Use of anti-human homeobox protein NKX-2.1 antibodies for immunohistochemical (IHC) regimen and for diagnosing cancer

Abstract

In alternative embodiments, provided are chimeric, synthetic or recombinant antibodies (Ab) against the human homologous box NKX-2. 1 (also known as NK2 homologous box 1, or thyroid transcription factor 1 (TTF-1)) protein, including articles of manufacture and kits comprising them, and methods of making and using them, including, for example, their use in the detection or diagnosis and treatment of cancer. In alternative embodiments, an anti-NKX-2. 1 (TTF-1) antibody provided herein is used with an agent for determining whether NKX-2. 1 (TTF-1) expression or activity is present, reduced or deleted; or the sensitivity of the specific binding of the Ab or the antigen (Ag) binding fragment or protein (ABP) or the monomer or dimer ABP provided by the invention with the squamous cell carcinoma cells is lower than the sensitivity of the specific binding with the adenocarcinoma cells (or binding with lower affinity), so that false positives of the squamous cell carcinoma are less, and the positive recognition of the lung adenocarcinoma is better.

Description

[0001] Related Applications

[0002] This PCT International Utility Patent Application claims the benefit of priority under 35 U.S. Patent Act Section 119(e) to U.S. Provisional Patent Application Serial No. (USSN) 63 / 411,913, filed on September 30, 2022, which is incorporated herein by reference in its entirety and for all purposes. Technical Field

[0003] The present invention generally relates to immunohistochemistry (IHC) and cancer diagnosis and treatment. In alternative embodiments, provided are chimeric, synthetic or recombinant anti-human homeobox NKX-2.1 (anti-human homeobox NKX-2.1, also known as NK2 homeobox 1, or thyroid transcription factor 1 (TTF-1)) protein antibodies (Abs), including articles and kits containing them, and methods of making and using them, including, for example, their use in detecting or diagnosing and treating cancer or other diseases or conditions involving TTF-1 expression. In alternative embodiments, the anti-NKX-2.1 (TTF-1) antibodies provided herein are used together with reagents for determining the presence, absence, reduction or lack of NKX-2.1 (TTF-1) expression or activity. In alternative embodiments, the antibodies (Ab) provided herein may help distinguish between adenocarcinoma and squamous cell carcinoma in non-small cell lung cancer; the Ab or its antigen (Ag) binding fragment or protein (ABP) or monomeric or dimeric ABP provided herein specifically binds to lung squamous cell carcinoma cells with lower sensitivity than that of specifically binding to adenocarcinoma cells (or binds to lung squamous cell carcinoma cells with lower affinity than that of specifically binding to adenocarcinoma cells), thereby resulting in fewer false positives for lung squamous cell carcinoma and better positive recognition of lung adenocarcinoma. Background Art

[0004] Thyroid transcription factor-1 (TTF-1) or homeobox NKX-2.1 (also known as NK2 homeobox 1) belongs to the homeodomain transcription factor family and is selectively expressed in the thyroid, lung, and diencephalon. TTF-1 has been identified as a transcriptional regulator of thyroid-specific genes and has also been shown to play an important role in the activation of lung-specific differentiation genes (4,5).

[0005] Although known anti-human TTF-1 antibodies can be used for the identification and diagnosis of certain cancers, currently used anti-human TTF-1 antibodies may not be able to distinguish lung adenocarcinoma from squamous cell carcinoma with high sensitivity and / or specificity.

[0006] Alternative names (aliases) for the homeobox protein Nkx-2.1 and gene NKX2-1 are: Homeobox protein NK-2 homolog A; Thyroid nuclear factor 1; Thyroid transcription factor 1, abbreviated as "TTF-1"; Thyroid-specific enhancer-binding protein, abbreviated as T / EBP. Summary of the invention

[0007] In an alternative embodiment, provided is an isolated or purified antibody (Ab), or a synthetic or recombinant Ab, or an antigen (Ag) binding fragment thereof, or a monomeric or dimeric antigen binding protein (ABP), which is capable of specifically binding to human homeobox NKX-2.1 (also known as NK2 homeobox 1, or thyroid transcription factor 1 (TTF-1)) protein or polypeptide,

[0008] Wherein, the isolated or purified Ab, or synthetic or recombinant Ab, or its Ag binding fragment, or monomeric or dimeric ABP comprises:

[0009] (a) a heavy chain variable region (VH) capable of specifically binding to a human homeobox NKX-2.1 protein or polypeptide, comprising:

[0010] (1) an amino acid sequence comprising the three complementarity determining regions (CDRs) CDR1, CDR2 and CDR3 of SEQ ID NO: 1, or CDR1 amino acid (aa) residues GFSLSSYA (residues 25 to 32 of SEQ ID NO: 1), CDR2 aa residues INSYGNA (residues 50 to 56 of SEQ ID NO: 1), and CDR3 aa residues TTTHSYGFGAFDP (residues 95 to 107 of SEQ ID NO: 1), or

[0011] (2) an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% sequence identity, or about 70%-100% sequence identity to each of the three complementarity determining regions (CDRs) CDR1, CDR2, and CDR3 of SEQ ID NO: 1, or CDR1 amino acid (aa) residues GFSLSSYA (residues 25 to 32 of SEQ ID NO: 1), CDR2 aa residues INSYGNA (residues 50 to 56 of SEQ ID NO: 1), and CDR3 aa residues TTTHSYGFGAFDP (residues 95 to 107 of SEQ ID NO: 1),

[0012] And optionally, the heavy chain variable region (VH) that can specifically bind to the human homeobox NKX-2.1 protein or polypeptide is or comprises the following amino acid sequence:

[0013] QSLKESEGGLFKPTDTLTLTCTVS GFSLSSYA MIWVRQAPGNGLEYIGA INSYGNA YYASWAKSSRSTITRDTNENTVTLKMTSLTAADTATYFC TTTHSYGFGAFDP WGPGTLVTVSS (SEQ ID NO: 1); or

[0014] (3) an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% sequence identity, or about 70%-100% sequence identity to SEQ ID NO: 1, or an amino acid sequence having complete sequence identity to SEQ ID NO: 1;

[0015] (b) a light chain variable region (VL) capable of specifically binding to a human homeobox NKX-2.1 protein or polypeptide, comprising:

[0016] (1) an amino acid sequence comprising the following three CDRs: CDR1, CDR2 and CDR3 of SEQ ID NO: 2, or CDR1 amino acid (aa) residues ESAYNNR (residues 27 to 33 of SEQ ID NO: 2), CDR2 aa residues DAS (residues 51 to 53 of SEQ ID NO: 2), and CDR3 aa residues QGVYSSVSDVYA (residues 112 to 123 of SEQ ID NO: 2), or

[0017] (2) an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% sequence identity, or about 70%-100% sequence identity to each of the following three CDRs: CDR1, CDR2, and CDR3 of SEQ ID NO:2, or CDR1 amino acid (aa) residues ESAYNNR (residues 27 to 33 of SEQ ID NO:2), CDR2 aa residues DAS (residues 51 to 53 of SEQ ID NO:2), and CDR3 aa residues QGVYSSVSDVYA (residues 112 to 123 of SEQ ID NO:2),

[0018] The light chain variable region (VL) that can optionally specifically bind to the human homeobox NKX-2.1 protein or polypeptide is or comprises the following amino acid sequence:

[0019] AAVLTQTPSPVSAAVGGTVSISCQSS ESAYNNRLAWYQQKPGQPPKLLIY DAS KLASGVPSRFKGSGSGTPFTLTISGVQCADAATYYC QGVYSSVSDVYA FGGGTEVVVK (SEQ ID NO: 2); or

[0020] (3) an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% sequence identity, or about 70%-100% sequence identity to SEQ ID NO: 2, or an amino acid sequence having complete (100%) sequence identity to SEQ ID NO: 2; or

[0021] (c) A heterodimer capable of specifically binding to a human homeobox NKX-2.1 protein or polypeptide, comprising the heavy chain variable region (VH) of (a) and the light chain variable region (VL) of (b).

[0022] In alternative embodiments, the isolated or purified Ab, or synthetic or recombinant Ab, or Ag-binding fragment thereof, or monomeric or dimeric ABP provided herein, is manufactured or is in the form of:

[0023] An antigen binding fragment, wherein optionally the antigen binding fragment comprises a FAb or an Ab fragment having at least one constant domain and one variable domain of each of the Ab heavy and light chains.

[0024] F(ab')2 (or Ab digested by pepsin, which produces two fragments: F(ab')2 fragment and pFc' (pepsin cleaved Fc) fragment,

[0025] Fab' (single chain of F(ab')2 fragment),

[0026] A single chain variable fragment (scFv) (or a fusion protein in which the variable regions of the heavy and light chains of an Ab are connected by a linker peptide, optionally with a length of about 10 to about 25 amino acids),

[0027] (scFv) 2, or dual scFv or di-scFv, or a single peptide chain having two variable heavy regions and two variable light regions to produce a tandem scFv,

[0028] A minibody, or a fusion protein comprising an Ab heavy chain variable region and a light chain variable region linked by or to an alkyl group, wherein optionally the alkyl group comprises a methyl group or an ethyl group,

[0029] Diabodies (or scFvs in which the linker peptide is too short (optionally about 5 amino acids) to allow the two variable regions to fold together, forcing the scFv to dimerize), triabodies or tetrabodies (or scFvs in which the linker peptide is too short (optionally about 1 or 2 amino acids) to allow the two variable regions to fold together, forcing the scFv to trimerize or tetramerize),

[0030] Single domain antibodies (dAB) (or a single variable region of an Ab heavy chain or an Ab light chain),

[0031] Multiple complementarity determining region (CDR) fragments, or

[0032] A multispecific antibody formed from two or more antibody fragments.

[0033] In alternative embodiments of the isolated or purified Ab, or synthetic or recombinant Ab, or Ag-binding fragment thereof, or monomeric or dimeric ABP provided herein:

[0034] - the heavy chain variable region (VH), if present:

[0035] (a) further comprises a carboxyl terminal constant domain, or further comprises the amino acid sequence of SEQ ID NO: 3, or comprises SEQ ID NO: 4;

[0036] (b) further comprises an amino-terminal signal peptide, or further comprises an amino-terminal signal peptide, wherein the signal peptide comprises: METGLRWLLLVAVLKGVQC (SEQ ID NO: 5), or comprises:

[0037] A heavy chain variable region having a signal peptide comprising the following amino acid sequence:

[0038] METGLRWLLLVAVLKGVQC QSLKESEGGLFKPTDTLTLTCTVSGFSLSSYAMIWVRQAPGNGLEYIGA INSYGNAYYASWAKSRSTITRDTNENTVTLKMTSLTAADTATYFCTTTHSYGFGAFDPWGPGTLVTVSS (SEQ ID NO: 6), or

[0039] A heavy chain variable region plus a constant region having a signal peptide comprising the following amino acid sequence:

[0040] METGLRWLLLVAVLKGVQC QSLKESEGGLFKPTDTLTLTCTVSGFSLSSYAMIWVRQAPGNGLEYIGA INSYGNAYYASWAKSRSTITRDTNENTVTLKMTSLTAADTATYFCTTTHSYGFGAFDPWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTA RPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:7); or

[0041] (c) comprising SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:7, having one or more amino acid substitutions, additions (insertions) or deletions, and the recombinant Ab or its Ag-binding fragment or monomeric or dimeric ABP retains its ability to specifically bind to human homeobox NKX-2.1 protein or polypeptide,

[0042] and optionally one or more amino acid substitutions, said amino acid substitutions comprising one or more conservative amino acid substitutions, and said isolated or purified Ab, or synthetic or recombinant Ab, or Ag-binding fragment thereof, or monomeric or dimeric ABP, retains its ability to specifically bind to human homeobox NKX-2.1 protein or polypeptide.

