Anti-thyroid peroxidase antibodies and immunoassays

Abstract

Disclosed herein are immunoassays for detecting anti-thyroid peroxidase antibodies in a biological sample from a subject and / or diagnosing a thyroid disease in a subject. The disclosed immunoassays employ recombinant thyroid peroxidase (rTPO) and anti-TPO monoclonal antibodies to assess the level of anti-thyroid peroxidase antibody-induced formation or disruption of complexes comprising a solid support and the rTPO. Also disclosed are methods and kits thereof.

Description

ANTI-THYROID PEROXIDASE ANTIBODIES AND IMMUNOASSAYS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No.63 / 709,503, filed October 20, 2024, and U.S. Provisional Patent Application Serial No.63 / 745,347, filed January 15, 2025, which are hereby incorporated by reference in their entirety. FIELD

[0002] Disclosed herein are anti-thyroid peroxidase (aTPO) antibodies, recombinant TPO antigens, and immunoassays for the detection of autoantibodies against TPO, including methods, compositions, and kits employing monoclonal antibodies and methods for diagnosis of autoimmune thyroid diseases. SEQUENCE LISTING STATEMENT

[0003] This application contains a computer readable Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file was created on September 25, 2025, is named 137519016402.xml and is 162,885 bytes in size. BACKGROUND

[0004] In the developed world in general, the prevalence of undiagnosed thyroid disease is likely falling due to widespread thyroid function testing and relatively low thresholds for treatment initiation. Taylor et al., “Global Epidemiology of Hyperthyroidism and Hypothyroidism,” Nat. Rev. Endocrinol.14(5):301-316 (2018). High prevalence of thyroid disease is found in countries such as India, China, and Brazil. Taylor et al., “Global Epidemiology of Hyperthyroidism and Hypothyroidism,” Nat. Rev. Endocrinol.14(5):301- 316 (2018).

[0005] Thyroid diseases affect an estimated 200 million people worldwide. Zhang et al., “Prevalence and Trends of Thyroid Disease Among Adults, 1999-2018,” Endocrine Practice 29(11):875-880 (2023). Two billion people are at risk for iodine deficiency that may lead to thyroid disease. Hatch-McChesney & Lieberman, “Iodine and Iodine Deficiency: A Comprehensive Review of a Re-Emerging Issue,” Nutrients 14(17):3474 (2022). Thyroid diseases are more prevalent in women than in men, at a ratio of 8:1.

[0006] Hypothyroidism is characterized by low levels of T3 (tri-iodothyronine) and T4 (thyroxine). Patient assessment, beginning with the measurement of TSH, is the best indicator of hypothyroidism. Klee & Hay, “Biochemical Testing of Thyroid Function,” Endocrinol. Metab. Clin. North Am.26:763-775 (1997). Possible complications of untreated hypothyroidism include increased risk of heart disease, pregnancy complications, infertility, and depression.

[0007] Hyperthyroidism results from overproduction of T3 and T4 hormones. The endogenous condition occurs relatively infrequently, while exogenous hyperthyroidism states may be triggered by excess medication. Graves’ disease is a common form of hyperthyroidism. Excess thyroid hormones impact cardiac performance, the renal and skeletal systems, liver function, and the female reproductive system.

[0008] Thyroid peroxidase (TPO), a protein found in thyroid follicular cells, is a catalyst in the synthesis of key thyroid hormones. Autoimmune thyroid diseases are characterized by the presence of anti-thyroid peroxidase antibodies (aTPO).

[0009] Elevated levels of anti-TPO antibodies are a risk factor for autoimmune thyroid disease, including Hashimoto’s thyroiditis, Graves’ disease, atrophic thyroiditis, primary myxedema, and postpartum thyroiditis in women.

[0010] Critical thyroid glycoprotein is essential for thyroxine (T4) and triiodothyronine (T3) synthesis. Anti-thyroid peroxidase antibodies attach to TPO and affect hormone levels. High levels of these antibodies are a sign of autoimmune thyroid disease (AITD).

[0011] aTPO testing is an important aspect of laboratory testing for the diagnosis of AITD. Ragusa et al., “Hashimotos’ Thyroiditis: Epidemiology, Pathogenesis, Clinic and Therapy,” Best Pract. Res. Clin. Endocrinol. Metab.33(6):101367 (2019). In addition to this role in diagnosis, aTPO may potentially play a role in the pathogenesis of AITD. Rifai et al., Textbook of Clinical Chemistry and Molecular Diagnostics, 6th ed., 2018. Studies have demonstrated that patients with subclinical hypothyroidism and elevated aTPO progress to overt hypothyroidism at a rate greater than patients who are below the cut-off for aTPO. Soh & Aw, “Laboratory Testing in Thyroid Conditions - Pitfalls and Clinical Utility,” Ann Lab Med.39(1):3–14 (2019). In this context, monitoring of aTPO is recommended. There is a need for rapid reliable assays to detect aTPO in patient samples.

[0012] The present disclosure is directed at overcoming these and other deficiencies in the art.SUMMARY

[0013] A first aspect of the present disclosure relates to an anti-thyroid peroxidase (TPO) antibody (anti-TPO antibody), or antigen-binding fragment thereof, wherein the antibody comprises: (1) a heavy chain variable region comprising a heavy chain complementarity determining region 1 (CDR1H1), a heavy chain complementarity determining region 2 (CDRH2), and a heavy chain complementarity determining region 3 (CDRH3) and (2) a light chain variable region comprising a light chain complementarity determining region 1 (CDR1L1), a light chain complementarity determining region 2 (CDRL2), and a light chain complementarity determining region 3 (CDRL3), wherein the CDRH1 comprises the sequence TYVIH (SEQ ID NO:22), the CDRH2 comprises the sequence YIIPYNDRTRYYEKFKD (SEQ ID NO:24), the CDRH3 comprises the sequence GGGFTTIGDWYFDV (SEQ ID NO:26), the CDRL1 comprises the sequence KASQDINNYLS (SEQ ID NO:40), the CDRL2 comprises the sequence RANRLID (SEQ ID NO:42), and the CDRL3 comprises the sequence LQYAEYPFM (SEQ ID NO:44).

[0014] Another aspect of the present disclosure relates to an anti-thyroid peroxidase (TPO) antibody (anti-TPO antibody), or antigen-binding fragment thereof, wherein the antibody comprises: (1) a heavy chain variable region comprising a heavy chain complementarity determining region 1 (CDR1H1), a heavy chain complementarity determining region 2 (CDRH2), and a heavy chain complementarity determining region 3 (CDRH3), (2) a first light chain variable region comprising a light chain complementarity determining region 1 (CDR1L1), a light chain complementarity determining region 2 (CDRL2), and a light chain complementarity determining region 3 (CDRL3), and (3) a second light chain variable region comprising a light chain complementarity determining region 1 (CDR1L2-1), a light chain complementarity determining region 2 (CDRL2-2), and a light chain complementarity determining region 3 (CDRL2-3), wherein the CDRH1 comprises the sequence TSGMGVS (SEQ ID NO:58), the CDRH2 comprises the sequence HIYWDDDKRYNPSLMN (SEQ ID NO:60), the CDRH3 comprises the sequence RDGGNFGNNYYALDY (SEQ ID NO:62), the CDRL1 comprises the sequence RASKSVSTSGYNFMH (SEQ ID NO:76), the CDRL2 comprises the sequence LASYLKS (SEQ ID NO:78), the CDRL3 comprises the sequence QHSREFPWT (SEQ ID NO:80), the CDRL2-1 comprises the sequence RASKSVSTSGYSYMH (SEQ ID NO:94),the CDRL2-2 comprises the sequence LASYLKS (SEQ ID NO:96), and the CDRL2-3 comprises the sequence QHSREFPWT (SEQ ID NO:98).

[0015] A further aspect of the present disclosure relates to a method of detecting an anti-thyroid peroxidase (TPO) antibody in a biological sample from a subject. This method involves incubating the biological sample from the subject with (i) a solid support, (ii) an unlabeled anti-TPO antibody or antigen-binding fragment thereof as described herein, (iii) a recombinant TPO (rTPO), and (iv) a labeled anti-TPO antibody or antigen binding fragment thereof as described herein; and detecting the anti-thyroid peroxidase antibody in the biological sample, said detecting comprising analyzing a decrease in the formation of a complex comprising the solid support, the unlabeled anti-TPO antibody, the rTPO, and the labeled anti-TPO antibody.

[0016] Another aspect of the present disclosure relates to a method of diagnosing a thyroid disease in a subject. This method involves incubating a biological sample from a subject with (i) a solid support, (ii) an unlabeled anti-TPO antibody or antigen-binding fragment thereof of an antibody described herein, (iii) a recombinant TPO (rTPO), and (iv) a labeled anti-TPO antibody or antigen binding fragment thereof of an antibody described herein; and diagnosing the subject with the thyroid disease if the formation of a complex comprising the solid support, the unlabeled anti-TPO antibody, the rTPO, and the labeled anti-TPO antibody is decreased.

[0017] Novel monoclonal antibodies described herein may be used in conjunction with a recombinant TPO antigen, in a fully automated 1-step competitive immunoassay using acridinium ester (AE) chemiluminescent technology. The first antibody, in the Solid Phase, is a biotinylated mouse monoclonal anti-TPO antibody that is bound to streptavidin-coated paramagnetic microparticles. The second antibody is a mouse monoclonal anti-TPO antibody labeled with acridinium ester in a preformed complex with unlabeled recombinant thyroid peroxidase (rTPO).

[0018] Successful bridging of the Solid Phase antibody with the AE-labeled antibody and rTPO in the Lite Reagent will result in signal generation. Anti-TPO autoantibodies present in the sample will compete with the Solid Phase biotinylated anti-TPO monoclonal antibody for binding to rTPO and reduce the signal.

[0019] An inverse relationship exists between the amount of anti-TPO autoantibody present in the patient sample and the amount of relative light units (RLUs) detected by the system.

[0020] Advantages of the immunoassay described herein include the advantages of specific immunological properties tied to a recombinant antigen.

[0021] The anti-thyroid peroxidase antibodies described herein enhance assay architecture to leverage a recombinant antigen and enable improved analytical performance including precision, specificity, and sensitivity, while supporting better sustainability of supply. Other benefits include improved shelf-life, on-board stability, and calibration intervals. The antibodies and assays described herein are a REACH compliant formulation that protects the environment by eliminating the use of hazardous materials and ensure safer product waste management.

[0022] The anti-thyroid peroxidase assays described herein (also called anti-TPO II assays, herein) are for in vitro diagnostic use in the quantitative measurement of autoantibodies against thyroid peroxidase in human serum and plasma. In some embodiments, the immunoassays use the Atellica® Immunoassay Analyzer, ADVIA Centaur® XP, and ADVIA Centaur® XPT systems. In some embodiments, anti-thyroid peroxidase (aTPO) measurements are used, in conjunction with a clinical assessment, as an aid in the diagnosis of, e.g., autoimmune thyroiditis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG.1 is a schematic illustration of one embodiment of an immunoassay of the present disclosure. A timeline of an exemplary competitive assay format performed on the ADVIA Centaur® system is shown at the top. Shown below the timeline is an immune complex including an anti-TPO IgG Fab’2 antibody fragment (derived from SHC 194) in green that is labeled with acridinium ester in a complex with a recombinant TPO antigen (pink). Also shown is a biotinylated mouse monoclonal anti-TPO antibody (SHC 195; purple) bound to a streptavidin-coated paramagnetic microparticle. SHC 194 is also referred to herein as 7G11. SHC 195 is also referred to herein as 12F11.

[0024] FIG.2 is a schematic illustration of one embodiment of an immunoassay of the present disclosure. A timeline of an exemplary competitive assay performed on the Atellica® Immunoassay Analyzer system is shown at the top. Shown below the timeline is an immune complex including an anti-TPO IgG Fab’2 antibody fragment (derived from SHC 194) in green that is labeled with acridinium ester in a complex with a recombinant TPO antigen (pink). Also shown is a biotinylated mouse monoclonal anti-TPO antibody (SHC 195; purple) bound to a streptavidin-coated paramagnetic microparticle.

