Anti-human PAX8 antibody for in vitro diagnostic use by immunohistochemistry

Abstract

The present disclosure provides antibodies, including but not limited to monoclonal antibodies, capable of specifically binding the human PAX8 protein. Also provided are nucleic acids encoding the anti-PAX8 antibodies described herein, as well as expression vectors comprising these nucleic acids, host cells transformed with the expression vectors comprising the nucleic acids encoding the anti-PAX8 antibodies, and methods of making the antibodies. The anti-PAX8 antibodies described herein are useful for immunohistochemical detection of human PAX8 protein in tissue samples.

Description

Anti-human PAX8 antibody for in vitro diagnostic use by immunohistochemistryBy Morten Alder Schulz, Lasse Ramsgaard, and Tine Hagedorn-OlsenAssigned to Agilent Technologies, Inc.REFERENCE TO CROSS-RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Patent Application Serial No 63 / 563,209 by Morten Alder Schulz, Lasse Ramsgaard, and Tine Hagedorn- Olsen, entitled ‘Anti-human PAX8 antibody for in vitro diagnostic use by immunohistochemistry’ filed on March 8, 2024, the disclosure of which is incorporated herein by reference in it’s entirety for all purposes.REFERENCE TO A SEQUENCE LISTING SUBMITTED AS AN ASCII TEXT FILE

[0002] Filed herewith and expressly incorporated herein by reference in its entirety is a Sequence Listing submitted electronically as an XML file to file via Patent Center. The XML copy, in ST.26 format, was created on March 4, 2025, is named AGI_20230170_01_PCT and is 17,000 bytes in size.FIELD

[0003] This invention relates generally to antibodies useful for immunohistochemistry, and more particularly to novel anti-PAX8 antibodies useful for immunohistochemistry -based diagnostics and the like.BACKGROUND

[0004] The following includes information that may be useful in understanding the present inventions. It is not an admission that any of the information provided herein is prior art, or relevant, to the presently described or claimed inventions, or that any publication or document that is specifically or implicitly referenced is prior art.

[0005] PAX8 is a nuclear transcription regulator in the paired box family, expressed during and crucial for the organogenesis of the thyroid gland, kidney, and Mullerian tract (fallopian tube, uterus, cervix, and upper part of vagina). It is localized on chromosome 2ql3 and the molecular weight of the unprocessed precursor is 48 kDa, of five isoforms 31-42kDa. (NordiQC (1, 4)). PAX8 is crucial for organogenesis of the thyroid gland, kidney, and Mullerian system (fallopian tube, uterus, cervix, and upper part of vagina). Hence, PAX8 is a sensitive and specific marker fortumors of renal, Mullerian, thyroid origin in both primary and metastatic sites (1, 3, 4). Immunoreactivity of PAX8 has thus been detected in 91 % of thyroid tumors, in 90% of renal cell carcinomas, in 79% of ovarian cancers (93-99% of serous ovarian cancer), in 87-98% of endometrial adenocarcinomas and in 80-83% of cervical adenocarcinomas (1, 4), whereas tumors of the bladder, lung, breast, colon, stomach, prostate, head and neck et al are PAX8 negative for most cases (1, 4).

[0006] A primary use for PAX8 immunohistochemistry is thus within the diagnosis of renal cell carcinoma, ovarian tumors, and tumors of unknown origin. Primary renal epithelial tumors (like renal clear cell tumors) arising from renal parenchyma are usually positive for PAX8, whereas urothelial tumors (tumors arising from bladder, urethra, ureter, and renal pelvis, also called transitional cell carcinoma) are negative (1, 4, 5). Metastatic renal cell carcinoma may enter the differential diagnosis of tumors of unknown origin and PAX8 is the most useful antibody target to establish a diagnosis of metastatic renal cell carcinoma (3). Since PAX8 is expressed in most primary ovarian cancers it is useful to establish ovarian origin as opposed to metastasis to the ovary from other organs. PAX8 is especially useful for distinguishing between primary serous ovarian carcinoma from metastatic breast carcinoma (1, 2, 4).

[0007] A problem with anti-PAX8 antibodies currently available is cross reactivity in other cell types and to other isotypes of the PAX protein other than PAX8. Another problem with current anti-PAX8 antibodies can be a high background noise that limits sensitivity. In some embodiments, the antibodies described herein overcome these unsolved challenges and needs. Novel embodiments of anti-PAX8 antibodies which selectively recognize human PAX8 are described in detail below.BRIEF SUMMARY

[0008] The inventions described and claimed herein have many attributes and embodiments including, but not limited to, those set forth or described or referenced in this Brief Summary. The inventions described and claimed herein are not limited to, or by, the features or embodiments identified in this Summary, which is included for purposes of illustration only and not restriction. As a first aspect of the invention, an antibody or antigen binding fragment thereof capable of specifically binding the human PAX8 protein is provided. The PAX8 antigen used herein was designed as a synthetic peptide of human PAX8 protein. Peptides of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 consecutive amino acids, are potentially suitable antigens. The human PAX8 protein amino acid sequence is provided below for reference.

[0009] MPHNSIRSGHGGLNQLGGAFVNGRPLPEVVRQRIVDLAHQGVRPCDISRQ LRVSHGCVSKILGRYYETGSIRPGVIGGSKPKVATPKVVEKIGDYKRQNPTMFAWEIRD RLLAEGVCDNDTVPSVSSINRIIRTKVQQPFNLPMDSCVATKSLSPGHTLIPSSAVTPPESP QSDSLGSTYSINGLLGIAQPGSDKRKMDDSDQDSCRLSIDSQSSSSGPRKHLRTDAFSQH HLEPLECPFERQHYPEAYASPSHTKGEQGLYPLPLLNSTLDDGKATLTPSNTPLGRNLST HQT YP VVADPHSPF AIKQETPEVSS S S STPS SLS S SAFLDLQQ VGSGVPPFNAFPHAAS VY GQFTGQALLSGREMVGPTLPGYPPHIPTSGQGSYASSAIAGMVAGSEYSGNAYGHTPYS SYSEAWRFPNSSLLSSPYYYSSTSRPSAPPTTATAFDHL (SEQ ID NO: 1)

[0010] A first exemplary non-limiting example of an antibody or antigen binding fragment thereof capable of specifically binding the human PAX8 protein includes the following: i) a heavy chain variable region comprising heavy chain CDR1 comprising the amino acid sequence GIDFSGSYY (SEQ ID NO: 3), heavy chain CDR2 comprising the amino acid IYTYTTGRV (SEQ ID NO: 4), and heavy chain CDR3 comprising the amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5), and / or ii) a light chain variable region comprising light chain CDR1 comprising the amino acid sequence QSISSY (SEQ ID NO: 8), light chain CDR2 comprising the amino acid sequence KAS, YKAS (SEQ ID NO:6), or KAST (SEQ ID NO: 9), and light chain CDR3 comprising the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10). In one particular variation of this embodiment, the light chain CDR2 comprises the amino acid sequence KAS.

[0011] In some embodiments, such an antibody has both i) a heavy chain variable region comprising heavy chain CDR1 comprising the amino acid sequence GIDFSGSYY (SEQ ID NO: 3), heavy chain CDR2 comprising the amino acid IYTYTTGRV (SEQ ID NO: 4), and heavy chain CDR3 comprising the amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5), and ii) a light chain variable region comprising light chain CDR1 comprising the amino acid sequence QSISSY (SEQ ID NO: 8), light chain CDR2 comprising the amino acid sequence KAS, YKAS (SEQ ID NO:6), or KAST (SEQ ID NO: 9), and light chain CDR3 comprising the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10). In one particular variation of this embodiment, the light chain CDR2 comprises the amino acid sequence KAS.

[0012] In some variations of the first embodiment, the heavy chain variable region has an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence: QEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVRQAPGKGLEWIACIYTYTTGR VYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCARNSGPYTTIALSIWGPGTLVT VSS (SEQ ID NO: 2).

[0013] In some embodiments, the heavy chain variable region has an amino acid sequence with 100% identity to the amino acid sequence: QEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVRQAPGKGLEWIACIYTYTTGR VYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCARNSGPYTTIALSIWGPGTLVT VSS (SEQ ID NO: 2). In some variations of the first embodiment, the light chain variable region has an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence: DVVMTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYKASTLASGVP SRFSGSGSGTEFIFTISDLECADGATYYCQNNYGIASNYGVAFGGGTEVVVK (SEQ ID NO: 7). In one embodiment, the light chain variable region has an amino acid sequence with 100% identity to the amino acid sequence: DVVMTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYKASTLASGVP SRFSGSGSGTEFIFTISDLECADGATYYCQNNYGIASNYGVAFGGGTEVVVK (SEQ ID NO: 7).