[0043] In alternative embodiments of the isolated or purified Ab, or synthetic or recombinant Ab, or Ag-binding fragment thereof, or monomeric or dimeric ABP provided herein: the light chain variable region (VL), if present:

[0044] (a) further comprises a carboxyl terminal constant domain, or further comprises the amino acid sequence of SEQ ID NO: 8, or comprises SEQ ID NO: 9,

[0045] (b) further comprises an amino-terminal signal peptide, or further comprises an amino-terminal signal peptide consisting of MDTRAPTQLLGLLLLWLPGATF (SEQ ID NO: 12), or comprises:

[0046] A light chain variable region having a signal peptide comprising the following amino acid sequence:

[0047] MDTRAPTQLLGLLLLWLPGATFAAVLTQTPSPVSAAVGGTVSISCQSSESAYNNRLAWYQQKPGQPPK LLIYDASKLASGVPSRFKGSGSGTPFTLTISGVQCADAATYYCQGVYSSVSDVYAFGGGTEVVVK (SEQ ID NO: 10), or

[0048] A light chain variable region and a constant region, wherein the light chain variable region and the constant region have a signal peptide comprising the following amino acid sequence:

[0049] MDTRAPTQLLGLLLLWLPGATF AAVLTQTPSPVSAAVGGTVSISCQSSESAYNNRLAWYQQKPGQPPK LLIYDASKLASGVPSRFKGSGSGTPFTLTISGVQCADAATYYCQGVYSSVSDVYAFGGGTEVVVK GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO:11); or

[0050] (c) comprising SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, with one or more amino acid substitutions, additions (insertions) or deletions, and the recombinant Ab or its Ag-binding fragment or monomeric or dimeric ABP retains its ability to specifically bind to human homeobox NKX-2.1 protein or polypeptide,

[0051] and optionally one or more amino acid substitutions, said amino acid substitutions comprising one or more conservative amino acid substitutions, and said isolated or purified Ab, or synthetic or recombinant Ab, or Ag-binding fragment thereof, or monomeric or dimeric ABP, retains its ability to specifically bind to human homeobox NKX-2.1 protein or polypeptide.

[0052] In alternative embodiments: the one or more amino acid substitutions comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen conservative amino acid substitutions, and the recombinant Ab, or its Ag-binding fragment, or monomeric or dimeric ABP retains its ability to specifically bind to human homeobox NKX-2.1 protein or polypeptide.

[0053] In alternative embodiments of the isolated or purified Ab, or synthetic or recombinant Ab, or Ag-binding fragment thereof, or monomeric or dimeric ABP provided herein:

[0054] - the light chain variable region further comprises at least a portion of a light chain constant region;

[0055] -The light chain constant region comprises the following amino acid sequence:

[0056] GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO:8);

[0057] - the heavy chain variable region further comprises at least a portion of a heavy chain constant region;

[0058] -The heavy chain constant region comprises the following amino acid sequence:

[0059] GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTA RPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:3);

[0060] - the light chain variable region further comprises at least a portion of a light chain constant region; and the heavy chain variable region further comprises at least a portion of a heavy chain constant region;

[0061] -The isolated or purified or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, comprising:

[0062] A light chain comprising the following amino acid sequence (SEQ ID NO:9):

[0063] AAVLTQTPSPVSAAVGGTVSISCQSSESAYNNRLAWYQQKPGQPPKLLIYDASKLASGVPSRFKGSGS GTPFTLTISGVQCADAATYYCQGVYSSVSDVYAFGGGTEVVVK GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQID NO:9), and

[0064] A heavy chain comprising the following amino acid sequence (SEQ ID NO:4):

[0065] QSLKESEGGLFKPTDTLTLTCTVSGFSLSSYAMIWVRQAPGNGLEYIGAINSYGNAYYASWAKSRSTI TRDTNENTVTLKMTSLTAADTATYFCTTTHSYGFGAFDPWGPGTLVTVSS GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTA RPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ IDNO:4);

[0066] - the heavy chain constant region comprises an amino acid sequence from an IgG, IgM, IgA, IgD or IgE isotype, or the heavy chain constant region essentially comprises an amino acid sequence from an IgG, IgM, IgA, IgD or IgE isotype;

[0067] - the light chain constant region comprises an amino acid sequence from a kappa (κ) or lambda (λ) isotype, or the light chain constant region substantially comprises an amino acid sequence from a kappa (κ) or lambda (λ) isotype;

[0068] - at least a portion of the heavy chain constant region, at least a portion of the light chain constant region, or at least a portion of the heavy chain constant region and the light chain constant region is or comprises an amino acid sequence of human, rabbit, mouse or rat origin, or comprises a constant region amino acid sequence derived from human, rabbit, mouse or rat or substantially derived from human, rabbit, mouse or rat;

[0069] - at least a portion of the heavy chain constant region, at least a portion of the light chain constant region, or at least a portion of the heavy chain constant region and the light chain constant region is or comprises a synthetic amino acid sequence;

[0070] - the recombinant Ab, its Ag-binding fragment, or monomeric or dimeric ABP, or heavy chain constant region, or light chain constant region, or heavy chain constant region and light chain constant region further comprise or are combined with a heterologous protein, peptide, or compound or composition;

[0071] - the heterologous protein or peptide, or compound or composition comprises a detectable protein, a detectable agent or a binding moiety, or the heterologous protein or peptide comprises a carrier protein;

[0072] - the heterologous protein, peptide or compound or composition is covalently conjugated to a recombinant antibody (Ab) or its Ag-binding fragment or a monomeric or dimeric ABP;

[0073] - The detectable agent or binding moiety comprises biotin, a fluorescent or chemiluminescent tag, a fluorophore, perylene, fluorenyl, coumarin, 7-methoxycoumarin (Mca), 4-(dimethylaminoazo)benzene-4-carboxylic acid (4-(dimethylaminoazo)benzene-4-carboxylic acid, dabcyl), Tamra, boron-dipyrromethene (BODIPY) or a derivative thereof, a dye, a radioisotope, a quantum dot or a photoluminescent aqueous nanocrystal, a hapten or an antibody binding epitope or domain, and optionally the dye is or comprises rhodamine, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium ([2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium, NBD), nile red or nile blue, or is a fluorescent dye comprising sulfoindocyanine, and optionally the fluorophore is or comprises a dansyl, fluorescein, carboxyfluorescein (FAM) or 6-FAM moiety, and optionally the dye is or comprises a cyanine dye ( dye), Cy3 or Cy5, and optionally the hapten is or comprises biotin, theophylline, digoxigenin, carborane, fluorescein or a bromodeoxyuridine moiety; and / or

[0074] - The Ab, or Ag-binding fragment thereof, or monomeric or dimeric ABP is a recombinant Ab, or Ag-binding fragment thereof, or monomeric or dimeric ABP, or comprises a peptide or polypeptide produced by recombinant technology.

[0075] In alternative embodiments, provided are chimeric, synthetic or recombinant nucleic acids comprising: a nucleic acid sequence encoding an Ab or Ag-binding fragment thereof or a monomeric or dimeric ABP provided herein.

[0076] In alternative embodiments of the chimeric, synthetic or recombinant nucleic acids provided herein:

[0077] - the chimeric or recombinant nucleic acid further comprises and is operably linked to a transcriptional regulatory element, and optionally the transcriptional regulatory element comprises a promoter, and optionally the promoter is an inducible promoter or a constitutive promoter; and / or

[0078] - the chimeric or recombinant nucleic acid further comprises a sequence encoding an amino-terminal signal peptide, and optionally the amino-terminal signal peptide comprises the amino acid sequence: METGLRWLLLVAVLKGVQC (SEQ ID NO: 5); MDTRAPTQLLGLLLLWLPGATF (SEQ ID NO: 12), and optionally the amino-terminal signal peptide SEQ ID NO: 5 is the amino-terminus of the VH chain SEQ ID NO: 1, or the amino-terminus of SEQ ID NO: 4; or the amino-terminal signal peptide SEQ ID NO: 12 is the amino-terminus of the VL chain SEQ ID NO: 2, or the amino-terminus of SEQ ID NO: 9.

[0079] In alternative embodiments, provided are expression cassettes, vectors, recombinant viruses, artificial chromosomes, cosmids or plasmids comprising the chimeric, synthetic or recombinant nucleic acids provided herein.

[0080] In alternative embodiments, provided is a cell comprising a chimeric, synthetic or recombinant antibody or dimeric antigen binding protein provided herein, a chimeric or recombinant nucleic acid provided herein, or an expression cassette, vector, recombinant virus, artificial chromosome, cosmid or plasmid provided herein. The cell can be a bacterium, fungus, mammal, yeast, insect or plant cell, or a mammalian cell or a human cell.

[0081] In an alternative embodiment, provided is a method for detecting the presence of a human homeobox NKX-2.1 protein or polypeptide in or on a cell, tissue, organ or part of any of the foregoing, the method comprising:

[0082] (a) contacting the cell, tissue or organ, or any part of the foregoing, with at least one of the isolated or purified, or synthetic or recombinant Ab, or Ag-binding fragment thereof, or monomeric or dimeric ABP provided herein, and

[0083] (b) detecting specific binding of at least one Ab or its Ag-binding fragment or monomeric or dimeric ABP to human homeobox NKX-2.1 protein or polypeptide in or on a cell, tissue or organ or any part of the foregoing,

[0084] Thus, the presence of human homeobox NKX-2.1 protein or polypeptide in or on the cells, tissues, organs or any part thereof is detected.

[0085] In an alternative embodiment of the method provided herein for detecting the presence of human homeobox NKX-2.1 protein or polypeptide:

[0086] - said at least one Ab or Ag-binding fragment thereof, or monomeric or dimeric ABP comprises a variable heavy chain (VH) and a variable light chain (VL), said variable heavy chain (VH) having an amino acid sequence comprising SEQ ID NO: 1, and said variable light chain (VL) having an amino acid sequence comprising SEQ ID NO: 2;

[0087] - said at least one Ab or Ag-binding fragment thereof, or monomeric or dimeric ABP comprises a variable heavy chain (VH) and a variable light chain (VL), said variable heavy chain (VH) having an amino acid sequence comprising SEQ ID NO: 4, and said variable light chain (VL) having an amino acid sequence comprising SEQ ID NO: 9;

[0088] - said at least one Ab or Ag-binding fragment thereof, or monomeric or dimeric ABP comprises a variable heavy chain (VH) and a variable light chain (VL), said variable heavy chain (VH) having an amino acid sequence comprising SEQ ID NO: 7, and said variable light chain (VL) having an amino acid sequence comprising SEQ ID NO: 11;

[0089] - the method comprises contacting the cell, tissue or organ, or any part of the foregoing, with a mixture of two or at least two Abs, or Ag-binding fragments thereof, or monomeric or dimeric ABPs, or two or at least two different Abs, or Ag-binding fragments thereof, or monomeric or dimeric ABPs, wherein the two or at least two Abs, or Ag-binding fragments thereof, or monomeric or dimeric ABPs, or the two or at least two different Abs, or Ag-binding fragments thereof, or monomeric or dimeric ABPs comprise an Ab, or Ag-binding fragments thereof, or monomeric or dimeric ABP provided herein;

[0090] - said contacting comprises the use of an immunohistochemistry (IHC) assay;

[0091] - the method further comprises contacting the Ab or its Ag-binding fragment or monomer or dimeric ABP that specifically binds to the human homeobox NKX-2.1 polypeptide or peptide with a detectable agent to indicate or signal the specific binding of the Ab or its Ag-binding fragment or monomer or dimeric ABP to the human homeobox NKX-2.1 polypeptide or peptide, and optionally the detectable agent specifically binds to the Ab or its Ag-binding fragment or monomer or dimeric ABP;

[0092] - the cell, tissue, organ or part of any of the foregoing is or comprises or is derived from:

[0093] cancer cell,

[0094] wherein optionally the cancer cell is a thyroid cancer cell, a lung cancer cell or a cancer cell,

[0095] wherein optionally the cancer cell is a large cell carcinoma, a small cell carcinoma (optionally a small cell lung cancer or an extrapulmonary small cell carcinoma), a lung cancer cell or an adenocarcinoma cell, and optionally the adenocarcinoma cell is a lung adenocarcinoma cell or a thyroid adenocarcinoma cell,

[0096] wherein optionally the cancer cell is a squamous cell carcinoma (SCC) cell, a leukemia cell, a carcinoid tumor, a breast cancer cell, a colon cancer cell, a malignant melanoma cell or a multiple myeloma cell, a plasmacytoma cell or a lymphoma cell or a cancer derived from a lymphocyte,

