Antibodies and methods for ptk7 detection

Abstract

The present disclosure provides antibodies that specifically bind to protein tyrosine kinase 7 (PTK7), and methods of using such antibodies. In some cases, the PTK7 antibodies are used in an immunohistochemistry (IHC) assay, which can detect the presence of cells expressing PTK7 in a tissue from a patient.

Description

Technical Field

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. US63 / 603,097, filed November 27, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] sequence list

[0004] This application contains an electronically submitted sequence list in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy was created on November 12, 2023, named 2023-11-12-Sequence listing-20896-0005PR00.xml, and is 33,079 bytes in size.

[0005] This disclosure generally relates to antibodies and related methods for PTK7, and specifically to PTK7 antibodies and IHC methods for detecting PTK7 and PTK7-positive cells and tissues. Background Technology

[0006] Immunohistochemistry (IHC) is a widely used adjunctive diagnostic method in diagnostics and surgical pathology. The primary and initial purpose of IHC was for cell classification and organ origin determination. Its application has expanded to include the study of predictive and prognostic biomarkers in many malignancies, including but not limited to those in the breast, gastrointestinal tract, liver, lung, ovary, blood lymph nodes, and central nervous system. IHC utilizes antibodies to target specific antigens in particular tissues and cells to facilitate the identification of cells of interest.

[0007] The selection of a suitable target antigen and the appropriate construction of an antibody that binds to the target antigen determine the specificity and sensitivity of IHC. Protein tyrosine kinase 7 (PTK7), also known as colon cancer kinase 4 (CCK4), is found to be highly expressed in many types of cancer cells and exhibits limited expression in normal human tissues. Therefore, it is an attractive biomarker for detecting cancer cells and further for cancer diagnosis. However, progress in constructing effective PTK7 antibodies has been slow. There is an urgent need for PTK7 antibodies that can be used for the detection of PTK7-positive cells and serve as a key component of a reliable IHC kit. The embodiments disclosed herein address this and related needs. Summary of the Invention

[0008] This article provides protein tyrosine kinase 7 (PTK7) binders (e.g., antibodies) and their methods of use. The PTK7 binder may comprise a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity-determining regions HCDR1, HCDR2, and HCDR3 disposed within the heavy chain variable region framework, and the VL region comprises LCDR1, LCDR2, and LCDR3 disposed within the light chain variable region framework, wherein the VH and VL CDRs have amino acid sequences selected from the set of amino acid sequences shown below: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, LVS, and SEQ ID NO: 7, respectively; SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, QMS, and SEQ ID NO: 14, respectively; and SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, RVS, and SEQ ID NO: 21, respectively.

[0009] In some embodiments, the VH and VL regions have amino acid sequences selected from the following amino acid sequence pairs: SEQ ID NO: 1 and SEQ ID NO: 2, respectively; SEQ ID NO: 8 and SEQ ID NO: 9, respectively; and SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

[0010] In some embodiments, the VH and VL regions have amino acid sequences selected from the following amino acid sequence pairs: SEQ ID NO: 1 and SEQ ID NO: 2, respectively; SEQ ID NO: 8 and SEQ ID NO: 9, respectively; and SEQ ID NO: 15 and SEQ ID NO: 16, respectively, wherein the heavy chain and light chain framework regions are optionally modified by substitution, deletion, or insertion of 1 to 8 amino acids in the framework regions.

[0011] In some implementations, the framework region of the binder is the human framework region.

[0012] In some implementations, the framework region of the binder is the mouse framework region.

[0013] In some implementations, the binding agent is an antibody. In some implementations, the binding agent is a monoclonal antibody.

[0014] In some implementations, the binder further comprises a heavy chain constant region and / or a light chain constant region.

[0015] In some embodiments, the heavy chain constant region is an IgG isotype. In some embodiments, the heavy chain constant region is a human IgG1 constant region or a human IgG4 constant region. In some embodiments, the heavy chain constant region is a mouse IgG1 constant region, a mouse IgG2a constant region, a mouse IgG2c constant region, or a mouse IgG3 constant region. In some embodiments, the heavy chain constant region has the amino acid sequence shown in SEQ ID NO: 22, 24, or 26.

[0016] In some embodiments, the light chain constant region has the amino acid sequence shown in SEQ ID NO: 23, 25 or 27.

[0017] In some embodiments, the binder is a monoclonal antibody comprising a VH region having the amino acid sequence shown in SEQ ID NO:1, a VL region having the amino acid sequence shown in SEQ ID NO:2, a heavy chain constant region having the amino acid sequence shown in SEQ ID NO:22, and a light chain constant region having the amino acid sequence shown in SEQ ID NO:23.

[0018] In some embodiments, the binder is a monoclonal antibody comprising a VH region having the amino acid sequence shown in SEQ ID NO: 8, a VL region having the amino acid sequence shown in SEQ ID NO: 9, a heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 24, and a light chain constant region having the amino acid sequence shown in SEQ ID NO: 25.

[0019] In some embodiments, the binder is a monoclonal antibody comprising a VH region having the amino acid sequence shown in SEQ ID NO: 15, a VL region having the amino acid sequence shown in SEQ ID NO: 16, a heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 26, and a light chain constant region having the amino acid sequence shown in SEQ ID NO: 27.

[0020] This disclosure also provides a method for detecting cells expressing PTK7 in a sample, comprising: contacting the sample with a detection agent comprising a binding agent of the present disclosure, and detecting the formation of a binding complex between the detection agent and PTK7, wherein the detection of the binding complex indicates the presence of cells expressing PTK7 in the sample.

[0021] In some embodiments, the detection reagent further comprises a detectable biomarker that labels the binding agent. In some embodiments, the detectable biomarker includes biotin or an enzyme biomarker. In some embodiments, the enzyme biomarker includes horseradish peroxidase (HRP) or alkaline phosphatase (AP).

[0022] In some embodiments, detecting the formation of a binding complex between the detectant and PTK7 includes: adding a mating agent of the detectable marker; and detecting the reaction between the detectable marker and the mating agent as an indication of the formation of the binding complex.

[0023] In some embodiments, the detectable marker includes a fluorescent tag. In some embodiments, detecting the formation of a binding complex between the detector and PTK7 includes detecting a signal from the fluorescent tag as an indication of the formation of the binding complex.

[0024] In some embodiments, detecting the formation of a binding complex between the detection agent and PTK7 includes: contacting the sample with a labeled secondary antibody configured to bind the binding complex; and detecting a signal from the secondary antibody as an indication of the formation of the binding complex.

[0025] In some embodiments, the secondary antibody is labeled with a detectable biomarker. In some embodiments, the detectable biomarker includes biotin or an enzyme biomarker. In some embodiments, detecting the formation of a binding complex between the assay agent and PTK7 includes: adding a partner agent of the detectable biomarker; and detecting the reaction between the detectable biomarker and the partner agent as an indication of the formation of the binding complex. In some embodiments, the detectable biomarker includes a fluorescent tag. In some embodiments, detecting the formation of a binding complex between the assay agent and PTK7 includes: detecting a signal from the fluorescent tag as an indication of the formation of the binding complex. In some embodiments, the fluorescent tag includes fluorescein (FITC), phycoerythrin (PE), preferably R-PE, or allophycocyanin (APC).

[0026] In some embodiments, the sample is derived from the subject's tissue. In some embodiments, the tissue is selected from the group consisting of tonsils, appendix, breast, ovary, colon, prostate, skin, lung, uterus, cervix, kidney, pancreas, bladder, brain, thyroid gland, ear, nose, larynx, and esophagus. In some embodiments, the tissue is selected from the group consisting of breast, ovary, colon, prostate, skin, lung, uterus, esophagus, and bladder. In some embodiments, the tissue is breast tissue. In some embodiments, the tissue is ovary tissue.

