KAWASAKI DISEASE ANTIBODIES IDENTIFY HEPACIVIRUS PEPTIDES
20220119502 · 2022-04-21
Inventors
- Anne H. Rowley (Oak Park, IL, US)
- Standford L. Shulman (Evanston, IL, US)
- Susan C. Baker (Elmhurst, IL, US)
Cpc classification
C12N2770/24222
CHEMISTRY; METALLURGY
C07K2317/34
CHEMISTRY; METALLURGY
C07K2317/40
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
International classification
Abstract
The present disclosure provides monoclonal antibodies that target intracytoplasmic inclusion bodies and/or hepacivirus C NS4A and methods for their use.
Claims
1. An isolated intracytoplasmic inclusion body (ICI) antibody or antigen binding fragment thereof comprising: a heavy chain variable domain comprising a CDRH1 region selected from the group consisting of 272, 278, 284, 290, 295, 301, 306, 311, 317, 322, 328, 334, and 340; a CDRH2 region selected from the group consisting of 273, 279, 285, 291, 296, 302, 307, 312, 318, 323, 329, 335, and 341; and a CDRH3 region selected from the group consisting of 274, 280, 286, 299, 297, 303, 308, 313, 319, 324, 330, 336, and 342; and a light chain variable domain comprising a CDRL1 region selected from the group consisting of 269, 275, 281, 287, 293, 298, 304, 309, 314, 320, 325, 331, and 337; a CDRL2 region selected from the group consisting of 270, 276, 282, 288, 299, 315, 321, 326, 332, and 338; and a CDRL3 region selected from the group consisting of 271, 277, 283, 289, 294, 300, 305, 310, 316, 327, 333, and 339.
2. The isolated ICI antibody or antigen binding fragment of claim 1, comprising: (a) a heavy chain variable domain comprising a CDRH1 region consisting of SEQ ID NO:272, a CDRH2 region consisting of SEQ ID NO:273, and a CDRH3 region consisting of SEQ ID NO:274 and a light chain variable domain comprising a CDRL1 region consisting of SEQ ID NO:269, a CDRL2 region consisting of SEQ ID NO:270, and a CDRL3 region consisting of SEQ ID NO:271, or (b) a heavy chain variable domain comprising a CDRH1 region consisting of SEQ ID NO:278, a CDRH2 region consisting of SEQ ID NO:279, and a CDRH3 region consisting of SEQ ID NO:280 and a light chain variable domain comprising a CDRL1 region consisting of SEQ ID NO:275, a CDRL2 region consisting of SEQ ID NO:276, and a CDRL3 region consisting of SEQ ID NO:277, or (c) a heavy chain variable domain comprising a CDRH1 region consisting of SEQ ID NO:284, a CDRH2 region consisting of SEQ ID NO:285, and a CDRH3 region consisting of SEQ ID NO:286 and a light chain variable domain comprising a CDRL1 region consisting of SEQ ID NO:281, a CDRL2 region consisting of SEQ ID NO:282, and a CDRL3 region consisting of SEQ ID NO:283, or (d) a heavy chain variable domain comprising a CDRH1 region consisting of SEQ ID NO:290, a CDRH2 region consisting of SEQ ID NO:291, and a CDRH3 region consisting of SEQ ID NO:292 and a light chain variable domain comprising a CDRL1 region consisting of SEQ ID NO:287, a CDRL2 region consisting of SEQ ID NO:288, and a CDRL3 region consisting of SEQ ID NO:289, or (e) a heavy chain variable domain comprising a CDRH1 region consisting of SEQ ID NO:295, a CDRH2 region consisting of SEQ ID NO:296, and a CDRH3 region consisting of SEQ ID NO:297 and a light chain variable domain comprising a CDRL1 region consisting of SEQ ID NO:293, a CDRL2 region consisting of SEQ ID NO:282, and a CDRL3 region consisting of SEQ ID NO:294, or (f) a heavy chain variable domain comprising a CDRH1 region consisting of SEQ ID NO:301, a CDRH2 region consisting of SEQ ID NO:302, and a CDRH3 region consisting of SEQ ID NO:303 and a light chain variable domain comprising a CDRL1 region consisting of SEQ ID NO:298, a CDRL2 region consisting of SEQ ID NO:299, and a CDRL3 region consisting of SEQ ID NO:300, or (g) a heavy chain variable domain comprising a CDRH1 region consisting of SEQ ID NO:306, a CDRH2 region consisting of SEQ ID NO:307, and a CDRH3 region consisting of SEQ ID NO:308 and a light chain variable domain comprising a CDRL1 region consisting of SEQ ID NO:304, a CDRL2 region consisting of SEQ ID NO:282, and a CDRL3 region consisting of SEQ ID NO:305, or (h) a heavy chain variable domain comprising a CDRH1 region consisting of SEQ ID NO:311, a CDRH2 region consisting of SEQ ID NO:312, and a CDRH3 region consisting of SEQ ID NO:313 and a light chain variable domain comprising a CDRL1 region consisting of SEQ ID NO:309, a CDRL2 region consisting of SEQ ID NO:276, and a CDRL3 region consisting of SEQ ID NO:310, or (i) a heavy chain variable domain comprising a CDRH1 region consisting of SEQ ID NO:317, a CDRH2 region consisting of SEQ ID NO:318, and a CDRH3 region consisting of SEQ ID NO:319 and a light chain variable domain comprising a CDRL1 region consisting of SEQ ID NO:314, a CDRL2 region consisting of SEQ ID NO:315, and a CDRL3 region consisting of SEQ ID NO:316, or (j) a heavy chain variable domain comprising a CDRH1 region consisting of SEQ ID NO:322, a CDRH2 region consisting of SEQ ID NO:323, and a CDRH3 region consisting of SEQ ID NO:324 and a light chain variable domain comprising a CDRL1 region consisting of SEQ ID NO:320, a CDRL2 region consisting of SEQ ID NO:321, and a CDRL3 region consisting of SEQ ID NO:316, or (k) a heavy chain variable domain comprising a CDRH1 region consisting of SEQ ID NO:328, a CDRH2 region consisting of SEQ ID NO:329, and a CDRH3 region consisting of SEQ ID NO:330 and a light chain variable domain comprising a CDRL1 region consisting of SEQ ID NO:325, a CDRL2 region consisting of SEQ ID NO:326, and a CDRL3 region consisting of SEQ ID NO:327, or (l) a heavy chain variable domain comprising a CDRH1 region consisting of SEQ ID NO:334, a CDRH2 region consisting of SEQ ID NO:335, and a CDRH3 region consisting of SEQ ID NO:336 and a light chain variable domain comprising a CDRL1 region consisting of SEQ ID NO:331, a CDRL2 region consisting of SEQ ID NO:332, and a CDRL3 region consisting of SEQ ID NO:333, or (m) a heavy chain variable domain comprising a CDRH1 region consisting of SEQ ID NO:340, a CDRH2 region consisting of SEQ ID NO:341, and a CDRH3 region consisting of SEQ ID NO:342 and a light chain variable domain comprising a CDRL1 region consisting of SEQ ID NO:337, a CDRL2 region consisting of SEQ ID NO:338, and a CDRL3 region consisting of SEQ ID NO:339.
3. The isolated ICI antibody or antigen binding fragment of claim 1, comprising: (a) a light chain variable domain selected from the group consisting of SEQ ID NOs:165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, and 189; and (b) a heavy chain variable domain selected from the group consisting of SEQ ID NOs:166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, and 190.
4. The isolated ICI antibody of any of claims 1-3, wherein the heavy chain variable domain comprises a CDRH1 region consisting of SEQ ID NO:272, a CDRH2 region consisting of SEQ ID NO:273, and a CDRH3 region consisting of SEQ ID NO:274 and the light chain variable domain comprises a CDRL1 region consisting of SEQ ID NO:269, a CDRL2 region consisting of SEQ ID NO:270, and a CDRL3 region consisting of SEQ ID NO:271.
5. The isolated ICI antibody of claim 4, wherein the light chain variable domain is SEQ ID NO:165 and the heavy chain variable domain is SEQ ID NO:166.
6. The isolated ICI antibody of any of claims 1-3, wherein the heavy chain variable domain comprises a CDRH1 region consisting of SEQ ID NO:278, a CDRH2 region consisting of SEQ ID NO:279, and a CDRH3 region consisting of SEQ ID NO:280 and the light chain variable domain comprises a CDRL1 region consisting of SEQ ID NO:275, a CDRL2 region consisting of SEQ ID NO:276, and a CDRL3 region consisting of SEQ ID NO:277.
7. The isolated ICI antibody of claim 6, wherein the light chain variable domain is SEQ ID NO:167 and the heavy chain variable domain is SEQ ID NO:168.
8. The isolated ICI antibody of any of claims 1-3, wherein the heavy chain variable domain comprises a CDRH1 region consisting of SEQ ID NO:284, a CDRH2 region consisting of SEQ ID NO:285, and a CDRH3 region consisting of SEQ ID NO:286 and a light chain variable domain comprises a CDRL1 region consisting of SEQ ID NO:281, a CDRL2 region consisting of SEQ ID NO:282, and a CDRL3 region consisting of SEQ ID NO:283.
9. The isolated ICI antibody of claim 8, wherein the light chain variable domain is SEQ ID NO:169 and the heavy chain variable domain is SEQ ID NO:170.
10. The isolated ICI antibody of any of claims 1-3, wherein the heavy chain variable domain comprises a CDRH1 region consisting of SEQ ID NO:290, a CDRH2 region consisting of SEQ ID NO:291, and a CDRH3 region consisting of SEQ ID NO:292 and the light chain variable domain comprises a CDRL1 region consisting of SEQ ID NO:287, a CDRL2 region consisting of SEQ ID NO:288, and a CDRL3 region consisting of SEQ ID NO:289.
11. The isolated ICI antibody of claim 10, wherein the light chain variable domain is SEQ ID NO:171 and the heavy chain variable domain is SEQ ID NO:172.
12. The isolated ICI antibody of any of claims 1-3, wherein the heavy chain variable domain comprises a CDRH1 region consisting of SEQ ID NO:295, a CDRH2 region consisting of SEQ ID NO:296, and a CDRH3 region consisting of SEQ ID NO:297 and the light chain variable domain comprises a CDRL1 region consisting of SEQ ID NO:293, a CDRL2 region consisting of SEQ ID NO:282, and a CDRL3 region consisting of SEQ ID NO:294.
13. The isolated ICI antibody of claim 12, wherein the light chain variable domain is SEQ ID NO:173 and the heavy chain variable domain is SEQ ID NO:174.
14. The isolated ICI antibody of any of claims 1-3, wherein the heavy chain variable domain comprises a CDRH1 region consisting of SEQ ID NO:301, a CDRH2 region consisting of SEQ ID NO:302, and a CDRH3 region consisting of SEQ ID NO:303 and the light chain variable domain comprises a CDRL1 region consisting of SEQ ID NO:298, a CDRL2 region consisting of SEQ ID NO:299, and a CDRL3 region consisting of SEQ ID NO:300.
15. The isolated ICI antibody of claim 14, wherein the light chain variable domain is SEQ ID NO:175 and the heavy chain variable domain is SEQ ID NO:176.
16. The isolated ICI antibody of any of claims 1-3, wherein the heavy chain variable domain comprises a CDRH1 region consisting of SEQ ID NO:306, a CDRH2 region consisting of SEQ ID NO:307, and a CDRH3 region consisting of SEQ ID NO:308 and the light chain variable domain comprises a CDRL1 region consisting of SEQ ID NO:304, a CDRL2 region consisting of SEQ ID NO:282, and a CDRL3 region consisting of SEQ ID NO:305.
17. The isolated ICI antibody of claim 16, wherein the light chain variable domain is SEQ ID NO:177 and the heavy chain variable domain is SEQ ID NO:178.
18. The isolated ICI antibody of any of claims 1-3, wherein the heavy chain variable domain comprises a CDRH1 region consisting of SEQ ID NO:311, a CDRH2 region consisting of SEQ ID NO:312, and a CDRH3 region consisting of SEQ ID NO:313 and the light chain variable domain comprises a CDRL1 region consisting of SEQ ID NO:309, a CDRL2 region consisting of SEQ ID NO:276, and a CDRL3 region consisting of SEQ ID NO:310.
19. The isolated ICI antibody of claim 18, wherein the light chain variable domain is SEQ ID NO:179 and the heavy chain variable domain is SEQ ID NO:180.
20. The isolated ICI antibody of any of claims 1-3, wherein the heavy chain variable domain comprises a CDRH1 region consisting of SEQ ID NO:317, a CDRH2 region consisting of SEQ ID NO:318, and a CDRH3 region consisting of SEQ ID NO:319 and the light chain variable domain comprises a CDRL1 region consisting of SEQ ID NO:314, a CDRL2 region consisting of SEQ ID NO:315, and a CDRL3 region consisting of SEQ ID NO:316.
21. The isolated ICI antibody of claim 20, wherein the light chain variable domain is SEQ ID NO:181 and the heavy chain variable domain is SEQ ID NO:182.
22. The isolated ICI antibody of any of claims 1-3, wherein the heavy chain variable domain comprises a CDRH1 region consisting of SEQ ID NO:322, a CDRH2 region consisting of SEQ ID NO:323, and a CDRH3 region consisting of SEQ ID NO:324 and the light chain variable domain comprises a CDRL1 region consisting of SEQ ID NO:320, a CDRL2 region consisting of SEQ ID NO:321, and a CDRL3 region consisting of SEQ ID NO:316.
23. The isolated ICI antibody of claim 22, wherein the light chain variable domain is SEQ ID NO:183 and the heavy chain variable domain is SEQ ID NO:184.
24. The isolated ICI antibody of any of claims 1-3, wherein the heavy chain variable domain comprises a CDRH1 region consisting of SEQ ID NO:328, a CDRH2 region consisting of SEQ ID NO:329, and a CDRH3 region consisting of SEQ ID NO:330 and the light chain variable domain comprises a CDRL1 region consisting of SEQ ID NO:325, a CDRL2 region consisting of SEQ ID NO:326, and a CDRL3 region consisting of SEQ ID NO:327.
25. The isolated ICI antibody of claim 24, wherein the light chain variable domain is SEQ ID NO:185 and the heavy chain variable domain is SEQ ID NO:186.
26. The isolated ICI antibody of any of claims 1-3, wherein the heavy chain variable domain comprises a CDRH1 region consisting of SEQ ID NO:334, a CDRH2 region consisting of SEQ ID NO:335, and a CDRH3 region consisting of SEQ ID NO:336 and the light chain variable domain comprises a CDRL1 region consisting of SEQ ID NO:331, a CDRL2 region consisting of SEQ ID NO:332, and a CDRL3 region consisting of SEQ ID NO:333.
27. The isolated ICI antibody of claim 26, wherein the light chain variable domain is SEQ ID NO:187 and the heavy chain variable domain is SEQ ID NO:188.
28. The isolated ICI antibody of any of claims 1-3, wherein the heavy chain variable domain comprises a CDRH1 region consisting of SEQ ID NO:340, a CDRH2 region consisting of SEQ ID NO:341, and a CDRH3 region consisting of SEQ ID NO:342 and the light chain variable domain comprises a CDRL1 region consisting of SEQ ID NO:337, a CDRL2 region consisting of SEQ ID NO:338, and a CDRL3 region consisting of SEQ ID NO:339.
29. The isolated ICI antibody of claim 28, wherein the light chain variable domain is SEQ ID NO:189 and the heavy chain variable domain is SEQ ID NO:190.
