Methods of treating or preventing cancer by targeting the extracellular portion of keratin 14 (KRT14) residing on cancer cells

Abstract

Disclosed herein are methods, uses, and compositions for treating or preventing cancer in a mammalian subject, comprising administering to the subject an amount of an agent that targets an extracellular portion of KRT14 or a functional homolog thereof or a variant thereof that resides on a cancer cell, or an agent that induces production of an antagonist of an extracellular portion of KRT14 or a functional homolog or variant thereof on a cancer cell. The disclosure also extends to methods of monitoring and / or diagnosing cancer in a subject.

Description

[0001] TECHNICAL FIELD

[0002] The present invention relates generally to cancer therapy, including the treatment, prevention or delay of the development or metastasis of cancer, and to medicaments useful therefor.

[0003] BACKGROUND

[0004] The bibliographic details of the publications referred to in this specification have been collected at the end of the specification.

[0005] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0006] Cancer remains one of the most important diseases affecting humans and animals, with high morbidity and mortality. For example, ovarian cancer is the ninth most common cancer diagnosed in women. In fact, ovarian cancer is the most lethal of all gynecological cancers. Considerable resources have been spent on early diagnosis and treatment of ovarian cancer. Despite improvements in surgical and chemotherapeutic intervention, ovarian cancer survival rates have remained stable at about 25% (Vaughan et al. (2011) Nat Rev Cancer 11(10): 719-725).

[0007] More than 75% of ovarian cancer patients are diagnosed with advanced metastatic disease at the first clinical presentation. Treatment is largely limited to aggressive surgical and chemotherapy. However, more than 90% of patients relapse, often within the first year after treatment. In the vast majority of these patients, the recurrent tumours exhibit chemoresistance. This phenomenon limits further treatment options and underlies the very high mortality rate of ovarian cancer.

[0008] Despite the enormous efforts, attempts at genetic screening to identify potential therapeutic targets have been largely unsuccessful. This can be due to the highly heterogeneous nature of ovarian cancer tissue.

[0009] Keratin-14 (KRT14) is an intracellular protein component of the cytoskeleton, normally expressed in the primitive lineage of progenitor cells residing in the myoepithelial cells and epithelial niches of healthy adult tissues (Chu et al. (2001) Histopathology 39(1):9-16; Paraskevopoulou et al. (2016) Cell Cycle 15(23):3161-3162). In tumor tissues, KRT14 marks a population of specialized cells (alternatively described as “leader cells,” “tumor stem cells,” or “tumor initiating cells”) that control the invasion of tumor deposits into healthy tissues. The control of tumor invasion in a range of solid tumor types, including breast, bladder, and lung solid tumors, mechanistically involves cells expressing KRT14. In these tumors, the presence of cells expressing KRT14 was directly associated with reduced tumor invasive potential as well as reduced disease-free survival and overall survival (Chu et al. (2001) above; Cheah et al. (2015) Proc Natl Acad Sci USA 112(15):4725-4730; Volkmer et al. (2012) Proc Natl Acad Sci USA 109(6):2078-2083; Ho et al. (2012) Nat Rev Urol 9(10):583-594; Cheung et al. (2016) Proc Natl Acad Sci USA 113(7):E854-863; Cheung et al. (2013) Cell 155(7):1639-1651; Papafotiou et al. (2016) Nat Commun 7:11914).

[0010] There is an urgent need for treatments that can improve patient outcomes and quality of life.

[0011] Overview

[0012] According to the present invention, KRT14 has been determined to be essential for the in vitro invasion of ovarian cancer cells through the mesocortex and for successful implantation into ovarian tumors in vivo. KRT14 has also been determined to play a role in the migration and invasion of other cancer cell types, including colorectal cancer cells, endometrial cancer cells, brain cancer cells, breast cancer cells, and lung cancer cells. KRT14 is not expressed in healthy tissues, including in normal reproductive systems such as the ovaries and fallopian tubes. Importantly, it has been determined that KRT14 has an extracellular portion present in a range of cancers in both male and female subjects. The term “extracellular” should be understood to mean a portion, segment, or domain of KRT14 located outside the cell, exposed to the outside of the cell, or otherwise accessible from the outside of the cell.

[0013] Therefore, this paper teaches a method for treating or preventing cancer in mammalian subjects (including human and animal subjects), comprising administering to the subject an amount of an agent that targets the extracellular portion of KRT14 residing on cancer cells or a functional homolog or variant thereof, or an agent that induces in vivo production of an antagonist of the extracellular portion of KRT14 on cancer cells, said amount effectively preventing cancer cell invasion, migration, and / or metastasis. "Administering" to the subject includes contact with cancer cells. The "subject" can be male or female.

[0014] In the implementation plan, the cancer is a gynecological cancer, including but not limited to ovarian cancer or its form or stage. In the implementation plan, the cancer is endometrial cancer or colorectal cancer. In the implementation plan, the cancer is selected from brain cancer, bladder cancer, liver cancer, breast cancer, lung cancer, pancreatic cancer, intestinal cancer, colon cancer, gastrointestinal cancer, stomach cancer, laryngeal cancer, endometrial cancer, and colorectal cancer.

[0015] In the embodiments, the agent is an antibody that targets an epitope contained in a protein comprising the peptide sequence (coded in single letters) GFGGGYGGGLGAGLGGGFGGGFAGGDGL (SEQ ID NO:1), or a functional homolog thereof, or a variant having at least 80% similarity to SEQ ID NO:1 after optimal alignment. Other agents that act as antagonists or targets of SEQ ID NO:1 are also considered herein. SEQ ID NO:1 represents a human sequence. Homologs from other species are also considered herein as therapeutic and diagnostic targets. “Antibody” includes monoclonal antibodies, polyclonal antibodies, and antiserum binding to KRT14, as well as recombinant forms, fragments, and derivatives of exogenous portions or portions thereof that bind to KRT14.

[0016] In the embodiments disclosed herein, the agent comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH comprises: a complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence of SEQ ID NO:6, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:7, and a VHCDR3 comprising the amino acid sequence of SEQ ID NO:8; and wherein the VL comprises: a complementarity-determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO:9, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a VLCDR3 comprising the amino acid sequence of SEQ ID NO:11.

[0017] In the implementation scheme, VH includes:

[0018] (a) VH frame region 1 (FR1), wherein the VH frame region 1 (FR1) contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO:12;

[0019] (b) VH FR2, wherein the VH FR2 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:13;

[0020] (c) VH FR3, wherein VH FR3 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:14; and

[0021] (d) VH FR4, wherein the VH FR4 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:15;

[0022] And VL includes:

[0023] (e) VL FR1, wherein VL FR1 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:16;

[0024] (f) VL FR2, wherein VL FR2 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:17;

[0025] (g) VL FR3, wherein the VL FR3 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:18; and

[0026] (h)VL FR4, wherein the VL FR4 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:19.

[0027] In the implementation, VH contains an amino acid sequence that has at least 80% sequence identity with SEQ ID NO:3, and VL contains an amino acid sequence that has at least 80% sequence identity with SEQ ID NO:5.

[0028] In the embodiments, the mammalian subjects are human females or human males. However, the invention extends to veterinary applications in non-human male or female mammals.

[0029] As taught in this paper, a novel function of KRT14 as a key early regulator of ovarian cancer invasion and deposition was identified. Cells lacking a functional copy of the KRT14 gene are invasion-incompetent and cannot establish ovarian tumors in vivo. Targeting the epitope identified by SEQ ID NO:1 with an exogenously added agent completely eliminated the invasive ability of cancer cells in vitro, mimicking the effect of functional KRT14 loss. Similarly, inducing in vivo responses, such as a specific immune response against cells carrying SEQ ID NO:1, also effectively reduced cancer development.

[0030] In the implementation scheme, this specification teaches a method for treating ovarian cancer in a human subject, the method comprising administering to the subject an amount of an antibody targeting the extracellular portion of KRT14, defined by SEQ ID NO:1, residing on ovarian cancer cells, said amount being effective in preventing or reducing invasion, migration, and / or metastasis of ovarian cancer cells.

[0031] Many other solid tumor types (e.g., breast, bladder, lung, and others) have been shown to utilize this KRT14-mediated invasion mechanism, demonstrating that anti-KRT14 targeted therapy is broadly applicable across a range of solid tumor types.

[0032] This article describes a drug that targets SEQ ID NO:1 or its functional homologs or variants to eliminate cancer cell invasion, migration, and / or metastasis. In embodiments, the drug comprises an antibody specific to SEQ ID NO:1 or its functional homologs or variants. As indicated above, the antibody may be a polyclonal or monoclonal antibody or an antiserum containing a KRT14-binding antibody, or it may be a synthetic (e.g., recombinant) antibody or a KRT14-binding fragment or derivative of any of the foregoing. The antibody may also be an antibody derived from chondrocytes or its KRT14-binding fragment or derivative. In addition to being an antibody, the drug may be any affinity agent, including but not limited to aptamers, monobodies, anticalins, DARPin, and nanobodies.

[0033] This invention extends to combination therapies in which an agent targeting KRT14 or an agent inducing KRT14 antagonism in vivo is administered together with another anticancer agent and / or radiation therapy and / or surgical intervention. Examples of additional agents include chemotherapeutic agents such as one or more of the following: dactinomycin, daunorubicin, doxorubicin (doxorubicin), idarubicin, and mitoxantrone, or platinum-based agents or antimetabolites. Antimetabolites are substances that interfere with the body's chemical processes, such as the production of proteins, DNA, and other chemicals required for cell growth and reproduction; in cancer treatment, antimetabolites disrupt DNA production, which in turn inhibits cell division. Examples include azaserine, D-cyclic serine, mycophenolic acid, trimethoprim, 5-fluorouracil, capecitabine, methotrexate, gemcitabine, cytarabine (ara-C), and fludarabine. Other immunomodulatory agents, such as sensitized T cells and cytokines, may be administered. Combination therapies can be provided simultaneously, or in any order and sequentially over seconds, minutes, hours, days, or weeks.

[0034] This disclosure also extends to agents or KRT14-binding fragments thereof that specifically bind to the extracellular portion of KRT14 on cancer cells, wherein the agent comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH comprises: a complementarity-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO:6, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:7, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:8; and wherein the VL comprises: a complementarity-determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO:9, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:11.

[0035] In the implementation scheme, VH includes:

[0036] (a) VH frame region 1 (FR1), wherein the VH frame region 1 (FR1) contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO:12;

[0037] (b) VH FR2, wherein the VH FR2 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:13;

[0038] (c) VH FR3, wherein VH FR3 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:14; and

[0039] (d) VH FR4, wherein the VH FR4 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:15;

[0040] And VL includes:

[0041] (e) VL FR1, wherein VL FR1 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:16;

[0042] (f) VL FR2, wherein VL FR2 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:17;

[0043] (g) VL FR3, wherein the VL FR3 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:18; and

[0044] (h)VL FR4, wherein the VL FR4 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:19.

[0045] In the implementation, VH contains an amino acid sequence that has at least 80% sequence identity with SEQ ID NO:3, and VL contains an amino acid sequence that has at least 80% sequence identity with SEQ ID NO:5.

[0046] The amino acid sequence is represented by a sequence identifier (SEQ ID NO). The SEQ ID NO numerically corresponds to the sequence identifier. <400> 1(SEQ ID NO:1), <400> 2 (SEQ ID NO:2), etc. A sequence listing is provided after the claims.

[0047] Table 1 provides a summary of the sequence identifiers used throughout the subject description.

[0048] Table 1

[0049] Summary of sequence identifiers

[0050] SEQ ID NO: Description 1 Amino acid sequence of the extracellular portion of human KRT14 comprising the epitope 2 Nucleic acid sequence encoding the heavy chain variable region of monoclonal antibody (mAb) AN-17 3 Amino acid sequence of the heavy chain variable region of monoclonal antibody AN-17 4 Nucleic acid sequence encoding the light chain variable region of monoclonal antibody AN-17 5 Amino acid sequence of the light chain variable region of monoclonal antibody AN-17 6 Amino acid sequence of the heavy chain CDR1 of monoclonal antibody AN-17 7 Amino acid sequence of the heavy chain CDR2 of monoclonal antibody AN-17 8 Amino acid sequence of the heavy chain CDR3 of monoclonal antibody AN-17 9 Amino acid sequence of the light chain CDR1 of monoclonal antibody AN-17 10 Amino acid sequence of the light chain CDR2 of monoclonal antibody AN-17 11 Amino acid sequence of the light chain CDR3 of monoclonal antibody AN-17 12 Amino acid sequence of the heavy chain FR1 of monoclonal antibody AN-17 13 Amino acid sequence of the heavy chain FR2 of monoclonal antibody AN-17 14 Amino acid sequence of the heavy chain FR3 of monoclonal antibody AN-17 15 Amino acid sequence of the heavy chain FR4 of monoclonal antibody AN-17 16 Amino acid sequence of the light chain FR1 of monoclonal antibody AN-17 17 Amino acid sequence of the light chain FR2 of monoclonal antibody AN-17 18 Amino acid sequence of the light chain FR3 of monoclonal antibody AN-17 19 Amino acid sequence of the light chain FR4 of monoclonal antibody AN-17

[0051] Amino acids can be represented by their names or by single-letter or three-letter codes (Table 2).

[0052] Table 2

[0053] amino acid tri-letter and single-letter

[0054] Brief description of the attached diagram

[0056] Some of the accompanying drawings include color representations or figures. Color photographs may be obtained upon request from the patentee or from the appropriate patent office. If obtained from the patent office, a fee may be charged.

[0057] Figure 1 Photographs A through C show the identification of KRT14 at the leading edge of invasive ovarian cancer deposits. (A) Ovarian cancer spheroids were cultured using a peritoneal microenvironment model, and invasion across the mesothelial barrier was monitored. (B) Frozen sections containing spheroids actively breaching a monolayer of LP9 mesothelial cells were evaluated by MALDI imaging mass spectrometry, which identified KRT14 among several proteins at the invasion interface. (C) Immunostaining of KRT14 in diffuse ovarian cancer cells (OVCAR4) showing localization to invasive pseudopodia. A magnified area is shown (top right). KRT14 staining appears green against a black background. Nuclear staining by DAPI is blue.

[0058] Figure 2 Figures A and B are diagrams and photographic representations illustrating KRT14 expression required for migration and invasion of ovarian cancer cells in vitro. KRT14 gene expression was disrupted using CRISPR technology in the ovarian cancer cell lines OVCAR4 and CaOV3. Representative experiments are shown. (A) Proliferation and invasion were measured using xCELLigence. Loss of KRT14 expression had no effect on proliferation but completely eliminated invasion through the mesothelial monolayer in vitro. (B) Using an in vitro scratch assay, cells lacking KRT14 failed to repair wounds overnight.

[0059] Figure 3 Images A through C are photographs and diagrams illustrating KRT14 expression required for successful tumor implantation in vivo. KRT14 expression (“wild type; WT”) or lack of KRT14 expression (“KRT14…”) are also shown. KO Intrabursally, ID8 ovarian cancer cells were implanted into wild-type C57BL / 6 mice. Tumor growth was monitored in real time using in vivo fluorescence. (A) ID8 cells were successfully transplanted into a single ovary of each mouse and were detectable and localized to the transplantation site. Four weeks later, K14 cells were implanted. KO The cells in mice lost fluorescence. (B) At 3–4 weeks, fluorescence was enhanced in mice carrying wild-type tumor cells; in mice implanted with KRT14... KOIn mice implanted with wild-type ID8 cells, no similar increase in fluorescence was detected. (C) Mice were sacrificed at approximately 7 weeks of age and subjected to necropsy. Mice implanted with wild-type ID8 cells developed large primary ovarian tumors with metastases to the contralateral ovary, peritoneal wall, liver, intestine, and diaphragm, and showed significant accumulation of ascites in the peritoneal cavity. In contrast, mice implanted with KRT14 cells... KO The mice that produced the cells did not develop ascites, and no tumors were observed. No tumor cells were detected during necropsy in these mice.

[0060] Figure 4 Figures A and B are schematic and photographic representations showing the N-terminus of KRT14 exposed on the cell surface and accessible to exogenously added antibodies. (A) Flow cytometry was performed on intact, non-permeabilized ovarian cancer cells (as indicated) using a polyclonal antibody targeting the N-terminal region of KRT14. Between 30% and 50% of the cells were positively stained for KRT14 on their cell surface. Immunostaining of intact cells in the culture confirmed staining of a subset of cells with anti-KRT14 antibody. (B) The ability of antibodies targeting either the N-terminus or C-terminus of KRT14 to inhibit ovarian cancer cell invasion in vitro was tested. The anti-C-term antibody (C-term) had no effect on invasion, while the anti-N-term antibody (N-term) completely blocked invasion. Exogenously added full-length recombinant KRT14 protein (rK14) alone or in combination with the C-term antibody had no effect. However, rK14 successfully competed with N-term antibodies to restore its in vitro invasive ability.

[0061] Figure 5 Figures A through C are diagrams and photographic representations showing that a single antigenic region in the N-terminus of KRT14 is exposed and can be targeted to block in vitro invasion. (A) Using the publicly available IEDB portal ( http: / / tools.iedb.org / bcell / (A) The antigenicity and hydrophobicity of the N-terminus of KRT14 were predicted using computer analysis. Five potentially antigenic regions were predicted, and six corresponding peptides were synthesized. (B) Competitive assays using individual peptides were used to map the relevant regions of KRT14 recognized by polyclonal antibodies. Two peptides containing amino acids 83-110 (human sequence) successfully restored invasive ability in the Xcelligence assay. (C) In parallel wound healing assays, the same two peptides (#4 and #5) successfully competed with anti-N-terminal KRT14 antibodies to restore cell migration and achieve complete wound closure after 16 hours.

[0062] Figure 6Figures A and B are schematic representations illustrating the effectiveness of antiserum AN-17O20023 in blocking cancer cell invasion in vitro. (A) Antiserum targeting a specific KRT14 epitope effectively inhibited invasion, with efficacy comparable to that of a commercial polyclonal antibody (Sigma SAB4501657). (B) The inhibition of invasion by anti-KRT14 had no effect on cell viability.

[0063] Figure 7 This is a photographic representation illustrating the in vitro disruption of migration in non-ovarian cancer cell types by anti-KRT14 antibodies. Anti-KRT14 antibodies prevent wound closure in cell monolayers composed of endometrial or colorectal cancer cells. Short peptides (peptide 4 and peptide 5) mimicking the KRT14 epitope of interest can effectively compete with antibody binding to re-establish migration in vitro.

[0064] Figure 8 Photographs and diagrams in Figures A and B illustrate the disruption of mouse ovarian cancer cell migration in vitro by anti-KRT14 antibody. (A) Anti-KRT14 antibody prevents wound closure in a cell monolayer composed of mouse ID8 ovarian cancer cells. Short peptides (peptide 4 and peptide 5) mimicking the KRT14 epitope of interest can effectively compete with antibody binding to re-establish migration in vitro. (B) RTCA analysis confirms that anti-human KRT14 antibody blocks mouse ovarian cancer cell invasion in vitro.

[0065] Figure 9 This study demonstrates that the monoclonal anti-KRT14 antibody AN-17 (mAb AN-17) increases sensitivity to platinum chemotherapy in vitro. OVCAR4 ovarian cancer cells were incubated with mAb AN-17, cisplatin, or a combination of both, and cell proliferation was monitored over a 72-hour period. Cells treated with the combination of mAb AN-17 and cisplatin showed a significantly lower IC50 compared to cisplatin alone. In particular, mAb AN-17 significantly enhanced the toxicity of cisplatin at sublethal doses (n = 3 / treatment, mean cell index).

[0066] Figure 10 The results show that mAb AN-17 exhibits no cross-reactivity with a variety of protein antigens in vitro. A protein array was used to identify any cross-reactive proteins that might be recognized by mAb AN-17. The absence of obvious cross-reactivity demonstrates the high specificity of mAb AN-17 for KRT14.

[0067] Figure 11 This demonstrates the detection of KRT14 using mAb AN-17. Antibody dilutions from 1:1000 to 1:10,000 were successfully detected by Western blotting.

