WO2024081915A1 - Capture agents for detection of kawasaki disease - Google Patents

Abstract

The present disclosure provides capture proteins for detection of antibodies specific for Kawasaki disease, methods of using the antibodies, and kits comprising the same.

Description

CAPTURE AGENTS FOR DETECTION OF KAWASAKI DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63/379,408 filed on October 13, 2022, the content of which is incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant number All 50719 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO A SEQUENCE LISTING

[0003] A Sequence Listing accompanies this application and is submitted as an xml file of the sequence listing named “702581.02398.xml” which is 5,013 bytes in size and was created on October 11, 2023. The sequence listing is electronically submitted via Patent Center with the application and is incorporated herein by reference in its entirety.

BACKGROUND

[0004] Kawasaki Disease (KD) is a febrile illness of young childhood that has clinical and epidemiologic features of an infectious disease including epidemics with geographic wavelike spread. KD can result in potentially severe or even fatal coronary artery aneurysms in infants and children. First described by Dr. Tomisaku Kawasaki in Japan in the 1960s, and now recognized worldwide, the etiology remains elusive. Diagnosis of KD, particularly of incomplete cases who can have prolonged fever but few other clinical manifestations, is a major clinical problem in pediatrics because potentially severe, lifelong sequelae can be reduced by timely administration of intravenous gammaglobulin. High attack rates of KD are observed in Asian children, most likely because of genetic predisposition to the inciting agent. In Japan 1 in 65 children develop the disease by the age of 5 years. Accordingly, there is a need in the art to develop novel diagnostic tools and therapies to better timely diagnose and treat KD. SUMMARY OF THE DISCLOSURE

[0005] In an aspect, provided herein is an isolated capture protein comprising a fragment of SEQ ID NO: 2 or a sequence having at least 95% identity thereto. In embodiments, the fragment comprises SEQ ID NO: 1 or a sequence having at least 95% identity thereto. In embodiments, the capture protein further comprises an Fc domain, a His tag, or a FLAG tag. In embodiments, the isolated capture protein is linked to a solid support.

[0006] In another aspect, provided herein is a construct configured to express the capture protein disclosed herein.

[0007] In another aspect, provided herein is a host cell comprising the capture protein or construct disclosed herein.

[0008] In another aspect, provided herein is a method of detecting Kawasaki disease in a subj ect, the method comprising contacting a sample obtained from the subject with a capture protein comprising a fragment of SEQ ID NO:2 or a sequence having at least 95% identity thereto; and detecting the presence of a capture protein-antibody complex formed from binding of an antibody in the sample to the capture protein. In embodiments, the fragment comprises SEQ ID NO: 1 or a sequence having at least 95% identity thereto. In embodiments, the method further comprises treating the subject with Kawasaki disease with intravenous immunoglobulin (IV Ig) if the capture protein-antibody complex is detected. In embodiments, the capture protein further comprises an Fc domain, a His tag, or a FLAG tag. In embodiments, the capture protein is linked to a solid support. In embodiments, the method further comprises contacting the capture protein-antibody complex with a secondary antibody, wherein the secondary antibody is linked to a detectable label. In embodiments, the sample is a serum sample. In embodiments, the capture protein-antibody complex is detected using at least one of Western blot, ELISA, immunostaining, immunoprecipitation, flow cytometry, sensor chips, magnetic beads, and lateral flow. In embodiments, the capture protein-antibody complex is detected using Western blot.

[0009] In another aspect, provided herein is a kit comprising the isolated capture protein or the construct disclosed herein; and a detection reagent. In embodiments, the kit further comprises a solid support, wherein the isolated capture protein is linked to the solid support. In embodiments, the solid support comprises a lateral flow device. In embodiments, the detection reagent comprises a secondary antibody optionally linked to a detectable label. BRIEF DESCRIPTION OF DRAWINGS

[0010] FIG. 1 is a schematic depicting a model of KD pathogenesis subsequent to infection of the subject with a ubiquitous viral pathogen which may cause mild or asymptomatic infection in other subjects.

[0011] FIG. 2 shows IgM Western blots showing candidate KD genome fragment-mouse Fc fusion protein (FP) recognized by pretreatment KD sera.

[0012] FIG. 3 shows IgG Western blots showing candidate KD genome fragment-mouse Fc fusion protein (FP) recognized by pretreatment KD sera.

