Anti-human pax8 antibodies for in vitro immunohistochemical diagnostic use

Abstract

The present disclosure provides antibodies, including but not limited to monoclonal antibodies, that are capable of specifically binding to 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 expression vectors comprising nucleic acids encoding the anti-PAX8 antibodies described herein, 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

[0001] Cross-reference to related applications This application claims priority and benefit to U.S. Provisional Patent Application Serial No. 63 / 563,209, filed March 8, 2024, entitled ‘Anti-human PAX8 antibody for in vitro immunohistochemical diagnostic purposes’, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] Reference to a sequence list submitted as an ASCII text file The sequence list of electronic submissions via the Patent Centre as ASCII text files is attached hereto and is expressly incorporated herein by reference in its entirety. An XML copy in ST.26 format, created on March 4, 2025, is named AGI_20230170_01_PCT and is 17,000 bytes in size. Technical Field

[0003] The present invention generally relates to antibodies that can be used in immunohistochemistry, and more specifically to novel anti-PAX8 antibodies that can be used in immunohistochemistry-based diagnostics, etc. Background Technology

[0004] The following includes information that may be useful for understanding this invention. Any information provided herein does not constitute an admission that it is prior art or related to the invention described or claimed herein, nor does it constitute an admission that any express or implied references to any publication or document are prior art.

[0005] PAX8 is a nuclear transcriptional regulator in the pair-box family, expressed during and crucial for organogenesis in the thyroid, kidneys, and Müllerian ducts (fallopian tubes, uterus, cervix, and upper vagina). It is located on chromosome 2q13 and has an unprocessed precursor with a molecular weight of 48 kDa, with five isoforms ranging from 31 to 42 kDa (NordiQC(1, 4)). PAX8 is essential for organogenesis in the thyroid, kidneys, and Müllerian duct system (fallopian tubes, uterus, cervix, and upper vagina). Therefore, PAX8 is a sensitive and specific marker for primary and metastatic tumors of kidney, Müllerian duct, and thyroid origin (1, 3, 4). Therefore, PAX8 immunoreactivity has been detected in 91% of thyroid tumors, 90% of renal cell carcinomas, 79% of ovarian cancers (93-99% of serous ovarian cancers), 87-98% of endometrial adenocarcinomas, and 80-83% of cervical adenocarcinomas (1, 4), while tumors of the bladder, lungs, breasts, colon, stomach, prostate, head, and neck are PAX8 negative in most cases (1, 4).

[0006] Therefore, the primary use of PAX8 immunohistochemistry is in the diagnosis of renal cell carcinoma, ovarian tumors, and tumors of unknown origin. Primary renal epithelial tumors originating from the renal parenchyma (such as clear cell renal tumors) are typically PAX8 positive, while urothelial tumors (tumors originating from the bladder, urethra, ureter, and renal pelvis, also known as transitional cell carcinomas) are negative (1, 4, 5). Metastatic renal cell carcinoma may be included in the differential diagnosis of tumors of unknown origin, and PAX8 is the most useful antibody target for confirming metastatic renal cell carcinoma (3). Because PAX8 is expressed in most primary ovarian cancers, it can be used to establish ovarian origin rather than metastasis from other organs to the ovary. PAX8 is particularly useful in differentiating primary serous ovarian cancer from metastatic breast cancer (1, 2, 4).

[0007] A problem with currently available anti-PAX8 antibodies is their cross-reactivity with other cell types and with other PAX protein isotypes besides PAX8. Another issue with current anti-PAX8 antibodies is the high background noise that limits sensitivity. In some embodiments, the antibodies described herein overcome these unresolved challenges and needs. Novel embodiments of anti-PAX8 antibodies that selectively recognize human PAX8 are described in detail below. Summary of the Invention

[0008] The invention described and claimed herein has various attributes and embodiments, including but not limited to those set forth, described, or referenced in the summary. The invention described and claimed herein is not limited to or restricted to the features or embodiments specified in the summary, which is included for illustrative purposes only and not for limitation. As a first aspect of the invention, an antibody or antigen-binding fragment thereof capable of specifically binding to the human PAX8 protein is provided. The PAX8 antigen used herein is designed as a synthetic peptide of the human PAX8 protein. Peptides having 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 amino acid sequence of the human PAX8 protein is provided below for reference.

[0009] MPHNSIRSGHGGLNQLGGAFVNGRPLPEVVRQRIVDLAHQGVRPCDISRQLRVSHGCVSKILGRYYETGSIRPGVIGGSKPKVATPKVVEKIGDYKRQNPTMFAWEIRDRLLA EGVCDNDTVPSVSSINRIIRTKVQQPFNLPMDSCVATKSLSPGHTLIPSSAVTPPESPQSDSLGSTYSINGLLGIAQPGSDKRKMDDSDQDSCRLSIDSQSSSSGPRKHLRTDA FSQHHLEPLECPFERQHYPEAYASPSHTKGEQGLYPLPLLNSTLDDGKATLTPSNTPLGRNLSTHQTYPVVADPHSPFAIKQETPEVSSSSTPSSLSSSAFLDLQQVGSGVP PFNAFPHAASVYGQFTGQALLSGREMVGPTLPGYPPHIPTSGQGSYASSAIAGMVAGSEYSGNAYGHTPYSSYSEAWRFPNSSLLSSPYYYSSTSRPSAPPTTATAFDHL (SEQ ID NO: 1) A first illustrative non-limiting example of an antibody or antigen-binding fragment thereof capable of specifically binding to human PAX8 protein comprises the following: i) a heavy chain variable region comprising a heavy chain CDR1 containing the amino acid sequence GIDFSGSYY (SEQ ID NO: 3), a heavy chain CDR2 containing the amino acid sequence IYTYTTGRV (SEQ ID NO: 4), and a heavy chain CDR3 containing the amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5), and / or ii) a light chain variable region comprising a light chain CDR1 containing the amino acid sequence QSISSY (SEQ ID NO: 8), a light chain CDR2 containing the amino acid sequences KAS, YKAS (SEQ ID NO: 6), or KAST (SEQ ID NO: 9), and a light chain CDR3 containing the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10). In a particular variant of this embodiment, light chain CDR2 comprises the amino acid sequence KAS.

[0010] In some embodiments, such antibodies have both of the following: i) a heavy chain variable region comprising a heavy chain CDR1 containing the amino acid sequence GIDSGSYY (SEQ ID NO: 3), a heavy chain CDR2 containing the amino acid sequence IYTYTTGRV (SEQ ID NO: 4), and a heavy chain CDR3 containing the amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5); and ii) a light chain variable region comprising a light chain CDR1 containing the amino acid sequence QSISSY (SEQ ID NO: 8), a light chain CDR2 containing the amino acid sequences KAS, YKAS (SEQ ID NO: 6), or KAST (SEQ ID NO: 9), and a light chain CDR3 containing the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10). In a particular variant of this embodiment, light chain CDR2 comprises the amino acid sequence KAS.

[0011] In some variations of the first embodiment, the heavy chain variable region has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the following amino acid sequence: QEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVRQAPGKGLEWIACIYTYTTGRVYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCARNSGPYTTIALSIWGPGTLVTVSS (SEQ ID NO: 2).

[0012] In some embodiments, the heavy chain variable region has an amino acid sequence that is 100% identical to the following amino acid sequence: QEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVRQAPGKGLEWIACIYTYTTGRVYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCARNSGPYTTIALSIWGPGTLVTVSS (SEQ ID NO: 2). In some variations of the first embodiment, the light chain variable region has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the following amino acid sequence: DVVMTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTEFIFTISDLECADGATYYCQNNYGIASNYGVAFGGGTEVVVK (SEQ ID NO: 7). In one embodiment, the light chain variable region has an amino acid sequence that is 100% identical to the following amino acid sequence: DVVMTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTEFIFTISDLECADGATYYCQNNYGIASNYGVAFGGGTEVVVK (SEQ ID NO: 7).