[0027] In some implementations, the sample is taken from the subject, and the presence of cells expressing PTK7 indicates that the subject has a disease.

[0028] In some implementations, the sample is from a subject, and the method further includes: quantifying the binding of PTK-expressing cells in the sample to obtain a quantitative result, wherein a quantitative result exceeding a predetermined threshold indicates that the subject has a disease.

[0029] In some implementations, the disease is cancer.

[0030] In some embodiments, the disease is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, melanoma, lung cancer, esophageal cancer, gastric cancer, endometrial cancer, head and neck cancer, and bladder cancer. In some embodiments, the disease is breast cancer. In some embodiments, the disease is ovarian cancer. In some embodiments, the disease is squamous cell carcinoma of the lung or adenocarcinoma of the lung.

[0031] In some embodiments, this disclosure provides a method for determining whether a subject has a disease, comprising: obtaining a sample from the subject; contacting the sample with a detection agent comprising a binder of this disclosure; determining whether the subject has the disease by detecting and / or quantifying the formation of a binding complex between the detection agent and PTK7, wherein the presence of the binding complex or a level of the binding complex exceeding a predetermined threshold indicates that the subject has the disease. In some embodiments, the disease is cancer.

[0032] In some embodiments, this disclosure provides a method for treating a disease in a patient who has been diagnosed with the disease by the detection methods described herein. In one embodiment, the detection method includes obtaining a sample from a subject; contacting the sample with a detection agent comprising a binder of this disclosure; and determining whether the subject has the disease by detecting and / or quantifying the formation of a binding complex between the detection agent and PTK7, wherein the presence of the binding complex or a level of the binding complex exceeding a predetermined threshold indicates that the subject has the disease. In some embodiments, the binder comprises VH and VL regions containing the amino acid sequences shown below: SEQ ID NO: 1 and SEQ ID NO: 2, respectively; SEQ ID NO: 8 and SEQ ID NO: 9, respectively; or SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

[0033] In some implementation schemes, the disease is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, melanoma, and lung cancer.

[0034] In some implementations, the treatment includes administering chemotherapy to the subject.

[0035] In some implementations, the treatment includes administering hormone therapy to the subject.

[0036] In some implementations, the treatment includes administering radiation therapy to the subject.

[0037] In some implementations, the treatment includes administering immunotherapy to the subject.

[0038] In some implementations, the treatment includes administering stem cell therapy to the subject.

[0039] In some implementations, treatment includes administering a targeted therapy to the subject.

[0040] In some implementations, the treatment includes performing surgery on the subject.

[0041] In some implementations, the disease is breast cancer, and the treatment includes removal of the breast tumor or mastectomy.

[0042] In some implementations, the disease is ovarian cancer, and the treatment includes hysterectomy or salpingo-oophorectomy.

[0043] An anticancer agent is also provided for use in a method of treating a patient with cancer, the method comprising: contacting a sample from a patient with a detection agent comprising a binding agent of the present disclosure; determining whether the subject has the disease by detecting and / or quantifying the formation of a binding complex between the detection agent and PTK7, wherein the presence of the binding complex or a level of the binding complex exceeding a predetermined threshold indicates that the subject has the disease; and if the subject is determined to have a level above the predetermined threshold, and if so, treating the disease with the anticancer agent. Possible anticancer agents include any anticancer agents mentioned herein, such as chemotherapeutic agents or immunotherapeutic agents.

[0044] Also provided is an anticancer agent for preparing a medicament for treating cancer by comprising: contacting a sample from a patient with a detection agent comprising a binding agent of the present disclosure; determining whether the subject has the disease by detecting and / or quantifying the formation of a binding complex between the detection agent and PTK7, wherein the presence of the binding complex or the level of the binding complex exceeding a predetermined threshold indicates that the subject has the disease; and treating the disease with the anticancer agent if the subject is determined to have a level above the predetermined threshold.

[0045] In some embodiments, this disclosure provides a method for monitoring the progression of a disease in a subject, comprising the steps of: obtaining a sample from the subject; contacting the sample with a detection agent containing a binding agent of this disclosure; assessing the formation of a binding complex between the detection agent and PTK7 to obtain a first assessment result; repeating steps (a), (b), and (c) using samples from the subject at subsequent time points to obtain a second assessment result; and comparing the second assessment result with the first assessment result to determine the progression of the disease in the subject. In some embodiments, the disease is cancer.

[0046] In some embodiments, this disclosure provides a kit for detecting cells expressing PTK7 in a sample, the kit comprising a detection agent containing a binding agent of this disclosure. In some embodiments, the kit further comprises a secondary antibody labeled and configured to bind to the binding complex formed by the detection agent and PTK7. In some embodiments, the kit further comprises a specification for carrying out the methods of the invention.

[0047] These and other aspects of this disclosure can be more fully understood by referring to the following detailed description, non-limiting examples of specific embodiments, and accompanying drawings. Attached Figure Description

[0048] This disclosure is further described with reference to exemplary embodiments. These exemplary embodiments are described in detail with reference to the accompanying drawings. It should be noted that the drawings are not drawn to scale. These embodiments are non-limiting exemplary embodiments, wherein the same reference numerals denote similar structures in several views of the drawings, and wherein:

[0049] Figure 1A This graph illustrates the results of PTK7 antibody hybridoma screening of tumor tissue samples 26, 28, 42, 50, 61, 161, 170, and 175 by IHC assay. The positive control is located in the upper left corner of the graph.

[0050] Figure 1B This graph illustrates the results of PTK7 antibody hybridoma screening of tumor tissue samples 204, 206, 218, 222, 226, 227, 231, and 233 by IHC assay. The positive control is located in the upper left corner of the graph.

[0051] Figure 1C This graph illustrates the results of PTK7 antibody hybridoma screening of tumor tissue samples 309, 312, 316, 331, 333, and 363 by IHC assay. The positive control is located in the upper left corner of the graph.

[0052] Figure 1D This graph illustrates the results of PTK7 antibody hybridoma screening of tumor tissue samples 204, 206, 218, 222, 226, 227, 231, and 233 by IHC assay. The positive controls are located in the upper left and lower right corners of the graph.

[0053] Figure 2A This is a graph illustrating the IHC staining results of the PTK7 antibody subclone M6-9H5 (sample 26) in breast cancer.

[0054] Figure 2B This is a diagram illustrating the IHC staining results of the PTK7 antibody subclone M6-9H5 (sample 26) in ovarian cancer.

[0055] Figure 3A This is a diagram illustrating the IHC staining results of the PTK7 antibody subclone M6-2C6 (sample 222) in breast cancer.

[0056] Figure 3B This is a diagram illustrating the IHC staining results of the PTK7 antibody subclone M6-2C6 (sample 222) in ovarian cancer.

[0057] Figure 4A This is a diagram illustrating the IHC staining results of the PTK7 antibody subclone M6-14H10 (sample 235) in breast cancer.

[0058] Figure 4B This is a diagram illustrating the IHC staining results of the PTK7 antibody subclone M6-14H10 (sample 235) in ovarian cancer.

[0059] Figure 5A This is a diagram illustrating the IHC staining results of the PTK7 antibody subclone M10-5F7 (sample 312) in breast cancer.

[0060] Figure 5B This is a diagram illustrating the IHC staining results of the PTK7 antibody subclone M10-5F7 (sample 312) in ovarian cancer.

