Methods for developing virus protein specific capture agents, capture agents, and methods of using the capture agents
Inventors
Assignees
United States Department of the Army
Publication Number
US-11613746-B2
Publication Date
2023-03-28
Expiration Date
2039-05-09
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
A method for developing capture agents for target proteins employs a compound library to find cyclic peptide sequences that bind the target protein. The target protein is also reacted with a clickable group-provider reagent to provide the protein with clickable groups. The compounds in the library are provided with complementary clickable groups that bind the clickable group on the target protein when the peptide sequences bind the target protein. In some embodiments, the cyclic peptide sequences that bind the target protein are incorporated into constructs having one or more arms that can serve as capture agents or potential treatments against the pathogens from which the target protein is derived. Some embodiments provide pharmaceutical compositions for immunoassays, diagnostics, therapeutics or the like, that employ the constructs.
Core Innovation
The invention provides a method for developing synthetic capture agents for target proteins by utilizing a compound library to identify cyclic peptide sequences that bind the target protein. The method involves reacting the target protein with a clickable group-provider reagent, which imparts clickable groups onto the protein. Compounds in the library are functionalized with complementary clickable groups, allowing them to bind the clickable groups on the target protein through a click chemistry reaction when the peptide sequences bind the protein. These cyclic peptide sequences can be incorporated into constructs having one or more arms, which serve as capture agents or potential therapeutics against pathogens from which the target protein is derived.
The disclosed screening method allows rapid development of capture agents by functionalizing the protein target with alkynes and using an azide-functionalized cyclic peptide library, enabling selection of strong binders without requiring structural information or pre-existing molecules against the target. The cyclic peptide sequences obtained, exemplified by sequences specific to the Chikungunya virus (CHIKV) E2 protein, are relatively small peptides with unique binding properties compared to typical large monoclonal or polyclonal antibodies. Multi-arm constructs of these peptides improve target binding affinity and selectivity significantly, sometimes outperforming commercial antibodies.
The invention addresses the public health problem of detecting and treating infections such as Chikungunya virus, for which no approved antiviral or vaccine currently exists in the United States. Alphaviruses like CHIKV pose public health and national security threats due to their infectivity, stability, and prior use in biological weapons programs. Existing detection agents for CHIKV lack cyclic peptides and require structural knowledge or prior molecules. The invention provides a method and composition for rapidly developing stable, selective, and high-affinity capture agents without prior structural protein knowledge, suitable for diagnostics, immunoassays, and therapeutics.
Claims Coverage
The patent claims cover a method for developing capture agents that bind target proteins using a modified entire protein with clickable groups, cyclic peptide libraries, and multi-arm constructs. The inventive features include specific aspects of the capture agent development process, constructs, and their compositions.
Use of whole protein modified with click handle moieties
The method uses a whole target protein modified with at least one click handle moiety precursor without prior knowledge of the protein's structure, enabling binding to synthetic antigens comprising the modified protein and click handles.
Employing a compound library with complementary clickable groups having peptide sequences
Providing and incubating a compound library whose members each have complementary clickable groups and variable peptide sequences that can bind to the synthetic antigen via click chemistry, allowing selection of binding compounds.
Selecting and sequencing compounds binding the synthetic antigen for capture agent use
Selecting one or more compounds that bind the synthetic antigen with simultaneous click handle binding and sequencing each to obtain peptide sequences for use as capture agents or in their constructs.
Forming constructs with one or more arms incorporating selected compounds
Constructs comprising one or more polymer arms incorporating selected cyclic peptide compounds can be made, including one-arm or multi-arm constructs connected optionally to solid supports like beads or ELISA plates.
Specific structural forms of one-arm and multi-arm constructs with polymeric linkers
The patent claims specific chemical structures for one-arm and multi-arm constructs, where arms include polyethylene oxide-type linkers and cyclic peptides incorporating selected peptide sequences (such as SEQ ID NOs: 1-4 and D-amino acid versions).
Screening method involving pre-clear, target screen, and anti-screen steps
The method may include pre-clear screening to remove library compounds binding assay reagents, target screen with synthetic antigen, and anti-screening using complex biological solutions to select candidates with low non-specific binding.
Use of the CHIKV E2 protein as a target protein
The target protein in some claims is specifically the Chikungunya virus E2 protein, and the cyclic peptide sequences identified include specific sequences (e.g., WIYYI, YWHWS, IYLRY, FWQIL).
Assay tags and immunoassaying techniques for identifying suitable capture agents
Construct molecules include assay tags (e.g., beads, fluorophores, nanoparticles) and are used in various immunoassays to determine affinity and selectivity for the target protein, identifying suitable capture agents for diagnostics or therapeutics.
Overall, the claims cover a novel method of creating capture agents by chemically modifying whole proteins to add clickable groups, screening cyclic peptide libraries functionalized with complementary clickable groups, selecting and sequencing active compounds, forming multi-arm constructs incorporating these compounds, and using immunoassays for identification and application, particularly for CHIKV E2 protein detection and treatment.
Stated Advantages
The method enables rapid screening of full protein targets without requiring structural information, facilitating development of strong, specific capture agents for emerging or poorly characterized proteins.
Multi-arm cyclic peptide constructs exhibit improved affinity and selectivity, with over two orders of magnitude better performance than one-arm constructs and surpassing commercial antibodies in some cases.
The cyclic peptides demonstrate high thermal stability, maintaining binding activity after heating, thereby eliminating cold chain requirements for storage and shipment.
The approach allows the development of highly stable, selective, and small synthetic capture agents suitable for use in diagnostic assays, therapeutics, and potential in vivo applications.
Documented Applications
Use of capture agent constructs in immunoassays such as enzyme-linked immunosorbent assay (ELISA), LUMINEX® binding assays, Western blot, surface plasmon resonance, lateral flow, and other assay formats for detection and quantification of target proteins.
Pharmaceutical compositions comprising one-arm or multi-arm cyclic peptide constructs for use as therapeutics or inhibitors against infections by pathogens expressing the target proteins, such as the Chikungunya virus.
Application for early biological threat detection, diagnostics, and treatment of infectious diseases by identifying target proteins in biological samples including human serum, bodily fluids, tissues, and environmental samples.
Potential use of peptide constructs as targeted in vivo or in vitro inhibitors or therapeutics without the need for delivery of DNA or other agents, including fusion to therapeutic proteins to increase antigen binding avidity and efficacy.
Interested in licensing this patent?