Methods for determining DPP3 and therapeutic methods
Inventors
Assignees
Publication Number
US-11726094-B2
Publication Date
2023-08-15
Expiration Date
2037-04-20
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Abstract
The present invention is directed to methods for determining active DPP3 in a bodily fluid sample, an assay or kit for determining active DPP3 in a bodily fluid sample, a method for diagnosing a disease or condition in a subject accompanied by or related to necrotic processes and methods of treating or preventing said disease.
Core Innovation
The invention provides methods for the specific determination of active dipeptidyl peptidase III (DPP3) in bodily fluid samples. This includes assays and kits designed to bind specifically to full-length DPP3 and measure its enzymatic activity by quantifying the conversion of a substrate specific to DPP3. A particular embodiment is the enzyme capture assay (ECA), which uses a capture-binder that binds full-length DPP3 with minimal inhibition of its activity to enable accurate measurement of DPP3 enzymatic conversion products in the sample.
The methods extend to diagnosing diseases or conditions in subjects that are accompanied by or related to necrotic processes, by determining levels of active or total DPP3 and comparing them to predefined thresholds to assess disease presence. Moreover, therapeutic approaches include administering inhibitors of DPP3 activity, specifically antibodies or antibody fragments that selectively bind to full-length DPP3, to treat or prevent diseases linked to necrosis and elevated DPP3 activity.
The problem addressed is the lack of specific and reliable methods to determine active DPP3 in bodily fluids, differentiating it from other aminopeptidases, and the need for diagnostic tools to detect diseases with necrotic involvement. Also, it addresses the therapeutic gap by providing selective DPP3 inhibitors that can be used to treat such diseases, overcoming issues such as non-specific inhibition and the inability to target extracellular DPP3 effectively.
Claims Coverage
The patent includes four main independent claims covering methods of treating diseases related to necrotic processes using specific DPP3 inhibitors produced by particular immunization and cell fusion techniques, defining the processes for producing these inhibitors and their characteristics.
Method of treating necrotic diseases with DPP3 inhibitors produced by immunization with SEQ ID No. 2 peptides and trioma cell technology
A method for treating necrosis-related diseases by administering an effective amount of an inhibitor provided through immunizing a mammal with peptides containing SEQ ID No. 2, producing trioma cells by infecting or fusing human B cells with myeloma or hybridoma cells, testing immunoglobulins from trioma cells for DPP3 binding, and selecting DPP3-binding immunoglobulins as inhibitors.
Method of treating necrotic diseases with inhibitors produced by immunizing mammals and fusing splenocytes with myeloma or hybridoma cells
A method of treating necrotic disease conditions by administering an effective inhibitor obtained by immunizing a mammal with SEQ ID No. 2 peptides, fusing splenocytes from immunized mammals with myeloma or hybridoma cells, selecting and testing immunoglobulins from these fused cells that bind to DPP3 as the inhibitor.
Method of treating necrotic diseases using humanized antibodies derived from non-human mammals binding to SEQ ID No. 2
A method involving humanizing complementarity determining regions (CDRs) from a non-human immunoglobulin that binds to DPP3 (SEQ ID No. 2) and combining them with human framework regions (FRs) to produce a humanized antibody comprising humanized light and heavy chains for treatment of necrosis-related diseases.
Introduction of amino acid variations in CDRs or FRs to restore structural interactions after humanization
The method includes introducing amino acid variations in the CDR or FR sequences during humanization to recover necessary structural interactions disrupted by species changes in the framework regions, improving inhibitor efficacy.
The independent claims collectively cover methods of treating necrosis-associated diseases by administering DPP3 inhibitors prepared through specific immunization and cell fusion techniques, including humanized antibodies targeting DPP3 sequences (particularly SEQ ID No. 2), and incorporating strategies for optimizing antibody binding through amino acid sequence variations.
Stated Advantages
Provides specific and selective detection of active DPP3 in bodily fluids, distinguishing it from other aminopeptidases.
Enables reliable diagnosis of diseases associated with necrotic processes by measuring DPP3 levels or activity against predetermined thresholds.
Therapeutic inhibitors are selective for DPP3, do not cross cell membranes or blood-brain barrier, reducing off-target effects.
Developed antibodies strongly inhibit DPP3 activity and are safe for use, showing efficacy in reducing mortality and improving heart function in sepsis animal models.
Methods enable extracorporeal removal of excess DPP3 from plasma using affinity chromatography techniques.
Documented Applications
Determining active DPP3 levels in bodily fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusions.
Diagnosing diseases or conditions related to necrotic processes, including heart failure (acute and chronic), myocardial infarction, stroke, liver failure, burn and traumatic injuries, severe infections (microbial, viral like AIDS, parasitic like Malaria), sepsis, cancer, acute kidney injury, central nervous system disorders, autoimmune and vascular diseases, and hypotension.
Monitoring disease progression by repeated measurement of total or active DPP3.
Treating necrosis-related diseases using DPP3 inhibitors such as specific anti-DPP3 antibodies or fragments.
Extracorporeal removal of DPP3 from plasma by apheresis and affinity chromatography using immobilized anti-DPP3 antibodies.
Use of DPP3 enzyme capture assays or immunoassays for patient diagnostics and prognosis assessment in various diseases.
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