30. The isolated ICI antibody of any of claims 1-29, wherein the antibody is a chimeric antibody and the heavy chain constant domain is from rabbit, mouse, rat, or nonhuman primate.
31. The isolated ICI antibody of any of claims 1-30, wherein the light chain constant domain is a kappa light chain constant domain or a lambda light chain constant domain.
32. The isolated ICI antibody of any of claims 1-31, wherein the antibody is biotinylated.
33. A method of diagnosing Kawasaki Disease in a subject, comprising the steps of: i) obtaining a sample from a subject suspected of having Kawasaki Disease; ii) contacting the sample with the antibody of any of claims 1-31; and ii) detecting the binding of the antibody in the sample, whereby binding of the antibody indicates the presence of Kawasaki Disease.
34. The method of claim 33, wherein the sample is a blood sample.
35. The method of claim 33, wherein detecting the binding of the antibody in the sample is carried out using ELISA, Western blot, immunostaining, immunoprecipitation, flow cytometry, sensor chips, or magnetic beads.
36. A method of detecting intracytoplasmic inclusion bodies in a subject, comprising the steps of: i) obtaining a sample from a subject suspected of having Kawasaki Disease; ii) contacting the sample with the antibody of any of claims 1-31; and ii) detecting the binding of the antibody in the sample, whereby binding of the antibody indicates the presence of intracytoplasmic inclusion bodies.
37. The method of claim 36, where in the sample is a blood sample.
38. A method of detecting hepacivirus C in a subject, comprising the steps of: i) obtaining a sample form a subject suspected of having a hepacivirus C infection; ii) contacting the sample with the antibody of any of claims 1-31; and iii) detecting the binding of the antibody in the sample, wherein binding of the antibody indicates the presence of hepacivirus C infection.
Description
BRIEF DESCRIPTION OF DRAWINGS
[0017] The patent or patent application file contains at least one drawing in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
[0018]
[0019]
[0020]
[0021]
[0022]
[0023]
[0024]
[0025]
[0026]
DETAILED DESCRIPTION OF THE INVENTION
[0027] The present disclosure describes monoclonal antibodies that can bind to intracytoplasmic inclusion bodies (ICI) and/or hepacivirus C NS4A. The present disclosure also describes methods of using the disclosed monoclonal antibodies to diagnose and treat Kawasaki Disease (KD) in a subject.
[0028] 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, chimeric antibodies, humanized antibodies, and antibody fragments, including single chain variable fragments (ScFv), single domain antibody, and antigen-binding fragments, genetically engineered antibodies, among others, as long as the characteristic properties (e.g., ability to bind CD30) 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.
[0029] As stated above, the term “antibody” includes “antibody fragments” or “antibody-derived fragments” and “antigen binding fragments” which comprise an antigen binding domain. Furthermore, 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.
[0030] Antibodies can be genetically engineered from the CDRs and monoclonal antibody sequences described herein into antibodies and antibody fragments by using conventional techniques such as, for example, synthesis by recombinant techniques or chemical synthesis. Techniques for producing antibody fragments are well known and described in the art.
[0031] One may wish to engraft one or more CDRs from the monoclonal antibodies described herein into alternate scaffolds. For example, standard molecular biological techniques can be used to transfer the DNA sequences encoding the antibody's CDR(s) to (1) full IgG scaffold of human or other species; (2) a scFv scaffold of human or other species, or (3) other specialty vectors. If the CDR(s) have been transferred to a new scaffold all of the previous modifications described can also be performed. For example, one could consult Biotechnol Genet Eng Rev, 2013, 29:175-86 for a review of useful methods.
[0032] The antibodies or antibody fragments can be wholly or partially synthetically produced. Thus the antibody may be from any appropriate source, for example recombinant sources and/or produced in transgenic animals or transgenic plants. Thus, the antibody molecules can be produced in vitro or in vivo. The antibody or antibody fragment can be made that comprises all or a portion of a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM or IgD constant region.
[0033] Furthermore, the antibody or antibody fragment can 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.
[0034] The term “fragment” as used herein refers to fragments of biological relevance (functional fragment), e.g., fragments which can contribute to or enable antigen binding, e.g., form part or all of the antigen binding site or can contribute to the prevention of the antigen interacting with its natural ligands. Fragments in some embodiments comprise a heavy chain variable region (VH domain) and light chain variable region (VL) of the disclosure. In some embodiments, the fragments 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.
[0035] 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. Sixty CDRs can be found on a pentameric IgM molecule.
[0036] 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.
[0037] As used herein, the terms “proteins” and “polypeptides” are used interchangeably herein to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein” and “polypeptide” 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” are used interchangeably herein when referring to an encoded gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. The antibodies of the present invention are polypeptides, as well the antigen-binding fragments and fragments thereof.
[0038] The terms “monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of a single amino acid composition that specifically binds to a single epitope of the antigen.
[0039] 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.” Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art. In some embodiments, the antibodies are chimeric antibodies including heavy chain constant domains from non-human mammals (e.g., mouse, rat, rabbit, or non-human primate). In some embodiments, the antibodies disclosed in the present invention are chimeric antibodies including constant regions from rabbit heavy chain immunoglobulin sequences. Suitable heavy chain constant region sequences from non-human mammals, including mouse, rat, rabbit, and non-human primate are known in the art.
[0040] The antibodies disclosed in the present invention are human antibodies, as they include the constant region from human germline immunoglobulin sequences. 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, NS0 or CHO cell (like CHO Kl) 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.
[0041] ICI and KD specific monoclonal antibodies described herein include the following (Tables 1A and 1B). The sequences referenced in Table 1B are nucleotide sequences, whereby the nucleotide sequence encodes for the amino acid sequence of the light or heavy chain variable region.
TABLE-US-00001 TABLE 1A Heavy and Light Chain Variable Region nucleotide sequences, including CDR1, CDR2, and CDR3. Nucleotide Sequence Nucleotide Sequence Antibody Light Chain Encoding the Light Heavy Chain Encoding the Heavy name Variable Region Chain Variable Region Variable Region Chain Variable Region KD4-2H4 QSALTQPRSVS CAGTCTGCCCTGACTCAGCCTCGCT EVQLVESGGGLV GAGGTGCAGCTGGTGGAGTCCGG GSPGQSVTISCT CAGTGTCCGGGTCTCCTGGACAGTC QPGGSLRLSCAA GGGAGGCTTAGTTCAGCCTGGGGG GTSSDVGGYNY AGTCACCATCTCCTGCACTGGAACC SGFTFSSYWMYW GTCCCTGAGACTCTCCTGTGCAGC VSWYQQHPGK AGCAGTGATGTTGGTGGTTATAACT VRQAPGKGLVW CTCTGGATTCACCTTCAGTAGCTA APKLMIYDVSK ATGTCTCCTGGTACCAACAGCACCC VSRINSDGSTTNY CTGGATGTACTGGGTCCGCCAAGC RPSGVPDRFSG AGGCAAAGCCCCCAAACTCATGATT ADSVKGRFTISRD TCCAGGGAAGGGGCTGGTGTGGG SKSGNTASLTIS TATGATGTCAGTAAGCGGCCCTCAG NAKNTLYLQMNS TCTCACGTATTAATAGTGATGGGA GLQAEDEADY GGGTCCCTGATCGCTTCTCTGGCTCC LRAEDTAVYFCA GTACCACGAACTACGCGGACTCCG YCCSYAGSYTL AAGTCTGGCAACACGGCCTCCCTGA RYAHLGIGWYF TGAAGGGCCGATTCACCATCTCCA LFGGGTKLTVL CCATCTCTGGGCTCCAGGCTGAGGA DLWGRGTLVTVS GAGACAACGCCAAGAACACGCTG (SEQ ID NO: 165; TGAGGCTGATTATTACTGCTGCTCA S TATCTGCAAATGAACAGTCTGAGA CDR1 SEQ ID TATGCAGGCAGCTACACTTTGTTAT (SEQ ID NO: 166; GCCGAGGACACGGCTGTGTATTTC NO: 269; CDR2 TCGGCGGAGGGACCAAGCTGACCGT CDR1 SEQ ID TGTGCAAGATATGCCCACCTGGGG SEQ ID NO: 270; CCTAG NO: 272; CDR2 ATAGGTTGGTACTTCGATCTCTGG CDR3 SEQ ID (SEQ ID NO: 103) SEQ ID NO: 273; GGCCGTGGCACCCTGGTCACCGTC NO: 271) CDR3 SEQ ID TCCTCAG NO: 274) (SEQ ID NO: 102) KD6-2B2 DIQMTQSPSTLS GACATCCAGATGACCCAGTCTCCTT EVQLVESGGGVA GAGGTGCAGCTGGTGGAGTCTGG ASAGDRVTITC CCACCCTGTCTGCATCTGCAGGAGA QPGTSLRLSCEAS GGGAGGCGTGGCCCAGCCTGGGA RASQSVSKYLA CAGAGTCACCATCACTTGCCGGGCC GFTFRDYAMRW CGTCTCTGAGACTCTCCTGTGAAG WYQQKPGKAP AGTCAGAGTGTTAGTAAGTACTTGG VRQAPGKGLEW CGTCTGGATTCACCTTCCGTGACT RLLIYKASSLQS CCTGGTATCAGCAGAAACCAGGGA VAFIWNDGSKKY ATGCCATGCGCTGGGTCCGCCAGG GVPSRFSGSGS AAGCCCCTAGGCTCCTGATCTATAA YTDSVRGRFTISR CTCCAGGCAAGGGGCTGGAGTGG GTEFTLTISSLQ GGCATCTAGTTTACAAAGTGGGGTC DNSKNMLYLQM GTGGCATTTATATGGAATGATGGA PDDFATYYCQ CCATCAAGGTTCAGTGGCAGTGGAT DSLRAEDTALYY AGTAAGAAATATTACACAGACTCC HYNTYPSWTF CTGGGACAGAATTCACTCTCACCAT CARKMSEDDAF GTGAGGGGCCGATTCACCATCTCC GQGTKVEIK CAGCAGCCTGCAGCCTGATGATTTT DLWGQGTMVTV AGAGACAATTCCAAGAACATGCT (SEQ ID NO: 167; GCTACTTATTACTGCCAACACTATA (SEQ ID NO: 168; GTATCTGCAAATGGACAGCCTGAG CDR1 SEQ ID ATACTTATCCTTCGTGGACGTTCGG