[0097] Wherein optionally the lymphocyte is an early B cell, a pro-B cell, a pre-B lymphocyte, a mature B lymphocyte, a T lymphocyte, a parafollicular T lymphocyte, a cell derived from a parafollicular T lymphocyte subset,

[0098] lung cell, wherein optionally the lung cell is a lung pneumocyte cell (optionally a type II alveolar cell) or a Clara (or nonciliated bronchiolar secretory) cell,

[0099] Thyroid cells, optionally thyroid follicular cells or thyroid parafollicular cells,

[0100] follicle center cells, or cells in the tonsils, lymph node germinal centers, bone marrow stem cells, myelopoietic cells, liverbile canalicular cells, renal glomerular cells, proximal tubular cells, breast myoepithelial cells, stromal cells around or associated with an infiltrating tumor cell, kidney cells, epithelial cells, cerebellar cells, prostate cells, kidney cells, pancreatic cells, bone marrow cells,

[0101] and optionally the epithelial cell is or is derived from a brain, lung, intestinal, kidney, breast or placental epithelial cell, and optionally the organ is liver, prostate or lung; and / or

[0102] - the Ab or its Ag-binding fragment or monomeric or dimeric ABP specifically binds to lung squamous cell carcinoma cells with less sensitivity than to adenocarcinoma cells (or binds to lung squamous cell carcinoma cells with lower affinity than to adenocarcinoma cells), thereby resulting in fewer false positives for lung squamous cell carcinoma and better positive identification of lung adenocarcinoma;

[0103] In an alternative embodiment, provided are methods for detecting or diagnosing cancer,

[0104] The method comprises detecting the expression or presence of a human homeobox NKX-2.1 polypeptide or peptide in or on a cell, tissue or organ sample using the method provided herein,

[0105] Wherein, the detection of specific binding of the Ab or its Ag binding fragment or monomer or dimer ABP with the human homeobox NKX-2.1 polypeptide or peptide in or on the cell, tissue or organ or any part of the foregoing detects or diagnoses cancer or assists in the detection or diagnosis of cancer.

[0106] In alternative embodiments of the methods provided herein for detecting or diagnosing cancer:

[0107] - The cancer is:

[0108] Thyroid cancer cells, lung cancer cells or cancer cells,

[0109] wherein optionally the cancer cell is a large cell carcinoma, a small cell carcinoma (optionally a small cell lung cancer or an extrapulmonary small cell carcinoma), a lung cancer cell or an adenocarcinoma cell, and optionally the adenocarcinoma cell is a lung adenocarcinoma cell or a thyroid adenocarcinoma cell,

[0110] wherein optionally the cancer cell is a squamous cell carcinoma (SCC), a leukemia cell, a carcinoid cell, a breast cancer cell, a colon cancer cell, a malignant melanoma cell or a multiple myeloma cell, a plasma cell or a lymphoma cell or a cancer derived from a lymphocyte,

[0111] wherein optionally the lymphocytes are early B cells, pro-B cells, pre-B lymphocytes, mature B lymphocytes, T lymphocytes, parafollicular T lymphocytes, cells derived from a subpopulation of parafollicular T lymphocytes,

[0112] - the Ab or its Ag-binding fragment or monomeric or dimeric ABP specifically binds to lung squamous cell carcinoma cells with less sensitivity than to adenocarcinoma cells (or binds to lung squamous cell carcinoma cells with lower affinity than to adenocarcinoma cells), thereby resulting in fewer false positives for lung squamous cell carcinoma and better positive identification of lung adenocarcinoma;

[0113] - the cell, tissue or organ sample is from an individual in need;

[0114] - the detecting comprises performing an immunohistochemistry (IHC) assay;

[0115] - at least two Abs or Ag binding fragments thereof, or monomeric or dimeric ABPs are used to contact the cell, tissue or organ sample; and / or

[0116] - One of the two Abs or Ag-binding fragments thereof or monomeric or dimeric ABPs comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the variable heavy chain (VH) has an amino acid sequence comprising SEQ ID NO: 1, and the variable light chain (VL) has an amino acid sequence comprising SEQ ID NO: 2.

[0117] In an alternative embodiment, provided are methods for treating, ameliorating or preventing cancer (e.g., further growth or spread of cancer), comprising first detecting or diagnosing the cancer using the methods provided herein, and then treating (or treating or ameliorating) an individual in need thereof who has cancer.

[0118] In alternative embodiments of the methods provided herein for treating, ameliorating or preventing cancer:

[0119] - The cancer is:

[0120] Thyroid cancer, lung cancer or carcinoma,

[0121] wherein optionally the cancer is large cell carcinoma, small cell carcinoma (optionally small cell lung cancer or extrapulmonary small cell carcinoma), lung cancer or adenocarcinoma, and optionally the adenocarcinoma is lung adenocarcinoma or thyroid adenocarcinoma,

[0122] wherein optionally the cancer is squamous cell carcinoma (SCC), leukemia, carcinoid, breast cancer, colon cancer, malignant melanoma or multiple myeloma, plasmacytoma or lymphoma or a cancer derived from lymphocytes,

[0123] wherein optionally the lymphocytes are early B cells, pro-B cells, pre-B lymphocytes, mature B lymphocytes, T lymphocytes, parafollicular T lymphocytes, cells derived from a subpopulation of parafollicular T lymphocytes, and / or

[0124] - the Ab or its Ag-binding fragment or monomeric or dimeric ABP specifically binds to lung squamous cell carcinoma cells with less sensitivity than to adenocarcinoma cells (or binds to lung squamous cell carcinoma cells with lower affinity than to adenocarcinoma cells), thereby resulting in fewer false positives for lung squamous cell carcinoma and better positive identification of lung adenocarcinoma;

[0125] In an alternative embodiment, there is provided a use of at least one isolated or purified, or synthetic or recombinant antibody (Ab) provided herein, or an antigen (Ag) binding fragment thereof, or a monomeric or dimeric antigen binding protein (ABP), for detecting or diagnosing cancer, or treating, ameliorating or preventing cancer, wherein optionally the cancer is:

[0126] Thyroid cancer, lung cancer or carcinoma,

[0127] wherein optionally the cancer is large cell carcinoma, small cell carcinoma (optionally small cell lung cancer or extrapulmonary small cell carcinoma), lung cancer or adenocarcinoma, and optionally the adenocarcinoma is lung adenocarcinoma or thyroid adenocarcinoma,

[0128] wherein optionally the cancer is squamous cell carcinoma (SCC), leukemia, carcinoid tumor, breast cancer, colon cancer cell, malignant melanoma or multiple myeloma, plasmacytoma or lymphoma or a cancer derived from lymphocytes,

[0129] Optionally, the lymphocytes are early B cells, pro-B cells, pre-B lymphocytes, mature B lymphocytes, T lymphocytes, parafollicular T lymphocytes, or cells derived from a subpopulation of parafollicular T lymphocytes.

[0130] In alternative embodiments of the uses provided herein:

[0131] - the Ab or its Ag-binding fragment or monomeric or dimeric ABP specifically binds to lung squamous cell carcinoma cells with less sensitivity than to adenocarcinoma cells (or binds to lung squamous cell carcinoma cells with lower affinity than to adenocarcinoma cells), thereby resulting in fewer false positives for lung squamous cell carcinoma and better positive identification of lung adenocarcinoma; and / or

[0132] - said detecting comprises performing an immunohistochemistry (IHC) assay.

[0133] In an alternative embodiment, provided is an isolated or purified, or synthetic or recombinant antibody (Ab) provided herein, or an antigen (Ag) binding fragment thereof, or a monomeric or dimeric antigen binding protein (ABP), for use in detecting or diagnosing cancer, or treating, ameliorating or preventing cancer; and optionally the cancer is:

[0134] Thyroid cancer, lung cancer or

[0135] wherein optionally the cancer is large cell carcinoma, small cell carcinoma (optionally small cell lung cancer or extrapulmonary small cell carcinoma), lung cancer or adenocarcinoma, and optionally the adenocarcinoma is lung adenocarcinoma or thyroid adenocarcinoma,

[0136] wherein optionally the cancer is squamous cell carcinoma (SCC), leukemia, carcinoid tumor, breast cancer, colon cancer cell, malignant melanoma or multiple myeloma, plasmacytoma or lymphoma or a cancer derived from lymphocytes,

[0137] wherein optionally the lymphocytes are early B cells, pro-B cells, pre-B lymphocytes, mature B lymphocytes, T lymphocytes, parafollicular T lymphocytes, cells derived from a subpopulation of parafollicular T lymphocytes,

[0138] In alternative embodiments, in the isolated or purified, or synthetic or recombinant antibodies (Ab), or antigen (Ag) binding fragments thereof, or monomeric or dimeric antigen binding proteins (ABPs) provided herein: the Ab or its Ag binding fragment or monomeric or dimeric ABP specifically binds to lung squamous cell carcinoma cells with lower sensitivity than that to adenocarcinoma cells (or binds to lung squamous cell carcinoma cells with lower affinity than that to adenocarcinoma cells), thereby resulting in fewer false positives for lung squamous cell carcinoma and better positive identification of lung adenocarcinoma; or the detection or diagnosis comprises performing an immunohistochemistry (IHC) assay.

[0139] In alternative embodiments, provided is a kit comprising: an isolated or purified, or synthetic or chimeric or recombinant antibody provided herein; a chimeric or recombinant nucleic acid provided herein; or an expression cassette, vector, recombinant virus, artificial chromosome, cosmid or plasmid provided herein; or a cell provided herein; or a recombinant antibody (Ab) provided herein, or an antigen (Ag) binding fragment thereof, or a monomer or dimeric antigen binding protein (ABP). In alternative embodiments of the kit provided herein, the kit includes components required for immunohistochemistry (IHC) assays, and / or includes instructions for implementing the methods provided herein.

[0140] The details of one or more exemplary embodiments of the invention are shown in the accompanying drawings and the following description. Other features, objects, and advantages of the invention will be apparent from the description, drawings, and from the claims.

[0141] All publications, patents, and patent applications cited herein are hereby expressly incorporated by reference in their entirety for all purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0142] The patent or application file contains at least one drawing printed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0143] The drawings shown herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims.

[0144] Figure 1 Shown are IHC images of normal lung, showing specific staining of type II cells (arrows), as discussed in detail in Example 1 below.

[0145] Figure 2 Shown are IHC images of lung bronchi stained using the clone 6F6 antibody, as discussed in detail in Example 1 below.

[0146] Figure 3 Shown are IHC images of normal thyroid stained using the clone 6F6 antibody, as discussed in detail in Example 1 below.

[0147] Figure 4 An IHC image of negative normal tissue (normal liver) is shown, showing hepatocytes (arrows), as discussed in detail in Example 1 below.

[0148] Figure 5 IHC images of lung adenocarcinoma are shown, showing specific staining of tumor cells, as discussed in detail in Example 1 below.

[0149] Figure 6 IHC images of lung squamous cell carcinoma are presented, showing non-specific staining of tumor cells, as discussed in detail in Example 1 below.

[0150] Figure 7 IHC images of colon adenocarcinoma are presented, showing non-specific staining of tumor cells, as discussed in detail in Example 1 below.

[0151] Figure 8 Data showing specificity for different tumor types are presented graphically: Specificity was tested on a multi-tumor array (MTU951) using antibody clones 6F6 and SPT24, as discussed in detail in Example 1 below.

[0152] Figure 9 Data showing sensitivity to lung adenocarcinoma are presented, where specificity was tested on 22 cases of lung adenocarcinoma using clones 6F6, SPT24 and 8G7G3 / 1, and the results showed that clones 6F6 and SPT24 were more sensitive than clone 8G7G3 / 1, with clone SPT24 being the most sensitive, as discussed in detail in Example 1 below.

[0153] Figure 10 Data showing specificity for lung squamous cell carcinoma are shown: Specificity was tested on 32 lung squamous cell carcinomas using clones 6F6, SPT24 and 8G7G3 / 1, and the results showed that clone SPT24 was the least specific clone, as discussed in detail in Example 1 below.