[0014] A second non-limiting exemplary embodiment of an antibody or antigen binding fragment thereof capable of specifically binding the human PAX8 protein has i) a heavy chain variable region comprising heavy chain CDR1 comprising the amino acid sequence GIDFSGSYY (SEQ ID NO: 3) or an amino acid sequence at least 50% identical thereto, CDR2 comprising the amino acid IYTYTTGRV (SEQ ID NO: 4) or an amino acid sequence at least 50% identical thereto, and heavy chain CDR3 comprising the amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5) or an amino acid sequence at least 50% identical thereto, and / or ii) a light chain variable region comprising light chain CDR1 comprising the amino acid sequence QSISSY (SEQ ID NO: 8) or an amino acid sequence at least 50% identical thereto, light chain CDR2 comprising any one of the amino acid sequences KAS, YKAS (SEQ ID NO: 6), or KAST (SEQ ID NO: 9) or an amino acid sequence at least 50% identical thereto, and light chain CDR3 comprising the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10) or an amino acid sequence at least 50% identical thereto. In one particular variation of this embodiment, the light chain CDR2 comprises the amino acid sequence KAS.

[0015] In one variation of the second embodiment, the antibody has both i) a heavy chain variable region comprising heavy chain CDR1 comprising the amino acid sequence GIDFSGSYY (SEQ ID NO: 3) or an amino acid sequence at least 50% identical thereto, CDR2 comprising the amino acid IYTYTTGRV (SEQ ID NO: 4) or an amino acid sequence at least 50% identical thereto, and heavy chain CDR3 comprising the amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5) or an amino acid sequence at least 50% identical thereto, and ii) a light chain variable region comprising light chain CDR1 comprising the amino acid sequence QSISSY (SEQ ID NO:8) or an amino acid sequence at least 50% identical thereto, light chain CDR2 comprising any one of the amino acid sequences KAS, YKAS (SEQ ID NO: 6), or KAST (SEQ ID NO: 9) or an amino acid sequence at least 50% identical thereto, and light chain CDR3 comprising the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10) or an amino acid sequence at least 50% identical thereto. In one particular variation of this embodiment, the light chain CDR2 comprises the amino acid sequence KAS.

[0016] Some variants of the second embodiment have a heavy chain variable region comprising heavy chain CDR1 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to GIDFSGSYY (SEQ ID NO: 3), CDR2 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to IYTYTTGRV (SEQ ID NO: 4), and heavy chain CDR3 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to ARNSGPYTTIALSI (SEQ ID NO: 5). Some variants of the second embodiment have a light chain variable region comprising light chain CDR1 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to QSISSY (SEQ ID NO: 8), light chain CDR2 comprising the an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to KAS, YKAS (SEQ ID NO: 6), or KAST (SEQ ID NO: 9), and light chain CDR3 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to QNNYGIASNYGVA (SEQ ID NO: 10).

[0017] Typically, the antibody or antigen binding fragment thereof described herein is a rabbit, murine, rat, shark, llama, chicken, human, or chimeric antibody. For example, one particular antibody embodiment capable of specifically binding the human PAX8 protein described herein in greater detail is a rabbit antibody.

[0018] In preferred embodiments, an antibody described herein is capable of specifically binding the human PAX8 protein and shows substantially no cross-reactivity to other human PAX- family members; PAX2, PAX5, or PAX6.

[0019] Any of the antibodies described herein include an antigen binding fragment of an antibody that is typically selected from Fab, a Fab', a F(ab')2, a variable fragment (Fv), a triabody, a tetrabody, a minibody, a nanobody, a bispecific F(ab')2, a trispecific F(ab')2, a diabody, a bispecific diabody, a single chain variable fragment (scFv), a scFv-Fc, a Fab-Fc, a VHH, or a bispecific scFv, or a conjugate of one of the preceding further comprising a drug or moiety to facilitate imaging.

[0020] In another aspect of the invention, a DNA molecule and / or gene encoding the antibodies described herein are also provided. For example, in one variation of this embodiment, the DNA molecule or gene encodes an antibody wherein the light chain variable region has an amino acid sequence with 100% identity to the amino acid sequence:DVVMTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYKASTLASGVP SRFSGSGSGTEFIFTISDLECADGATYYCQNNYGIASNYGVAFGGGTEVVVK (SEQ ID NO: 7).

[0021] In another aspect, expression vectors comprising the DNA molecules described herein and an expression regulatory sequence operably linked to these DNA sequences are provided.

[0022] In another aspect, the expression vectors comprising DNA molecules described herein encoding antibodies are transfected into a recombinant host cell which can be used to make anti- PAX8 protein antibody or an antigen binding fragments described herein.

[0023] Thus, in another aspect, methods of producing an anti-PAX8 protein antibody or an antigen binding fragment that binds to the human PAX8 protein are provided in yet another aspect of the invention. In an exemplary embodiment, such a method has the steps of i) culturing a host cell comprising a nucleic acid sequence encoding an antibody described herein under conditions in which the host cell expresses the antibody or antigen binding fragment thereof, and ii) harvesting a preparation of the antibody or antigen binding fragment thereof expressed by the cell. In some variants of this embodiment, the host cell is transformed with an expression construct comprising a nucleic acid sequence encoding an antibody heavy chain variable region and an expression construct comprising a nucleic acid sequence encoding an antibody light chain variable region, and where the expression constructs are present in one or more expression vectors.

[0024] In other variations of embodiments of methods of making an anti-PAX8 protein antibody, the heavy chain variable region has i) a heavy chain CDR1 comprising the amino acid sequence GIDFSGSYY (SEQ ID NO: 3) or an amino acid sequence at least 50% identical thereto, the heavy chain CDR2 comprises the amino acid IYTYTTGRV (SEQ ID NO: 4) or an amino acid sequence at least 50% identical thereto, and the heavy chain CDR3 comprises the amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5) or an amino acid sequence at least 50% identical thereto; and / or ii) a light chain variable region has light chain CDR1 comprising the amino acid sequence QSISSY (SEQ ID NO: 8) or an amino acid sequence at least 50% identical thereto, light chain CDR2 comprising any one of the amino acid sequences KAS, YKAS (SEQ ID NO:6), or KAST (SEQ ID NO: 9) or an amino acid sequence at least 50% identical thereto, and light chain CDR3 comprising the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10) or an amino acid sequence at least 50% identical thereto. In one particular variation of this embodiment, the light chain CDR2 comprises the amino acid sequence KAS.

[0025] In other variations of embodiments of methods of making an anti- PAX8 protein antibody, the light chain variable region comprises a light chain CDR1 comprising the amino acid sequence QSISSY (SEQ ID NO: 8) or an amino acid sequence at least 50% identical thereto, lightchain CDR2 comprising any one of the amino acid sequences KAS, YKAS (SEQ ID NO:6), or KAST (SEQ ID NO: 9) or an amino acid sequence at least 50% identical thereto, and light chain CDR3 comprising the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10) or an amino acid sequence at least 50% identical thereto. In one particular variation of this embodiment, the light chain CDR2 comprises the amino acid sequence KAS.

[0026] In some variations of embodiments of methods of making an anti- PAX8 protein antibody, the heavy chain variable region comprises a heavy chain CDR1 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to GIDFSGSYY (SEQ ID NO: 3), CDR2 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to IYTYTTGRV (SEQ ID NO: 4), and heavy chain CDR3 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to ARNSGPYTTIALSI (SEQ ID NO: 5).

[0027] In some variations of embodiments of methods of making an anti- PAX8 protein antibody, the light chain variable region has a light chain CDR1 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to QSISSY (SEQ ID NO: 8), a light chain CDR2 comprising the an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to KAS, YKAS (SEQ ID NO: 6), or KAST (SEQ ID NO: 9), and a light chain CDR3 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to QNNYGIASNYGVA (SEQ ID NO: 10).