CDR1 SEQ ID AGCCGAGGACACGGCTCTTTATTA NO: 275; CDR2 CCAAGGGACCAAGGTGGAAATCAA NO: 278; CDR2 CTGTGCCAGAAAGATGAGTGAAG SEQ ID NO: 276; A SEQ ID NO: 279; ATGATGCTTTTGATCTGTGGGGCC CDR3 SEQ ID (SEQ ID NO: 128) CDR3 SEQ ID AAGGGACAATGGTCACCGTCT NO: 277) NO: 280) (SEQ ID NO: 127) KD8-1D4 DIQMTQSPSSLS GACATCCAGATGACCCAGTCTCCAT EVQLVESGGGLV GAGGTGCAGCTGGTGGAGTCTGG ASVGDRVTITC CCTCCCTGTCTGCATCCGTAGGAGA QPGGSLRLSCAA GGGAGGCTTGGTCCAGCCTGGAG RASQGIRNDLG CAGAGTCACCATCACTTGCCGGGCA SGFTFSDYYMDW GGTCCCTGAGACTCTCCTGTGCAG WYQQKPGKAP AGTCAGGGCATTAGAAATGATTTAG VRQAPGKGLEW CCTCTGGATTCACCTTCAGTGACT KLLIYGASSLQS GCTGGTATCAGCAGAAACCAGGGA VGRTRNKANSYTT ACTACATGGACTGGGTCCGCCAGG GVPSRFSGSGS AAGCCCCTAAGCTCCTGATCTATGG EYAASVKGRFTIS CTCCAGGGAAGGGGCTGGAGTGG GTDFTLTISSLQ TGCATCCAGTTTACAAAGTGGGGTC RDDSKNSLYLQM GTTGGCCGCACTAGAAACAAAGCT PEDIATYYCLQ CCATCAAGATTCAGCGGCAGTGGAT NSLKTEDTAVYY AACAGTTACACCACAGAATACGCC EYTYPLTFGGG CTGGCACAGATTTCACTCTCACCAT CARFLLVADAFD GCGTCTGTGAAAGGCAGATTCACC TKVEIK CAGCAGCCTGCAGCCTGAAGATATT IWGQGTMVTV ATCTCAAGAGATGATTCAAAGAAC (SEQ ID NO: 169; GCAACCTATTACTGTCTACAAGAAT (SEQ ID NO: 170; TCACTGTATCTGCAAATGAACAGC CDR1 SEQ ID ACACTTACCCGCTCACTTTCGGCGG CDR1 SEQ ID CTGAAAACCGAGGACACGGCCGT NO: 281; CDR2 AGGGACCAAGGTGGAAATCAAA NO: 284; CDR2 GTATTACTGTGCTAGATTTCTGCT SEQ ID NO: 282; (SEQ ID NO: 150) SEQ ID NO: 285; GGTTGCGGATGCTTTTGATATCTG CDR3 SEQ ID CDR3 SEQ ID GGGCCAAGGGACAATGGTCACCG NO: 283) NO: 286) TCT (SEQ ID NO: 149) KD6-1A2 QTVVTQEPSLT CAGACTGTGGTGACTCAGGAGCCCT EVQLVQSGAEVK GAGGTGCAGCTGGTGCAGTCTGGG VSPGETVTLTC CACTGACTGTGTCCCCAGGAGAGAC KTGSSVRLSCKTS GCTGAGGTGAAGAAGACTGGGTC GSNSGAVTSGH AGTCACTCTCACCTGTGGCTCCAAC GYTFTYRYLHWV CTCAGTGAGGCTTTCCTGCAAGAC HPHWFQQKPG AGTGGAGCTGTCACCAGTGGTCATC RQAPGHALEWLG CTCCGGATACACCTTCACATACCG QAPRTLIYDTTN ATCCCCACTGGTTCCAGCAGAAGCC WITIYNGNTKYAQ CTACCTGCACTGGGTGCGACAGGC KVSWTPARFSA TGGCCAAGCACCCAGGACTCTCATT EFQDRVTITREMS CCCCGGACACGCGCTTGAGTGGCT SLLGGKAALTL TATGACACAACCAACAAGGTCTCCT LTAVYMELRSLR GGGGTGGATTACGATTTATAATGG SGAQPEDEADY GGACACCTGCCCGCTTCTCAGCCTC SEDTAMYYCARS CAACACCAAGTATGCACAGGAATT YCLVSYSGGR CCTCCTTGGGGGCAAAGCTGCCCTG ALSGDNAFEFW CCAGGACAGAGTCACCATTACCAG MVFGGGTKLT ACCCTTTCGGGTGCGCAGCCTGAGG AQGTMVTV GGAAATGTCCTTGACCGCAGTATA VL ATGAGGCTGACTACTATTGCTTGGT (SEQ ID NO: 172; CATGGAGTTGAGGAGCCTAAGATC (SEQ ID NO: 171; CTCCTATAGTGGTGGTCGTATGGTC CDR1 SEQ ID TGAGGACACAGCCATGTATTACTG CDR1 SEQ ID TTCGGCGGAGGGACCAAGCTGACCG NO: 290; CDR2 TGCAAGGTCCGCGTTGTCTGGCGA NO: 287; CDR2 TCCTAC SEQ ID NO: 291; TAATGCCTTTGAATTCTGGGCCCA SEQ ID NO: 288; (SEQ ID NO: 122) CDR3 SEQ ID AGGGACAATGGTCACCGTCT CDR3 SEQ ID NO: 292) (SEQ ID NO: 121) NO: 289) KD8-1B10 DIQMTQSPSSLS GACATCCAGATGACCCAGTCTCCAT EVQLVESGGGLV GAGGTGCAGCTGGTGGAGTCTGG ASLGDRVTITC CCTCCCTGTCTGCATCTTTAGGAGA QPGGSLRLSCAA GGGAGGCTTGGTCCAGCCTGGAG RASQGIRHDLG CAGAGTCACCATCACTTGCCGGGCA SGFTFSDHYMDW GGTCCCTGAGACTCTCCTGTGCAG WYQQKPGKAP AGTCAGGGCATTAGACATGATTTAG VRQVPRKGLEW CCTCTGGATTCACCTTCAGTGACC KLLIYGASILQT GCTGGTATCAGCAGAAACCAGGGA VGRARNKANSYTT ACTATATGGACTGGGTCCGCCAGG GVPSRFSGSGS AAGCCCCTAAGCTCCTCATCTATGG EYAASVKGRFTIS TTCCAAGGAAGGGGCTGGAGTGG GTDFTLTISSLQ TGCATCCACTTTACAAACTGGGGTC RDDSKNSLYLQM GTTGGCCGTGCTAGAAACAAAGCT PEDFATYYCLQ CCATCAAGGTTCAGCGGCAGTGGGT NSLKSEDTAVYY AACAGTTACACCACAGAATACGCC KYNYPLTFGG CTGGCACAGATTTCACTCTCACCAT CARFLLIADAFDI GCGTCTGTGAAAGGCAGATTCACC GTKVEIK CAGCAGCCTGCAGCCTGAAGATTTT WGQGTMVTV ATCTCAAGAGATGATTCAAAGAAC (SEQ ID NO: 173; GCAACTTATTACTGTCTACAAAAGT (SEQ ID NO: 174; TCACTGTATCTACAAATGAACAGC CDR1 SEQ ID ACAACTACCCGCTCACTTTCGGCGG CDR1 SEQ ID CTGAAAAGCGAGGACACGGCCGT NO: 293; CDR2 AGGGACCAAGGTGGAGATCAAA NO: 295; CDR2 GTATTATTGTGCTAGATTTCTGCTT SEQ ID NO: 282; (SEQ ID NO: 148) SEQ ID NO: 296; ATTGCGGATGCTTTTGATATCTGG CDR3 SEQ ID CDR3 SEQ ID GGCCAAGGGACAATGGTCACCGT NO: 294) NO: 297) CT (SEQ ID NO: 147) KD6-1A10 QSVLTQPPSAS CAGTCTGTGCTGACGCAGCCACCCT VQLVESGGGVVQ GTGCAGCTGGTGGAGTCGGGGGG GTPGQGVTISCS CAGCGTCTGGGACCCCCGGGCAGGG PGRSLRLSCAASG AGGCGTGGTCCAGCCTGGGAGGTC GSRSNIGSNTV CGTCACCATCTCTTGTTCTGGAAGC FSFSNYFMHWVR CCTGAGACTCTCCTGTGCAGCCTC NWYRQFPGTAP AGGTCCAACATCGGAAGTAATACTG HVPGKGLEWLAT TGGATTCAGCTTCAGTAACTATTT KLLIYKNNQRPS TTAACTGGTACCGGCAGTTCCCAGG IWYDGSDKYYVD CATGCACTGGGTCCGCCATGTTCC GVPDRFSGSKP AACGGCCCCCAAACTCCTCATCTAT SVKGRFTISRDNS AGGCAAGGGGCTGGAGTGGCTGG GTSAYLAITGL AAGAACAATCAGCGGCCCTCAGGG KNTLYLQMGSLR CAACCATCTGGTATGATGGAAGTG QSDDQADYYC GTCCCTGACCGATTCTCTGGCTCCA AEDTAMYYCAK ATAAATACTATGTAGACTCCGTGA AAWDDSLKGV AGCCTGGCACCTCAGCCTACCTGGC GRQRGYYYDM AGGGCCGATTCACCATCTCCAGAG VFGGGTKLTVL CATCACTGGGCTCCAGTCTGACGAT GGYYVFDSWGQ ACAACTCCAAGAACACGCTCTATC (SEQ ID NO: 175; CAGGCTGATTATTACTGTGCAGCAT GTLVTVSS TACAAATGGGCAGCCTGAGAGCT CDR1 SEQ ID GGGATGACAGCCTGAAAGGTGTGGT (SEQ ID NO: 176; GAGGACACGGCTATGTATTACTGT NO: 298; CDR2 GTTCGGCGGAGGGACCAAGCTGACC CDR1 SEQ ID GCGAAAGGGAGACAACGAGGATA SEQ ID NO: 299; GTCCTAA NO: 301; CDR2 TTACTACGATATGGGTGGTTATTA CDR3 SEQ ID (SEQ ID NO: 120) SEQ ID NO: 302; TGTTTTTGACTCCTGGGGCCAGGG NO: 300) CDR3 SEQ ID AACCCTGGTCACCGTCTCCTCAG NO: 303) (SEQ ID NO: 119) KD1-2G11 DIQMTQSPSSLS GACATCCAGATGACCCAGTCTCCAT EVQLVESGGGLV GAGGTGCAGCTGGTGGAGTCTGG ASVGDRVTITC CCTCCCTGTCTGCATCTGTGGGGGA KPGGSLRLSCAA GGGAGGCTTGGTAAAGCCTGGGG RASQSVSRYLN CAGAGTCACCATCACTTGCCGGGCA SGFTFSSAWLSW GGTCCCTTAGACTCTCCTGTGCAG WYQQKPGRAP AGTCAGAGCGTTAGCAGGTATCTAA VRQDPGKGLEWL CCTCTGGATTCACTTTCAGTAGCG RLLIFGASSLRS ATTGGTATCAGCAGAAACCAGGGA GRIKSKAAGGTAD CCTGGTTGAGCTGGGTCCGTCAGG GVPSRFSGSGS GAGCCCCTAGGCTCCTGATCTTTGG HAASVKGRFTISR ATCCAGGGAAGGGGCTGGAGTGG GTDFSLTINSLQ TGCATCCAGTTTACGTAGTGGAGTC DDSKNTLYLQMN CTTGGCCGTATTAAAAGTAAAGCT PEDFATYYCQQ CCATCCAGGTTCAGTGGCAGTGGAT SLKIEDTAVYYC GCTGGTGGGACAGCAGACCACGC TYSTFGPFGPG CTGGGACAGATTTCTCTCTCACCAT TTGSLEGTITHA TGCATCCGTGAAAGGCAGATTCAC TKVDIK CAACAGTCTGCAACCTGAAGATTTT EDHWGQGTLVT CATCTCAAGAGATGATTCAAAAAA (SEQ ID NO: 177; GCAACTTACTACTGTCAACAGACTT VSS CACGTTATATCTGCAAATGAACAG CDR1 SEQ ID ACAGTACTTTCGGACCTTTCGGCCC (SEQ ID NO: 178; CCTGAAAATCGAGGACACAGCCG NO: 304; CDR2 TGGGACCAAAGTGGATATCAAA CDR1 SEQ ID TCTATTACTGTACCACAGGGTCAT SEQ ID NO: 282; (SEQ ID NO: 34) NO: 306; CDR2 TGGAGGGTACAATTACACACGCG CDR3 SEQ ID SEQ ID NO: 307; GAGGACCACTGGGGCCAGGGAAC NO: 305) CDR3 SEQ ID CCTGGTCACCGTCTCCTCA NO: 308) (SEQ ID NO: 33) KD7-1D3 DIQMTQSPSTLS GACATCCAGATGACCCAGTCTCCTT EVQLLESGGGLV GAGGTGCAGCTGTTGGAGTCTGGG ASVGERVTITC CCACCCTGTCTGCATCTGTAGGAGA QPAGSLRLACAA GGAGGCCTGGTACAGCCGGCGGG RASQSISSWLA GAGAGTCACCATCACTTGCCGGGCC SGFTFSNYAMTW GTCCCTGAGACTCGCCTGTGCAGC WYQQKPGKAP AGTCAGAGTATTAGTAGCTGGTTGG IRQAPGKGLEWV CTCTGGATTCACCTTTAGCAACTA NLLIYKASSLES CCTGGTATCAGCAGAAACCAGGGA STISSGGYNTNYA TGCCATGACTTGGATCCGCCAGGC GVPSRFSGSGS AAGCCCCTAACCTCCTGATCTATAA DSVKGRFTISRDN TCCAGGGAAGGGGCTGGAGTGGG GTEFTLTISGLQ GGCGTCTAGTTTAGAAAGTGGGGTC SKNTVYLQMSSL TCTCAACTATTAGTAGTGGTGGTT PDDFATYYCQ CCATCAAGGTTCAGCGGCAGTGGAT RVEDTAVYYCVT ATAATACAAACTACGCAGACTCCG QYNSYSLTFGQ CTGGGACAGAATTCACTCTCACCAT RRAGSYYAYWG TGAAGGGCCGGTTCACCATTTCTA GTKVEIK CAGCGGCCTGCAGCCTGATGATTTT QGTLVTV GAGACAATTCCAAGAACACGGTG (SEQ ID NO: 179; GCAACTTATTACTGCCAACAGTATA (SEQ ID NO: 180; TATCTGCAAATGAGCAGCCTGAGA CDR1 SEQ ID ATAGTTATTCATTGACGTTCGGCCA CDR1 SEQ ID GTCGAGGACACGGCCGTATATTAC NO: 309; CDR2 AGGGACCAAGGTGGAAATCAAA NO: 311; CDR2 TGTGTGACTCGGAGAGCTGGGAGC SEQ ID NO: 276; (SEQ ID NO: 138) SEQ ID NO: 312; TACTATGCCTACTGGGGCCAGGGA CDR3 SEQ ID CDR3 SEQ ID ACCCTGGTCACCGTCT NO: 310) NO: 313) (SEQ ID NO: 137) KD11-1C2 DIVMTQSPLSLP GATATTGTGATGACTCAGTCTCCAC QVQLVQSGAEVK CAGGTGCAGCTGGTGCAGTCTGGG VTLGQPASISCR TCTCCCTGCCCGTCACCCTTGGACA KPGAPVKVSCKA GCTGAGGTGAAGAAGCCTGGGGC SSQSLAYSDGN GCCGGCCTCCATCTCCTGCAGGTCT SGYTFTNYYMH CCCAGTGAAGGTTTCCTGCAAGGC TYLNWFHQRP AGCCAAAGCCTCGCATACAGTGATG WVRQAPGQGLE ATCTGGATACACCTTCACCAACTA GHSPRRLIYKVF GAAACACCTACCTGAATTGGTTTCA WLGLINAGSGRTR CTATATGCACTGGGTGCGACAGGC NRDSGVPDRFS CCAGAGGCCAGGCCACTCTCCAAGG YAPKFQGRVTMT CCCTGGACAAGGACTTGAGTGGCT GSGSGTDFTLKI CGCCTAATTTATAAGGTTTTTAATCG RDTSTSTLYIEVD GGGACTAATCAACGCTGGTTCTGG SRVEAEDVGVY GGACTCTGGGGTCCCAGACAGATTC SLRSDDTAVYYC TAGGACAAGGTACGCACCGAAGT YCMQGTHWPI AGCGGCAGTGGGTCAGGCACTGATT ARGSAHIEAGGS TCCAGGGCAGAGTCACCATGACCA TFGQGTRLEIK TCACACTGAAAATCAGCAGGGTGGA AFDKWGQGTLV GGGACACGTCCACGAGCACACTCT (SEQ ID NO: 181; GGCTGAGGATGTTGGGGTTTATTAC TV ACATAGAGGTGGACAGCCTGAGA CDR1 SEQ ID TGCATGCAAGGTACACACTGGCCGA (SEQ ID NO: 182; TCCGACGACACGGCCGTTTATTAC NO: 314; CDR2 TCACCTTCGGCCAAGGGACACGACT CDR1 SEQ ID TGTGCGAGAGGATCCGCCCATATA SEQ ID NO: 315; GGAGATTAAAC NO: 317; CDR2 GAAGCGGGTGGTTCCGCCTTTGAC CDR3 SEQ ID (SEQ ID NO: 63) SEQ ID NO: 318; AAGTGGGGCCAGGGAACCCTGGT NO: 316) CDR3 SEQ ID CACCGTCTC NO: 319) (SEQ ID NO: 62) KD11-1E9 DIVMTQSPLSLP GATATTGTGATGACTCAGTCTCCAC QVQLVQSGAEVK CAGGTGCAGCTGGTGCAGTCTGGG VILGQSASISCR TGTCCCTGCCCGTCATCCTTGGACA KPGAPVKVSCQA GCTGAGGTGAAGAAGCCTGGGGC SSQSLIHSDGNI GTCGGCCTCCATCTCCTGCAGGTCT SGFIFINYYFHWV CCCAGTGAAGGTTTCCTGCCAGGC FLNWFQQRPGQ AGTCAAAGCCTCATACACAGTGATG RQAPGQGLEWM ATCTGGATTCATTTTCATCAATTAC SPRRLIYKVSNR GGAACATCTTCTTGAATTGGTTTCA GVINPTSGKSKLA TATTTCCACTGGGTGCGGCAGGCC DSGVPDRFSGS GCAGAGGCCAGGCCAATCTCCAAG PTLENRVTMTSD CCTGGACAAGGCCTGGAGTGGAT GSGTDFTLKISR GCGCCTAATTTATAAGGTTTCTAAC TSTRTLYIEVTSL GGGAGTAATAAACCCCACTAGTG VEAEDVGVYY CGGGACTCTGGGGTCCCAGACAGAT TSDDTAIYYCAR GTAAGTCAAAACTCGCGCCGACGT CMQGTHWPIT TCAGCGGCAGTGGGTCAGGCACTGA GSARAEAAGSAF TGGAGAATCGAGTCACCATGACCA FGQGTRLEIK TTTCACACTGAAAATCAGCAGGGTG DHWGQGTLVTV GCGACACGTCCACGAGAACACTTT (SEQ ID NO: 183; GAGGCTGAGGATGTTGGCGTTTATT (SEQ ID NO: 184; ACATAGAGGTGACCAGCCTGACAT CDR1 SEQ ID ACTGCATGCAAGGTACACACTGGCC CDR1 SEQ ID CTGACGACACGGCCATATATTACT NO: 320; CDR2 GATCACCTTCGGCCAAGGGACACGA NO: 322; CDR2 GTGCGAGAGGGTCCGCCCGCGCA SEQ ID NO: 321; CTGGAGATTAAAC SEQ ID NO: 323; GAAGCGGCTGGTTCCGCCTTTGAC CDR3 SEQ ID (SEQ ID NO: 67) CDR3 SEQ ID CACTGGGGCCAGGGAACCCTGGTC NO: 316) NO: 