[0061] Figure 6A This is a graph illustrating the IHC staining results of PTK7 antibody subclone M1-11E5 (sample 58) in breast cancer.

[0062] Figure 6B This is a diagram illustrating the IHC staining results of PTK7 antibody subclone M1-11E5 (sample 58) in ovarian cancer.

[0063] Figure 7A This is a diagram illustrating the IHC staining results of the PTK7 antibody subclone M6-17F7 (sample 134) in breast cancer.

[0064] Figure 7B This is a diagram illustrating the IHC staining results of the PTK7 antibody subclone M6-17F7 (sample 134) in ovarian cancer.

[0065] Figure 8A This is a graph illustrating the IHC staining results of the PTK7 antibody subclone M6-8G5 (sample 236) in breast cancer.

[0066] Figure 8B This is a diagram illustrating the IHC staining results of the PTK7 antibody subclone M6-8G5 (sample 236) in ovarian cancer.

[0067] Figure 9AThis is a diagram illustrating the IHC staining results of the PTK7 antibody subclone M6-11E2 (sample 259) in breast cancer.

[0068] Figure 9B This is a diagram illustrating the IHC staining results of the PTK7 antibody subclone M6-11E2 (sample 259) in ovarian cancer.

[0069] Figure 10 This is a graph illustrating the IHC staining results of PTK7 antibody subclone M6-11E2 (sample 259) at different magnifications: both ovarian cancer and breast cancer showed positive results in the medial membrane and tumor stroma.

[0070] Figure 11 This is a graph illustrating the IHC staining results of PTK7 antibody subclone M6-8G5 (sample 236) at different magnifications: both ovarian cancer and breast cancer showed positive results in the medial membrane and tumor stroma.

[0071] Figure 12 This is a graph illustrating the IHC staining results of PTK7 antibody subclone M10-5F7 (sample 312) at different magnifications: positive for ovarian cancer media, negative for breast cancer media, and positive for tumor stroma.

[0072] Figure 13 This is a diagram illustrating the results of cell staining technique (CST) IHC staining at different magnifications: both ovarian cancer and breast cancer showed positive results in the medial membrane and tumor stroma.

[0073] Figure 14A This is a graph illustrating the IHC titration results of the PTK7 antibody subclone M6-11E2 (sample 259) in the tonsils.

[0074] Figure 14B This is a graph illustrating the IHC titration results of the PTK7 antibody subclone M6-11E2 (sample 259) in ovarian cancer-1.

[0075] Figure 14C This is a graph illustrating the IHC titration results of the PTK7 antibody subclone M6-11E2 (sample 259) in ovarian cancer-2.

[0076] Figure 14D This is a graph illustrating the IHC titration results of the PTK7 antibody subclone M6-11E2 (sample 259) in breast cancer.

[0077] Figure 15A This is a graph illustrating the IHC titration results of the PTK7 antibody subclone M6-8G5 (sample 236) in the tonsils.

[0078] Figure 15B This is a graph illustrating the IHC titration results of the PTK7 antibody subclone M6-8G5 (sample 236) in ovarian cancer-1.

[0079] Figure 15C This is a graph illustrating the IHC titration results of the PTK7 antibody subclone M6-8G5 (sample 236) in ovarian cancer-2.

[0080] Figure 15D This is a graph illustrating the IHC titration results of the PTK7 antibody subclone M6-8G5 (sample 236) in breast cancer.

[0081] Figure 16A This is a graph illustrating the IHC titration results of the PTK7 antibody subclone M10-5F7 (sample 312) in the tonsils.

[0082] Figure 16B This is a graph illustrating the IHC titration results of the PTK7 antibody subclone M10-5F7 (sample 312) in ovarian cancer-1.

[0083] Figure 16C This is a graph illustrating the IHC titration results of the PTK7 antibody subclone M10-5F7 (sample 312) in ovarian cancer-2.

[0084] Figure 16D This is a graph illustrating the IHC titration results of the PTK7 antibody subclone M10-5F7 (sample 312) in breast cancer.

[0085] Figure 17A This is a figure illustrating the results of IHC tissue microarrays of PTK7 antibody subclone M6-11E2 (sample 259) in various types of normal and cancerous tissues by manual staining.

[0086] Figure 17B This is a figure illustrating the results of IHC tissue microarray analysis of PTK7 antibody subclone M6-11E2 (sample 259) in various types of normal and cancerous tissues using Leica autostainer.

[0087] Figure 18A This is a figure illustrating the results of IHC tissue microarrays of PTK7 antibody subclone M6-8G5 (sample 236) in various types of normal and cancerous tissues by manual staining.

[0088] Figure 18B This is a figure illustrating the results of IHC tissue microarray analysis of PTK7 antibody subclone M6-8G5 (sample 236) in various types of normal and cancerous tissues using Leica autostainer.

[0089] Figure 19A This is a figure illustrating the results of IHC tissue microarrays of PTK7 antibody subclone M10-5F7 (sample 312) in various types of normal and cancerous tissues by manual staining.

[0090] Figure 19B This is a figure illustrating the results of IHC tissue microarray analysis of PTK7 antibody subclone M10-5F7 (sample 312) in various types of normal and cancerous tissues using Leica autostainer.

[0091] Figure 20 This graph illustrates the different levels of PTK7 antigen expression in cell lines PA-1, MDA-MB-453, MCF-7, and Raji (negative control) stained with IHC using subclone M6-11E2 (sample 259) as the primary antibody. The results are displayed clockwise from the bottom left, showing the MCF-7, PA-1, MDA-MB-453, and Raji cell lines.

[0092] Figure 21 This graph illustrates the different levels of PTK7 antigen expression in cell lines PA-1, MDA-MB-453, MCF-7, and Raji (negative control) stained with IHC using subclone M6-8G5 (sample 236) as the primary antibody. The results are displayed clockwise from the bottom left, showing the MCF-7, PA-1, MDA-MB-453, and Raji cell lines.

[0093] Figure 22 This graph illustrates the different levels of PTK7 antigen expression in cell lines PA-1, MDA-MB-453, MCF-7, and Raji (negative control) after IHC staining using subclone M10-5F7 (sample 312) as the primary antibody. The results are displayed clockwise from the bottom left, showing the MCF-7, PA-1, MDA-MB-453, and Raji cell lines.

[0094] Additional features will be set forth in part in the description which follows, and will become apparent in part to those skilled in the art upon examination of the following and accompanying drawings, or may be learned by the generation or operation of examples. The features of this disclosure can be realized and obtained by practice or by using various aspects of the methods, instruments, and combinations set forth in the detailed examples discussed below. Detailed Implementation

[0095] The following description is presented to enable any person skilled in the art to make and use this disclosure, and is provided in the context of a particular application and its requirements. Various modifications to the disclosed embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of this disclosure. Therefore, this disclosure is not limited to the embodiments shown, but should be accorded the widest scope consistent with the claims.

[0096] The terminology used herein is for the purpose of describing particular example implementations only and is not intended to be restrictive. As used herein, the singular forms “a,” “an,” and “the” may also be intended to include the plural forms unless the context clearly indicates otherwise. It will be further understood that, when used in this specification, the terms “comprises,” “comprising,” “includes,” and / or “including” specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups of these.

[0097] These and other features and characteristics of this disclosure, as well as the operation and function of related structural elements and the economy of combination and manufacture of components, will become more apparent once the following description is considered with reference to the accompanying drawings, all of which form part of this specification. However, it should be clearly understood that the drawings are for illustrative and descriptive purposes only and are not intended to limit the scope of this disclosure. It should be understood that the drawings are not drawn to scale.