[0025] FIG.3 is a graph showing precision across the assay range for an aTPO assay of the present disclosure (also called an aTPOII assay herein) performed on the Atellica® system.

[0026] FIG.4 is a graph showing precision across the assay range for an aTPO assay of the present disclosure (also called an aTPOII assay herein) performed on the ADVIA Centaur® system.

[0027] FIG.5 shows the evaluation of aTPO mouse monoclonal antibody SHC 194 (7G11). FIG.5 (left) shows a table of the number of clones sequenced and the sequence identity for each of the sequences of SHC 194. FIG.5 (right) shows an illustration of an antibody.

[0028] FIG.6 shows the evaluation of aTPO mouse monoclonal antibody SHC 195 (12F11). FIG.6 (left) shows a table of the number of clones sequenced and the sequence identity for each of the sequences of SHC 195. FIG.6 (right) shows an illustration of an antibody.

[0029] FIGs.7A-B are graphs showing the kinetics of aTPO antibody 7G11 (SHC 194) Fab response over time.

[0030] FIGs.8A-B are graphs showing the kinetics of aTPO antibody 12F11 (SHC 195) Fab response over time. DETAILED DESCRIPTION

[0031] The disclosed antibodies, methods, and kits may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that the disclosed antibodies, methods, and kits are not limited to the specific antibodies, methods, and kits described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed antibodies, methods, and kits.

[0032] Unless specifically stated otherwise, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosed antibodies, methods, and kits are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.

[0033] Throughout this text, the descriptions refer to methods of detecting an antibody and methods of diagnosing a thyroid disease. Where the disclosure describes or claims a feature or embodiment associated with a method of detecting an antibody, such a feature or embodiment is equally applicable to the methods of diagnosing a thyroid disease. Likewise, where the disclosure describes or claims a feature or embodiment associated with a method of diagnosing a thyroid disease, such a feature or embodiment is equally applicable to the methods of detecting an antibody.

[0034] Where a range of numerical values is recited or established herein, the range includes the endpoints thereof and ail the individual integers and fractions within the range, and also includes each of the narrower ranges therein formed by all the various possible combinations of those endpoints and internal integers and fractions to form subgroups of the larger group of values within the stated range to the same extent as if each of those narrower ranges was explicitly recited. Where a range of numerical values is stated herein as being greater than a stated value, the range is nevertheless finite and is bounded on its upper end by a value that is operable within the context of the invention as described herein. Where a range of numerical values is stated herein as being less than a stated value, the range is nevertheless bounded on its lower end by a non-zero value. It is not intended that the scope of the invention be limited to the specific values recited when defining a range. All ranges are inclusive and combinable.

[0035] When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.

[0036] It is to be appreciated that certain features of the disclosed products, methods, and kits which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed products, methods, and kits that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

[0037] As used herein, the singular forms “a,” “an,” and “the” include the plural.

[0038] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0039] The term “comprising” is intended to include examples encompassed by the terms “consisting essentially of” and “consisting of”; similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.”

[0040] A “reference sequence” means a nucleic acid or amino acid used as a comparator for another nucleic acid or amino acid, respectively, when determining sequence identity. A reference sequence can be a wild-type sequence. “Sequence identity,” “percent identity,” or “% identical” refers to the exactness of a match between a reference sequence and a sequence being compared to it when optimally aligned. For example, sequence alignments and percent identity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the Multalin program (Corpet, “Multiple Sequence Alignment with Hierarchical Clustering,” Nucleic Acids Res.16:10881-90 (1988), which is hereby incorporated by reference in its entirety) or the Megalign®program of the LASERGENE®bioinformatics computing suite (DNASTAR®Inc., Madison, Wis.). Sequences may also be aligned using algorithms known in the art including, but not limited to, CLUSTAL V algorithm or the BLASTN or BLAST 2 sequence programs.

[0041] Disclosed herein are antibodies, immunoassays, and methods for detecting anti-thyroid peroxidase (anti-TPO) antibodies in a biological sample from a subject and / or diagnosing a thyroid disease in a subject.

[0042] The terms “antibody” and “antigen-binding fragment thereof” encompass any modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The term “complementarity determining region” or “CDR” is defined as the portion(s) of an antibody variable chain that bind to its specific antigen.

[0043] Thyroid peroxidase autoantibody immunoassays are described in U.S. Patent No.11,327,074 and U.S. Patent No.11,835,518, which are hereby incorporated by reference in their entirety. Previous versions of commercialized diagnostic devices employed monoclonal antibodies with native, human sourced antigen and / or anti-human secondary antibodies.

[0044] A first aspect of the present disclosure relates to an anti-thyroid peroxidase (TPO) antibody (anti-TPO antibody), or antigen-binding fragment thereof, wherein the antibody comprises: (1) a heavy chain variable region comprising a heavy chaincomplementarity determining region 1 (CDR1H1), a heavy chain complementarity determining region 2 (CDRH2), and a heavy chain complementarity determining region 3 (CDRH3) and (2) a light chain variable region comprising a light chain complementarity determining region 1 (CDR1L1), a light chain complementarity determining region 2 (CDRL2), and a light chain complementarity determining region 3 (CDRL3), and wherein the CDRH1 comprises the sequence TYVIH (SEQ ID NO:22), the CDRH2 comprises the sequence YIIPYNDRTRYYEKFKD (SEQ ID NO:24), the CDRH3 comprises the sequence GGGFTTIGDWYFDV (SEQ ID NO:26), the CDRL1 comprises the sequence KASQDINNYLS (SEQ ID NO:40), the CDRL2 comprises the sequence RANRLID (SEQ ID NO:42), and the CDRL3 comprises the sequence LQYAEYPFM (SEQ ID NO:44).

[0045] In some embodiments, the anti-TPO antibody is SHC 194, comprising the following sequences: Heavy chain: DNA sequence (1401 bp) (SEQ ID NO:3) Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region-Stop codon ATGGAATGGAGTTGGATATTTCTCTTTCTCCTGTCAGGAACTGCAGGTGTCCACTCTGAGGT CCAGCTGCAACAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCA AGGCCTCTGGATACACATTCACTACCTATGTTATACACTGGGTGAAGCAGAAGCCTGGGCAG GGCCTTGAGTGGATTGGATATATTATTCCTTACAATGATCGGACTAGGTATTATGAGAAGTT CAAAGACAAGGCCACACTGACTTCAGACAAATCCTCCAGTACAGCCTACATGGAACTCAGCA GCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGGTGGGGGATTCACTACAATA GGGGACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGCCAAAAC GACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGA CCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGA TCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAG CAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCC ACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCT TGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGT GCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATC CCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCC CGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGA CTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCT CCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCC TGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTC AGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGC AACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCA TACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA Heavy chain: Amino acid sequence (466 aa) (SEQ ID NO:1): Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region-Stop codon MEWSWIFLFLLSGTAGVHSEVQLQQSGPELVKPGASVKMSCKASGYTFTTYVIHWVKQKPGQ GLEWIGYIIPYNDRTRYYEKFKDKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGGFTTI GDWYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSG SLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKP CICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQP REEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPP PKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKS NWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK- Light chain: DNA sequence (705 bp) (SEQ ID NO:4): Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region-Stop codon ATGAGGACCCCTGCTCAGTTTCTTGGAATCTTGTTGCTCTGGTTTCCAGGTATCAAATGTGA CATCAAGATGACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTATCA CTTGCAAGGCGAGTCAGGACATTAATAACTATTTAAGCTGGTTCCAGCAGAAACCAGGGAAA TCTCCTAAGACCCTGATCTATCGTGCAAACAGATTGATAGATGGGGTCCCATCAAGGTTCAG TGGCAGTGGATCTGGGCAAGATTATTCTCTCACCATCAGCAGCCTAGAATATGAAGATGTGG GAATTTATTATTGTCTACAGTATGCTGAATATCCATTCATGTTCGGCTCGGGGACAAAGTTG GAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTT AACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATG TCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAG GACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGA ACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGA GCTTCAACAGGAATGAGTGTTAGLight chain: Amino acid sequence (234 aa) (SEQ ID NO:2): Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region-Stop codon MRTPAQFLGILLLWFPGIKCDIKMTQSPSSMYASLGERVTITCKASQDINNYLSWFQQKPGK SPKTLIYRANRLIDGVPSRFSGSGSGQDYSLTISSLEYEDVGIYYCLQYAEYPFMFGSGTKL EIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ DSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC-

[0046] In some embodiments the SHC 194 heavy chain sequence does not comprise a signal peptide: Heavy chain without the signal sequence and stop codon: Amino acid sequence (447 aa) (SEQ ID NO:101): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region EVQLQQSGPELVKPGASVKMSCKASGYTFTTYVIHWVKQKPGQGLEWIGYIIPYNDRTRYYE KFKDKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGGFTTIGDWYFDVWGAGTTVTVSSA KTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYT LSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPK DVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMH QDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDF FPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHN HHTEKSLSHSPGK

[0047] In some embodiments the SHC 194 light chain sequence does not comprise a signal peptide: Light chain: Amino acid sequence without the signal sequence and stop codon (214 aa) (SEQ ID NO:102): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region DIKMTQSPSSMYASLGERVTITCKASQDINNYLSWFQQKPGKSPKTLIYRANRLIDGVPSRF SGSGSGQDYSLTISSLEYEDVGIYYCLQYAEYPFMFGSGTKLEIKRADAAPTVSIFPPSSEQ LTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEY ERHNSYTCEATHKTSTSPIVKSFNRNEC

[0048] In some embodiments, the anti-TPO antibody, or antigen-binding fragment thereof according to this aspect further comprises: (3) a heavy chain variable region further comprising a heavy chain framework region 1 (FRH1), a heavy chain framework region 2 (FRH2), a heavy chain framework region 3 (FRH3), and a heavy chain framework region 4(FRH4) and (4) a light chain variable region comprising a light chain framework region 1 (FRL1), a light chain comprising a light chain framework region 2 (FRL2), a light chain comprising a light chain framework region 3 (FRL3), and a light chain comprising a light chain framework region 4 (FRL4), and wherein the FRH1 comprises the sequence EVQLQQSGPELVKPGASVKMSCKASGYTFT (SEQ ID NO:21), the FRH2 comprises the sequence WVKQKPGQGLEWIG (SEQ ID NO:23), the FRH3 comprises the sequence KATLTSDKSSSTAYMELSSLTSEDSAVYYCAR (SEQ ID NO:25), the FRH4 comprises the sequence WGAGTTVTVSS (SEQ ID NO:27), the FRL1 comprises the sequence DIKMTQSPSSMYASLGERVTITC (SEQ ID NO:39), the FRL2 comprises the sequence WFQQKPGKSPKTLIY (SEQ ID NO:41), the FRL3 comprises the sequence GVPSRFSGSGSGQDYSLTISSLEYEDVGIYYC (SEQ ID NO:43), and the FRL4 comprises the sequence FGSGTKLEIK (SEQ ID NO:45).

[0049] In some embodiments, the anti-TPO antibody, or antigen-binding fragment thereof, according to this aspect of the disclosure, comprises: (i) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is identical, to the sequence of EVQLQQSGPELVKPGASVKMSCKASGYTFTTYVIHWVKQKPGQGLEWIGYIIPYND RTRYYEKFKDKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGGFTTIGDWYFDV WGAGTTVTVSS (SEQ ID NO:108), provided that the heavy chain variable region comprises the sequences of CDRH1, CDRH2, and CDRH3 of SEQ ID NOs: 22, 24, and 26, respectively; and (ii) a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is identical, to the sequence of DIKMTQSPSSMYASLGERVTITCKASQDINNYLSWFQQKPGKSPKTLIYRANRLIDGV PSRFSGSGSGQDYSLTISSLEYEDVGIYYCLQYAEYPFMFGSGTKLEIK (SEQ ID NO:110), provided that the light chain variable region comprises the sequences of CDRL1, CDRL2, and CDRL3 of SEQ ID NOs: 40, 42, and 44, respectively.