324) ACCGTCTC (SEQ ID NO: 66) KD11-2E10 DIQMTQSPSSLS GACATCCAGATGACCCAGTCTCCAT QVQLQESGPGLV CAGGTGCAGCTGCAGGAGTCGGG ASVGDRVTITC CCTCCCTGTCTGCATCTGTAGGAGA KPSETLSLTCTVS CCCAGGACTGGTGAAGCCTTCGGA QASLDIKNYLN CAGAGTCACCATCACTTGCCAGGCG GASMNGYYWN GACCCTGTCCCTCACCTGCACTGT WYQQKPGEAP AGTCTGGACATTAAAAACTATCTAA WIRQPPGKGLEW CTCTGGTGCCTCCATGAATGGTTA KLLISDASTLET ATTGGTATCAGCAGAAACCAGGGG IGHIHYVGTTMKA CTACTGGAACTGGATCCGGCAGCC GVPSRFSGRGS AAGCCCCCAAGCTCCTGATCTC TNDSPSLRSRVTI CGACCCAGGGAAGGGACTGGAGTGGA GTHFTVTIRSLQ TGCATCCACCTTGGAAACCGGGGTC SVDTSRSQISLKL TTGGACATATCCATTACGTTGGGA SEDVATYYCQ CCATCAAGGTTCAGTGGAAGGGGGT TSVTAADTAVYY CTACCATGAAGGCCACCAACGAC QYGKLPFTFGP CTGGGACACATTTCACTGTCACCAT CARRRYHFWSD AGCCCCTCCCTCAGGAGTCGAGTC GTKVDIK CCGCAGCCTGCAGTCTGAAGATGTT PGTFDIWGQGT ACCATATCAGTGGACACGTCCAGG (SEQ ID NO: 185; GCAACATATTACTGTCAACAATATG MVTV AGCCAGATCTCCCTGAAGCTGACG CDR1 SEQ ID GTAAACTCCCATTCACTTTCGGCCCT (SEQ ID NO: 186; TCTGTGACCGCCGCGGACACGGCC NO: 325; CDR2 GGGACCAAAGTGGATATCAAAC CDR1 SEQ ID GTGTATTACTGTGCGAGAAGGCGG SEQ ID NO: 326; (SEQ ID NO: 77) NO: 328; CDR2 TACCATTTTTGGAGTGACCCCGGA CDR3 SEQ ID SEQ ID NO: 329; ACGTTTGATATCTGGGGCCAAGGG NO: 327) CDR3 SEQ ID ACAATGGTCACCGTCTC NO: 330) (SEQ ID NO: 76) KD7-2C1 QLVLTQPSSLS CAGCTTGTGCTGACTCAGCCGTCTT EVQLLESGGGLV GAGGTGCAGCTGTTGGAGTCTGGG ASPGASASLTC CCCTGTCTGCATCTCCTGGAGCATC QPGGSLRLSCAA GGAGGCTTGGTACAGCCTGGGGG TLRSGINVGIYS AGCCAGTCTCACCTGCACCTTACGC SGFSFSTYAMSW GTCCCTGAGACTCTCCTGTGCAGC IYWYQQKPGSP AGTGGCATCAATGTTGGTATTTATA VRQAPGKGLQW CTCTGGATTCAGCTTTAGCACTTA PQYLLRFKSDS GCATTTATTGGTACCAGCAGAAGCC VSSISGNGYSSYY TGCCATGAGCTGGGTCCGCCAGGC DKRQGSGVPSR AGGGAGTCCTCCCCAGTATCTCCTG ADSVKGRFHISR TCCAGGAAAGGGGCTGCAGTGGG FSGSKDGSANA AGGTTCAAATCAGACTCAGATAAAC DNSKNTLSLEVN TCTCAAGCATTAGTGGGAATGGTT AILVISGVQSED GTCAGGGCTCTGGAGTCCCCAGCCG SLGAEDTAVYFC ACAGCTCATATTATGCAGACTCCG EADYYCMMW CTTCTCTGGATCCAAAGATGGTTCG AKGDGGSGWFG TGAAGGGCCGCTTCCACATCTCCA HSSAWVFGGG GCCAATGCAGCGATTTTAGTCATCT FDYWGQGTLVT GAGACAATTCCAAGAACACGCTGT TKLTVL CTGGGGTCCAGTCTGAGGATGAGGC V CTCTGGAAGTGAACAGTCTGGGCG (SEQ ID NO: 187; TGACTACTACTGTATGATGTGGCAC (SEQ ID NO: 188; CCGAGGACACGGCCGTCTATTTCT CDR1 SEQ ID AGCAGCGCTTGGGTGTTCGGCGGAG CDR1 SEQ ID GTGCGAAAGGTGACGGTGGCAGT NO: 331; CDR2 GGACCAAGCTGACCGTCCTAC NO: 334; CDR2 GGCTGGTTTGGTTTTGACTATTGG SEQ ID NO: 332; (SEQ ID NO: 144) SEQ ID NO: 335; GGCCAGGGAACCCTGGTCACCGTC CDR3 SEQ ID CDR3 SEQ ID T NO: 333) NO: 336) (SEQ ID NO: 143) KD9-1E10 QSVLTQPPSAS CAGTCTGTGCTGACGCAGCCACCCT EVQLVQSGAEVK GAGGTGCAGCTGGTGCAGTCTGGG GTPGQRVIISCS CAGCGTCTGGGACCCCCGGGCAGAG RPGASMKLSCKT GCTGAGGTGAAACGGCCTGGGGC GGNSNIGSYTV GGTCATTATTTCTTGTTCTGGGGGG FGYIFTTYYIHWV CTCAATGAAGCTTTCTTGCAAGAC QWYHQVPGTA AACTCCAACATCGGAAGTTATACTG RQAPGQGLEYLG ATTTGGATACATCTTCACCACCTA PKLLIYRSNQRP TCCAGTGGTACCATCAAGTCCCAGG IINPAVGTTNFAQ CTACATACACTGGGTGCGGCAGGC SGVPDRFSGSK AACGGCCCCCAAACTCCTCATCTAT KFKGRLSMTRDT CCCTGGACAAGGTCTTGAATACTT SGTSASLAISGL AGGAGTAATCAGCGGCCCTCAGGG STSTVYMELSSPT GGGAATAATCAACCCTGCTGTTGG QSEDEADYYCS GTCCCTGACCGATTCTCTGGCTCCA SDDSAVYYCARE TACCACAAACTTCGCACAGAAGTT AWDDSLNAYV AGTCTGGCACCTCAGCCTCCCTGGC LHATHFFDFWG CAAGGGCAGGCTCAGCATGACCA FGTGTRVAVL CATCAGTGGGCTCCAGTCTGAAGAT QGTLVTV GGGACACGTCCACGAGCACAGTCT (SEQ ID NO: 189; GAGGCTGATTATTACTGTTCAGCAT (SEQ ID NO: 190; ACATGGAACTGAGCAGCCCGACA CDR1 SEQ ID GGGATGACAGCCTGAATGCTTATGT CDR1 SEQ ID TCTGACGACTCGGCCGTCTATTAC NO: 337; CDR2 CTTCGGAACTGGGACCAGGGTCGCC NO: 340; CDR2 TGTGCGAGGGAACTACACGCTACA SEQ ID NO: 338; GTCCTAG SEQ ID NO: 341; CACTTCTTCGACTTCTGGGGCCAG CDR3 SEQ ID (SEQ ID NO: 160) CDR3 SEQ ID GGAACCCTGGTCACCGTCT NO: 339) NO :342) (SEQ ID NO: 159)
TABLE-US-00002 TABLE 1B Monoclonal Antibody Nucleotide Sequences. Sequence names ending in “L” of “K” indicate a nucleotide sequence that encodes a light chain variable domain and the remainder of the sequence names indicate a nucleotide sequence that encodes a heavy chain variable domain. KD Patient# Sequence Name (SEQ ID NO:) KD1 1A4 1 KD1 1A5 2 KD1 1A7 3 KD1 1A9 4 KD1 1B2 5 KD1 1B8 6 KD1 1C4 7 KD1 1C5 8 KD1 1C6 9 KD1 1C6K 10 KD1 1F1 11 KD1 1F1K 12 KD1 1F3 13 KD1 1F3K 14 KD1 1F6H 15 KD1 1F6L 16 KD1 1F7 17 KD1 1G8 18 KD1 1H10 19 KD1 1H12 20 KD1 2A10 21 KD1 2A3 22 KD1 2A5 23 KD1 2A5L 24 KD1 2A8 25 KD1 2A8K 26 KD1 2B11 27 KD1 2B9 28 KD1 2C2 29 KD1 2C2K 30 KD1 2F3 31 KD1 2G10 32 KD1 2G11 33 KD1 2G11K 34 KD1 2G6 35 KD1 2H10 36 KD1 2H10K 37 KD10 1A10 38 KD10 1A10K 39 KD10 1B1 40 KD10 1B1K 41 KD10 1B8 42 KD10 1B8K 43 KD10 1E9 44 KD10 1E9L 45 KD10 1F11 46 KD10 1F11L 47 KD10 1G12 48 KD10 1G12L 49 KD10 2A1 50 KD10 2A1L 51 KD10 2E5 52 KD10 2G12 53 KD11 1A12 54 KD11 1A12L 55 KD11 1B12 56 KD11 1B12K 57 KD11 1B7 58 KD11 1B7L 59 KD11 1C1 60 KD11 1C1K 61 KD11 1C2 62 KD11 1C2K 63 KD11 1D6 64 KD11 1D6L 65 KD11 1E9 66 KD11 1E9K 67 KD11 1F6 68 KD11 1F6K 69 KD11 2A12 70 KD11 2A12K 71 KD11 2D10 72 KD11 2D10K 73 KD11 2D5 74 KD11 2D5K 75 KD11 2E10 76 KD11 2E10K 77 KD11 2E7 78 KD11 2E7K 79 KD11 2F12 80 KD11 2F12K 81 KD11 2G1 82 KD11 2G1K 83 KD11 2H6 84 KD11 2H6L 85 KD2 1A7 86 KD2 1A7L 87 KD2 2B6 88 KD2 2B6L 89 KD2 2C7 90 KD2 2C7L 91 KD2 2F8 92 KD2 2F8L 93 KD3 1G8 94 KD3 1G8L 95 KD3 1G9 96 KD3 1G9L 97 KD4 1F5 98 KD4 1F5L 99 KD4 2C5 100 KD4 2C5K 101 KD4 2H4 102 KD4 2H4L 103 KD5 1C10 104 KD5 1C10K 105 KD5 1E2 106 KD5 1E2K 107 KD5 1E2L 108 KD5 2C1 109 KD5 2C10 110 KD5 2C10L 111 KD5 2C1K 112 KD5 2C6 113 KD5 2C6K 114 KD5 2D3 115 KD5 2D3K 116 KD5 2F6 117 KD5 2F6K 118 KD6 1A10 119 KD6 1A10L 120 KD6 1A2 121 KD6 1A2L 122 KD6 1D1 123 KD6 1D1L 124 KD6 2A12 125 KD6 2A12L 126 KD6 2B2 127 KD6 2B2K 128 KD6 2F4 129 KD6 2F4L 130 KD6 2G3 131 KD6 2G3K 132 KD6 2H3 133 KD6 2H3K 134 KD7 1A12 135 KD7 1A12K 136 KD7 1D3 137 KD7 1D3K 138 KD7 1D9 139 KD7 1D9L 140 KD7 2A9 141 KD7 2A9K 142 KD7 2C1 143 KD7 2C1L 144 KD7 2F9 145 KD7 2F9K 146 KD8 1B10 147 KD8 1B10K 148 KD8 1D4 149 KD8 1D4K 150 KD8 2F3 151 KD8 2F3L 152 KD9 1A10 153 KD9 1A10K 154 KD9 1B5 155 KD9 1B5L 156 KD9 1D2 157 KD9 1D2L 158 KD9 1E10 159 KD9 1E10L 160 KD9 2C5 161 KD9 2C5L 162 KD9 2C8 163 KD9 2C8L 164
TABLE-US-00003 TABLE 1C Strong Binding Monoclonal Antibodies: KDPt # Sequence Name SEQ ID NO: KD1 2G11 33 KD1 2G11K 34 KD11 1C2 62 KD11 1C2K 63 KD11 1E9 66 KD11 1E9K 67 KD11 2E10 76 KD11 2E10K 77 KD4 2H4 102 KD4 2H4L 103 KD6 1A10 119 KD6 1A10L 120 KD6 2B2 127 KD6 2B2K 128 KD7 1D3 137 KD7 1D3K 138 KD7 2C1 143 KD7 2C1L 144 KD9 1E10 159 KD9 1E10L 160
TABLE-US-00004 TABLE 1D Weak Binding Monoclonal Antibodies: KDPt # Sequence Name SEQ ID NO: KD1 2H10 36 KD1 2H10K 37 KD10 1A10 38 KD10 1A10K 39 KD10 2A1 50 KD10 2A1L 51 KD11 1B12 56 KD11 1B12K 57 KD11 1C1 60 KD11 1C1K 61 KD11 1D6 64 KD11 1D6L 65 KD11 1F6 68 KD11 1F6K 69 KD11 2E7 78 KD11 2E7K 79 KD11 2F12 80 KD11 2F12K 81 KD11 2G1 82 KD11 2G1K 83 KD11 2H6 84 KD11 2H6L 85 KD5 2C1 109 KD5 2C1K 112 KD6 1A2 121 KD6 1A2L 122 KD6 2A12 125 KD6 2A12L 126 KD6 2H3 133 KD6 2H3K 134 KD7 2A9 141 KD7 2A9K 142 KD8 1B10 147 KD8 1B10K 148 KD8 1D4 149 KD8 1D4K 150 KD9 1A10 153 KD9 1A10K 154 KD9 1B5 155 KD9 1B5L 156 KD9 1D2 157 KD9 1D2L 158 KD9 2C5 161 KD9 2C5L 162
[0042] In some aspects, a monoclonal antibody described herein includes a light chain variable region selected from the group consisting of SEQ ID NOs:165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, and 189, or a sequence substantially identical thereto, and a heavy chain comprising a rabbit heavy chain constant domain and a heavy chain variable region selected from the group consisting of SEQ ID NOs:166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, and 190, or a sequence substantially identical thereto.
[0043] In some aspects, an antibody as described herein includes the light chain variable region of SEQ ID NO:165 and the heavy chain variable region of SEQ ID NO:166, the light chain variable region of SEQ ID NO:167 and the heavy chain variable region of SEQ ID NO:168, the light chain variable region of SEQ ID NO:169 and the heavy chain variable region of SEQ ID NO:170, the light chain variable region of SEQ ID NO:171 and the heavy chain variable region of SEQ ID NO:172, the light chain variable region of SEQ ID NO: 173 and the heavy chain variable region of SEQ ID NO: 174, the light chain variable region of SEQ ID NO: 175 and the heavy chain variable region of SEQ ID NO: 176, the light chain variable region of SEQ ID NO:177 and the heavy chain variable region of SEQ ID NO: 178, the light chain variable region of SEQ ID NO:179 and the heavy chain variable region of SEQ ID NO:180, the light chain variable region of SEQ ID NO:181 and the heavy chain variable region of SEQ ID NO:182, the light chain variable region of SEQ ID NO:183 and the heavy chain variable region of SEQ ID NO:184, the light chain variable region of SEQ ID NO:185 and the heavy chain variable region of SEQ ID NO:186, the light chain variable region of SEQ ID NO:187 and the heavy chain variable region of SEQ ID NO:188, or the light chain variable region of SEQ ID NO:189 and the heavy chain variable region of SEQ ID NO: 190.
[0044] In some aspects, provided herein is a nucleic acid or polynucleotide encoding an antibody described herein.
[0045] Protein and nucleic acid sequence identities are evaluated using the Basic Local Alignment Search Tool (“BLAST”) which is well known in the art (Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268; Altschul et al., 1997, Nucl. Acids Res. 25: 3389-3402). The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as “high-scoring segment pairs,” between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula (Karlin and Altschul, 1990), the disclosure of which is incorporated by reference in its entirety. The BLAST programs can be used with the default parameters or with modified parameters provided by the user.