[0098] In the context of the treatment methods described herein, products mentioned in said treatment methods are also provided. The use of the mentioned products in the preparation of medicaments for treating the mentioned conditions is also provided. A pharmaceutical composition comprising the products for use in said methods is also provided.

[0099] All references mentioned herein are incorporated herein by reference in their entirety. Priority claims in this application are also incorporated herein by reference in their entirety.

[0100] definition

[0101] For convenience, certain terms are defined herein in the specification, embodiments, and claims. Unless otherwise stated or implied by context, the following terms and phrases have the meanings provided below. Definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed disclosure, as the scope of this disclosure is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0102] As used herein, and unless otherwise stated, the terms “a” and “one” mean “an,” “at least one,” or “one or more.” Unless the context requires otherwise, singular terms used herein shall include plural terms, and plural terms shall include singular terms.

[0103] Unless the context explicitly requires otherwise, throughout the specification and claims, the terms “comprising”, “including”, etc., shall be interpreted in an inclusive sense, rather than an exclusive or exhaustive sense; that is, in the sense of “including but not limited to”. Where the language “comprising” or “including” is used herein to describe something, embodiments “consisting of the described content” are also provided.

[0104] The terms “reduced,” “lower,” “reduced,” “reduced,” “reduced,” and “inhibited” are generally used in this article to refer to a statistically significant reduction relative to a reference.

[0105] The terms “increased,” “increased,” “enhanced,” or “activated” are generally used in this document to refer to an increase in the degree of static significance relative to a reference.

[0106] As used herein, the terms “protein” and “polypeptide” are used interchangeably to refer to a series of amino acid residues linked together by peptide bonds between adjacent α-amino and carboxyl groups. The terms “protein” and “polypeptide” also refer to polymers of amino acids, including modified amino acids (e.g., phosphorylated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. “Protein” and “polypeptide” are generally used to refer to relatively large polypeptides, while the term “peptide” is generally used to refer to small polypeptides; however, the usage of these terms overlaps in the art. When referring to encoded gene products and fragments thereof, the terms “protein” and “polypeptide” are used interchangeably herein. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the foregoing.

[0107] PTK7 (protein tyrosine kinase-like 7) is a member of the receptor protein tyrosine kinase family, also known as colon cancer kinase 4 (CCK4). In humans, this protein is encoded by the PTK7 gene. PTK7 is overexpressed on the surface of many types of cancer cells, including but not limited to solid tumors. These solid tumors include, but are not limited to, breast cancer (BC), lung cancer (LC), esophageal cancer (ESC), gastric cancer (GC), bladder cancer (BLC), endometrial cancer (EC), ovarian cancer (OVC), head and neck cancer (HNC), and hematologic malignancies. Human PTK7 peptides include, but are not limited to, those having the amino acid sequences listed in UniProt identification number Q13308 and those having the amino acid sequences listed in GenBank, such as, but not limited to, GenBank accession numbers NP_002812.2, NP_690619.1, NP_690620.1, NP_690621.1, NP_001257327.1, XP_011513067.1, XP_011513068.1, XP_047275113.1, XP_054212039.1, XP_054212040.1 and XP_054212041.1, which are incorporated herein by reference. Despite lacking detectable catalytic tyrosine kinase activity, PTK7 overexpression is closely associated with Wnt signaling and promotes cancer cell stemness, survival, and tumor progression (see, for example, Atasaven et al., 2013; Cui et al., 2021; Gärtner, 2014; Chen et al., 2014; Jiang et al., 2020; Shin et al., 2013; Liu et al., 2017; Lin et al., 2012; Özçelik et al., 2020; Xiang et al., 2022; Wang et al., 2014; Prebet et al., 2010; Jiang et al., 2012; Damelin et al., 2017). PTK7 expression is typically low in normal tissues such as the bladder, kidney, breast, lung, ovary, uterus, and dendritic cells, although some levels of protein expression have been reported in the digestive tract (Damelin et al., 2017). Therefore, PTK7 is a promising target for novel anticancer therapies.

[0108] As used herein, an epitope is an amino acid typically bound by immunoglobulin VH / VL pairs (such as antibodies, their antigen-binding moieties, and other binding agents described herein). Epitopes can be formed on a polypeptide by consecutive amino acids or by discontinuous amino acids juxtaposed through the ternary folding of a protein. Epitopes formed by consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed by ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically contain at least 3, more usually at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. An epitope defines the minimum binding site for antibodies, their antigen-binding moieties, and other binding agents, and thus represents a specific target for antibodies, their antigen-binding moieties, or other immunoglobulin-based binding agents. In the case of a single-domain antibody, the epitope represents a structural unit bound by a separate variable domain. In addition to the specific binding agents described herein, particularly antibodies, binding agents, particularly antibodies, that bind the same epitopes as the specific antibodies described herein are also provided. Binding agents, particularly antibodies, that cross-block the specific binding agents described herein, particularly antibodies, are also provided.

[0109] As used in this article, "binding agent" is a general term that refers to a 10 -5 M (10000 nM) or smaller KD, such as 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12M or smaller KD molecules that bind to targets such as human PTK7 protein. The conjugate can be an antibody, such as, but not limited to: Fab, Fab', F(ab')2, scFab, Fv, scFv, VH, VHH, VL, VLR, etc., diabodies, monoclonal antibodies (mAb), polyclonal antibodies (pAb), mAbdAb, phage display-derived conjugates, affinity antibodies, heteroconjugated antibodies, bispecific antibodies, and evibodies. In one embodiment, the conjugate is an antibody. In one embodiment, the conjugate is an antibody comprising two heavy chains and two light chains. In one embodiment, the conjugate is a monoclonal antibody. Specific binding may be affected by, for example, the affinity and coercivity of the antibody, antigen-binding moiety, or other conjugate, as well as the concentration of the target peptide. Those skilled in the art can use any suitable method, such as titrating the antibody or other conjugate in a suitable cell binding assay, to determine suitable conditions for the selective binding of the antibodies, antigen-binding moiety, and other conjugates described herein to PTK7. A PTK7-specific binder cannot be replaced by a dissimilar competitor. In some embodiments, a PTK7-specific binder is defined as one that preferentially recognizes its target antigen PTK7 in a complex mixture of proteins and / or macromolecules.

[0110] The antibodies mentioned in this article specifically cover antibody fragments that retain the ability to target PTK-7. Therefore, when referring to antibodies in this article, their antigen-binding fragments are also provided. Examples of antibody fragments include Fab, Fab', F(ab')2, scFab, Fv, scFv, VH, VHH, VL, and VLR antibodies.

[0111] This document discloses variant binding agents, such as binding agents with a limited number of sequence alterations. The variant antibody will still be able to bind the PTK7 protein. Therefore, in one embodiment, the variant antibody will still be able to bind the PTK7 protein. In one embodiment, the variant antibody will still give the same or similar results in IHC. For example, the variant antibody will still be usable for performing one of the IHC methods described herein. In one embodiment, in addition to being able to bind PTK7, the variant will retain at least one other specific function mentioned herein.

[0112] As used herein, a "detectable biomarker" refers to a molecule that is attached to or used to label a binding agent or secondary antibody and can be used to generate a detectable signal that can be detected by colorimetry or fluorescence (e.g., visualization) or otherwise evaluated. Such detectable biomarkers may include, but are not limited to, enzyme substrates (e.g., biotin), enzymes (e.g., horseradish peroxidase (HRP), alkaline phosphatase, glucose oxidase, and β-galactosidase), and fluorescent tags (e.g., fluorescent dyes, such as, but not limited to, fluorescein (FITC), phycoerythrin (PE; e.g., R-PE), or allophycocyanin (APC)). Detectable signals can be generated directly from detectable biomarkers such as fluorescent proteins; detectable signals can also be colorimetric products of a chemical reaction between a detectable biomarker and its conjugate agent.