[0154] In different drawings, the same reference symbols denote the same elements. DETAILED DESCRIPTION

[0155] In alternative embodiments, antibodies that can specifically bind to lung adenocarcinoma cells are provided, including providing chimeric, synthetic or recombinant anti-human homeobox NKX-2.1 (anti-human homeobox NKX-2.1, also known as NK2 homeobox 1, or thyroid transcription factor 1 (TTF-1)) protein antibodies (Abs), including products and kits containing them, and methods of making and using them, including, for example, their use in detecting or diagnosing and treating cancer or other diseases or conditions involving TTF-1 expression. In alternative embodiments, the anti-TTF-1 antibodies provided herein are used in assays (such as immunohistochemistry (IHC) assays), optionally with detection agents or detectable agents, for determining whether TTF-1 expression or activity is present, reduced or absent on cells, for example, detecting TTF-1 expression on cells in a biopsy or other tissue or cell sample.

[0156] In alternative embodiments, the Ab provided herein, or its antigen (Ag) binding fragment or protein (ABP), or monomeric or dimeric ABP specifically binds to lung squamous cell carcinoma cells with lower sensitivity than to adenocarcinoma cells (or binds to lung squamous cell carcinoma cells with lower affinity than to adenocarcinoma cells), thereby resulting in fewer false positives for lung squamous cell carcinoma and better positive recognition of lung adenocarcinoma.

[0157] In an alternative embodiment, the clinical use of TTF-1 detection, such as TTF-1 IHC detection, is to distinguish adenocarcinoma from squamous cell carcinoma in non-small cell lung cancer, as described, for example, by Yatabe et al. (1). Yatabe et al. discussed how the diagnostic sensitivity and specificity of the anti-TTF-1 antibody (Ab) produced by Agilent Dako clone 8G7G3 / 1 (IR056) differ from the anti-TTF-1 antibody produced by clone SPT24. The difference in sensitivity and specificity between the two anti-TTF-1 antibodies has been reported many times (2,3). Provided herein is a novel and improved anti-TTF-1 antibody produced by a new clone, wherein the novel anti-TTF-1 antibody provided herein has a diagnostic sensitivity comparable to that of SPT24 anti-TTF-1Ab, and a diagnostic specificity comparable to that of 8G7G3 / 1 anti-TTF-1Ab.

[0158] Expression of Recombinant Chimeric Antibodies

[0159] In alternative embodiments, the chimeric or recombinant Abs provided herein, including the exemplary chimeric or recombinant anti-human TTF-1 Abs (with or without a signal peptide), can be expressed as recombinant Abs using plasmids (or any expression vectors) encoding the respective heavy and light chains, or the heavy and light chains can be encoded in different expression vectors.

[0160] In some embodiments, the heavy and light chains can be expressed (in cis or trans) in HEK293-6E cells from pTT5 TM Vector (National Research Council Canada, NRC-CNRC, Canada) expression. In alternative embodiments, the (one or more) vectors expressing the heavy chain and / or light chain are episomal, or are chromosomally integrated, for example, integrated in a stable cell line capable of synthesizing (optionally inducible synthesis) the heavy chain and / or light chain.

[0161] In alternative embodiments, provided is the nucleic acid of the chimeric or recombinant Ab provided herein. The nucleic acid provided herein can be manufactured, separated and / or operated by methods such as cloning and expressing of cDNA libraries, by PCR amplification information or genomic DNA. For the implementation of the nucleic acid of the embodiment provided herein, whether it is RNA, cDNA, genomic DNA, vector, virus or its heterozygote, can be separated from various sources, genetically engineered, amplified and / or recombinantly expressed / generated. The recombinant polypeptide generated by these nucleic acids can be separated or cloned independently, and the test of required activity is carried out. Any recombinant expression system can be used, including bacteria, fungi, mammals, yeast, insects or plant cell expression systems.

[0162] Alternatively, the nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described, for example, in Adams (1983) J. Am. Chem. Soc. 105: 661; Belousov (1997) Nucleic Acids Res. 25: 3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19: 373-380; Blommers (1994) Biochemistry 33: 7886-7896; Narang (1979) Meth. Enzymol. 68: 90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; U.S. Pat. No. 4,458,066.

[0163] Techniques for nucleic acid manipulation, e.g., subcloning, labeling probes (e.g., labeling with random primers using Klenow polymerase, nick translation, amplification), sequencing, hybridization, and the like, are fully described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULARBIOLOGY, Ausubel, ed., John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed., Elsevier, NY (1993).

[0164] Other useful means for obtaining and manipulating nucleic acids for implementing the embodiments provided herein include screening and recloning of inserts isolated or amplified from, for example, genomic clones or cDNA clones. The source of nucleic acid includes a recombinant nucleic acid sequence, genomic or cDNA library contained or expressed in, for example, a mammalian artificial chromosome (MAC), see, for example, U.S. Patent No. 5,721,118; 6,025,155; a human artificial chromosome, see, for example, Rosenfeld (1997) Nat. Genet. 15: 333-335; a yeast artificial chromosome (YAC); a bacterial artificial chromosome (BAC); a P1 artificial chromosome, see, for example, Woon (1998) Genomics 50: 306-316; a vector (PAC) derived from P1, see, for example, Kern (1997) Biotechniques 23: 120-124; a cosmid, a recombinant virus, a phage, a phagemid or a plasmid.

[0165] In alternative embodiments, the nucleic acids provided herein are operably linked to transcriptional regulatory elements, including a promoter, which can be a constitutive or inducible transcriptional regulatory element.

[0166] In alternative aspects, provided is an "expression cassette" comprising a nucleotide sequence as provided herein, e.g., a nucleotide sequence encoding a chimeric or recombinant antibody as provided herein. The expression cassette may include at least one transcriptional regulatory element, e.g., a promoter, which is operably connected to the antibody coding sequence and may optionally also include a transcription termination signal. Additional factors necessary for expression or conducive to expression may also be used, e.g., enhancers.

[0167] In alternative aspects, the expression cassette for implementing the embodiments provided herein includes plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vectors, etc. In alternative aspects, "carriers" for implementing the embodiments provided herein may include nucleic acids that can infect, transfect, transiently or permanently transduce cells. In alternative aspects, the carrier for implementing the embodiments provided herein may be naked nucleic acids, or nucleic acids compounded with proteins or lipids. In alternative aspects, the carrier for implementing the embodiments provided herein may include viral or bacterial nucleic acids and / or proteins and / or membranes (e.g., cell membranes, viral lipid envelopes, etc.). In alternative aspects, the carrier for implementing the embodiments provided herein may include but is not limited to replicons (e.g., RNA replicons, bacteriophages), and DNA fragments may be attached to the replicons and replicated. Therefore, carriers include but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, etc., see, e.g., U.S. Patent No. 5,217,879), and may include both expression and non-expression plasmids. In alternative aspects, vectors used to practice the embodiments provided herein can be stably replicated by the cell as an autonomous structure during mitosis, or can be integrated into the host's genome.

[0168] In alternative aspects, "promoters" for implementing the embodiments provided herein include all sequences capable of driving transcription of coding sequences in cells (e.g., bacteria, yeast, fungi, plants, insects (e.g., baculovirus) or mammalian cells). Therefore, the promoter used in the construct includes cis-acting transcription control elements and regulatory sequences, which participate in regulating or regulating the time and / or rate of gene transcription. For example, the promoter used to implement the embodiments provided herein can be a cis-acting transcription control element, including enhancers, promoters, transcription terminators, origins of replication, chromosome integration sequences, 5' and 3' untranslated regions or intron sequences, which are all involved in transcriptional regulation. These cis-acting sequences can interact with proteins or other biomacromolecules to perform (turn on / off, regulate, regulate, etc.) transcription.

[0169] "Constitutive" promoters for implementing the embodiments provided herein can be those promoters that drive expression continuously under most environmental conditions and development or cell differentiation states. "Inducible" or "adjustable" promoters for implementing the embodiments provided herein can instruct the expression of nucleic acids provided herein under the influence of environmental conditions or developmental conditions. Examples of environmental conditions that may affect transcription for implementing the inducible promoters for implementing the embodiments provided herein include the presence of inducing factors applied to cells.

[0170] In alternative embodiments, the peptides and polypeptides used to implement the embodiments provided herein may include any "mimetic" and / or "peptide mimetic" form. In alternative embodiments, the peptides and polypeptides used to implement the embodiments provided herein may include synthetic chemical compounds having substantially the same structural and / or functional properties as natural polypeptides, for example, chimeric or recombinant antibodies provided herein. The mimetic used to implement the embodiments provided herein may be a chimeric molecule composed entirely of synthetic, non-natural analogs of amino acids, or a chimeric molecule composed of a portion of natural peptide amino acids and a portion of non-natural amino acid analogs. The mimetic may also incorporate any number of natural amino acid conservative substitutions, as long as the substitution does not substantially change the structure and / or activity of the mimetic. Routine experiments will determine whether the mimetic is effective for implementing the present invention, for example, whether the mimetic composition is effective in specifically binding to TTF-1 protein. The methods described in detail herein and other methods known to those skilled in the art can be used to select or guide those skilled in the art to select effective mimetic for implementing the compositions and / or methods of the present invention.

[0171] The polypeptide mimetic compositions for implementing the embodiments provided herein can include any combination of non-natural structural components. In alternative aspects, the mimetic compositions for implementing the embodiments provided herein can include one or all of the following three structural groups: a) residue linking groups other than natural amide bonds ("peptide bonds") connected; b) non-natural residues that replace naturally occurring amino acid residues; or c) residues that induce secondary structure mimicry, i.e., induce or stabilize secondary structure (e.g., β-turn, γ-turn, β-fold, α-helical conformation, etc.). For example, when all or part of the residues of a polypeptide are linked together by chemical means other than natural peptide bonds, this polypeptide can be characterized as a mimetic.

[0172] Summary of the Amino Acid Sequences of the Polypeptides and Peptides Provided herein:

[0173] Heavy chain variable domain

[0174] QSLKESEGGLFKPTDTLTLTCTVS GFSLSSYA MIWVRQAPGNGLEYIGA INSYGNAYYASWAKSRSTITRDTNENTVTLKMTSLTAADTATYFC TTTHSYGFGAFDP WGPGTLVTVSS(SEQ ID NO:1)

[0175] Light chain variable domain

[0176] AAVLTQTPSPVSAAVGGTVSISCQSS ESAYNNR LAWYQQKPGQPPKLLIY DAS KLASGVPSRFKGSGSGTPFTLTISGVQCADAATYYC QGVYSSVSDVYA FGGGTEVVVK(SEQ ID NO:2)

[0177] VH constant

[0178] GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:3)

[0179] Full H chain

[0180] QSLKESEGGLFKPTDTLTLTCTVSGFSLSSYAMIWVRQAPGNGLEYIGAINSYGNAYYASWAKSRSTITRDTNENTVTLKMTSLTAADTATYFCTTTHSYGFGAFDPWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:4)

[0181] VH signal P

[0182] METGLRWLLLVAVLKGVQC(SEQ ID NO:5)

[0183] VH+ signal P

[0184] METGLRWLLLVAVLKGVQC QSLKESEGGLFKPTDTLTLTCTVSGFSLSSYAMIWVRQAPGNGLEYIGA INSYGNAYYASWAKSRSTITRDTNENTVTLKMTSLTAADTATYFCTTTHSYGFGAFDPWGPGTLVTVSS (SEQ IDNO:6)

[0185] Full H chain + signal P

[0186] METGLRWLLLVAVLKGVQC QSLKESEGGLFKPTDTLTLTCTVSGFSLSSYAMIWVRQAPGNGLEYIGA INSYGNAYYASWAKSSRSTITRDTNENTVTLKMTSLTAADTATYFCTTTHSYGFGAFDPWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:7)

[0187] VL constant

[0188] GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO:8)

[0189] Full L chain

[0190] AAVLTQTPSPVSAAVGGTVSISCQSSESAYNNRLAWYQQKPGQPPKLLIYDASKLASGVPSRFKGSGS GTPFTLTISGVQCADAATYYCQGVYSSVSDVYAFGGGTEVVVK GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQID NO:9)

[0191] VL + signal P

[0192] MDTRAPTQLLGLLLLWLPGATF AAVLTQTPSPVSAAVGGTVSISCQSSESAYNNRLAWYQQKPGQPPK LLIYDASKLASGVPSRFKGSGSGTPFTLTISGVQCADAATYYCQGVYSSVSDVYAFGGGTEVVVK (SEQ ID NO:10)