[0028] In some variations of embodiments of methods of making an anti- PAX8 protein antibody, the nucleic acid sequence encoding an antibody heavy chain variable region has a nucleic acid sequence encoding heavy chain HCDR1, HCDR2, and HCDR3 regions of the amino acid sequence: METGLRWLLLVAVLKGVQCQEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVR QAPGKGLEWIACIYTYTTGRVYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCA RNSGPYTTIALSIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPV TVTWNSGTLTNGVRTFPS VRQS SGL YSLS S VVS VTS S SQP VTCNVAHP ATNTKVDKT VA PSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINN EQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARG QPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDS DGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 11), and / or the nucleic acid sequence encoding an antibody light chain variable region has a nucleic acid sequence encoding light chain LCDR1, LCDR2, and LCDR3 regions of the amino acid sequence:MDTRAPTQLLGLLLLWLPGARCADVVMTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTEFIFTISDLECADGATYYCQNNYGIAS NYGVAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVD GTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRG DC (SEQ ID NO: 12), wherein HCDR1 is the amino acid sequence GIDFSGSYY (SEQ ID NO: 3), HCDR2 is the amino acid sequence IYTYTTGRV (SEQ ID NO: 4), and HCDR3 is the amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5), and where LCDR1 is the amino acid sequence QSISSY (SEQ ID NO: 8), LCDR2 is any one of the amino acid sequences KAS, YKAS (SEQ ID NO:6), or KAST (SEQ ID NO: 9), and LCDR3 is the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10). In one particular variation of this embodiment, the LCDR2 comprises the amino acid sequence KAS.

[0029] In some variations of embodiments of methods of making an anti- PAX8 protein antibody, the nucleic acid sequence encoding an antibody heavy chain variable region has both i) a nucleic acid sequence encoding the heavy chain HCDR1, HCDR2, and HCDR3 regions of the amino acid sequence: METGLRWLLLVAVLKGVQCQEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVR QAPGKGLEWIACIYTYTTGRVYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCA RNSGPYTTIALSIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPV TVTWNSGTLTNGVRTFPS VRQS SGL YSLS S VVS VTS S SQP VTCNVAHP ATNTKVDKT VA PSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINN EQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARG QPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDS DGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 11), and ii) the nucleic acid sequence encoding an antibody light chain variable region has a nucleic acid sequence encoding light chain LCDR1, LCDR2, and LCDR3 regions of the amino acid sequence MDTRAPTQLLGLLLLWLPGARCADVVMTQTPSSVEAAVGGTVTIKCQASQSISSYLAW YQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTEFIFTISDLECADGATYYCQNNYGIAS NYGVAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVD GTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRG DC (SEQ ID NO: 12), where HCDR1 is the amino acid sequence GIDFSGSYY (SEQ ID NO: 3), HCDR2 is the amino acid sequence IYTYTTGRV (SEQ ID NO: 4), and HCDR3 is the amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5), and where LCDR1 is the amino acid sequence QSISSY (SEQ ID NO: 8), LCDR2 is any one of the amino acid sequences KAS, YKAS (SEQ ID NO:6), or KAST (SEQ ID NO: 9), and LCDR3 is the amino acid sequenceQNNYGIASNYGVA (SEQ ID NO: 10). In one particular variation of this embodiment, the LCDR2 comprises the amino acid sequence KAS.

[0030] In some variations of embodiments of methods of making an anti- PAX8 protein antibody, the nucleic acid sequence encoding the antibody heavy chain variable region comprises a nucleic acid sequence encoding the heavy chain variable region having an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence: QEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVRQAPGKGLEWIACIYTYTTGR VYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCARNSGPYTTIALSIWGPGTLVT VSS (SEQ ID NO: 2).

[0031] In some variations of embodiments of methods of making an anti- PAX8 protein antibody, the nucleic acid sequence encoding the antibody heavy chain variable region has the nucleic acid sequence: ATGGAGACCGGTCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGT CAGGAGCAGCTGGTGGAGTCCGGGGGAGGCCTGGTCCAGCCTGAGGGATCCCTGAC ACTCACCTGCAAAGCCTCTGGAATCGACTTCAGTGGCAGCTATTATGGTTGCTGGGT CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATTTATACTTATACTAC TGGTAGGGTGTACTACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAATCT CGTCAGCTACGGTGACTCTGCAAATGACCAGTCTGACAGCCGCGGACACGGCCACC TATTTCTGTGCGAGAAATTCTGGTCCTTATACTACTATTGCCCTCAGCATCTGGGGCC CAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCAC TGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCA AAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACAGCGGCACCCTGACAAAT GGCGTGCGGACCTTTCCTAGCGTGCGGCAGTCTAGCGGCCTGTACTCTCTGAGCAGC GTGGTGTCCGTGACCAGCAGCTCTCAGCCAGTGACCTGCAACGTGGCCCACCCCGCC ACCAATACCAAGGTGGACAAGACCGTGGCCCCCAGCACCTGTAGCAAGCCTACCTG TCCTCCACCCGAACTGCTGGGCGGACCCAGCGTGTTCATCTTCCCACCCAAGCCCAA GGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGT CCCAGGACGACCCCGAGGTGCAGTTCACCTGGTACATCAACAACGAACAAGTGCGG ACCGCCAGACCCCCCCTGAGAGAGCAGCAGTTCAACAGCACCATCCGGGTGGTGTC CACCCTGCCTATCGCCCACCAGGATTGGCTGCGGGGCAAAGAGTTCAAGTGCAAGG TGCACAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAGAGGC CAGCCCCTGGAACCCAAAGTGTATACAATGGGACCTCCCAGAGAGGAACTGAGCAG CAGATCCGTGTCCCTGACCTGCATGATCAACGGCTTCTACCCCAGCGACATCAGCGT GGAATGGGAGAAGAACGGCAAGGCCGAGGACAACTACAAGACCACCCCTGCCGTG CTGGACAGCGACGGCAGCTACTTCCTGTACTCCAAGCTGAGCGTGCCCACCAGCGAATGGCAGAGGGGCGACGTGTTCACATGCAGCGTGATGCACGAGGCCCTGCACAACC ACTACACCCAGAAGTCCATCAGCCGCAGCCCCGGCAAATGA (SEQ ID NO: 13), and the nucleic acid sequence encoding an antibody light chain variable region has the nucleic acid sequence: ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGT GCCAGATGTGCCGATGTTGTGATGACCCAGACTCCATCCTCCGTGGAGGCAGCTGTG GGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAGTAGCTACTTAGC CTGGTATCAGCAGAAACCAGGACAGCCTCCCAAGCTCCTGATCTACAAGGCATCCA CTCTGGCATCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCA TTTTCACCATCAGCGACCTGGAGTGTGCCGATGGTGCCACTTACTACTGTCAAAACA ATTATGGTATTGCTAGTAATTATGGTGTTGCTTTCGGCGGAGGTACCGAGGTGGTGG TCAAAGGCGATCCTGTGGCCCCTACTGTCCTCATCTTCCCGCCGGCTGCTGATCAGG TGGCCACAGGCACAGTGACCATCGTGTGCGTGGCCAACAAGTACTTCCCCGACGTG ACCGTGACCTGGGAAGTCGATGGCACCACACAGACCACCGGCATCGAGAACAGCAA GACCCCTCAGAACAGCGCCGACTGCACCTACAACCTGAGCAGCACCCTGACACTGA CCAGCACACAGTACAACAGCCACAAAGAGTACACCTGTAAAGTCACCCAGGGCACC ACCAGCGTGGTGCAGAGCTTCAATAGAGGCGACTGCTGA (SEQ ID NO: 14).

[0032] In another aspect of the invention, methods of detecting a human PAX8 protein in a tissue or organ are provided. These embodiments often include a step of contacting a sample obtained from an individual with an antibody or antigen binding fragment thereof that is described herein and detecting binding of the selected antibody or antigen binding fragment thereof to a sample (for example, from a subject).