[0046] “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
[0047] The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 85% sequence identity to the SEQ ID. Alternatively, percent identity can be any integer from 85% to 100%. More preferred embodiments include at least: 85%, 86%, 87% 88%, 89%, 90%, 91% 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described. These values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.
[0048] “Substantial identity” of amino acid sequences for purposes of this invention normally means polypeptide sequence identity of at least 85%. Preferred percent identity of polypeptides can be any integer from 85% to 100%. More preferred embodiments include at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
[0049] In some embodiments, the isolated antibody or fragment thereof is directly or indirectly linked to a tag or agent. In some embodiments, the antibody or fragment thereof is conjugated to the tag or agent. In other embodiments, the tag or agent is a polypeptide, wherein the polypeptide is translated concurrently with the antibody polypeptide sequence.
[0050] The term “tag” or “agent” as used herein includes any useful moiety that allows for the purification, identification, detection, diagnosing, imaging, or therapeutic use of the antibody of the present invention. The terms tag or agent includes epitope tags, detection markers and/or imaging moieties, including, for example, enzymatic markers, fluorescence markers, radioactive markers, among others. Additionally, the term tag or agent includes therapeutic agents, small molecules, and drugs, among others. The term tag or agent also includes diagnostic agents. In some embodiments, the tag is a biotinylated tag.
Methods of Detection, Imaging and Diagnosing
[0051] The antibodies disclosed herein can be used for methods of assaying, detecting, imaging, and diagnosing Kawasaki Disease (KD) or the presence of intracytoplasmic inclusion bodies (ICI).
[0052] One embodiment provides a method of detecting ICI in a sample, wherein the method comprises contacting the sample with an antibody described herein, and detecting the binding of the antibody in the sample. An increase in binding of the antibody to the sample as compared to binding of the antibody to a negative control sample detects ICI within the sample.
[0053] The term “contacting” or “exposing,” as used herein refers to bringing a disclosed antibody and a cell, a target receptor, a biological sample, or other biological entity, together in such a manner that the antibody can detect and/or affect the activity of the target, either directly; i.e., by interacting with the target itself, or indirectly; i.e., by interacting with another molecule, co-factor, factor, or protein that is attached to said target.
[0054] Another embodiment provides a method of diagnosing KD in a subject by contacting a sample from the subject with the antibody disclosed herein and detecting the binding of the antibody to the sample. In some embodiments, the sample from the subject is compared to a control sample. An increased binding of the antibody in the sample as compared to the control sample confirms the diagnosis of KD. A negative signal, e.g. no binding of the antibody, signals that KD is not present.
[0055] Suitable methods of detection are known in the art and include, but are not limited to, for example, ELISA, Western blot, immunostaining, immunoprecipitation, flow cytometry, sensor chips, magnetic beads, and the like.
[0056] As used herein “sample” or “biological sample” refers to a sample taken from a subject, such as but not limited to, a blood or tissue sample.
[0057] The present invention has been described in terms of one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention.
Example 1
[0058] The embodiment described here demonstrates the development of monoclonal antibodies based on plasmablasts isolated from subjects diagnosed with KD. The embodiment described in this example also demonstrates the binding of the monoclonal antibodies to hepacivirus C NS4A.
[0059] Traditional approaches of cultivating an infectious agent from patient tissues, as well as molecular biology approaches such as deep sequencing of KD tissues (7), have been unrevealing, indicating that alternative approaches to solve this problem are needed. Analysis of peripheral blood plasmablasts is emerging as a powerful tool in studies of pathogenesis, diagnosis, and therapeutic discovery in infectious diseases (8-16), vaccine science (16-22), and autoimmune disease (23, 24). Multiple studies have shown that >75% of peripheral blood plasmablasts express antibodies specific to antigenic targets of an ongoing immune response (25-28). Identification of specific KD antigens would facilitate diagnostic test development and improved therapies.
[0060] We discovered an oligoclonal IgA response in KD arterial tissues (29), and made “first generation” synthetic antibodies comprised of oligoclonal alpha heavy chains paired with random light chains (30, 31) as tools to identify the target antigen(s). These antibodies identified ICI in KD but not in infant control ciliated bronchial epithelium by immunohistochemistry (30-32). However, the antibodies did not yield specific antigen by Western blot, immunoprecipitation, or screening of poly A-selected KD arterial, spleen, or liver expression cDNA libraries.
[0061] We hypothesized that our original KD synthetic antibodies were not optimal for specific antigen identification because they did not include in vivo cognate light and heavy chains. We thought it likely that antigen-specific antibody-producing cells were present in the blood of KD children, since these cells were present in the coronary arteries (29, 30). Therefore, we isolated peripheral blood plasmablasts from children with KD 1-3 weeks after fever onset using single cell sorting by flow cytometry, analyzed their cognate light and heavy chain sequences, and prepared “second generation” synthetic antibodies from oligoclonal plasmablasts. Our goal was to use the KD-specific B cell response to identify antigens important in KD pathogenesis.
Results
[0062] Plasmablast isolation from selected KD patients˜KD is a clinical diagnosis, but can be considered confirmed in a young child with a prolonged febrile illness who develops coronary artery aneurysms (3). To be certain that we included children with definite KD in this study, we preferentially enrolled those with coronary artery aneurysms at diagnosis and those who appeared to be at high risk for such complications (3). By convention, the first day of illness in KD is defined as the first day of fever (3). Plasmablasts from 11 KD patients (
TABLE-US-00005 TABLE 2A Clinical and plasmablast data for Kawasaki disease patients in this study. Day of Day of illness Total # Race/ illness IVIG Incomplete Zmax Plasmablasts Patient Age Gender Ethnicity of sample given KD RCA/LAD (isotype) KD1 4 yr F Hispanic 17 13 No RCA 4.7 119 (49A, 28G, 42M) KD2 4 yr F Caucasian 24 8 No NL 115 (64A, 22G, 29M) KD3 5 yr M Black 20 19 Yes NL 67 (33A, 31G, 3M) KD4 3 mo M Hispanic 8 6 Yes RCA 3.0, 153 (102G, LAD 3.5 30A, 21M) KD5 11 mo F ½ 14 10 Yes NL 115 (63G, Caucasian, 39A, 13M) ½ Asian (Vietnamese) KD6 4 yr F Black 13 5 and 8 No RCA 3.0 80 (43A, 23G, 14M) KD7 17 mo M Caucasian 13 9 No RCA 9.8, 121 (56A, LAD 5.7 50G, 15M) KD8 17 mo M Caucasian 14 12 Yes LAD 3.5 85 (41A, 34G, 10M) KD9 3 yr M Asian 9 6 No NL 81 (30A, (Indian) 38G, 13M) KD10 5 yr F Asian (½ 11 7 No RCA 5.4, 88 (41A, Japanese, LAD 7.4, 41G, 6M) ½ Korean) distal large R, L CAA
[0063] Genetic characterization of KD plasmablasts reveals an oligoclonal response—Forty-two sets of clonally related plasmablasts were identified in ten patients (
TABLE-US-00006 TABLE 2B Genetic characteristics of KD plasmablasts in this study CDR3 Number of CDR3 Number of Number of Patient # Heavy chain length mutations Light chain length mutations plasmablasts (total PB) mAb IGVH IGVD IGVJ (aa) (aa) IGV IGJ (aa) (aa) (G, A, M) KD1 (119) 1F3 IGHV3- IGHD3- IGHJ4*02 10 5 IGKV2- IGKJ4*01 8 2 12 (1, 10, 1) 7*01 10*01 29*02 2H10 IGHV3- IGHD1- IGHJ4*02 10 6 IGKV2- IGKJ4*01 8 2 7*01 26*01 29*02 2A8 IGHV3- IGHD3- IGHJ5*02 10 10 IGKV2- IGKJ1*01 9 7 1 (0, 1, 0) 7*01 16*01 30*02 1F1 IGHV3- IGHD7- IGHJ4*02 16 8 IGKV3- IGKJ1*01 10 3 2 (0, 2, 0) 21*01 27*01 15*01 1C6 IGHV4- IGHD1- IGHJ5*02 13 10 IGKV4- IGKJ1*01 10 2 2 (1, 1, 0) 39-*01 1*01 1*01 2G11 IGHV3- IGHD5- IGHJ4*02 15 10 IGKV1- IGKJ3*01 9 10 2 (0, 1, 1) 15*01 24*01 39*01 KD2 (115) 2B6 IGHV3- IGHD4- IGHJ4*02 12 18 IGLV3- IGLJ2*01 10 9 2 (0, 1, 1) 15*05 23*01 10*01 2F8 IGHV3- IGHD3- IGHJ4*02 15 12 IGLV3- IGLJ3*02 11 9 1 (0, 1, 0) 73*02 16*01 25*02 1A7 IGHV3- IGHD3- IGHJ4*02 15 8 IGLV3- IGLJ3*02 12 13 1 (0, 1, 0) 73*02 22*01 25*03 2C7 IGHV6- IGHD5- IGHJ4*02 13 6 IGLV1- IGLJ1*01 11 8 3 (1, 2, 0) 1*01 12*01 51*02 KD3 (67) 1G8 IGHV5- IGHD3- IGHJ4*02 10 6 IGLV2- IGLJ3*02 10 13 2 (0, 2, 0) 10-1*03 10*01 14*01 1G9 IGHV1- IGHD3- IGHJ4*02 9 10 IGLV8- IGLJ2*01 10 10 2 (2, 0, 0) 18*04 9*01 61*01 KD4 (153) 2C5 IGHV1- IGHD2- IGHJ4*02 14 1 IGKV3- IGKJ2*01 10 0 2 (2, 0, 0) 3*01 2*01 20*01 2H4 IGHV3- IGHD7- IGHJ2*01 14 4 IGLV2- IGLJ2*01 10 0 2 (1, 0, 1) 74*01 27*01 11*01 1F5 IGHV5- IGHD4- IGHJ6*03 19 0 IGLV2- IGLJ3*02 10 0 2 (2, 0, 0) 51*01 11*01 14*01 KD5 (115) 1E2 IGHV5- IGHD6- IGHJ4*02 13 4 IGLV4- IGLJ3*02 9 0 2 (2, 0, 0) 51*03 19*01 60*03 1C10 IGHV5- IGHD3- IGHJ3*02 16 5 IGKV3- IGKJ5*01 9 3 2 (2, 0, 0) 51*01 9*01 20*01 2C1 IGHV4- IGHD6- IGHJ4*02 16 1 IGK3- IGKJ2*01 11 0 3 (2, 1, 0) 61*02 13*01 20*01 2C6 IGHV3- IGHD3- IGHJ4*02 13 7 IGKV1- IGKJ1*01 9 1 2 (0, 1, 1) 23*01 22*01 5*03 2C10 IGHV3- IGHD3- IGHJ4*02 15 15 IGLV4- IGLJ3*02 10 4 2 (2, 0, 0) 23*01 10*01 69*01 2D3 IGHV4- IGHD3- IGHJ5*02 15 7 IGKV4- IGKJ1*01 9 3 2 (0, 2, 0) 61*02 22*01 1*01 2F6 IGHV1- IGHD3- IGHJ4*02 13 7 IGKV1- IGKJ2*04 8 4 2 (0, 1, 1) 8*01 10*01 39*01 KD6 (80) 1A2 IGHV1- IGHD3- IGHJ3*01 13 17 IGLV7- IGLJ2*01 10 12 3 (0, 3, 0) 45*02 9*01 46*01 2A12 IGHV1- IGHD2- IGHJ3*01 13 17 SAME SAME 10 11 45*02 8*01 1A10 IGHV3- IGHD3- IGHJ4*02 20 11 IGLV1- IGLJ3*02 10 11 2 (0, 2, 0) 33*03 22*01 44*01 2F4 IGHV1- IGHD2- IGHJ4*02 17 9 IGLV2- IGLJ2*01 10 8 2 (0, 2, 0) 45*02 2*01 11*01 2G3 IGHV3- IGHD5- IGHJ2*01 18 11 IGKV3- IGKJ4*01 8 11 2 (2, 0, 0) 21*01 12*01 20*01 2H3 IGHV3- IGHD3- IGHJ3*01 19 9 IGKV3- IGKJ4*01 8 5 2 (1, 0, 1) 15*01 10*01 15*01 2B2 IGHV3- IGHD3- IGHJ3*01 12 15 IGKV1- IGKJ1*01 10 9 2 (0, 2, 0) 33*01 3*01 5*03 KD7 (121) 1A12 IGHV3- IGHD4- IGHJ4*02 9 9 IGKV1- IGKJ1*01 9 4 1 (0, 1, 0) 7*03 17*01 5*03 1D3 IGHV3- IGHD1- IGHJ4*02 11 15 IGKV1- IGKJ1*01 9 3 1 (0, 1, 0) 23/ 26*01 5*03 23D*01 1D9 IGHV5- IGHD6- IGHJ4*02 14 10 IGLV1- IGLJ1*01 11 7 1 (0, 1, 0) 10-1*03 25*01 44*01 2A9 IGHV5- IGHD5- IGHJ4*02 13 20 IGKV1- IGKJ1*01 9 11 1 (0, 1, 0) 10-1*03 24*01 5*03 2C1 IGHV3- IGHD6- IGHJ4*02 14 13 IGLV5- IGLJ3*02 9 10 1 (0, 1, 0) 23/ 19*01 45*02 23D*01 2F9 IGHV3- IGHD2- IGHJ4*02 12 17 IGKV2- IGKJ2*01 9 5 1 (0, 1, 0) 23/ 21*01 24*01 23D*01 KD8 (85) 1B10 IGHV3- IGHD3- IGHJ3*02 12 3 IGKV1- IGKJ4*01 9 6 2 (0, 2, 0) 72*01 16*01 6*01 1D4 IGHV3- IGHD3- IGHJ3*02 12 1 IGKV1- IGKJ4*01 9 4 72*01 9*01 6*01 KD9 (81) 1D2 IGHV1- IGHD5- IGHJ4*02 11 5 IGLV7- IGLJ3*02 9 8 2 (1, 1, 0) 46*01 18*01 43*01 2C8 IGHV1- IGHD2- IGHJ4*02 11 5 IGLV7- IGLJ3*02 9 5 46*01 15*01 43*01 1E10 IGHV1- IGHD4- IGHJ4*02 12 22 IGLV1- IGLJ1*01 11 10 1 (0, 1, 0) 46*01 11*01 44*01 2C5 IGHV4- IGHD3- IGHJ6*02 14 4 IGLV2- IGLJ1*01 10 7 2 (0, 0, 2) 59*08 10*01 23*01 1B5 IGHV4- IGHD3- IGHJ6*02 14 2 IGLV2- IGLJ1*01 10 6 59*08 10*01 23*01 1A10 IGHV3- IGHD3- IGHJ4*02 16 20 IGLKV3- IGKJ2*01 9 13 1 (0, 1, 0) 30*02 10*01 11*01 KD10 (88) 1F11# IGHV3- IGHD3- IGHJ4*02 12 14 IGLV2- IGLJ1*01 11 6 5 (5, 0, 0) 7*03 10*01 11*01 1B1# IGHV3- IGHD6- IGHJ4*02 9 17 IGKV2- IGKJ1*01 9 5 2 (2, 0, 0) 21*01 19*01 30*02 2A1 IGHV4- IGHD7- IGHJ4*02 10 14 IGLV7- IGLJ3*02 11 6 2 (0, 2, 0) 31*03 27*01 46*01 1A10 IGHV4- IGHD3- IGHJ4*02 15 20 IGKV3- IGKJ2*01 9 12 1 (0, 1, 0) 34*01 10*01 20*01 1B8 IGHV3- IGHD2- IGHJ4*02 10 19 IGKV3- IGKJ1*01 9 13 1 (0, 1, 0) 7*01 2*01 15*01 1E9# IGHV3- IGHD6- IGHJ4*02 18 18 IGLV3- IGLJ3*02 11 13 1 (0, 1, 0) 11*06 13*01 25*02 1G12 IGHV3- IGHD6- IGHJ4*02 18 15 IGLV1- IGLJ3*02 11 6 1 (0, 1, 0) 11*06 19*01 44*01 KD11 (132) 1E9 IGHV1- IGHD6- IGHJ4*02 16 28 IGKV2- IGKJ5*01 9 6 2 (1, 1, 0) 46*01 13*01 30*02 1C2 IGHV1- IGHD6- IGHJ4*02 16 15 IGKV2- IGKJ5*01 9 5 46*01 25*01 30*01 2A12 IGHV3- IGHD6- IGHJ4*02 13 0 IGKV3- IGKJ2*01 10 0 2 (0, 2, 0) 21*01 13*01 20*01 2H6 IGHV3- IGHD6- IGHJ4*02 15 11 IGLV9- IGLJ3*02 13 4 2 (2, 0, 0) 21/J4 6*01 49*01 1B7# IGHV3- IGHD2- IGHJ6*02 20 1 IGLV2- IGLJ2*01 10 2 6 (0, 0, 6) 23*01 2*01 8*01 1B12 IGHV3- IGHD3- IGHJ6*02 25 10 IGKV3- IGKJ1*01 8 8 2 (2, 0, 0) 49*03 3*01 15*01 2E7 IGHV4- IGHD3- IGHJ5*02 18 15 IGKV1- IGKJ1*01 9 8 2 (2, 0, 0) 4*02 10*01 17*01 1C1 IGHV4- IGHD3- IGHJ6*02 14 1 IGKV3- IGKJ2*01 9 1 2 (0, 0, 2) 34*01 10*01 20*01 1F6 IGHV4- IGHD1- IGHJ5*02 18 16 IGKV3- IGKJ4*01 8 16 5 (5, 0, 0) 39*07 7*01 20*01 2G1 IGHV4- IGHD1- IGHJ5*02 18 17 IGKV3- IGKJ4*01 8 8 39*07 7*01 20*01 2E10 IGHV4- IGHD3- IGHJ3*02 16 16 IGKV1- IGKJ3*01 9 13 2 (2, 0, 0) 59*01 3*01 33*01 2F12 IGHV4- IGHD3- IGHJ3*01 16 20 IGKV1- IGKJ3*01 9 10 59*01 3*02 33*01 1A12# IGHV5- IGHD1- IGHJ6*02 20 7 IGLV1- IGLJ2*01 11 3 2 (0, 2, 0) 51*01 7*01 51*01 1D6 IGHV6- IGHD5- IGHJ4*02 13 0 IGLV1- IGLJ1*01 11 0 2 (0, 2, 0) 1*01 18*01 47*01 2D5 IGHV3- IGHD3- IGHJ4*02 22 11 IGKV1- IGKJ3*01 9 12 2 (2, 0, 0) 23*01 10*01 39*01 #Low yield of antibody prohibited further testing.