[0068] Figure 12This study demonstrates that mAb AN-17 can identify KRT14+ cells in human and mouse ovarian cancer cells. OVCAR3, CAOV4, ID8, and patient-derived 3.1937-07 cell lines were analyzed in KRT14+ cell populations using mAb AN-17 or a commercially available polyclonal antibody against KRT14 (Sigma SAB4501657) on a BD LSRFortessa X-20 (BD Biosciences) flow cytometer.

[0069] Figure 13 This diagram illustrates the detection of circulating tumor cells in mice bearing epithelial ovarian tumors using mAb AN17. Cardiac blood was obtained from 12-week-old mice bearing ID8 iRFP720+ epithelial ovarian tumors and stained with anti-CD45 and mAb AN-17. Circulating ID8 tumor cells were identified as KRT14+CD45- cells and confirmed by their iRFP720+ status. Spiked iRFP720+ ID8 cells were used as a positive control.

[0070] Figure 14 The image shows the detection of KRT14+ cells by immunofluorescence staining. Cancer cells were incubated with mAb AN-17 after being intact (left) or permeabilized (right) to label surface KRT14 or intracellular KRT14, respectively.

[0071] Figure 15 Immunohistochemical staining of tumor tissue using mAb AN-17 is shown. Staining is limited to the tumor epithelium and is similar to that of a commercially available polyclonal anti-KRT14 antibody (Sigma SAB4501657).

[0072] Figure 16 This study illustrates the nonspecific tissue uptake and clearance of mAb AN-17 over a 7-day period. Tumor-free mice were injected with 0.5 mg / kg (ip) of mAb AN-17, and tissue distribution over time was assessed by monitoring fluorescence. Comparisons were made with a non-targeted IgG-κ isotype control antibody. No nonspecific retention of mAb AN-17 was observed, and both antibodies (mAb AN-17 and control IgG-κ) were barely detectable after 7 days. Tissues examined included reproductive organs (ovaries, fallopian tubes, uterus); intestine; liver; kidney; spleen; lung; heart; and brain (n = 2 animals / group, mean + / - SD). Measurements at each time point were offset against axes to clarify overlapping datasets.

[0073] Figure 17This study illustrates the nonspecific tissue uptake and clearance of mAb AN-17 over a 7-day period. Tumor-free mice were injected with mAb AN-17 at doses of 0.5 mg / kg, 1.0 mg / kg, 2.5 mg / kg, 5.0 mg / kg, or 10.0 mg / kg (ip), and tissue distribution over time was assessed by monitoring fluorescence. Comparisons were made with a non-targeted IgG-κ isotype control antibody. No nonspecific retention of mAb AN-17 was observed in any of the tissues evaluated, and mAb AN-17 was largely undetectable after 7 days. Tissues examined included reproductive organs (ovaries, fallopian tubes, uterus); intestine; liver; kidney; spleen; lung; heart; and brain (n = 2 animals / group, mean + / - SD).

[0074] Figure 18 This demonstrates the high specificity of mAb AN-17 for tumor tissue. Mice with established primary ovarian tumors (n = 2 / group / time point) were administered mAb AN-17 via intraperitoneal injection at doses of 5 mg / kg or 10 mg / kg. Control animals received the same dose of isotype-matched control antibody. Mice were sacrificed on days 1, 3, 5, and 7 post-administration, and antibody localization was assessed by fluorescence (as above). Fluorescence is expressed as mean radiative fluorescence intensity per unit tissue area over time. (A) Tumor-specific fluorescence signal. (B) Fluorescence in non-tumor reproductive tissues shows no specific signal. (C) Images of mAb AN-17 fluorescence (red) in postmortem isolated tumors and non-tumor reproductive tissues used for comparison (n = 2 / group / time point; mean + / - SD).

[0075] Figure 19 Administration of mAb AN-17 resulted in the direct regression of established tumor masses in mice. Mice with established primary ovarian tumors (n=10 / group) were administered mAb AN-17 twice weekly (Monday and Thursday) via intraperitoneal injection at a dose of 5 mg / kg. Control animals received either an isotype-matched control antibody or only PBS mediator. After 3 weeks of continuous treatment, all animals were slaughtered and examined, and tumor mass was measured postmortem. 60% of mice receiving either the mediator or the isotype control antibody had primary ovarian tumors at slaughter. In contrast, no tumors were identifiable in mice treated with mAb AN-17 (mean + / - SD).

[0076] Figure 20The nucleic acid and amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of (A) monoclonal antibody AN-17 (from clone AN-17A RG4.E5b.A7.B4) and the VH and VL amino acid sequences of (B) mAb AN-17 are shown as the % similarity between these sequences and the sequences of unrearranged germline mouse antibodies (using the IMGT / V-Quest program). N / A = Not applicable; nt = nucleotide.

[0077] Figure 21 The nucleic acid and amino acid sequences of VH and VL of mAb AN-17 are shown.

[0078] Figure 22 The VH(A) and VL(B) amino acid sequences of mAb AN-17 are shown, with annotations in bold and underlined text to highlight the frame region (FWR) and complementarity-determining region (CDR).

[0079] Figure 23 The migration of non-ovarian cancer cells (BT16 atypical teratoid rhabdomyosarcoma (brain) carcinoma, NCI-H1573 lung adenocarcinoma, SJ-GBM2 primary glioblastoma multiforme, AN3CA endometrial carcinoma, SW620 colorectal cancer and MDA-MB-468 breast cancer cell line) in vitro was impaired by mAb AN-17.

[0080] Detailed description

[0081] Throughout this specification, unless the context otherwise requires, the word “comprise” or variations thereof such as “comprises” or “comprising” shall be understood to implicitly include the stated element or integer or method step, or group of elements or integers or method steps, but not exclude any other element or integer or method step, or group of elements or integers or method steps.

[0082] Unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” as used in this subject matter description include plural aspects. Thus, for example, reference to “cancer cell” includes a single cancer cell as well as two or more cancer cells; reference to “epitope” includes a single epitope as well as two or more epitopes; reference to “this disclosure” includes one or more aspects taught in this disclosure; and so on. The term “invention” covers the aspects taught and implemented herein. The “form” of the invention covers any variations and derivatives contemplated herein. All aspects of the invention are realized across the breadth of the claims.

[0083] This invention relates to treatment regimens for treating, preventing, or otherwise alleviating the progression of cancer in mammalian subjects. Subjects may be male or female. Alleviation of progression includes preventing or reducing the invasion, migration, and / or metastasis of cancer cells, thereby treating, preventing, or delaying the development of cancer or reducing its metastatic potential. The terms "cancer" and "tumor" are used interchangeably herein.

[0084] Therefore, this paper implements a method for treating or preventing cancer in mammalian subjects. The method includes administering an agent to the mammalian subject, the agent being:

[0085] (i) Directly targeting the extracellular portion of KRT14 on cancer cells or its functional homologs or variants; examples of agents include antibodies, including fragments and derivatives that directly target the extracellular portion of KRT14, and appropriately deimmunized antibodies or other targeting portions or ligands; or

[0086] (ii) An endogenous agent that induces in vivo targeting of the extracellular portion of KRT14 or its functional homologs or variants on cancer cells; examples of endogenous agents include, but are not limited to, antibodies, T cells and macrophages.

[0087] In either case, the agent or endogenous agent induces cytotoxicity or cytostasis in cancer cells carrying the extracellular portion of KRT14, thereby preventing or reducing cancer cell invasion, migration, and / or metastasis. For example, regarding (i), the antibody may bind to induce complement-mediated or macrophage- or cytokine-mediated cell lysis or senescence. Optionally, the antibody or other targeting agent may be conjugated to a cytotoxic molecule or used to sensitize lymphocytes. References to "antibody" include monoclonal antibodies, polyclonal antibodies, antiserum containing KRT-14-binding antibodies, and synthetic or recombinant forms, fragments, and derivatives that bind to the exogenous portion or a portion thereof of KRT14. In addition to being an antibody, the drug may be any affinity agent, including but not limited to aptamers, monospecific antibodies, anticalins, DARPin, and nanobodies.

[0088] In the implementation scheme, the extracellular portion of human KRT14 is defined by the amino acid sequence (in single-letter code) NH2-GFGGGYGGGLGAGLGGGFGGGFAGGDGL (SEQ ID NO:1).

[0089] This document includes functional homologs and / or variants in humans or non-human mammals. In examples, functional homologs or variants of SEQ ID NO:1 include proteins containing an amino acid sequence that, after optimal alignment, has at least 80% similarity to SEQ ID NO:1. "At least 80% similarity" includes having at least 80%, 81%, 82%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% similarity or identity to SEQ ID NO:1. Such sequences are homologs or variants of the extracellular portion of KRT14.

[0090] Table 3 shows examples of homologs that have at least about 80% similarity to SEQ ID NO:1.

[0091] Table 3

[0092]

[0093]

[0094]

[0095]

[0096] As used herein, the term "similarity" includes exact identity between sequences compared at the amino acid level. However, when non-identity exists at the amino acid level, "similarity" includes amino acids that are related to each other at the structural, functional, biochemical, and / or conformational levels. In embodiments, amino acid and sequence comparisons are performed at the level of identity rather than similarity.

[0097] Terms used to describe sequence relationships between two or more polypeptides include “reference sequence,” “comparison window,” “sequence similarity,” “sequence identity,” “percentage of sequence similarity,” “percentage of sequence identity,” “substantially similar,” and “substantially identical.” A “reference sequence” is 5 to 20 amino acids in length. A “comparison window” is a conceptual segment of typically 5–20 consecutive residues compared to the reference sequence. For optimal alignment of two sequences, the comparison window may contain approximately 20% or less of additions or deletions (i.e., vacancies) compared to the reference sequence (which contains no additions or deletions). Optimal alignment of sequences used to align the comparison window can be achieved through computerized algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection, selecting the best alignment (i.e., the highest percentage of homology on the comparison window) produced by any of the various methods. See also the BLAST family of programs, for example, published by Altschul et al. (1997) Nucl. Acids. Res. 25:3389. A detailed discussion of sequence analysis can be found in section 19.3 of Ausubel et al. (1994-1998) In: Current Protocols in Molecular Biology, John Wiley & Sons Inc.

[0098] As used in this paper, the terms “sequence similarity” and “sequence identity” refer to the degree to which sequences are identical, functionally similar, or structurally similar, based on amino acid-by-amino acid comparison windows. Therefore, the “sequence identity percentage” is calculated, for example, by comparing two best-aligned sequences on a comparison window, determining the number of positions in both sequences where the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the sequence identity percentage.

[0099] Therefore, the terms "variant" and "derivative" refer to amino acid sequences that exhibit identity or similarity to the basic sequence of a reference amino acid sequence (i.e., SEQ ID NO:1 or a subsequence thereof). The terms "variant" and "derivative" also include naturally occurring allelic variants.

[0100] "Derivatives" also include mutants, fragments, parts, portions, or hybrid molecules relating to SEQ ID NO:1 or its functional homologs. Derivatives typically carry, but are not limited to, a single or more amino acid substitution, addition, and / or deletion.

[0101] "Homologous" refers to similar polypeptides that have at least about 80% similar amino acid sequences and originate from another animal species or from different loci within the same species.

[0102] Variants also include “analogs” that are typically chemical analogs. Chemical analogs of SEQ ID NO:1 considered herein include, but are not limited to, modifications of side chains, incorporation of non-natural amino acids and / or their derivatives during peptide, polypeptide, or protein synthesis, and other methods using cross-linking agents and imposing conformational restrictions on protein molecules or their analogs.

[0103] Examples of side-chain modifications contemplated by the present invention include modifications of the amino group by means of: reductive alkylation by reaction with acetaldehyde followed by reduction with NaBH4; amidation by methyl acetylimine ester; acylation by acetic anhydride; carbamylation of the amino group by cyanic acid; trinitrobenzoylation of the amino group by 2,4,6-trinitrobenzenesulfonic acid (TNBS); acylation of the amino group by succinic anhydride and tetrahydrophthalic anhydride; and pyridoxine pyridoxine by pyridoxal-5-phosphate followed by reduction with NaBH4.

[0104] The guanidine group of arginine residues can be modified by forming heterocyclic condensation products using reagents such as 2,3-butanedione, phenylglyoxal, and glyoxal.

[0105] The carboxyl group can be modified by activating the carbodiimide via O-acylisourea and then derivatizing it, for example, to become the corresponding amide.

[0106] The thiol group can be modified by methods such as carboxylation with iodoacetic acid or iodoacetamide; oxidation of performic acid to sulfoalanine; formation of mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercury derivatives using 4-chloromercuric benzoic acid, 4-chloromercuric benzenesulfonic acid, phenylmercuric chloride, 2-chloromercuric-4-nitrophenol and other mercury preparations; and carbamylation with cyanic acid at alkaline pH.

[0107] Tryptophan residues can be modified, for example, by oxidation with N-bromosuccinimide or by alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfonic acid phenyl halides. Alternatively, tyrosine residues can be altered by nitration with tetranitromethane to form 3-nitrotyrosine derivatives.

[0108] The modification of the imidazole ring of histidine residues can be accomplished by alkylation with iodoacetic acid derivatives or by N-carbethoxylation with diethyl pyrocarbonate.

[0109] Crosslinking agents can be used, for example, to stabilize 3D conformations, using the same bifunctional crosslinking agent, such as one with (CH2) n Spacer groups, bifunctional imino esters (n=1 to n=6), glutaraldehyde, N-hydroxysuccinimide esters, and heterobifunctional reagents that typically contain an amino reactive moiety such as N-hydroxysuccinimide and another specifically reactive moiety such as maleimide or a disulfide moiety (SH) or carbodiimide (COOH). Furthermore, peptides can be conformally restricted by, for example, C α and N α The incorporation of methyl amino acids, the C of amino acids α and C β The introduction of double bonds between atoms, and the formation of cyclic peptides or analogs by introducing covalent bonds, such as amide bonds between the N-terminus and C-terminus, between two side chains, or between a side chain and the N-terminus or C-terminus.

[0110] Such analogues can be used to synthesize vaccines or generate antibodies for use as targeting agents. Analogues may possess properties such as increased serum half-life. Antibodies can also be found in antiserum containing KRT14-binding antibodies.

[0111] This article teaches a method for treating or preventing cancer in mammalian subjects, comprising administering to the subject an amount of an agent that targets the extracellular portion of KRT14 residing on cancer cells or a functional homolog or variant thereof, or an agent that induces in vivo production of an antagonist of the extracellular portion of KRT14 on cancer cells or a functional homolog or variant thereof, said amount effectively preventing or reducing cancer cell invasion, migration and / or metastasis.

[0112] This article describes a method for treating or preventing cancer in mammalian subjects, comprising administering to the subject an agent that targets an extracellular portion of an amino acid sequence listed in SEQ ID NO:1 or an amino acid sequence having at least about 80% similarity to SEQ ID NO:1 after optimal alignment, or an agent that induces in vivo production of an antagonist of the extracellular portion of KRT14 on cancer cells, or an agent that effectively prevents or reduces cancer cell invasion, migration, and / or metastasis.

[0113] This article also teaches a method for treating or preventing cancer in mammalian subjects, comprising administering to the subject an amount of an agent that targets the extracellular portion of cancer cells as defined by the amino acid sequence listed in SEQ ID NO:1 or a functional homolog or variant thereof, or an agent that induces in vivo production of an antagonist of the extracellular portion of KRT14 on cancer cells or a functional homolog or variant thereof on cancer cells, thereby effectively preventing or reducing cancer cell invasion, migration and / or metastasis by said amount.

[0114] This invention also provides a method for treating ovarian cancer in human subjects, comprising administering to the subject an amount of an antibody targeting the extracellular portion of KRT14, as defined in SEQ ID NO:1, residing on ovarian cancer cells, said amount effectively preventing or reducing ovarian cancer cell invasion, migration, and / or metastasis. The drug can be any affinity agent besides an antibody, including but not limited to aptamers, monospecific antibodies, anticalins, DARPin, and nanobodies.

[0115] The term "administration to a subject" includes contact with cancer cells by any means, such that the agent comes into contact with the extracellular portion of KRT14 on the cancer cells. Mammal subjects can be male or female and can be of any age.

[0116] This study identified short epitopes of the KRT14 protein exposed on the cell surface, which can interact with exogenously added agents or specifically induced endogenous molecules. Targeting this region using antibodies or other targeting agents that recognize the exposed sequence completely prevented cancer cell invasion in vitro, mimicking the effect of KRT14 gene ablation. These data suggest that KRT14 represents a previously unrecognized and highly specific target on tumor cells for targeted antagonist therapy.

[0117] Failure to form solid tumors was also observed in mice. Implanted cancer cells lacking the functional KRT14 gene became undetectable after several weeks. This suggests that these cancer cells may be removed from animals through, for example, immune-mediated clearance. Therefore, this paper proposes that the use of anti-KRT14 therapy can promote tumor stabilization (through impaired implantation / invasion) and tumor regression. Targeted anti-KRT14 therapy has high potential for cancer treatment for the following reasons:

[0118] (i) Anti-KRT14 therapy is expected to be non-toxic because KRT14 is not widely expressed and antagonists that are non-toxic to cells have been selected.

[0119] ii) Anti-KRT14 therapy is applicable to all stages of the disease and is suitable for targeting both primary and metastatic deposits;

[0120] iii) Over time and in response to chemotherapy, cells expressing KRT14 are specifically enriched; therefore, anti-KRT14 therapy is highly suitable for patients who have developed relapsed, chemotherapy-resistant disease and have no other conventional treatment options available.

[0121] iv) In addition to stabilizing existing disease, anti-KRT14 therapy may also potentially promote tumor regression.

[0122] Anti-KRT14 therapy also has several other applications, including:

[0123] i) Conjugated with a cytotoxic "payload" for targeted therapies against invasive cancer stem cell populations;

[0124] ii) For use in CAR-T therapy, it is designed to guide anti-tumor cytotoxic T cells to destroy the tumor-initiating cell population;

[0125] iii) Therapeutic or preventative vaccination;

[0126] iv) Generation of “dehumanized” antibodies for veterinary applications;

[0127] v) Thermo-analytical applications, for example, to predict treatment response / chemotherapy tolerance / tumor recurrence or progression.

[0128] KRT14-dependent tumor progression has also been identified as a key mechanism involved in several other solid tumor types and may represent a conserved mechanism underlying tumor spread. Therefore, the application of anti-KRT14 therapy extends far beyond ovarian cancer and may prove applicable to the treatment of a wide range of solid tumors. As used herein, “cancer” refers to a group of diseases and conditions characterized by uncontrolled cell growth (e.g., tumor formation) without any differentiation into specialized or distinct cell types. Cancers considered for treatment in this article include, but are not limited to, and in addition to gynecological conditions such as ovarian cancer, ABL1 proto-oncogene disorders, AIDS-related cancers, acoustic neuroma, acute lymphoblastic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adrenocortical carcinoma, and idiopathic myeloid metaplasia. Metaplasia, hair loss, alveolar soft tissue sarcoma, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancer, colon cancer, brainstem glioma, brain and CNS tumors, breast cancer, CNS tumors, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia. Leukemia, chronic myeloid leukemia, colorectal cancer, cutaneous T-cell lymphoma, fibrosarcoma protuberans, small round cell tumor of connective tissue proliferative disease, ductal carcinoma, endocrine carcinoma, endometrial cancer, ependymoma, esophageal cancer, Ewing sarcoma, extrahepatic bile duct cancer, ocular cancer, melanoma, retinoblastoma, fallopian tube cancer, Fanconi anemia, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, genitourinary cancer. Cancer), germ cell tumors, gestational trophoblastic disease, glioma, gynecological cancers, hematological malignancies, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, vesicular birth defects, hypercalcemia, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Levi-Flaumene syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphoma Edema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breast cancer, malignant rhabdoid tumor of the kidney, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, oral cancer, multiple endocrine tumors, mycosis fungoides, myelodysplastic syndrome, myeloma, myeloproliferative disorders, nasal cancer, nasopharyngeal carcinoma, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen's rupture syndrome, non-melanoma skin cancer, non-small cell lung cancer (NSCLC), ocular cancer.Cancers including: esophageal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, pancreatic cancer, paranasal cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral neuroectodermal tumor, pituitary cancer, polycythemia vera, prostate cancer, rare cancers and related conditions, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, salivary gland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma, spinal cord tumors, squamous cell carcinoma (skin), gastric cancer, synovial sarcoma, testicular cancer, thymic cancer, thyroid cancer, transitional cell carcinoma (bladder), transitional cell carcinoma (renal pelvis / ureter), trophoblastic carcinoma, urethral cancer, urinary system cancers, uroplakins, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms' tumor. Endometrial cancer and colorectal cancer can also be treated. These cancers can affect male or female subjects and can also be treated according to the present invention.