[0050] In some embodiments, the anti-TPO antibody, or antigen-binding fragment thereof, according to this aspect of the disclosure, comprises: (i) a heavy chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is identical, to the sequence of EVQLQQSGPELVKPGASVKMSCKASGYTFTTYVIHWVKQKPGQGLEWIGYIIPYND RTRYYEKFKDKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGGFTTIGDWYFDV WGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGS LSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCG CKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVH TAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRP KAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMD TDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:101) provided that the heavy chain comprises the sequences of CDRH1, CDRH2, and CDRH3 of SEQ ID NOs: 22, 24, and 26, respectively; and (ii) a light chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is identical, to the sequence of DIKMTQSPSSMYASLGERVTITCKASQDINNYLSWFQQKPGKSPKTLIYRANRLIDGV PSRFSGSGSGQDYSLTISSLEYEDVGIYYCLQYAEYPFMFGSGTKLEIKRADAAPTVSI FPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYS MSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:102), provided that the light chain comprises the sequences of CDRL1, CDRL2, and CDRL3 of SEQ ID NOs: 40, 42, and 44, respectively.

[0051] The amino acid and DNA sequences for the SHC 194 antibody are provided in Tables 1-6 below. Table 1. SHC 194 Antibody Heavy Chain and Light Chain Amino Acid Sequences SHC 194 Antibody Sequences (Amino Acid) SEQ ID NO(s):SDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIV PRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCTable 2. SHC 194 Antibody Heavy Chain and Light Chain DNA Sequences SHC 194 Antibody Sequences (DNA) SEQ ID NO(s):GACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGT CCTGCAAGGCCTCTGGATACACATTCACTACCTATGTTACTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCA CTATCACTTGCAAGGCGAGTCAGGACATTAATAACTATTa e . n oy eavy an equences SHC 194 Heavy Chain Sequences (DNA) SEQ ID NO.HC FR4TGGGGCGCAGGGACCACGGTCACCGTCTCCTCASEQ ID NO:18HC GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTAAAGCC SEQ ID NO:107Table 4. SHC 194 Antibody Heavy Chain Amino Acid Sequences SHC 194 Heavy Chain Sequences (Amino Acid) SEQ ID NO. HC SignalMEWSWIFLFLLSGTAGVHSSEQ ID NO:20Table 5. SHC 194 Antibody Light Chain DNA Sequences SHC 194 Light Chain Sequences (DNA) SEQ ID NO.LC FR3GGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGASEQ ID NO:34 TTATTCTCTCACCATCAGCAGCCTAGAATATGAAGATGTGGTable 6. SHC 194 Antibody Light Chain Amino Acid Sequences SHC 194 Light Chain Sequences (Amino Acid) SEQ ID NO.LC FR4FGSGTKLEIKSEQ ID NO:45LC DIKMTQSPSSMYASLGERVTITCKASQDINNYLSWFQQKPG SEQ ID NO:110e (TPO) antibody (anti-TPO antibody), or antigen-binding fragment thereof, wherein the antibody comprises: (1) a heavy chain variable region comprising a heavy chain complementarity determining region 1 (CDR1H1), a heavy chain complementarity determining region 2 (CDRH2), and a heavy chain complementarity determining region 3 (CDRH3), (2) a first light chain variable region comprising a light chain complementarity determining region 1 (CDR1L1), a light chain complementarity determining region 2 (CDRL2), and a light chain complementarity determining region 3 (CDRL3), and (3) a second light chain variable region comprising a light chain complementarity determining region 1 (CDR1L2-1), a light chain complementarity determining region 2 (CDRL2-2), and a light chain complementarity determining region 3 (CDRL2-3), wherein the CDRH1 comprises the sequence TSGMGVS (SEQ ID NO:58), the CDRH2 comprises the sequence HIYWDDDKRYNPSLMN (SEQ ID NO:60), the CDRH3 comprises the sequence RDGGNFGNNYYALDY (SEQ ID NO:62), the CDRL1 comprises the sequence RASKSVSTSGYNFMH (SEQ ID NO:76), the CDRL2 comprises the sequence LASYLKS (SEQ ID NO:78), the CDRL3 comprises the sequence QHSREFPWT (SEQ ID NO:80), the CDRL2-1 comprises the sequence RASKSVSTSGYSYMH (SEQ ID NO:94), the CDRL2-2 comprises the sequence LASYLKS (SEQ ID NO:96), and the CDRL2-3 comprises the sequence QHSREFPWT (SEQ ID NO:98).

[0053] In some embodiments, the anti-TPO antibody is SHC 195 comprising the following sequences: Heavy chain: DNA sequence (1407 bp) (SEQ ID NO:8) Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region-Stop codonATGGACAGGCTTACTTCCTCATTCCTGCTGCTGATTGTCCCTGCATATGTCCTTTCCCAAGT TACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTT CTTTTTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTGAGTTGGATTCGTCAGCCTTCA GGAAAGGGTCTGGAGTGGCTGGCACACATTTATTGGGATGATGACAAGCGCTACAATCCATC TCTGATGAACCGGCTCACAATCTCCAAGGATACCTCCAGAAACCACGTTTTCCTCAAGATCA CCAGTGTGGATTTGGCAGATACTGCCACATACTACTGTGCTCGAAGAGACGGAGGTAACTTC GGTAATAATTACTATGCTTTGGACTACTGGGGTCAAGGAATCTCCGTCACCGTCTCCTCAGC CAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCA TGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAAC TCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACAC TCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACG TTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGT AAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAA GGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGG ATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACG CAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCA CCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCC CCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATT CCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTT CTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGA ACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAG AAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAA CCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA Heavy chain: Amino acid sequence (468 aa) (SEQ ID NO:5) Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region-Stop codon MDRLTSSFLLLIVPAYVLSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPS GKGLEWLAHIYWDDDKRYNPSLMNRLTISKDTSRNHVFLKITSVDLADTATYYCARRDGGNF GNNYYALDYWGQGISVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWN SGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGC KPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQT QPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTI PPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQ KSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK-Light chain 1: DNA sequence (717 bp) (SEQ ID NO:9) Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region-Stop codon ATGGAGACAGACACACTCCTGTTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGA CATTGTGCTGACACAGTCTCCTGCTTCCTTAACTGTATCTCTGGGGCAGAGGGCCACCATCT CATGCAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAATTTTATGCACTGGTATCAACAG AAACCAGGACAGTCACCCAAACTCCTCATCTATCTTGCATCCTACCTAAAATCTGGGGTCCC TGCCAGGTTCACTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCTTTCCTGTGGAGG GGGAGGATGCTGCAACCTACTACTGTCAGCACAGTAGGGAGTTTCCGTGGACGTTCGGTGGA GGCACCAACCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATC CAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCA AAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGT TGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAA GGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCAC CCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG Light chain 1: Amino acid sequence (238 aa) (SEQ ID NO:6) Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region-Stop codon METDTLLLWVLLLWVPGSTGDIVLTQSPASLTVSLGQRATISCRASKSVSTSGYNFMHWYQQ KPGQSPKLLIYLASYLKSGVPARFTGSGSGTDFTLNIFPVEGEDAATYYCQHSREFPWTFGG GTNLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNS WTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC- Light chain 2: DNA sequence (717 bp) (SEQ ID NO:10) Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region-Stop codon ATGGAGACAGACACACTCCTGTTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGA CATTGTGCTGACACAGTCTCCTGCTTCCTTAACTGTATCTCTGGGGCAGAGGGCCACCATCT CATGCAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAG AAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGCATCCTACCTAAAATCTGGGGTCCC TGCCAGGTTCACTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCTTTCCTGTGGAGG GGGAGGATGCTGCAACCTACTACTGTCAGCACAGTAGGGAGTTTCCGTGGACGTTCGGTGGA GGCACCAACCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATC CAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCA AAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAA GGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCAC CCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG Light chain 2: Amino acid sequence (238 aa) (SEQ ID NO:7) Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region-Stop codon METDTLLLWVLLLWVPGSTGDIVLTQSPASLTVSLGQRATISCRASKSVSTSGYSYMHWNQQ KPGQPPRLLIYLASYLKSGVPARFTGSGSGTDFTLNIFPVEGEDAATYYCQHSREFPWTFGG GTNLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNS WTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC-

[0054] In some embodiments the SHC 195 heavy chain sequence does not comprise a signal peptide: Heavy chain without the signal peptide: Amino acid sequence (449 aa) (SEQ ID NO:103) FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYN PSLMNRLTISKDTSRNHVFLKITSVDLADTATYYCARRDGGNFGNNYYALDYWGQGISVTVS SAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDL YTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPK PKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPI MHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMIT DFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGL HNHHTEKSLSHSPGK

[0055] In some embodiments the SHC 195 light chain 1 sequence does not comprise a signal peptide: Light chain 1: Amino acid sequence (218 aa) (SEQ ID NO:104) FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region DIVLTQSPASLTVSLGQRATISCRASKSVSTSGYNFMHWYQQKPGQSPKLLIYLASYLKSGV PARFTGSGSGTDFTLNIFPVEGEDAATYYCQHSREFPWTFGGGTNLEIKRADAAPTVSIFPP SSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLT KDEYERHNSYTCEATHKTSTSPIVKSFNRNEC In some embodiments the SHC195 light chain 2 sequence does not comprise a signal peptide: Light chain 2: Amino acid sequence (218 aa) (SEQ ID NO:105)FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region DIVLTQSPASLTVSLGQRATISCRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLASYLKSGV PARFTGSGSGTDFTLNIFPVEGEDAATYYCQHSREFPWTFGGGTNLEIKRADAAPTVSIFPP SSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLT KDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0056] In some embodiments, the anti-TPO antibody, or antigen-binding fragment thereof, according to this aspect of the disclosure, further comprises: (4) a heavy chain variable region further comprising a heavy chain framework region 1 (FRH1), a heavy chain framework region 2 (FRH2), a heavy chain framework region 3 (FRH3), and a heavy chain framework region 4 (FRH4), (5) a first light chain variable region comprising a light chain framework region 1 (FRL1), a light chain comprising a light chain framework region 2 (FRL2), a light chain comprising a light chain framework region 3 (FRL3), and a light chain comprising a light chain framework region 4 (FRL4), and (6) a second light chain variable region comprising a light chain framework region 1 (FRL2-1), a light chain comprising a light chain framework region 2 (FRL2-2), a light chain comprising a light chain framework region 3 (FRL2-3), and a light chain comprising a light chain framework region 4 (FRL2-4), wherein the FRH1 comprises the sequence QVTLKESGPGILQPSQTLSLTCSFSGFSLS (SEQ ID NO:57), the FRH2 comprises the sequence WIRQPSGKGLEWLA (SEQ ID NO:59), the FRH3 comprises the sequence RLTISKDTSRNHVFLKITSVDLADTATYYCAR (SEQ ID NO:61), the FRH4 comprises the sequence WGQGISVTVSS (SEQ ID NO:63), the FRL1 comprises the sequence DIVLTQSPASLTVSLGQRATISC (SEQ ID NO:75), the FRL2 comprises the sequence WYQQKPGQSPKLLIY (SEQ ID NO:77), the FRL3 comprises the sequence GVPARFTGSGSGTDFTLNIFPVEGEDAATYYC (SEQ ID NO:79), the FRL4 comprises the sequence FGGGTNLEIK (SEQ ID NO:81), the FRL2-1 comprises the sequence DIVLTQSPASLTVSLGQRATISC (SEQ ID NO:93), the FRL2-2 comprises the sequence WNQQKPGQPPRLLIY (SEQ ID NO:95),the FRL2-3 comprises the sequence GVPARFTGSGSGTDFTLNIFPVEGEDAATYYC (SEQ ID NO:97), and the FRL2-4 comprises the sequence FGGGTNLEIK (SEQ ID NO:99).