[0013] A second non-limiting illustrative embodiment of an antibody or antigen-binding fragment thereof capable of specifically binding to human PAX8 protein has: i) a heavy chain variable region comprising a heavy chain CDR1 containing the amino acid sequence GIDSGSYY (SEQ ID NO: 3) or an amino acid sequence at least 50% identical thereto, a heavy chain CDR2 containing the amino acid sequence IYTYTTGRV (SEQ ID NO: 4) or an amino acid sequence at least 50% identical thereto, and a heavy chain CDR3 containing 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 a light chain CDR1 containing the amino acid sequence QSISSY (SEQ ID NO: 8) or an amino acid sequence at least 50% identical thereto, a light chain CDR2 containing the amino acid sequence KAS, YKAS (SEQ ID NO: 6) or KAST (SEQ ID NO: 9) or an amino acid sequence at least 50% identical thereto, and a heavy chain CDR3 containing the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 8) or an amino acid sequence at least 50% identical thereto, and a heavy chain CDR2 containing the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 9). The light chain CDR3 is an amino acid sequence that is at least 50% identical to ID NO: 10. In a particular variant of this embodiment, the light chain CDR2 contains the amino acid sequence KAS.

[0014] In a variant of the second embodiment, the antibody has both of the following: i) a heavy chain variable region comprising a heavy chain CDR1 containing the amino acid sequence GIDSGSYY (SEQ ID NO: 3) or an amino acid sequence at least 50% identical thereto, a heavy chain CDR2 containing the amino acid sequence IYTYTTGRV (SEQ ID NO: 4) or an amino acid sequence at least 50% identical thereto, and a heavy chain CDR3 containing 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 a light chain CDR1 containing the amino acid sequence QSISSY (SEQ ID NO: 8) or an amino acid sequence at least 50% identical thereto, a light chain CDR2 containing 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 a light chain CDR2 containing the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 8). 10) or a light chain CDR3 having at least 50% identical amino acid sequence. In a particular variant of this embodiment, the light chain CDR2 contains the amino acid sequence KAS.

[0015] Some variations of the second embodiment have heavy chain variable regions comprising heavy chain CDR1 containing an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% identity with GIDSGSYY (SEQ ID NO: 3), heavy chain CDR2 containing an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% identity with IYTYTTGRV (SEQ ID NO: 4), and heavy chain CDR3 containing an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% identity with ARNSGPYTTIALSI (SEQ ID NO: 5). Some variations of the second embodiment have a light chain variable region comprising a light chain CDR1 containing an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% identity with QSISSY (SEQ ID NO: 8), a light chain CDR2 containing an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% identity with KAS, YKAS (SEQ ID NO: 6), or KAST (SEQ ID NO: 9), and a light chain CDR3 containing an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% identity with QNNYGIASNYGVA (SEQ ID NO: 10).

[0016] Typically, the antibodies or antigen-binding fragments described herein are rabbit antibodies, mouse antibodies, rat antibodies, shark antibodies, llama antibodies, chicken antibodies, human antibodies, or chimeric antibodies. For example, a specific antibody embodiment capable of specifically binding to the human PAX8 protein, described in more detail herein, is a rabbit antibody.

[0017] In a preferred embodiment, the antibody described herein is capable of specifically binding to the human PAX8 protein and exhibits substantially no cross-reactivity with other PAX family members, PAX2, PAX5, or PAX6.

[0018] Any antibody described herein contains an antigen-binding fragment typically selected from Fab, Fab', F(ab')2, variable fragment (Fv), trisomic antibody, tetrasomic antibody, mini antibody, nanobody, bispecific F(ab')2, trispecific F(ab')2, bisomic antibody, bispecific bisomic antibody, single-chain variable fragment (scFv), scFv-Fc, Fab-Fc, VHH, or bispecific scFv, or conjugates of the foregoing, wherein the conjugate further contains a drug or a portion for enhancing imaging.

[0019] In another aspect of the invention, a DNA molecule and / or gene encoding the antibody described herein is also provided. For example, in a variant of the embodiment, the DNA molecule or gene encodes an antibody, wherein the light chain variable region has an amino acid sequence that is 100% identical to the following amino acid sequence: DVVMTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTEFIFTISDLECADGATYYCQNNYGIASNYGVAFGGGTEVVVK (SEQ ID NO: 7).

[0020] In another aspect, an expression vector is provided that comprises the DNA molecule described herein and an expression regulatory sequence operatively linked to those DNA sequences.

[0021] In another aspect, an expression vector containing a DNA molecule encoding an antibody as described herein is transfected into a recombinant host cell, which can be used to prepare the anti-PAX8 protein antibody or its antigen-binding fragment as described herein.

[0022] Therefore, in another aspect, and still in another aspect of the invention, a method for generating an anti-PAX8 protein antibody or an antigen-binding fragment thereof that binds to the human PAX8 protein is provided. In an exemplary embodiment, such a method comprises the steps of: i) culturing host cells containing a nucleic acid sequence encoding the antibody under conditions in which the host cells express the antibody or the antigen-binding fragment thereof described herein; and ii) harvesting a preparation of the antibody or the antigen-binding fragment thereof expressed by the cells. In some variations of this embodiment, the host cells are transformed with an expression construct containing a nucleic acid sequence encoding a variable region of the antibody heavy chain and an expression construct containing a nucleic acid sequence encoding a variable region of the antibody light chain, and wherein the expression construct is present in one or more expression vectors.

[0023] In other variations of the method for preparing anti-PAX8 protein antibodies, the heavy chain variable region comprises: i) a heavy chain CDR1 containing the amino acid sequence GIDSGSYY (SEQ ID NO: 3) or an amino acid sequence at least 50% identical thereto; a heavy chain CDR2 containing the amino acid sequence IYTYTTGRV (SEQ ID NO: 4) or an amino acid sequence at least 50% identical thereto; and a heavy chain CDR3 containing 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 a light chain CDR1 containing the amino acid sequence QSISSY (SEQ ID NO: 8) or an amino acid sequence at least 50% identical thereto; a light chain CDR2 containing 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 a light chain CDR2 containing the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 8). 10) or a light chain CDR3 having at least 50% identical amino acid sequence. In a particular variant of this embodiment, the light chain CDR2 contains the amino acid sequence KAS.

[0024] In other variations of the method for preparing anti-PAX8 protein antibodies, the light chain variable region comprises a light chain CDR1 containing the amino acid sequence QSISSY (SEQ ID NO: 8) or an amino acid sequence at least 50% identical thereto; a light chain CDR2 containing 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 a light chain CDR3 containing the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10) or an amino acid sequence at least 50% identical thereto. In a particular variation of this embodiment, light chain CDR2 contains the amino acid sequence KAS.

[0025] In some variations of the method for preparing anti-PAX8 protein antibodies, the heavy chain variable region comprises heavy chain CDR1 containing an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% identity with GIDSGSYY (SEQ ID NO: 3), heavy chain CDR2 containing an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% identity with IYTYTTGRV (SEQ ID NO: 4), and heavy chain CDR3 containing an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% identity with ARNSGPYTTIALSI (SEQ ID NO: 5).

[0026] In some variations of the method for preparing anti-PAX8 protein antibodies, the light chain variable region comprises a light chain CDR1 containing an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% identity with QSISSY (SEQ ID NO: 8), a light chain CDR2 containing an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% identity with KAS, YKAS (SEQ ID NO: 6), or KAST (SEQ ID NO: 9), and a light chain CDR3 containing an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% identity with QNNYGIASNYGVA (SEQ ID NO: 10).

[0027] In some variations of the method for preparing anti-PAX8 protein antibodies, the nucleic acid sequence encoding the variable region of the antibody heavy chain has nucleic acid sequences encoding the heavy chain HCDR1, HCDR2, and HCDR3 regions with the following amino acid sequences: METGLRWLLLVAVLKGVQCQEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVRQAPGKGLEWIACIYTYTTGRVYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCARNSGPYTTIALSIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTF PSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWL RGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 11), and / or the nucleic acid sequence encoding the antibody light chain variable region having nucleic acid sequences encoding the light chain LCDR1, LCDR2, and LCDR3 regions encoding the following amino acid sequences: MDTRAPTQLLGLLLLWLPGARCADVVMTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTEFIFTISDLECADGATYYCQNNYGIASNYGVAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 12), wherein HCDR1 is the amino acid sequence GIDFSGSYY (SEQ ID NO: 3), and HCDR2 is the amino acid sequence IYTYTTGRV (SEQ ID NO: 12). 4), and HCDR3 is the amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5).Furthermore, 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 a particular variant of this embodiment, LCDR2 contains the amino acid sequence KAS.