[0129] In the implementation scheme, the cancer is a gynecological cancer. In the implementation scheme, the gynecological cancer is ovarian cancer or a stage or form of ovarian cancer. Optionally, the cancer is liver cancer, bladder cancer, lung cancer, colon cancer, gastrointestinal cancer, intestinal cancer, pancreatic cancer, and / or laryngeal cancer and other cancers, particularly in male or female subjects. The invention extends to combination therapies, wherein an agent targeting KRT14 or an agent inducing KRT14 antagonism in vivo is administered together with another anticancer agent and / or radiotherapy and / or surgical intervention. In the implementation scheme, the methods disclosed herein also include administering additional anticancer agents to the subject and / or exposing the patient to immunotherapy, radiotherapy, and / or surgical intervention. Illustrative examples of additional anticancer agents include chemotherapeutic agents such as one or more of the following: daunorubicin, doxorubicin, doxorubicin (adriamycin), idarubicin, and mitoxantrone, or platinum-based agents or antimetabolites. Antimetabolites are substances that interfere with the body's chemical processes, such as the production of proteins, DNA, and other chemicals required for cell growth and reproduction; in cancer treatment, antimetabolites disrupt DNA production, which in turn inhibits cell division. Examples include diazoserine, D-cycloserine, mycophenolic acid, trimethoprim, 5-fluorouracil, capecitabine, methotrexate, gemcitabine, cytarabine (ara-C), and fludarabine. Other immunomodulatory agents, such as sensitized T cells and cytokines, may be administered. Combination therapy may be provided simultaneously or sequentially, within seconds, minutes, hours, days, or weeks. In the implementation scheme, additional anticancer agents are selected from the group consisting of: daunorubicin, doxorubicin, idarubicin (doxorubicin), idarubicin, and mitoxantrone, platinum-based agents, antimetabolites, sensitized T cells, and cytokines. In the implementation scheme, the antimetabolite is selected from the group consisting of: diazoserine, D-cyclic serine, mycophenolic acid, trimethoprim, 5-fluorouracil, capecitabine, methotrexate, gemcitabine, cytarabine (ara-C), and fludarabine.

[0130] In the implementation plan, the mammalian subject is a human. For gynecological cancers, the subject is a human female. However, for all other cancers, the subject can be a human male or a human female.

[0131] Therefore, this paper teaches a method for treating or preventing cancer in human subjects, the method comprising administering to the subject an amount of an agent that targets the extracellular portion of KRT14 residing on cancer cells or a functional homolog or variant thereof, or an agent that induces in vivo production of an antagonist of the extracellular portion of KRT14 on cancer cells or a functional homolog or variant thereof, said amount effectively preventing or reducing cancer cell invasion, migration and / or metastasis.

[0132] This article implements a method for treating or preventing cancer in human subjects, the method comprising administering to the subject an amount of an agent that targets an extracellular portion of cancer cells defined by the amino acid sequence listed in SEQ ID NO:1 or a functional homolog or variant thereof, or an agent that induces an antagonist of an extracellular portion of KRT14 or a functional homolog or variant thereof produced by the subject on cancer cells, said amount effectively preventing or reducing cancer cell invasion, migration and / or metastasis.

[0133] This article also teaches a method for treating or preventing cancer in human subjects, comprising administering to the subject an agent that targets an extracellular portion of an amino acid sequence residing on cancer cells, defined by an amino acid sequence having at least about 80% similarity to SEQ ID NO:1 after optimal alignment, or an agent that induces in vivo production of an antagonist of the extracellular portion of KRT14 on cancer cells, or an agent that effectively prevents or reduces cancer cell invasion, migration, and / or metastasis.

[0134] In the implementation plan, the cancer is ovarian cancer, and the subjects are human female subjects.

[0135] Therefore, this paper teaches a method for treating or preventing ovarian cancer in human female subjects, the method comprising administering to the subject an amount of an agent that targets the extracellular portion of KRT14 or a functional homolog or variant thereof residing on cancer cells, or an agent that induces the in vitro production of an antagonist of the extracellular portion of KRT14 or a functional homolog or variant thereof on cancer cells, said amount effectively preventing or reducing cancer cell invasion, migration and / or metastasis.

[0136] This article describes a method for treating or preventing ovarian cancer in human female subjects, comprising administering to the subject an amount of an agent that targets the extracellular portion of cancer cells defined by the amino acid sequence listed in SEQ ID NO:1 or a functional homolog or variant thereof, or an agent that induces the subject to produce an antagonist of the extracellular portion of KRT14 or a functional homolog or variant thereof on cancer cells, said amount effectively preventing or reducing cancer cell invasion, migration and / or metastasis.

[0137] This article also teaches a method for treating or preventing ovarian cancer in human female subjects, comprising administering to the subject an agent that targets an extracellular portion of an amino acid sequence listed in SEQ ID NO:1 or an amino acid sequence having at least about 80% similarity to SEQ ID NO:1 after optimal alignment, or an agent that induces in vivo production of an antagonist of the extracellular portion of KRT14 on cancer cells, or an agent that effectively prevents or reduces cancer cell invasion, migration, and / or metastasis.

[0138] This invention also has veterinary applications, such as treating cancer in companion animals (e.g., dogs and cats) or other non-human animals such as farm animals (e.g., equines, pigs, sheep, cattle, goats, llamas, and alpacas), laboratory test animals (e.g., mice, rabbits, guinea pigs, hamsters), and wild captive animals (e.g., Tasmanian devils). Other animals considered for treatment include gibbons, chimpanzees, rhesus monkeys, green monkeys, orangutans, baboons, shrews, gorillas, hedgehogs, baby monkeys, kangaroo rats, wild yaks, Philippine tarsiers, ferrets, elephants, and bats. Regarding equines, these include horses, Przewalski's horses, zebras, and donkeys. Regarding horses, these include Thoroughbreds, warmbloods, quarter horses, and standard horses, as well as equestrian horses and performance horses.

[0139] Therefore, this paper teaches a method for treating or preventing cancer in non-human mammal subjects, the method comprising administering to the subject an amount of an agent that targets the extracellular portion of KRT14 residing on cancer cells or a functional homolog or variant thereof, or an agent that induces in vivo production of an antagonist of the extracellular portion of KRT14 on cancer cells or a functional homolog or variant thereof, said amount effectively preventing or reducing cancer cell invasion, migration and / or metastasis.

[0140] This article also teaches a method for treating or preventing cancer in non-human mammal subjects, comprising administering to the subject an agent that targets an extracellular portion of an amino acid sequence listed in SEQ ID NO:1 or an amino acid sequence having at least about 80% similarity to SEQ ID NO:1 after optimal alignment, or an agent that induces in vivo production of an antagonist of the extracellular portion of KRT14 on cancer cells, said amount effectively preventing or reducing cancer cell invasion, migration, and / or metastasis.

[0141] This article describes a method for treating or preventing cancer in non-human mammal subjects, comprising administering to the subject an amount of an agent that targets the extracellular portion of cancer cells defined by the amino acid sequence listed in SEQ ID NO:1 or a functional homolog or variant thereof, or an agent that induces in vivo production of an antagonist of the extracellular portion of KRT14 on cancer cells or a functional homolog or variant thereof, said amount effectively preventing or reducing cancer cell invasion, migration and / or metastasis.

[0142] The non-human mammalian subject can be male or female. In the implementation scheme, the agent is an antibody. The antibody can be a human-derived antibody, a deimmunized antibody, or a mammalianized antibody suitable for a specific mammalian subject. For example, mouse antibodies can be humanized for human use. Therefore, antibodies can be generated in a mammalian species for use in that mammal, or can be mammalianized or deimmunized as appropriate. For the avoidance of doubt, "antibody" can be a polyclonal or monoclonal antibody or an antiserum comprising: KRT14-binding antibody or KRT14-binding variant or derivative or fragment of these antibodies, or in synthetic or recombinant form, including F'(ab)-binding fragments. In addition to being an antibody, the drug can be any affinity agent, including but not limited to aptamers, monospecific antibodies, anticalins, DARPins, and nanobodies.

[0143] Therefore, the present invention also provides the application of biochemical techniques to render antibodies derived from one animal substantially non-immunogenic in another animal of the same or different species. This biochemical process is referred to herein as “deimmunization.” “Deimmunization” as mentioned herein includes processes such as complementarity-determining region (CDR) grafting, “remodeling” of the framework region of an immune-interacting molecule, and variable (v) region mutation, all aimed at reducing the immunogenicity of an immune-interacting molecule (e.g., an antibody) in a specific host (e.g., a human subject). In embodiments, preferred immune-interacting molecules are antibodies, such as polyclonal or monoclonal antibodies specific to cancer cells carrying the extracellular portion of KRT14. In embodiments, the immune-interacting molecule is a monoclonal antibody derived from one animal that exhibits reduced immunogenicity in another animal of the same or different species, such as, but not limited to, humans.

[0144] The phrase "substantially non-immunogenic" includes reduced immunogenicity compared to the parental antibody, i.e., the antibody exposed prior to the deimmunization process. The term "immunogenicity" includes the ability to initiate, induce, or otherwise promote humoral and / or T cell-mediated responses in a host animal. Practical immunogenicity criteria include the ability of an amino acid sequence derived from the antibody's variable (v) region to interact with MHC class II molecules, thereby stimulating or promoting T cell-mediated responses, including T cell-assisted humoral responses.

[0145] "Antibody" refers to a protein in the immunoglobulin family that can combine, interact, or otherwise associate with an antigen. Therefore, an antibody is an antigen-binding molecule. "Antibody" is an example of an immune-interacting molecule and includes polyclonal or monoclonal antibodies or antiserum. In embodiments, the immune-interacting molecule of the present invention is a monoclonal antibody.

[0146] The term “antigen” is used in its broadest sense herein to refer to a substance capable of reacting in and / or inducing an immune response. References to “antigen” include antigenic determinants or epitopes or cancer cells as defined by SEQ ID NO:1 or its functional homologs or variants.

[0147] "Antigen-binding molecule" refers to any molecule that has a binding affinity for the target antigen (i.e., SEQ ID NO:1). It should be understood that this term extends to immunoglobulins (e.g., polyclonal or monoclonal antibodies), immunoglobulin fragments, and non-immunoglobulin-derived protein frameworks that exhibit antigen-binding activity. The terms "antibody" and "antigen-binding molecule" include deimmunized forms of these molecules.

[0148] "Antigenic determinant" or "epitope" refers to a KRT14 portion that has an extracellular domain that can be directed by an immune response.

[0149] Although the antibodies of this invention are typically deimmunized forms of mouse monoclonal antibodies used in humans, the subject matter is extended to deimmunized antibodies from any source for use in any host. Examples of animal sources and hosts include humans, primates, livestock (e.g., sheep, cattle, horses, pigs, donkeys), laboratory test animals (e.g., mice, rabbits, guinea pigs, hamsters), and companion animals (e.g., dogs, cats).

[0150] Immunization and subsequent monoclonal antibody production can be performed using standard protocols, such as those derived from... The methods described are as follows: Milstein (Kohler et al. (1975) Nature 256:495-499 and Kohler et al. (1976) Eur. J. Immunol. 6(7):511-519; Coligan et al. Current Protocols in Immunology, 1991-1997 or Toyama et al. (1987) Monoclonal Antibody, Experiment Manual, published by Kodansha Scientific). Essentially, animals are immunized with an antigen (i.e., a protein or protein analog containing SEQ ID NO:1 or a functional homolog or variant thereof) using standard methods to generate antibody-producing cells, particularly antibody-producing somatic cells (e.g., B lymphocytes). These cells can then be removed from the immunized animal for immortalization. The antigen may need to be associated with a vector first.

[0151] "Carrier" refers to any substance that typically has a high molecular weight, which is naturally or artificially linked to a non-immunogenic or poorly immunogenic substance (such as a hapten) to enhance the immunogenicity of that substance.

[0152] Immortification of antibody-producing cells can be performed using methods well known in the art. For example, immortalization can be achieved using a transformation method with Epstein-Barr virus (EBV) [Kozbor et al. (1986) Methods in Enzymology 121:140]. In a preferred embodiment, antibody-producing cells are immortalized using a cell fusion method widely used for producing monoclonal antibodies (described above in [Coligan et al. (1991-1997)]). In this method, antibody-producing somatic cells, particularly B cells, with the potential to produce antibodies are fused with a myeloma cell line. These somatic cells can be derived from lymph nodes, spleen, and peripheral blood of sensitized animals such as rodents, including mice and rats. In an exemplary embodiment of the invention, mouse spleen cells are used. However, it is possible to use rat, rabbit, sheep, or goat cells, or cells from other animal species, instead.

[0153] Specialized myeloma cell lines for fusion programs to generate hybridomas have been developed from lymphocytic tumors (Kohler et al. (1976) above; Kozbor et al. (1986) above; and Volk et al. (1982) J.Virol. 42(1):220-227).

[0154] Many myeloma cell lines can be used to generate fusion cell hybrids, including, for example, P3X63-Ag8, P3X63-AG8.653, P3 / NS1-Ag4-1 (NS-1), Sp2 / O-Ag14, and S194 / 5.XXO.Bu.1. and The P3X63-Ag8 and NS-1 cell lines have been described (Kohler et al. (1976) above). Shulman et al. (1978) Nature 276:269-270 developed the Sp2 / O-Ag14 myeloma cell line. Trowbridge (1982) J.Exp.Med.148(1):220-227 reported the S194 / 5.XXO.Bu.1 line.

[0155] Methods for generating antibody-producing hybrids of spleen or lymph node cells and myeloma cells typically involve mixing somatic cells and myeloma cells at a ratio of 10:1 (although this ratio can vary from about 20:1 to about 1:1) in the presence of an agent that promotes cell membrane fusion (chemical, viral, or electrochemical). Fusion methods have been described (Kohler et al. (1975), Kohler et al. (1976), Gefter et al. (1977) Somatic Cell Genet. 3:231-236, and Volk et al. (1982)). The fusion promoters used by these researchers were Sendai virus and polyethylene glycol (PEG).

[0156] Because the fusion process produces viable hybrids at a very low frequency (e.g., when the spleen is used as a source of somatic cells, approximately every 1 x 10^6 cells). 5Only one hybrid is obtained from each spleen cell, so having means to select fusion cell hybrids from the remaining unfused cells, particularly unfused myeloma cells, is preferred. Means to detect the desired antibody-producing hybridoma among the other obtained fusion cell hybrids are also necessary. Typically, the selection of fusion cell hybrids is accomplished by culturing cells in a medium that supports hybridoma growth but prevents the growth of unfused myeloma cells, which would normally continue to divide indefinitely. Somatic cells used in fusion cannot maintain long-term viability in vitro and therefore do not pose a problem. In an example of the invention, myeloma cells lacking hypoxanthine phosphoribosyltransferase (HPRT-negative) are used. Selection of these cells is performed in a hypoxanthine / aminopterin / thymidine (HAT) medium in which fusion cell hybrids survive due to the HPRT-positive genotype of the spleen cells. It is also possible to use myeloma cells with different genetic defects (drug sensitivity, etc.), and selection can be made in a medium supporting the growth of hybrids that are genotypically competent for the genetic defects.

[0157] Selective culture of fusion cell hybrids takes several weeks. Early in this period, it is necessary to identify those hybrids that produce the desired antibodies so that they can be subsequently cloned and propagated. Typically, about 10% of the obtained hybrids produce the desired antibodies, although a range of about 1% to about 30% is not uncommon. Detection of antibody-producing hybrids can be achieved by any of several standard assays, including enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays, as described, for example, in Kennet et al. (Chou et al., U.S. Patent No. 6,056,957).

[0158] After selecting the desired fusion cell hybrid and cloning it into a single antibody-producing cell line, each cell line can be propagated using either of two standard methods. A suspension of hybridoma cells can be injected into a histocompatibility animal. The injected animal then develops a tumor that secretes specific monoclonal antibodies produced by the fusion cell hybrid. Body fluids from the animal, such as serum or ascites, can be extracted to provide high concentrations of monoclonal antibodies. Alternatively, the single cell line can be propagated in vitro in a laboratory culture vessel. The culture medium containing high concentrations of single-specific monoclonal antibodies can be harvested by decanting, filtration, or centrifugation followed by purification.

[0159] The cell lines are tested for specificity against the antigen of interest using any suitable immunoassay technique. For example, the cell lines can be aliquoted into multiple wells and incubated, and the supernatant from each well can be analyzed using enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody technology, etc. One or more cell lines that produce monoclonal antibodies that recognize the target antigen but not non-target epitopes are identified, and then directly cultured in vitro or injected into histocompatibility animals to form tumors, and the desired antibodies are produced, collected, and purified.

[0160] Therefore, the present invention first provides a monoclonal antibody that specifically interacts with a protein comprising an extracellular portion of SEQ ID NO:1 or a variant thereof or an epitope thereof.

[0161] The monoclonal antibody is then typically subjected to deimmunization. Such a process can take any of many forms, including the preparation of chimeric antibodies with the same or similar specificity as the monoclonal antibody prepared according to the invention. Chimeric antibodies are antibodies whose light and heavy chain genes are typically constructed through genetic engineering from the variable and constant regions of immunoglobulins belonging to different species. Therefore, according to the invention, after obtaining a hybridoma that produces the desired monoclonal antibody, techniques are used to produce interspecies monoclonal antibodies, where the binding region of one species is combined with the antibody non-binding region of another species (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443). For example, a CDR derived from a non-human (e.g., mouse) monoclonal antibody can be grafted onto a human antibody, thereby “humanizing” the mouse antibody (European Patent Publication No. 0 239 400, Jones et al. (1986) Nature 321:522-525, Verhoeyen et al. (1988) Science 239:1534-1536, and Richmann et al. (1988) Nature 332:323-327). In this case, the deimmunization process is specific to humans. More specifically, a CDR can be grafted onto a variable region of a human antibody, with or without a human constant region. The non-human antibody providing the CDR is generally referred to as the “donor,” and the human antibody providing the framework is generally referred to as the “recipient.” The constant region does not need to be present, but if it is present, it must be substantially identical to the constant region of human immunoglobulins, i.e., at least about 85%–90%, preferably about 95% or more identical. Therefore, all parts of a humanized antibody, except for the possible CDR, are substantially identical to the corresponding parts of the natural human immunoglobulin sequence. Thus, a "humanized antibody" is an antibody containing both humanized light and heavy chain immunoglobulins. Through the process of "humanization," the donor antibody is called "humanized" because the resulting humanized antibody is expected to bind to the same antigen as the donor antibody that provided the CDR. The term "humanization" as used herein includes antibodies that deimmunize against a specific host (in this case, the human host).

[0162] It should be understood that deimmunizing antibodies may have additional conserved amino acid substitutions that have essentially no effect on antigen binding or the function of other immunoglobulins.

[0163] Exemplary methods for generating deimmunizing antibodies according to the present invention are described, for example, in Richmann et al. (1988) above, Chou et al. (US Patent No. 6,056,957), Queen et al. (US Patent No. 6,180,377), Morgan et al. (US Patent No. 6,180,377) and Chothia et al. (1987) J. Mol. Biol. 196:901.