[0057] In some embodiments, the anti-TPO antibody, or antigen-binding fragment thereof, according to this aspect comprises: (i) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is identical, to the sequence of QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDD KRYNPSLMNRLTISKDTSRNHVFLKITSVDLADTATYYCARRDGGNFGNNYYALDY WGQGISVTVSS (SEQ ID NO:112), provided that the heavy chain variable region comprises the sequences of CDRH1, CDRH2, and CDRH3 of SEQ ID NOs: 58, 60, and 62, respectively; (ii) a first light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is identical, to the sequence of DIVLTQSPASLTVSLGQRATISCRASKSVSTSGYNFMHWYQQKPGQSPKLLIYLASYL KSGVPARFTGSGSGTDFTLNIFPVEGEDAATYYCQHSREFPWTFGGGTNLEIK (SEQ ID NO:114), provided that the first light chain variable region comprises the sequences of CDRL1, CDRL2, and CDRL3 of SEQ ID NOs: 76, 78, and 80, respectively; and (iii) a second light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is identical, to the sequence of DIVLTQSPASLTVSLGQRATISCRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLASYL KSGVPARFTGSGSGTDFTLNIFPVEGEDAATYYCQHSREFPWTFGGGTNLEIK (SEQ ID NO:116), provided that the second light chain variable region comprises the sequences of CDRL2-1, CDRL2-2, and CDRL2-3 of SEQ ID NOs: 94, 96, and 98, respectively.

[0058] In some embodiments, the anti-TPO antibody, or antigen-binding fragment thereof, according to this aspect comprises: (i) a heavy chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is identical, to the sequence of QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEW LAHIYWDDDKRYNPSLMNRLTISKDTSRNHVFLKITSVDLADTATYYCARRDGGNFGNNYYALDYWGQGISVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEP VTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKV DKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFS WFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIE KTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAEN YKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG K (SEQ ID NO:103) provided that the heavy chain comprises the sequences of CDRH1, CDRH2, and CDRH3 of SEQ ID NOs: 58, 60, and 62, respectively; (ii) a first light chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is identical, to the sequence of DIVLTQSPASLTVSLGQRATISCRASKSVSTSGYNFMHWYQQKPGQSPKLLIYLASYL KSGVPARFTGSGSGTDFTLNIFPVEGEDAATYYCQHSREFPWTFGGGTNLEIKRADA APTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDS KDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:104), provided that the first light chain comprises the sequences of CDRL1, CDRL2, and CDRL3 of SEQ ID NOs: 76, 78, and 80, respectively; and (iii) a second light chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is identical, to the sequence of DIVLTQSPASLTVSLGQRATISCRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLASYL KSGVPARFTGSGSGTDFTLNIFPVEGEDAATYYCQHSREFPWTFGGGTNLEIKRADA APTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDS KDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:105), provided that the second light chain comprises the sequences of CDRL2-1, CDRL2-2, and CDRL2-3 of SEQ ID NOs: 94, 96, and 98, respectively.

[0059] The amino acid and DNA sequences for the SHC 195 antibody are provided in Tables 7-14 below. Table 7. SHC 195 Antibody Heavy Chain and Light Chain I and II Amino Acid Sequences SHC 195 Antibody Sequences (Amino Acid) SEQ ID NO(s):GGNFGNNYYALDYWGQGISVTVSSAKTTPPSVYPLAPGSA AQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVsignal IFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ peptide NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEASHC 195 Antibody Sequences (DNA) SEQ ID NO(s): Heavy ChainATGGACAGGCTTACTTCCTCATTCCTGCTGCTGATTGTCCSEQ ID NO:8TTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACT GGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCGGGGTCCCTGCCAGGTTCACTGGCAGTGGGTCTGGGACAG ACTTCACCCTCAACATCTTTCCTGTGGAGGGGGAGGATGC. SHC 195 Heavy Chain Sequences (DNA) SEQ ID NO.CAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCACACATTT ATTGGGATGATGACAAGCGCTACAATCCATCTCTGATGAATable 10. SHC 195 Antibody Heavy Chain Amino Acid Sequences SHC 195 Heavy Chain Sequences (Amino Acid) SEQ ID NO. HC SignalMDRLTSSFLLLIVPAYVLSSEQ ID NO:56Table 11. SHC 195 Antibody Light Chain I DNA Sequences SHC 195 Light Chain I Sequences (DNA) SEQ ID NO.LC I FR2TGGTATCAACAGAAACCAGGACAGTCACCCAAACTCCTCASEQ ID NO:68 TCTATTable 12. SHC 195 Antibody Light Chain I Amino Acid Sequences SHC 195 Light Chain I Sequences (Amino Acid) SEQ ID NO.LC I CDR1RASKSVSTSGYNFMHSEQ ID NO:76LC I FR2WYQQKPGQSPKLLIYSEQ ID NO:77. SHC 195 Light Chain II Sequences (DNA) SEQ ID NO.LC II GACATTGTGCTGACACAGTCTCCTGCTTCCTTAACTGTAT SEQ ID NO:115 Variable CTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAGCAATable 14. SHC 195 Antibody Light Chain II Amino Acid Sequences SHC 195 Light Chain II Sequences (Amino Acid) SEQ ID NO.LC IIQHSREFPWTSEQ ID NO:98CDR3monoclonal antibodies, or antigen-binding fragment thereof. In some embodiments, the anti- TPO antibodies described herein are Fab’2 fragments of monoclonal antibodies. In some embodiments, the labeled anti-TPO antibody or antigen-binding fragment thereof comprises a Fab’2 fragment. In some embodiments, the unlabeled anti-TPO antibody or antigen-binding fragment thereof comprises a Fab’2fragment.

[0061] Antibodies that may be used in embodiments of the present disclosure may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“intrabodies”), antibody fragments (e.g. Fv, Fab and Fab’2), half-antibodies, hybrid derivatives, as well as single chain antibodies (scFv), chimeric antibodies and de-immunized or humanized antibodies (Ed Harlow and David Lane, USING ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 1999); Houston et al., “Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli,” Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Bird et al, “Single-Chain Antigen-Binding Proteins,” Science 242:423-426 (1988), each of which is hereby incorporated by reference in its entirety).

[0062] Methods for monoclonal antibody production may be carried out using the techniques described herein or are well-known in the art (MONOCLONAL ANTIBODIES – PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONS (Mary A. Ritter and Heather M. Ladyman eds., 1995), which is hereby incorporated by reference in its entirety). Generally, the process involves obtaining immune cells (lymphocytes) from the spleen of amammal which has been previously immunized with the antigen of interest either in vivo or in vitro.

[0063] The monoclonal antibodies of the present disclosure can be humanized antibodies. Humanized antibodies are antibodies that contain minimal sequences from non- human (e.g., murine) antibodies within the variable regions. Such antibodies are used therapeutically to reduce antigenicity and human anti-mouse antibody responses when administered to a human subject. In practice, humanized antibodies are typically human antibodies with minimal to no non-human sequences. A human antibody is an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human.

[0064] In addition to whole antibodies, the present disclosure encompasses antigen binding portions of such antibodies. Such binding portions include the monovalent Fab fragments, Fv fragments (e.g., single-chain antibody, scFv), and single variable VH and VL domains, and F(ab’)2 fragments (also called Fab’2fragments, herein), Bis-scFv, diabodies, triabodies, minibodies, etc. These antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in James Goding, MONOCLONAL ANTIBODIES:PRINCIPLES AND PRACTICE 98-118 (Academic Press, 1983) and Ed Harlow and David Lane, ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor Laboratory, 1988), both of which are hereby incorporated by reference in their entirety, or other methods known in the art. In some embodiments, the anti-TPO antibody is a Fab’2fragment of SHC 194. In some embodiments, the anti-TPO antibody is a Fab’2fragment of SHC 195. In some embodiments, the anti-TPO antibody is a Fab fragment of SHC 194. In some embodiments, the anti-TPO antibody is a Fab fragment of SHC 195.

[0065] It may further be desirable, especially in the case of antibody fragments, to modify the antibody in order to increase its serum half-life. This can be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle (e.g., by DNA or peptide synthesis).

[0066] Other aspects of the present disclosure pertain to nucleic acids encoding the antibodies disclosed herein, vectors comprising the nucleic acids described herein, and host cells comprising the nucleic acids or the vectors described herein.

[0067] Antibodies may be generated using recombinant DNA technology, such as, for example, an antibody or fragment thereof expressed by a bacteriophage. Alternatively, the synthetic antibody is generated by the synthesis of a DNA molecule encoding and expressing the antibody of the present disclosure or the synthesis of an amino acid sequence specifying the antibody, where the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

[0068] Monoclonal antibodies can be made using recombinant DNA methods as described in U.S. Patent No.4,816,567 to Cabilly et al, which is hereby incorporated by reference in its entirety. The polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cells, for example, by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, monoclonal antibodies are generated by the host cells. In some embodiments, the host cell is a hybridoma. In some embodiments, the hybridoma cell is capable of producing the monoclonal antibody of SHC 194. In some embodiments, the hybridoma cell is capable of producing the monoclonal antibody of SHC 195. In some embodiments, the antibody or antigen-binding fragment is recombinantly produced.

[0069] Also, recombinant monoclonal antibodies or fragments thereof of the desired species can be isolated from phage display libraries (McCafferty et al., “Phage Antibodies: Filamentous Phage Displaying Antibody Variable Domains,” Nature 348:552-554 (1990); Clackson et al., “Making Antibody Fragments using Phage Display Libraries,” Nature 352:624-628 (1991); and Marks et al., “By-Passing Immunization. Human Antibodies from V-Gene Libraries Displayed on Phage,” J. Mol. Biol.222:581-597 (1991), all of which are hereby incorporated by reference in their entirety).

[0070] Polynucleotide(s) encoding a monoclonal antibody can further be modified using recombinant DNA technology to generate alternative antibodies or derivatives. For example, the constant domains of the light and heavy chains of a mouse monoclonal antibody can be substituted by those regions derived from a human antibody to generate a chimeric antibody. Alternatively, the constant domains of the light and / or heavy chains of a monoclonal antibody can be substituted by a non-immunoglobulin polypeptide to generate afusion antibody. In other embodiments, the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. Furthermore, site-directed or high-density mutagenesis of the variable region can be used to optimize specificity and affinity of a monoclonal antibody.

[0071] “Specifically binds” or “binds specifically” (or derivatives thereof) when used in the context of antibodies, or antibody fragments, represents binding via domains encoded by immunoglobulin genes or fragments of immunoglobulin genes to one or more epitopes of a protein of interest, without preferentially binding other molecules in a sample containing a mixed population of molecules. Typically, an antibody binds to a cognate antigen with a Kd of less than about 1x10-8M, as measured by a surface plasmon resonance assay or a cell- binding assay. Phrases such as “[antigen]-specific” antibody (e.g., rTPO-specific antibody) are meant to convey that the recited antibody specifically binds the recited antigen.

[0072] In some embodiments, the SHC 194 mAb or antigen-binding fragment thereof binds specifically to rTPO (SEQ ID NO:106) with a KDof 5 x 10-8M or less, a KDof 1 x 10-9M or less, or a KD of 5 x 10-9M or less. In some embodiments, the SHC 195 mAb or antigen-binding fragment thereof binds specifically to rTPO (SEQ ID NO:106) with a KD of 5 x 10-8M or less, a KD of 1 x 10-9M or less, or a KD of 5 x 10-9M or less.

[0073] A further aspect of the present disclosure relates to a method of detecting an anti-thyroid peroxidase (TPO) antibody in a biological sample from a subject. This method involves incubating the biological sample from the subject with (i) a solid support, (ii) an unlabeled anti-TPO antibody or antigen-binding fragment thereof as described herein, (iii) a recombinant TPO (rTPO), and (iv) a labeled anti-TPO antibody or antigen binding fragment thereof as described herein; and detecting the anti-thyroid peroxidase antibody in the biological sample, said detecting comprising analyzing a decrease in the formation of a complex comprising the solid support, the unlabeled anti-TPO antibody, the rTPO, and the labeled anti-TPO antibody.