[0064] Expression of KD monoclonal antibodies—We preferentially made human light chain-rabbit heavy chain antibodies from the plasmablasts described in Table 2B, because they allowed for testing of the antibodies on human KD tissues using anti-rabbit secondary reagents. If sufficient quantities of antibody for testing were not produced using the rabbit heavy chain construct, fully human antibodies were made and biotinylated for immunohistochemistry assays. For four sets of clonally related sequences and one highly mutated IgA plasmablast, antibody production was not sufficient for further testing. For the other 38 clonally related sets and for the other antibodies made from mutated single IgA plasmablasts, approximately 300 ug to 1 mg of human/rabbit or human/human antibodies were produced in one 60 ml culture flask per assay. We tested 60 monoclonal antibodies from 11 patients. Of the 60 antibodies, 52 had entirely different VH/VL sequences and eight were members of clonally related plasmablast sets in which the related antibodies had 1-4 amino acid mutations in the CDR3 sequence within the set.
[0065] Immunohistochemistry shows binding of KD monoclonal antibodies to cytoplasmic inclusion bodies in KD ciliated bronchial epithelium—Our prior studies demonstrated binding of synthetic antibodies with non-cognate VDJ and VJ pairs to intracytoplasmic inclusion bodies in ciliated bronchial epithelial cells of children who died from acute KD but not of infant controls who died of non-KD illnesses (29-31, 35). We therefore initially performed immunohistochemistry assays to determine if the newly synthesized KD monoclonal antibodies with in vivo cognate VDJ and VJ partners identified the inclusion bodies in acute KD formalin-fixed, paraffin-embedded lung tissue. Failure to bind in this assay does not preclude synthetic antibodies from being KD antigen-specific, because antigens can be disrupted by formalin fixation and paraffin embedding. However, strong positive results from this assay were useful to prioritize antibodies for additional assays that could specifically identify target KD antigen(s). Initial studies with antibodies KD1-2G11 and KD4-2H4 revealed strong binding of the antibody to KD lung tissues from the United States (n=3) and Japan (n=2) (
TABLE-US-00007 TABLE 2C Genetic characteristics of Kawasaki disease (KD) monoclonal antibodies (Mab) that bind strongly to KD intracytoplasmic inclusion bodies. #mutations CDR3 from length germline KD Original Mab Heavy chain (amino acids) (amino acids) Light chain patient isotype 1-2G11 IGHV3-15/JH4 15 10 IGKV1-30/JK3 1 IgA 4-2H4 IGHV3-74/JH2 14 4 IGLV2-14/JL3 4 IgG 6-1A10 IGHV3-33/JH4 20 11 IGLV1-44/JL3 6 IgA 6-2B2 IGHV3-33/JH3 12 15 IGKV1-5/JK1 6 IgA 7-1D3 IGHV3-23/JH4 11 15 IGKV1-5/JK1 7 IgA 7-2C1 IGHV3-23/JH4 14 13 IGLV5-45/JL3 7 IgA 9-1E10 IGHV1-46/JH4 12 22 IGLV1-44/JL1 9 IgA 11-1E9* IGHV1-46/JH4 16 28 IGKV2-30/JK5 11 IgG 11-1C2* IGHV1-46/JH4 16 15 IGKV2-30/JK5 11 IgA 11-2E10 IGHV4-59/JH3 16 16 IGKV1-33/JK3 11 IgG *Clonally related plasmablasts from KD patient 11
[0066] Screening an animal virus peptide array reveals KD4-2H4 recognizes hepacivirus peptides—To determine whether KD monoclonal antibodies bind to an epitope that is shared with a known animal virus, we created a custom array (PEPperPRINT, Table S4) of peptides reported to be B cell epitopes of animal viruses and included in the Immune Epitome Database and Analysis Resource (available on the World Wide Web at iedb.org). Monoclonal antibody KD4-2H4 showed binding to multiple similar peptides from a short region of the C-terminal end of the NS4A protein of hepacivirus C (
[0067] Substitution matric analysis demonstrates amino acids required for antibody KD4-2H4 binding to a hepacivirus peptide—To determine critical amino acids for KD4-2H4 binding to hepacivirus NS4A peptide 1, we tested a peptide substitution array, in which each position of the reactive peptide AIIPDREALYQEFDEME (SEQ ID NO:219) was sequentially replaced by each of 20 amino acids (PEPperPRINT). This peptide was chosen from the reactive peptides on the animal virus peptide array to place the highly significant motif LYQxFDE in the mid-region of the peptide. The substitution array showed that amino acids 9L and 11Q of this peptide were essential for antibody binding (
[0068] Multiple KD monoclonal antibodies recognize an optimized KD peptide by ELISA—Initial ELISA experiments showed that monoclonal antibody KD4-2H4 reacted with hepacivirus C NS4Apeptide 1. This peptide was derived from the strongest binding peptide on the animal virus peptide array (
TABLE-US-00008 TABLE 3 C-terminal region amino acid sequences of some hepaciviruses and comparison with the NS4A peptides recognized by KD monoclonal antibodies. Hepacivirus NS4A sequence SEQ Accession Hepacivirus C (human) KPAVIPDREVLYQAFDEMEEC 193 AFX78101 Hepacivirus A (non-primate, KVVVVPHKGVLYADFDEMEEC 194 KP325401 canine, equine) Hepacivirus K (bat) KIFISQDRDNLYTILDEMEEC 195 KC796074 Hepacivirus M (bat) KIFLSPDKDHLYGWFEEMEEC 196 KC796078 Hepacivirus L (bat) TTLEDADMPAYLTDMGELEEC 197 KC796077 Hepacivirus E (rodent) TTAARVVQPPEDDITENLEEC 198 KC815310 Hepacivirus F (rodent) STAARVVEFEPLDTEEVLEEC 199 KC411784 Hepacivirus J (rodent) FVNTLPPEVLFELEPTEAEEC 200 KC411777 Hepacivirus I (rodent) CRPELRLNETIDEPAIELEEC 201 KC411806 Hepacivirus G (Norway rat 1) DSSAALNPAPEMSILEEVEEC 202 MF113386 Hepacivirus H (Norway rat 2) AKGAEPXPLEQEMDTAGMEEC 203 KJ950939 Hepacivirus N (bovine) DSGEPPNAPEPADDDDGFEEC 204 MH027953 Hepacivirus D (Guereza, Old QAERAQVMEDFIGDLIETEEC 205 KC551800 World monkey) Sifaka hepacivirus (lemur) TTSALHAPPPPISLEGALEEC 206 MH824539 Hepacivirus B (GB virus-B; SVPTGATVAPVVDEEEIVEEC 207 U22304 human) Hepacivirus P (ground squirrel) STASLASQPIDDPLTSGLEEC 208 MG211815 Peptides used in this study NS4A peptide 1 KPAVIPDREVLYQGFDEMEEC 209 NS4A peptide 2 KPAVIPDREALYQDIDEMEEC-BSA conjugated 210 Scrambled peptide IYPLEDMAEPKVERIDAQEDC-BSA conjugated 211
TABLE-US-00009 TABLE 4 Genetic characteristics of Kawasaki disease (KD) monoclonal antibodies (Mab) that recognize KD peptide #mutations Binding CDR3 from to KD Heavy length germline Light KD Original inclusion Mab chain (aacids) (aacids) chain patient isotype bodies 4-2H4 IGHV3- 14 4 IGLV2- 4 IgG Strong 74/JH2 14/JL3 6-1A10 IGHV3- 20 11 IGLV1- 6 IgA Strong 33/JH4 44/JL3 6-2B2 IGHV3- 12 15 IGKV1- 6 IgA Strong 33/JH3 5/JK1 8-1B10* IGHV3- 12 3 IGKV1- 8 IgA Weak 72/JH3 6/JK4 8-1D4* IGHV3- 12 1 IGKV1- 8 IgA Weak 72/JH3 6/JK4 *Clonally related plasmablasts from KD patient 8
[0069] Monoclonal antibodies KD4-2H4 and KD6-2B2 share a common epitope—KD36-2B32 showed strong reactivity with KID peptide by ELISA (
[0070] Blocking experiments show that the KD peptide epitope is present in KD inclusion bodies—Pre-incubation of monoclonal antibodies KD4-2H4 and KD6-2B2 with KD peptide blocked binding of the antibodies to intracytoplasmic inclusion bodies in KD ciliated bronchial epithelium (
[0071] Human protein array analyses and immunohistochemistry studies of monoclonal antibodies KD4-2H4 and KD6-2B2 do not yield a human protein as the target of the antibodies m—We performed human protein array analysis using both KD4-2H4 and KD6-2B2. The array covers ˜80% of the canonical proteome. The only human protein that showed reactivity with both antibodies on this array was integral membrane protein 2B (ITM2B), a transmembrane protein whose C-terminal end is processed by proteases to release a peptide that inhibits the deposition of beta-amyloid; mutations in this protein are associated with neurodegenerative diseases (39). This reactivity could be explained by a partially shared epitope between KD peptide and the ITM2B protein (-ALYQ-I-, (SEQ ID NO: 192)). Immunohistochemistry of KD lung with polyclonal anti-ITM2B revealed a different pattern of staining of bronchial epithelium compared with KD4-2H4 and KD6-2B2, and anti-ITM2B did not block the binding of the antibodies to inclusion bodies in KD ciliated bronchial epithelium (
[0072] Sera from additional KD patients recognize KD peptide—To determine if sera from KD patients recognize KD peptide, we performed Western blot analyses for IgG antibody using glutathione sulfur transferase (GST)-KD peptide multimer fusion protein and GST as the antigens. We screened KD and control patient sera at a dilution of 1:5000 in phosphate buffered saline, which reduced background from non-specific binding. A minority of sera (both KD and control) exhibited non-specific binding (reactivity with GST alone) and were excluded from the study. Treatment of KD children with intravenous gammaglobulin (IVIG) at the time of diagnosis could potentially complicate analysis for IgG seroconversion to the identified epitope. Therefore, we tested sera from KD patients who presented on days 8-14 after fever, prior to receiving IVIG therapy (Table 5), when IgG antibody to the agent might have developed. We also tested serum taken on day 28 after onset from a KD patient who presented prior to the time that IVIG was standard treatment for KD. These results showed that sera from 5/8 KD children who had not received IVIG therapy had IgG antibody to the KD peptide epitope during or after the second week of illness (Table 5,
TABLE-US-00010 TABLE 5 Kawasaki Disease (KD) patient sera (before IVIG treatment) tested by Western blot assay for IgG antibody to KD peptide multimer. Day of Coronary Western Sample Age Gender Illness Abnormalities result Year KD12 10 yr M 11 Yes Positive 1994 KD13 4 yr F 14 Yes Positive 2003 KD14 6 yr F 8 Yes Positive 2003 KD15 3 yr M 9 No Positive 2002 KD16 6 mo M 28 Yes Positive 1984 KD17 3 yr M 8 No Negative 2002 KD18 15 yr M 9 Yes Negative 1991 KD19 3 yr M 8 Yes Negative 2011 KD4 3 mo M 3 Yes Negative 2017 KD20 7 mo F 5 Yes Negative 2013 KD21 7 mo F 5 Yes Negative 2005 KD22 3 yr F 5 Yes Negative 2002 KD23 2 yr F 5 No Negative 2008 KD24 16 mo M 5 No Negative 2009 KD25 20 mo M 5 No Negative 2017 KD26 17 mo M 5 No Negative 2017 KD27 4 mo F 4 No Negative 1991 KD28 3 yr M 4 No Negative 1991 KD29 2 yr M 4 No Negative 1991 KD30 7 yr F 4 No Positive 1990
TABLE-US-00011 TABLE 6 Control sera tested by Western blot assay for IgG antibody to KD peptide multimer. Western Sample Age Gender Diagnosis result Febrile control 1 9 yr F Staphylococcus Negative aureus septic arthritis Febrile control 2 7 mo M Haemophilus Negative influenzae type a meningitis Febrile control 3 11 mo M Staphylococcus Negative aureus septic arthritis Febrile control 4 21 mo M Group A Negative streptococcal septic arthritis Febrile control 5 14 mo F E. coli Negative pyelonephritis Febrile control 6 10 yr F Staphylococcus Negative aureus osteomyelitis Febrile control 7 4 yr F Viral-induced Negative leukopenia and thrombocytopenia Febrile control 8 4 yr F Pneumonia with Negative effusion Febrile control 9 5 yr M Pneumococcal Positive pneumonia Infant 5-9 mo Unknown Unknown N = 17 controls 10-26 Negative (deidentified)
Discussion
[0073] Here, we identified a peptide that is recognized by antibodies that develop during acute KD. Our data demonstrate that monoclonal antibodies derived from clonally expanded plasmablasts in the peripheral blood of children with KD identify intracytoplasmic inclusion bodies in the bronchial epithelium of fatal KD cases. A subset of these antibodies bind to a specific protein epitope that is similar to one found in the NS4A protein of hepacivirus C. Furthermore, the inclusion bodies contain a protein with this epitope. We also demonstrate that IgG antibody to this epitope is much more prevalent during or after the second week of illness than in the first week of KD, and we did not detect antibody in 17 infants 5-9 months of age. Our report of an epitope targeted by the immune response to KD demonstrates that preparing antibodies from plasmablast responses to acute inflammatory diseases of unknown etiology can be useful in identifying the inciting antigens.