[0113] As used herein, "detector" refers to a molecule that includes a binder that binds PTK7. In some embodiments, the detector is such a binder. In some embodiments, the detector further includes one or more auxiliary portions (e.g., detectable markers) attached to (e.g., covalently bound) the binder.

[0114] As used herein, a “partner agent” refers to a molecule that can bind to a detectable biomarker, wherein the partner agent and the detectable biomarker react to produce a detectable signal. Examples of partner agents include, but are not limited to, avidin, streptavidin, NeutrAvidin, and enzyme substrates including, but not limited to, 3,3',5,5'-tetramethylbenzidine (TMB), 3,3'-diaminobenzidine (DAB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3-amino-9-ethylcarbazole (AEC), o-phenylenediamine dihydrochloride (OPD), homovanillic acid, AmplexRed, aminoethylcarbazole (AEC), nitroblue tetrazolium chloride (NBT), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (BCIG or X-Gal).

[0115] The binders and antibodies described herein are particularly useful in IHC. They can be used for any type of antibody assay or other purpose. In one embodiment, the binders of the present invention can be used in flow cytometry. In another embodiment, the binders of the present invention can be used in ELISA.

[0116] Antibodies and binders

[0117] This document provides PTK7-binding antibodies (also known as PTK7 antibodies) that specifically bind to human PTK7, along with their antigen-binding moieties and other binding agents. In some embodiments, the PTK7 antibody, antigen-binding moieties, and / or other binding agents specifically bind to PTK7+ cells in a subject and can be used to identify / detect / quantify such PTK7+ cells. In some embodiments, the PTK7 antibody, antigen-binding moieties, and / or other binding agents specifically bind to PTK7+ cancer cells in a subject and can be used to identify / detect / quantify such PTK+ cancer cells. In some embodiments, the PTK7 antibody, antigen-binding moieties, and / or other binding agents specifically bind to PTK7+ cells associated with a disease or condition in a subject, such as cancer or an autoimmune disease. In some embodiments, the PTK7 antibody, antigen-binding moieties, and / or other binding agents can be used to identify / detect / diagnose such diseases or conditions in a subject (such as a human or animal).

[0118] In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences selected from the following amino acid sequence pairs: SEQ ID NO:1 and SEQ ID NO:2, respectively; SEQ ID NO:8 and SEQ ID NO:9, respectively; and SEQ ID NO:15 and SEQ ID NO:16, respectively. In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO:8 and SEQ ID NO:9, respectively. In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region, the VH region and the VL region having amino acid sequences as shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

[0119] In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region having an amino acid sequence selected from the following amino acid sequence pairs: SEQ ID NO:1 and SEQ ID NO:2, respectively; SEQ ID NO:8 and SEQ ID NO:9, respectively; and SEQ ID NO:15 and SEQ ID NO:16, respectively; wherein the heavy chain and light chain variable frame regions are optionally modified by substitution of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conserved amino acids in the frame regions, and wherein the CDR of the heavy chain or light chain variable region is unmodified. In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region having an amino acid sequence selected from the following amino acid sequence pairs: SEQ ID NO: 1 and SEQ ID NO: 2, respectively; SEQ ID NO: 8 and SEQ ID NO: 9, respectively; and SEQ ID NO: 15 and SEQ ID NO: 16, respectively; wherein the heavy chain and light chain variable frame regions are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids in the frame regions, and wherein the CDR of the heavy chain or light chain variable region is unmodified. The phrase "wherein the CDR of the heavy chain or light chain variable region is unmodified" means that the VH and VL CDRs do not have amino acid substitutions, deletions, or insertions.

[0120] In some embodiments, the PTK7 antibody or its antigen-binding portion includes a heavy chain variable (VH) region having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% similarity to the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 8, or SEQ ID NO: 15. In some embodiments, the PTK7 antibody or its antigen-binding portion includes a light chain variable (VL) region having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% similarity to the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 9, or SEQ ID NO: 16. In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% similar to the following amino acid sequence pairs: SEQ ID NO: 1 and SEQ ID NO: 2, respectively; SEQ ID NO: 8 and SEQ ID NO: 9, respectively; and SEQ ID NO: 15 and SEQ ID NO: 16, respectively. The level of similarity is achieved by amino acid substitutions, deletions, or insertions in the VH and VL sequences as follows: SEQ ID NO: 1 and SEQ ID NO: 2, respectively; SEQ ID NO: 8 and SEQ ID NO: 9, respectively; and SEQ ID NO: 15 and SEQ ID NO: 16, respectively. The variant antibody will still be able to bind PTK7.

[0121] In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; wherein the heavy chain and light chain variable framework regions are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conserved amino acids in the framework regions, and wherein the CDR of the heavy chain or light chain variable regions is unmodified. Variant antibodies or their antigen-binding portions will still be able to bind PTK7.

[0122] In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a heavy chain variable (VH) region sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% as shown in SEQ ID NO: 1. In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a light chain variable (VL) region of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% as shown in SEQ ID NO: 2. In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH and VL regions have amino acid sequences of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Variant antibodies or their antigen-binding portions will still be able to bind PTK7.

[0123] In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively; wherein the heavy chain and light chain variable framework regions are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conserved amino acids in the framework regions, and wherein the CDR of the heavy chain or light chain variable regions is unmodified. Variant antibodies or their antigen-binding portions will still be able to bind PTK7.

[0124] In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a heavy chain variable (VH) region sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% as shown in SEQ ID NO: 8. In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a light chain variable (VL) region of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% as shown in SEQ ID NO: 9. In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH and VL regions have amino acid sequences of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% as shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively. Variant antibodies or their antigen-binding portions will still be able to bind PTK7.

[0125] In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively; wherein the heavy chain and light chain variable framework regions are optionally modified by substitution of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conserved amino acids in the framework regions, and wherein the CDR of the heavy chain or light chain variable regions is unmodified. In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively; wherein the heavy chain and light chain variable framework regions are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids in the framework regions, and wherein the CDR of the heavy chain or light chain variable regions is unmodified. Variant antibodies or their antigen-binding portions will still be able to bind to PTK7.

[0126] In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a heavy chain variable (VH) region sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the sequence shown in SEQ ID NO: 15. In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a light chain variable (VL) region of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the sequence shown in SEQ ID NO: 16. In some embodiments, the PTK7 antibody or its antigen-binding portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region, the VH region and VL region having amino acid sequences of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% as shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively. Variant antibodies or their antigen-binding portions will still be able to bind PTK7.