[0164] Another form of antibody includes the immunoglobulin neoantigen receptor (IgNAR), an antibody isotype found only in the cartilage of marine animals (sharks and rays), which has evolved over hundreds of millions of years to be stably expressed in the high-urea environment of the bloodstream (Greenberg et al. (1995) Nature 374:168-173; Nuttall et al. (2001) Mol Immunol 38:313-326). In sharks, the IgNAR response is antigen-driven, and both immunological and naive molecular libraries of the variable domains of IgNAR have been constructed and successfully screened for antigen-specific binding agents (Greenberg et al. (1995) above; Nuttall et al. (2001) above). IgNARs are bivalent, but target antigens by a single immunoglobulin variable domain (~14 kDa) exhibiting two complementarity-determining region (CDR) loops attached to different numbers of constant domains (Nuttall et al. (2003) Eur J Biochem 270:3543-3554; Roux et al. (1998) Proc Natl Acad Sci USA 95:11804-11809). In contrast, conventional immunoglobulin (Ig) antibodies have variable weight (V... H )+Variable Lightness (V) L The IgNAR variable domain (V) is in the form of a domain (~26 kDa) and binds to the antigen through up to 6 CDRs (Chothia et al. (1989) Nature 342:877-883; Padlan (1994) Mol Immunol 31:169-217). NAR The small size and thermodynamic and chemical stability of the V1 antibody offer significant advantages over conventional antibodies. Furthermore, the small V1 antibody... NARThe size allows this special antibody domain to reach occult antigenic epitopes via a particularly long and variable CDR3 loop (Greenberg et al. 1995 above; Ewert et al. (2002) Biochemistry 41:3628-2636; Nuttall et al. (2004) Proteins 55:187-197; Stanfield et al. (2004) Science 305:1770-1773; Streltsov et al. (2004) Proc Natl Acad Sci USA 101:12444-12449; Streltsov et al. (2005) Protein Sci 14:2901-2909). IgNAR domains that recognize a variety of target antigens have been identified, including: apical membrane protein 1 (AMA-1) from Plasmodium falciparum (Nuttall et al., 2004, above); Kgp protease from Porphyromonas gingivalis (Nuttall et al., 2002, FEBS Lett 516:80-86); cholera toxin (Goldman et al., Anal Chem 78:8245-8255, 2006); Tom70 mitochondrial transmembrane protein (Nuttall et al., 2003, above) and lysozyme (Streltsov et al., 2004, above).

[0165] IgNARs, or more conventional antibodies, can be used as therapeutic agents or to deliver cytotoxic molecules to cancer cells. They can also be used for diagnosis.

[0166] Precise and sensitive binding reagents are the cornerstone of the protein-based therapeutics and diagnostics industry. Given the high global cancer rates, there is an urgent need for such reagents targeting cancer antigens for therapeutic and diagnostic purposes. The identification of the extracellular portion of KRT14 and the function of KRT14 have enabled the development of these therapeutic and diagnostic applications.

[0167] In another embodiment, the agent is a vaccine comprising a peptide portion containing an extracellular portion of KRT14 sufficient to generate an immune response against cancer cells carrying an extracellular portion of KRT14.

[0168] The vaccine may be a peptide vaccine or a complex or conjugate comprising an extracellular portion of KRT14 or a KRT14 analog and / or SEQ ID NO:1. In embodiments, the vaccine comprises a peptide portion and one or more pharmaceutically acceptable carriers, diluents, or excipients, said peptide portion comprising an amino acid sequence listed in SEQ ID NO:1 or a functional homologue thereof or a variant thereof, comprising a peptide sequence having at least 80% similarity to SEQ ID NO:1 after optimal alignment. The present invention also realizes pharmaceutical compositions comprising antibodies against an extracellular portion of KRT14.

[0169] The term "pharmaceutically acceptable" refers to a physiologically and pharmaceutically acceptable form of a carrier, diluent, or excipient.

[0170] This invention also includes pharmaceutical compositions and formulations comprising antisense or sense compounds that downregulate KRT14 expression. The pharmaceutical compositions of this invention can be administered in various ways, depending on whether local or systemic treatment is desired and depending on the area to be treated. Administration can be local (including vaginal and rectal delivery), pulmonary (e.g., by inhalation or blowing of powders or aerosols, including via nebulizers), intratracheal, intranasal, epidermal and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, such as intrathecal or intraventricular administration. Pharmaceutical compositions and formulations for local application may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional drug carriers, aqueous, powdered, or oily bases, thickeners, etc., may be necessary or desired. The pharmaceutical formulations of this invention can be readily available in unit dosage forms and can be prepared using conventional techniques well known in the pharmaceutical industry. Such techniques include the step of associating the active ingredient with a pharmaceutical carrier or excipient. Typically, formulations are prepared by uniformly and tightly associating the active ingredient with a liquid carrier or a subdivided solid carrier, or both, and then shaping the product, if necessary. In embodiments, the pharmaceutical composition also comprises additional anticancer agents, illustrative examples of which are known to those skilled in the art and described elsewhere herein. In embodiments, the additional anticancer agents are selected from the group consisting of: dextrin, daunorubicin, doxorubicin (doxorubicin), idarubicin and mitoxantrone, platinum-based agents, antimetabolites, sensitized T cells, and cytokines. In embodiments, the antimetabolites are selected from the group consisting of: diazoserine, D-cycloserine, mycophenolic acid, trimethoprim, 5-fluorouracil, capecitabine, methotrexate, gemcitabine, cytarabine (ara-C), and fludarabine.

[0171] It also provides a rapid, effective, and sensitive assay for identifying cancers, including gynecological cancers such as ovarian cancer. The assay enables the early detection of cancer, and particularly ovarian cancer. However, the invention is not limited to the early detection of ovarian cancer, as the assay can be used for any stage of gynecological cancer, or for its treatment, or for any complications arising therefrom.

[0172] The term "cancer" in relation to "gynecological conditions" includes ovarian cancer and its subtypes, such as mucinous or endometrial ovarian cancer, or its stages, such as stage I, II, III, or IV. Terms such as "ovarian cancer," "epithelial ovarian cancer," and "malignant ovarian tumor" are used interchangeably herein. This invention is useful when applied to the diagnosis of symptomatic women, but it is equally applicable to the diagnosis of asymptomatic women and / or women at high risk of developing gynecological conditions. However, this invention encompasses a wide range of cancers in both male and female subjects.

[0173] This invention extends to the terms "ligand" or "binding agent" and other similar terms, referring to any compound, composition, or molecule capable of binding to epitopes on KRT14 with specific or substantially specific (i.e., with limited cross-reactivity). A "binding agent" typically has single specificity. However, binding agents with multispecificity to two or more epitopes are also considered herein. Binding agents (or ligands) are typically antibodies, such as monoclonal antibodies or their derivatives or analogues, but also include, but are not limited to: Fv fragments; single-chain Fv (scFv) fragments; Fab' fragments; F(ab')2 fragments; humanized antibodies and antibody fragments; camelized antibodies and antibody fragments; and the aforementioned multivalent forms. If appropriate, multivalent binding agents may also be used, including but not limited to: monospecific or bispecific antibodies; such as disulfide-stabilized Fv fragments, scFv tandems [(scFv)2 fragments], bispecific antibodies (diabodies), trispecific antibodies (tribodies), or tetraspecific antibodies (tetrabodies), which are typically covalently linked or otherwise stabilized (i.e., leucine zipper or helical stabilized) scFv fragments. "Binding agent" also includes aptamers, as described in the art.

[0174] Other non-limiting examples of suitable antigen-binding fragments for antibodies include: (i) Fd fragments; (ii) dAb fragments; and (iii) minimal recognition units consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., isolated CDRs, such as CDR3 peptides), or restricted FR3-CDR3-FR4 peptides. This disclosure also extends to other engineered molecules such as domain-specific antibodies, monodomain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-grafted antibodies, single-arm antibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.

[0175] In this embodiment, the antigen-binding antibody fragment comprises at least one immunoglobulin variable domain. The variable domain can contain amino acid sequences of any suitable length or composition, and typically contains at least one CDR adjacent to or in the same frame as one or more frame sequences. When the antigen-binding fragment contains V H Domain and V L In the case of a structural domain, V H and V L Domains can be positioned relative to each other in any suitable arrangement. For example, a variable region can be dimer and contain V. H -V H V H -V L or V L -V L Dimer. Optionally, the antigen-binding fragment of the antibody may contain monomer V. H or V L Structural domain.

[0176] In some embodiments, the antigen-binding antibody fragment may include at least one variable domain covalently linked to at least one constant domain. Non-limiting configurations of the variable and constant domains that can be found in the antigen-binding fragment include: (i) V H -C H 1; (ii)V H -C H 2; (iii)V H -C H 3; (iv)V H -C H 1-C H 2; (v)V H -C H 1-C H 2-C H 3, (vi)VH -C H 2-C H 3;(vii)V H -C L (viii)V L -C H 1; (ix)V L -C H 2, (x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3; (xiii)V L -C H 2-C H 3; and (xiv)V L -C L In any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly connected to each other or connected via full or partial hinge or connector regions. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, resulting in flexible or semi-flexible connections between adjacent variable and / or constant domains in a single polypeptide molecule. In some embodiments, as described herein, the antigen-binding fragment may comprise any of the variable and constant domain configurations listed above that are non-covalently associated with each other and / or with one or more monomers V H or V L A homodimer or heterodimer (or other multimer) with non-covalently associated domains (e.g., via one or more disulfide bonds). Multispecific antigen-binding molecules will typically contain at least two distinct variable domains, each capable of specifically binding to a different antigen or a different epitope on the same antigen. Any form of multispecific antigen-binding molecule, including bispecific antigen-binding molecule forms, can be adapted using conventional techniques available in the art for use in the context of antigen-binding fragments of antibodies of this disclosure.

[0177] The term "variable region" or "variable domain" refers to a domain of the heavy or light chain of an immunoglobulin that participates in binding to a target antigen. The variable domains of the heavy and light chains of natural immunoglobulin molecules (respectively, V...) H and V LThey typically have similar structures, with each domain comprising four conserved framework regions and three highly variable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single V H or V L The domain may be sufficient to confer antigen binding specificity.

[0178] For therapeutic applications, it may be desirable to modify the binder to be compatible with the target species; that is, the species to which the binder is to be applied. In an implementation, the binder is a humanized binder.

[0179] In some embodiments, as described herein, the FR of the binder (including an antibody or its antigen-binding fragment) may be identical to the FR of the germline sequence of the target species (i.e., the species to which the binder is to be administered). In some embodiments, the FR may be natural or artificially modified. While it is generally desirable that each FR sequence be identical to the FR sequence of one or more immunoglobulin molecules derived from the target species, including minimizing the immune response against the binder molecule upon administration to a subject of the target species, in some embodiments, the binder may contain one or more foreign amino acid residues that are foreign at corresponding positions in one or more FRs from the target species across one or more of its FR sequences. Preferably, when the binder contains one or more foreign amino acid residues that are foreign at corresponding positions in the target species across one or more of its FR sequences, these “foreign” amino acid residues will not (i) adversely affect the binding specificity of the binder to its target antigen (KRT14), and / or (ii) induce an immune response against the binder upon administration to a subject of the target species.

[0180] In the embodiments disclosed herein, the agent (including an antibody binding to the extracellular portion of KRT14 and its KRT14-binding fragment, as described herein) comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH comprises: a complementarity-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO:6, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:7, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:8; and wherein the VL comprises: a complementarity-determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO:9, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:11.

[0181] In the implementation scheme, VH includes:

[0182] (a) VH frame region 1 (FR1), wherein the VH frame region 1 (FR1) contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO:12;

[0183] (b) VH FR2, wherein the VH FR2 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:13;

[0184] (c) VH FR3, wherein VH FR3 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:14; and

[0185] (d) VH FR4, wherein the VH FR4 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:15;

[0186] And VL includes:

[0187] (e) VL FR1, wherein VL FR1 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:16;

[0188] (f) VL FR2, wherein VL FR2 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:17;

[0189] (g) VL FR3, wherein the VL FR3 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:18; and

[0190] (h)VL FR4, wherein the VL FR4 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:19.

[0191] In the implementation, VH contains an amino acid sequence that has at least 80% sequence identity with SEQ ID NO:3, and VL contains an amino acid sequence that has at least 80% sequence identity with SEQ ID NO:5.

[0192] This disclosure also extends to agents or KRT14-binding fragments thereof that specifically bind to the extracellular portion of KRT14 on cancer cells, wherein the agent comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH comprises: a complementarity-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO:6, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:7, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:8; and wherein the VL comprises: a complementarity-determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO:9, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:11.

[0193] In the implementation scheme, VH includes:

[0194] (a) VH frame region 1 (FR1), wherein the VH frame region 1 (FR1) contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO:12;

[0195] (b) VH FR2, wherein the VH FR2 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:13;

[0196] (c) VH FR3, wherein VH FR3 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:14; and

[0197] (d) VH FR4, wherein the VH FR4 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:15;

[0198] And VL includes:

[0199] (e) VL FR1, wherein VL FR1 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:16;

[0200] (f) VL FR2, wherein VL FR2 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:17;

[0201] (g) VL FR3, wherein the VL FR3 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:18; and

[0202] (h)VL FR4, wherein the VL FR4 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:19.

[0203] In the implementation, VH contains an amino acid sequence that has at least 80% sequence identity with SEQ ID NO:3, and VL contains an amino acid sequence that has at least 80% sequence identity with SEQ ID NO:5.

[0204] Ligands and binding agents can be used to detect the presence of cells carrying KRT14 in an assay. ECLIA, ELISA, and Luminex LabMAP immunoassays are examples of suitable assays for detecting biomarker levels. In one example, a first binding agent / antibody is attached to a surface, and a second binding agent / antibody containing a detectable group binds to the first antibody. Examples of detectable groups include, but are not limited to, fluorescent dyes, enzymes, epitopes for binding to the second binding agent (e.g., when the second binding agent / antibody is a mouse antibody detected by a fluorescently labeled anti-mouse antibody), such as antigens or members of a binding pair, such as biotin. The surface can be a flat surface, such as in the case of a typical grid array (e.g., but not limited to 96-well plates and flat microarrays), or a non-flat surface, such as coated bead array technology, where each “type” of bead is labeled with, for example, a fluorescent dye (such as the Luminex technology described in U.S. Patents 6,599,331, 6,592,822, and 6,268,222) or a quantum dot technology (e.g., as described in U.S. Patent 6,306,610). Such measurements can also be considered as laboratory information management systems (LIMS).

[0205] As used in this article, “immunoassay” refers to immunoassays that can detect and quantify the desired biomarker, namely the extracellular portion of KRT14, typically but not limited to sandwich assays.

[0206] A method for diagnosing gynecological conditions or other cancers is provided, which involves determining the presence of the extracellular portion of KRT14 and using the level of the extracellular portion as a second knowledge base in an algorithm generated using a first knowledge base of known amounts of KRT14 in patients with known diseases. A method for detecting preclinical ovarian cancer or other cancers is also provided, which includes determining the presence and / or velocity of KRT14 in a subject's sample. "Velocity" refers to the change in KRT14 concentration in a patient's sample over time.

[0207] As indicated above, gynecological conditions include cancer or its complications. The term "cancer" as used herein includes all cancers, including but not limited to "gynecological cancers." In one embodiment, gynecological cancers include, but are not limited to, tubal metaplasia, ovarian serous borderline neoplasms, serous adenocarcinoma, low-grade mucinous neoplasms, and endometrial tumors. In a specific embodiment, a gynecological cancer is an ovarian neoplasm undergoing abnormal Mullerian epithelial differentiation. Other gynecological conditions considered herein include inflammatory conditions such as endometriosis. As indicated above, the invention extends to a wide range of cancers in both male and female subjects.

[0208] As used herein, the term "sample" means any sample containing cancer cells that people wish to detect, including but not limited to biological fluids (including blood, plasma, serum, ascites), tissue extracts, freshly harvested cells, and lysates of cells that have been incubated in cell cultures. In a particular embodiment, the sample is gynecological tissue, blood, serum, plasma, or ascites.

[0209] As indicated above, a “subject” can be any mammal, typically a human, suspected of having or having a gynecological condition or other cancer. A subject can be referred to as a patient and is a mammal suspected of having or having cancer or at risk of developing cancer. The term “condition” also includes complications arising from it.

[0210] The term "control sample" includes any sample that can be used to build a first knowledge base of data from subjects with known disease states.

[0211] The methods of this invention can be used for the diagnosis and staging of cancers such as gynecological cancers, including ovarian cancer. The invention can also be used to monitor the progression of a condition and to monitor the effectiveness of a particular treatment. In particular, the methods can be used to confirm the absence or relief of symptoms of a condition, such as after surgery, chemotherapy, immunotherapy, and / or radiotherapy. The methods can also be used to monitor chemotherapy and the recurrence of abnormal tissue.

[0212] As indicated above, antibodies can be used in any of many immunoassays that depend on the binding interaction between the antigenic determinant of a biomarker and the antibody. Examples of such assays include radioimmunoassays, enzyme immunoassays (e.g., ECLIA, ELISA), immunofluorescence, immunoprecipitation, latex agglutination, hemagglutination, and histochemical assays. Antibodies can be used to detect and quantify the levels of biomarkers in samples to determine their role in cancer and to diagnose cancer.

[0213] In particular, the antibodies of the present invention can also be used for immunohistochemical analysis, for example at the cellular and subcellular levels, to detect biomarkers, localize them to specific cells and tissues, and at specific subcellular locations, and quantify their expression levels. In addition to antibodies, the drug can be any affinity reagent, including but not limited to aptamers, monospecific antibodies, anticalins, DARPin, and nanobodies.

[0214] Cytochemical techniques known in the art for locating antigens using light and electron microscopy can be used to detect cells carrying the KRT14 extracellular domain. Typically, the antibodies of the present invention can be labeled with detectable substances, and based on the presence of the detectable substances, biomarker proteins can be localized in tissues and cells. Examples of detectable substances include, but are not limited to, the following: radioactive isotopes (e.g., 3 H, 14 C 35 S, 125 I, 131 1) Fluorescent markers (e.g., FITC, rhodamine, lanthanide phosphors, luminescent markers such as luminol); enzyme markers (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase); biotinylate groups (detectable by labeled avidin, e.g., streptavidin containing fluorescent markers or enzyme activity detectable by optical or calorimetric methods); and predetermined polypeptide epitopes recognized by a second reporter (e.g., leucine zipper pairs, binding sites of secondary antibodies, metal-binding domains; epitope tags). In embodiments, the markers are attached via spacer arms of various lengths to reduce potential steric hindrance. Antibodies may also be conjugated to electron-dense substances, such as ferritin or colloidal gold, which are easily visualized by electron microscopy.

[0215] Antibodies or samples can be immobilized on carriers or solid supports capable of immobilizing cells, antibodies, etc. For example, carriers or supports can be nitrocellulose or glass, polyacrylamide, gabbro, and magnetite. Support materials can have any possible configuration, including spherical (e.g., beads), cylindrical (e.g., the inner surface of a test tube or well, or the outer surface of a rod), or flat (e.g., sheets, test strips). Indirect methods can also be used, where the initial antigen-antibody reaction is amplified by introducing a secondary antibody specific to the antibody that reacts with the biomarker protein. For example, if the antibody specific to the KRT14 extracellular domain is a rabbit IgG antibody, the secondary antibody could be goat anti-rabbit gamma globulin labeled with the detectable substance described herein.

[0216] When radiolabeled substances are used as detectable materials, the KRT14 biomarker can be located via autoradiography. The results of autoradiography can be quantified by determining the density of particles in the autoradiography using various optical methods or by counting the particles.

[0217] Labeled antibodies against KRT14 can be used to locate tumor tissue in patients undergoing surgery, i.e., for imaging. Typically, for in vivo application, the antibodies are labeled with radiolabels (e.g., iodine-123, iodine-125, iodine-131, gallium-67, technetium-99, and indium-111). The labeled antibody preparation can be administered intravenously to the patient in a suitable carrier several hours to four days prior to tissue imaging. During this period, unbound portions are cleared from the patient, leaving only those antibodies that have associated with the tumor tissue. The presence of the isotope is detected using a suitable gamma camera. The labeled tissue can be correlated with known markers on the patient's body, allowing the surgeon to precisely locate the tumor.