[0074] Whether the protein epitope identified in this study derives from a previously unidentified hepacivirus or related virus remains to be determined. Although some propose an autoimmune etiology of KD, the marked severity of the disease in very young infants (41) and the peak incidence at 10 months of age (42) are not supportive of this hypothesis, because autoimmune diseases rarely arise in the first year of life. Moreover, the short 1-3 week duration of the clinical illness even in untreated patients (3) and the rarity of disease recurrence (43) argue against the autoimmune theory. In contrast, many lines of evidence support a ubiquitous viral agent as the cause of KD in genetically susceptible children. These include the young age group affected (42), well-documented epidemics of illness (2, 44-46), the self-limited nature of the clinical illness (3), the lack of clinical response to antibiotic therapy (3, 43), the high prevalence of the condition in Japan, where 1 in 65 children develop KD by the age of 5 (42) (a prevalence rate similar to that of many ubiquitous viral infections), the upregulation of interferon response genes in KD lung and coronary arteries (35, 38), the identification of virus-like particles in close proximity to KD inclusion bodies in ciliated bronchial epithelium (35), and the prominent IgA immune response suggesting a mucosal portal of entry of the putative ubiquitous causative agent (28, 30, 47, 48).
[0075] Hepaciviruses are enveloped, spherical RNA viruses that are ˜50 nm in diameter and can result in persistent infection. Since 2010, when hepatitis C virus was the sole confirmed member of the Hepacivirus genus, there has been a steady increase in the number of new hepaciviruses identified in various animal species (49). The identified epitope recognized by the KD monoclonal antibodies reported here is most similar to that of hepacivirus C (˜90% identity), non-primate hepacivirus (˜60% identity), and hepacivirus M and K (bat, ˜50% identity) and less homologous to that of rodent, bovine, Old World monkey, and lemur hepaciviruses (Table 3). It differs substantially from the NS4A sequence of hepacivirus B (GB virus-B) and pegiviruses.
[0076] Although hepaciviruses are primarily hepatotropic, RNA of non-primate hepacivirus has been identified in the cytoplasm of ciliated bronchial epithelial cells of infected dogs and horses by in situ hybridization (50, 51). Our prior studies indicated that KD intracytoplasmic inclusion bodies contain RNA and can persist in at least a subset of KD patients (36). Moreover, using transmission electron microscopy, we found that inclusion bodies in KD ciliated bronchial epithelial cells were located in the endoplasmic reticulum/Golgi region, with spherical, 50 nm virus-like particles in close proximity (35), consistent with but not confirmatory of infection with a virus in this family or one with a viral ancestry shared with the hepaciviruses. However, the epitope identified is short, and could derive from a non-hepacivirus source.
[0077] We are presently using genomic and proteomic approaches to determine the gene sequence from which the immunogenic epitope identified in this study arises. We hypothesize that the source is an RNA virus whose genome is present in very low quantity in KD clinical samples, such as blood at the time of clinical presentation, and in the target tissues of fatal cases by the time of death occurring weeks to years after illness onset. We are working to identify the targets of KD monoclonal antibodies that bind ICI but do not bind to KD peptide, because these antibodies may bind other epitopes of the putative viral agent. We are also developing more sensitive assays for multiple antibody isotypes that could facilitate KD diagnosis and could be evaluated in worldwide multicenter studies.
[0078] This study is limited by its investigation of the plasmablast response to KD in a single US center over a 16-month period from 2017-18. However, KD monoclonal antibodies prepared in this study react with tissue samples from KD children from other geographic areas of the US and Japan who died during different decades, and sera from KD children in Chicago from the 1980s, 1990s and 2000s react with the identified protein epitope, suggesting that the results may be applicable to additional KD patients in other locations and over other time periods.
[0079] Further multicenter collaborative studies are needed to investigate the relevance of these findings to KD patients around the world. Despite these limitations, we believe our results may provide the most promising direction for etiologic studies of KD and the development of serologic tests that has emerged in the more than 50 years since Dr. Kawasaki described the clinical features of the disease (52). Identification of the etiology of KD is a pediatric research priority that will enable diagnostic test development, improved therapy, and ultimately prevention of this serious, increasingly recognized worldwide childhood illness.
Methods
[0080] Patients and specimens. Peripheral blood (3 ml) was obtained from 11 KD patients on day 8-24 after fever onset (
[0081] Flow cytometry. Peripheral blood mononuclear cells obtained by Ficoll gradient centrifugation were stained with antihuman CD3-FITC (Fisher), CD19-Pacific Blue, CD38-AlexaFluor647, and CD27-PE (Biolegend). CD3-CD19+CD38++CD27++ cells were gated (
[0082] Amplification, sequencing and cloning of immunoglobulin variable regions. Reverse transcription and PCR of heavy and light chain variable genes were performed according to a published protocol (54, 55), and PCR products were directly sequenced. Heavy chain sequences were analyzed for VH family and CDR3 sequence and clonally related sequences identified and prioritized for antibody synthesis. Plasmablasts were determined to be clonally related when their sequences encoded identical functional heavy and light chain variable families and had a CDR3 sequence of identical length with the same amino acid sequence or 1-4 amino acid differences. Sequences that were out-of-frame or had stop codons were not further investigated. In some cases, IgA plasmablasts showing substantial somatic mutation of their immunoglobulin variable region sequences were prioritized for production, even if other clonally related plasmablasts were not identified in the dataset from that patient. Light chains were cloned into human immunoglobulin kappa or lambda light chain expression vectors (55) and heavy chains were cloned into human gamma1 and rabbit gamma (pFUSEss vectors, InvivoGen, San Diego, Calif.) heavy chain expression vectors, to enable production of human and rabbit versions of the antibodies.
[0083] Antibody production. Antibodies were produced by transfection of 293F suspension cells using a 1.5:1 ratio of light chain:heavy chain DNAs and Freestyle MAX reagent, and were purified using protein A agarose beads (ThermoFisher Scientific).
[0084] Immunohistochemistry using KD monoclonal antibodies. Immunohistochemistry was performed on KD and control infant tissues as previously reported (29, 47) using a screening dilution of 1:100 of each synthetic antibody and either biotinylated human antibody or rabbit antibody followed by biotinylated goat anti-rabbit secondary antibody using the Vectastain ABC Elite kit (Vector Laboratories). If background staining was high at 1:100, the antibodies were further diluted.
[0085] Monoclonal antibody reactivity with animal virus peptides. A custom Animal Virus Discovery peptide array was designed (PEPperPRINT, available on the World Wide web at pepperprint.com) made up of 29,939 peptides derived from 13,123 B cell animal virus epitopes reported in the Immune Epitope Database and Analysis Resource (available on the World Wide Web at iedb.org), control hemagglutinin peptides (769 spots), and 1,261 random peptides to fill the microarray. Peptides >4 amino acids were printed either in full length or translated into overlapping 17 amino acid peptides and were printed in duplicate. The array was blocked with blocking buffer MB-070 (Rockland) for 30 minutes, and washing buffer was PBS, pH 7.4 with 0.05% Tween 20. The array was pre-stained with the secondary antibodies as described below, to determine background staining. Rabbit KD4-2H4 (2.5 ug/ml) was incubated with the array for 16 hours at 4° C. in washing buffer with 10% blocking buffer with shaking at 140 rpm. Secondary antibody incubations were then performed with sheep anti-rabbit IgG (H+L) DyLight 680 (1:5000) for 45 minutes in washing buffer with 10% blocking buffer at room temperature. Control antibody was added to the secondary antibody incubations [mouse monoclonal anti-hemaggutinin (12CA5) DyLight800 (1:2000)]. Fluorescence intensity was read on a LI-COR Odessey Imaging System (scanning offset 0.65 mm, resolution 21 um, scanning intensities of 7/7 (red=700 nm/green=800 nm). Microarray image analysis was done with PepSlide® Analyzer. To identify common motifs MEME bioinformatics analysis was performed on the top hits with the highest spot intensities (available on the World Wide Web at meme.nbcr.net/meme/intro.html). MEME represents motifs as position-dependent letter-probability matrices which describe the probability of each possible letter at each position in the pattern. The MEME pre-settings were a maximum of one motif each sequence and of maximum three different motifs, as well as a minimum motif length of 5 amino acids. The e-value corresponds to the statistical significance of a consensus motif with the given log likelihood ratio (or higher) and with the same width and site count that one would find in a similarly sized set of random sequences. An e-value of 1 would be expected for the identification of a certain motif in a set of random peptides by chance.
[0086] Substitution analysis. Substitution analysis was performed on viral peptides recognized by antibodies KD4-2H4 and KD6-2B2 by creating a peptide array that includes stepwise substitution of all amino acid positions of the peptide with all 20 amino acids, to determine the amino acids that yielded optimal binding to the antibody (PEPperPRINT). Methods were identical to those described for the animal virus peptide array, with the exception that antibody KD4-2H4 was tested at 10 ug/ml and antibody KD6-2B2 was tested at 100 ug/ml.
[0087] ELISA for binding of peptides to KD monoclonal antibodies. Maxisorp Nunc Immuno 96-well plates were coated with 1 ug of synthetic peptides (Anaspec) per well and incubated with rabbit KD monoclonal antibodies at 10, 1, and 0.1p g/ml followed by horseradish peroxidase-labelled goat anti-rabbit antibody at 1:3000 (Fisher). Absorbance at 450 nm was determined on a Multiskan FC spectrophotometer after addition of Ultra 3,3′,5,5′-tetramethylbenzidine followed by 1.5M sulfuric acid solution. Absorbance of the KD peptide (KPAVIPDREALYQDIDEMEEC, SEQ ID NO:210) assays were recorded after subtraction of absorbance with the scrambled peptide (IYPLEDMAEPKVERIDAQEDC, SEQ ID NO:211). An OD reading >0.4 was arbitrarily determined as a positive; all negative antibodies had values of ≤0.04.
[0088] Blocking experiments to determine specificity of peptide binding. Monoclonal antibodies showing binding to animal virus peptides were incubated with a five-fold excess (by weight) of representative peptide or a scrambled peptide at 37° C. for 45 minutes, and each mixture was then applied to KD lung tissue for immunohistochemistry.
[0089] Testing of monoclonal antibodies for polyreactivity. ELISA was performed on monoclonal antibodies at 10, 1, and 0.1 ug/ml in ELISA plates coated with 1 ug/ml per well single stranded DNA (Sigma D8899), insulin (Sigma I2643), and bovine serum albumin (Fraction V, Fisher BP1600) (56). As a positive control, antibody KD11-1C1 was used. This VH4-34 antibody was encoded by an IgM plasmablast isolated from KD patient 11 (Table 2B). It was produced as an IgG antibody and was found to be polyreactive in ELISA against multiple diverse peptides, as is typical of many VH4-34 IgM antibodies (57).
[0090] Human protein array analyses and human protein immunohistochemistry. KD monoclonal antibodies human KD4-2H4 and rabbit KD6-2B2 were incubated at a concentration of 1 μg/ml at 4° C. overnight on a human proteome array (HuProt™v4.0, CDI Laboratories) which includes 16,793 genes, covering ˜80% of the canonical proteome as defined by the Human Protein Atlas (available on the World Wide Web at proteinatlas.org). Arrays were then probed with Alexa-647-anti-human IgG Fc gamma and Alexa-555-anti-rabbit secondary antibodies and imaged by CDI laboratories. Polyclonal rabbit antibody to integral membrane protein 2B (ITM2B) validated by the Human Protein Atlas (available on the World Wide Web at proteinatlas.org, HPA029292, Sigma) was used at the recommended 1:200 dilution for immunohistochemistry experiments including blocking experiments.
[0091] Western blot assay using glutathione sulfur transferase (GST)-concatemerized KD peptide fusion protein. We optimized the nucleotide sequence that codes for the KD peptide sequence for expression in E. coli and prepared a multimer with three copies of the peptide linked by short spacers [GST3X:AGKPAVIPDREALYQDIDEMEECLDEAGKPAVIPDREALYQDIDEMEECLDEAGKPA VIPDREALYQDIDEMEECLD (SEQ ID NO:191)]. This method of using a concatemerized fusion protein was shown to enhance diagnostic assays for Zika virus infection (58). The multimer sequence was cloned into the pGEX-KG plasmid (ATCC #77103) and fusion protein expression was induced from the recombinant vector and the original expression vector with 100 nM isopropyl β-d-1-thiogalactopyranoside (Goldbio, I248C) for 1.5 hrs at 30° C. (59). Bacteria were pelleted and resuspended in phosphate buffered saline and subjected to sonication (Branson digital sonifier SFX 550, Emerson), with cycles of 10 second sonication, 30 seconds of rest, for 15 cycles. The cell debris was pelleted by centrifugation at 15,000×g for 10 minutes. The cell-free supernatant was filtered using a 0.45p filter. The GST protein in the filtrate was purified using GSTrap FF (GE, 17513001) and eluted with 10 mM reduced glutathione (Sigma G4251), 50 mM Tris-HCl buffer pH 8.0. Purified proteins were quantified by BCA assay (Cat #23225, Thermofisher) and visualized by Coomassie staining. Western blot assays were performed following electrophoresis on 12% Tris-Glycine gels (Biorad) and transfer to PDVF membrane (Fisher IPVH00010). Blocking was with 5% nonfat dry milk in Tris buffered saline with 1% Tween 20 (TBST). Serum samples from KD patients and controls were diluted in TBST containing 5% nonfat dry milk at 1:5000 and incubated with membranes overnight at 4° C. Following incubation, membranes were washed five times in TBST, followed by incubation with horseradish peroxidase labelled goat anti-human IgG (Thermo, A18811) at a dilution of 1:5000 in phosphate buffered saline. After washing again five times in TBST, Supersignal West Femto Substrate (ThermoFisher, 34096) was applied, and blots were imaged using a ChemiDoc MP Imaging System (Biorad). Human immunoglobulin for intravenous use (IVIG) (Gammagard 10%, lot LE12T120AB) was assayed under the same conditions, but results were indeterminate because of reactivity with both GST and GST-3X. On each blot, lane 1 was GST alone (500 ng), lane 2 was GST-3× (500 ng), and lane 3 was human IgG (10-20 ng, Sigma 12511) used as a positive control for the binding of the secondary antibody. Blots were stripped and incubated with polyclonal rabbit anti-GST antibody (Fisher #717500, 1:1000) to ensure that both GST proteins were strongly detected after transfer. Sera found to be reactive with both GST and GST-3× were considered indeterminate and excluded from analyses (˜25% of all KD and control sera tested).