[0127] In some embodiments, this document provides a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region having an amino acid sequence selected from the following amino acid sequence pairs: SEQ ID NO: 1 and SEQ ID NO: 2, respectively; SEQ ID NO: 8 and SEQ ID NO: 9, respectively; and SEQ ID NO: 15 and SEQ ID NO: 16, respectively; wherein the binder specifically binds to human PTK7. In some embodiments, the binder of this disclosure specifically binds to human PTK7 with a higher or similar binding affinity (lower Kd) than the antibody coftotuzumab. In some embodiments, this document provides a binder comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having an amino acid sequence selected from the following amino acid sequence pairs: SEQ ID NO: 1 and SEQ ID NO: 2, respectively; SEQ ID NO: 8 and SEQ ID NO: 9, respectively; and SEQ ID NO: 15 and SEQ ID NO: 16, respectively; wherein the heavy chain and light chain variable framework regions are optionally modified by substitution of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conserved amino acids in the framework regions, and wherein the CDR of the heavy chain or light chain variable region is unmodified. In some embodiments, this document provides a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region having an amino acid sequence selected from the following amino acid sequence pairs: SEQ ID NO: 1 and SEQ ID NO: 2, respectively; SEQ ID NO: 8 and SEQ ID NO: 9, respectively; and SEQ ID NO: 15 and SEQ ID NO: 16, respectively; wherein the heavy chain and light chain variable frame regions are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids in the frame regions, and wherein the CDR of the heavy chain or light chain variable region is unmodified. As described herein, the binder comprises a PTK7 antibody or its antigen-binding moiety, and may optionally comprise other peptides or polypeptides covalently attached to the PTK7 antibody or its antigen-binding moiety. In any of these embodiments, the binder specifically binds to human PTK7.

[0128] In some embodiments, a binding agent is provided herein comprising a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; wherein the binding agent specifically binds to human PTK7. In some embodiments, a binding agent is provided herein comprising a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; wherein the heavy chain and light chain variable framework regions are optionally modified by substitution of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conserved amino acids in the framework regions, and wherein the CDR of the heavy chain or light chain variable region is unmodified. In some embodiments, this document provides a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; wherein the heavy chain and light chain variable framework regions are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids in the framework regions, and wherein the CDR of the heavy chain or light chain variable region is unmodified.

[0129] In some embodiments, a binding agent is provided herein comprising a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively; wherein the binding agent specifically binds to human PTK7. In some embodiments, a binding agent is provided herein comprising a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively; wherein the heavy chain and light chain variable framework regions are optionally modified by substitution of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conserved amino acids in the framework regions, and wherein the CDR of the heavy chain or light chain variable region is unmodified. In some embodiments, this document provides a binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively; wherein the heavy chain and light chain variable framework regions are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids in the framework regions, and wherein the CDR of the heavy chain or light chain variable region is unmodified. The variant antibody or its antigen-binding portion will still be able to bind PTK7.

[0130] In some embodiments, a binding agent is provided herein comprising a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively; wherein the binding agent specifically binds to human PTK7. In some embodiments, a binding agent is provided herein comprising a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively; wherein the heavy chain and light chain variable framework regions are optionally modified by substitution of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conserved amino acids in the framework regions, and wherein the CDR of the heavy chain or light chain variable region is unmodified. In some embodiments, this document provides a binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively; wherein the heavy chain and light chain variable framework regions are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids in the framework regions, and wherein the CDR of the heavy chain or light chain variable region is unmodified. The variant antibody or its antigen-binding moiety will still be able to bind PTK7.

[0131] In some embodiments, an antibody or antigen-binding moiety is provided comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity-determining regions HCDR1, HCDR2, and HCDR3 disposed within the heavy chain variable region frame region, and the VL region comprises LCDR1, LCDR2, and LCDR3 disposed within the light chain variable region frame region, wherein the VH and VLCDR have amino acid sequences selected from the set of amino acid sequences shown below: (i) SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, LVS, and SEQ ID NO:7, respectively; (ii) SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, QMS, and SEQ ID NO:14, respectively; and (iii) SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, RVS, and SEQ ID NO:21, respectively. In some embodiments, the CDR sequences described herein are based on the IMGT unique numbering scheme, Lefranc, MP The Immunologist, 7, 132-136 (1999). In some embodiments, each VH and VL region contains a humanized frame region. In some embodiments, each VH and VL region contains a human frame region. In some embodiments, each VH and VL region contains a mouse frame region.

[0132] In some embodiments, an antibody or antigen-binding moiety is provided comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region contains complementarity-determining regions HCDR1, HCDR2, and HCDR3 disposed within the heavy chain variable region frame region, and the VL region contains LCDR1, LCDR2, and LCDR3 disposed within the light chain variable region frame region, wherein the VH and VLCDR have amino acid sequences as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, LVS, and SEQ ID NO: 7, respectively. In some embodiments, the CDR sequences described herein are numbered according to the IMGT unique numbering scheme. Lefranc, MP The Immunologist, 7, 132-136 (1999). In some embodiments, each VH and VL region contains a humanized frame region. In some embodiments, each VH and VL region contains a human frame region. In some embodiments, each VH and VL region contains a mouse frame region.

[0133] In some embodiments, an antibody or antigen-binding moiety is provided comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region contains complementarity-determining regions HCDR1, HCDR2, and HCDR3 disposed within the heavy chain variable region frame region, and the VL region contains LCDR1, LCDR2, and LCDR3 disposed within the light chain variable region frame region, wherein the VH and VLCDR have amino acid sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, QMS, and SEQ ID NO: 14, respectively. In some embodiments, the CDR sequences described herein are based on the IMGT unique numbering scheme Lefranc, MP The Immunologist, 7, 132-136 (1999). In some embodiments, each VH and VL region contains a humanized frame region. In some embodiments, each VH and VL region contains a human frame region. In some embodiments, each VH and VL region contains a mouse frame region. In some implementations, each VH and VL region contains a mouse frame region.

[0134] In some embodiments, an antibody or antigen-binding moiety is provided comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region contains complementarity-determining regions HCDR1, HCDR2, and HCDR3 disposed within the heavy chain variable region frame region, and the VL region contains LCDR1, LCDR2, and LCDR3 disposed within the light chain variable region frame region, wherein the VH and VLCDR have amino acid sequences as shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, RVS, and SEQ ID NO: 21, respectively. In some embodiments, the CDR sequences described herein are numbered according to the IMGT unique numbering scheme. Lefranc, MP The Immunologist, 7, 132-136 (1999). In some embodiments, each VH and VL region contains a humanized frame region. In some embodiments, each VH and VL region contains a human frame region. In some embodiments, each VH and VL region contains a mouse frame region.

[0135] In some embodiments, a binder is provided comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity-determining regions HCDR1, HCDR2, and HCDR3 disposed within the heavy chain variable region frame region, and the VL region comprises LCDR1, LCDR2, and LCDR3 disposed within the light chain variable region frame region, wherein the VH and VL CDRs have amino acid sequences as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, LVS, and SEQ ID NO: 7, respectively. In some embodiments, the CDR sequences described herein are according to the IMGT unique numbering scheme. Lefranc, MP The Immunologist, 7, 132-136 (1999). In some embodiments, each VH and VL region comprises a humanized frame region. In some embodiments, each VH and VL region comprises a human frame region. In some embodiments, each VH and VL region comprises a mouse frame region.

[0136] In some embodiments, a binder is provided comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity-determining regions HCDR1, HCDR2, and HCDR3 disposed within the heavy chain variable region frame region, and the VL region comprises LCDR1, LCDR2, and LCDR3 disposed within the light chain variable region frame region, wherein the VH and VL CDRs have amino acid sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, QMS, and SEQ ID NO: 14, respectively. In some embodiments, the CDR sequences described herein are numbered according to the IMGT unique numbering scheme. Lefranc, MP The Immunologist, 7, 132-136 (1999). In some embodiments, each VH and VL region comprises a humanized frame region. In some embodiments, each VH and VL region comprises a human frame region. In some embodiments, each VH and VL region comprises a mouse frame region.