[0074] In some embodiments, the decrease in the formation of a complex comprising the solid support, the unlabeled anti-TPO antibody, the rTPO, and the labeled anti-TPO antibody is compared to the amount of complex formation in a negative control sample (e.g., a sample with no or low amounts of aTPO autoantibodies). In some embodiments, the negative control sample is a buffer, or a biological sample from a subject without anti-TPO antibodies. In some embodiments, the negative control comprises aTPO levels below the 95thpercentile of an apparently healthy population. In some embodiments, the decrease in theformation of a complex comprising the solid support, the unlabeled anti-TPO antibody, the rTPO, and the labeled anti-TPO antibody is compared to the amount of complex formation in a positive control sample (e.g., a sample comprising aTPO autoantibodies). In some embodiments, the positive control sample is a buffer that is spiked with an anti-TPO monoclonal antibody or a biological sample from a subject with aTPO autoantibodies. In some embodiments, the positive control can be a native sample from one or more subjects with specific ranges of aTPO autoantibodies. In some embodiments, the positive control comprises aTPO levels above the 95thpercentile of an apparently healthy population.

[0075] Another aspect of the present disclosure relates to a method of diagnosing a thyroid disease in a subject. This method involves incubating a biological sample from a subject with (i) a solid support, (ii) an unlabeled anti-TPO antibody or antigen-binding fragment thereof as described herein, (iii) a recombinant TPO (rTPO), and (iv) a labeled anti- TPO antibody or antigen binding fragment thereof as described herein; and diagnosing the subject with the thyroid disease if the formation of a complex comprising the solid support, the unlabeled anti-TPO antibody, the rTPO, and the labeled anti-TPO antibody is decreased.

[0076] In some embodiments, the methods can comprise a “competition” immunoassay or an “inhibition” immunoassay, an exemplary reaction scheme for which is illustrated in FIG.4 of U.S. Patent No.11,327,074, which is hereby incorporated by reference in its entirety. As used herein, a “competition” immunoassay refers to an immunoassay in which two or more binding molecules compete for binding to a target molecule. The binding molecules can be antibodies, for example, and the target molecule can be an antigen. The binding molecules can compete, for example, for binding to the same epitope(s) on the antigen. An “inhibition” immunoassay refers to an immunoassay in which one or more binding molecules inhibit binding of one or more other binding molecules to a target molecule. The binding molecules can be antibodies, for example, and the target molecule can be an antigen. Inhibition can be caused by steric hindrance or other known mechanisms of binding inhibition. Whether the claimed assays are classified as a “competition” or “inhibition” immunoassay will depend, for example, on the rTPO epitope recognized by the anti-thyroid peroxidase antibody in the biological sample and the labeled anti-TPO antibody. The claimed methods encompass both competition and inhibition immunoassays, and thus the terms “competition” and “inhibition” are not intended to limit the scope of the claimed methods.

[0077] In some embodiments of the competition / inhibition immunoassay, the labeled anti-TPO antibody and the rTPO can be in a pre-formed immune complex prior to their addition to the reaction mixture. In some embodiments, the unlabeled anti-TPO antibody and solid support, can be in pre-formed complex prior to their addition to the reaction mixture. In some embodiments, the labeled anti-TPO antibody, the rTPO, the unlabeled anti-TPO antibody, and the solid support are not in a pre-formed complex prior to their addition to the reaction mixture. In yet other embodiments, the labeled anti-TPO antibody, the rTPO, the unlabeled anti-TPO antibody, and the solid support are all present in a pre-formed complex prior to their addition to the reaction mixture. Whether the various components are in one or more pre-formed complexes prior to their addition to the reaction mixture will depend, in part, on the dissociation constants (KD) of the labeled and unlabeled antibodies and the rTPO. The disclosed methods are not limited by the type or extent of complex formation added to the reaction mixture. It is to be understood that the biological sample, the labeled anti-TPO antibody, the rTPO, the unlabeled anti-TPO antibody, and the solid support can be added to the reaction mixture in any order.

[0078] FIGs.1 and 2 disclose one exemplary embodiment of the competition / inhibition immunoassays described herein, in which a labeled anti-TPO antibody and an rTPO are incubated with a solid support having an unlabeled anti-TPO antibody bound thereto. In the absence of the anti-TPO antibody from the biological sample, the labeled anti-TPO antibody will bind to the rTPO and in turn bind to or interact with the solid support having unlabeled anti-TPO antibody bound thereto, thereby forming a complex comprising the labeled anti-TPO antibody, the rTPO, and the solid support. Thus, in the absence of the anti-TPO antibody from the biological sample, the labeled anti-TPO antibody will be coupled to the solid support, isolation of which will result in isolation of the labeled anti-TPO antibody. When anti-TPO antibodies are present in the biological sample, the anti- TPO antibodies will compete with or otherwise inhibit the labeled anti-TPO antibody for binding to the rTPO or will compete with or otherwise inhibit the unlabeled anti-TPO antibody bound to the solid support for binding to the rTPO, thereby preventing the complex from forming or displacing the labeled anti-TPO antibody from the complex. Thus, in the presence of anti-TPO antibodies from the biological sample, the presence of complex comprising the labeled anti-TPO antibody, the rTPO, and the solid support, is decreased.

[0079] In some embodiments, the labeled anti-TPO antibody, the unlabeled rTPO, and the unlabeled anti-TPO antibody bound to the solid support can be incubated in areaction mixture for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, or any number or range therein. In some embodiments, the labeled anti-TPO antibody, the unlabeled rTPO, and the unlabeled anti-TPO antibody bound to the solid support can be incubated in a reaction mixture for less than about 10 minutes. In some embodiments, the labeled anti-TPO antibody, the unlabeled rTPO, and the unlabeled anti-TPO antibody bound to the solid support can be incubated in a reaction mixture for 1 minute to 30 minutes, or any number or range therein. The biological sample known to have, or suspected of having, anti-TPO antibodies can be added and incubated in the reaction mixture for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, or less than about 10 minutes or any number or range therein. In some embodiments, the incubating steps are performed in a total of about 10 minutes to about 20 minutes. In some embodiments, the incubating steps are performed in a total of 10 minutes to 20 minutes or any number or range therein. The subsequent detecting can be performed in less than about 5 minutes. It is to be understood that the amount of time needed for the assay may vary based upon several factors including the level of the anti-TPO antibody(ies) in the biological sample and the affinity of the anti- TPO antibody(ies) for the rTPO. Thus, the disclosed methods can be performed for any suitable amount of time.

[0080] The unlabeled anti-TPO antibody can be directly or indirectly coupled to the solid support. Suitable techniques for directly coupling the unlabeled anti-TPO antibody to the solid support include, for example, covalent attachment, adsorption, noncovalent interaction, or combinations thereof. Suitable means for indirectly coupling the unlabeled anti-TPO antibody to the solid support include, for example, linking through a peptide, a protein, an antibody, a linker, or a combination thereof. In some embodiments, the unlabeled anti-TPO antibody can be indirectly coupled to the solid support through streptavidin and biotin. For example, the unlabeled anti-TPO antibody can be biotinylated and the solid support can comprise streptavidin. The labeled and / or the unlabeled anti-TPO antibody can be any anti-TPO antibody. In some embodiments, the unlabeled antibody is SHC 195 or a fragment thereof. In some embodiments, the labeled antibody is SHC 194 or a fragment thereof. In some embodiments, the unlabeled antibody is SHC 194 or a fragment thereof. In some embodiments, the labeled antibody is SHC 195 or a fragment thereof.

[0081] In some embodiments, the solid support having unlabeled anti-TPO antibody bound thereto is present at about 10 ng / ml to about 10 μg / ml, or any number or range therein. In some embodiments, the solid support having unlabeled anti-TPO antibody bound thereto is present at 10 ng / ml to 10 μg / ml, or any number or range therein. In some embodiments, the solid support having unlabeled anti-TPO antibody bound thereto is present at 6 μg / ml. In some embodiments, the solid support having unlabeled anti-TPO antibody bound thereto is present at 3 μg / ml to 10 μg / ml or any number or range therein. In some embodiments, the solid support having unlabeled anti-TPO antibody bound thereto is present at 5.7 μg / ml to 6.3 μg / ml or any number or range therein.

[0082] Exemplary solid supports include, but are not limited to, a column matrix material, a culture plate, a tube, a dish, a flask, a microtiter plate, a bead / particle, heat-killed formalin-(or other chemically)-fixed prokaryotic or eukaryotic cells, microscope slides, ACLAR® Film, or any other optically transparent polymer, or a combination thereof. The solid support can be fully or partially composed of plastic, cellulose, cellulose derivatives, nitrocellulose, glass, fiberglass, latex, or a combination thereof. In some embodiments, the solid support comprises a magnetic bead / particle. In some embodiments, the magnetic bead / particle is a paramagnetic particle (PMP). In some embodiments, the magnetic bead / particle is a latex magnetic particle (LMP). In some embodiments, the magnetic bead / particle is coated with streptavidin. In some embodiments, the streptavidin coated magnetic bead / particle is present at about 0.05 mg / mL to about 150 mg / mL, or any number or range therein. In some embodiments, the streptavidin coated magnetic bead / particle is present at 0.05 mg / mL to 150 mg / mL, or any number or range therein. In some embodiments, the streptavidin coated magnetic bead / particle is present at 0.05 mg / mL to 1 mg / mL or any number or range therein. In some embodiments, the streptavidin coated magnetic bead / particle is present at 0.3 mg / mL. In some embodiments, the magnetic bead / particle is immobilized using a magnet.

[0083] The label can be any suitable label known to those skilled in the art to be useful for creating a detectable signal. Suitable detectable labels include, but are not limited to, enzyme conjugates (e.g., horseradish peroxidase (HRP), alkaline phosphatase, glucose oxidase, and β-galactosidase), fluorescent probes, radioactive isotopes, chemiluminescent compounds, bioluminescent compounds, or combination thereof. In some embodiments, the label is an acridinium ester (AE) or an analog thereof. Suitable AE analogs include: dimethyl acridinium ester (DMAE), N-sulfopropyl dimethyl acridinium ester (NSP-DMAE), highquantum yield acridinium ester (HQYAE), Zwitterionic acridinium ester (ZAE), N- sulfopropyl-2-isopropoxy dimethyl acridinium ester (Iso-Di-ZAE), trisuifopropyl acridinium ester (TSP-AE), or N-sulfopropyl dimethyl acridinium ester with hexa ethylene glycol linker (HEG-GLU-AE).

[0084] In some embodiments, the labeled anti-TPO antibody or fragment thereof is present at about 10 ng / ml to about 2 μg / ml, or any number or range therein. In some embodiments, the labeled anti-TPO antibody is present at 10 ng / ml to 2 μg / ml, or any number or range therein. In some embodiments, the labeled anti-TPO antibody is present at 30 ng / ml. In some embodiments, the labeled anti-TPO antibody is a labeled Fab’2 fragment and is present at 30 ng / ml. In some embodiments, the labeled anti-TPO antibody is a labeled Fab’2fragment and is present at 10-50 ng / ml, or any number or range therein. In some embodiments, the labeled anti-TPO antibody is a labeled Fab’2fragment and is present at 28.5-31.5 ng / ml.

[0085] The solid support having unlabeled anti-TPO antibody bound thereto and / or the unlabeled rTPO and / or the labeled anti-TPO antibody can be present in a buffer comprising, for example, one or more of phosphate buffer, NaCl, EDTA, pluronic F-127, sodium azide, sorbitol, sulfhydryl modified bovine serum, or any combinations, variations, or equivalents thereof. In one embodiment, the buffer comprises about 100 mM phosphate buffer, about 400 mM NaCl, about 1.9 g / L EDTA, about 0.2% (v / v) pluronic F-127, about 0.9 g / L sodium azide, about 10% sorbitol, and about 10 g / L sulfahydryl modified bovine serum albumin.