[0218] Therefore, in another embodiment, the present invention provides a method for detecting cancer in a patient, the method comprising:

[0219] (a) Provide samples from patients;

[0220] (b) The sample is contacted with an agent that binds to the KRT14 cellular epitopes to determine its level, and the level is algorithmically processed to provide a probability index of the patient having cancer; and

[0221] (c) Diagnose the risk of a patient having cancer based on a probability index.

[0222] In another embodiment, the present invention provides a method for detecting circulating KRT14-positive cancer cells in a patient, the method comprising:

[0223] (a) Provide blood samples from the patient;

[0224] (b) Contact the blood sample with an agent that binds to the outer surface of KRT14 cells to determine the presence of KRT14-positive cancer cells in the sample.

[0225] The methods described herein can be performed using pre-packaged diagnostic kits containing the necessary reagents for carrying out any of the methods of the invention. For example, the kit may include at least one specific antibody against the extracellular portion of KRT14, which can be conveniently used, for example, in a clinical setting to screen and diagnose patients, and to screen and identify individuals exhibiting a predisposition to cancer development. The kit also includes a detailed description of the methods for carrying out the invention.

[0226] This invention also provides an algorithm-based screening assay for screening samples from patients. Typically, input data is collected based on KRT14 levels and processed by an algorithm to assess the statistical significance of any increase or decrease in levels; this information is then the output data. This invention includes computer software and hardware for evaluating the input data.

[0227] The assays of this invention allow for integration into existing or newly developed pathology architectures or platform systems. For example, this invention contemplates a method that allows a user to determine a subject's status regarding cancer or its subtype or stage, the method comprising:

[0228] (a) Data on the presence of the extracellular portion of KRT14 is received via a communication network;

[0229] (b) Processing subject data via an algorithm that provides disease index values;

[0230] (c) Determine the subject's condition based on the comparison of disease index values ​​with predetermined values; and

[0231] (d) Transmitting instructions on the subject's status to the user via a communication network.

[0232] In another embodiment disclosed herein, a method for monitoring cancer in a patient is provided, the method comprising:

[0233] (a) Provide the patient with a blood sample at the first point in time;

[0234] (b) The sample from (a) was contacted with an agent that binds to the outer surface of KRT14 cells to determine the level of KRT14-positive cancer cells in the sample;

[0235] (c) Provide a blood sample from the patient at a second time point, where the first time point is different from the second time point;

[0236] (d) Contact the sample from (c) with an agent that binds to the KRT14 cellular epitopes to determine the level of KRT14-positive cancer cells in the sample; and

[0237] (e) Determine whether there is a change in the level of KRT14 positive cancer cells in patients between the first and second time points;

[0238] The change in the level of KRT14 positive cancer cells in patients between the first and second time points indicates a change in the cancer status of the patients.

[0239] Such methods can be appropriately used to monitor changes in cancer status or stage (e.g., in response to therapy) or to detect recurrence (e.g., after tumor removal).

[0240] In one implementation plan, the cancer is a gynecological cancer. In another implementation plan, the gynecological cancer is ovarian cancer or a stage or form of ovarian cancer. In yet another implementation plan, the cancer is selected from brain cancer, bladder cancer, liver cancer, breast cancer, lung cancer, pancreatic cancer, intestinal cancer, colon cancer, gastrointestinal cancer, stomach cancer, laryngeal cancer, endometrial cancer, and colorectal cancer.

[0241] The "algorithm" or "algorithmic function" mentioned above includes the performance of multivariate analysis functions. In addition to the architectures and platforms described above, a range of different architectures and platforms can be implemented. It should be understood that any architecture suitable for implementing this invention can be used. Example

[0242] The aspects disclosed herein are further described through the following non-limiting embodiments. The following materials and methods may be used.

[0243] Cell culture. Cell lines OVCAR-4 (NIH-OVCAR4) and CaOV-3#HTB-75 were purchased from ATCC and NIH. OVCAR-4 cells were maintained in Roswell Park Memorial Institute medium-1640 (RPMI) (Life Technologies, 21870092); CaOV-3 cells were maintained in Dulbecco modified Eagle medium (DMEM) (Thermo Scientific, #11965118); SKOV3 cells (… #HTB-77 TMCells were maintained in Dulbecco modified Eagle medium (DMEM) / Ham's F-12 (DMEM / F12) (Thermo Scientific, #11965118); COV362.4 and Sigma-Aldrich (Sigma #07071904) were maintained in high-glucose Dulbecco modified Eagle medium (DMEM-HG); BT-16 atypical teratoid / rhabdoid tumor (CVCL_M156) and NCI-H1573 lung adenocarcinoma (NCI-H1573) were both maintained at Rosswel Park Memorial Institute (RPMI); MDA-MB-468 triple-negative breast cancer cells (CVCL_0419) were maintained in DMEM; ANE CA (ATCC HTB-11) were maintained in Eagle minimum essential medium (EMEM); and SW 620 cells (ATCC CCL-227) were maintained in Leibovitz's L-15 medium. All media were supplemented with 10% fetal bovine serum (FCS) (Thermo Fisher, #16000044) and 1% penicillin-streptomycin (Thermo Scientific, #15240062). The ID8 mouse epithelial OC cell line (Dr. Kathy Roby, Kansas University Medical Center, Kansas City, KA, USA) was grown in Gibco DMEM (Thermo Fisher Scientific) containing 4% fetal bovine serum (FBS), 1% insulin-transferrin-selenite (ITS), and 1% penicillin / streptomycin (PS). The human mesothelial cell line LP9 (Coriell Institute Cell Repository #AG07086) was maintained in HamsF12 / 199 medium containing 10% v / v FCS, 1% v / v penicillin-streptomycin, 10 ng / ml EGF, and 0.4 μg / ml hydrocortisone. All cell lines were maintained at 37°C with 5% v / v CO2 and cell viability was counted using a Countess IIFL automated cell counter prior to all assays. With patient consent, non-adhesive tumor cells were obtained from malignant ascites using an established purification method (Latifi et al. (2012) PLoS ONE 7(10)) and maintained under hypoadhesive conditions prior to analysis by culturing on hypoadhesive plates in MCDB:F12 medium and 10% v / v FCS.

[0244] CRISPR-mediated gene silencing was performed using KRT14 targeting and overexpression. CRISPR-mediated gene silencing was conducted according to the Zhang laboratory protocol (Cong et al. (2013) Science 339(6121):819-23), using three guide strands per gene. In DMEM, Lipofectamine 2000 transfection reagent (Invitrogen, #11668019) was used with guide strands (1-3), a non-targeted control, or the KRT14 overexpression construct KRT14, according to the manufacturer's protocol. OE Cells were transfected with Origene (RC214907). After transfection and a 12-hour recovery period, cells were passaged into selective medium and maintained under selective pressure by adding 1 μg / ml puromycin (Sigma-Aldrich, #P8833) or genimycin (trademark) selective antibiotic (G418 sulfate) (LifeTechnologies Australia #10131-035). Cells were subjected to limiting dilutions, with the selective medium changed every two days for approximately two weeks. Single colonies were amplified, and the knockdown of the target gene was measured by Western blot analysis and validated by Sanger sequencing.

[0245] Human tissue arrays and immunohistochemistry. Immunohistochemistry was performed on tissue microarray (TMA) sections (tumors and fallopian tubes) purchased from USBIOMAX (#ov2085, #ov20811) or generated internally, as previously described [Bilandzic et al. (2014) Cancer Lett 354(1):107-114; Rainczuk et al. (2013) J Proteome Res; Salamonsen et al. (2013) Fertil Steril 99(4):1086-92] (Supplementary data 6 and 7). For antigen retrieval, sections were incubated in 50 mM glycine (pH 3.5) at 90 °C for 10 min. Sections were incubated overnight at 4°C with Rb-KRT14 antibody (1:100, Sigma, SAB4501657) and mAb AN-17 (1:500) in 0.1% w / v BSA / PBS. Subsequent steps were performed at room temperature, with washing with PBS between incubations. Sections were incubated for 1 hour with goat anti-rabbit IgG peroxidase conjugate (1:1000, Dako, Glostrup, Denmark; catalog item PO448), biotinylated rabbit anti-goat IgG antibody (1:1000, Vector Laboratories Cat. No: BA-5000), or biotinylated rabbit anti-mouse IgG antibody (1:1000, Vector Laboratories Cat. No: BA-9200), followed by incubation with the Vectastain Elite ABC kit according to the manufacturer's instructions (Vector Laboratories, Burlingame, California). After development with 3,3′-diaminobenzidine tetrahydrochloride, antibody binding detection showed a brown precipitate, and Harris hematoxylin was used as a counterstain. Sections were fixed under glass coverslips in Depex (BDH Laboratory Supplies, Poole, United Kingdom). Positive immunostaining was evaluated relative to parallel sections exposed to an isotype (IgG) control. Immunostaining in tumor and stromal tissues was evaluated using Aperio ImageScope (v 12.3.3), as described above (Rainczuk et al. (2013)).

[0246] Western blot analysis. SDS-PAGE and Western blot were performed as previously described in Bilandzic et al. (2013) Mol Endocrinol, 2013.27(3):466-79. The blots were detected using antibodies against KRT14 (1:1000, SAB4501657), mAb AN-17, and β-actin (1:20,000; Sigma-Aldrich, Castle Hill, Australia). HRP-conjugated goat anti-mouse, anti-rabbit, and donkey anti-goat secondary antibodies (1:50,000; Merck Millipore, Kilsyth, Australia) (Bilandzic et al. (2013) Mol Endocrinol, 2013.27(3):466-79). Protein bands were detected using Clarity Western ECL blot substrate (Biorad #1705061) and visualized using the ChemiDoc MP system (Bio-Rad #1708280).

[0247] xCELLigence Real-Time Cell Analysis (RTCA). Real-time cell analysis (RTCA) was performed using an xCELLigence RTCASP 96-well analyzer (ACEA Biosciences). Cell lines were synchronized to the G0 phase by overnight incubation in serum-free medium before starting. For proliferation assays, cells were fed at a rate of 0.5 × 10⁶ cells / well. 3 Cells were seeded at 0.14 ml / well (as outlined in the experimental text), and impedance readings were acquired every 5 minutes for 8 hours (to monitor cell adhesion), followed by impedance readings every 15 minutes for 24 hours (to monitor cell proliferation). For invasion assays, the upper chamber of the CIM-16 well plate was coated with Matrigel matrix (1:10 in SFM; BD Biosciences, San Jose, CA). Cells were seeded into the upper chamber (as described above), and + / - 10% v / v FBS was added to the lower chamber. All assays were performed in duplicate or triplicate, with at least three independent experiments.

[0248] Peritoneal microenvironment model. To establish a peritoneal microenvironment model, a two-compartment RTCA CIM plate was prepared by coating the upper compartment with Matrigel (1:10 in SFM; BDBiosciences), and then adding 7×10⁻⁶ ppm. 4LP9 cells / well were seeded and monitored until a confluent monolayer was formed (Domcke et al. (2013) Nat Commun 4:2126). Spheroids (obtained from fresh patient ascites; 10 spheroids / well) were seeded in SFM in the upper chamber, and medium ± 10% v / v FBS was added to the chamber. Real-time readings were used to determine the optimal time for use as the collection point for MALDI imaging analysis, with all samples prepared in either a two-well or three-well assay. As an additional control, we also performed concurrent endpoint invasion assays in parallel using a modified Boyden chamber. In this case, mesothelial LP9 cells were labeled with Cell Trace (trademark) CFSE prior to seeding of the ovarian cancer spheroids. Mesothelial invasion was assessed based on the retraction of CSFE-labeled mesothelial cells beneath the spheroids using a Cytation (trademark) 3 multimodal imager (BioTek Instruments, Winooski USA).

[0249] Samples were prepared for MALDI-IMS. The spheroid-mesothelial interface was co-cultured on Thermanox (trademark) sectionable coverslips and covered with agar at time points corresponding to pre-invasion, during-invasion, and post-invasion (measured by RTCA assay). Samples were sectioned at 5 μm, and the invasion interface was located by H&E staining of periodically selected sections. After identification, the two unstained portions across the interface were placed on indium tin oxide (ITO) slides for MALDI treatment (Bruker Daltonik, GmbH).

[0250] MALDI IMS. Trypsin-hydrolyzed peptides at the ovarian cancer-globulus-mesothelial interface were identified using the ImageID workflow (Bruker). Trypsin was applied to serial tissue sections by nebulization using an ImagePrep spray device (Bruker). Samples were digested in a humidified chamber for 90 min, followed by extraction and purification of peptides using C18 pipette tips. LC-MALDI analysis was performed using an ultrafleXtreme MALDI-TOF / TOF (Bruker) and a Dionex Ultimate 3000RSLC system (Thermo), as described (Rainczuk et al. (2014) Int J Cancer 134(3):530-41). MALDI images of subsequently digested serial sections were then acquired using flexImaging 4.1 (Bruker), as previously described (Rainczuk et al. (2014) above). ImageID software (Bruker) was used to compare and filter LC-MALDI and MALDI image data, and to match quality peaks between the image data and LC-MALDI analysis. The quality tolerance for peak matching was automatically calculated by ImageID software.

[0251] Methylcellulose covering and spheroid formation. Ovarian cancer cells were dissociated by trypsin digestion and resuspended in complete cell culture medium (98% minimum viability, as determined by a counteress cell counter). 2,500 cells / spheroid were covered in serum-free medium with 0.25% w / v methylcellulose (Sigma Aldrich, Castle Hill, Australia) and seeded into single wells of 96-well CELLSTAR (registered) U-bottom suspension plates (Greiner Bio One, Interpath Services PTY, Vic, Australia). Spheroid aggregation and formation for each cell line were observed using an optical microscope and imaged at regular intervals. The formed spheroids were collected using a large-bore pipette tip and centrifugation.

[0252] Mesothelial displacement assay. Human mesothelial cell line LP9 was seeded as described above and incubated at 37°C until a confluent monolayer formed. Ovarian cancer globules were collected (as described above), and 16 globules were seeded into wells containing the confluent mesothelial monolayer. Phase-contrast microscopy was used to image mesothelial displacement and outgrowth at regular intervals.

[0253] In vitro wound repair assay. Ovarian cancer cells (or other cancer cell lines—BT16, NCI-H1573, AN3CA, SW620, and MDA-MB-468) were grown to confluence in complete medium in 12-well plates, followed by serum starvation overnight to synchronize with G0. The next day, the cell culture medium was removed, and the cell monolayer was injured by scraping with a pipette tip attached to the pipette. Non-adherent cells were removed by gentle washing with PBS and replaced with complete growth medium with or without mAb AN-17 (1 μg / ml) or commercial KRT14 polyclonal antibody (1 μg / ml). Wound areas were imaged at regular intervals ranging from 0 to 72 h under a phase-contrast microscope (Leica). Wound closure was measured in the image series using AnalySIS LS Research Software (Olympus) to determine the wound area each day. Experiments were repeated in triplicate, with at least six wound areas observed for each growth condition.

[0254] Matrigel and Collagen I Growth Halo Formation Assay: Staining and Imaging. The protocol was adapted from Nguyen-Ngoc et al. (2012) Proc Natl Acad Sci US A.109(39):E2595-604. Briefly, ovarian cancer spheroids were collected to produce a suspension of 6 spheroids / matrix. The spheroids were embedded in 3D Matrigel (354230; BD Biosciences) or rat tail collagen I (354236; BD Biosciences). Cultures were set in 8 wells on a coverslip slide (94.6190.802, Starstedt) as described above by Nguyen-Ngoc et al. (2012). For antibody staining, spheres cultured in Matrigel or Collagen I were fixed with 4% w / v paraformaldehyde for 30 min, rinsed twice in PBS for 10 min each time, permeabilized with 0.5% v / v Triton X-100 in PBS for 20 min, rinsed twice in PBS for 10 min each time, and blocked in 10% v / v FBS in PBS at room temperature for 2 h. They were then incubated overnight at 4°C with primary antibodies (1:1000 anti-N cadherin antibody [5D5]ab98952 AbCam and 1:500 KRT14). The next day, the samples were washed three times with PBS and incubated for 3 h at room temperature with secondary antibodies (1:2000 goat anti-rabbit IgG Alexa 467, ab150083 and 1:2000 goat anti-mouse IgG Alexa 488, ab150117), followed by three rinses in PBS for 10 min each time. The samples were imaged using a Cytation 3 multi-mode imager (BioTek Instruments, Winooski USA) with Gen5 Image+ software or a Nikon C1 confocal microscope (Monash MicroImaging Facility, Monash).

[0255] Real-time PCR. Total RNA was extracted from primary high-grade serous ovarian tumors (n=3) and entire normal ovaries (n=3) using the Tissue Lyser LT system with 5mm stainless steel beads and the RNeasy Mini Kit (Qiagen). Total RNA was extracted from the following samples grown as monolayers or spheroids (KRT14) using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. KOOvarian cancer cell lines OVCAR4 and CaOV3 (and wild-type lines); ovarian cancer (n=3) or benign fibroadenoma (n=2) spheroids derived from ascites; and the target peritoneal cell layer LP-9. Sensitive and antisense oligonucleotide primers targeting KRT14, HNRN, FNDC3B, 18S, and CDCA8 were designed against published human sequences and validated as previously described (Bilandzic et al. (2009) Mol Endocrinol, 23(4):539-48). cDNA was synthesized using Superscript III reverse transcriptase (Life Technologies, Grand Island, NY). Real-time PCR samples were prepared to a final volume of 10 μl using Applied Biosystems ABISYBR mix (Scoresby, Victoria, Australia). As previously described (Bilandzic et al. (2009) above), quantitative real-time PCR was performed using an Applied Biosystems ABI 7900HT Fast real-time PCR machine, with all reactions performed in triplicate. Based on the standard curve for each PCR product, the yield was converted to femtograms, and the resulting mRNA levels were normalized to 18S mRNA levels per sample.

[0256] Statistical analysis. Statistical analysis was performed using GraphPad Prism (version 6; GraphPad Software Inc., San Diego, CA). As indicated, for data derived from cell assays, means were compared using one-way or two-way ANOVA with Bonferroni, Dunnett, or Tukey post-hoc tests. To determine whether there were significant differences in mRNA expression between samples, the Mann-Whitney U test or unpaired t-test was performed. Means were considered significant if p < 0.05. All experiments were independently repeated at least three times.

[0257] Kaplan-Meier curves. The Kaplan-Meier online plotting tool (http: / / kmplot.com / analysis / ) was used to generate survival curves using mRNA data from patients with serous ovarian cancer from 15 public ovarian cancer datasets, where the optimal cutoff value was automatically selected by the plotting tool, and the log-rank, p-value, and hazard ratio (and 95% confidence interval) were calculated (Lanczky et al. (2016) Breast Cancer Res Treat. 3(160): 439-446).

[0258] Example 1

[0259] Adhesion and proliferation cannot predict a cell's invasive ability.

[0260] Metastatic ovarian cancer cells interact with the mesothelial monolayer that serves as the lining of the peritoneum and organs, invading and attaching to the underlying stroma to establish secondary nodules (Kenny et al. (20017) Int J Cancer 121(7):1463-72; Burleson et al. (2006) J Transl Med 4:6; Sodek et al. (2012) Cancer Metastasis Rev 31(1-2):397-414). Mesothelial translocation was assessed using tumor cells derived from primary ascites to evaluate the emergence of invasive filopodia from globules over a prolonged timeframe in vitro. At the start of the assay, globules from benign or malignant samples were of similar size and showed no significant morphological differences. Extensive growth halo formation and clearance of the underlying mesothelial layer occurred within 24 hours in all malignant samples; in contrast, benign globules showed no clear evidence of growth halo formation or invasion. The lack of invasion was not due to failed adhesion or reduced cell proliferation; in fact, in the RTCA assay, benign cells showed relatively increased adhesion to both uncoated and fibronectin-coated culture plates and obtained a higher proliferation index than malignant cell samples. These data demonstrate that only malignant cells exhibit invasive ability; and that invasive potential cannot be predicted from cell adhesion or proliferation ability in vitro.

[0261] Example 2

[0262] Proteomic profiling identifies proteins with unique invasion interfaces.