[0137] In some embodiments, a binder is provided comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity-determining regions HCDR1, HCDR2, and HCDR3 disposed within the heavy chain variable region frame region, and the VL region comprises LCDR1, LCDR2, and LCDR3 disposed within the light chain variable region frame region, wherein the VH and VL CDRs have amino acid sequences as shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, RVS, and SEQ ID NO: 21, respectively. In some embodiments, the CDR sequences described herein are based on the IMGT unique numbering scheme Lefranc, MP The Immunologist, 7, 132-136 (1999). In some embodiments, each VH and VL region comprises a humanized frame region. In some embodiments, each VH and VL region comprises a human frame region. In some embodiments, each VH and VL region comprises a mouse frame region.

[0138] In some embodiments, the molecules, compositions, and methods described herein relate to the identification / detection / quantification of PTK7+ cells in a subject in vitro or in vivo using a PTK7 antibody, its antigen-binding portion, and / or other conjugates (e.g., identification / detection / quantification of PTK7+ cells in cancer or tumors, or PTK7+ cells associated with autoimmune diseases or conditions). In some embodiments, the compositions and methods described herein relate to treating PTK7+ cancer in a subject by administering a PTK7 antibody, its antigen-binding portion, other conjugates, or conjugates thereof. Therefore, the present invention also provides pharmaceutical compositions comprising the conjugates described herein and pharmaceutically acceptable carriers or excipients.

[0139] As used herein, the term "antibody" refers to immunoglobulin molecules and the immunoactive portion of immunoglobulin molecules, i.e., molecules containing antigen-binding sites that specifically bind to antigens (e.g., human PTK7). The term generally refers to antibodies composed of two immunoglobulin heavy chain variable regions and two immunoglobulin light chain variable regions, including full-length antibodies (which have both heavy and light chain constant regions).

[0140] Each heavy chain consists of a variable region (abbreviated as VH) and a constant region. The heavy chain constant region may contain three domains CH1, CH2, and CH3, and optionally a fourth domain CH4. Each light chain consists of a variable region (abbreviated as VL) and a constant region. The light chain constant region is the CL domain. The VH and VL regions can be further divided into highly variable regions called complementarity-determining regions (CDRs) and conservative regions scattered with regions called frames (FRs). Thus, each VH and VL region consists of three CDRs and four FRs arranged in the following order from the N-end to the C-end: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. This structure is well known to those skilled in the art.

[0141] As used herein, the “antigen-binding moiety” of a PTK7 antibody refers to a portion of a PTK7 antibody as described herein that has the VH and VL sequences of the PTK7 antibody or the CDR of the PTK7 antibody and specifically binds to PTK7. Examples of antigen-binding moieties include Fab, Fab', F(ab')2, Fv, scFv, disulfide-linked Fv, single-domain antibodies (also known as VHH, VNAR, sdAb, or nanobodies), or bispecific antibodies (see, for example, Huston et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883 (1988) and Bird et al., Science 242, 423-426 (1988), which are incorporated herein by reference). As used herein, in each case of PTK7 antibody, the terms Fab, F(ab')2, and Fv refer to: (i) a Fab fragment, i.e., a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked to each other in the hinge region by disulfide bridges; and (iii) an Fv fragment consisting of VL and VH domains. Although the two domains of the Fv fragment, VL and VH, are encoded by separate coding regions, they can be further linked to each other using synthetic linkers, for example, multi-G4S amino acid sequences (as disclosed in SEQ ID NO: 58 as “(G4S)n”, where n = 1 to 5), making it possible to prepare them as a single protein chain in which the VL and VH regions bind to form a monovalent molecule (referred to as a single-chain Fv or scFv). The term “antigen-binding portion” for antibody is also intended to include such single-chain antibodies. Other forms of single-chain antibodies, such as “bi-antibodies,” are also included herein. Biantibodies are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker connecting the VH and VL domains is too short, so that the two domains cannot combine on the same chain. This forces the VH and VL domains to pair with complementary domains (VL and VH, respectively) on different chains and form two antigen-binding sites (see, for example, Holliger, R et al., (1993) Proc. Natl. Acad. Sci. USA 90:64446448; Poljak, R. J et al., (1994) Structure 2:1121-1123).

[0142] A single-domain antibody is an antibody moiety composed of a single monomeric variable antibody domain. Single-domain antibodies can be derived from variable domains of antibody heavy chains from camels (e.g., nanobodies or VHH moieties). Furthermore, the term single-domain antibody also includes autonomous human heavy chain variable domains (aVH) or VNAR moieties derived from sharks (see, for example, Hasler et al., Mol. Immunol. 75:28-37, 2016).

[0143] Techniques for generating single-domain antibodies (e.g., DABs or VHHs) are known in the art, as disclosed, for example, in Cossins et al. (2006, Prot Express Purif 51:253-259) and Li et al. (Immunol. Lett. 188:89-95, 2017). Single-domain antibodies can be obtained, for example, from camels, alpacas, or llamas using standard immunization techniques. (See, for example, Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75, 2003; and Maass et al., J Immunol Methods 324:13-25, 2007). VHHs can have efficient antigen-binding capabilities and can interact with novel epitopes that are inaccessible to conventional VH-VL pairs (see, for example, Muyldermans et al., 2001). Alpaca serum IgG contains approximately 50% camel heavy chain IgG antibody (HCAb) only (see, e.g., Maass et al., 2007). Alpacas can be immunized with antigens, and VHHs that bind to and neutralize the target antigen can be isolated (see, e.g., Maass et al., 2007). PCR primers for amplifying the coding sequence of alpaca VHHs have been identified and can be used to construct alpaca VHH phage display libraries, which can be used for antibody fragment isolation using standard biopanning techniques well known in the art (see, e.g., Maass et al., 2007).

[0144] In some embodiments, the PTK7 antibody or its antigen-binding portion is part of a bispecific or multispecific binder. Bispecific and multispecific antibodies include the following: scFv1-ScFv2, scFv12-Fc-scFv22, IgG-scFv, DVD-Ig, triomab / quadroma, dual-IgG, scFv2-Fc, TandAb, and scFv-HSA-scFv. In some embodiments, IgG-scFv is IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG, or IgG-2scFv. See, for example, Brinkmann and Kontermann, MAbs 9(2):182-212 (2017); Wang et al., Antibodies, 2019, 8, 43; Dong et al., 2011, MAbs 3:273-88; Natsume et al., J. Biochem.140(3):359-368, 2006; Cheal et al., Mol. Cancer Ther. 13(7):1803-1812, 2014; and Bates and Power, Antibodies, 2019, 8, 28. Therefore, the present invention further provides bispecific antibodies comprising at least one pair of VH and VL regions as described herein. The present invention also provides bispecific antibodies comprising at least one antigen-binding site comprising a set of six CDRs as described herein, namely, a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as described herein.

[0145] Modification of VH and VL zones

[0146] Regarding the amino acid sequences of VH and VL, those skilled in the art will recognize that individual substitutions, deletions, or additions (insertions) of the nucleic acids encoding VH or VL, or changes in a single amino acid or a small percentage of amino acids in the coding sequence of the polypeptide, are “conservatively modified variants” in which the modification results in the substitution of the amino acid with a chemically similar amino acid (conservative amino acid substitution), and the modified polypeptide retains the ability to specifically bind PTK7.

