Identification and medical applications of anti-citrullinated-protein antibodies in rheumatoid arthritis
Inventors
Labaer, Joshua • Qiu, Ji • Karthikeyan, Kailash • Buckner, Jane • Nepom, Gerald
Assignees
Virginia Mason Medical Center • Arizona State University ASU • Arizona State University Downtown Phoenix campus
Publication Number
US-12235268-B2
Publication Date
2025-02-25
Expiration Date
2037-06-14
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Abstract
Compositions and methods for detection of anti-citrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) patients. Patient samples known or suspected of containing ACPAs were probed against citrullinated proteins, and antibody responses to 190 citrullinated proteins in 20 RA patients were investigated. Unique antibody reactivity patterns in both clinical anti-cyclic citrullinated peptide assay positive (CCP+) and negative (CCP−) RA patients were observed. At individual antigen levels, six novel antibody/antigen complexes were discovered and validated against specific citrullinated antigens (Myelin Basic Protein (MBP), osteopontin (SPP1), flap endonuclease (FENI), insulin like growth factor binding protein 6 (IGFBP6), insulin like growth factor I (IGF1) and stanniocalcin-2 (STC2)) in RA patients. Identification of immune-dominant epitope(s) for citrullinated MBP was also performed. The identified biomarkers have high specificity, especially MBP.
Core Innovation
This invention introduces compositions and methods for detecting anti-citrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) patients using a high-throughput platform. By leveraging a nucleic acid programmable protein array (NAPPA) that enables simultaneous post-translational modification of many proteins, antibody responses to 190 citrullinated proteins were profiled among RA patients. The invention identified unique patterns of antibody reactivity in both anti-cyclic citrullinated peptide assay positive (CCP+) and negative (CCP−) RA patients, including the discovery and validation of six novel antibody/antigen complexes: Myelin Basic Protein (MBP), osteopontin (SPP1), flap endonuclease (FEN1), insulin like growth factor binding protein 6 (IGFBP6), insulin like growth factor I (IGF1), and stanniocalcin-2 (STC2).
The problem addressed is the limitation of current ACPA assays, which measure generalized reactivity with citrulline-containing peptides but do not provide information about disease-specific RA antigens or the specificity of autoantibody responses. RA presents with clinical heterogeneity and differing autoantibody profiles, making it difficult to achieve sensitive and specific diagnostics using existing tests. There is a need to identify more citrullinated antigens and their immunodominant epitopes to improve diagnostic sensitivity and specificity, as well as to elucidate the cause of RA.
The invention further includes mapping immune-dominant epitopes for citrullinated MBP, identifying two reactive epitope patterns using deletion mutants and tiling fragments. These identified biomarkers, especially MBP and its epitopes, were found to have high specificity and sensitivity in distinguishing RA patient samples, providing new diagnostic targets. The platform and methods described allow unbiased, high-throughput identification and validation of novel ACPAs and their target antigens, addressing unmet needs in RA diagnosis and research.
Claims Coverage
The patent includes one independent claim comprising multiple inventive features for detecting anti-citrullinated antibodies in samples from rheumatoid arthritis patients.
Combinatorial use of multiple in vitro citrullinated antigens including MBP peptide fragments and additional RA-associated antigens immobilized on a substrate
A method is described wherein a plasma or serum sample from a RA patient is contacted with a substrate that contains: - At least one in vitro citrullinated antigen selected from MBP peptide fragments (SEQ ID NO. 1 or SEQ ID NO. 2) - At least one additional in vitro citrullinated antigen from the group SPP1, FEN1, IGFBP6, IGF1, and STC2 Both the MBP fragments and the additional antigen(s) are linked to haloalkane dehalogenase, and the substrate is coated with a reactive chloroalkane ligand capable of binding to the haloalkane dehalogenase, thus immobilizing the antigens.
Detection of antibody-antigen complexes formed between citrullinated antigens and anti-citrullinated protein antibodies
After immobilizing the antigens, the method involves: 1. Contacting the patient sample with the immobilized citrullinated antigens to allow antibody-antigen complex formation. 2. Contacting the complexes with a label (e.g., a dye-labeled secondary antibody). 3. Detecting the presence of antibody-antigen complexes by detecting the label, thereby identifying anti-citrullinated antibodies in the sample.
Post-translational citrullination and on-substrate immobilization of antigens using a high-throughput protein array process
The method, in some embodiments, includes: - Depositing plasmid DNA encoding the citrullinated antigens linked to haloalkane dehalogenase into microwells, combined with in vitro protein expression lysates to express the proteins. - Immobilizing the expressed antigens on a substrate coated with the reactive chloroalkane ligand. - Citrullinating the immobilized antigens post-translationally on the substrate prior to exposing them to patient samples. This process allows high-throughput preparation and modification of multiple citrullinated antigens for antibody detection.
In summary, the claims cover a high-throughput method for detecting anti-citrullinated antibodies in RA involving immobilization of specific citrullinated antigens (including MBP epitopes and other novel RA biomarkers) on a reactive substrate, formation of antibody-antigen complexes, and sensitive detection based on labeled secondary antibodies.
Stated Advantages
Allows high-throughput, unbiased identification and screening of many citrullinated antigens simultaneously for improved diagnostic sensitivity and specificity.
Enables detection of antibody responses to novel antigens that are missed by traditional CCP-based assays, addressing patient heterogeneity.
Provides a platform for mapping immunodominant epitopes, which can aid in the development of more specific and sensitive diagnostic assays.
The method avoids the need to express, purify, and modify proteins individually, streamlining the screening and validation process.
Supports stratification of rheumatoid arthritis subtypes based on individual antibody reactivity patterns.
Documented Applications
Diagnosis of rheumatoid arthritis through detection of anti-citrullinated protein antibodies specific to novel and known citrullinated antigens.
Patient differentiation and stratification by profiling the specificity of ACPA responses among RA patients.
Research tool for studying the pathogenesis of rheumatoid arthritis by mapping immunodominant epitopes and antibody reactivity patterns.
Potential use in monitoring therapeutic efficacy by measuring changes in detected antibody-antigen complexes during treatment.
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