[0028] In some variations of the method for preparing anti-PAX8 protein antibodies, the nucleic acid sequence encoding the variable region of the antibody heavy chain has both of the following: i) a nucleic acid sequence encoding the heavy chain HCDR1, HCDR2, and HCDR3 regions with the following amino acid sequences: METGLRWLLLVAVLKGVQCQEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVRQAPGKGLEWIACIYTYTTGRVYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCARNSGPYTTIALSIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGV RTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQD WLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 11), and the nucleic acid sequence encoding the antibody light chain variable region described in ii) has nucleic acid sequences encoding the light chain LCDR1, LCDR2, and LCDR3 regions with the following amino acid sequences: MDTRAPTQLLGLLLLWLPGARCADVVMTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTEFIFTISDLECADGATYYCQNNYGIASNYGVAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 12), wherein HCDR1 is the amino acid sequence GIDFSGSYY (SEQ ID NO: 3), and HCDR2 is the amino acid sequence IYTYTTGRV (SEQ ID NO: 12). 4), and HCDR3 is the amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5).Furthermore, 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 a particular variant of this embodiment, LCDR2 contains the amino acid sequence KAS.

[0029] In some variations of the method for preparing anti-PAX8 protein antibodies, the nucleic acid sequence encoding the antibody heavy chain variable region includes a nucleic acid sequence encoding a heavy chain variable region having an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the following amino acid sequence: QEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVRQAPGKGLEWIACIYTYTTGRVYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCARNSGPYTTIALSIWGPGTLVTVSS (SEQ ID NO: 2).

[0030]

[0031] In another aspect of the invention, methods for detecting human PAX8 protein in tissues or organs are provided. These embodiments typically include the steps of: contacting a sample obtained from an individual with an antibody or antigen-binding fragment thereof described herein; and detecting the binding of the selected antibody or antigen-binding fragment thereof to the sample (e.g., a sample from a subject).

[0032] In some variations of the method for detecting human PAX8 protein in tissues or organs, samples are obtained from one or more of the following tissues of the subject: organs of Müllerian origin, such as the fallopian tubes, uterus, cervix, and vagina; or other organs, such as the ovary, kidney, thyroid gland, bladder, lung, colon, breast, stomach, prostate, head, and neck; or any other organ or tissue from which binding or non-binding is detected, indicating the origin of cancer or tumor formation in the tissue from which the sample was obtained. In some variations of the method provided herein, detection of human PAX8 protein is used to detect or diagnose renal cell carcinoma, ovarian tumors, or tumors of unknown origin. In some variations of the method provided herein, detection of human PAX8 protein is used to detect or diagnose ovarian cancer. In some variations of this embodiment, the method is used to establish ovarian origin, rather than metastasis from other organs to the ovary. In some variations of the method provided herein, detection of human PAX8 protein is used to differentiate between renal epithelial tumors and urothelial tumors. In some variations of the method provided herein, detection of human PAX8 protein is used to diagnose metastatic renal cell carcinoma. In some variations of the method presented in this paper, detection of human PAX8 protein was used to differentiate between primary serous ovarian cancer and metastatic breast cancer.

[0033] In another aspect of the invention, a composition comprising the anti-PAX8 antibody described herein or an antigen-binding fragment thereof is provided.

[0034] In another aspect, a kit or system is provided for detecting the presence of human PAX8 protein in a sample. An exemplary kit or system includes an anti-PAX8 antibody or antigen-binding fragment as described herein, and reagents for detecting complexes of anti-PAX8 antibodies bound to human PAX8 protein from a tissue sample. The system provided herein also includes instruments (e.g., Dako Omnis (Agilent)) for automating sample processing to detect complexes of anti-PAX8 antibodies bound to human PAX8 protein from a tissue sample. Attached Figure Description

[0035] Figure 1 Immunohistochemistry of the fallopian tubes using monoclonal rabbit anti-human PAX8 from clone 1G2 is illustrated. Using the same antibody, inserted secretory epithelial cells showed moderate to strong nuclear staining, while ciliated epithelial cells showed weak to moderate nuclear staining.

[0036] Figure 2 Immunohistochemistry was performed on positive clinical tissues of ovarian serous carcinoma using monoclonal rabbit anti-human PAX8 from clone 1G2. Neoplastic cells showed moderate to strong nuclear staining.

[0037] Figure 3 Immunohistochemistry was performed on positive clinical tissues of clear cell renal cell carcinoma using a monoclonal rabbit anti-human PAX8 from clone 1G2. The neoplastic cells showed weak to moderate nuclear staining.

[0038] Figure 4 Immunohistochemistry was performed on positive clinical tissues for thyroid cancer using a monoclonal rabbit anti-human PAX8 from clone 1G2. Tumor cells showed a moderate to strong staining reaction.

[0039] Figure 5 Immunohistochemistry was performed on negative clinical tissues of metastatic invasive ductal carcinoma using monoclonal rabbit anti-human PAX8 from clone 1G2. No obvious staining was observed in tumor cells.

[0040] Figure 6 Immunohistochemistry of transitional cell carcinoma-negative clinical tissues using monoclonal rabbit anti-human PAX8 from clone 1G2 is illustrated. As shown in the figure, tumor cells are unstained, but remnants of normal tubules show some staining.

[0041] Figure 7 Immunohistochemistry of clinical tissues negative for malignant mesothelioma carcinoma using monoclonal rabbit anti-human PAX8 from clone 1G2 is illustrated. As shown in the figure, tumor cells are unstained.

[0042] Figure 8 Immunohistochemistry of negative clinical tissues from colorectal adenocarcinoma was illustrated using monoclonal rabbit anti-human PAX8 from clone 1G2. Tumor cells were not stained.

[0043] Figure 9 Immunohistochemistry of lung adenocarcinoma (a negative clinical tissue) using monoclonal rabbit anti-human PAX8 from clone 1G2 is illustrated. As shown in the figure, the tumor cells are unstained.

[0044] Figure 10 Immunohistochemistry was performed on clinical tissues negative for medullary thyroid carcinoma using monoclonal rabbit anti-human PAX8 from clone 1G2. Thyroid carcinoma is reported to be positive in approximately 50% of cases. Positive nuclear staining of residual portions of normal thyroid tissue is visible.

[0045] Figure 11Immunohistochemistry of normal liver tissue samples using a monoclonal rabbit anti-human PAX8 from clone 1G2 is illustrated. Hepatocytes in normal livers were reported to be negative and could be used as a negative control structure.

[0046] Figure 12 Immunohistochemistry of colon tissue samples using monoclonal rabbit anti-human PAX8 from clone 1G2 is illustrated. Colonic and appendiceal epithelium were reportedly negative and could be used as negative control structures. Unstained neuroendocrine cells in the crypt epithelium indicated no cross-reactivity to PAX6.

[0047] Figure 13 Immunohistochemistry of normal tonsil tissue samples using monoclonal rabbit anti-human PAX8 from clone 1G2 is illustrated. No B lymphocytes were stained in the germinal centers, indicating no cross-reactivity to PAX5 or PAX2.

[0048] Figure 14 Immunohistochemistry of a normal testicular tissue sample using a monoclonal rabbit anti-human PAX8 from clone 1G2 is illustrated. Diffuse nonspecific cytoplasmic staining of epithelial cells was observed, which was unexpected.

[0049] Figure 15 Immunohistochemistry of a normal ovarian tissue sample using a monoclonal rabbit anti-human PAX8 from clone 1G2 is illustrated. Nuclear staining of epithelial cells was observed, which was unexpected. Detailed Implementation

[0050] Various aspects of the invention will now be described with reference to the following sections, which should be understood to be provided by way of illustration only and not to limit the scope of the invention.