[0193] Full L chain + signal P

[0194] MDTRAPTQLLGLLLLWLPGATF AAVLTQTPSPVSAAVGGTVSISCQSSESAYNNRLAWYQQKPGQPPK LLIYDASKLASGVPSRFKGSGSGTPFTLTISGVQCADAATYYCQGVYSSVSDVYAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO:11)

[0195] VL signal P

[0196] MDTRAPTQLLGLLLLWLPGATF(SEQ ID NO:12)

[0197] Immunohistochemistry (IHC)

[0198] In alternative embodiments, immunohistochemistry (IHC) methodology and / or reagents for practicing the compositions, articles, kits or methods provided herein may include or comprise the use of any IHC protocol, IHC device, apparatus and / or image or data analysis system known in the art for practicing IHC or IHC reagents, for example, as described in: U.S. Pat. No. (USPN) 11,321,881 (describing IHC imaging protocols and apparatus); 11,249,085, describing the use of click chemistry for signal amplification in IHC and ISH assays; 11,222,424 (describing IHC imaging apparatus and computer programs); 11,143,648, describing the use of polychromatin quinone carboxamide and tyrosine kinase; Amine conjugates for chromogenic IHC and ISH staining; 11,047,774 (describing an automated IHC sample processing system); 11,028,044, describing a process for preparing 3,3'5,5'-tetramethylbenzidine (TMB) and its salts on a commercial scale; 10,977,791, describing a computer-implemented method for the analysis of tissue samples; 10,816,443, describing an automated batch stainer for staining biological samples on microscope slides; 10,718,773, describing a method for determining the eligibility of a subject with a malignant tumor to receive a therapeutic agent; 10,565,479 (describing a method for identifying blurred areas in a digital image of stained tissue); 10,564,076 (describing a system for analytical (or IHC) sample preparation); 10,551,395 (describing an automated histological staining system); 10,551,378 (describing a method for tissue staining); 10,504,224 (describing a digital tissue image analysis system for IHC); 10,501,777 (describing simultaneous, multiplexed detection and quantification of protein expression in IHC); 10,488,340 (describing a method for extracting an image of a target fluorophore in a biological material); 10,453,195 (describing a method for detecting a tissue region of interest using digital pathology imaging); 10,438,381 (describing a method for generating digital images of tissue sections); 10,416,176 (describing methods for processing specimens in automated histological staining systems); 10,393,633 (describing methods for processing and inhibiting degradation of IHC samples); 10,217,011 (describing processing of IHC slides); 10,209,165 (describing automated or semi-automated methods for assessing the staining quality of specimens containing cells); 10,126,216 (describing methods for fixing tissue specimens for IHC); 9,423,322; or as described in U.S. Patent Application Publication No. US2021 / 0239683A1, describing compositions that form porous hydrogels around cells, which are suitable for immunostaining cells within the hydrogel;or USPN11,112,413 or US2022 / 0057408 A1, describing a method for detecting one or more targets in an IHC tissue sample using an antibody with an epitope tag; or US2021 / 0048432 A1, describing direct immunohistochemistry (IHC) staining and direct immunocytochemistry (ICC) techniques; or US2020 / 0292536, describing synthetic controls useful in immunohistochemistry (IHC) assays; or US20220057408, describing the use of antibodies with epitope tags in immunohistochemistry (IHC) assays; or US20210201485, describing a computer-implemented method for analyzing tissue samples; US2019 / 0178867A1 (describing the detection of specific tissue objects in thin sections of tissue samples imaged under bright field microscopy without the use of chromogenic stains specific for these tissue objects); US2019 / 0156510 A1 (describing an image analysis method for analyzing IHC tissue samples); US2019 / 0293637A1 (methods and systems for quantitative immunohistochemistry (IHC) of targeted protein molecules); US2019 / 0080450 A1 (describing a method for automatically determining the staining quality of IHC-stained biological samples); or, US2020 / 0316589 A1 (describing a porous solid support container for processing and testing fixed biological materials). ;

[0199] In alternative embodiments, the chimeric or recombinant antibodies, antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins in the IHC protocols or kits provided herein are substantially purified or isolated, or are in the form of unpurified or partially purified culture supernatants.

[0200] In alternative embodiments, the methods provided herein can use or comprise reagents for detecting or visualizing antibody-antigen interactions using any product or method known in the art (eg, IHC protocols or reagents).

[0201] In alternative embodiments, the methods provided herein include the use of chromogenic immunohistochemistry (CIH) techniques, wherein a primary antibody (e.g., a chimeric or recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein provided herein) or a secondary antibody (e.g., the secondary antibody binds to the primary antibody after the primary antibody (i.e., a chimeric or recombinant antibody (Ab), an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein provided herein) has specifically bound, paired or associated with a TTF-1 epitope or polypeptide) is conjugated / coupled to an enzyme (e.g., a peroxidase, such as horseradish peroxidase (HRP)) that is capable of catalyzing a color-producing reaction.

[0202] In alternative embodiments, the methods provided herein include the use of immunofluorescence, wherein the primary or secondary antibody is labeled with a fluorophore, such as fluorescein or fluorescein isothiocyanate (FITC), a triarylmethane dye such as rhodamine or a rhodamine derivative (e.g., tetramethylrhodamine (TRITC), rhodamine 6G, rhodamine 123, rhodamine B, carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR), sulforhodamine 101), aminomethylcoumarin acetate / salt (AMCA), Alexa Fluor®, TM or Dylight TM Fluorescent substance. 3,3'-Diaminobenzidine (DAB) can also be used.

[0203] In alternative embodiments, the methods provided herein include the use of a direct method or a one-step staining method, wherein a primary antibody (e.g., a chimeric or recombinant antibody (Ab) provided herein, or an antigen-binding fragment or monomer thereof, or a dimeric antigen-binding protein) is labeled and reacted directly with an antigen, e.g., in a tissue section. Although this technique uses only one antibody and is therefore simple and rapid, the sensitivity may be low due to the lesser degree of signal amplification.

[0204] In alternative embodiments, the methods provided herein include the use of an indirect method, in which an unlabeled primary antibody (first layer) is bound to a target antigen (e.g., TTF-1), for example in a tissue or organ, and then a labeled secondary antibody (second layer) is reacted with the primary antibody. The secondary antibody can be an isotype antibody, such as IgG, directed against the animal species from which the primary antibody is derived. This method can be more sensitive than a direct detection strategy because if the secondary antibody is conjugated / coupled to a detection agent such as a fluorescent marker or an enzyme reporter, then several secondary antibodies will bind to each primary antibody to achieve signal amplification.

[0205] In an alternative embodiment, further amplification can be achieved if the secondary antibody is conjugated / coupled to multiple detection molecules (e.g., biotin molecules) that are capable of recruiting the secondary antibody mediated by avidin, streptavidin, or NeutrAvidin. TM A complex of protein-bound enzyme.

[0206] In alternative embodiments, IHC is performed on tissue sections or tissue biopsies, e.g., paraformaldehyde (PFA) fixed tissues or organs, or formalin fixed paraffin embedded tissues. In alternative embodiments, tissues are sliced ​​or used intact. Prior to slicing, tissue samples may be embedded in a medium such as paraffin or a cryogenic medium. Tissue sections may be sliced ​​using a variety of instruments, most commonly a microtome, cryostat, or vibrating microtome. Samples may be sliced ​​into approximately 3 μm to 5 μm sections. μmSections can be mounted on slides, dehydrated using increasing concentrations of alcohol washes (e.g., 50%, 75%, 90%, 95%, 100%), and cleared with a detergent like xylene, and then imaged under a microscope.

[0207] Depending on the method of fixation and tissue preservation, samples may require additional steps to make TTF-1 epitopes available for antibody binding, including deparaffinization and antigen retrieval. Antigen retrieval is usually necessary for formalin-fixed paraffin-embedded tissues and may include heating or protease pretreatment of sections.

[0208] In an alternative embodiment, IHC is performed using the EnVision DuoFLEX Doublestain System TM (EnVision DuoFLEX dual staining system) (Agilent, San Jose, CA), which allows staining of two or more markers on a single slide. In an alternative embodiment, IHC is performed using the EnVision FLEXHRPMagenta, high pH (Dako Omnis TM ) system, and the combination can be performed by EnVisionFLEXHRP TM Magenta Chromogen was used for visualization. In an alternative embodiment, IHC was performed using the EnVision FLEXMiniKiT TM High pH, ​​a highly sensitive visualization system that can be used with the Dako Autostainer TM instruments for IHC; this Dual Link system detects mouse and rabbit primary antibodies and visualizes the reaction with a 3,3'-diaminobenzidine (DAB) chromogen (DAB forms a water-insoluble brown precipitate when oxidized (e.g. by peroxidase)).

[0209] Products and kits

[0210] Provided are articles of manufacture and kits for practicing the methods provided herein, e.g., comprising a chimeric or recombinant anti-TTF-1 polypeptide Ab provided herein; and optionally the articles of manufacture and kits may further comprise some or all of the reagents required to practice IHC, and optionally may include instructions for practicing the methods provided herein.

[0211] In alternative embodiments, the article of manufacture or kit includes a mixture or cocktail of antibodies (Ab) provided herein, for example, a mixture or cocktail including two, three or more anti-human TTF-1 Ab antibodies.

[0212] In alternative embodiments, the article of manufacture or kit includes a mixture or cocktail of antibodies (Ab), including antibodies containing the heavy chain and / or light chain CDRs of the antibodies provided herein, or antibodies produced by antibody-producing clones provided herein.

[0213] In alternative embodiments, the article of manufacture or kit comprises an antibody producing clone provided herein.

[0214] Any of the above aspects and embodiments may be combined with any other aspects or embodiments disclosed herein in the Summary, Figures, and / or Detailed Description sections.

[0215] As used in this specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0216] Unless specifically stated or obvious from the context, the term "or" as used herein should be construed as inclusive and encompassing both "or" and "and."

[0217] Unless specifically stated or apparent from the context, the term "about" as used herein should be understood to be within the normal tolerance range in the art, such as within 2 standard deviations of the mean. About (the use of the term "approximately") can be understood to be within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the recited value. Unless otherwise expressly provided, all numerical values ​​provided herein are modified by the term "about".

[0218] Unless specifically stated or obvious from the context, as used herein, the terms "substantially all," "substantially a majority," "substantially all," or "majority" encompass at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more of the referenced amount of the composition.

[0219] Each of the patents, patent applications, publications, and documents referred to herein is hereby incorporated by reference in their entirety. The citation of the above patents, patent applications, publications, and documents is not an admission that any of the above is relevant prior art, nor does it constitute any admission of the contents or dates of these publications or documents. The incorporation of these documents by reference should not, in itself, be construed as a claim or admission that any part of the contents of any document is deemed to be necessary material to satisfy the statutory disclosure requirements for patent applications in any country or region. Nevertheless, the right to rely on any such document is reserved, where appropriate, for the purpose of providing material that is determined by the examining body or court to be essential to the subject matter for which protection is sought.

[0220] Without departing from the basic aspects of the present invention, the foregoing may be modified. Although the present invention has been described in considerable detail with reference to one or more specific embodiments, it will be appreciated by those skilled in the art that changes may be made to the embodiments specifically disclosed in this application, and that these modifications and improvements are within the scope and spirit of the present invention. The present invention described herein in an exemplary manner may also be appropriately implemented in the absence of any elements not specifically disclosed herein. Thus, for example, in each example herein, any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with any of the other two terms. Therefore, the terms and expressions used are used as descriptive terms rather than restrictive terms, do not exclude equivalents of the features shown and described or parts thereof, and should be recognized that there are various modifications that may be made within the scope of the present invention.

[0221] The present invention will be further described with reference to the examples described herein; however, it should be understood that the present invention is not limited to these examples.

[0222] Example

[0223] Unless otherwise indicated in the examples, all recombinant DNA techniques were performed according to standard protocols, for example, as described in Sambrook et al., (2012) Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press, NY and Ausubel et al., (1994) Current Protocols in Molecular Biology, Current Protocols, USA, Volumes 1 and 2. Other references to standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, NY, Brown (1998) Molecular Biology LabFax, 2nd edition, Academic Press (UK), Volumes I and II. Standard materials and methods for polymerase chain reaction can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and McPherson et al., (2000) PCR-Basics: From Background to Bench, 1st edition, Springer Verlag, Germany.