[0013] FIG. 4 shows IgG, IgM, and IgA Western blots showing KD genome fragment-mouse Fc fusion protein (FP) recognized by pretreatment sera from day 11 of illness.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present disclosure describes a previously unidentified protein fragment that is recognized by antibodies in sera of patients having Kawasaki disease (KD). In an embodiment, the disclosure provides a use for this protein fragment as a capture agent specific for the detection of antibodies associated with KD in serum, and for detection of KD in children. This protein is recognized by pretreatment KD sera in the second week of illness, and is larger than previously reported peptides for serological assays for KD. Thus, the capture protein has improved practical use in diagnostic assays, which is urgently needed.

[0015] Kawasaki Disease is a febrile illness of young childhood that has clinical and epidemiologic features of an infectious disease including epidemics with geographic wavelike spread. In some cases, Kawasaki disease manifests only as prolonged fever, making timely diagnosis difficult. Furthermore, the exact cause of Kawasaki disease is not known. The inventors hypothesize that the cause of Kawasaki disease is a ubiquitous pathogen. Previously, the inventors discovered that serum samples from of KD patients taken in different geographic locations and from different times in history contained antibodies directed to similar antigens, supporting the inventors’ hypothesis.

[0016] Proteins specific for Kawasaki disease

[0017] The inventors discovered that patients diagnosed with Kawasaki disease produce antibodies that recognize a particular protein (see FIGs. 2 and 3). This protein may be used as a diagnostic tool to identify individuals having Kawasaki disease. [0018] Accordingly, in a first aspect, provided herein is a capture protein (KD1) for binding antibodies found in subjects with Kawasaki disease. The capture protein comprises a fragment of SEQ ID NO: 2 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. The fragment may comprise SEQ ID NO: 1 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. The capture protein may be an isolated protein.

[0019] The terms “proteins” and “polypeptides” are used interchangeably herein to refer to a polymer of protein amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein” and “polypeptide” also refer to encoded gene product and fragments thereof. Thus, exemplary proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0020] The phrases “percent identity” and “% identity,” as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases. [0021] Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, or at least 700 contiguous amino acid residues; or a fragment of no more than 15, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 amino acid residues; or over a range bounded by any of these values (e.g., a range of 500-600 amino acid residues) Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0022] The capture protein may comprise an Fc domain. The terms “Fc domain”, “Fc region,” and “Fc part” are used interchangeably to refer to a C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

[0023] The capture protein may further comprise a detectable label or tag. The term "tag" or “detectable label” as used herein includes any useful moiety that allows for the purification, identification, detection, diagnosing, imaging, or therapeutic use of the capture protein of the present invention and are readily known in the art. Suitable tags or detection labels include epitope tags, detection markers and/or imaging moieties, including, for example, enzymatic markers, fluorescence markers, radioactive markers, among others.

[0024] The term "additional moiety" includes other molecules that may be linked to the antibody or antibody fragment thereof. Suitable additional moieties include, but are not limited to, for example, therapeutic agents, small molecules, and drugs, among others. The additional moieties can also include diagnostic agents.

[0025] The capture protein may comprise a peptide tag, such as a His tag or a FLAG tag. The capture protein may comprise a biotin or a biotinylated tag. The capture protein may comprise a fluorescent protein, luciferase, a fluorescent compound, or a colorimetric reagent.

[0026] The capture protein may be linked to a solid support. As used herein, “solid support” refers to any suitable material for the immobilization peptides (including, but not limited to epitope fragments, antibodies, or antibody fragments thereof) of the instant disclosure. For example, the solid support may be beads, particles, tubes, wells, probes, dipsticks, pipette tips, slides, fibers, membranes, papers, natural and modified celluloses, polyacrylamides, agaroses, glass, polypropylene, polyethylene, polystyrene, dextran, nylon, amylases, plastics, magnetite or any other suitable material readily known to one of skill in the art.

[0027] In a second aspect, provided herein is a polynucleotide encoding the capture protein KD 1. The polynucleotide may comprise SEQ ID NO: 3 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. The polynucleotide may be comprised in a nucleic acid construct capable of expressing KD1. The construct may be an expression vector or vector capable of expressing the protein in a host cell. As used herein, the term “construct” refers to a recombinant polynucleotide, i.e., a polynucleotide that was formed artificially by combining at least two polynucleotide components from different sources (natural or synthetic). For example, the constructs described herein comprise a polynucleotide encoding the fusion protein disclosed herein, operably linked to a promoter that (1) is associated with another gene found within the same genome, (2) from the genome of a different species, or (3) is synthetic. Constructs can be generated using conventional recombinant DNA methods.