[0051] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids, and also to both naturally occurring and non-naturally occurring amino acid polymers. Methods for obtaining (e.g., producing, isolating, purifying, synthesizing, and recombinantly manufacturing) polypeptides are well known to those skilled in the art.

[0052] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to that of naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that have been modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds whose basic chemical structure is the same as that of naturally occurring amino acids (i.e., carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are compounds whose structure differs from the general chemical structure of amino acids but function in a manner similar to that of naturally occurring amino acids.

[0053] Amino acids in this article may be referred to by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides may be referred to by their generally accepted single-letter codes.

[0054] The compositions of this invention encompass amino acid substitutions in proteins and peptides that generally do not alter the activity of the protein or peptide (H. Neurath, RL Hill, The Proteins (Academic Press, New York, 1979). In one embodiment, these substitutions are "conserved" amino acid substitutions. The most common 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.

[0055] For the term "conserved variant" of an amino acid sequence, those skilled in the art will recognize that alterations, additions, or deletions of a single amino acid or a small percentage of amino acids in the coding sequence of a nucleic acid, peptide, polypeptide, or protein are considered "conserved variants," wherein the alteration results in the substitution of the amino acid by a chemically similar amino acid. Conservative representation of functionally similar amino acids is well known in the art. Such conserved variants complement, but do not exclude, the polymorphic variants, interspecific homologs, and alleles of the present invention.

[0056] The following eight groups each contain amino acids that are conserved substitutes for each other: 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)).

[0057] As used herein, an analogue refers to a peptide, polypeptide, or protein sequence that differs from that of a reference peptide, polypeptide, or protein. Such differences may include the addition, deletion, or substitution of amino acids; phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.; the use of non-natural amino acid structures; or other such modifications as known in the art.

[0058] As used in this article, "non-natural amino acids" refers to amino acids other than the 20 typical amino acids found in human proteins. Non-natural amino acids are either naturally occurring or chemically synthesized non-protein amino acids. These may include, but are not limited to, aminoisobutyric acid (Aib), β-amino acids (β... 3 and β 2 ), homoamino acids, proline and pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives, cyclically substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, and N-methyl amino acids.

[0059] An antibody is a polypeptide containing a framework region derived from an immunoglobulin gene or fragment thereof capable of specifically binding to and recognizing antigens. The recognized immunoglobulin genes include constant regions of κ, λ, α, γ, δ, ε, and μ, as well as numerous variable regions. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. Typically, the antigen-binding region of an antibody is the most critical for both the specificity and affinity of the binding.

[0060] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light" chain (approximately 25 kD) and a "heavy" chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of approximately 100 to 110 or more amino acids, primarily responsible for antigen recognition. The term variable light chain (V... L ) and variable heavy chain (V H These refer to the light chains and heavy chains, respectively.

[0061] Antibodies exist, for example, as intact immunoglobulins or as numerous well-defined fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies below the disulfide bonds in the hinge region to produce F(ab)'2, a dimer of Fab, which itself is a light chain linked to VH-CH1 by disulfide bonds. F(ab)'2 can be reduced under mild conditions to break the disulfide bonds in the hinge region, thereby converting the (Fab')2 dimer into Fab' monomers. Fab' monomers are essentially Fab with the hinge region intact (see Fundamental Immunology, Paul, ed., 3rd ed., 1993). Although various antibody fragments are defined in relation to the digestion of intact antibodies, those skilled in the art will understand that such fragments can be synthesized chemically or de novo using recombinant DNA methodologies. Therefore, as used herein, the term antibody also includes antibody fragments generated by modifying an intact antibody, antibody fragments synthesized de novo using recombinant DNA methodologies (e.g., single-stranded Fv), or antibody fragments identified using phage display libraries (see, for example, McCafferty et al.). Nature 348:552-554 (1990), the entire contents of which are incorporated herein by reference.

[0062] Therefore, in any aspect of the invention, the term antibody also encompasses nanobodies, microbodies, bivalent antibodies, tripois antibodies, etc. Bivalent antibodies are small, bivalent, biologically specific antibody fragments with high affinity and specificity. Due to their better specificity and rapid blood clearance, they generally have a better signal-to-noise ratio, thereby increasing their potential for diagnostic and therapeutic targeting of specific antigens (Sundaresan et al., J Nucl Med 44:1962-9 (2003)). Furthermore, these antibodies are advantageous because, if desired, they can be engineered into different types of antibody fragments ranging from small single-chain Fvs to complete IgGs with different isotypes (Wu & Senter, Nat. Biotechnol 23:1137-1146 (2005). In some embodiments, the antibody fragment is part of a bisomatic antibody. In some embodiments, in any aspect, the present invention provides a high-affinity antibody for use according to the present invention.

[0063] The CDR regions provided by this invention can be used to construct anti-PAX8 binding proteins, including but not limited to antibodies, scFv, trisomic antibodies, bisomic antibodies, mini antibodies, nanobodies, etc. In one embodiment, the anti-PAX8 protein of this invention will include 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 with the CDR region described herein. The anti-PAX8 binding protein may include, for example, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, or combinations thereof from the antibodies provided herein. In a particular embodiment of this invention, the anti-PAX8 binding protein may include all three CDR-H sequences of the antibodies provided herein, all three CDR-L sequences of the antibodies provided herein, or both. The anti-PAX8 antibody sequence can be used on an antibody backbone or fragment thereof, and may also include humanized antibodies or antibodies containing humanized sequences. In some implementations, the CDR area can be defined using Kabat definition, Chothia definition, AbM definition, contact definition, or any other suitable CDR numbering system.

[0064] In some embodiments, the present invention provides antibodies (e.g., bisomatic antibodies, mini antibodies, trisomatic antibodies) or fragments thereof having the CDR described herein or variants thereof. Bisomatic antibodies were first described by Hollinger et al. PNAS (USA) As described in 90(14): 6444-6448 (1993), it can be constructed using the heavy and light chains disclosed herein, as well as by using individual CDR regions disclosed herein. Typically, bisomal antibody fragments contain a heavy chain variable domain (VH) linked to a light chain variable domain (VL) via a linker that is too short to allow pairing between the two domains on the same chain. Therefore, the VH and VL domains of one fragment are forced to pair with complementary VH and VL domains of another fragment, thus forming two antigen-binding sites. Trisomal antibodies can also be constructed similarly to have three antigen-binding sites. Fv fragments contain complete antigen-binding sites comprising a VL domain and a VH domain held together by non-covalent interactions. The Fv fragments covered by this invention also include constructs in which the VH and VL domains are crosslinked via glutaraldehyde, intermolecular disulfide bonds, or other linkers. The variable domains of the heavy and light chains can fuse together to form single-chain variable fragments (scFvs), which retain the original specificity of the parent immunoglobulin. The single-chain Fv (scFv) dimer was first proposed by Gruber et al. J. Immunol.As described in 152(12):5368-74 (1994), the heavy and light chains disclosed herein can be used, as well as individual CDR regions disclosed herein, for construction. 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, both of which are incorporated herein by reference in their entirety for all purposes, particularly concerning the design of microantibodies and bisomatic antibodies).

[0065] Bispecific antibodies can be generated through chemical cross-linking or hybridoma technology. Alternatively, bispecific antibody molecules can be produced through recombinant technology. Dimerization can be promoted by reducing the length of the linkers that connect the VH and VL domains from about 15 amino acids, typically used to generate scFv fragments, to about 5 amino acids. These linkers facilitate intrachain assembly of the VH and VL domains. Any suitable short linker can be used. Thus, the two fragments assemble into a dimer molecule. Further shortening the linker length to 0-2 amino acids can generate trimer (trimeric antibody) or tetramer (tetrameric antibody) molecules.