[0224] Example 1: Antibodies for identifying and distinguishing lung adenocarcinoma from lung squamous cell carcinoma

[0225] In alternative embodiments, the Ab provided herein, or an antigen (Ag) binding fragment or protein (ABP) thereof, or a monomeric or dimeric ABP specifically binds to lung squamous cell carcinoma cells with less sensitivity than to adenocarcinoma cells (or binds to lung squamous cell carcinoma cells with a lower affinity than to adenocarcinoma cells), thereby resulting in fewer false positives for binding to lung squamous cell carcinoma cells and better identification of lung adenocarcinoma.

[0226] The study by Yatabe et al. (1) showed that the antibody of clone 8G7G3 / 1 (producing anti-human TTF-1 antibody) binds to TTF-1 with higher specificity than the anti-human TTF-1 antibody produced by clone SPT24, but the anti-human TTF-1 antibody produced by clone SPT24 has higher sensitivity.

[0227] Described herein is a new anti-human TTF-1 antibody TTF-1 assay that has the same specificity as the anti-human TTF-1 antibody produced by clone 8G7G3 / 1 and the same sensitivity as the anti-human TTF-1 antibody produced by clone SPT24. The goal is to identify as many positive lung adenocarcinomas as those identified using the anti-human TTF-1 antibody of SPT24 and to identify as few positive squamous cell carcinomas as seen using the anti-human TTF-1 antibody of clone 8G7G3 / 1.

[0228] Since three different assays, including clone SPT24, clone 8G7G3 / 1, and the new clone 6F6 described herein, require testing a large number of samples, the sensitivity and specificity of the antibodies were tested on tissue microarrays (TMA), specifically the lung cancer medium density tissue array LUC1501. TM and the multi-tumor array MTU951 from USBiomax (Derwood, MD) TM .

[0229] Anti-human TTF-1 antibody clone 8G7G3 / 1 is less sensitive for lung adenocarcinoma than anti-human TTF-1 antibody clone SPT24 (false negatives), but more specific for lung squamous cell carcinoma (fewer false positives). See, e.g., the following table from Yatabe et al. (1).

[0230]

[0231] The expected expression patterns in both normal and tumor tissues are summarized below.

[0232] Normal lung: Type II pneumocytes, Clara cells (or non-ciliated bronchial secretory cells), and basal cells of the terminal bronchi should show strong nuclear staining without significant cytoplasmic reaction. Columnar epithelial cells of the terminal bronchial walls may show moderate to strong nuclear staining, depending on the sensitivity of the assay.

[0233] In a normal thyroid Follicular epithelial cells should show strong nuclear staining reaction.

[0234] In normal liver Hepatocytes will be negative.

[0235] Tumor lung: In lung adenocarcinoma, tumor cells will show moderate to strong nuclear staining reaction. Tumor cells in lung squamous cell carcinoma should be negative.

[0236] Clone selection

[0237] Rabbits were immunized with a recombinant protein representing amino acids 1-156 of human TTF-1 expressed in E. coli. Rabbit blood was collected and evaluated by ELISA and IHC, and candidate rabbits were selected for B cell selection. Several candidate clones were identified; one B cell clone, clone 6F6, was subjected to IHC and selection to obtain functional heavy and light chain sequences. Recombinant antibodies were produced in HEK cells from clone 6F6 and tested by IHC, showing specificity for TTF-1. The new rabbit anti-human TTF-1 antibody clone 6F6 was tested on various normal and neoplastic tissues to determine specificity and sensitivity and compared to the specificity and sensitivity of clones SPT24 and 8G7G3 / 1.

[0238] These organizations include:

[0239] Tumor tissue

[0240] Lung adenocarcinoma (clinically relevant positive tissue)

[0241] Squamous cell carcinoma of the lung (clinically relevant negative tissue)

[0242] Colon adenocarcinoma (should be negative)

[0243] Lung carcinoid (may be positive)

[0244] Small cell carcinoma (may be positive)

[0245] Multi-Tumor Array (MTU951)

[0246] Normal tissue

[0247] Large multi block containing 12 different normal and neoplastic

[0248] 30 different normal tissues

[0249] Large multi-panel containing 12 different normal and tumor tissues

[0250] New determination of reaction mode

[0251] Weaker than clone SPT24 on low-expressing epithelial cells of the bronchus. The rationale for this is that the 6F6 clone would have the same problem as clone SPT24 with a more sensitive protocol, namely an increased risk of false-positive staining of squamous cell carcinomas.

[0252] Background staining of mucus-producing cells in the stomach. This staining was also observed, albeit to a lesser extent compared to the reference clone SPT24. Although this reaction is undesirable, it does not pose any diagnostic risk nor the risk of not being able to interpret the specific staining.

[0253] No reaction was observed in normal hepatocytes. Clone 8G7G3 / 1 did stain hepatocytes to a high degree.

[0254] There was some positivity on colon adenocarcinomas: some colon adenocarcinomas were weakly positive in the same areas where the SPT24 clone was positive. Therefore, the new clone does not seem to be more specific in this regard, but rather the staining intensity is lower with the new assay.

[0255] The multi-tumor array MTU951 showed specific staining in the expected tissue. Clone SPT24 had unexpected staining of one case of cutaneous squamous cell carcinoma.

[0256] Lung tumor tissue microarray LUC1501, the new assay showed lower staining intensity compared to the reference assay using SPT24. However, no false negative cases occurred (although the staining was weaker). The difference in intensity was generally in the range of 0.5 to 2. One clinical tissue stained more intensely with clone 6F6 than with SPT24, while all other tissues stained more weakly. Nine of 32 negative clinical tissues were positive for clone SPT24 and five of 32 were positive for clone 6F6.

[0257] The new clone 6F6 was tested in triplicate on 30 normal tissues (different cases). All reactions were as expected except for staining of mucus-producing cells in the stomach.

[0258] organize #Positive number / total number of cases organize #Positive number / total number of cases Peripheral nerves 0 / 3 Parathyroid gland 3 / 3 Adrenal glands 0 / 3 Pituitary gland 0 / 3 marrow 0 / 3 prostate 0 / 3 Cerebellum 0 / 3 Salivary glands 0 / 3 brain 0 / 3 Skeletal muscle 0 / 3 cervix 0 / 3 skin 0 / 3 colon 0 / 3 Small Intestine 0 / 3 esophagus 0 / 3 spleen 0 / 3 heart 0 / 3 Stomach* 0 / 3 kidney 0 / 3 testis 0 / 3 liver 0 / 3 Thymus 0 / 3 lung 3 / 3 thyroid 3 / 3 Mesothelial cells 0 / 3 tonsil 0 / 3 Ovaries 0 / 3 Uterus 0 / 3 pancreas 0 / 3 Breast / Mammary gland 0 / 3

[0259] Figure 1 Shown is an IHC image of normal lung showing specific staining of type II cells (arrows). Staining was performed using clone 6F6 antibody.

[0260] Figure 2 Shown are IHC images of lung bronchi stained with clone 6F6 antibody.

[0261] Figure 3 Shown are IHC images of normal thyroid stained with clone 6F6 antibody.

[0262] Figure 4 IHC images of negative normal tissue (normal liver) showing hepatocytes (arrows) are shown. Staining was performed using clone 6F6 antibody.

[0263] Figure 5Shown are IHC images of lung adenocarcinoma, showing specific staining of tumor cells. Staining was performed using clone 6F6 antibody.

[0264] Figure 6 Shown is an IHC image of a lung squamous cell carcinoma showing nonspecific staining of tumor cells. Staining was performed using the clone 6F6 antibody.

[0265] Figure 7 Shown is an IHC image of a colon adenocarcinoma showing nonspecific staining of tumor cells. Staining was performed using clone 6F6 antibody.

[0266] Figure 8 Demonstrated: Specificity for different tumor types: Specificity was tested on a multi-tumor array (MTU951) using antibody clones 6F6 and SPT24. Clone 6F6 showed the expected positive reaction, while clone SPT24 showed non-specific positive staining in one cutaneous squamous cell carcinoma.

[0267] Fig. 9 Demonstrated: Sensitivity for lung adenocarcinoma: Specificity was tested on 22 lung adenocarcinomas using clones 6F6, SPT24, and 8G7G3 / 1. The results showed that clones 6F6 and SPT24 were more sensitive than clone 8G7G3 / 1, with clone SPT24 being the most sensitive. Clone 8G7G3 / 1 was the least sensitive clone.

[0268] Fig.10 Demonstrated: Specificity for lung squamous cell carcinoma: Specificity was tested on 32 lung squamous cell carcinomas using clones 6F6, SPT24, and 8G7G3 / 1. The results showed that clone SPT24 was the least specific clone (9 out of 32 positive). Clone 8G7G3 / 1 was the most specific clone (4 out of 32 positive), followed by clone 6F6 (5 out of 32 positive).

[0269] References

[0270] 1.Yatabe Y, et al., Best Practices Recommendations for DiagnosticImmunohistochemistryin Lung Cancer. J Thorac Oncol. 2019; 14(3): 377-407.

[0271] 2.Comperat E, et al., Variable sensitivity and specificity of TTF-1antibodies in lungmetastatic adenocarcinoma of colorectal origin. Mod Pathol. 2005; 18(10): 1371-6.

[0272] 3.Matoso A, et al., Comparison of thyroid transcription factor-1expression by 2monoclonalantibodies in pulmonary and nonpulmonary primary tumors.ApplImmunohistochem MolMorphol.2010;18(2):142-9.

[0273] 4. Holzinger A, et al., Monoclonal antibody to thyroid transcription factor-1: production, characterization, and usefulness in tumordiagnosis. Hybridoma. 1996; 15(1): 49-53.

[0274] 5. Lazzaro D, et al., The transcription factor TTF-1 is expressed at theonset of thyroid and lung morphogenesis and in restricted regions of the foetal brain. Development. 1991; 113(4): 1093-104.

[0275] Several embodiments of the present invention have been described. However, it will be appreciated that various modifications can be made without departing from the spirit and scope of the present invention. Embodiments of the present invention are set forth in the appended claims.

Claims

1. An isolated or purified antibody (Ab), or a synthetic or recombinant Ab, or an antigen (Ag) binding fragment thereof, or a monomeric or dimeric antigen binding protein (ABP), which is capable of specifically binding to human homeobox NKX-2.1 (also known as NK2 homeobox 1, or thyroid transcription factor 1 (TTF-1)) protein or polypeptide, in, The isolated or purified Ab, or synthetic or recombinant Ab, or Ag-binding fragment thereof, or monomeric or dimeric ABP comprises: (a) can be combined with human homology box NKX- 2.1 A heavy chain variable region (VH) that specifically binds to a protein or polypeptide, comprising: (1) an amino acid sequence comprising the three CDR1, CDR2 and CDR3 complementarity determining regions (CDRs) of SEQ ID NO: 1, or CDR1 amino acid (aa) residues GFSLSSYA (residues 25 to 32 of SEQ ID NO: 1), CDR2 aa residues INSYGNA (residues 50 to 56 of SEQ ID NO: 1), and CDR3 aa residues TTTHSYGFGAFDP (residues 95 to 107 of SEQ ID NO: 1), or (2) an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% sequence identity, or about 70%-100% sequence identity to each of the three CDR1, CDR2, and CDR3 complementarity determining regions (CDRs) of SEQ ID NO: 1, or CDR1 amino acid (aa) residues GFSLSSYA (residues 25 to 32 of SEQ ID NO: 1), CDR2 aa residues INSYGNA (residues 50 to 56 of SEQ ID NO: 1), and CDR3 aa residues TTTHSYGFGAFDP (residues 95 to 107 of SEQ ID NO: 1), or (3) an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% sequence identity, or about 70%-100% sequence identity to SEQ ID NO: 1, or an amino acid sequence having complete sequence identity to SEQ ID NO: 1; (b) can bind to the human homology box NKX- 2.1 A light chain variable region (VL) that specifically binds to a protein or polypeptide, comprising: (1) an amino acid sequence comprising the three CDR1, CDR2 and CDR3 complementarity determining regions (CDRs) of SEQ ID NO: 2, or CDR1 amino acid (aa) residues ESAYNNR (residues 27 to 33 of SEQ ID NO: 2), CDR2 aa residues DAS (residues 51 to 53 of SEQ ID NO: 2) and CDR3 aa residues QGVYSSVSDVYA (residues 90 to 101 of SEQ ID NO: 2), or (2) an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% sequence identity, or about 70%-100% sequence identity to each of the three CDR1, CDR2, and CDR3 complementarity determining regions (CDRs) of SEQ ID NO:2, or CDR1 amino acid (aa) residues ESAYNNR (residues 27 to 33 of SEQ ID NO:2), CDR2 aa residues DAS (residues 51 to 53 of SEQ ID NO:2), and CDR3 aa residues QGVYSSVSDVYA (residues 90 to 101 of SEQ ID NO:2); or (3) an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% sequence identity, or about 70%-100% sequence identity to SEQ ID NO: 2, or an amino acid sequence having complete (100%) sequence identity to SEQ ID NO: 2; or (c) can be combined with human homology box NKX- 2.1 A protein or polypeptide-specific binding heterodimer comprising (a) a heavy chain variable region (VH) and (b) a light chain variable region (VL).

