. The compositions of the invention include molecules with the biochemical properties of 1) high affinity binding to fibrin receptors and 2) activation of cell-signaling systems comparable to that observed in vivo by fibrin. The fibrin compositions of the invention are compatible both in biochemical assays and cell-based assays, and thus useful for in vitro assays for screening of test agents that modulate cell activation and/or signaling pathways mediated by fibrin or associated with fibrin activity.
Core Innovation
The invention provides compositions exhibiting in vivo activity of fibrin but operable in an in vitro setting, in vitro assays comprising these compositions, methods of producing them, and methods for their use. These compositions include molecules characterized by high affinity binding to fibrin receptors and by activation of cell-signaling systems comparable to those seen in vivo. The fibrin compositions are compatible with both biochemical and cell-based assays, making them useful for screening test agents that modulate cell activation and signaling pathways mediated by fibrin or associated with fibrin activity.
The problem addressed by the invention arises because while fibrinogen is known to perform critical coagulation and inflammatory functions, there was no prior method to efficiently reproduce the in vivo configurations of fibrin and its interactions in vitro. In particular, no assay existed capable of predicting the functional activity of agents binding fibrin based on in vitro assays. The invention addresses the need for fibrin compositions and assays capable of predicting in vivo activity through demonstrated in vitro activity, thus enabling the identification and evaluation of agents that modulate fibrin-induced cell signaling and activation.
Claims Coverage
The claims encompass inventive features relating to assay methods involving fibrin compositions and their ability to activate immune cells, particularly macrophages or microglia.
Assay method for identifying test agents inducing macrophage or microglia activation
An assay method comprising immobilizing a fibrin composition that displays an exposed, pro-inflammatory, cryptic fibrin epitope onto a solid support surface, introducing macrophages or microglia to this surface, introducing test agents, and measuring the activation of said immune cells.
Use of central nervous system cells in activation assays
The assay method wherein the macrophages or microglia introduced are cells derived from the mammalian central nervous system.
Measurement of activation via gene expression
Measuring activation of macrophages or microglia by assessing expression of one or more genes known to be associated with their activation.
Measurement of activation via reactive oxidative species production
Measuring macrophage or microglia activation by detecting production of reactive oxidative species known to be linked to their activation.
These inventive features collectively define assay methods using fibrin compositions with exposed pro-inflammatory epitopes to induce and measure immune cell activation, facilitating the identification of test agents modulating such activation.
Stated Advantages
The compositions mimic the in vivo proinflammatory activity of fibrin in vitro, offering reproducible activity suitable for biochemical and cell-based assays.
Assays using these compositions can better predict in vivo efficacy of fibrin inhibitors and modulators, increasing the likelihood of identifying effective therapeutic agents.
The assays are compatible with high throughput screening, enabling screening of small molecule and antibody libraries.
The fibrin assay plates can be stored and scaled up, providing quality control and reproducibility.
The compositions present the pro-inflammatory cryptic fibrin epitope, improving functional screening and binding assay accuracy compared to fibrinogen alone.
Documented Applications
In vitro assays for screening test agents that modulate cell activation or signaling pathways mediated by fibrin.
Research tools to identify agents that modulate in vivo fibrin activity, particularly involving macrophages, microglia, and cells of the neuronal lineage.
Assay methods to identify test agents affecting CD11b/CD18 integrin receptor signaling via fibrin.
Identification of biomarkers indicative of fibrin-mediated pathological conditions in patient serum or cerebrospinal fluid.
High throughput screening for small molecules and antibody libraries targeting fibrin signaling pathways.
Diagnostic tools for detecting molecules associated with fibrin-mediated biological processes.
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