[0066] To prepare antibodies, such as recombinant, monoclonal, or polyclonal antibodies, many techniques known in the art can be used (see, for example, Kohler and Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., Monoclonal Antibodies and Cancer Therapy Alan R. Liss, Inc., pp. 77-96 (1985); Coligan, Current Protocols in Immunology (1991); Harlow and Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2nd edition, 1986, all of which are incorporated herein by reference in their entirety). For example, genes encoding the heavy and light chains of an antibody of interest can be cloned from cells; for instance, genes encoding monoclonal antibodies can be cloned from hybridomas and used to produce recombinant monoclonal antibodies. Libraries of heavy and light chains encoding monoclonal antibodies can also be prepared from hybridoma cells or plasma cells. Random combinations of heavy and light chain gene products generate a large pool of antibodies with different antigen specificities (see, for example, Kuby). Immunology(3rd edition, 1997). Techniques used for generating single-chain or recombinant antibodies (US Patent Nos. 4,946,778, 4,816,567) can be adapted to generate antibodies for the peptides of the present invention. Transgenic mice or other organisms (such as other mammals) can also be used to express humanized or human antibodies (see, for example, US Patent 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 et al., 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 and Huszar, Intern. Rev. Immunol . 13:65-93 (1995)). Alternatively, phage display technology can be used to identify antibodies and heteropolymer Fab fragments that specifically bind to specific 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, that is, capable of recognizing 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, such as two covalently conjugated antibodies, or immunotoxins (see, for example, U.S. Patent Nos. 4,676,980, WO 91 / 00360; and WO 92 / 200373). All references cited in the foregoing paragraphs are incorporated herein by reference in their entirety.

[0067] Methods for humanizing or primate-izing nonhuman antibodies are well known in the art. Typically, humanized antibodies have one or more amino acid residues introduced from a nonhuman source. These nonhuman amino acid residues are often referred to as importing residues, which are typically derived from importing variable domains. Humanization can be performed essentially according to the methods of Winter and colleagues (see, for example, Jones et al.). Nature321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol References 2:593-596 (1992), all of which are incorporated herein by reference in their entirety, are used to replace the corresponding sequences of human antibodies with rodent CDRs or CDR sequences. Accordingly, such humanized antibodies are chimeric antibodies (US Patent No. 4,816,567, the entire disclosure of which is incorporated herein by reference), in which substantially less than the complete human variable domain has been replaced by a 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 replaced by residues from similar sites in rodent antibodies.

[0068] "Chimeric antibody" is an antibody molecule in which (a) a constant region or a portion thereof is altered, replaced or exchanged such that the antigen-binding site (variable region) is linked to a completely different molecule of a different or altered class, effector function and / or species, or which endows the chimeric antibody with new properties, such as the constant region of an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) a variable region or a portion thereof is altered, replaced or exchanged by a variable region with a different or altered antigen specificity.

[0069] When referring to proteins or peptides, the phrase "specifically or selectively binds" to antibodies or "specifically or selectively reacts with" refers to the determination of the presence of a protein, typically a binding reaction present in a heterogeneous population of proteins and other biological agents that are not detected by cross-reactivity. Most assays used herein to determine selectivity are qualitative, where the lack of immunoreactivity cannot be easily seen or detected by the methods used. Various immunoassays described herein or otherwise known in the art can be used to select antibodies that have a specific immunoreactivity with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that are specifically immunoreactive to proteins (for a description of immunoassays and conditions that can be used to determine specific immunoreactivity, see, for example, Harlow and Lane). Using Antibodies, A Laboratory Manual (1998) (The full text of the cited reference is incorporated herein by reference).

[0070] The construction of suitable vectors containing the desired and control sequences employs standard ligation and restriction endonuclease techniques, which are well understood in the art (see Maniatis et al.). Molecular Cloning: A Laboratory Manual(Cold Spring Harbor Laboratory, New York (1982), the full text of which is incorporated herein by reference). Isolated plasmids, DNA sequences, or synthetic oligonucleotides are cut, trimmed, and rejoined into the desired form.

[0071] When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked." For example, if the DNA of a pre-sequence or secretory leader sequence is expressed as a pre-protein involved in polypeptide secretion, then the DNA of the pre-sequence or secretory leader sequence is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the sequence; or if a ribosome binding site is localized to promote translation, then the ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means that the linked DNA sequences are adjacent to each other, and in the case of a secretory leader sequence, are contiguous and within the reading frame. However, enhancers do not necessarily need to be contiguous. Linkage is accomplished by joining at a convenient restriction site. If such a site is not available, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.

[0072] The term "conserved variant" applies to both amino acid and nucleic acid sequences. For a specific nucleic acid sequence, a conserved variant refers to those nucleic acids that encode the same or substantially the same amino acid sequence, or, where the nucleic acid does not encode an amino acid sequence, substantially the same sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each alanine position specified by a codon, the codon can be changed to any of the corresponding codons without altering the encoded polypeptide. This nucleic acid variation is a "silent variant," which is a conserved variant. Each possible silent variant of the nucleic acid is also described in this document for each nucleic acid sequence encoding a polypeptide. Those skilled in the art will recognize that each codon in the nucleic acid (except AUG and TGG, where AUG is typically the only codon for methionine and TGG is typically the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Therefore, each silent variant of the nucleic acid encoding a polypeptide is implicit with respect to the expression product in each described sequence, but not with respect to the actual probe sequence.

[0073] In the context of two or more nucleic acid or polypeptide sequences, the term "identity" or "identity percentage" refers to two or more sequences or subsequences that are identical or have the same specified percentage of amino acid residues or nucleotides (i.e., approximately 60% identity within a specified region when comparing and aligning the largest correspondence within a comparison window or specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher), as measured by manual alignment and visual inspection using a BLAST or BLAST2.0 sequence comparison algorithm with the default parameters described below. Such sequences are referred to as "substantially identical." This definition also refers to, or can be applied to, complement in the test sequence. The definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. As described below, preferred algorithms may take into account vacancies, etc. Preferably, the identity exists in a region of at least about 25 amino acids or nucleotides in length, or more preferably in a region of 50-100 amino acids or nucleotides in length.

[0074] For sequence comparisons, typically one sequence serves as a reference sequence, and the test sequence is compared to the reference sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and subsequence coordinates are specified if necessary, along with the sequence algorithm program parameters. Preferably, default program parameters can be used, or alternative parameters can be specified. The sequence alignment algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the program parameters.

[0075] As used herein, a “comparison window” includes a reference to a segment having any of the following numbers of consecutive positions selected from: 20 consecutive positions to the full length of the reference sequence, typically about 25 to 100, or 50 to about 150 consecutive positions, more typically 100 to 150 consecutive positions, wherein the sequence can be compared with a reference sequence having the same number of consecutive positions after optimal alignment of the two sequences. Sequence alignment methods for comparison are well known in the art. Optimal alignment of sequences for comparison can be, for example, by Smith and Waterman… Adv. Appl. Math The local homology algorithm of 2:482 (1981), through Needleman and Wunsch, J. Mol. Biol The homology alignment algorithm of .48:443 (1970), through Pearson and Lipman, Proc. Nat'l. Acad. Sci. USASimilarity search methods, such as those described in 85:2444 (1988), can be implemented through computerized methods (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Dr., Madison, Wis.), or through manual comparison and visual inspection (see, for example...). Current Protocols in Molecular Biology (Edited by Ausubel et al., 1995 supplement) All references mentioned above are incorporated herein by full citation.

[0076] Preferred examples of algorithms suitable for determining sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, described by Altschul et al., respectively. Nuc. Acids Res 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol.215:403-410 (1990). The percentage of sequence identity of the nucleic acids and proteins of the present invention was determined using BLAST and BLAST 2.0, along with the parameters described herein. The software used to perform the BLAST analysis is publicly available from the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) in the query sequence by recognizing short words of length W that match or satisfy a threshold score T with a positive value when compared to words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., ibid.). These initial neighborhood word hits serve as seeds to initiate a search for longer HSPs containing them. The word hits are expanded in both directions along each sequence until the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for matching residue pairs; always >0) and N (penalty score for non-matching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate cumulative scores. Word hit expansion in each direction stops when: the cumulative alignment score decreases by an amount X from its maximum value; the cumulative score becomes zero or below zero due to the accumulation of one or more negatively scored residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the alignment sensitivity and speed. The BLASTN program (for nucleotide sequences) defaults to a word length (W) of 11, an expected value (E) of 10, M=5, N=-4, and compares two strands. For amino acid sequences, the BLASTP program defaults to a word length of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA The comparison results (B) were 50, the expected value (E) was 10, M=5, and N=-4, and the two chains were compared. All references mentioned in the preceding paragraphs are incorporated herein by reference in their entirety.