[0086] Another aspect of the present disclosure relates to a method of diagnosing a thyroid disease in a subject. This method involves incubating a biological sample from a subject with (i) a solid support, (ii) an unlabeled anti-TPO antibody or antigen-binding fragment thereof of an antibody described herein, (iii) a recombinant TPO (rTPO), and (iv) a labeled anti-TPO antibody or antigen binding fragment thereof of an antibody described herein; and diagnosing the subject with the thyroid disease if the formation of a complex comprising the solid support, the unlabeled anti-TPO antibody, the rTPO, and the labeled anti-TPO antibody is decreased.

[0087] In some embodiments, an unlabeled anti-TPO antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO:112, a first light variable region chain of SEQ ID NO:114, and a second light chain variable region of SEQ ID NO:116. In some embodiments, the labeled anti-TPO antibody or antigen binding fragmentthereof comprises the heavy chain variable region of SEQ ID NO:108, and a light chain variable region of SEQ ID NO:110.

[0088] The method of diagnosing can further comprise a step of detecting the complex. For example, detecting the complex may comprise taking a read-out of a signal from the label, wherein the intensity of the signal from the label indicates the amount of label present in the assay. In some embodiments, the intensity of the signal decreases if anti-TPO antibodies are present in the sample from the subject.

[0089] The thyroid disease can be an autoimmune disorder. In some embodiments, the autoimmune disorder is Hashimoto’s thyroiditis or Graves’ disease. In some embodiments, the autoimmune disorder is Hashimoto’s thyroiditis, Graves’ disease, atrophic thyroiditis, primary myxedema, and / or postpartum thyroiditis in women.

[0090] Similar to the methods of detecting described above, in the methods of diagnosing, the labeled anti-TPO antibody, the unlabeled rTPO, or the unlabeled anti-TPO antibody bound can be directly or indirectly linked / coupled to the solid support. Suitable techniques for direct linking / coupling include, for example, covalent attachment, adsorption, noncovalent interaction, or combinations thereof. In some embodiments, the direct linking / coupling can be achieved by glutaraldehyde fixation, N-hydroxysuccinimide (MTS) chemistry, or 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) NHS chemistry. Suitable means for indirect linking / coupling include, for example, linking / coupling through a peptide, a protein, an antibody, a linker, or a combination thereof. In some embodiments, the indirect linking / coupling to the solid support is via streptavidin and biotin. For example, the unlabeled rTPO, the labeled anti-TPO antibody, or the unlabeled anti-TPO antibody can be biotinylated and the solid support can comprise streptavidin. In some embodiments, the anti- TPO antibody is a biotin conjugate.

[0091] Suitable solid supports and labeled antibodies are described herein supra.

[0092] Suitable times for performing the methods of diagnosing an autoimmune disease include those provided above for the various methods of detecting an antibody.

[0093] The methods can further comprise determining a level of anti-thyroid peroxidase antibodies in the biological sample of the subject. In some embodiments, the level of anti-thyroid peroxidase antibodies in the biological sample of the subject is directly proportional to the level of the complex detected. Thus, the level of anti-TPO antibody can be determined by determining the level of solid support coupled to or in complex with the labeled component (i.e., the labeled rTPO or antibody). Determining the level of solidsupport coupled to or in complex with the labeled component can be performed, for example, by measuring the signal from the complex. Similarly, in embodiments where the method is used to diagnose a thyroid disease in a subject, the solid support coupled to or in complex with the labeled component is detected by measuring the signal or absence thereof from the labeled component linked to the solid support. In some embodiments, determining the level of the complex detected comprises comparing the level of decrease of the complex formation detected relative to (i) a negative control sample, e.g., a biological sample from a subject that does not have anti-TPO antibodies, and / or (ii) a positive control sample, e.g., a biological sample from a subject that does have anti-TPO antibodies. In some embodiments, the subject is diagnosed with a thyroid disease if the level of complex formation is decreased by a set threshold or cutoff value. In some embodiments, the set threshold or cutoff value is determined based on one or more control samples. In some embodiments, the threshold value or cutoff value is the amount of complex formation in normal healthy individuals, individuals of a similar age, individuals of a similar sex, and / or individuals of a similar physical condition in comparison to the subject. In some embodiments, the threshold value or cutoff value is determined by aTPO levels above the 95thpercentile of an apparently healthy population. In some embodiments, the subject is not diagnosed with thyroid disease if the sample from the subject has a similar level of signal as the negative control. In some embodiments, the subject is diagnosed with thyroid disease if the sample from the subject has a similar level of signal as the positive control or a level of signal less than the negative control.

[0094] The disclosed methods can be performed manually or can be automated. For example, the disclosed methods can be performed using an ADVIA CENTAUR® Immunoassay System or an ATELLICA® system.

[0095] Suitable biological samples for detecting the anti-TPO antibody include any biological sample from a subject that contains, or is suspected of containing, anti-TPO antibodies including, but not limited to, serum, plasma, whole blood, saliva, urine, semen, perspiration, tears, and body tissues.

[0096] The immunoassays disclosed herein employ a recombinant thyroid peroxidase (rTPO). In some embodiments, the rTPO is from cynomolgus monkey. In some embodiments, the rTPO comprises the amino acid sequence set forth in SEQ ID NO:106 below (also SEQ ID NO:1 of PCT Publication No. WO 2020 / 010007, which is hereby incorporated by reference in its entirety). In some embodiments, the rTPO further comprisesan epitope tag. The epitope tag can be at the N- terminus or at the C-terminus of the rTPO. The epitope tag can be any suitable tag known to persons skilled in the art including, but not limited to, a 6-histidine tag, a hemagglutinin tag, a glutathione-S-transferase, a maltose binding protein, or a chitin binding protein. In some embodiments, the rTPO comprises a C- terminal 6-histidine tag. In some embodiments, the rTPO comprises a N-terminal 6-histidine tag. Polynucleotide(s) encoding a rTPO can be generated using DNA synthesis and recombinant DNA technology known to those of skill in the art.

[0097] The amino acid sequence for the recombinant TPO (rTPO) from a cynomolgus monkey (Macaca fascicularis) is set forth as follows (SEQ ID NO:106):

[0098] ADPGYLLECTEAFFPFISRGKELLWGKPEESRVAGILEESKRLVDTAMYATM QRNLKKREILSPHQLLSFSKLPEPTSGEIARAAEIMETSIQAMKRKVNLKIQQSQHPTDALS EDLLSIIANMSGCLPYMLPPKCPNTCLANKYRPITGACNNRDHPRWGASNTALARWLPPVYE DGFSQPRGWNPSILHNGFPLPPVREVTRHVIQVSNEVVTDDDRYSDLLMAWGQYIDHDIAFT PQSTSKAAFRGGADCQVTCENQNPCFPIQLPEEARPAAGTACLPFYRSSAACGTGDQGALFG NLSTANPRQQMNGLTSFLDASTVYGSSPALERQLRNWTSAEGLLRVHARLRDSGRAYLPFAP PRAPAACAPEPGIPGETRGPCFLAGDGRASEVPSLTALHTLWLREHNRLAAALKALNAHWSA DAVYQEARKVVGALHQIITLRDYVPRILGPEAFQQYVGPYEGYDSAANPTVSNVFSTAAFRF GHATIHPLVRRLDAGFQEHPGLPGLWLHETFFSPWTLLHGGGLDPLIRGLLARPAKLQVQDQ LMNEELTERLFVLSNSSTLDLASINLQRGRDHGLPGYNEWREFCGLPRLETPADLSTAIASR SVADKILDLYKHPDNIDVWLGGLAENFLPRARTGPLFACLIGKQMKALRDGDWFWWENSHVF TDAQRHELEKHSLSRVICDNTGLTRVPVDAFRVGKFPEDFESCDSIPGMNLEAWRETFPQDD KCGFPESVENGDFVHCEESGRRVLVYSCRHGYELQGHEQLTCTQEGWDFQPPLCKDVNECAD GAHPPCHASARCRNTKGGFQCLCADPYELGDDGRTCVDSGRLPR

[0099] Further disclosed herein are kits. In some embodiments, the kits can comprise a solid support, an rTPO, and one or more anti-TPO antibodies of the present disclosure. In some embodiments, a portion of the anti-TPO antibodies are unlabeled and a portion of the anti-TPO antibodies are labeled. In some embodiments, the kit comprises reagents for determining levels of anti-TPO antibodies in a sample from a subject and instructions for comparing the expression level

[0100] Suitable solid supports and labels for any of the kits disclosed herein include those solid supports and labels disclosed for the methods above. In some embodiments, the kit comprises all necessary reagents and components for assay operation. In some embodiments, the kit may be configured for use on automated immunoassay platforms, suchas the Atellica® Immunoassay Analyzer and ADVIA Centaur® systems, and may include pre-measured, ready-to-use reagents. In some embodiments, the kit comprises comprehensive instructions for use and recommended storage conditions. EXAMPLES

[0101] The examples below are intended to exemplify the practice of embodiments of the disclosure but are by no means intended to limit the scope thereof. EXAMPLE 1 – Sequencing of aTPO Antibodies SHC-194 & SHC-195

[0102] For each of mouse monoclonal anti-TPO antibodies SHC 194 (7G11) and SHC 195 (12F11), total RNA was isolated from each of the hybridoma cells following the technical manual of RNA Isolation Kit (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China). Total RNA was then reverse transcribed into cDNA using isotype-specific anti-sense primers or universal primers following the technical manual of SMARTScribe Reverse Transcriptase (Takara Bio Inc., Kusatsu, Shiga, Japan). The antibody fragments of VH and VL were amplified using rapid amplification of cDNA ends (RACE) (GenScript Biotech Corporation, Piscataway, NJ). Amplified antibody fragments were cloned into a standard cloning vector separately. Colony PCR was performed to screen for clones with inserts of correct sizes. No less than five colonies with inserts of correct sizes were sequenced for each fragment (see FIGs.5 and 6). The sequences of different clones were aligned, and the consensus sequence of these clones is shown in SEQ ID NOs:1-4 for SHC 194 and SEQ ID NOs:5-10 for SHC 195.

[0103] Tables 15 and 16 show IgBLAST analysis of the V(D)J junctions of SHC 194 and SHC 195, respectively. The isotype of SHC 194 (7G11) is mouse IgG1 / kappa, as analyzed by the sequences of the constant region. The isotype of SHC 195 (12F11) is mouse IgG1 / kappa, as analyzed by the sequences of the constant region.Table 15. IgBLAST Analysis of V(D)J Junctions of Anti-TPO Antibody SHC 194 V- Seque V-GENE Function REGIO J-GENE D-GENE Juncti d ll l lit N d ll l d ll l AA Junction on e e eV- Seque V-GENE Functiona REGION J-GENE D-GENE Juncti nce and allele lit identit % and and allele AA Junction on e e e eEXAMPLE 2 – Evaluation of aTPOII Assays and aTPOII Assay Components

[0104] FIGs.1-2 show exemplary embodiments of the anti-TPOII antibody assay. These exemplary embodiments show a competitive immunoassay that uses chemiluminescent technology.

[0105] Table 17 shows concentrations of components of the assays of FIGs.1 and 2. Table 17. Components and Concentration of the aTPOII Assays Illustrated in FIGs.1 & 2 Component Concentration

[0106] Exemplary component amounts for an aTPOII product composition assay kit are shown in Table 18 below. This table includes non-limiting examples of the target amounts and high and low ranges for each component. Other amounts are contemplated. Table 18. Exemplary Components for an aTPOII Assay Kit Solid Phase Target Low High Streptavidin Coated Magnetic Beads 50mg / mL 45 55 0 0 h 0 0 h 5 5 5 hSodium Pyrithione 0.9g / L 0.855 0.945 Sodium Azide 0.5g / L 0.475 0.525 25 7 h 2 2 5 555

[0107] Results of the anti-TPOII immunoassays of FIGs.1 and 2 are shown in Tables 19-25 below. These results show that the anti-TPOII immunoassay a highly sensitive in vitro diagnostic test for the detection of anti-TPO Ab, giving laboratorians an accurate, quantitative determination of anti-TPO antibody in serum or plasma.