[0033] In some variations of the methods of detecting a human PAX8 protein in a tissue or organ, a sample is obtained from a subject from one or more of the following tissues: organs of Mullerian origin, such as fallopian tube, uterus, cervix, and vagina, or other organs such as ovary, kidney, thyroid, bladder, lung, colon, breast, stomach, prostate, head and neck, or any other organs or tissues where the detection of binding or no binding is indicative of the origin of a cancer or neoplasia in a tissue from which the sample was obtained. In some variations of the methods provided herein, detecting the human PAX8 protein is used for detecting or for a diagnosis of renal cell carcinoma, ovarian tumors, or tumors of unknown origin. In some variations of the methods provided herein, detecting human PAX8 protein is used for detecting or for a diagnosis of ovarian cancer. In some variations of this embodiment, the method is used to establish an ovarian origin as opposed to metastasis to the ovary from other organs. In some variations of the methods provided herein, detecting the human PAX8 protein is used for distinguishing between renal epithelial tumors and urothelial tumors. In some variations of themethods provided herein, the detecting the human PAX8 protein is used to establish a diagnosis of metastatic renal cell carcinoma. In some variations of the methods provided herein, the detecting the human PAX8 protein is used to distinguish between primary serous ovarian carcinomas and metastatic breast carcinomas.

[0034] In another aspect of the invention, compositions comprising an anti-PAX8 antibody or antigen binding fragment thereof described herein are provided.

[0035] In still another aspect, a kit or system for detecting the presence of the human PAX8 protein in a sample is provided. An exemplary kit or system includes an anti-PAX8 antibody or antigen binding fragment described herein, and reagents for detecting a complex of the anti-PAX8 antibody bound to human PAX8 protein from a tissue sample. In systems provided herein, also included is an instrument (for example Dako Omnis (Agilent)) for automatically processing the samples to detect the complex of the anti-PAX8 antibody bound to human PAX8 protein from a tissue sample.BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 illustrates immunohistochemistry using the monoclonal rabbit anti-human PAX8 from clone 1G2 on fallopian tube. The intercalated secretory epithelial cells show moderate to strong nuclear staining while the ciliated epithelial cells show weak to moderate nuclear staining using the same antibody.

[0037] Figure 2 illustrates immunohistochemistry using the monoclonal rabbit anti-human PAX8 from clone 1G2 on a positive clinical tissue of ovarian serous carcinoma. The neoplastic cells show a moderate to strong nuclear staining.

[0038] Figure 3 illustrates immunohistochemistry using the monoclonal rabbit anti-human PAX8 from clone 1G2 on a positive clinical tissue of renal clear cell carcinoma. The neoplastic cells show a weak to moderate nuclear staining reaction.

[0039] Figure 4 illustrates immunohistochemistry using the monoclonal rabbit anti-human PAX8 from clone 1G2 on the positive clinical tissue thyroid cancer. The neoplastic cells show a moderate to strong staining reaction.

[0040] Figure 5 illustrates immunohistochemistry using the monoclonal rabbit anti-human PAX8 from clone 1G2 on the negative clinical tissue metastatic invasive ductal breast carcinoma. There was no visible staining of the tumor cells.

[0041] Figure 6 illustrates immunohistochemistry using the monoclonal rabbit anti-human PAX8 from clone 1G2 on a negative clinical tissue transitional cell carcinoma. As shown, there is no staining of the tumor cells, but the remnants of normal tubules show some staining.

[0042] Figure 7 illustrates immunohistochemistry using the monoclonal rabbit anti-human PAX8 from clone 1G2 on malignant mesothelioma carcinoma, a negative clinical tissue. As shown, there is no staining of the tumor cells.

[0043] Figure 8 illustrates immunohistochemistry using the monoclonal rabbit anti-human PAX8 from clone 1G2 on the negative clinical tissue colorectal adenocarcinoma. There is no staining of the tumor cells.

[0044] Figure 9 illustrates immunohistochemistry using the monoclonal rabbit anti-human PAX8 from clone 1G2 on a lung adenocarcinoma, which is a negative clinical tissue. As shown, there is no staining of the tumor cells.

[0045] Figure 10 illustrates immunohistochemistry using the monoclonal rabbit antihuman PAX8 from clone 1G2 on a medullary thyroid carcinoma, a negative clinical tissue. Thyroid carcinomas have been reported to be positive in about 50% of cases. Positive nuclear staining of remnants of normal thyroid glands can be seen.

[0046] Figure 11 illustrates immunohistochemistry using the monoclonal rabbit antihuman PAX8 from clone 1G2 on a normal liver tissue sample. Hepatocytes in normal liver is reported to be negative and can be used as a negative control structure.

[0047] Figure 12 illustrates immunohistochemistry using the monoclonal rabbit antihuman PAX8 from clone 1G2 on a colon tissue sample. Colon and appendix epithelium has been reported to be negative and can be used as a negative control structure. No staining of neuroendocrine cells in crypt epithelium indicates no cross-reactivity towards PAX6.

[0048] Figure 13 illustrates immunohistochemistry using the monoclonal rabbit antihuman PAX8 from clone 1G2 on a normal tonsil tissue sample. There was no staining of B- lymphocytes in the germinal center which indicates no cross-reactivity towards PAX5 or PAX2.

[0049] Figure 14 illustrates immunohistochemistry using the monoclonal rabbit antihuman PAX8 from clone 1G2 on a normal testis tissue sample. Diffuse unspecific cytoplasmic staining of epithelial cells was observed, which was not expected.

[0050] Figure 15 illustrates immunohistochemistry using the monoclonal rabbit antihuman PAX8 from clone 1G2 on a normal ovary tissue sample. Nuclear staining of epithelial cells was observed, which was not expected.DETAILED DESCRIPTION

[0051] Various aspects of the invention will now be described with reference to the following section which will be understood to be provided by way of illustration only and not to constitute a limitation on the scope of the invention.

[0052] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. Methods for obtaining (for example, producing, isolating, purifying, synthesizing, and recombinantly manufacturing) polypeptides are well known to one of ordinary skill in the art.

[0053] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, for example, hydroxyproline, g- carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, for example, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (for example, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0054] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0055] The present composition encompasses amino acid substitutions in proteins and peptides, which do not generally alter the activity of the proteins or peptides (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). In one embodiment, these substitutions are “conservative” amino acid substitutions. The most commonly occurring substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly, in both directions

[0056] As to "conservatively modified variants" of amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds, or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in theart. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[0057] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, for example, Creighton, Proteins (1984)).

[0058] Analogue as used herein denotes a peptide, polypeptide, or protein sequence which differs from a reference peptide, polypeptide, or protein sequence. Such differences may be the addition, deletion, or substitution of amino acids, phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like, the use of nonnatural amino acid structures, or other such modifications as known in the art.

[0059] The term “unnatural amino acids” as used herein refers to amino acids other than the 20 typical amino acids found in the proteins in our human body. Unnatural amino acids are non-proteinogenic amino acids that either occur naturally or are chemically synthesized. They may include but are not limited to aminoisobutyric acid (Aib), P-amino acids (P3and p2), homoamino acids, proline and pyruvic acid derivatives, 3-substituted alanine derivatives. Glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, Linear core amino acids, diamino acids, D-amino acids and N-methyl amino acids.

[0060] "Antibody" refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.

[0061] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.

[0062] Antibodies exist, for example, as intact immunoglobulins or as a number of well- characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH— CHI by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology, Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (for example, single chain Fv) or those identified using phage display libraries (see, for example, McCafferty et al., Nature 348:552-554 (1990), incorporated by reference herein in its entirety).

[0063] Accordingly, in either aspect of the invention, the term antibody also embraces nanobodies, minibodies, diabodies, triabodies and the like. Diabodies are small bivalent biospecific antibody fragments with high avidity and specificity. Their high signal to noise ratio is typically better due to a better specificity and fast blood clearance increasing their potential for diagnostic and therapeutic targeting of specific antigen (Sundaresan et al., JNuclMed 44: 1962-9 (2003). In addition, these antibodies are advantageous because they can be engineered if necessary as different types of antibody fragments ranging from a small single chain Fv to an intact IgG with varying isoforms (Wu & Senter, Nat. Biotechnol. 23: 1137-1146 (2005)). In some embodiments, the antibody fragment is part of a diabody. In some embodiments, in either aspect, the invention provides high avidity antibodies for use according to the invention.

[0064] The CDR regions provided by the invention may be used to construct an anti-PAX8 binding protein, including without limitation, an antibody, a scFv, a triabody, a diabody, a minibody, a nanobody and the like. In a certain embodiment, an anti- anti-PAX8 protein of the invention will comprise at least one CDR region described herein or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the CDR regions described herein. Anti-PAX8 binding proteins may comprise, for example, a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, a CDR-L3, or combinations thereof, from an antibody provided herein. In particular embodiments of the invention, an anti-PAX8 binding protein may comprise all three CDR-H sequences of an antibody provided herein, all three CDR-L sequences of an antibody provided herein, or both. Anti-PAX8 antibody sequences may be used on an antibody backbone,or fragment thereof, and likewise may include humanized antibodies, or antibodies containing humanized sequences. In some embodiments, the CDR regions may be defined using the Kabat definition, the Chothia definition, the AbM definition, the contact definition, or any other suitable CDR numbering system.