[0028] The terms “nucleic acid,” “nucleic acid sequence,” “polynucleotide,” and “polynucleotide sequence,” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. A “polynucleotide” may refer to a polydeoxyribonucleotide (containing 2-deoxy-D-ribose), a polyribonucleotide (containing D- ribose), and to any other type of polynucleotide that is an N glycoside of a purine or pyrimidine base. There is no intended distinction in length between the terms “nucleic acid”, “oligonucleotide” and “polynucleotide”, and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA. For use in the present methods, an oligonucleotide also can comprise nucleotide analogs in which the base, sugar, or phosphate backbone is modified as well as non-purine or non-pyrimidine nucleotide analogs. These phrases also refer to DNA or RNA of genomic, natural, or synthetic origin (which may be single-stranded or double- stranded and may represent the sense or the antisense strand).

[0029] A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques known in the art. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell. The nucleic acids disclosed herein may be “substantially isolated or purified.” The term “substantially isolated or purified” refers to a nucleic acid that is removed from its natural environment, and is at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which it is naturally associated.

[0030] Suitable vectors are known in the art and contain the necessary elements in order for the gene encoded within the vector to be expressed as a protein in the host cell. The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, specifically exogenous DNA segments encoding the antibodies or fragments thereof. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome to be expressed in viral particles to be infected into cells and allow expression of the viral vectors carried within the viral particles. [0031] Certain vectors are capable of autonomous replication in a host cell into which they are introduced. Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., lentiviral vectors). Viral vectors include those known in the art, e.g., replication defective retroviruses (including lentiviruses), adenoviruses and adeno-associated viruses (rAAV)), which serve equivalent functions. Lentiviral vectors may be used to make suitable lentiviral vector particles by methods known in the art to transform cells in order to express the capture protein described herein.

[0032] In a third aspect, provided herein is a host cell comprising the capture proteins or nucleic acids disclosed herein. The host cell can be a prokaryotic or eukaryotic host cell. Suitable host cells include, but are not limited to, mammalian cells, bacterial cells and yeast cells. The term “host cell” includes a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells also include “transformants” and “transformed cells”, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity that was screened or selected for in the originally transformed cell are included herein. It should be appreciated that the host cell can be any cell capable of expressing proteins, for example fungi; mammalian cells; insect cells, using, for example, a baculovirus expression system; plant cells, such as, for example, corn, rice, Arcibidopsis and the like. See, generally, Verma, R. et al., J Immunol Methods. 1998 Jul. 1 ; 216(1-2): 165-81.

[0033] Methods of Detecting, Imaging and Diagnosing Kawasaki Disease

[0034] In a fourth aspect, provided herein is a method of detecting antibodies associated with Kawasaki disease in a subject, the method comprising contacting a sample obtained from the subject with a capture protein comprising a fragment of SEQ ID NO: 2 or a sequence having at least 95% identity thereto; and detecting the presence of a capture protein-antibody complex formed from binding of an antibody in the sample to the capture protein. The fragment may comprise SEQ ID NO: 1 or a sequence having at least 95% identity thereto. The subject may be suspected of having Kawasaki disease. Detecting the presence of the capture protein-antibody complex confirms that the subject has Kawasaki disease.

[0035] The capture protein may comprise an Fc domain. The terms “Fc domain”, “Fc region,” and “Fc part” are used interchangeably to refer to a C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

[0036] The capture protein may comprise a detectable label or tag. The capture protein may comprise a peptide tag, such as a His tag or a FLAG tag.

[0037] The capture protein may be linked to a solid support.

[0038] The method may further comprise contacting the capture protein-antibody complex with a secondary antibody, wherein the secondary antibody is linked to a detectable label or tag. Suitable secondary antibodies include, but are not limited to, anti-human antibodies that specifically bind to human Fc region of antibodies, particularly to IgG, IgM, and IgA antibodies. Other suitable anti-human antibodies that could specifically recognize human antibodies bound to the peptide are also contemplated. [0039] Methods for detecting the capture protein -anti body complex include, but are not limited to, enzyme-linked immunoabsorbent assay (ELISA), Western blot, immunostaining, immunoprecipitation, flow cytometry, sensor chips, magnetic beads, nanoparticles, lateral flow assay.