[0108] Table 19 below shows a comparison of the performance of the prior immunoassay (aTPOI) compared to the performance of the aTPOII immunoassay of the present disclosure. Both assays are performed on the Atellica® system. The aTPOI assay is described in PCT Publication No. WO 2020 / 010007, which is hereby incorporated by reference in its entirety. Table 19. Comparison of Atellica IM aTPOI and aTPOII Assay Performance aTPOI Assay aTPOII Assay (this dStandard for Anti-Thyroid Peroxidase Antibodies, NIBSCaTPOI Assay aTPOII Assay (this di l d C

[0110] Tables 21 and 22 below show a comparison of the sensitivity and specificity of the aTPOII assay of the present disclosure on the Atellica® and the ADVIA Centaur® systems.

[0111] For Table 21, sensitivity and specificity were determined using the ADVIA Centaur XP system in accordance with CLSI Document EP12-A2.16. The performance of the ADVIA Centaur aTPOII assay was compared to a commercially available anti TPO assay using 485 autoimmune thyroiditis and non-autoimmune thyroiditis patient samples. The Relative Sensitivity was 87.05% (168 / 193) [95% Confidence Interval 81.58%-91.07%]. The Relative Specificity was 100% (292 / 292) [95% Confidence Interval 98.70%-100%]. The Overall Agreement was 94.85% (460 / 485) [95% Confidence Interval 92.50%-96.48%]. Table 21. Sensitivity and Specificity of Advia Centaur aTPOII Assay Comparative Assay Positive Ne ative Totalo a e , e qua a e e o co pa so as es e acco a ce with the governing standard CLSI EP09C-ED3 and CLSI EP12-A2. The performance of the Atellica IM aTPOII assay was compared to a commercially available anti-TPO assay using 280 autoimmune thyroiditis and non-autoimmune thyroiditis patient samples. The Overall Percent Agreement was 97.50% [95% Confidence Interval 94.93%-98.78%]. The Positive Percent Agreement was 92.93% [95% Confidence Interval 86.12%-96.53%]. The Negative Percent Agreement was 100% [95% Confidence Interval 97.92%-100%]. Table 22. Atellica IM aTPOII Assay vs Third Party Assay Comparative Assay

[0113] Table 23 below shows a comparison of the aTPOII assay of the present disclosure versus the prior aTPOI assay, both performed on the Atellica® system. Testingincluded 244 patient samples using the Atellica IM aTPOII assay and the Atellica IM aTPOI assay described in PCT Publication No. WO 2020 / 010007, which is hereby incorporated by reference in its entirety. The Positive Percent Agreement was 99.2% (120 / 121) [95% Confidence Interval 95.5%-99.9%]. The Negative Percent Agreement was 98.4% (121 / 123) [95% Confidence Interval 94.3%-99.6%]. The Overall Percent Agreement was 98.8% (241 / 244) [95% Confidence Interval 96.5%-99.6%]. Table 23. Atellica IM aTPOII Assay vs Atellica IM aTPOI Assay Atellica IM aTPOI Assay > 60 IU / mL < 60 IU / mL Totaldisclosure versus the prior aTPOI assay, both performed on the ADVIA Centaur® system. Testing included 244 patient samples using the ADVIA Centaur aTPOII assay and the ADVIA Centaur aTPOI assay described in PCT Publication No. WO 2020 / 010007, which is hereby incorporated by reference in its entirety. The Positive Percent Agreement was 97.5% (118 / 121) [95% Confidence Interval 93.0%-99.2%]. The Negative Percent Agreement was 98.4% (121 / 123) [95% Confidence Interval 94.3%-99.6%]. The Overall Percent Agreement was 98.0% (239 / 244) [95% Confidence Interval 95.3%-99.1%]. Table 24. Advia Centaur aTPOII Assay vs Advia Centaur aTPOI Assay Advia Centaur aTPOI Assay

[0115] Table 25 below shows a summary of the aTPOII assay performance on the Atellica® and ADVIA Centaur® systems.Table 25. Atellica IM and Advia Centaur aTPOII Assay Performance Advia Centaur XP / XPT Atellica IM aTPOII Assay aTPOII AssayEXAMPLE 3 – Kinetics / Affinities of Anti-TPO Monoclonal Antibodies

[0116] The binding affinities of the anti-TPO mAbs for recombinant TPO antigen were measured on a BiacoreTMT200 instrument (GE Healthcare, Chicago, IL) using a Series S CM5 sensor chip, buffers, amine coupling kit and regeneration solutions from Cytiva (Marlborough, MA). The avidity effect associated with bivalency of the intact antibodies was avoided and the 1:1 binding model requirement was satisfied by conducting the experiment with the Fab fragments of the anti-TPO mAbs.

[0117] The Fab fragments were prepared by papain digestion of the anti-TPO mAbs in the presence of 10 mM L-cysteine in PBS, pH 7.0 containing 2 mM Na2EDTA at an enzyme / mAb ratio of 1:100. After incubation at 37ºC for 24 h papain hydrolysis was stopped with 7.5 mM iodoacetamide, and samples of digested anti-TPO mAbs were dialyzed against 10 mM Tris-HCl, pH 7.5. The Fab fragments were purified in flow-through mode by capturing of the Fc fragments with anion-exchange MonoQTM 5 / 50 GL column (GEHealthcare, # 17-5166-01) equilibrated with 10 mM Tris-HCl, pH 7.5. The bounded Fc fragments were eluted from the column with 0-0.5 M NaCl gradient. Homogeneity and integrity of the Fab fragments was confirmed by SDS-PAGE under reduced and non-reduced conditions (data not shown).

[0118] Kinetics / Affinity Surface Bound protocol for BiacoreTMT200 instrument was used to determine binding parameters of interaction between anti-TPO 7G11 (SHC 194) and 12F11 (SHC 195) Fab-fragments (7G11 Fab and 12F11 Fab, respectively) and recombinant TPO antigen (rTPO).

[0119] rTPO was immobilized at 1,115 RU density on the carboxymethyl dextran surface in flow cell 4 of the CM5 sensor chip using amino-coupling chemistry as follows. A new Series S CM5 sensor chip was washed with HBS-N buffer (10 mM HEPES, pH 7.4, 150 mM NaCl) and activated by 7-min injection of a solution of 200 mM 1-ethyl-3(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 50 mM N-hydroxy succinimide (NHS) at flow rate 10 uL / min. rTPO Antigen (1 μg / mL in 10 mM sodium acetate, pH 4.5) was immobilized on the EDC / NHS-activated chip surface during the 7 min injection, followed by a 7min injection of 1 M ethanolamine HCl (pH 8.5) to block the excess of active ester groups. The reference surface in flow cell 1 was prepared in the same manner using TPO-irrelevant Mab.

[0120] Kinetics / Affinity assays were performed in HBS-P+ running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% v / v Surfactant 20) at 25°C. Nine concentrations of the anti-TPO Fab fragment were injected over the surface of the chip for 7 min at 30 μL / min flow rate followed by a 30 min of dissociation. The buffer blanks were run in triplicate and used to double-reference the Fab-binding data before fitting.

[0121] After each cycle of binding the surface was regenerated by injection of 10 mM Glycine-HCL, pH 1.5 during 30s at 90 ml / min flow rate.

[0122] Kinetics data were collected over 0.25-32 nM anti-TPO Fab fragments. The response from reference surface was subtracted from the rTPO / Fab sensograms to remove the bulk effect, and the buffer blanks sensograms were used to double-reference the Fab-binding data before fitting. rTPO / Fab kinetics data were globally fitted to simple 1:1 interaction model, and BiacoreTM T200 Evaluation Software ver.3.2.1 (Cytiva) was used to determine the binding parameters of the rTPO / Fab-fragment interaction.

[0123] The binding parameters of anti-TPO Fab fragments interaction with recombinant TPO antigen are shown in Table 26 below:Table 26. Binding Parameters of anti-TPO Fab fragments Fab ka (1 / Ms) kd (1 / s) KD(M) Rmax (RU) TPO 7G11 2.54E+05 1.40E-04 5.50E-10 105.6 [binding to rTPO is shown in FIGs.7A-B and Tables 27-28 below. Table 27. Report Table for Kinetics of TPO 7G11 Fab 3’ Curve ka kd KD R Conc Tc Flow Kt (RU / RI Chi2U- (1 / Ms) (1 / s) (M) max (M) ml / Ms) (RU) (RU2) valu (RU) min eabe 8. arameers abe or ne cs o O G ab 3 Curve ka SE Kd SE Rmax SE Conc Tc SE f (^l / RI (1 / Ms) (ka) (1 / s) (kd) (RU) (Rmax (M) (tc) min) (RU

[0125] The antibody kinetics / affinity analysis of anti-TPO 12F11 (SHC 195) Fab binding to rTPO is shown in FIGs.8A-B and Tables 29-30 below.Table 29. Report Table for Kinetics of TPO 12F11 Fab 3’ Curve ka kd KD R Conc Tc Flow Kt RI Chi2U- (1 / Ms) (1 / s) (M) max (M) ^l / (RU / (RU) (RU2value (RU) min Ms) )Curve ka SE Kd SE Rmax SE Conc Tc SE f (^l / RI (1 / Ms) (ka) (1 / s) (kd) (RU) (Rmax (M) (tc) min) (RU)

[0126] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

WHAT IS CLAIMED IS:

1. An anti-thyroid peroxidase (TPO) antibody (anti-TPO antibody), or antigen-binding fragment thereof, wherein the antibody comprises: (1) a heavy chain variable region comprising a heavy chain complementarity determining region 1 (CDR1H1), a heavy chain complementarity determining region 2 (CDRH2), and a heavy chain complementarity determining region 3 (CDRH3), and (2) a light chain variable region comprising a light chain complementarity determining region 1 (CDR1L1), a light chain complementarity determining region 2 (CDRL2), and a light chain complementarity determining region 3 (CDRL3), wherein the CDRH1 comprises the sequence TYVIH (SEQ ID NO:22), the CDRH2 comprises the sequence YIIPYNDRTRYYEKFKD (SEQ ID NO:24), the CDRH3 comprises the sequence GGGFTTIGDWYFDV (SEQ ID NO:26), the CDRL1 comprises the sequence KASQDINNYLS (SEQ ID NO:40), the CDRL2 comprises the sequence RANRLID (SEQ ID NO:42), and the CDRL3 comprises the sequence LQYAEYPFM (SEQ ID NO:44).

2. The anti-TPO antibody, or antigen-binding fragment thereof, of claim 1, wherein the antibody further comprises: (3) a heavy chain variable region further comprising a heavy chain framework region 1 (FRH1), a heavy chain framework region 2 (FRH2), a heavy chain framework region 3 (FRH3), and a heavy chain framework region 4 (FRH4) and (4) a light chain variable region comprising a light chain framework region 1 (FRL1), a light chain comprising a light chain framework region 2 (FRL2), a light chain comprising a light chain framework region 3 (FRL3), and a light chain comprising a light chain framework region 4 (FRL4), and wherein the FRH1 comprises the sequence EVQLQQSGPELVKPGASVKMSCKASGYTFT (SEQ ID NO:21), the FRH2 comprises the sequence WVKQKPGQGLEWIG (SEQ ID NO:23), the FRH3 comprises the sequence KATLTSDKSSSTAYMELSSLTSEDSAVYYCAR (SEQ ID NO:25), the FRH4 comprises the sequence WGAGTTVTVSS (SEQ ID NO:27), the FRL1 comprises the sequence DIKMTQSPSSMYASLGERVTITC (SEQ ID NO:39),the FRL2 comprises the sequence WFQQKPGKSPKTLIY (SEQ ID NO:41), the FRL3 comprises the sequence GVPSRFSGSGSGQDYSLTISSLEYEDVGIYYC (SEQ ID NO:43), and the FRL4 comprises the sequence FGSGTKLEIK (SEQ ID NO:45).