2. The isolated or purified Ab, or synthetic or recombinant Ab, or Ag-binding fragment thereof, or monomeric or dimeric ABP according to claim 1, which is manufactured or is in the form of: Antigen binding fragment (Fab or Ab fragment having one constant domain and one variable domain each on the Ab heavy and light chains), F(ab')2 (or Ab digested by pepsin, which produces two fragments: F(ab')2 fragment and pFc' (pepsin cleaved Fc) fragment, Fab' (single chain of F(ab')2 fragment), a single chain variable fragment (scFv) (or a fusion protein of the variable regions of the Ab heavy and light chains linked together with a linker peptide, optionally with a length of about 10 to about 25 amino acids), (scFv) 2, or dual scFv or di-scFv, or a single peptide chain having two variable heavy regions and two variable light regions, the single peptide chain producing a tandem scFv, A small body (or a fusion protein in which the variable regions of the heavy and light chains of Ab are linked to an alkyl group, optionally the alkyl group is a methyl group or an ethyl group), Diabodies (or scFvs in which the linker peptide is too short (optionally about 5 amino acids) to allow the two variable regions to fold together, forcing the scFv to dimerize), triabodies or tetrabodies (or scFvs in which the linker peptide is too short (optionally about 1 or 2 amino acids) to allow the two variable regions to fold together, forcing the scFv to trimerize or tetramerize), Single domain antibodies (dAB) (or a single variable region of an Ab heavy chain or an Ab light chain), Multiple complementarity determining region (CDR) fragments, or A multispecific antibody formed from two or more antibody fragments.

3. The isolated or purified Ab, or synthetic or recombinant Ab, or Ag-binding fragment thereof, or monomeric or dimeric ABP according to claim 1 or claim 2, wherein the heavy chain variable region (VH), if present: (a) further comprises a carboxyl terminal constant domain, or further comprises the amino acid sequence of SEQ ID NO: 3, or comprises SEQ ID NO: 4; (b) further comprises an amino-terminal signal peptide, or further comprises an amino-terminal signal peptide, wherein the signal peptide comprises: METGLRWLLLVAVLKGVQC (SEQ ID NO: 5), or comprises SEQ ID NO: 6 or SEQ ID NO: 7; (c) comprising SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:7, with one or more amino acid substitutions, additions (insertions) or deletions, and the recombinant Ab or its Ag-binding fragment or monomeric or dimeric ABP retains its ability to specifically bind to human homeobox NKX-2.1 protein or polypeptide; or (d) one or more amino acid substitutions, wherein the amino acid substitutions include one or more conservative amino acid substitutions.

4. An isolated or purified Ab, or a synthetic or recombinant Ab, or an Ag-binding fragment thereof, or a monomeric or dimeric ABP according to any one of claims 1 to 3 or any of the preceding claims, wherein the light chain variable region (VL), if present: (a) further comprises a carboxyl terminal constant domain, or further comprises the amino acid sequence of SEQ ID NO: 8, or comprises SEQ ID NO: 9, (b) further comprises an amino-terminal signal peptide, or further comprises an amino-terminal signal peptide consisting of MDTRAPTQLLGLLLLWLPGATF (SEQ ID NO: 12), or comprises SEQ ID NO: 10 or SEQ ID NO: 11; (c) comprising SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11, having one or more amino acid substitutions, additions (insertions) or deletions, and the recombinant Ab or its Ag binding fragment or monomer or dimer ABP retains its specific binding to human homeobox NKX- 2.1 Capacity of proteins or peptides, (d) one or more amino acid substitutions, wherein the amino acid substitutions comprise one or more conservative amino acid substitutions, and the isolated or purified Ab, or synthetic or recombinant Ab, or Ag-binding fragment thereof, or monomeric or dimeric ABP, retains its specific binding to human homeobox NKX- 2.1 The capacity of the protein or polypeptide; or (e) the isolated or purified Ab, or synthetic or recombinant Ab, or Ag-binding fragment thereof, or monomeric or dimeric ABP has two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen conservative amino acid substitutions, and the recombinant Ab, or Ag-binding fragment thereof, or monomeric or dimeric ABP retains its homology to human homeobox NKX- 2.1 The ability of proteins or peptides to bind specifically; (f) the light chain variable region further comprises at least a portion of a light chain constant region; (g) the light chain constant region comprises the amino acid sequence: GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 8); (h) the heavy chain variable region further comprises at least a portion of a heavy chain constant region; (I) The heavy chain constant region comprises the amino acid sequence: GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTA RPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:3); (j) the light chain variable region further comprises at least a portion of a light chain constant region; and the heavy chain variable region further comprises at least a portion of a heavy chain constant region; or (k) The isolated or purified or recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, comprising: A light chain comprising the amino acid sequence (SEQ ID NO: 9); and A heavy chain comprising the amino acid sequence (SEQ ID NO: 4).

5. An isolated or purified Ab, or a synthetic or recombinant Ab, or an Ag-binding fragment thereof, or a monomeric or dimeric ABP according to any one of claims 1 to 4 or any of the preceding claims, wherein: (a) the heavy chain constant region comprises an amino acid sequence from an IgG, IgM, IgA, IgD or IgE isotype, or the heavy chain constant region substantially comprises an amino acid sequence from an IgG, IgM, IgA, IgD or IgE isotype; (b) the light chain constant region comprises an amino acid sequence from a kappa (κ) or lambda (λ) isotype, or the light chain constant region substantially comprises an amino acid sequence from a kappa (κ) or lambda (λ) isotype; (c) at least a portion of the heavy chain constant region, at least a portion of the light chain constant region, or at least a portion of the heavy chain constant region and the light chain constant region is or comprises an amino acid sequence derived from human, rabbit, mouse or rat, or comprises a constant region amino acid sequence derived from human, rabbit, mouse or rat or substantially derived from human, rabbit, mouse or rat; (d) at least a portion of the heavy chain constant region, at least a portion of the light chain constant region, or at least a portion of the heavy chain constant region and the light chain constant region is or comprises a synthetic amino acid sequence; (e) the recombinant Ab, its Ag-binding fragment, or monomeric or dimeric ABP, or heavy chain constant region, or light chain constant region, or heavy chain constant region and light chain constant region further comprise or are combined with a heterologous protein, peptide, or compound or composition; (f) the heterologous protein or peptide, or compound or composition comprises a detectable protein, detectable agent or binding moiety; (g) the heterologous protein or peptide comprises a carrier protein; (h) the heterologous protein, peptide or compound or composition is covalently conjugated to a recombinant antibody (Ab) or its Ag-binding fragment or a monomeric or dimeric ABP; (i) the detectable agent or binding moiety comprises biotin, a fluorescent or chemiluminescent tag, a fluorophore, perylene, fluorenyl, coumarin, 7-methoxycoumarin (Mca), 4-(dimethylaminoazo)benzene-4-carboxylic acid (dabcyl), Tamra, boron-dipyrromethene (BODIPY) or a derivative thereof, a dye, a radioisotope, a quantum dot or a photoluminescent aqueous nanocrystal, a hapten or an antibody binding epitope or domain; (j) the dye is or contains rhodamine, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (NBD), Nile red or Nile blue, or is a fluorescent dye containing sulfoindocyanine; (l) the fluorophore is or comprises a dansyl, fluorescein, carboxyfluorescein (FAM) or 6-carboxyfluorescein (6-FAM) moiety; (m) the dye is or comprises a cyanine dye, Cy3 or Cy5; (n) the hapten is or comprises a biotin, theophylline, digoxin, carborane, fluorescein or bromodeoxyuridine moiety; or (o) The Ab, or Ag-binding fragment thereof, or monomeric or dimeric ABP is a recombinant Ab, or Ag-binding fragment thereof, or monomeric or dimeric ABP, or comprises a peptide or polypeptide produced by recombinant technology.

6. A chimeric, synthetic or recombinant nucleic acid comprising: a nucleic acid sequence encoding the Ab or its Ag binding fragment or monomeric or dimeric ABP according to any one of claims 1 to 5.

7. The chimeric, synthetic or recombinant nucleic acid of claim 6, wherein: (a) the chimeric or recombinant nucleic acid further comprises and is operably linked to a transcriptional regulatory element; (b) the transcriptional regulatory element comprises a promoter; (c) the promoter is an inducible promoter or a constitutive promoter; (d) the chimeric or recombinant nucleic acid further comprises a sequence encoding an amino-terminal signal peptide; (e) the amino terminal signal peptide comprises the amino acid sequence: METGLRWLLLVAVLKGVQC (SEQ ID NO: 5); MDTRAPTQLLGLLLLWLPGATF (SEQ ID NO: 12); or (f) the amino-terminal signal peptide SEQ ID NO:5 is the amino-terminus of the VH chain SEQ ID NO:1, or the amino-terminus of SEQ ID NO:4; or the amino-terminal signal peptide SEQ ID NO:12 is the amino-terminus of the VL chain SEQ ID NO:2, or the amino-terminus of SEQ ID NO:

9.

8. An expression cassette, vector, recombinant virus, artificial chromosome, cosmid or plasmid comprising the chimeric, synthetic or recombinant nucleic acid according to claim 8.

9. A cell comprising or containing: (a) a chimeric, synthetic or recombinant antibody or dimeric antigen binding protein according to any one of claims 1 to 5, (b) the chimeric or recombinant nucleic acid according to claim 6 or 7, (c) The expression cassette, vector, recombinant virus, artificial chromosome, cosmid or plasmid according to claim 8.

10. The cell of claim 9, wherein the cell is a bacterial, fungal, mammalian, yeast, insect or plant cell, or the mammalian cell is a human cell.

11. A method for detecting the presence of a human homeobox NKX-2.1 protein or polypeptide in or on a cell, tissue, organ or any part thereof, the method comprising: (a) contacting a cell, tissue or organ, or part of any of the foregoing, with at least one isolated or purified, or synthetic or recombinant Ab, or Ag-binding fragment thereof, or monomeric or dimeric ABP according to any one of claims 1 to 27 or any of the preceding claims, and (b) detecting at least one Ab or its Ag-binding fragment or monomer or dimer ABP in or on a cell, tissue or organ or any part of the foregoing and binding to human homeobox NKX- 2.1 Specific binding of proteins or peptides, Thus, human homeobox NKX- 2.1 Presence of protein or polypeptide.