[0040] If the capture protein-antibody complex is detected within the sample, the method may further comprise: iv) treating the subject with intravenous immunoglobulin (IV Ig). Other treatment options for KD could also be employed and considered within the scope of this invention. [0041] As used herein, the terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and/or to prevent or slow the appearance or to reverse the progression or severity of symptoms of Kawasaki disease. As used herein, “intravenous Ig” refers to an effective amount of pooled IgG from donor subjects. Trade names of intravenous immunoglobulin formulations include Flebogamma", Gamunex®, Privigen®, Octagam®, and Gammagard®, while trade names of subcutaneous formulations include Cutaquig®, Cuvitru®, HyQvia®, Hizentra®, Gamunex-C®, and Gammaked®. [0042] The sample may be any biological sample obtained from the patient or subject that comprises antibodies, such as a blood sample or a serum sample.

[0043] Kits for Detecting Kawasaki Disease Antibodies and Diagnosing Kawasaki Disease

[0044] In a fifth aspect of the disclosure, a kit is provided. Any suitable kits comprising the components to carry out the methods described herein are contemplated.

[0045] The kit may comprise an isolated capture protein comprising a fragment of SEQ ID NO: 2 or a sequence having at least 95% identity thereto, or a construct for expressing the capture protein; and a detection reagent. The fragment may comprise SEQ ID NO: 1 or a sequence having at least 95% identity thereto. The kit may further comprise a solid support. The capture protein may be linked to the solid support. The solid support may be, but is not limited to being, the inner, bottom surface of a well of a microtiter plate or a substrate that is included as part of a lateral flow device, for example. The reagents employed in the methods of using the kit may be dried or immobilized onto the solid support, which may comprise a chromatographic support, contained within the device.

[0046] An exemplary lateral flow device is the lateral flow device that is described in U.S. Pat. No. 5,726,010, which is incorporated herein by reference in its entirety. The device for performing a lateral flow assay may be a SNAP® device, which is commercially available from IDEXX Laboratories, Tnc. of Westbrook, Me. However, it is to be understood that the skilled artisan will recognize that a large variety of other lateral flow devices that are not SNAP® devices or described by U.S. Pat. No. 5,726,010 allow for the immobilization of an antibody thereon, and therefore would be suitable for being used in the methods and kits device of the present invention.

[0047] Capture proteins used in the methods and kits of the invention may be immobilized on the solid support by any methodology known in the art, including, for example, covalently or non- covalently, directly or indirectly, attaching the proteins to the solid support. Therefore, while these proteins may be attached to the solid support by physical adsorption (i.e., without the use of chemical linkers), it is also true that these proteins may be immobilized to the solid support by any chemical binding (i.e., with the use of chemical linkers) method readily known to one of skill in the art.

[0048] A suitable kit may be an ELISA kit capable of detecting the binding of the capture protein to a human antibody, and therefore in some embodiments the kit further comprises a secondary antibody capable of binding the Fc portion of human antibodies. In some embodiments, the secondary antibody is linked to a detectable label. In some embodiments, the secondary antibody binds to IgG, IgM, or IgA antibodies.

[0049] The terms “antibody” or “antibody molecule” are used herein interchangeably and refer to immunoglobulin molecules or other molecules which comprise an antigen binding domain. The term “antibody” or “antibody molecule” as used herein is thus intended to include whole antibodies (e g., IgG, IgA, IgE, IgM, or IgD), monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, and antibody fragments, including single chain variable fragments (ScFv), single domain antibodies, and antigen-binding fragments, genetically engineered antibodies, among others, as long as the characteristic properties (e.g., ability to bind antigens derived from Kawasaki disease) are retained. The term “antibody fragment” as used herein is intended to include any appropriate antibody fragment that displays antigen binding function, for example, Fab, Fab’, F(ab’)2, scFv, Fv, dsFv, ds-scFv, Fd, mini bodies, monobodies, and multimers thereof and bispecific antibody fragments. Fragments may comprise a heavy chain variable region (VH domain) and light chain variable region (VL) of the disclosure. Fragments may comprise one or more of the heavy chain complementarity determining regions (CDRHs) of the antibodies or of the VH domains, and one or more of the light chain complementarity determining regions (CDRLs), or VL domains to form the antigen binding site. [0050] The term “complementarity determining regions” or “CDRs,” as used herein, refers to part of the variable chains in immunoglobulins (antibodies) and T cell receptors, generated by B- cells and T-cells respectively, where these molecules bind to their specific antigen. As the most variable parts of the molecules, CDRs are crucial to the diversity of antigen specificities generated by lymphocytes. There are three CDRs (CDR1, CDR2 and CDR3), arranged non-consecutively, on the amino acid sequence of a variable domain of an antigen binding site. Since the antigen binding sites are typically composed of two variable domains (on two different polypeptide chains, heavy and light chain), there are six CDRs for each antigen binding site that can collectively come into contact with the antigen. A single whole antibody molecule has two antigen binding sites and therefore contains twelve CDRs. For further example, sixty CDRs can be found on a pentameric IgM molecule.