3. The anti-TPO antibody, or antigen-binding fragment thereof, of claim 1, wherein the antibody comprises: (i) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is identical, to the sequence of EVQLQQSGPELVKPGASVKMSCKASGYTFTTYVIHWVKQKPGQGLEWIGYIIPYND RTRYYEKFKDKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGGFTTIGDWYFDV WGAGTTVTVSS (SEQ ID NO:108), provided that the heavy chain variable region comprises the sequences of CDRH1, CDRH2, and CDRH3 of SEQ ID NOs: 22, 24, and 26, respectively; and (ii) a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is identical, to the sequence of DIKMTQSPSSMYASLGERVTITCKASQDINNYLSWFQQKPGKSPKTLIYRANRLIDGV PSRFSGSGSGQDYSLTISSLEYEDVGIYYCLQYAEYPFMFGSGTKLEIK (SEQ ID NO:110), provided that the light chain variable region comprises the sequences of CDRL1, CDRL2, and CDRL3 of SEQ ID NOs: 40, 42, and 44, respectively.

4. The anti-TPO antibody, or antigen-binding fragment thereof, of any one of claims 1-3, wherein the antibody, or antigen-binding fragment thereof, is a monoclonal antibody, or antigen-binding fragment thereof.

5. The anti-TPO antibody, or antigen-binding fragment thereof, of any one of claims 1-4, wherein the antibody, or antigen-binding fragment thereof binds to rTPO with a KD of 1 x 10-9M or less.

6. A nucleic acid, comprising a nucleic acid sequence encoding an antibody, or antigen-binding fragment thereof, of any one of claims 1-5.

7. A vector comprising the nucleic acid of claim 6.

8. A host cell comprising the nucleic acid of claim 6 or the vector of claim 7.

9. An anti-thyroid peroxidase (TPO) antibody (anti-TPO antibody), or antigen-binding fragment thereof, wherein the antibody comprises: (1) a heavy chain variable region comprising a heavy chain complementarity determining region 1 (CDR1H1), a heavy chain complementarity determining region 2 (CDRH2), and a heavy chain complementarity determining region 3 (CDRH3), (2) a first light chain variable region comprising a light chain complementarity determining region 1 (CDR1L1), a light chain complementarity determining region 2 (CDRL2), and a light chain complementarity determining region 3 (CDRL3), and (3) a second light variable region chain comprising a light chain complementarity determining region 1 (CDR1L2-1), a light chain complementarity determining region 2 (CDRL2-2), and a light chain complementarity determining region 3 (CDRL2-3), wherein the CDRH1 comprises the sequence TSGMGVS (SEQ ID NO:58), the CDRH2 comprises the sequence HIYWDDDKRYNPSLMN (SEQ ID NO:60), the CDRH3 comprises the sequence RDGGNFGNNYYALDY (SEQ ID NO:62), the CDRL1 comprises the sequence RASKSVSTSGYNFMH (SEQ ID NO:76), the CDRL2 comprises the sequence LASYLKS (SEQ ID NO:78), the CDRL3 comprises the sequence QHSREFPWT (SEQ ID NO:80), the CDRL2-1 comprises the sequence RASKSVSTSGYSYMH (SEQ ID NO:94), the CDRL2-2 comprises the sequence LASYLKS (SEQ ID NO:96), and the CDRL2-3 comprises the sequence QHSREFPWT (SEQ ID NO:98).

10. The anti-TPO antibody, or antigen-binding fragment thereof, of claim 9, wherein the antibody further comprises: (4) a heavy chain variable region further comprising a heavy chain framework region 1 (FRH1), a heavy chain framework region 2 (FRH2), a heavy chain framework region 3 (FRH3), and a heavy chain framework region 4 (FRH4),(5) a first light chain variable region comprising a light chain framework region 1 (FRL1), a light chain comprising a light chain framework region 2 (FRL2), a light chain comprising a light chain framework region 3 (FRL3), and a light chain comprising a light chain framework region 4 (FRL4), and (6) a second light chain variable region comprising a light chain framework region 1 (FRL2-1), a light chain comprising a light chain framework region 2 (FRL2-2), a light chain comprising a light chain framework region 3 (FRL2-3), and a light chain comprising a light chain framework region 4 (FRL2-4), wherein the FRH1 comprises the sequence QVTLKESGPGILQPSQTLSLTCSFSGFSLS (SEQ ID NO:57), the FRH2 comprises the sequence WIRQPSGKGLEWLA (SEQ ID NO:59), the FRH3 comprises the sequence RLTISKDTSRNHVFLKITSVDLADTATYYCAR (SEQ ID NO:61), the FRH4 comprises the sequence WGQGISVTVSS (SEQ ID NO:63), the FRL1 comprises the sequence DIVLTQSPASLTVSLGQRATISC (SEQ ID NO:75), the FRL2 comprises the sequence WYQQKPGQSPKLLIY (SEQ ID NO:77), the FRL3 comprises the sequence GVPARFTGSGSGTDFTLNIFPVEGEDAATYYC (SEQ ID NO:79), the FRL4 comprises the sequence FGGGTNLEIK (SEQ ID NO:81), the FRL2-1 comprises the sequence DIVLTQSPASLTVSLGQRATISC (SEQ ID NO:93), the FRL2-2 comprises the sequence WNQQKPGQPPRLLIY (SEQ ID NO:95), the FRL2-3 comprises the sequence GVPARFTGSGSGTDFTLNIFPVEGEDAATYYC (SEQ ID NO:97), and the FRL2-4 comprises the sequence FGGGTNLEIK (SEQ ID NO:99).

11. The anti-TPO antibody, or antigen-binding fragment thereof, of claim 9, wherein the antibody comprises: (i) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is identical, to the sequence ofQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDD KRYNPSLMNRLTISKDTSRNHVFLKITSVDLADTATYYCARRDGGNFGNNYYALDY WGQGISVTVSS (SEQ ID NO:112), provided that the heavy chain variable region comprises the sequences of CDRH1, CDRH2, and CDRH3 of SEQ ID NOs: 58, 60, and 62, respectively; (ii) a first light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is identical, to the sequence of DIVLTQSPASLTVSLGQRATISCRASKSVSTSGYNFMHWYQQKPGQSPKLLIYLASYL KSGVPARFTGSGSGTDFTLNIFPVEGEDAATYYCQHSREFPWTFGGGTNLEIK (SEQ ID NO:114), provided that the first light chain variable region comprises the sequences of CDRL1, CDRL2, and CDRL3 of SEQ ID NOs: 76, 78, and 80, respectively; and (iii) a second light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is identical, to the sequence of DIVLTQSPASLTVSLGQRATISCRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLASYL KSGVPARFTGSGSGTDFTLNIFPVEGEDAATYYCQHSREFPWTFGGGTNLEIK (SEQ ID NO:116), provided that the second light chain variable region comprises the sequences of CDRL2-1, CDRL2-2, and CDRL2-3 of SEQ ID NOs: 94, 96, and 98, respectively.

12. The anti-TPO antibody, or antigen-binding fragment thereof, of any one of claims 9-11, wherein the antibody, or antigen-binding fragment thereof, is a monoclonal antibody, or antigen-binding fragment thereof.

13. The anti-TPO antibody, or antigen-binding fragment thereof, of any one of claims 9-12, wherein the antibody, or antigen-binding fragment thereof binds to rTPO with a KDof 1 x 10-9M or less.

14. A nucleic acid, comprising a nucleic acid sequence encoding an antibody, or antigen-binding fragment thereof, of any one of claims 9-13.

15. A vector comprising the nucleic acid of claim 14.

16. A host cell comprising the nucleic acid of claim 14 or the vector of claim 15.

17. A method of producing an antibody, or antigen-binding fragment thereof, comprising culturing the host cell of claim 8 or claim 16 under conditions suitable for expression of the antibody or antigen-binding fragment thereof.

18. A method of detecting an anti-thyroid peroxidase (TPO) antibody in a biological sample from a subject, the method comprising: incubating the biological sample from the subject with (i) a solid support, (ii) an unlabeled anti-TPO antibody or antigen-binding fragment thereof of any one of claims 9- 13, (iii) a recombinant TPO (rTPO), and (iv) a labeled anti-TPO antibody or antigen binding fragment thereof of any one of claims 1-5; and detecting the anti-thyroid peroxidase antibody in the biological sample, said detecting comprising analyzing a decrease in the formation of a complex comprising the solid support, the unlabeled anti-TPO antibody, the rTPO, and the labeled anti-TPO antibody.

19. The method of claim 18, wherein the label comprises an enzyme conjugate, a fluorescent probe, a radioactive isotope, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.

20. The method of claim 19, wherein the label comprises an acridinium ester (AE) or an analog thereof.

21. The method of claim 20, wherein the acridinium ester analog is DMAE, NSP-DMAE, HQYAE, ZAE, Iso-Di-ZAE, TSP-AE, or HEG-GLU-AE.

22. The method of claim 18, wherein the labeled anti-TPO antibody is present at about 10 ng / ml to about 2 μg / ml.

23. The method of claim 18, wherein the complex is in a buffer comprising one or more of phosphate buffer, NaCl, EDTA, pluronic F-127, sodium azide, sorbitol, and sulfahydryl modified bovine serum albumin.

24. The method of any one of claims 18 to 23, wherein the solid support comprises a column matrix material, a culture plate, a tube, a dish, a flask, a microtiter plate, a bead, or a combination thereof.

25. The method of claim 24, wherein the solid support comprises a paramagnetic particle (PMP) or a latex magnetic particle (LMP).

26. The method of any one of claims 18 to 25, wherein the rTPO comprises rTPO from a cynomolgus monkey.

27. The method of claim 26, wherein the rTPO from a cynomolgus monkey is glycosylated, deglycosylated, or a mixture thereof.

28. The method of any claims 18 to 27, wherein the rTPO comprises an epitope tag.

29. The method of claim 28, wherein the epitope tag is a 6-histidine tag, a hemagglutinin tag, a glutathione-S-transferase, a maltose binding protein, or a chitin binding protein.

30. The method of any one of claims 18 to 29, further comprising: determining a level of the anti-thyroid peroxidase antibody in the biological sample of the subject.

31. The method of claim 30, wherein the level of the anti-thyroid peroxidase antibody in the biological sample of the subject is directly proportional to the level of the complex detected.

32. The method of any one of claims 18 to 31, wherein the anti-thyroid peroxidase antibody is an autoantibody.

33. A method of diagnosing a thyroid disease in a subject, said method comprising:incubating a biological sample from a subject with (i) a solid support, (ii) an unlabeled anti-TPO antibody or antigen-binding fragment thereof of any one of claims 9-13, (iii) a recombinant TPO (rTPO), and (iv) a labeled anti-TPO antibody or antigen binding fragment thereof of any one of claims 1-5; and diagnosing the subject with the thyroid disease if the formation of a complex comprising the solid support, the unlabeled anti-TPO antibody, the rTPO, and the labeled anti-TPO antibody is decreased.

34. The method of claim 33, wherein the thyroid disease is an autoimmune disorder.

35. The method of claim 34, wherein the autoimmune disorder is Hashimoto’s thyroiditis or Graves’ disease.

36. A kit comprising: a solid support; a recombinant TPO (rTPO); and anti-TPO antibodies of any one of claims 1-5 and / or claims 9-13.

37. The kit of claim 36, wherein the solid support comprises a column matrix material, a culture plate, a tube, a dish, a flask, a microtiter plate, a bead, or a combination thereof.

38. The kit of claim 36, wherein a portion of the anti-TPO antibodies is unlabeled and a portion of the anti-TPO antibodies is labeled.

39. The kit of claim 38, wherein the label comprises an enzyme conjugate, a fluorescent probe, a radioactive isotope, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.

40. The kit of claim 38, wherein the label is an acridinium ester (AE) or an analog thereof.

Images