[0065] In some embodiments, the invention provides antibodies (for example, diabodies, minibodies, triabodies) or fragments thereof having the CDRs or variants thereof described herein. Diabodies, first described by Hollinger et al., PNAS (USA) 90(14): 6444-6448 (1993), may be constructed using heavy and light chains disclosed herein, as well as by using individual CDR regions disclosed herein. Typically, diabody fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VH and VL domains of another fragment, thereby forming two antigen-binding sites. Triabodies can be similarly constructed with three antigen-binding sites. An Fv fragment contains a complete antigen-binding site which includes a VL domain and a VH domain held together by non-covalent interactions. Fv fragments embraced by the present invention also include constructs in which the VH and VL domains are crosslinked through glutaraldehyde, intermolecular disulfides, or other linkers. The variable domains of the heavy and light chains can be fused together to form a single chain variable fragment (scFv), which retains the original specificity of the parent immunoglobulin. Single chain Fv (scFv) dimers, first described by Gruber et al., J. Immunol. 152(12):5368-74 (1994), may be constructed using heavy and light chains disclosed herein, as well as by using individual CDR regions disclosed herein. Many techniques known in the art can be used to prepare the specific binding constructs of the present invention (see, U.S. Patent Application Publication No. 20070196274 and U.S. Patent Application Publication No. 20050163782, which are each herein incorporated by reference in their entireties for all purposes, particularly with respect to minibody and diabody design).

[0066] Bispecific antibodies can be generated by chemical cross-linking or by the hybrid hybridoma technology. Alternatively, bispecific antibody molecules can be produced by recombinant techniques. Dimerization can be promoted by reducing the length of the linker j oining the VH and the VL domain from about 15 amino acids, routinely used to produce scFv fragments, to about 5 amino acids. These linkers favor intrachain assembly of the VH and VL domains. Any suitable short linker can be used. Thus, two fragments assemble into a dimeric molecule. Further reduction of the linker length to 0-2 amino acids can generate trimeric (triabodies) or tetrameric (tetrabodies) molecules.

[0067] For preparation of antibodies, for example, recombinant, monoclonal, or polyclonal antibodies, many techniques known in the art can be used (see, for example, Kohler & Milstein,Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986), all incorporated by reference herein in their entirety). The genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, for example, the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity (see, for example, Kuby, Immunology (3rd ed. 1997)). Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Pat. No. 4,946,778, U.S. Pat. No. 4,816,567) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized or human antibodies (see, for example, U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al., Bio / Technology 10:779-783 (1992); Lonberg etal., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, for example, McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can also be made bispecific, i.e., able to recognize two different antigens (see, for example, WO 93 / 08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Suresh et al., Methods in Enzymology 121 :210 (1986)). Antibodies can also be heteroconjugates, for example, two covalently joined antibodies, or immunotoxins (see, for example, U.S. Pat. No. 4,676,980, WO 91 / 00360; and WO 92 / 200373). All references described in the paragraph above are incorporated by reference herein in their entirety.

[0068] Methods for humanizing or primatizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (see, for example, Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), all incorporated by reference herein in their entirety), by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567, the disclosure of which is incorporated herein by reference in its entirety), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0069] A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and / or species, or an entirely different molecule which confers new properties to the chimeric antibody, for example, an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

[0070] The phrase "specifically or selectively binds" to an antibody or "specifically or selectively immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologies which are not detected by cross-reaction. Most of the assays used to determine selectivity herein are of a qualitative nature, where lack of immunoreactivity is simply not visible or detectable by the methods used. A variety of immunoassay formats described herein or otherwise known in the art, may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, for example, Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) (incorporated by reference herein in its entirety) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity) .

[0071] Construction of suitable vectors containing the desired sequences and control sequences employs standard ligation and restriction techniques, which are well understood in the art (see Maniatis el al., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982), incorporated by reference herein in its entirety). Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and re-ligated in the form desired.

[0072] Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are near each other, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0073] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

[0074] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0075] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0076] A "comparison window," as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to the full length of the reference sequence, usually about 25 to 100, or 50 to about 150, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. AppL Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'L Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel etal., eds. 1995 supplement)). All references described in the paragraph above are incorporated by reference herein in their entirety.

[0077] A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403- 410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. All references described in the paragraph above are incorporated by reference herein in their entirety.

[0078] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form and complements thereof. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0079] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (for example, degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. All references described in the paragraph above are incorporated by reference herein in their entirety.

[0080] Embodiments of the invention may provide an antibody described herein in the form of a composition, such composition typically formulated for stability and / or storage and foruse in immunohistochemistry. However, other compositions of the antibodies described herein including pharmaceutical formulations are contemplated.

[0081] Embodiments of the invention may provide a kit and methods of using the kit, including for example a diagnostic kit that includes an antibody described herein that may be contacted with an antigen in a biological sample such that the antigen-antibody interaction is detectable. A biological sample may include but is not limited to a normal tissue, neoplastic tissue, and any tissue described herein. Detection of the antibody-antigen binding may be made manually, automatically, via image analysis or the like and may even be made via an automated staining device. Such a kit is typically included with instructions for use. As discussed herein in the Examples, use of the antibody may be performed on an automated staining device such as the Dako Omnis instrument (Agilent) with methods including but not limited to immunoassay, immunohistochemistry (IHC), IHC of FFPE, I CH of frozen -tissue sections, and ELISA.Additional Embodiments

[0082] (1) A method of producing a producing an anti- PAX8 protein antibody or an antigen binding fragment that binds to the human PAX8 protein, the method comprising the steps of: culturing a host cell comprising a nucleic acid sequence encoding an antibody described herein under conditions in which the host cell expresses the antibody or antigen binding fragment thereof; and harvesting a preparation of the antibody or antigen binding fragment thereof expressed by the cell.

[0083] (2) A method according to embodiment 1 wherein the host cell is transformed with an expression construct comprising a nucleic acid sequence encoding an antibody heavy chain variable region and an expression construct comprising a nucleic acid sequence encoding an antibody light chain variable region, wherein the expression constructs are present in one or more expression vectors.

[0084] (3) A method according to embodiment 2 wherein the heavy chain variable region has i) a heavy chain CDR1 comprising the amino acid sequence GIDFSGSYY (SEQ ID NO: 3) or an amino acid sequence at least 50% identical thereto, the heavy chain CDR2 comprises the amino acid IYTYTTGRV (SEQ ID NO: 4) or an amino acid sequence at least 50% identical thereto, and the heavy chain CDR3 comprises the amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5) or an amino acid sequence at least 50% identical thereto; and / or ii) a light chain variable region has light chain CDR1 comprising the amino acid sequence QSISSY (SEQ ID NO: 8) or an amino acid sequence at least 50% identical thereto, light chain CDR2 comprising any one of the amino acid sequences KAS, YKAS (SEQ ID NO: 6), or KAST (SEQ ID NO: 9) or an amino acid sequenceat least 50% identical thereto, and light chain CDR3 comprising the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10) or an amino acid sequence at least 50% identical thereto.

[0085] (4) A method according to embodiment 3 wherein the light chain variable region comprises a light chain CDR1 comprising the amino acid sequence QSISSY (SEQ ID NO: 8) or an amino acid sequence at least 50% identical thereto, light chain CDR2 comprising any one of the amino acid sequences KAS, YKAS (SEQ ID NO: 6), or KAST (SEQ ID NO: 9) or an amino acid sequence at least 50% identical thereto, and light chain CDR3 comprising the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10) or an amino acid sequence at least 50% identical thereto.

[0086] (5) An method according to embodiment 3 or 4 wherein the heavy chain variable region comprises a heavy chain CDR1 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to GIDFSGSYY (SEQ ID NO: 3), CDR2 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to IYTYTTGRV (SEQ ID NO: 4), and heavy chain CDR3 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to ARNSGPYTTIALSI (SEQ ID NO: 5).