[0051] Within the variable domain, CDR1 and CDR2 may be found in the variable (V) region of a polypeptide chain, and CDR3 includes some of V, and all of diversity (D, heavy chains only) and joining (J) regions. Since most sequence variation associated with immunoglobulins and T cell receptors is found in the CDRs, these regions are sometimes referred to as hypervariable regions. Among these, CDR3 shows the greatest variability as it is encoded by a recombination of VJ in the case of a light chain region and VDJ in the case of heavy chain regions. The tertiary structure of an antibody is important to analyze and design new antibodies.

[0052] Although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), (see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al. , PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context.

[0053] Furthermore, the antibody or antibody fragment can further comprise all or a portion of a kappa light chain constant region or a lambda light chain constant region. All or part of such constant regions may be produced wholly or partially synthetic. Appropriate sequences for such constant regions are well known and documented in the art.

[0054] The term “monoclonal antibody” refers to an antibody molecule that specifically binds to a single epitope of an antigen. [0055] The term “chimeric antibody” refers to an antibody comprising a variable region, i.e., binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Other forms of “chimeric antibodies” are those in which the class or subclass has been modified or changed from that of the original antibody. Such “chimeric” antibodies are also referred to as “class-switched antibodies.”

[0056] The term “recombinant human antibody” includes all human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies isolated from a host cell such as an SP2-0, NSO or CHO cell (like CHO KI) or from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and, in some embodiments, constant regions derived from human germline immunoglobulin sequences in a rearranged form.

[0057] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” [0058] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0059] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0060] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0061] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0062] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. These aspects and embodiments are illustrative and non-exhaustive in nature. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the abovedescribed elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

EXAMPLE

[0063] Novel Kawasaki disease-associated protein

[0064] The inventors performed laser capture microdissection and sonication on ciliated bronchial epithelium containing inclusion bodies from formalin-fixed paraffin embedded (FFPE) lung tissue of two Kawasaki disease (KD) cases according to the method taught in Amini et al. BMC Molecular Biol (2017) 18:22. The sturdiness of KD inclusion bodies has previously been a barrier for extracting RNA using conventional methods. It is believed that the prolonged sonication in the Amini et al. method compared with the typical RNA extraction protocol allows for extraction of RNA having improved quality and quantity from the FFPE tissue.

[0065] The extracted RNA was then sequenced. A contig assembled from the reads revealed sequences that remained unassigned after BLAST analyses. Unassigned sequences having the longest open reading frames were selected for further study. A 708-nucleotide fragment (SEQ ID NO: 3) having no significant similarity to any known protein or nucleotide sequence was found to be of particular interest. The fragment was also not present in RNA sequencing datasets obtained from KD tissues that were not subjected to sonication. The fragment encodes a 215 amino acid open reading frame (KD2) (SEQ ID NO: 2, underlined portion) as shown in Table 1. The open reading frame includes a start codon, but no stop codon at the 3’ end, suggesting that the complete protein is larger.

[0066] Next, the inventors made a fusion protein corresponding to a particularly hydrophilic portion of the fragment (KD1) (SEQ ID NO: 1). This fragment was fused to a mouse Fc domain. The fusion protein was then tested for reactivity with KD and control sera in Western blot assays. [0067] In pre-treatment Kawasaki disease (KD) sera from week 2-3 following fever onset, 10/15 (67%) samples show either IgG (FIG. 3) or IgM (FIG. 2) antibody or both bind to the KDl-Fc fusion protein. This is compared with 1/11 (9%) infant control sera that were positive for IgG antibody and 2/21 (10%) infant control sera that were positive for IgM antibody (p=0.01 ). For each blot, lane 1 contains mouse Fc alone, lane 2 contains the fusion protein, and lane 3 contains the human IgG or IgM antibody Based on these results, the protein represents a segment of the proteome of the infectious agent that causes Kawasaki disease. Thus, this protein may be used for diagnosis of potential KD patients at an early stage of the illness as described herein.

[0068] Additionally, the inventors tested for reactivity with pre-treatment KD sera from day 11 of illness from a child with KD who died on day 13 of a ruptured giant coronary artery aneurysm. The sera were positive for IgG, IgM, and IgA antibodies, further indicating that KD1 may be used for Kawasaki Disease diagnosis.