[0263] Previous studies have not directly examined proteins at the interface between actively invasive cancer cells and mesothelial cells. To assess the abundance and localization of invasion-related proteins, spheroid / mesothelial co-cultures were harvested after attachment to the mesothelial cell line but before the onset of invasion (as determined by RTCA assays). Mesothelial attachment, but not invasion, was confirmed in samples used for MALDI IMS analysis using parallel endpoint Boyden chamber assays.

[0264] Cell-spheroid interface cultures were embedded in agarose, sliced, and localized by IHC. Figure 1A); then serial sections were analyzed by IMS to identify proteins located at the invasion interface. The analysis also included spheroids (not shown) derived from ascites in patients with benign fibromas to provide a control for inter-sample atypia. MALDI IMS and subsequent LC-MALDI-MS / MS identified 26 proteins uniquely present at the spheroid / mesothelial interface in co-cultures containing malignant rather than benign spheroids. Several proteins previously associated with ovarian cancer (e.g., HSP90, AMH, and OSM) were present [Vesci et al. (2014) Int J Oncol 45(4):1421-9; Liu et al. (2013) Clin Cancer Res 19(18):5053-67; Kim et al. (2014) Obstet Gynecol Sci. 57(5):343-57; Richards (2013) ISRN Inflammation 2013:23], validating the method and suggesting that these proteins may play an important role in the early stages of invasion. Further restrictions on the analysis were imposed to: (i) include only those proteins identified in each high-grade serous ovarian cancer (HGSC) sample; and (ii) exclude proteins also identified in the mesothelial cell monolayer. Following this highly rigorous method, four proteins (KRT14, HRNR, CDCA8, and FNDC3B) were identified as unique to HGSC cells from all patients at the cancer-mesothelial interface. Immunostaining for TMA and RT-PCR on fresh frozen tissue were also used to confirm the expression and localization of candidate proteins in independent tumor tissues compared to histologically normal ovarian tissue.

[0265] Example 3

[0266] KRT14 on the invasion interface is essential for the invasion of ovarian cancer cells.

[0267] The abundance of HRNR, KRT14, CDCA8, and FNDC3B was detected in several HGSC cell lines (OVCAR3, OVCAR4, and CaOV3) by Western blotting (Domcke et al. (2013) above). Consistent with proteomic analysis, HRNR, KRT14, and CDCA8 were detected in cancer cell lysates but not in mesothelial cell controls. FNDC3B was detected in LP9 mesothelial cells and excluded from further analysis. KRT14, CDCA8, and HRNR were then knocked out (CaOV3 and OVCAR4 cell lines) using CRISPR, and their specific loss in clonal populations was confirmed by sequencing PCR and Western blotting. The effect of functional KRT14, CDCA8, or HRNR loss on cell proliferation and invasion was tested by RTCA. Cells lacking HRNR or CDCA8 showed significantly reduced proliferation (not shown) compared to untreated or non-targeted controls; in contrast, KRT14 loss did not affect proliferation (…). Figure 2 This indicates that it is not essential for tumor cell viability or growth. Both CDCA8 knockout cells and HRNR knockout cells also retained invasive ability; CDCA8 knockout cells showed similar invasion dynamics to untreated or untargeted cells, while HRNR knockout cells showed a lag at the onset of invasion. However, cells lacking functional KRT14 showed a complete loss of invasive ability. Figure 2 A), no invasion was observed after 30 hours (or after an extended period of up to 7 days). KRT14-mediated loss of invasiveness was confirmed in a 2D wound healing assay. Figure 2 B), where KRT14 knockout (KRT14 KO The cells failed to repair the damaged monolayer after 48 hours. Therefore, further research focuses on KRT14 as a key gene controlling the invasive ability of malignant ovarian cancer cells.

[0268] Example 4

[0269] Peritoneal microenvironment model

[0270] Using a peritoneal microenvironment model, KRT14 expression was detected at the earliest stage of metastasis at the “leading edge” of invasive ovarian cancer cells. These cells were defined as “leading cells.” In this model, ovarian cancer spheroids covered a mesothelial monolayer on a matrigel matrix established in a CIM-16RTCA plate. Real-time monitoring of spheroid attachment and invasion through the mesothelial monolayer / matrix using the xCELLigence instrument provided a dynamic snapshot of invasive cell behavior.

[0271] Spheroids from patients with benign (ovarian fibroma) or malignant (HGSC) disease were isolated from ascites and their invasiveness was assessed. Malignant HGSC cells rapidly invaded the mesothelial monolayer, with all samples exhibiting active invasion within 4 hours of addition. In contrast, spheroids obtained from patients with benign fibroma failed to disrupt the mesothelial monolayer. Therefore, in vitro, the initiation of cancer cell invasion occurs rapidly upon contact with the mesothelial monolayer, indicating the timeframe for analyzing early events involved in invasion.

[0272] Example 5

[0273] Real-time in vitro invasion assay

[0274] Real-time in vitro invasion assay (Bilandzic and Stenvers (2014) J Vis Exp 87) was used to measure the invasion of ovarian cancer cells through the mesothelial monolayer, demonstrating KRT14 (K14 KO Genetic ablation completely eliminated the invasive ability of various ovarian cancer cell lines (CVAR4 and CaOV3). Figure 2 The invasive ability of primary ovarian cancer cells (n=5) recovered from ascites fluid was also assessed (data not shown). Loss of KRT14 expression had no effect on cell viability and proliferation, consistent with other studies (Papafoliou et al. (2016) above; Rock et al. (2009) Proc Natl Acad Sci USA 106(31):12771-12775). KRT14 KO Ovarian cancer cells also failed to repair damaged cell monolayers in wound healing assays, demonstrating a loss of migration ability. Figure 2 B). It was also observed that KRT14 cultured into multicellular spheroids... KO Ovarian cancer cells showed reduced binding to the mesothelial monolayer and failed to initiate mesothelial clearance (not shown), which is a key requirement for invasion in epithelial ovarian cancer (EOC) (Iwanicki et al. (2011) Cancer Discov 1(2):144-157). In vivo studies using an syngeneic mouse model of ovarian cancer (Roby et al. (2000) Carcinogenesis 21(4):585-591) demonstrated the effectiveness of intracystic implantation of KRT14 in mice. KO Ovarian cancer cells failed to establish a tumor ( Figure 3 ); and KRT14 was implanted. KO The mice that developed the cells did not develop bloating or any other symptoms.

[0275] Example 6

[0276] Migrating cells showed increased KRT14 expression.

[0277] KRT14 mRNA expression was measured in migrating cells from the following clinical samples: (i) primary tumor tissue; (ii) HGSC cells derived from ascites; and (iii) benign cells; (iv) histologically normal ovaries; and (v) isolated target peritoneal cell layer LP9 (n = 3 / group). All malignant cells expressed KRT14 at the start of the assay, while expression was not detected in benign fibromas, normal ovaries, or LP9 mesothelial cells. Consistent with cell lines, migrating cells were detected only in malignant samples (i.e., tumor tissue or ascites-derived); cells isolated from benign ascites, normal controls, or isolated LP9 cells failed to invade. Invasive cells that had migrated into the lower chamber were significantly enriched in KRT14 compared to pre-migrating samples from the primary tumor sample, with the highest KRT14 mRNA levels observed in the ascites-derived ovarian cancer cell population. In summary, the data indicate that although KRT14 has no effect on cell viability or proliferation, it is particularly needed to maintain the invasive potential of a subset of migratory cancer cells in vitro; and it is significantly enriched in actively invasive cells.

[0278] Example 7

[0279] KRT14 is restricted to a subset of HGSC cells that influence globule assembly and adhesion to the mesothelium.

[0280] Immunostaining was used to determine the abundance of KRT14 and its localization in ovarian cancer cells (both immortalized and primary ascites-derived). In monolayer cultures, KRT14 was confined to a few isolated cells, while in spheroids cultured under low-adhesion conditions, KRT14 immunostaining showed localization only at the outer edge of the spheroids. The absence of internal KRT14 staining was not due to antibody occlusion from the spheroids, as anti-N-cadherin antibodies effectively penetrated, staining the entire spheroid. KRT14 was examined... KO and KRT14 overexpression (KRT14) OE This study aimed to assess whether KRT14 expression is essential for in vitro spheroid formation. Wild-type OVCAR4 cells and cells transfected with a non-targeted CRISPR control formed spheroids after 12 hours in low-adhesion cultures; in contrast, KRT14 knockout cells remained largely dispersed after 12 hours. However, prolonged incubation (48 hours) resulted in the formation of spheroids with comparable size and morphology to the control. Conversely, KRT14... OE The strain rapidly forms dense and compact spheres, and after a 12-hour culture period, a visible growth halo of the original spheres is clearly visible. Compared to the untreated control, KRT14... KO The spheroids have a significantly reduced ability to adhere to the mesothelial monolayer in vitro.

[0281] Example 8

[0282] KRT14+ cells lead to invasive pseudopodia formation and mesothelial clearance.

[0283] When inoculated onto the mesothelial monolayer, wild-type HGSC spheroids exhibited growth halo formation, mesothelial clearance, and extensive deposition and migration within 24 hours. Cells overexpressing K14 caused rapid dispersion and translocation of the mesothelial layer; in contrast, KRT14... KO Cells failed to disrupt the mesothelial monolayer. To examine whether matrix type affected invasion, globules (both cell lines and ascites-derived HGSC cells) were embedded in Matrigel or collagen-I matrix, and invasive pseudopodia growth halo formation was monitored over time. KRT14+ invasive pseudopodia emerged from wild-type globules 12 hours after being placed in collagen-I matrix, but required 48–72 hours (depending on cell type) to become visible in Matrigel. Immunostaining showed that KRT14+ cells specifically localized to invasive pseudopodia, with the non-invasive globule core cells maintaining the KRT14- phenotype. This was confirmed by monolayer scratch assays, in which KRT14+ cells specifically localized to the wound closure area. Consistent with their lack of invasiveness, KRT14+ cells… KO Cells failed to form visible invasive pseudopodia in either matrix. In summary, the data suggest that KRT14 expression is a characteristic of actively invasive cells, and its loss significantly impairs the ability of globules to spread during tumor growth halo formation and to cause mesothelial translocation.

[0284] Example 9

[0285] KRT14 is associated with tumor stage and is negatively correlated with predicting progression-free survival in ovarian cancer patients.

[0286] To determine the clinical relevance of KRT14 expression, a tissue microarray (n=292) comprising various histological subtypes, ovarian cancer grades and stages, and normal ovarian and fallopian tube sections was stained to obtain KRT14 abundance and localization. KRT14 expression was generally undetectable in normal ovarian (5%, 1 / 20) or fallopian tube (0%, 0 / 8) tissues, but was pervasive in all examined ovarian cancer subtypes. Staining localized to the tumor epithelium, with little evidence of KRT14 in the stromal tissue. In particular, KRT14 was detected in 100% of HGSC tissues, and was significantly elevated compared to normal ovaries (p=0.0362, with a post-hoc unpaired t-test by Tukeys).

[0287] We then inquired about the potential association between KRT14 expression and patient prognosis in 15 publicly available ovarian cancer datasets (http: / / www.cbioportal.org / ) [Lanczky et al. (2016) above]. High KRT14 expression was associated with reduced progression-free survival (PFS) (HR 1.17; 95% CI 1.03–1.33, P < 0.015), particularly for patients diagnosed with early-stage (stage I–II) disease (HR 1.96; 95% CI 1.08–3.56, P < 0.025). High KRT14 expression was also associated with decreased PFS following platinum- and paclitaxel-based chemotherapy (HR 1.27; 95% CI 1.07–1.51, P < 0.006) and was a negative predictor of PFS after optimal debulking (HR 1.24; 95% CI 1.03–1.5, P < 0.026). Therefore, patients with shallow deletion of KRT14 are more sensitive to chemotherapy and show improved responses to primary therapy. Thus, KRT14 expression is an independent predictor of prognosis for patients with high-grade serous ovarian cancer.

[0288] Example 10

[0289] Implanted in mice

[0290] After KRT14 was implanted KO In mice with these cells, no tumor deposits or even fluorescent tumor cells were detected during necropsy, indicating that the tumor not only failed to implant but was subsequently removed postoperatively. Figure 3 Using flow cytometry and immunocytochemical staining of intact cells, the N-terminus of KRT14 was observed to be exposed on the cell surface. Figure 4 Consistent with other studies (Papafotiou et al. (2016) above; Rock et al. (2009) above), KRT14+ cells represent only a subset of the tumor cell population. Figure 4 A). Using polyclonal antibodies targeting the N-terminus or C-terminus of KRT14, it was confirmed that antibodies targeting the N-terminus could prevent in vitro invasion, mimicking the effect of KRT14 gene knockout. Figure 4 B). Anti-C-terminal antibodies had no effect on invasion and were consistent with the intracellular localization of the well-defined C-terminal region of KRT14. Figure 4 B). The exogenously added full-length recombinant KRT14 protein can competitively bind to antibodies and restore in vitro invasion ( Figure 4(B) Therefore, the loss of invasive ability is directly due to the binding of the KRT14 antibody. Furthermore, this effect is mediated by the binding of the antibody to the N-terminus of KRT14; and the N-terminus is exposed on the outer side of the cell, making it accessible for antibody binding.

[0291] Example 11

[0292] Antigenicity and hydrophobicity diagram

[0293] Antigenicity and hydrophobicity maps were used to examine the first 200 amino acids of KRT14 using a computer. Figure 5 A). Five potential antigenic regions were predicted, and six peptides containing these regions were synthesized for further competitive assays. Blockade of N-terminal antibodies by peptides #4 and #5 restored ovarian cancer cell invasion in vitro. Figure 5 B) and migration Figure 5 C). Despite being recognized by the polyclonal antibody used, peptides 1, 2, 3, and 6 failed to regain full invasive and / or migratory capabilities. Based on antibody-peptide competition assays, the surface-exposed region of the KRT14 protein was defined within the amino acid sequence NH2-GFGGGYGGGLGAGLGGGFGGGFAGGDGL (SEQ ID NO: 1). This amino acid sequence has been used to immunize mice to generate monoclonal antibodies targeting the N-terminus of KRT14 in intact cells.

[0294] Example 12

[0295] Antiserum for KRT14

[0296] Functional testing was used to evaluate the ability of the inventors' internal mouse antiserum AN-17 O20023, generated targeting a protein fragment containing a specific KRT14 epitope, to block cancer cell invasion in vitro.

[0297] Real-time invasion assays were performed using a xCELLigence Real-Time Cell Analysis (RTCA) DP 6-well instrument (ACEABiosciences). Ovarian cancer cells (SKOV3) were synchronized to the G0 phase by incubation in serum-free medium (SFM) and seeded (4 x 10⁻⁶ cells / well). 4Cells / well were placed in the upper chamber of a CIM-16 well plate coated with Matrigel matrix (1:10 in SFM; BD Biosciences, San Jose, CA). Medium containing 10% v / v FBS was added to the lower chamber as a chemoattractant. Antiserum from mouse AN-17 O20023 (diluted 1:100 in SFM), control serum, or a commercially available polyclonal antibody against KRT14 (Sigma SAB4501657; 1 μg / ml in SFM) was added to the upper chamber, and invasion measurements were taken every 15 minutes for 24 hours. Cell viability was assessed at the end of the assay by alpha blue staining. All assays were performed in duplicate. Antiserum AN-17O20023 was generated against a protein fragment containing a specific KRT14 epitope.

[0298] In untreated and serum-only control wells, invasion was observed within 5 hours of inoculation. Figure 6 A). Commercially available polyclonal anti-KRT14 (Sigma SAB4501657: which recognizes a specific KRT14 epitope) showed inhibition of invasion. Antiserum AN-17O20023 effectively blocked in vitro invasion, with potency similar to purified anti-KRT14 polyclonal antibodies. Figure 6 A) demonstrates its effective target recognition. There was no significant difference in viability at the endpoint between untreated cells and control or antiserum-treated cells. Figure 6 B) indicates that the lack of invasion is not due to impaired proliferative capacity.

[0299] The antiserum AN-17O20023 effectively blocked the in vitro invasion ability of ovarian cancer cells, with potency similar to commercially available formulations. This effect was not due to impaired cell proliferation, but rather to specificity against invasion, as previously observed. This antibody is suitable for characterization as a lead compound in ongoing studies.

[0300] Example 13

[0301] Effects on non-ovarian cancer cells

[0302] Anti-KRT14 antibodies were used to test whether they could prevent the invasion of cancer cells from other (non-ovarian) solid tumor types.

[0303] In vitro wound repair assays were performed using the AN3CA endometrial cancer and SW620 colorectal cancer cell lines. Cells were grown to confluence in complete culture medium in 12-well plates and then serum starved overnight to synchronize with G0. The next day, the cell culture medium was removed, and a monolayer of cells was injured by scraping with a pipette tip attached to the pipette. Non-adherent cells were removed by gentle washing with PBS. Cells were incubated in complete growth medium + / - alone with commercial KRT14 antibody (1 μg / ml) or in combination with a 1 μg / ml competing peptide. Wound areas were imaged at regular intervals ranging from 0 to 72 hours under a phase-contrast microscope (Leica). Wound closure was measured in the image series using AnalySIS LS Research Software (Olympus) to determine the wound area per day. Experiments were repeated in triplicate, with at least six wound areas observed for each growth condition, and data were collected from the presented 16-hour collection points.

[0304] After 16 hours of culture, both endometrial cancer and colorectal cancer cell lines showed impaired wound healing in the presence of anti-KRT14 antibody. Figure 7 In the presence of a competing KRT14 epitope, wound healing capacity was restored to the level observed in the untreated control.

[0305] Anti-KRT14 antibodies inhibited the migration behavior of colorectal cancer and endometrial cancer cell lines, similar to the inhibition observed in ovarian cancer cells. As demonstrated by competition assays, the inhibition was specific to a defined KRT14 epitope. Data suggest that antibody-mediated KRT14 inhibition may act as a pan-cancer mechanism to suppress invasion in multiple solid tumor types.

[0306] Example 14

[0307] Cross-species action of antibodies

[0308] The ability of anti-human KRT14 antibody to prevent invasion of non-human ovarian cancer cells was evaluated.

[0309] All experiments were performed using the mouse ID8 ovarian cancer cell line. Real-time invasion assays (according to Example 5) and in vitro wound repair assays (according to Example 6) were performed as described.

[0310] Anti-KRT14 antibodies specific to the human KRT14 epitope effectively blocked the migration of mouse cancer cells in vitro. Figure 8 A). Peptide competition assays (as described in Example 6) restored the migration ability of these cells. Figure 8 A) demonstrates specificity for the KRT14 epitope.

[0311] Real-time invasion assays confirmed that the anti-KRT14 antibody blocked the invasion of ID8 cells in vitro. Figure 8 B), whose efficacy is similar to that observed in human cell lines.

[0312] The KRT14 epitope exhibits high homology (>80%) across multiple species. Therefore, antibodies targeting the human epitope can effectively bind to non-human proteins to inhibit cell migration and invasion in vitro. These data suggest that anti-KRT14 therapy may be applicable across species.

[0313] Example 15

[0314] Synergistic effect of mAb AN-17 with standard care chemotherapy in vitro

[0315] This study was conducted to determine whether targeting the cell surface antigen KRT14 with the monoclonal antibody AN-17 (mAb AN-17), which has been shown to impair migration and invasion (as described elsewhere in this article), would sensitize ovarian cells to the standard first-line chemotherapy agent cisplatin.

[0316] Real-time cell analysis (RTCA) was performed as previously described (PMID: 31443478; Bilandzic et al., Cancers (Basel). 2019; 11(9), E1228) using an xCELLigence RTCA SP 96-well instrument (ACEABiosciences). Cell lines were synchronized to G0 by overnight incubation in serum-free medium and at 0.5 × 10⁻⁶ cells / well. 3 0.15 ml / well of cells were seeded. After 24 hours, cisplatin alone (1.25 mg / ml), mAb AN-17 alone (1 μg / ml), or a combination of cisplatin and mAb AN-17 were added. Impedance readings were acquired every 15 minutes during the experiment. mAb AN-17 contains the heavy chain variable region of SEQ ID NO:3 and the light chain variable region of SEQ ID NO:5, encoded by the nucleic acid sequences of SEQ ID NO:2 and SEQ ID NO:4, respectively (see also...). Figure 21 and Figure 22 ).