[0077] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides, polymers thereof in single-stranded or double-stranded form, and their complements. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, said nucleotides being synthetic, naturally occurring, or non-natural, having binding properties similar to a reference nucleic acid, and being metabolized in a manner similar to that of the reference nucleotide. Examples of such analogs include, but are not limited to, thiophosphates, aminophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acid (PNA).

[0078] Unless otherwise stated, specific nucleic acid sequences implicitly encompass variants of their conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases 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 terms nucleic acid, gene, cDNA, mRNA, oligonucleotide, and polynucleotide are used interchangeably. All references cited in the preceding paragraphs are incorporated herein by reference in their entirety.

[0079] Embodiments of the present invention can provide antibodies described herein in compositional form, such compositions being typically formulated for stability and / or storage and for use in immunohistochemistry. However, other compositions of the antibodies described herein are contemplated, including pharmaceutical formulations.

[0080] Embodiments of the present invention may provide kits and methods of using said kits, including, for example, diagnostic kits comprising antibodies described herein, which can contact antigens in a biological sample such that antigen-antibody interactions are detectable. Biological samples may include, but are not limited to, normal tissue, growth tissue, and any tissue described herein. Detection of antibody-antigen binding can be performed manually, automatically, via image analysis, and even via automated staining equipment. Such kits typically include instructions for use. As discussed herein in the examples, the use of antibodies can be performed on automated staining equipment such as Dako Omnis instruments (Agilent) using methods including, but not limited to, immunoassay, immunohistochemistry (IHC), IHC of FFPE, ICH of frozen tissue sections, and ELISA.

[0081] Additional Implementation Methods (1) A method for producing an anti-PAX8 protein antibody or an antigen-binding fragment thereof that binds to human PAX8 protein, the method comprising the steps of: culturing a host cell containing a nucleic acid sequence encoding the antibody under conditions in which the host cell expresses the antibody or the antigen-binding fragment thereof; and harvesting a preparation of the antibody or the antigen-binding fragment thereof expressed by the cell.

[0082] (2) The method according to Embodiment 1, wherein the host cell is transformed with an expression construct containing a nucleic acid sequence encoding a variable region of an antibody heavy chain and an expression construct containing a nucleic acid sequence encoding a variable region of an antibody light chain, wherein the expression construct is present in one or more expression vectors.

[0083] (3) The method according to Embodiment 2, wherein the heavy chain variable region comprises: i) a heavy chain CDR1 containing the amino acid sequence GIDFSGSYY (SEQ ID NO: 3) or an amino acid sequence that is at least 50% identical thereto, a heavy chain CDR2 containing the amino acid IYTYTTGRV (SEQ ID NO: 4) or an amino acid sequence that is at least 50% identical thereto, and a heavy chain CDR3 containing the amino acid sequence ARNSGPYTTIALSI (SEQ ID NO: 5) or an amino acid sequence that is at least 50% identical thereto; and / or ii) the light chain variable region comprises a light chain CDR1 containing the amino acid sequence QSISSY (SEQ ID NO: 8) or an amino acid sequence that is at least 50% identical thereto, a light chain CDR2 containing any one of the amino acid sequences KAS, YKAS (SEQ ID NO: 6) or KAST (SEQ ID NO: 9) or an amino acid sequence that is at least 50% identical thereto, and a light chain CDR3 containing the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10) or an amino acid sequence that is at least 50% identical thereto.

[0084] (4) According to the method of embodiment 3, the light chain variable region comprises a light chain CDR1 containing the amino acid sequence QSISSY (SEQ ID NO: 8) or an amino acid sequence that is at least 50% identical thereto, a light chain CDR2 containing any one of the amino acid sequences KAS, YKAS (SEQ ID NO: 6) or KAST (SEQ ID NO: 9) or an amino acid sequence that is at least 50% identical thereto, and a light chain CDR3 containing the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10) or an amino acid sequence that is at least 50% identical thereto.

[0085] (5) The method according to embodiment 3 or 4, wherein the heavy chain variable region comprises a heavy chain CDR1 containing an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity with GIDSGSYY (SEQ ID NO: 3), a heavy chain CDR2 containing an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity with IYTYTTGRV (SEQ ID NO: 4), and a heavy chain CDR3 containing an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity with ARNSGPYTTIALSI (SEQ ID NO: 5).

[0086] (6) The method according to any one of embodiments 3, 4 or 5, wherein the method includes a light chain variable region comprising a light chain CDR1 having an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity with QSISSY (SEQ ID NO: 8), a light chain CDR2 having an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity with KAS, YKAS (SEQ ID NO: 6) or KAST (SEQ ID NO: 9), and a light chain CDR3 having an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% identity with QNNYGIASNYGVA (SEQ ID NO: 10).

[0087] (7) The method according to any one of embodiments 3-6, wherein the nucleic acid sequence encoding the antibody heavy chain variable region comprises a nucleic acid sequence encoding the heavy chain HCDR1, HCDR2 and HCDR3 regions encoding the following amino acid sequences: METGLRWLLLVAVLKGVQCQEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVRQAPGKGLEWIACIYTYTTGRVYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCARNSGPYTTIALSIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFP SVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLR GKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQID NO: 11), and / or the nucleic acid sequence encoding the variable region of the antibody light chain includes nucleic acid sequences encoding the light chain LCDR1, LCDR2, and LCDR3 regions encoding the following amino acid sequences: MDTRAPTQLLGLLLLWLPGARCADVVMTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTEFIFTISDLECADGATYYCQNNYGIASNYGVAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 12), wherein HCDR1 consists of the amino acid sequence GIDFSGSYY (SEQ ID NO: 3), and HCDR2 consists of the amino acid sequence IYTYTTGRV (SEQ ID NO: 12). 4) Composition, and HCDR3 is composed of the amino acid sequence ARNSPYTTIALSI (SEQ ID NO: 5).Furthermore, LCDR1 is composed of the amino acid sequence QSISSY (SEQ ID NO: 8), LCDR2 is composed of the amino acid sequences KAS, YKAS (SEQ ID NO: 6), or KAST (SEQ ID NO: 9), and LCDR3 is composed of the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10).

[0088] (8) The method according to any one of embodiments 3-7, wherein the nucleic acid sequence encoding the variable region of the antibody heavy chain comprises both of the following: i) a nucleic acid sequence encoding the heavy chain HCDR1, HCDR2 and HCDR3 regions with the following amino acid sequences: METGLRWLLLVAVLKGVQCQEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVRQAPGKGLEWIACIYTYTTGRVYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCARNSGPYTTIALSIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGV RTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQD WLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 11), and the nucleic acid sequence encoding the variable region of the antibody light chain described in ii) includes nucleic acid sequences encoding the light chain LCDR1, LCDR2, and LCDR3 regions encoding the following amino acid sequences: MDTRAPTQLLGLLLLWLPGARCADVVMTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTEFIFTISDLECADGATYYCQNNYGIASNYGVAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 12), wherein HCDR1 consists of the amino acid sequence GIDFSGSYY (SEQ ID NO: 3), and HCDR2 consists of the amino acid sequence IYTYTTGRV (SEQ ID NO: 12). 4) Composition, and HCDR3 is composed of the amino acid sequence ARNSPYTTIALSI (SEQ ID NO: 5).Furthermore, LCDR1 is composed of the amino acid sequence QSISSY (SEQ ID NO: 8), LCDR2 is composed of the amino acid sequences KAS, YKAS (SEQ ID NO: 6), or KAST (SEQ ID NO: 9), and LCDR3 is composed of the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 10).

[0089] (9) The 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 the heavy chain, wherein the heavy chain variable region has an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the following amino acid sequence: QEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVRQAPGKGLEWIACIYTYTTGRVYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCARNSGPYTTIALSIWGPGTLVTVSS (SEQ ID NO: 2).