12. The method according to claim 11, wherein: (a) the at least one Ab or Ag-binding fragment thereof, or monomeric or dimeric ABP comprises a variable heavy chain (VH) and a variable light chain (VL), the variable heavy chain (VH) having an amino acid sequence comprising SEQ ID NO: 1, and the variable light chain (VL) having an amino acid sequence comprising SEQ ID NO: 2; (b) the at least one Ab or Ag-binding fragment thereof, or monomeric or dimeric ABP comprises a variable heavy chain (VH) and a variable light chain (VL), the variable heavy chain (VH) having an amino acid sequence comprising SEQ ID NO: 4, and the variable light chain (VL) having an amino acid sequence comprising SEQ ID NO: 9; (c) the at least one Ab or Ag-binding fragment thereof, or monomeric or dimeric ABP comprises a variable heavy chain (VH) and a variable light chain (VL), the variable heavy chain (VH) having an amino acid sequence comprising SEQ ID NO: 7, and the variable light chain (VL) having an amino acid sequence comprising SEQ ID NO: 11; (d) the method comprises contacting a cell, tissue or organ, or a part of any of the foregoing, with a mixture of two or at least two Abs, or Ag-binding fragments thereof, or monomeric or dimeric ABPs, or two or at least two different Abs, or Ag-binding fragments thereof, or monomeric or dimeric ABPs, and wherein the two or at least two Abs, or Ag-binding fragments thereof, or monomeric or dimeric ABPs, or two or at least two different Abs, or Ag-binding fragments thereof, or monomeric or dimeric ABPs comprise the Ab, or Ag-binding fragments thereof, or monomeric or dimeric ABPs according to any one of claims 1 to 5; (e) The method of (d), wherein the contacting comprises the use of an immunohistochemistry (IHC) assay; (f) The method of any one of (a) to (d), further comprising contacting the antibody or its Ag-binding fragment or monomer or dimeric ABP that specifically binds to the human homeobox NKX-2.1 polypeptide or peptide with a detectable agent to indicate or signal the specific binding of the Ab or its Ag-binding fragment or monomer or dimeric ABP to the human homeobox NKX-2.1 polypeptide or peptide; (g) The method of (f), wherein the detectable agent specifically binds to the Ab or its Ag-binding fragment or monomeric or dimeric ABP; (h) the cell, tissue, organ or any part thereof is or comprises or is derived from a cancer cell, (i) wherein the cancer cell is a thyroid cancer cell, a lung cancer cell or a cancer cell; (j) the cancer cells are large cell carcinoma, small cell carcinoma, lung cancer cells or adenocarcinoma cells; (k) the small cell carcinoma is small cell lung cancer or extrapulmonary small cell carcinoma, or the adenocarcinoma cell is a lung adenocarcinoma cell or a thyroid adenocarcinoma cell; (l) the cancer cells are squamous cell carcinoma (SCC) cells, leukemia cells, carcinoid tumor cells, breast cancer cells, colon cancer cells, malignant melanoma cells, multiple myeloma cells, plasmacytoma cells or lymphoma cells, or cancers derived from lymphocytes; (m) the lymphocytes are early B cells, pro-B cells, pre-B lymphocytes, mature B lymphocytes or T lymphocytes, or the T lymphocytes are parafollicular T lymphocytes or a subpopulation of parafollicular T lymphocytes; (n) the cell is a lung cell, a thyroid cell, a follicular center cell, or a cell in the tonsil, a lymph node germinal center, a bone marrow stem cell, a bone marrow hematopoietic cell, a liver bile tubule cell, a kidney glomerular cell, a proximal tubule cell, a breast myoepithelial cell, a stromal cell surrounding or associated with an infiltrating tumor cell, a kidney cell, an epithelial cell, a cerebellar cell, a prostate cell, a kidney cell, a pancreatic cell, or a bone marrow cell; (o) the epithelial cell is or is derived from an epithelial cell of the brain, lung, intestine, kidney, breast or placenta; the lung cell is a lung alveolar cell (and optionally the lung alveolar cell may be a type II alveolar cell), or a Clara cell or a non-ciliated bronchiolar secretory cell; or the thyroid cell is a thyroid follicular cell or a thyroid parafollicular cell; (p) the organ is liver, prostate or lung; (q) the cell, tissue, organ or any part thereof is or is derived from lung adenocarcinoma; or (r) The Ab or its Ag-binding fragment or monomeric or dimeric ABP specifically binds to lung squamous cell carcinoma cells with lower sensitivity than to adenocarcinoma cells (or binds to lung squamous cell carcinoma cells with lower affinity than to adenocarcinoma cells), thereby resulting in fewer false positives for lung squamous cell carcinoma and better positive identification of lung adenocarcinoma.

13. A method for detecting or diagnosing cancer, wherein the method comprises detecting the expression or presence of a human homeobox NKX-2.1 polypeptide or peptide in or on a cell, tissue or organ sample using the method according to any one of claims 11 to 12, The detection of specific binding of the Ab or its Ag binding fragment or monomer or dimer ABP to the human homeobox NKX-2.1 polypeptide or peptide in or on the cell, tissue or organ or any part of the foregoing detects or diagnoses cancer or assists in the detection or diagnosis of cancer.

14. The method according to claim 13, wherein: (a) the cancer is a thyroid cancer cell, a lung cancer cell or a cancer cell, (b) the cancer is a cancer cell, or is a large cell cancer, a small cell cancer (optionally a small cell lung cancer or an extrapulmonary small cell cancer), a lung cancer cell, or an adenocarcinoma cell, and optionally the adenocarcinoma cell is a lung adenocarcinoma cell or a thyroid adenocarcinoma cell, (c) the cancer is a squamous cell carcinoma (SCC) cell, a leukemia cell, a carcinoid tumor, a breast cancer cell, a colon cancer cell, a malignant melanoma cell, a multiple myeloma cell, a plasmacytoma cell or a lymphoma cell, or is a cancer derived from a lymphocyte, wherein optionally the lymphocyte is an early B cell, a pro-B cell, a pre-B lymphocyte, a mature B lymphocyte, a T lymphocyte, a parafollicular T lymphocyte, or a cell derived from a parafollicular T lymphocyte subset; (d) the cancer is lung cancer or lung adenocarcinoma, (e) the Ab or its Ag-binding fragment or monomeric or dimeric ABP specifically binds to lung squamous cell carcinoma cells with lower sensitivity than to adenocarcinoma cells (or binds to lung squamous cell carcinoma cells with lower affinity than to adenocarcinoma cells), thereby resulting in fewer false positives for lung squamous cell carcinoma and better positive identification of lung adenocarcinoma; (f) the cell, tissue or organ sample is from an individual in need; (g) the detection comprises performing an immunohistochemistry (IHC) assay, (h) at least two Abs or Ag-binding fragments thereof, or monomeric or dimeric ABPs are used to contact the cell, tissue or organ sample; or (i) One of the two Abs or their Ag-binding fragments or monomeric or dimeric ABPs comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the variable heavy chain (VH) has an amino acid sequence comprising SEQ ID NO: 1, and the variable light chain (VL) has an amino acid sequence comprising SEQ ID NO:

2.

15. A method for treating, improving or preventing cancer, the method comprising first detecting or diagnosing the cancer using the method according to any one of claims 13 to 14, and then treating an individual in need thereof for treating, improving or preventing the cancer.

16. The method of claim 15, wherein: (a) the cancer is a thyroid cancer cell, a lung cancer cell or a cancer cell, (b) the cancer cell is a cancer cell, or is a large cell carcinoma, a small cell carcinoma (optionally a small cell lung cancer or an extrapulmonary small cell carcinoma), a lung cancer cell, or an adenocarcinoma cell, and optionally the adenocarcinoma cell is a lung adenocarcinoma cell or a thyroid adenocarcinoma cell, (c) the cancer cell is a squamous cell carcinoma (SCC) cell, a leukemia cell, a carcinoid tumor, a breast cancer cell, a colon cancer cell, a malignant melanoma cell, a multiple myeloma cell, a plasmacytoma cell or a lymphoma cell, or a cancer derived from a lymphocyte, (d) the lymphocytes are early B cells, pro-B cells, pre-B lymphocytes, mature B lymphocytes, T lymphocytes, parafollicular T lymphocytes, or cells derived from a subpopulation of parafollicular T lymphocytes, (e) the cancer is lung cancer, or the lung cancer is lung adenocarcinoma, or (f) The Ab or its Ag-binding fragment or monomeric or dimeric ABP specifically binds to lung squamous cell carcinoma cells with lower sensitivity than to lung adenocarcinoma cells (or binds to lung squamous cell carcinoma cells with lower affinity than to adenocarcinoma cells), thereby resulting in fewer false positives for lung squamous cell carcinoma and better positive identification of lung adenocarcinoma.

17. Use of at least one of the isolated or purified, or synthetic or recombinant antibodies (Ab) according to any one of claims 1 to 5 or according to any one of the preceding claims, or antigen (Ag) binding fragments thereof, or monomeric or dimeric antigen binding proteins (ABPs) for detecting or diagnosing cancer, or treating, ameliorating or preventing cancer.

18. The use according to claim 17, wherein: (a) the cancer is a thyroid cancer cell, a lung cancer cell or a cancer cell, (b) wherein optionally the cancer cell is a large cell carcinoma, a small cell carcinoma (optionally a small cell lung cancer or an extrapulmonary small cell carcinoma), a lung cancer cell or an adenocarcinoma cell, and optionally the adenocarcinoma cell is a lung adenocarcinoma cell or a thyroid adenocarcinoma cell, (c) the cancer cell is a squamous cell carcinoma (SCC) cell, a leukemia cell, a carcinoid tumor, a breast cancer cell, a colon cancer cell, a malignant melanoma cell or a multiple myeloma cell, a plasmacytoma cell or a lymphoma cell or a cancer derived from a lymphocyte, (d) the lymphocytes are early B cells, pro-B cells, pre-B lymphocytes, mature B lymphocytes, T lymphocytes, parafollicular T lymphocytes, or cells derived from a subpopulation of parafollicular T lymphocytes, (e) the cancer is lung cancer, or the lung cancer is lung adenocarcinoma, (f) the Ab or its Ag-binding fragment or monomeric or dimeric ABP specifically binds to lung squamous cell carcinoma cells with lower sensitivity than to adenocarcinoma cells (or binds to lung squamous cell carcinoma cells with lower affinity than to adenocarcinoma cells), thereby resulting in fewer false positives for lung squamous cell carcinoma and better positive identification of lung adenocarcinoma; (g) The detecting comprises performing an immunohistochemistry (IHC) assay.

19. An isolated or purified, or synthetic or recombinant antibody (Ab) according to any one of claims 1 to 5 or according to any one of the preceding claims, or an antigen (Ag) binding fragment thereof, or a monomeric or dimeric antigen binding protein (ABP), for use in detecting or diagnosing cancer, or for treating, ameliorating or preventing cancer.

20. The isolated or purified, or synthetic or recombinant antibody (Ab), or antigen (Ag) binding fragment thereof, or monomeric or dimeric antigen binding protein (ABP) according to claim 19, wherein: (a) the cancer is a thyroid cancer cell, a lung cancer cell or a cancer cell, (b) the cancer is a cancer cell, or a large cell cancer, a small cell cancer (optionally a small cell lung cancer or an extrapulmonary small cell cancer), a lung cancer cell, or an adenocarcinoma cell, and optionally the adenocarcinoma cell is a lung adenocarcinoma cell or a thyroid adenocarcinoma cell, (c) the cancer cell is a squamous cell carcinoma (SCC) cell, a leukemia cell, a carcinoid tumor, a breast cancer cell, a colon cancer cell, a malignant melanoma cell or a multiple myeloma cell, a plasmacytoma cell or a lymphoma cell or a cancer derived from a lymphocyte, (d) the lymphocytes are early B cells, pro-B cells, pre-B lymphocytes, mature B lymphocytes, T lymphocytes, parafollicular T lymphocytes, or cells derived from a subpopulation of parafollicular T lymphocytes, (e) the cancer is lung cancer, or the lung cancer is lung adenocarcinoma, (f) the Ab or its Ag-binding fragment or monomeric or dimeric ABP specifically binds to lung squamous cell carcinoma cells with lower sensitivity than to adenocarcinoma cells (or binds to lung squamous cell carcinoma cells with lower affinity than to adenocarcinoma cells), thereby resulting in fewer false positives for lung squamous cell carcinoma and better positive identification of lung adenocarcinoma; (g) The detecting or diagnosing comprises performing an immunohistochemistry (IHC) assay.

21. A kit, comprising: (a) an isolated or purified or synthetic or chimeric or recombinant antibody according to any one of claims 1 to 5; a chimeric or recombinant nucleic acid according to any one of claims 6 to 7; or an expression cassette, vector, recombinant virus, artificial chromosome, cosmid or plasmid according to claim 8; or a cell according to any one of claims 9 to 10; or a recombinant antibody (Ab) or antigen (Ag) binding fragment thereof, or a monomeric or dimeric antigen binding protein (ABP) according to any one of claims 19 to 20; and / or (b) components required for an immunohistochemistry (IHC) assay, and / or instructions for carrying out the method of any preceding claim.

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