[0317] Treatment of cells with cisplatin at 15 μg / ml or 20 μg / ml resulted in complete cell death, while a dose of 1.25 μg / ml was sublethal, and cells continued to proliferate. Figure 9 As previously demonstrated, mAb AN-17 alone had no effect on cell viability or proliferation. However, at each dose tested, co-incubation of cells with mAb AN-17 and cisplatin significantly reduced cell viability and proliferation compared to chemotherapy alone. Figure 9The most pronounced effect was observed when cisplatin was used at a sublethal dose of 1.25 μg / ml; co-incubation with mAb AN-17 resulted in complete cell death within ~50 hours. Figure 9 This is similar to the effect of a 12-fold higher dose of cisplatin alone. Therefore, mAb An-17 exerts an effect that increases the sensitivity of cells to standard platinum chemotherapy by at least 10-fold in vitro.

[0318] Example 16

[0319] mAb AN-17 showed no detectable cross-reactivity to a group of up to 10,000 antigens.

[0320] The following experiments were conducted to determine whether mAb AN-17 showed any cross-reactivity to a group of protein antigens.

[0321] Invitrogen was used, containing recombinant human protein from 9,184 individuals sampled in duplicate. TM ProtoArray TM Human protein microarray v5.0 (ThermoFisher Scientific, WalthamMA) was used for proteomic analysis of mAb AN-17 antibody reactivity. All procedures were performed as previously described (PMID: 29141850; Wilson et al. Cancer Epidemiol Biomarkers Prev. 2018; 27(2): 183-192). mAb AN-17 probe arrays were used with a 1:500 dilution in wash buffer. Fluorescent detection antibodies against IgG heavy and light chains were derived from Abcam (#ab150119 goat anti-mouse IgG H&L Alexa). 647 (pre-adsorbed) is diluted to 2 mg / ml in wash buffer before use. A single array incubated with a separate detection antibody is used as a control for nonspecific antibody binding.

[0322] Array imaging was performed using a Fuji FLA5100 multiwavelength scanner as described (PMID: 29141850; Wilson et al., Cancer Epidemiol Biomarkers, Prev. 2018; 27(2): 183-192), with a 635 nm excitation laser and dual bandpass Cy3 / Cy5 filters. Images were acquired at a resolution of 10 μm, with the PMT set at 1000 V. Array alignment, feature extraction, and data normalization were performed as previously described (PMID: 29141850; Wilson et al., Cancer Epidemiol Biomarkers, Prev. 2018; 27(2): 183-192). Array coordinates were obtained from a downloadable .GAL file provided by the manufacturer (ThermoFisher Scientific; http: / / www.lifetechnologies.com / au / en / home / life- science / protein-expression-and-analysis / biomarker-discovery / protoarray / resources / lot-specific-information.html Obtained from ).

[0323] Figure 10 The image shows individual array images illustrating the reactivity of the antigen with mAb AN-17 or a secondary antibody alone. No significant differences were found between the control and mAb AN-17-treated arrays, indicating that mAb AN-17 does not react with any proteins present on the tested arrays.

[0324] These data demonstrate the high specificity of mAb AN-17 for its target antigen (KRT14) in vitro. mAb AN-17 does not react with any antigens present on the array, including several associated keratins. Therefore, mAb AN-17 exhibits high affinity for its target antigen.

[0325] Example 17

[0326] mAb AN-17 was used to detect KRT14 via Western blotting.

[0327] A 1 μg aliquot of the full-length recombinant KRT14 protein (Abcam#ab73637) was separated by SDS-PAGE and transferred to a PVDF membrane using a standard procedure (PMID:23952987). The membrane was probed with mAb AN-17 at dilutions of 1:1000, 1:5000, and 1:10,000. The secondary antibody was a goat anti-mouse HRP conjugate (1:50,000 dilution), in which KRT14 was detected using chemiluminescence according to a standard protocol (PMID:23952987; Rainczuk et al., J Proteome Res. 2013; 12(9):4074-88).

[0328] Western blotting using mAb AN-17 as the primary antibody successfully detected the full-length KRT14 protein. Figure 11 Multiple dilutions yielded strong signals.

[0329] Example 18

[0330] mAb AN-17 was used to identify KRT14+ leader cells and circulating tumor cells by flow cytometry.

[0331] Flow cytometry was performed according to the standard protocol (PMID: 30602661), with data acquisition using a BD LSRFortessa X-20 (BD Biosciences) and analysis using FlowJo software v10.5.0 (BD Biosciences). Samples used to evaluate the KRT14+ cell population included OVCAR3, CAOV4, and ID8 mouse ovarian cancer cells, ascites-derived ovarian cancer cells from clinical samples (specified #3.1937-07), and cardiac blood from mice bearing epithelial ovarian tumors at 12 weeks of age. For cell lines, 1x102 cells were used. 6 Cells / cell lines were incubated with mAb AN-17 (1:200 dilution) or a commercially available anti-KRT14 polyclonal antibody (Sigma SAB4501657; 1:50 dilution) as a positive control in non-immune serum at room temperature for 45 min. Secondary antibodies, goat anti-mouse Alexa647 (mAb AN-17) or goat anti-rabbit IgG Alexa 488 (commercial Ab) diluted 1:500, were incubated for 30 min. For blood samples, 2 x 10⁻⁶ cells / cell lines were incubated in non-immune serum. 6 individual cells and Alexa Cells were incubated with 488-conjugated mAbAN-17 antibody for 45 minutes. After washing with PBS, the cells were resuspended in PBS / 2% FBS, and data were acquired using a BD LSRFortessa X-20 (BD Biosciences) flow cytometer.

[0332] Flow cytometry analysis of KRT14-expressing cells, in cell lines (human-OVCAR4, CAOV4; mouse-ID8) and clinically acquired ovarian cancer cells (3:19367-03), showed similar results using mAb AN-17 compared to the commercially available polyclonal antibody Sigma SAB4501657. Figure 12 ).

[0333] Extracellular KRT14 expression also marked the circulating tumor cell (CTC) population. Using a mouse ovarian cancer model previously described in PMID:30602661 (Wilson et al., Cancers (Basel). 2018; 11(1), E32), CTCs specifically expressed the near-infrared fluorophore iRFP720. Whole blood from mice with ovarian cancer was probed using mAb AN-17, and KRT14+iRFP+ cells were analyzed by flow cytometry. mAb AN-17 was able to correctly identify iRFP+ cells from whole blood, demonstrating its utility in identifying and capturing CTCs.

[0334] mAb AN-17 identified KRT14+ cells by flow cytometry with sensitivity comparable to commercially available anti-KRT14 polyclonal antibodies (Sigma SAB4501657). Furthermore, mAb AN-17 was able to detect and isolate KRT14+ CTCs from mice bearing ID8iRFP720+ epithelial ovarian tumors by flow cytometry. Therefore, anti-KRT14 binders, such as mAb AN-17, have the potential for diagnostic and / or prognostic testing, including the potential for capturing and analyzing CTCs. Data also suggest that anti-KRT14 binders, such as mAb AN-17, could be used for in vivo therapeutic targeting of these cells.

[0335] Example 19

[0336] mAb AN-17 was detected in KRT14+ cells by immunofluorescence staining.

[0337] Ovarian cancer cell lines SKOV-3, Ovcar4, and Cov362.3 were seeded onto 96-well black fluorescent imaging plates. Intact or fixed (1% paraformaldehyde; PFA) and permeabilized (0.1% Triton X) cells were stained with mAb AN-17. For fixed cells, samples were blocked in 10% fetal bovine serum (FBS) and then stained with 1 μg / ml mAb AN-17 at room temperature for 1 hour. Cells were then washed with PBS and stained with Alexa at a dilution of 1:2000. Alexa-647 goat anti-mouse secondary antibody was used for staining at room temperature for 1 hour. For intact cells, samples were stained with 1 μg / ml mAb AN-17 conjugated with Alexa-647 in PBS containing 1% FBS at 37°C for 2 hours. The medium was replaced with fresh PBS / 1% FBS, and the cells were imaged immediately. Samples were imaged at 4X magnification using a Cytation 3 multimode reader.

[0338] Specific intracellular cytoplasmic localization of mAb AN-17 was observed in permeabilized ovarian cancer cell line samples. Figure 14In intact living cells, major cell surface localization of mAb AN-17 was observed.

[0339] These data demonstrate that mAb AN17 can be effectively used for staining KRT14 in cells via immunofluorescence. Staining is effective in both live and intact cells as well as in fixed and permeabilized cells.

[0340] Example 20

[0341] mAb AN-17 was used to detect KRT14+ cells in cancer tissue sections by immunohistochemical staining.

[0342] Immunohistochemistry of formalin-fixed paraffin-embedded (FFPE) samples was performed using a standard protocol (PMID: 31443478; Bilandzic et al., Cancers (Basel). 2019; 11(9), E1228). After overnight incubation, the potency of mAb AN-17 across a concentration range (2 μg / ml–0.25 μg / ml) was tested and compared with a commercially available anti-KRT14 antibody (Sigma SAB4501657, 1:100 dilution) and a mouse IgG control. As described, after incubation with the secondary antibody, antibody binding and localization were visualized as a brown precipitate using the Vectastain Elite ABC kit according to the manufacturer's instructions (Vector Laboratories, Burlingame, CA, USA).

[0343] Tissue stained with mAb A-17 showed the same localization as the commercial anti-KRT14 polyclonal antibody (Sigma SAB4501657), with staining specifically observed in tumor epithelium. Figure 15 There was little evidence of staining in the stromal tissue, and no staining was observed in the mouse IgG control. Signal abundance was less diffuse compared to a commercially available polyclonal antibody (SigmaSAB4501657).

[0344] These data show that mAb AN-17 can be effectively used to locate KRT14 in tumor tissue sections.

[0345] Example 21

[0346] mAb AN-17 showed no detectable acute toxicity in vivo.

[0347] Induction of ovarian tumors; mouse ID8 ovarian tumor cells were implanted into C57BL / 6 cysts in mice and the primary tumor was allowed to develop over a period of ~4 weeks, as previously described (PMID:30602661; Wilson et al. Cancers (Basel). 2018; 11(1), E32)).

[0348] Assessment of mAb AN-17 toxicity: Mice (n = 2 / dose / time point) received a single dose of mAb AN-17 or an isotype-matched control IgGκ antibody (Ultra-LEAF™ purified mouse IgG1, κ isotype control, Biolegend #401411) via intraperitoneal injection, and were then monitored for any clinical signs of toxicity over a period of up to 7 days. Doses of 0.5 mg / kg, 1.0 mg / kg, 2.5 mg / kg, 5.0 mg / kg, and 10 mg / kg were evaluated. After 7 days, mice were sacrificed, and any evidence of toxicity (macroscopic histological appearance, presence of inflammation, or any obvious lesions) was examined postmortem.

[0349] Evidence of toxicity was assessed in all mice (tumor and non-tumor) throughout the experimental period (initial reactions after injection, signs of distress), as well as post-mortem evidence of toxicity (as described above). No evidence of toxicity was observed at any time point or at any dose tested.

[0350] Example 22

[0351] mAb AN-17 specifically locates in tumor tissues in vivo and is not retained in healthy tissues.

[0352] Label mAb AN-17 or an allotype-matched control IgGκ antibody (Ultra-LEAF) at a 1:10 ratio with ALEXA647 or ALEXA750 fluorescent dye (Thermo Fischer Scientific) according to the manufacturer's instructions. TMPurified mouse IgG1,κ isotype control (Biolegend#401411). Mice (n=2 / group) received ALEXA-labeled mAb AN-17 or isotype control IgGκ (as described above) via intraperitoneal injection (100 μl). Control mice received a separate medium (PBS). Mice were euthanized and selected tissues (liver, kidney, spleen, intestine, ovary + fallopian tube / ovarian tumor (as applicable), brain, heart, lung) were harvested at time points from 4 hours to 7 days post-injection. Tissue distribution and clearance were assessed using an IVIS Lumina III imaging system (Perkin Elmer) via ALEXA647 or ALEXA750 fluorescence. Bright-field (auto-exposure) and fluorescence imaging (ALEXA 647 ex 640, em 670; or ALEXA 750 ex 740, em 790 nm) were performed. Spectral unmixing and image analysis were performed as previously described (PMID: 30602661; Wilson et al., Cancers (Basel). 2018 Dec 31; 11(1), E32). In each case, the background mean radiative efficiency from the animal treated with the medium was subtracted from the fluorescence measurements, and the resulting data were plotted for comparison.

[0353] Following a single dose of 0.5 mg / kg, the localization of mAb AN-17 in healthy tissues (using mice without ovarian tumors) over a 7-day period was compared with that of a non-targeting isotype control antibody. In each case, the antibody was fluorescently labeled for post-mortem detection. Antibody-related fluorescence was observed in the intestine, reproductive organs, liver, kidney, spleen, lung, and heart; no fluorescence was detected in the brain. Figure 16 However, in mAb AN-17 and isotype control IgG... K There were no significant differences between the antibodies, demonstrating that mAb AN-17 did not specifically accumulate in any of the healthy tissues examined. Furthermore, by day 7, each antibody had been substantially cleared from all organs (as judged by the loss of fluorescence signal), indicating that mAb AN-17 is not long-term persistent. Figure 16 ).

[0354] Since antibody clearance appeared to be completed on day 7 at low doses, clearance of mAb AN-17 from healthy tissues was evaluated in a similar manner at multiple escalating doses (0.5 mg / kg to 10 mg / kg). Even at the highest dose (10 mg / kg), mAb AN-17 was undetectable in almost all organs after 7 days and was generally present at levels lower than the control antibody. Figure 17These data indicate that mAb AN-17 does not exhibit non-selective accumulation or retention in healthy tissues.

[0355] The distribution of mAb AN-17 in mice with primary ovarian tumors was then evaluated using single doses of 5 mg / kg and 10 mg / kg. Evaluation was performed over a 7-day period. At 24 hours post-administration, mAb AN-17 was strongly localized to tumor tissue. Figure 18 Unlike healthy tissue, mAb AN-17 persists in tumor tissue and can be detected even after 7 days. Figure 18 High fluorescence yields were initially observed in mice receiving a 10 mg / kg dose; however, by day 3 post-injection, similar levels of mAb AN-17 were detected regardless of the dose, suggesting that tumors may have been saturated at a 5 mg / kg dose. Furthermore, the initial levels of mAb AN-17 detected in tumor tissue were two orders of magnitude higher than the initial levels detected in the reproductive tract of tumor-free mice (compare). Figure 18 (Figures A, B, and C)

[0356] These data show that mAb AN-17 is specific to tumor tissue and persists at the tumor site for at least 1 week after administration.

[0357] The data presented in this article demonstrate that mAb AN-17 exhibits high specificity for its target (KRT14), minimal off-target effects, and low retention in non-target tissues. In healthy, non-tumor-bearing mice, mAb AN-17 showed no specific retention in healthy organs and appeared to be rapidly cleared. In contrast, mAb AN-17 was specifically detected in association with ovarian tumors and persisted in ovarian tumors for at least 1 week. No toxicity was observed at any dose or at any time point, indicating that the high specificity of mAb AN-17 confers a favorable safety profile at injection. Furthermore, no maximum tolerated dose was reached. Therefore, mAb AN-17 demonstrates a favorable safety profile and high in vivo specificity for tumor tissues.

[0358] Example 23

[0359] Treatment with mAb AN-17 successfully induced regression of established ovarian tumors in vivo.

[0360] The aim of these experiments was to determine whether mAb AN-17 could be used to influence tumor progression in established primary malignant tumor models in syngeneic mouse models.

[0361] Mouse ID8 ovarian tumor cells were intracystically implanted into C57BL / 6 mice, allowing primary tumor development for a period of approximately 4 weeks, as previously described (PMID: 30602661). Mice with primary tumors (n = 10 / group) received 5 mg / kg mAb AN-17 twice weekly via intraperitoneal injection for 3 weeks. Control animals received an equal dose of isotype-matched control IgGκ antibody (control group 1); or / and an equal volume of a separate mediator (PBS) (control group 2). After 3 weeks of treatment twice weekly (Monday and Thursday), all mice were sacrificed and tumor size and weight were assessed. Two additional non-tumor-bearing animals served as non-surgical controls.

[0362] Twice-weekly administration of mAb AN-17 had no observable adverse effects on the animals, as previously indicated by the single-dose experiment (above). After 3 weeks of continuous treatment, all mice were sacrificed, and the effect of mAb AN-17 administration on tumor size was assessed. In mice treated with the medium alone (control group 2), 6 / 10 animals (60%) had primary ovarian tumors. Figure 19 Similarly, 6 out of 10 mice (60%) treated with the same type of control antibody (control group 1) also developed primary tumors, demonstrating that the non-targeted antibody had no effect on tumor progression.

[0363] When mice receiving mAb AN-17 were analyzed, tumors could not be identified in the right ovary (implantation site), on the right ovary, or in any other location in the animals. Figure 19 Furthermore, the ovaries extracted from these mice appeared healthy and showed no observable morphological differences compared to untreated, non-surgical controls.

[0364] Treatment of mice with mAb AN-17 resulted in complete regression of established primary ovarian tumors to undetectable levels after 3 weeks. No adverse events were observed during the treatment period, indicating that mAb AN-17 administration is a safe and highly effective treatment for established solid tumors in vivo.

[0365] Example 24

[0366] mAb AN-17 blocks migration and invasion in various cancer cells.

[0367] The experiments summarized above demonstrate the antibody-specific inhibition of cell migration and invasion by mAb An-17 in ovarian cancer, colorectal cancer, and endometrial cancer cell lines. The following experiments were conducted to investigate the effects of mAb AN-17 on the migration and invasion of other cancer cell types.

[0368] In vitro wound repair assays were performed on the following cancer cell lines: BT16 atypical teratoid rhabdomyosarcoma (brain) carcinoma, NCI-H1573 lung adenocarcinoma, SJ-GBM2 primary glioblastoma multiforme, AN3CA endometrial carcinoma, SW620 colorectal cancer, and MDA-MB-468 breast cancer. Cells were grown to confluence in complete culture medium in 12-well plates and subsequently serum starved overnight to synchronize with G0. The next day, cell culture medium was removed, and cell monolayers were injured by scraping with pipette tips attached to pipettes. Non-adhesive cells were removed by gentle washing with phosphate-buffered saline (PBS) in the absence or presence of mAb AN-17 (at 1 μg / ml), and complete growth medium was added to each well. Wound areas were imaged hourly from 0 to 24 hours. Wound closure was measured in the image series using AnalySIS LS Research Software (Olympus) to determine the wound area per day. The experiment was repeated in triplicate, with at least six wound areas observed under each growth condition.

[0369] like Figure 23 As shown, when challenged using an in vitro wound healing assay, untreated cells were able to migrate and close the wound after 16 hours. In contrast, under the same conditions, all cell lines treated with mAb AN-17 failed to close the wound. These data demonstrate that mAb AN-17 inhibits cell migration and invasion of cancer cells, consistent with similar effects previously shown in colorectal cancer, endometrial cancer, and various ovarian cancer cell lines.

[0370] These data demonstrate that KRT14 antagonists inhibit the migration behavior of multiple cancer cell types, including at least ovarian cancer, endometrial cancer, brain cancer, lung cancer, and breast cancer cells. The distinct nature of these cancer types suggests that KRT14 antagonists, such as mAb AN-17, target highly conserved pathways in cancer cells and are therefore suitable for the diagnosis, prognosis, and treatment of various tumor types.

[0371] Those skilled in the art will understand that, apart from those specifically described herein, the disclosure described herein is susceptible to change and modification. It should be understood that all such changes and modifications are taken into account in this disclosure.

[0372] This disclosure also implements all steps, features, compositions, and compounds individually or collectively mentioned or indicated in this specification, as well as any and all combinations of any two or more steps or features or compositions or compounds.