[0090]

[0091] (11) A method for detecting human PAX8 protein in a tissue or organ, the method comprising contacting a sample obtained from an individual with an antibody or an antigen-binding fragment thereof provided therein, and detecting the binding of the antibody or the antigen-binding fragment thereof to the sample.

[0092] (12) The method according to embodiment 11, wherein the sample is selected from the group consisting of: organs from which Müllerian ducts are derived, such as fallopian tubes, uterus, cervix and vagina; or other organs, such as ovary, kidney, thyroid, bladder, lung, colon, breast, stomach, prostate, head and neck; or other organs from which binding or non-binding is detected, indicating the origin of cancer or tumor formation in the tissue from which the sample was obtained.

[0093] (13) The method according to embodiment 11 or 12, wherein the detection of the human PAX8 protein is used to detect or diagnose renal cell carcinoma, ovarian tumor or tumor of unknown origin.

[0094] (14) The method according to embodiment 11 or 12, wherein the detection of the human PAX8 protein is used to detect or diagnose ovarian cancer.

[0095] (15) The method according to embodiment 14, wherein the method is used to establish the origin of the ovary, rather than the transfer from other organs to the ovary.

[0096] (16) The method according to embodiment 11 or 12, wherein the detection of the human PAX8 protein is used to distinguish between renal epithelial tumors and urothelial tumors.

[0097] (17) The method according to embodiment 11 or 12, wherein the detection of the human PAX8 protein is used to diagnose metastatic renal cell carcinoma.

[0098] (18) The method according to embodiment 11 or 12, wherein the detection of the human PAX8 protein is used to differentiate between primary serous ovarian cancer and metastatic breast cancer.

[0099] (19) A composition comprising the anti-PAX8 antibody or an antigen-binding fragment thereof provided herein.

[0100] (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 an antigen-binding fragment thereof according to any one of claims 1-14; ii) a reagent for detecting a complex of the anti-PAX8 antibody bound to the human PAX8 protein from a tissue sample; and optionally iii) an instrument for automatically processing the sample to detect the complex of the anti-PAX8 antibody bound to the human PAX8 protein from a tissue sample.

[0101] The following examples are included for illustrative purposes and not for limitation.

[0102] Example 1: Antibody Methods and Selection This embodiment describes the preparation of specific monoclonal antibodies. A synthetic peptide representing a portion of the human PAX8 protein was KLH conjugated and used for rabbit immunization. Animal blood was subsequently collected for IHC confirmation of serum reactivity to human PAX8 protein in several clinically relevant positive and negative human tissues. Several B-cell selections were performed using blood from rabbits exhibiting optimal immune responses to the human PAX8 antigen. B cells expressing antigen-specific antibodies were isolated into monoclonal antibodies and screened by immunoassays. Human PAX8 binding of the recombinant antibodies was tested on a BLItz instrument using biolayer interferometry (BLI), and subsequently on normal and clinical tissues using the standard FLEX protocol in IHC. Some antibodies were identified as exhibiting human PAX8-specific staining in IHC. Clone 1G2, derived from rabbits immunized with a synthetic peptide antigen representing human PAX8, showed very promising results on the tested tissues. The antibodies have been specifically tested on various clinical tissues and have shown usability for in vitro diagnostics via immunohistochemistry.

[0103] Cloning selection Rabbit blood was evaluated using IHC, and candidate rabbits expressing PAX8-specific antibodies were selected for B cell selection. B cell selection was performed using blood samples from relevant rabbits. The most promising B cells were selected for molecular cloning and sequencing of the variable heavy and light chain antibody (Ab) chains. Antibody stability was tested and validated using IHC assays.

[0104] Example 2: Immunohistochemistry PAX8 is a nuclear transcriptional regulator in the pairbox family, expressed during and crucial for organogenesis in the thyroid, kidneys, and Müllerian ducts (fallopian tubes, uterus, cervix, and upper vagina). It is located on chromosome 2q13 and has an unprocessed precursor with a molecular weight of 48 kDa, with five isoforms ranging from 31 to 42 kDa (NordiQC(1,4)). PAX8 is known to be expressed in fallopian tube epithelium (both ciliated non-secreting and intercalated secretory epithelium), renal epithelium (proximal and distal tubules, loops of Henle, collecting ducts, and parietal epithelium of Bowman's bursa), and thyroid epithelial cells.

[0105] The 1G2 antibody described in Example 1 was used for immunohistochemistry of various tissues in this example (including those previously known clinically as negative or positive controls) and to verify that the antibody implementation described herein is specific to the human PAX8 protein. Results are shown in Table 1. Various tissue types were tested using the anti-PAX8 antibody, and results were compared to expected results for those tissue types. The ready-to-use FLEX monoclonal rabbit anti-human PAX8 clone DAK-PAX8 (Dako Omnis, Agilent) was used for IHC. This was intended for use with the Dako Omnis instrument (Agilent) for qualitative immunohistochemical (IHC) detection of formalin-fixed, paraffin-embedded tissue sections.

[0106] Table 1—Comparison of expected response patterns with observed response patterns in various tissues High-expression structures are the secretory epithelial cells of the intercalated tubules of the fallopian tubes, and they are expected to show moderate to strong nuclear staining; however, weaker cytoplasmic staining may also be seen, and this is acceptable. Low-expression structures are the ciliated epithelial cells of the fallopian tubes, and they are expected to show weak to moderate nuclear staining; however, this may vary depending on the histological condition and may not be distinguishable in all tissues. In liver tissue, nuclear staining should not be seen in hepatocytes. In colon and appendix tissue, nuclear staining should not be seen in columnar epithelial cells. Colon tissue shows no staining in neuroendocrine tissue expressing PAX6. This indicates that the antibody does not cross-react with PAX6 in the qualitative assay we used. In tonsillar tissue, no B lymphocyte staining should be seen, indicating that the antibody does not react with PAX5 or PAX2.

[0107] Optimized IHC assays were performed in triplicate on 30 normal tissues (different cases). The reactions were as expected, but cells in lung macrophages, bone marrow, and breast were stained, and granular staining was observed in the cerebellum. The results were largely consistent with those reported for other antibodies. The main difference from the reference antibody clone SP348 (6) was that the new assay was positive in lung macrophages, ovaries, and testes. The main difference from the reference antibody clone MRQ-50 (7) was that MRQ-50 was positive in tonsils, spleen, salivary glands, parathyroid glands, and pancreas, while the new clone (1G2) was positive in lung macrophages, ovaries, and testes. Overall, the main difference was that the new assay was positive in ovaries and testes, while both reference assays were negative in ovaries and testes. Upon closer examination of the staining in these two tissues, the epithelial staining in the ovary appeared to be specific (nuclear staining), while the staining in the testes was diffuse cytoplasmic and may be associated with tissue dysplasia. Epithelial cells are sparse in the ovary and therefore may not be representative of normal ovarian tissue. See the accompanying illustrations of representative images of various tissues, including the ovary as described in the assay. Figure 15 ) and testes ( Figure 14 )dyeing.

[0108] Table 2 summarizes the number of positive results for each type of tissue tested / total number of tests.

[0109] Example 3: Specificity In some implementations of the antibody, it is desirable that the anti-PAX8 antibody exhibits substantially no cross-reactivity with human PAX2, PAX5, or PAX6. In this embodiment, the cross-reactivity of the antibody implementations was tested to see if they reacted with other PAX proteins besides PAX8. Negative control tissues were used to illustrate that the anti-PAX8 antibody exhibited substantially no cross-reactivity with human PAX2, PAX5, or PAX6. Tonsils were stained to show no cross-reactivity with PAX5 and PAX2, as evidenced by, for example, no staining observed in squamous epithelial cells and lymphocytes (positive B cell nuclear staining indicates cross-reactivity with PAX5, while positive nuclear staining of mantle B cells, germinal center B cells, and interfollicular peripheral B cells indicates cross-reactivity with PAX2) (see [link to relevant documentation]). Figure 13 The colon was stained to indicate the absence of PAX6 cross-reactivity because no neuroendocrine cells were observed staining in the crypts of the colonic epithelium (see [link to original text]). Figure 12 (8) (Reference: NordiQC PAX8 assessment run 68).