[0092] Statistical analysis. GraphPad Prism 8 was used to plot ELISA results. Comparison of serologic results between groups was performed using a two-tailed Fisher exact test using the function fishertest in R 3.6.1.
[0093] Human subjects. This study was approved by the Institutional Review Board of the Ann & Robert H. Lurie Children's Hospital of Chicago, and patients were enrolled following informed consent.
REFERENCES
[0094] 1. Yanagawa H, Nakamura Y, Yashiro M, Fujita Y, Nagai M, Kawasaki T, et al. A nationwide incidence survey of Kawasaki disease in 1985-1986 in Japan. J Infect Dis. 1988; 158(6):1296-301. [0095] 2. Yanagawa H, Nakamura Y, Kawasaki T, and Shigematsu I. Nationwide epidemic of Kawasaki disease in Japan during winter of 1985-86. Lancet. 1986; 2(8516):1138-9. [0096] 3. McCrindle B W, Rowley A H, Newburger J W, Burns J C, Bolger A F, Gewitz M, et al. Diagnosis, Treatment, and Long-Term Management of Kawasaki Disease: A Scientific Statement for Health Professionals From the American Heart Association. Circulation. 2017; 135(17):e927-e99. [0097] 4. Newburger J W, Takahashi M, Beiser A S, Burns J C, Bastian J, Chung K J, et al. A single intravenous infusion of gamma globulin as compared with four infusions in the treatment of acute Kawasaki syndrome. N Engl J Med. 1991; 324(23):1633-9. [0098] 5. Onouchi Y, Gunji T, Burns J C, Shimizu C, Newburger J W, Yashiro M, et al. ITPKC functional polymorphism associated with Kawasaki disease susceptibility and formation of coronary artery aneurysms. Nat Genet. 2008; 40(1):35-42. [0099] 6. Makino N, Nakamura Y, Yashiro M, Sano T, Ae R, Kosami K, et al. Epidemiological observations of Kawasaki disease in Japan, 2013-2014. Pediatr Int. 2018; 60(6):581-7. [0100] 7. Fan Q, Nelson C S, Bialas K M, Chiuppesi F, Amos J, Gurley T C, et al. Plasmablast Response to Primary Rhesus Cytomegalovirus (CMV) Infection in a Monkey Model of Congenital CMV Transmission. Clin Vaccine Immunol. 2017; 24(5). [0101] 8. Chai N, Swem L R, Park S, Nakamura G, Chiang N, Estevez A, et al. A broadly protective therapeutic antibody against influenza B virus with two mechanisms of action. Nat Commun. 2017; 8:14234. [0102] 9. Kauffman R C, Bhuiyan T R, Nakajima R, Mayo-Smith L M, Rashu R, Hoq M R, et al. Single-Cell Analysis of the Plasmablast Response to Vibrio cholerae Demonstrates Expansion of Cross-Reactive Memory B Cells. MBio. 2016; 7(6). [0103] 10. Garcia M, Iglesias A, Landoni V I, Bellomo C, Bruno A, Cordoba M T, et al. Massive plasmablast response elicited in the acute phase of hantavirus pulmonary syndrome. Immunology. 2017; 151(1):122-35. [0104] 11. Tanko R F, Soares A P, Muller T L, Garrett N J, Samsunder N, Abdool Karim Q, et al. Effect of Antiretroviral Therapy on the Memory and Activation Profiles of B Cells in HIV-Infected African Women. J Immunol. 2017; 198(3):1220-8. [0105] 12. Priyamvada L, Quicke K M, Hudson W H, Onlamoon N, Sewatanon J, Edupuganti S, et al. Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus. Proc Natl Acad Sci USA. 2016; 113(28):7852-7. [0106] 13. Priyamvada L, Cho A, Onlamoon N, Zheng N Y, Huang M, Kovalenkov Y, et al. B Cell Responses during Secondary Dengue Virus Infection Are Dominated by Highly Cross-Reactive, Memory-Derived Plasmablasts. J Virol. 2016; 90(12):5574-85. [0107] 14. Carter M J, Mitchell R M, Meyer Sauteur P M, Kelly D F, and Truck J. The Antibody-Secreting Cell Response to Infection: Kinetics and Clinical Applications. Frontiers in immunology. 2017; 8:630. [0108] 15. Chen Y Q, Wohlbold T J, Zheng N Y, Huang M, Huang Y, Neu K E, et al. Influenza Infection in Humans Induces Broadly Cross-Reactive and Protective Neuraminidase-Reactive Antibodies. Cell. 2018; 173(2):417-29 e10. [0109] 16. Ellebedy A H, Jackson K J, Kissick H T, Nakaya H I, Davis C W, Roskin K M, et al. Defining antigen-specific plasmablast and memory B cell subsets in human blood after viral infection or vaccination. Nat-Immunol. 2016; 17(10):1226-34. [0110] 17. Regules J A, Cicatelli S B, Bennett J W, Paolino K M, Twomey P S, Moon J E, et al. Fractional Third and Fourth Dose of RTS,S/AS01 Malaria Candidate Vaccine: A Phase 2a Controlled Human Malaria Parasite Infection and Immunogenicity Study. J Infect Dis. 2016; 214(5):762-71. [0111] 18. Shaw J M, Miller-Novak L K, Mohanram V, McKinnon K, Demberg T, Vargas-Inchaustegui D A, et al. Influence of Plasma Cell Niche Factors on the Recruitment and Maintenance of IRF4hi Plasma Cells and Plasmablasts in Vaccinated, Simian Immunodeficiency Virus-Infected Rhesus Macaques with Low and High Viremia. J Virol. 2017; 91(4). [0112] 19. Stanfield B A, Pahar B, Chouljenko V N, Veazey R, and Kousoulas K G. Vaccination of rhesus macaques with the live-attenuated HSV-1 vaccine VC2 stimulates the proliferation of mucosal T cells and germinal center responses resulting in sustained production of highly neutralizing antibodies. Vaccine. 2017; 35(4):536-43. [0113] 20. Pitisuttithum P, Boonnak K, Chamnanchanunt S, Puthavathana P, Luvira V, Lerdsamran H, et al. Safety and immunogenicity of a live attenuated influenza H5 candidate vaccine strain A/17/turkey/Turkey/05/133 H5N2 and its priming effects for potential pre-pandemic use: a randomised, double-blind, placebo-controlled trial. Lancet Infect Dis. 2017. [0114] 21. Kim J H, Mishina M, Chung J R, Cole K S, Nowalk M P, Martin J M, et al. Cell-Mediated Immunity Against Antigenically Drifted Influenza A (H3N2) Viruses in Children During a Vaccine Mismatch Season. J Infect Dis. 2016; 214(7):1030-8. [0115] 22. Mei H E, Hahne S, Redlin A, Hoyer B F, Wu K, Baganz L, et al. Plasmablasts with a mucosal phenotype contribute to plasmacytosis in SLE. Arthritis Rheumatol. 2017. [0116] 23. Kinslow J D, Blum L K, Deane K D, Demoruelle M K, Okamoto Y, Parish M C, et al. Elevated IgA Plasmablast Levels in Subjects at Risk of Developing Rheumatoid Arthritis. Arthritis Rheumatol. 2016; 68(10):2372-83. [0117] 24. Wrammert J, Smith K, Miller J, Langley W A, Kokko K, Larsen C, et al. Rapid cloning of high-affinity human monoclonal antibodies against influenza virus. Nature. 2008; 453(7195):667-71. [0118] 25. Tan Y C, Blum L K, Kongpachith S, Ju C H, Cai X, Lindstrom T M, et al. High-throughput sequencing of natively paired antibody chains provides evidence for original antigenic sin shaping the antibody response to influenza vaccination. Clin Immunol. 2014; 151(1):55-65. [0119] 26. Lu D R, Tan Y C, Kongpachith S, Cai X, Stein E A, Lindstrom T M, et al. Identifying functional anti-Staphylococcus aureus antibodies by sequencing antibody repertoires of patient plasmablasts. Clin Immunol. 2014; 152(1-2):77-89. [0120] 27. Wrammert J, Onlamoon N, Akondy R S, Perng G C, Polsrila K, Chandele A, et al. Rapid and massive virus-specific plasmablast responses during acute dengue virus infection in humans. J Virol. 2012; 86(6):2911-8. [0121] 28. Rowley A H, Shulman S T, Spike B T, Mask C A, and Baker S C. Oligoclonal IgA response in the vascular wall in acute Kawasaki disease. J Immunol. 2001; 166(2):1334-43. [0122] 29. Rowley A H, Baker S C, Shulman S T, Garcia F L, Guzman-Cottrill J A, Chou P, et al. Detection of antigen in bronchial epithelium and macrophages in acute Kawasaki disease by use of synthetic antibody. J Infect Dis. 2004; 190(4):856-65. [0123] 30. Rowley A H, Shulman S T, Garcia F L, Guzman-Cottrill J A, Miura M, Lee H L, et al. Cloning the arterial IgA antibody response during acute Kawasaki disease. J Immunol. 2005; 175(12):8386-91. [0124] 31. Rowley A H, Baker S C, Shulman S T, Fox L M, Takahashi K, Garcia F L, et al. Cytoplasmic inclusion bodies are detected by synthetic antibody in ciliated bronchial epithelium during acute Kawasaki disease. J Infect Dis. 2005; 192(10):1757-66. [0125] 32. Fink K. Origin and Function of Circulating Plasmablasts during Acute Viral Infections. Frontiers in immunology. 2012; 3:78. [0126] 33. DeKosky B J, Lungu O I, Park D, Johnson E L, Charab W, Chrysostomou C, et al. Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires. Proc Natl Acad Sci USA. 2016; 113(19):E2636-45. [0127] 34. Georgiou G, Ippolito G C, Beausang J, Busse C E, Wardemann H, and Quake S R. The promise and challenge of high-throughput sequencing of the antibody repertoire. Nat Biotechnol. 2014; 32(2):158-68. [0128] 35. Rowley A H, Baker S C, Shulman S T, Rand K H, Tretiakova M S, Perlman E J, et al. Ultrastructural, immunofluorescence, and RNA evidence support the hypothesis of a “new” virus associated with Kawasaki disease. J Infect Dis. 2011; 203(7):1021-30. [0129] 36. Rowley A H, Baker S C, Shulman S T, Garcia F L, Fox L M, Kos I M, et al. RNA-containing cytoplasmic inclusion bodies in ciliated bronchial epithelium months to years after acute Kawasaki disease. PLoS ONE. 2008; 3(2):e1582. [0130] 37. Forsstrom B, Axnas B B, Stengele K P, Buhler J, Albert T J, Richmond T A, et al. Proteome-wide epitope mapping of antibodies using ultra-dense peptide arrays. Molecular & cellular proteomics: MCP. 2014; 13(6):1585-97. [0131] 38. Rowley A H, Wylie K M, Kim K Y, Pink A J, Yang A, Reindel R, et al. The transcriptional profile of coronary arteritis in Kawasaki disease. BMC Genomics. 2015; 16(1):1076. [0132] 39. Matsuda S, and Senda T. BRI2 as an anti-Alzheimer gene. Med Mol Morphol. 2019; 52(1):1-7. [0133] 40. Rowley A H, and Shulman S T. The Epidemiology and Pathogenesis of Kawasaki Disease. Front Pediatr. 2018; 6:374. [0134] 41. Cameron S A, Carr M, Pahl E, DeMarais N, Shulman S T, and Rowley A H. Coronary artery aneurysms are more severe in infants than in older children with Kawasaki disease. Arch Dis Child. 2019; 104(5):451-5. [0135] 42. Makino N, Nakamura Y, Yashiro M, Kosami K, Matsubara Y, Ae R, et al. Nationwide epidemiologic survey of Kawasaki disease in Japan, 2015-2016. Pediatr Int. 2019; 61(4):397-403. [0136] 43. Maddox R A, Holman R C, Uehara R, Callinan L S, Guest J L, Schonberger L B, et al. Recurrent Kawasaki disease: USA and Japan. Pediatr Int. 2015; 57(6):1116-20. [0137] 44. Bell D M, Brink E W, Nitzkin J L, Hall C B, Wulff H, Berkowitz I D, et al. Kawasaki syndrome: description of two outbreaks in the United States. N Engl J Med. 1981; 304(26):1568-75. [0138] 45. Dean A G, Melish M E, Hicks R, and Palumbo N E. An epidemic of Kawasaki syndrome in Hawaii. J Pediatr. 1982; 100(4):552-7. [0139] 46. Salo E, Pelkonen P, and Pettay O. Outbreak of Kawasaki syndrome in Finland. Acta Paediatr Scand. 1986; 75(1):75-80. [0140] 47. Rowley A H, Eckerley C A, Jack H M, Shulman S T, and Baker S C. IgA plasma cells in vascular tissue of patients with Kawasaki syndrome. J Immunol. 1997; 159(12):5946-55. [0141] 48. Rowley A H, Shulman S T, Mask C A, Finn L S, Terai M, Baker S C, et al. IgA plasma cell infiltration of proximal respiratory tract, pancreas, kidney, and coronary artery in acute Kawasaki disease. J Infect Dis. 2000; 182(4):1183-91. [0142] 49. Hartlage A S, Cullen J M, and Kapoor A. The Strange, Expanding World of Animal Hepaciviruses. Annu Rev Virol. 2016; 3(1):53-75. [0143] 50. El-Attar L M, Mitchell J A, Brooks Brownlie H, Priestnall S L, and Brownlie J. Detection of non-primate hepaciviruses in UK dogs. Virology. 2015; 484:93-102. [0144] 51. Pfaender S, Cavalleri J M, Walter S, Doerrbecker J, Campana B, Brown R J, et al. Clinical course of infection and viral tissue tropism of hepatitis C virus-like nonprimate hepaciviruses in horses. Hepatology. 2015; 61(2):447-59. [0145] 52. Kawasaki T. [Acute febrile mucocutaneous syndrome with lymphoid involvement with specific desquamation of the fingers and toes in children]. Arerugi. 1967; 16(3):178-222. [0146] 53. Kobayashi T, Saji T, Otani T, Takeuchi K, Nakamura T, Arakawa H, et al. Efficacy of immunoglobulin plus prednisolone for prevention of coronary artery abnormalities in severe Kawasaki disease (RAISE study): a randomised, open-label, blinded-endpoints trial. Lancet. 2012; 379(9826):1613-20. [0147] 54. Ho I Y, Bunker T T, Erickson S A, Neu K E, Huang M, Cortese M, et al. Refined protocol for generating monoclonal antibodies from single human and murine B cells. J Immunol Methods. 2016; 438:67-70. [0148] 55. Smith K, Garman L, Wrammert J, Zheng N Y, Capra J D, Ahmed R, et al. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat Protoc. 2009; 4(3):372-84. [0149] 56. Wardemann H, Yurasov S, Schaefer A, Young J W, Meffre E, and Nussenzweig M C. Predominant autoantibody production by early human B cell precursors. Science. 2003; 301(5638):1374-7. [0150] 57. Pugh-Bernard A E, Silverman G J, Cappione A J, Villano M E, Ryan D H, Insel R A, et al. Regulation of inherently autoreactive VH4-34 B cells in the maintenance of human B cell tolerance. J Clin Invest. 2001; 108(7):1061-70. [0151] 58. Mishra N, Caciula A, Price A, Thakkar R, Ng J, Chauhan L V, et al. Diagnosis of Zika Virus Infection by Peptide Array and Enzyme-Linked Immunosorbent Assay. MBio. 2018; 9(2). [0152] 59. Guan K L, and Dixon J E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991; 192(2):262-7.