[0373] bibliography:

[0374] Altschul et al.(1997)Nucl.Acids.Res.25:3389

[0375] Ausubel et al.(1994-1998)In:Current Protocols in Molecular Biology,John Wiley&Sons Inc.

[0376] Bilandzic et al.(2009)Mol Endocrinol,23(4):539-48

[0377] Bilandzic et al.(2013)Mol Endocrinol,2013.27(3):466-79

[0378] Bilandic et al.(2014)Cancer Lett 354(1):107-114

[0379] J Vis Exp 87 , Bilandic and Stenvers ( 2014 )

[0380] Burleson et al.(2006)J Transl Med 4:6

[0381] Proc Natl Acad Sci USA 112(15):4725-4730

[0382] Cheung et al.(2013)Cell 155(7):1639-1651

[0383] Cheung et al.(2016)Proc Natl Acad Sci USA 113(7):E854-863

[0384] Chothia et al.(1987)J.Mol.Biol.196:901

[0385] Chothia et al.(1989)Nature 342:877-883

[0386] Chu et al.(2001)Histopathology 39(1):9-16

[0387] Coligan et al.Current Protocols in Immunology,1991-1997

[0388] Cong et al.(2013)Science 339(6121):819-23

[0389] Domcke et al.(2013)Nat Comnun 4:2126

[0390] Ewert et al.(2002)Biochemistry 41:3628-2636

[0391] Greenberg et al.(1995)Nature 374:168-173

[0392] Gefter et al.(1977)Somatic Cell Genet.3:231-236

[0393] Ho et al.(2012)Nat Rev Urol 9(10):583-594

[0394] Iwanicki et al.(2011)Cancer Discov 1(2):144-157

[0395] Jones et al.(1986)Nature 321:522-525

[0396] Kenny et al.(20017)Ini J Cancer 121(7):1463-72

[0397] Kim et al.(2014)Obstet Gynecol Sci.57(5):343-57

[0398] Kohler et al.(1975)Nature 256:495-499

[0399] Kohler et al.(1976)Eur.J.Immunol.6(7):511-519

[0400] Kozbor et al.(1986)Methods in Enzymology 121:140

[0401] Lanczky et al.(2016)Breast Cancer Res Treat.3(160):439-446

[0402] Latifi et al.(2012)PLoS ONE 7(10)

[0403] Liu et al.(1987)Proc.Natl.Acad.Sci.USA 84:3439-3443

[0404] Liu et al. (2013) Clin Cancer Res 19(18): 5053-67

[0405] Nguyen-Ngoc et al.(2012)Proc Natl Acad Sci US A.109(39):E2595-604

[0406] Nuttall et al.(2001)Mol Immuunol 38:313-326

[0407] Nuttall et al.(2002)FEBS Lett 516:80-86

[0408] Nuttall et al.(2003)Eur J Biochem 270:3543-3554

[0409] Nuttall et al.(2004)Proteins 55:187-197

[0410] Padlan(1994)Mol Immunol 31:169-217

[0411] Papafotiou et al.(2016)Nat Commun 7:11914

[0412] Paraskevopoulou et al. (2016) Cell Cycle 15(23): 3161-3162

[0413] Rainczuk et al.(2013)J Proteome Res

[0414] Rainczuk et al.(2014)Int J Cancer 134(3):530-41

[0415] Richmann et al.(1988)Nature 332:323-327

[0416] Richards(2013)ISRN Inflammation 2013:23

[0417] Roby et al.(2000)Carcinogenesis 21(4):585-591

[0418] Rock et al.(2009)Proc Natl Acad Sci USA 106(31):12771-12775

[0419] Roux et al.(1998)Proc Natl Acad Sci USA 95:11804-11809

[0420] Salamonsen et al.(2013)Fertil Steril 99(4):1086-92

[0421] Shulman et al.(1978)Nature 276:269-270

[0422] Sodek et al.(2012)Cancer Metastasis Rev 31(1-2):397-414

[0423] Stanfield et al.(2004)Science 305:1770-1773

[0424] Streltsov et al.(2004)Proc Natl Acad Sci USA 101:12444-12449

[0425] Streltsov et al(2005)Protein Sci 14:2901-2909

[0426] Toyama et al.(1987)Monoclonal Antibody,Experiment Manual,published byKodansha Scientific)

[0427] Trowbridge(1982)J.Exp.Med.148(1):220-227

[0428] Verhoeyen et al. (1988) Science 239: 1534-1536

[0429] Vesci et al. (2014) Int J Oncol 45(4): 1421-9

[0430] Volk et al.(1982)J.Virol.42(1):220-227

[0431] Volkmer et al. (2012) Proc Natl Acad Sci USA 109(6):2078-2083. sequence list <110> Hudson Institute of Medicine <120> Treatment and prevention methods <130> 35538517 <150> 2019900382 <151> 2019-02-07 <160> 19 <170> PatentIn version 3.5 <210> 1 <211> 28 <212> PRT <213> Homo sapiens <400> 1 Gly Phe Gly Gly Gly Tyr Gly Gly Gly Leu Gly Ala Gly Leu Gly Gly 1 5 10 15 Gly Phe Gly Gly Gly Phe Ala Gly Gly Asp Gly Leu 20 25 <210> 2 <211> 354 <212> DNA <213> House mouse (Mus musculus) <400> 2 caggtgcagc tgaaggagtc aggacctggc ctggtggcac cctcacagag cctgtccatc 60 acatgcactg tctctgggtt ctcattatcc agatatagtg tacactgggt tcgccagcct 120 ccaggaaagg gtctggagtg gctgggaatg atatggggtg gtggaagcac agactataat 180 tcagctctca aatccagact gagcatcagc aaggacaact ccaagagcca agttttctta 240 aaaatgaaca gtctgcaaac tgatgacaca gccatgtact actgtgccag aaaagactac 300 ggctactcct actttgacta ctggggccaa ggcaccactc tcacagtctc ctca 354 <210> 3 <211> 118 <212> PRT <213> Mus musculus <400> 3 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Arg Tyr 20 25 30 Ser Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Gly Gly Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Arg Lys Asp Tyr Gly Tyr Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> 4 <211> 363 <212> DNA <213> Mus musculus <400> 4 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 120 tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcca 300 ttcacgttcg gctcggggac aaagttggaa ataaaacggg ctgatgctgc accaactgta 360 tcc 363 <210> 5 <211> 121 <212> PRT <213> Mus musculus <400> 5 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser 115 120 <210> 6 <211> 8 <212> PRT <213> Mus musculus <400> 6 Gly Phe Ser Leu Ser Arg Tyr Ser 1 5 <210> 7 <211> 7 <212> PRT <213> House mouse (Mus musculus) <400> 7 Ile Trp Gly Gly Gly Ser Thr 1 5 <210> 8 <211> 12 <212> PRT <213> House mouse (Mus musculus) <400> 8 Ala Arg Lys Asp Tyr Gly Tyr Ser Tyr Phe Asp Tyr 1 5 10 <210> 9 <211> 11 <212> PRT <213> House mouse (Mus musculus) <400> 9 Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 10 <211> 3 <212> PRT <213> House mouse (Mus musculus) <400> 10 Lys Val Ser 1 <210> 11 <211> 9 <212> PRT <213> House mouse (Mus musculus) <400> 11 Phe Gln Gly Ser His Val Pro Phe Thr 1 5 <210> 12 <211> 25 <212> PRT <213> House mouse (Mus musculus) <400> 12 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser 20 25 <210> 13 <211> 17 <212> PRT <213> Mus musculus <400> 13 Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly 1 5 10 15 Met <210> 14 <211> 38 <212> PRT <213> Mus musculus <400> 14 Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu Ser Ile Ser Lys Asp Asn 1 5 10 15 Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp 20 25 30 Thr Ala Met Tyr Tyr Cys 35 <210> 15 <211> 11 <212> PRT <213> Mus musculus <400> 15 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 1 5 10 <210> 16 <211> 26 <212> PRT <213> House mouse (Mus musculus) <400> 16 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser 20 25 <210> 17 <211> 17 <212> PRT <213> House mouse (Mus musculus) <400> 17 Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 18 <211> 36 <212> PRT <213> House mouse (Mus musculus) <400> 18 Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly 20 25 30 Val Tyr Tyr Cys 35 <210> 19 <211> 19 <212> PRT <213> House mouse (Mus musculus) <400> 19 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro 1 5 10 15 Thr Val Ser

Claims

1. Use of an agent that targets an extracellular portion of keratin 14 (KRT14) residing on a cancer cell, or an agent that induces production of an antagonist of the extracellular portion of KRT14 on a cancer cell, in the manufacture of a medicament for treating or preventing cancer in a mammalian subject, wherein the agent prevents or reduces cancer cell invasion, migration, and / or metastasis of the cancer cell in the mammalian subject, wherein the extracellular portion of KRT14 is defined by SEQ ID NO: 1; wherein the cancer is one or more of ovarian cancer, brain cancer, lung adenocarcinoma, primary glioblastoma multiforme, endometrial cancer, colorectal cancer, and breast cancer, and wherein the agent comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein: (i) the VH comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 6, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 7, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 8, and (ii) the VL comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO: 9, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 10, and a CDR3 consisting of the amino acid sequence of SEQ ID NO:

11.

2. The use of claim 1, wherein the cancer is ovarian cancer or a stage of ovarian cancer.

3. The use of claim 1, wherein the mammalian subject is a human.

4. The use of claim 3, wherein the agent is SEQ ID NO: 1 or a functional homolog thereof.

5. The use of claim 4, wherein the antagonist is an antibody specific for an epitope within SEQ ID NO:

1.

6. The use of claim 5, wherein the antibody is a monoclonal antibody or a deimmunized form thereof.

7. The use of claim 1, wherein the agent is a vaccine that induces an immune response specific for a cancer cell comprising the extracellular portion of KRT14.

8. The use of claim 7, wherein the vaccine comprises an antagonist molecule that induces an antibody specific for SEQ ID NO:

1.

9. The use of claim 3, wherein the agent is an antibody specific for SEQ ID NO: 1 conjugated to a cytotoxic molecule.

10. Use of an antibody that targets an extracellular portion of keratin (KRT14) residing on an ovarian cancer cell, as determined by SEQ ID NO: 1, in the manufacture of a medicament for treating ovarian cancer in a human subject, wherein the antibody comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein: ​ ​ ​ ​ (i) the VH comprises a complementarity-determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO:6, a CDR2 consisting of the amino acid sequence of SEQ ID NO:7, and a CDR3 consisting of the amino acid sequence of SEQ ID NO:8, and (ii) the VL comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO:9, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 10, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 11, wherein the antibody is effective to prevent or reduce ovarian cancer cell invasion, migration, and / or metastasis of the ovarian cancer cell in the human subject.

11. A diagnostic reagent comprising an antibody specific for an extracellular portion of keratin 14 (KRT14) on keratin 14 (KRT14) positive cancer cells in a patient suffering from one or more of ovarian cancer, brain cancer, lung adenocarcinoma, primary glioblastoma multiforme, endometrial cancer, colorectal cancer, and breast cancer, wherein the extracellular portion of KRT14 is defined by SEQ ID NO: 1; and wherein the antibody comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein: (i) the VH comprises a complementarity-determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO:6, a CDR2 consisting of the amino acid sequence of SEQ ID NO:7, and a CDR3 consisting of the amino acid sequence of SEQ ID NO:8, and (ii) the VL comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO:9, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 10, and a CDR3 consisting of the amino acid sequence of SEQ ID NO:

11.

12. Use of an agent that binds to an extracellular epitope of KRT14 in the manufacture of a medicament for detecting keratin 14 (KRT14) positive cancer in a patient suffering from one or more of ovarian cancer, brain cancer, lung adenocarcinoma, primary glioblastoma multiforme, endometrial cancer, colorectal cancer, and breast cancer, wherein the detection comprises: (a) providing a sample from the patient; (b) contacting the sample with the medicament to determine its level, and algorithmically processing the level to provide an index of the probability that the patient has cancer of one or more of ovarian cancer, brain cancer, lung adenocarcinoma, primary glioblastoma multiforme, endometrial cancer, colorectal cancer, and breast cancer, the medicament binds to an extracellular epitope of KRT14 comprising SEQ ID NO: 1, and wherein the medicament comprises an agent comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein: (i) the VH comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO:6, a CDR2 consisting of the amino acid sequence of SEQ ID NO:7, and a CDR3 consisting of the amino acid sequence of SEQ ID NO:8, and (ii) the VL comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO:9, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 10, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 11; and (c) diagnosing the patient at risk of having cancer based on the probability index.

13. Use of an agent that binds to a KRT14 extracellular epitope in the manufacture of a medicament for detecting circulating keratin 14 (KRT14) positive cancer cells in a patient suffering from one or more of ovarian cancer, brain cancer, lung adenocarcinoma, primary glioblastoma multiforme, endometrial cancer, colorectal cancer, and breast cancer, and wherein the medicament comprises an agent comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein: (i) the VH comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO:6, a CDR2 consisting of the amino acid sequence of SEQ ID NO:7, and a CDR3 consisting of the amino acid sequence of SEQ ID NO:8, and (ii) the VL comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO:9, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 10, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 11; wherein the detecting comprises: (a) providing a blood sample from the patient; (b) contacting the blood sample with the medicament to determine the presence of KRT14 positive cancer cells in the sample, the medicament binding to an extracellular epitope of KRT14 comprising SEQ ID NO:

1.

14. Use of an agent that binds to a KRT14 extracellular epitope and comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL) in the manufacture of a medicament for monitoring keratin 14 (KRT14) positive cancer in a patient, wherein: (i) the VH comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO:6, a CDR2 consisting of the amino acid sequence of SEQ ID NO:7, and a CDR3 consisting of the amino acid sequence of SEQ ID NO:8, and (ii) the VL comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO:9, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 10, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 11, wherein the monitoring comprises: (a) providing a blood sample from the patient; (b) contacting the blood sample with the medicament to determine the presence of KRT14 positive cancer cells in the sample, the medicament binding to an extracellular epitope of KRT14 comprising SEQ ID NO:

1. said patient is afflicted with one or more of ovarian cancer, brain cancer, lung adenocarcinoma, primary glioblastoma multiforme, endometrial cancer, colorectal cancer, and breast cancer, wherein said monitoring comprises: (a) providing a blood sample of a patient at a first time point; (b) contacting the sample of (a) with the drug to determine the level of KRT14 positive cancer cells in the sample, the drug binding to an extracellular epitope of KRT14 comprising SEQ ID NO: 1; (c) providing a blood sample of a patient at a second time point, wherein the first time point is different from the second time point; (d) contacting the sample of (c) with the drug to determine the level of KRT14 positive cancer cells in the sample; and (e) determining whether there is a change in the level of KRT14 positive cancer cells in the patient between the first time point and the second time point; wherein a change in the level of KRT14 positive cancer cells in the patient between the first time point and the second time point is indicative of a change in the state of cancer in the patient.

15. An agent that specifically binds to an extracellular portion of a keratin (KRT14) on a cancer cell, wherein the agent comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH comprises a complementarity determining region 1 (VH CDR1) consisting of the amino acid sequence of SEQ ID NO: 6, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 7, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 8; and wherein the VL comprises a complementarity determining region 1 (VL CDR1) consisting of the amino acid sequence of SEQ ID NO: 9, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 10, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO:

11.

16. The agent of claim 15, wherein the VH comprises: (a) a VH framework region 1 (FR1) comprising the amino acid sequence of SEQ ID NO: 12; (b) a VH FR2 comprising the amino acid sequence of SEQ ID NO: 13; (c) a VH FR3 comprising the amino acid sequence of SEQ ID NO: 14; and (d) a VH FR4 comprising the amino acid sequence of SEQ ID NO: 15; and the VL comprises: (e) a VL FR1 comprising the amino acid sequence of SEQ ID NO: 16; (f) a VL FR2 comprising the amino acid sequence of SEQ ID NO: 17; (g) a VL FR3 comprising the amino acid sequence of SEQ ID NO: 18; and (h) a VL FR4 comprising the amino acid sequence of SEQ ID NO:

19. (h) VL FR4, which comprises the amino acid sequence of SEQ ID NO:

19.

17. The agent or KRT14-binding fragment thereof of claim 16, wherein: (a) the VH comprises the amino acid sequence of SEQ ID NO: 3, and (b) the VL comprises the amino acid sequence of SEQ ID NO:

5.

18. The use of any one of claims 4-6, 9, or 12-14, wherein the agent comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH comprises: a complementarity determining region 1 (VH CDR1) consisting of the amino acid sequence of SEQ ID NO: 6, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 7, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 8; and wherein the VL comprises: a complementarity determining region 1 (VL CDR1) consisting of the amino acid sequence of SEQ ID NO: 9, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 10, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

11.

19. The use of claim 18, wherein the VH comprises: (a) a VH framework region 1 (FR1) comprising the amino acid sequence of SEQ ID NO: 12; (b) a VH FR2 comprising the amino acid sequence of SEQ ID NO: 13; (c) a VH FR3 comprising the amino acid sequence of SEQ ID NO: 14; and (d) a VH FR4 comprising the amino acid sequence of SEQ ID NO: 15; and the VL comprises: (e) a VL FR1 comprising the amino acid sequence of SEQ ID NO: 16; (f) a VL FR2 comprising the amino acid sequence of SEQ ID NO: 17; (g) a VL FR3 comprising the amino acid sequence of SEQ ID NO: 18; and (h) a VL FR4 comprising the amino acid sequence of SEQ ID NO:

19.

20. The use of claim 19, wherein: (a) the VH comprises the amino acid sequence of SEQ ID NO: 3, and (b) the VL comprises the amino acid sequence of SEQ ID NO:

5.

21. The diagnostic agent of claim 11, wherein the antibody comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH comprises a complementarity determining region 1 (VH CDR1) consisting of the amino acid sequence of SEQ ID NO: 6, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 7, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 8; and wherein the VL comprises a complementarity determining region 1 (VL CDR1) consisting of the amino acid sequence of SEQ ID NO: 9, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 10, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO:

11.

22. The diagnostic agent of claim 21, wherein the VH comprises: (a) a VH framework region 1 (FR1) comprising the amino acid sequence of SEQ ID NO: 12; (b) a VH FR2 comprising the amino acid sequence of SEQ ID NO: 13; (c) a VH FR3 comprising the amino acid sequence of SEQ ID NO: 14; and (d) a VH FR4 comprising the amino acid sequence of SEQ ID NO: 15; and the VL comprises: (e) a VL FR1 comprising the amino acid sequence of SEQ ID NO: 16; (f) a VL FR2 comprising the amino acid sequence of SEQ ID NO: 17; (g) a VL FR3 comprising the amino acid sequence of SEQ ID NO: 18; and (h) a VL FR4 comprising the amino acid sequence of SEQ ID NO:

19.

23. The diagnostic agent of claim 22, wherein: (a) the VH comprises the amino acid sequence of SEQ ID NO: 3, and (b) the VL comprises the amino acid sequence of SEQ ID NO:

5.

24. The use of any one of claims 12-14 or the agent of any one of claims 15-17, wherein the cancer is one or more selected from ovarian cancer, brain cancer, lung adenocarcinoma, primary glioblastoma multiforme, endometrial cancer, colorectal cancer, and breast cancer.

25. The use or agent of claim 24, wherein the cancer is ovarian cancer or a stage or form of ovarian cancer.

26. The use of any one of claims 1-10 or 18-25, wherein the agent is formulated for administration simultaneously or sequentially with an additional anti-cancer agent and / or an additional therapy selected from the group consisting of immunotherapy, radiation therapy, and / or surgical intervention.

27. The use of claim 26, wherein the additional anti-cancer agent is selected from the group consisting of dactinomycin, daunorubicin, doxorubicin (adriamycin), idarubicin and mitoxantrone, platinum-based agents, antimetabolites, sensitized T cells and cytokines.

28. The use of claim 27, wherein the antimetabolite is selected from the group consisting of diazomycin, D-cycloserine, mycophenolic acid, trimethoprim, 5-fluorouracil, capecitabine, methotrexate, gemcitabine, cytarabine (ara-C), and fludarabine.

Images