[0110] All patents, publications, scientific articles, websites, and other documents and materials mentioned or cited herein demonstrate the skill level of a person skilled in the art to which this invention pertains, and each such cited document and material is incorporated herein by reference to the extent that it is incorporated in its entirety herein or set forth herein. The applicant reserves the right to incorporate any and all materials and information from any such patents, publications, scientific articles, websites, electronically available information, and other reference materials or documents into this specification.

[0111] The specific methods and compositions described herein represent preferred embodiments and are illustrative, and are not intended to limit the scope of the invention. Other objects, aspects, and embodiments will arise in those skilled in the art upon consideration of this specification, and said objects, aspects, and embodiments are covered within the spirit of the invention as defined by the scope of the claims. It will be apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention described herein can be suitably practiced without the presence of any one or more elements or one or more limitations not specifically disclosed herein as necessary. Thus, for example, in each instance herein, in embodiments or examples of the invention, any of the terms “comprising,” “substantially consisting of,” and “consisting of” can be replaced by any of the other two terms in the specification. Also, the terms “comprising,” “including,” “containing,” etc., should be read broadly and without limitation. The methods and processes described herein can be suitably practiced in different sequences of steps, and they are not necessarily limited to the sequence of steps indicated herein or in the claims. It should also be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “described” include multiple indicators unless the context clearly indicates otherwise. In no event shall this patent be construed as limited to the specific examples, embodiments, or methods specifically disclosed herein. In no event shall this patent be construed as being limited by any statement made by any examiner or any other officer or employee of the Patent and Trademark Office, unless the applicant expressly and unconditionally accepts such statement in their written response.

[0112] The terms and expressions used are descriptive rather than restrictive, and their use is not intended to exclude any equivalents of the features shown and described or portions thereof. However, it should be recognized that various modifications are possible within the scope of the claimed invention. Therefore, it should be understood that although the invention has been specifically disclosed by way of preferred embodiments and optional features, modifications and variations of the concepts disclosed herein can be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

[0113] This invention has been described extensively and generally. Each narrower category and subgroup falling into the higher disclosure also forms part of this invention. This includes general descriptions of the invention that remove any subject matter from said category using some kind of restrictive or negative limitation, regardless of whether the excluded content specifically falls into that general description. Other embodiments are within the scope of the following claims. Furthermore, where features or aspects of the invention are described in the Markush group, those skilled in the art will recognize that the invention is also described in the form of any individual member or subgroup of the Markush group.

[0114] References 1. Laury AR, Perets R, Piao H, Krane JF, Barletta JA, French C, etal. A comprehensive analysis of PAX8 expression in human epithelial tumors. AmJ Surg Pathol. 2011;35(6):816-26.

[0115] 2. Nonaka D, Chiriboga L, Soslow RA. Expression of pax8 as a usefulmarker in distinguishing ovarian carcinomas from mammary carcinomas. Am J SurgPathol. 2008;32(10):1566-71.

[0116] 3. Reuter VE, Argani P, Zhou M, Delahunt B, Members of the IIiDUPG. Best practices recommendations in the application of immunohistochemistry in the kidney tumors: report from the International Society of Urologic Pathology conference. Am J Surg Pathol. 2014;38(8):e35-49.

[0117] 4. Tacha D, Zhou D, Cheng L. Expression of PAX8 in normal and neoplastic tissues: a comprehensive immunohistochemical study.ApplImmunohistochem Mol Morphol.2011;19(4):293-9.

[0118] 5. Tong GX, Yu WM, Beaubier NT, Weeden EM, Hamele-Bena D, MansukhaniMM, O'Toole KM. Expression of PAX8 in normal and neoplastic renal tissues: animmunohistochemical study. Mod Pathol. 2009;22(9):1218-27.

[0119] 6. Cell Marque spec. sheet CMC36333000 Rev.0.0v1.

[0120] 7. Cell Marque spec. sheet CMC46180020 Rev.2.0.

[0121] 8. NordiQC PAX8 assessment Run 68 2023.

[0122] All references mentioned in the 'References' section above are incorporated in full into this paper by way of citation.

Claims

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

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

3. The antibody according to claim 1 or 2, wherein the heavy chain variable region has an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the following amino acid sequence: QEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVRQAPGKGLEWIACIYTYTTGRVYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCARNSGPYTTIALSIWGPGTLVTVSS| (SEQ ID NO: 2).

4. The antibody according to claim 4, wherein the heavy chain variable region has an amino acid sequence that is 100% identical to the following amino acid sequence: QEQLVESGGGLVQPEGSLTLTCKASGIDFSGSYYGCWVRQAPGKGLEWIACIYTYTTGRVYYANWAKGRFTISKISSATVTLQMTSLTAADTATYFCARNSGPYTTIALSIWGPGTLVTVSS (SEQ ID NO:2).

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

6. The antibody according to claim 5, wherein the light chain variable region has an amino acid sequence that is 100% identical to the following amino acid sequence: DVVMTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTEFIFTISDLECADGATYYCQNNYGIASNYGVAFGGGTEVVVK (SEQ ID NO: 7).

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

8. The antibody according to claim 7, wherein the antibody comprises: i) a heavy chain variable region comprising a heavy chain CDR1 containing the amino acid sequence GIDSGSYY (SEQ ID NO: 3) or an amino acid sequence at least 50% identical thereto, a heavy chain CDR2 containing the amino acid sequence IYTYTTGRV (SEQ ID NO: 4) or an amino acid sequence at least 50% identical thereto, and a heavy chain CDR3 containing 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 a light chain CDR1 containing the amino acid sequence QSISSY (SEQ ID NO: 8) or an amino acid sequence at least 50% identical thereto, a light chain CDR2 containing the amino acid sequence KAS, YKAS (SEQ ID NO: 6) or KAST (SEQ ID NO: 9) or an amino acid sequence at least 50% identical thereto, and a light chain CDR2 containing the amino acid sequence QNNYGIASNYGVA (SEQ ID NO: 8). 10) or a light chain CDR3 that has at least 50% the same amino acid sequence as it.

9. The antibody according to claim 7 or 8, wherein the antibody has a heavy chain variable region comprising a heavy chain CDR1 containing an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% identity with GIDSGSYY (SEQ ID NO: 3), a heavy chain CDR2 containing an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% identity with IYTYTTGRV (SEQ ID NO: 4), and a heavy chain CDR3 containing an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% identity with ARNSGPYTTIALSI (SEQ ID NO: 5).

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

11. The antibody according to any one of claims 1 to 10, wherein the antibody or its antigen-binding fragment is rabbit, mouse, rat, shark, llama, chicken, human, or chimeric.

12. The antibody according to claim 11, wherein the antibody or its antigen-binding fragment is a rabbit antibody.

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

14. The antibody according to any one of claims 1-13, wherein the antibody is an antigen-binding fragment selected from the group consisting of: Fab, Fab', F(ab')2, variable fragment (Fv), trisomic antibody, tetrasomic antibody, mini antibody, bispecific F(ab')2, trispecific F(ab')2, bisomic antibody, bispecific bisomic antibody, single-chain variable fragment (scFv), scFv-Fc, Fab-Fc, VHH, or bispecific scFv, or a conjugate thereof, said conjugate further comprising a drug or a portion for enhancing imaging.

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

16. The DNA molecule or gene of claim 15, wherein the DNA molecule or gene encodes the antibody of claim 6.

17. An expression vector comprising a DNA molecule according to claim 15 or 16 and an expression regulatory sequence operatively linked to the DNA sequence.

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

19. A method for producing an anti-PAX8 protein antibody or an antigen-binding fragment thereof that binds to human PAX8 protein, the method comprising the following steps: i) Culture host cells containing a nucleic acid sequence encoding the antibody under conditions in which the host cells express the antibody or an antigen-binding fragment thereof according to any one of claims 1-13; ii) Harvest preparations of the antibody or its antigen-binding fragment expressed by the cells.

20. The method of 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 construct is present in one or more expression vectors.

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