[0087] (6) An method according to any one of embodiments 3, 4, or 5 comprising a light chain variable region comprising light chain CDR1 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to QSISSY (SEQ ID NO: 8), light chain CDR2 comprising the an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to KAS, YKAS (SEQ ID NO:6), or KAST (SEQ ID NO: 9), and light chain CDR3 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to QNNYGIASNYGVA (SEQ ID NO: 10).

[0088] (7) A method according to any one of embodiments 3-6, wherein the nucleic acid sequence encoding an antibody heavy chain variable region comprises a nucleic acid sequence encoding heavy chain HCDR1, HCDR2, and HCDR3 regions of the amino acid sequence METGLRWLLLVAVLKGVQCQEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVR QAPGKGLEWIACIYTYTTGRVYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCA RNSGPYTTIALSIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPV TVTWNSGTLTNGVRTFPS VRQS SGL YSLS S VVS VTS S SQP VTCNVAHP ATNTKVDKT VA PSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINN EQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARG QPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDS DGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 11), and / or the nucleic acid sequence encoding an antibody light chain variable region comprises a nucleic acid sequence encoding light chain LCDR1, LCDR2, and LCDR3 regions of the aminoacid sequenceMDTRAPTQLLGLLLLWLPGARCADVVMTQTPSSVEAAVGGTVTIKCQASQSISSYLAW YQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTEFIFTISDLECADGATYYCQNNYGIAS NYGVAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVD GTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRG DC (SEQ ID NO: 12), wherein HCDR1 consists of amino acid sequence GIDFSGSYY (SEQ ID NO: 3), HCDR2 consists of amino acid sequence IYTYTTGRV (SEQ ID NO: 4), and HCDR3 consists of amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5), and wherein LCDR1 consists of amino acid sequence QSISSY (SEQ ID NO: 8), LCDR2 consists of amino acid sequence KAS, YKAS (SEQ ID NO:6), or KAST (SEQ ID NO: 9), and LCDR3 consists of amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10).

[0089] (8) A method according to any one of embodiments 3-7, wherein the nucleic acid sequence encoding an antibody heavy chain variable region comprises both i) a nucleic acid sequence encoding the heavy chain HCDR1, HCDR2, and HCDR3 regions of the amino acid sequence METGLRWLLLVAVLKGVQCQEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVR QAPGKGLEWIACIYTYTTGRVYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCA RNSGPYTTIALSIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPV TVTWNSGTLTNGVRTFPS VRQS SGL YSLS S VVS VTS S SQP VTCNVAHP ATNTKVDKT VA PSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINN EQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARG QPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDS DGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 11), and ii) the nucleic acid sequence encoding an antibody light chain variable region comprises a nucleic acid sequence encoding light chain LCDR1, LCDR2, and LCDR3 regions of the amino acid sequenceMDTRAPTQLLGLLLLWLPGARCADVVMTQTPSSVEAAVGGTVTIKCQASQSISSYLAW YQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTEFIFTISDLECADGATYYCQNNYGIAS NYGVAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVD GTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRG DC (SEQ ID NO: 12), wherein HCDR1 consists of amino acid sequence GIDFSGSYY (SEQ ID NO: 3), HCDR2 consists of amino acid sequence IYTYTTGRV (SEQ ID NO: 4), and HCDR3 consists of amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5), and wherein LCDR1 consists of amino acid sequence QSISSY (SEQ ID NO: 8), LCDR2 consists of amino acidsequence KAS, YKAS (SEQ ID N0:6), or KAST (SEQ ID NO: 9), and LCDR3 consists of amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10).

[0090] (9) A method according to any one of embodiments 3-8, wherein the nucleic acid sequence encoding the antibody heavy chain variable region comprises a nucleic acid sequence encoding heavy chain wherein the heavy chain variable region having an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence: QEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVRQAPGKGLEWIACIYTYTTGR VYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCARNSGPYTTIALSIWGPGTLVT VSS (SEQ ID NO: 2).

[0091] (10) A method according to embodiment 3, wherein the nucleic acid sequence encoding the antibody heavy chain variable region comprises the nucleic acid sequence ATGGAGACCGGTCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGT CAGGAGCAGCTGGTGGAGTCCGGGGGAGGCCTGGTCCAGCCTGAGGGATCCCTGAC ACTCACCTGCAAAGCCTCTGGAATCGACTTCAGTGGCAGCTATTATGGTTGCTGGGT CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATTTATACTTATACTAC TGGTAGGGTGTACTACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAATCT CGTCAGCTACGGTGACTCTGCAAATGACCAGTCTGACAGCCGCGGACACGGCCACC TATTTCTGTGCGAGAAATTCTGGTCCTTATACTACTATTGCCCTCAGCATCTGGGGCC CAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCAC TGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCA AAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACAGCGGCACCCTGACAAAT GGCGTGCGGACCTTTCCTAGCGTGCGGCAGTCTAGCGGCCTGTACTCTCTGAGCAGC GTGGTGTCCGTGACCAGCAGCTCTCAGCCAGTGACCTGCAACGTGGCCCACCCCGCC ACCAATACCAAGGTGGACAAGACCGTGGCCCCCAGCACCTGTAGCAAGCCTACCTG TCCTCCACCCGAACTGCTGGGCGGACCCAGCGTGTTCATCTTCCCACCCAAGCCCAA GGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGT CCCAGGACGACCCCGAGGTGCAGTTCACCTGGTACATCAACAACGAACAAGTGCGG ACCGCCAGACCCCCCCTGAGAGAGCAGCAGTTCAACAGCACCATCCGGGTGGTGTC CACCCTGCCTATCGCCCACCAGGATTGGCTGCGGGGCAAAGAGTTCAAGTGCAAGG TGCACAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAGAGGC CAGCCCCTGGAACCCAAAGTGTATACAATGGGACCTCCCAGAGAGGAACTGAGCAG CAGATCCGTGTCCCTGACCTGCATGATCAACGGCTTCTACCCCAGCGACATCAGCGT GGAATGGGAGAAGAACGGCAAGGCCGAGGACAACTACAAGACCACCCCTGCCGTG CTGGACAGCGACGGCAGCTACTTCCTGTACTCCAAGCTGAGCGTGCCCACCAGCGA ATGGCAGAGGGGCGACGTGTTCACATGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCATCAGCCGCAGCCCCGGCAAATGA (SEQ ID NO: 13) and the nucleic acid sequence encoding an antibody light chain variable region comprises the nucleic acid sequence ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGT GCCAGATGTGCCGATGTTGTGATGACCCAGACTCCATCCTCCGTGGAGGCAGCTGTG GGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAGTAGCTACTTAGC CTGGTATCAGCAGAAACCAGGACAGCCTCCCAAGCTCCTGATCTACAAGGCATCCA CTCTGGCATCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCA TTTTCACCATCAGCGACCTGGAGTGTGCCGATGGTGCCACTTACTACTGTCAAAACA ATTATGGTATTGCTAGTAATTATGGTGTTGCTTTCGGCGGAGGTACCGAGGTGGTGG TCAAAGGCGATCCTGTGGCCCCTACTGTCCTCATCTTCCCGCCGGCTGCTGATCAGG TGGCCACAGGCACAGTGACCATCGTGTGCGTGGCCAACAAGTACTTCCCCGACGTG ACCGTGACCTGGGAAGTCGATGGCACCACACAGACCACCGGCATCGAGAACAGCAA GACCCCTCAGAACAGCGCCGACTGCACCTACAACCTGAGCAGCACCCTGACACTGA CCAGCACACAGTACAACAGCCACAAAGAGTACACCTGTAAAGTCACCCAGGGCACC ACCAGCGTGGTGCAGAGCTTCAATAGAGGCGACTGCTGA (SEQ ID NO: 14).

[0092] (H) A method of detecting the human PAX8 protein in a tissue or organ, the method comprising contacting a sample obtained from an individual with an antibody or antigen binding fragment thereof provided and detecting binding of said antibody or antigen binding fragment thereof to said sample.

[0093] (12) A method according to embodiment 11, wherein the sample is selected from a group consisting of a sample from organs of Mullerian origin, such as fallopian tube, uterus, cervix, and vagina, or other organs such as ovary, kidney, thyroid, bladder, lung, colon, breast, stomach, prostate, head and neck or other organs wherein the detection of binding or no binding is indicative of the origin of a cancer or neoplasia in a tissue from which said sample was obtained.