[0069] Materials and Methods

[0070] The protein

VPGQASAFSPAIAVTADPAPDQTASSDGSMIKPDLSGIVTEEAASADPIIPVAPAPVRILNE DGSVGLAPEPQPQGNDTEPKASPRKAAAMKKVQESPEDPQAAQEAAAASADPEPKVDL P AGVYD ANAPD YD S S VRTDL SPKSP ADRATKQGLTPTLIAEITNPQP STS YKL was cloned into the Invitrogen vector pInfuse-mIgG2B-Fc (https://www.invivogen.com/pinfuse- migg2b-fc) at the multiple cloning site and transfected into 293F cells in tissue culture. The expressed protein was purified from tissue culture supernatant using protein A beads. Protein was also made from the vector without an insert, and the mouse Fc protein was similarly purified and run as a control next to the fusion protein in Western blot assays. For Western analysis, 250 ng of protein was run on 12% Mini Protein TGX Stain Free Gels (Bio-Rad). After transfer to PDVF membrane and blocking in 5% carnation nonfat dry milk in TBST, the blots were incubated with 1 :2500 dilutions of KD or control sera in 5% milk TBST overnight at 4°C. After washing, the blots were incubated for one hour at room temperature with HRP-labelled goat anti-human IgG, IgA, or IgM (Thermo Fisher) at 1 :50,000 for IgM and 1 : 10000 for IgG and IgA in PBS. Chemiluminescent imaging was performed following incubation in Supersignal West Femto (Thermo Fisher). Following imaging, blots were stripped and reblocked, then incubated with HRP-labelled goat anti-mouse-IgG (Thermo Fisher) to check protein loading.

[0071] Table 1 : Protein and Nucleotide Sequences for Candidate KD Fragment

Claims

CLAIMS We claim:

1. An isolated capture protein comprising a fragment of SEQ ID NO: 2 or a sequence having at least 95% identity thereto.

2. The isolated capture protein of claim 1, wherein the fragment comprises SEQ ID NO: 1 or a sequence having at least 95% identity thereto.

3. The isolated capture protein of claim 1 or 2, wherein the capture protein further comprises an Fc domain, a His tag, or a FLAG tag.

4. The isolated capture protein of any one of claims 1-3, wherein the isolated capture protein is linked to a solid support.

5. A construct configured to express the capture protein of any one of claims 1-3.

6. A host cell comprising the capture protein of any one of claims 1-3, or the construct of claim 5.

7. A method of detecting Kawasaki disease in a subject, the method comprising: contacting a sample obtained from the subject with a capture protein comprising a fragment of SEQ ID NO: 2 or a sequence having at least 95% identity thereto; and detecting the presence of a capture protein-antibody complex formed from binding of an antibody in the sample to the capture protein.

8. The method of claim 7, wherein the fragment comprises SEQ ID NO: 1 or a sequence having at least 95% identity thereto.

9. The method of claim 8, further comprising treating the subject with Kawasaki disease with intravenous immunoglobulin (IV Ig) if the capture protein-antibody complex is detected.

10. The method of any one of claims 7-9, wherein the capture protein further comprises an Fc domain, a His tag, or a FLAG tag.

11. The method of any one of claims 7-10, wherein the capture protein is linked to a solid support.

12. The method of any one of claims 7-11, further comprising contacting the capture proteinantibody complex with a secondary antibody, wherein the secondary antibody is linked to a detectable label.

13. The method of any one of claims 7-12, wherein the sample is a serum sample.

14. The method of any one of claims 7-13, wherein the capture protein-antibody complex is detected using at least one of Western blot, ELISA, immunostaining, immunoprecipitation, flow cytometry, sensor chips, magnetic beads, and lateral flow.

15. The method of claim 14, wherein the capture protein-antibody complex is detected using Western blot.

16. A kit compri si ng : the isolated capture protein of any one of claims 1-3 or the construct of claim 5; and a detection reagent.

17. The kit of claim 16, further comprising a solid support, wherein the isolated capture protein is linked to the solid support.

18. The kit of claim 17, wherein the solid support comprises a lateral flow device.

19. The kit of any one of claims 16-18, wherein the detection reagent comprises a secondary antibody optionally linked to a detectable label.

20. A capture protein-antibody complex comprising: an isolated capture protein comprising a fragment of SEQ ID NO: 2 or a sequence having at least 95% identity thereto; and an antibody to which the isolated capture protein binds.