[0094] (13) A method according to embodiment 11 or 12, wherein the detecting the humanPAX8 protein is used for detecting or for a diagnosis of renal cell carcinoma, ovarian tumors, or tumors of unknown origin.

[0095] (14) A method according to embodiment 11 or 12, wherein the detecting the humanPAX8 protein is used for detecting or for a diagnosis of ovarian cancer.

[0096] (15) A method of embodiment 14, wherein the method is used to establish an ovarian origin as opposed to metastasis to the ovary from other organs.

[0097] (16) A method according to embodiment 11 or 12, wherein the detecting the humanPAX8 protein is used for distinguishing between renal epithelial tumors and urothelial tumors.

[0098] (17) A method according to embodiment 11 or 12, wherein the detecting the humanPAX8 protein is used to establish a diagnosis of metastatic renal cell carcinoma.

[0099] (18) A method according to embodiment 11 or 12, wherein the detecting the humanPAX8 protein is used to distinguish between primary serous ovarian carcinomas and metastatic breast carcinomas.

[0100] (19) A composition comprising an anti-PAX8 antibody or antigen binding fragment thereof provided herein.

[0101] (20) A kit or system for detecting the presence of the human PAX8 protein in a sample, the kit or system comprising i) an anti-PAX8 antibody or antigen binding fragment thereof of any one of claims 1-14, ii) reagents for detecting a complex of the anti-PAX8 antibody bound to human PAX8 protein from a tissue sample, and, optionally, iii) an instrument for automatically processing the samples to detect said complex of the anti-PAX8 antibody bound to human PAX8 protein from a tissue sample.

[0102] The following Examples are included for illustration and not limitation.EXAMPLE 1: Antibody Methods and Selection

[0103] The making of specific monoclonal antibodies is described in this example. A synthetic peptide representing a portion of the human PAX8 protein was KLH-conjugated and used for immunization of rabbits. Animal bleeds were later taken to confirm serum reactivity to human PAX8 protein in several positive and negative clinically relevant human tissues by H4C. Blood from rabbits with best immune responses against human PAX8 antigen was used to carry out several B-cell selections. B-cells expressing antigen-specific antibodies were isolated as monoclonals and screened by immunoassays. Recombinant antibodies were tested for human PAX8 binding by biolayer interferometry (BLI) on a BLItz instrument, and subsequently tested in H4C by standard FLEX protocols on normal and clinical tissues. Some antibodies were identified to show human PAX8 specific staining in H4C. Clone 1G2 derived from rabbits immunized with the synthetic peptide antigen representing human PAX8 showed very promising results on tested tissues. The antibody has been tested for specificity on different clinical tissues and shows usefulness for in vitro diagnostics by immunohistochemistry.Clone selection

[0104] Rabbit bleeds were evaluated by IHC and candidate rabbits expressing PAX8 specific antibodies were chosen for B-cell selections. B-cell selections were performed by using 1blood samples from relevant rabbits. The most promising B-cells were selected for molecular cloning and sequencing of variable heavy and light Ab chains. The stability of antibodies was tested and proved in IHC tests.EXAMPLE 2: Immunohistochemistry

[0105] PAX8 is a nuclear transcription regulator in the paired box family, expressed during and crucial for the organogenesis of the thyroid gland, kidney, and Mullerian tract (fallopian tube, uterus, cervix, and upper part of vagina). It is localized on chromosome 2ql3 and the molecular weight of the unprocessed precursor is 48 kDa, of five isoforms 3 l-42kDa. (NordiQC (1, 4)). PAX8 is known to be expressed in fallopian tube epithelia, both ciliated non-secretory epithelia and intercalated secretory epithelia, in kidney epithelia (proximal and distal tubules, loops of Henle, collecting ducts, and the parietal epithelia of the Bowman’s capsule), and in thyroid epithelial cells.

[0106] The 1G2 antibody described in Example I was used for immunohistochemistry in various tissues in this Example, including those previously known clinically to be either negative or positive controls and to validate that the antibody embodiments described herein are specific to human PAX8 protein. The results are shown in Table 1. Various tissue types are tested and compared to what would be expected in that tissue type using an anti-PAX8 antibody. FLEX Monoclonal Rabbit Anti-Human PAX8, Clone DAK-PAX8, Ready-to-Use (Dako Omnis, Agilent) was used for the IHC. This is a qualitative assay intended for use in immunohistochemistry (IHC) together with the Dako Omnis instrument (Agilent) on formalin-fixed, paraffin- embedded tissue sections.

[0107] Table 1 - Expected reaction pattern vs observed in various tissues

[0108] High expressing structures are intercalated secretory epithelial cells of the fallopian tube and they are expected to show moderate to strong nuclear staining; however, a weak cytoplasmatic stain may be seen and is accepted. Low expressing structures are ciliated epithelial cells of the fallopian tube and they are expected to show weak to moderate nuclear staining; however, this may vary with different tissue cases and may not be distinguishable in all tissues. For liver tissue, no nuclear staining should be seen in the liver cells. For colon and appendix tissue, no nuclear staining should be seen in the columnar epithelial cells. Colon tissue showed no staining in the neuroendocrine tissues, which express PAX6. This shows that the antibody does not cross react with PAX6 in the qualitative assay we utilized. For tonsil tissue, no staining of B-lymphocytes should be seen, this shows that the antibody does not react with PAX5 or PAX2.

[0109] An optimized IHC assay was tested on 30 normal tissues in triplicate (different cases). The reaction was as expected, but with some staining of pulmonary macrophages, cells in bone marrow and breast, and granular staining in cerebellum. The results are mostly in agreement with what has been reported for other antibodies. The main differences to reference antibody clone SP348 (6) were positivity with new assay in pulmonary macrophages, ovary, and testis. The main differences to reference antibody clone MRQ-50 (7) were positivity with MRQ-50 in tonsil, spleen, salivary gland, parathyroid, and pancreas, while new clone (1G2) was positive in pulmonary macrophages, ovary, and testis. Overall, the main differences are positivity with the new assay in ovary and testis, which are negative with both reference assays. Looking more carefully at the staining in the two tissues, the epithelium staining of ovary looks specific (nuclear) while the testis staining is diffuse cytoplasmic and may be related to poor tissue. The epithelium in ovary is scarce and may not represent normal ovary tissue as such. See the Figures for representative images of various tissues, including ovary (Fig. 15) and testis (Fig. 14) staining with the assay.

[0110] Table 2 Summarizes the number of positives / total tested for each tissue tested.EXAMPLE 3: Specificity[oni] In some embodiments of the antibody, it is desirable that the anti-PAX8 antibody has substantially no cross-reactivity to human PAX2, PAX5, or PAX6. In this Example, the cross reactivity of antibody embodiments was tested to see if they react with other PAX proteins other than PAX8. Negative control tissue was used to illustrate, that the anti-PAX8 antibody has substantially no cross-reactivity to human PAX2, PAX5, or PAX6. Tonsil was stained to illustrate no cross reactivity to PAX5 and PAX2, as no staining was seen in for example squamous epithelial cells and lymphocytes (positive nuclear staining in B-cells indicate cross reaction with PAX5, positive nuclear staining of mantle zone B-cells, the germinal center B-cells and the interfollicular peripheral B-cells indicate cross reactivity with PAX2) (See figure 13). Colon was stained to illustrate no PAX6 cross-reaction as no staining in the neuroendocrine cells was seen in the crypt of colon epithelium (see Figure 12) (8) (Reference: NordiQC PAX8 assessment run 68).

[0112] All patents, publications, scientific articles, web sites, and other documents and materials referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced document and material is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such patents, publications, scientific articles, web sites, electronically available information, and other referenced materials or documents.

[0113] The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. Thus, for example, in each instance herein, in embodiments or examples of the present invention, any of the terms “comprising”, “consisting essentially of’, and “consisting of’ may be replaced with either of the other two terms in the specification. Also, the terms “comprising”, “including”, containing”, etc. are to be read expansively and without limitation. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims. It is also that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the contextclearly dictates otherwise. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.

[0114] The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0115] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0116] Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0117] REFERENCES1. Laury AR, Perets R, Piao H, Krane JF, Barletta JA, French C, et al. A comprehensive analysis of PAX8 expression in human epithelial tumors. Am J Surg Pathol. 2011;35(6):816-26.2. Nonaka D, Chiriboga L, Soslow RA. Expression of pax8 as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas. Am J Surg Pathol.2008;32(10):1566-71.3. Reuter VE, Argani P, Zhou M, Delahunt B, Members of the IliDUPG. Best practices recommendations in the application of immunohistochemistry in the kidney tumors: report from the International Society of Urologic Pathology consensus conference. Am J Surg Pathol. 2014;38(8):e35-49.4. Tacha D, Zhou D, Cheng L. Expression of PAX8 in normal and neoplastic tissues: a comprehensive immunohistochemical study. Appl Immunohistochem Mol Morphol. 2011; 19(4): 293-9.5. Tong GX, Yu WM, Beaubier NT, Weeden EM, Hamele-Bena D, Mansukhani MM, O'Toole KM. Expression of PAX8 in normal and neoplastic renal tissues: an immunohistochemical study. Mod Pathol. 2009;22(9): 1218-27.6. Cell Marque spec, sheet CMC36333000 Rev.O.Ovl.7. Cell Marque spec, sheet CMC46180020 Rev.2.0.8. NordiQC PAX8 assessment Run 68 2023.

[0118] All references described above in the ‘Reference’ section are incorporated by reference herein in their entirety.

Claims

What is claimed is:

1. An antibody or antigen binding fragment thereof capable of specifically binding the human PAX8 protein comprising: i) a heavy chain variable region comprising heavy chain CDR1 comprising the amino acid sequence GIDFSGSYY (SEQ ID NO: 3), heavy chain CDR2 comprising the amino acid IYTYTTGRV (SEQ ID NO: 4), and heavy chain CDR3 comprising the amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5), and / or ii) a light chain variable region comprising light chain CDR1 comprising the amino acid sequence QSISSY (SEQ ID NO: 8), light chain CDR2 comprising any one of the amino acid sequences KAS, YKAS (SEQ ID NO: 6), or KAST (SEQ ID NO: 9), and light chain CDR3 comprising the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10).

2. An antibody of claim 1 comprising both i) a heavy chain variable region comprising heavy chain CDR1 comprising the amino acid sequence GIDFSGSYY (SEQ ID NO: 3), heavy chain CDR2 comprising the amino acid IYTYTTGRV (SEQ ID NO: 4), and heavy chain CDR3 comprising the amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5), and ii) a light chain variable region comprising light chain CDR1 comprising the amino acid sequence QSISSY (SEQ ID NO: 8), light chain CDR2 comprising any one of the amino acid sequences KAS, YKAS (SEQ ID NO:6), or KAST (SEQ ID NO: 9), and light chain CDR3 comprising the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10).

3. An antibody of claim 1 or 2, wherein the heavy chain variable region has an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence: QEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVRQAPGKGLEWIACIYTYTTGR VYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCARNSGPYTTIALSIWGPGTLVT VSS (SEQ ID NO: 2).

4. An antibody of claim 4, wherein the heavy chain variable region has an amino acid sequence with 100% identity to the amino acid sequence: QEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVRQAPGKGLEWIACIYTYTTGR VYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCARNSGPYTTIALSIWGPGTLVT VSS (SEQ ID NO: 2).

5. An antibody of any one of claims 1-4, wherein the light chain variable region has an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence:DVVMTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTEFIFTISDLECADGATYYCQNNYGIASNYGVAFGGGTEVVVK (SEQ ID NO: 7).

6. An antibody of claim 5, wherein the light chain variable region has an amino acid sequence with 100% identity to the amino acid sequence: DVVMTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYKASTLASGVP SRFSGSGSGTEFIFTISDLECADGATYYCQNNYGIASNYGVAFGGGTEVVVK (SEQ ID NO: 7).

7. An antibody or antigen binding fragment thereof capable of specifically binding the human PAX8 protein comprising: i) a heavy chain variable region comprising heavy chain CDR1 comprising the amino acid sequence GIDFSGSYY (SEQ ID NO: 3) or an amino acid sequence at least 50% identical thereto, CDR2 comprising the amino acid IYTYTTGRV (SEQ ID NO: 4) or an amino acid sequence at least 50% identical thereto, and heavy chain CDR3 comprising the amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5) or an amino acid sequence at least 50% identical thereto, and / or ii) a light chain variable region comprising light chain CDR1 comprising the amino acid sequence QSISSY (SEQ ID NO: 8) or an amino acid sequence at least 50% identical thereto, light chain CDR2 comprising any one of the amino acid sequences KAS, YKAS (SEQ ID NO: 6), or KAST (SEQ ID NO: 9) or an amino acid sequence at least 50% identical thereto, and light chain CDR3 comprising the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10) or an amino acid sequence at least 50% identical thereto.

8. An antibody of claim 7 comprising both i) a heavy chain variable region comprising heavy chain CDR1 comprising the amino acid sequence GIDFSGSYY (SEQ ID NO: 3) or an amino acid sequence at least 50% identical thereto, CDR2 comprising the amino acid IYTYTTGRV (SEQ ID NO: 4) or an amino acid sequence at least 50% identical thereto, and heavy chain CDR3 comprising the amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5) or an amino acid sequence at least 50% identical thereto, and ii) a light chain variable region comprising light chain CDR1 comprising the amino acid sequence QSISSY (SEQ ID NO: 8) or an amino acid sequence at least 50% identical thereto, light chain CDR2 comprising any one of the amino acid sequences KAS, YKAS (SEQ ID NO: 6), or KAST (SEQ ID NO: 9) or an amino acid sequence at least 50% identical thereto, and light chain CDR3 comprising the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10) or an amino acid sequence at least 50% identical thereto.

9. An antibody of claim 7 or 8 having a heavy chain variable region comprising heavy chain CDR1 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or95% identity to GIDFSGSYY (SEQ ID NO: 3), CDR2 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to IYTYTTGRV (SEQ ID NO: 4), and heavy chain CDR3 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to ARNSGPYTTIALSI (SEQ ID NO: 5).

10. An antibody of any one of claim 7-9 having a light chain variable region comprising light chain CDR1 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to QSISSY (SEQ ID NO: 8), light chain CDR2 comprising the an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to KAS, YKAS (SEQ ID NO:6), or KAST (SEQ ID NO: 9), and light chain CDR3 comprising an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity to QNNYGIASNYGVA (SEQ ID NO: 10).

11. An antibody of any one of claims 1-10, wherein the antibody or antigen binding fragment thereof is a rabbit, murine, rat, shark, llama, chicken, human, or chimeric.

12. An antibody of claim 11, wherein the antibody or antigen binding fragment thereof is a rabbit antibody.

13. An antibody of any one of claims 1-12, wherein the antibody capable of specifically binding the human PAX8 protein shows substantially no cross-reactivity to human PAX-2, PAX-5, or PAX-6.

14. An antibody of any one of claims 1-13, wherein the antibody is an antigen binding fragment of an antibody is selected from Fab, a Fab', a F(ab')2, a variable fragment (Fv), a triabody, a tetrabody, a minibody, a bispecific F(ab')2, a trispecific F(ab')2, a diabody, a bispecific diabody, a single chain variable fragment (scFv), a scFv-Fc, a Fab-Fc, a VHH, or a bispecific scFv, or a conjugate of one of the preceding further comprising a drug or moiety to facilitate imaging.

15. An isolated DNA molecule or gene encoding an antibody or antigen binding fragment thereof according to any one of claims 1-14.

16. A DNA molecule or gene according to claim 15, wherein the DNA molecule or gene encodes an antibody according to claim 6.

17. An expression vector comprising the DNA molecule of claim 15 or 16 and an expression regulatory sequence operably linked to the DNA sequence.

18. A recombinant host cell, wherein the recombinant host cell comprises the expression vector of claim 17.

19. A method of producing a producing an anti- PAX8 protein antibody or an antigen binding fragment that binds to the human PAX8 protein, the method comprising the steps of:i) culturing a host cell comprising a nucleic acid sequence encoding an antibody of any one of Claims 1-13 under conditions in which the host cell expresses the antibody or antigen binding fragment thereof; and ii) harvesting a preparation of the antibody or antigen binding fragment thereof expressed by the cell.

20. A method according to claim 19 wherein the host cell is transformed with an expression construct comprising a nucleic acid sequence encoding an antibody heavy chain variable region and an expression construct comprising a nucleic acid sequence encoding an antibody light chain variable region, wherein the expression constructs are present in one or more expression vectors.

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