[0147] In some embodiments, conserved modified variants of the PTK7 antibody or its antigen-binding portion may have alterations in the frame region (i.e., in addition to the CDR), for example, conserved modified variants of the PTK7 antibody may have amino acid sequences of VH and VL CDRs (as shown in the following set of amino acid sequences: (i) SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, LVS, and SEQ ID NO:7, respectively; (ii) SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, QMS, and SEQ ID NO:14, respectively; and (iii) SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, RVS, and SEQ ID NO:14, respectively). NO:21, and has at least one conserved amino acid substitution in the frame region (FR). In some embodiments, the VH and VL amino acid sequences in the FR have no more than 8, 6, 4, 2, or 1 conserved amino acid substitutions compared to the unmodified VH and VL amino acid sequences. In some embodiments, the VH and VL amino acid sequences in the FR have 8 to 1, 6 to 1, 4 to 1, or 2 to 1 conserved amino acid substitutions compared to the unmodified VH and VL amino acid sequences. In a further aspect of any of these embodiments, conserved modified variants of PTK7 antibodies, their antigen-binding moieties, or other binding agents exhibit specific binding to PTK7. Variant antibodies or their antigen-binding moieties will retain the ability to bind PTK7.

[0148] For conserved amino acid substitutions, a given amino acid can be replaced by residues with similar physicochemical characteristics, for example, replacing one aliphatic residue with another (such as Ile, Val, Leu, or Ala replacing each other), or replacing one polar residue with another (such as between Lys and Arg; between Glu and Asp; or between Gln and Asn). Other such conserved amino acid substitutions, such as substitutions of entire regions with similar hydrophobic characteristics, are well known. Peptides containing conserved amino acid substitutions can be tested in any of the assays described herein to confirm retention of the desired activity (i.e., for PTK7) of the native or reference peptide, such as antigen-binding activity and specificity.

[0149] In some embodiments, this document provides a PTK7 antibody or its antigen-binding moiety or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; wherein the heavy chain and light chain variable framework regions are optionally modified by substitution of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conserved amino acids in the framework regions, and wherein the CDR of the heavy chain or light chain variable regions is unmodified. In some embodiments, this document provides a PTK7 antibody or its antigen-binding moiety or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; wherein the heavy chain and light chain variable framework regions are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids in the framework regions, and wherein the CDR of the heavy chain or light chain variable regions is unmodified. Variant antibodies or their antigen-binding portions will retain the ability to bind PTK7.

[0150] In some embodiments, this document provides a PTK7 antibody or its antigen-binding moiety or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively; wherein the heavy chain and light chain variable framework regions are optionally modified by substitution of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conserved amino acids in the framework regions, and wherein the CDR of the heavy chain or light chain variable regions is unmodified. In some embodiments, this document provides a PTK7 antibody or its antigen-binding moiety or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively; wherein the heavy chain and light chain variable framework regions are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids in the framework regions, and wherein the CDR of the heavy chain or light chain variable regions is unmodified. Variant antibodies or their antigen-binding portions will retain the ability to bind PTK7.

[0151] In some embodiments, this document provides a PTK7 antibody or its antigen-binding moiety or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively; wherein the heavy chain and light chain variable framework regions are optionally modified by substitution of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conserved amino acids in the framework regions, and wherein the CDR of the heavy chain or light chain variable regions is unmodified. In some embodiments, this document provides a PTK7 antibody or its antigen-binding moiety or other binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region having amino acid sequences as shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively; wherein the heavy chain and light chain variable frame regions are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids in the frame regions, and wherein the CDR of the heavy chain or light chain variable region is unmodified. The variant antibody or its antigen-binding moiety will retain the ability to bind PTK7.

[0152] In any of these embodiments, the functional activity of the PTK7-binding antibody or its antigen-binding moiety, or other binding agent, includes specific binding to PTK7. Additional functional activities include the identification / detection / quantification of PTK7+ cells (e.g., cancer cells or autoimmune cells). Where dose dependence is indeed present, it does not need to be the same as the dose dependence of the reference antibody or its antigen-binding moiety, but rather substantially similar to or better than the dose dependence of the reference antibody or its antigen-binding moiety described herein in relation to a given activity (i.e., the candidate peptide will exhibit greater activity relative to the reference antibody). Therefore, in one embodiment, the variant binding agent, particularly the antibody or its antigen-binding fragment, may retain the specific functions mentioned herein.

[0153] For conserved substitutions, amino acids can be grouped according to the similarity of their side chain properties (see Allhninger, in Biochemistry, 2nd edition, pp. 73-75, Worth Publishers, New York (1975)): (1) Nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) Acidic: Asp (D), Glu (E); and (4) Basic: Lys (K), Arg (R), His (H).

[0154] Alternatively, for conserved substitutions, naturally occurring residues can be grouped based on common side-chain properties into several groups: (1) hydrophobic: leucine, Met, Ala, Val, Leu, Ile; (2) neutral-hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions would require exchanging members of one or more of these categories.

[0155] Specific conservative substitutions include, for example: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, Gln or to Glu; Met to Leu, Tyr or to Ile; Phe to Met, Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile or to Leu.

[0156] In some embodiments, the conserved modified variants of the PTK7 antibody or its antigen-binding moiety are preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher identical to a reference VH or VL sequence, wherein the VH and VL CDRs are unmodified. The degree of homology (identity percentage) between the reference and modified sequences can be determined, for example, by comparing the two sequences using freely accessible computer programs (e.g., BLASTp or BLASTn with default settings) commonly used for this purpose on the World Wide Web.

[0157] In some embodiments, compared to the unmodified VH and VL amino acid sequences, the VH and VL amino acid sequences in the frame region have no more than 8, 6, 4, 2, or 1 conserved amino acid substitutions. In some embodiments, compared to the unmodified VH and VL amino acid sequences, the VH and VL amino acid sequences in the frame region have 8 to 1, 6 to 1, 4 to 1, or 2 to 1 conserved amino acid substitutions. In some embodiments, compared to the unmodified VH and VL amino acid sequences, the VH and VL amino acid sequences in the frame region have no more than 8, 6, 4, 2, or 1 amino acid substitutions, deletions, or insertions. In some embodiments, compared to the unmodified VH and VL amino acid sequences, the VH and VL amino acid sequences in the frame region have 8 to 1, 6 to 1, 4 to 1, or 2 to 1 conserved amino acid substitutions. In some embodiments, the VH and VL amino acid sequences together have no more than 8, 6, 4, 2, or 1 amino acid substitutions, deletions, or insertions compared to the unmodified VH and VL regions.

[0158] In some embodiments, the binder, particularly the antibody or its antigen-binding fragment, may have a small number of amino acid substitutions, deletions, or insertions in the listed CDR sequence, provided that the ability to bind PTK7 is retained. In one embodiment, a binder as described herein is also provided having a total of no more than six amino acid sequence variations in a specific CDR used with the binder described herein. In one embodiment, a total of no more than four amino acid sequence variations. In one embodiment, a total of no more than two amino acid sequence variations. In one embodiment, no more than a single amino acid sequence variation. In any such embodiment, the amino acid sequence variations may be conserved.

[0159] Modification of the natural (or reference) amino acid sequence can be accomplished using any of a variety of techniques known to those skilled in the art. Mutation can be introduced at a specific locus, for example, by synthesizing an oligonucleotide containing a restriction site flanking the desired mutated sequence that can be linked to a fragment of the natural sequence. After linking, the resulting reconstructed sequence encodes a variant with the desired amino acid insertion, substitution, or deletion. Alternatively, an oligonucleotide-guided site-specific mutagenesis procedure can be used to provide a nucleotide sequence with changes to specific codons altered according to the desired substitution, deletion, or insertion. The techniques for making such modifications are well-established and include, for example, those disclosed in Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Patent Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entirety.

[0160] constant region

[0161] In some embodiments, the PTK7 antibody or its antigen-binding portion or other binder has a fully human constant region. In some embodiments, the PTK7 antibody or its antigen-binding portion or other binder has a humanized constant region. In some embodiments, the PTK7 antibody or its antigen-binding portion or other binder has a non-human constant region. In some embodiments, the PTK7 antibody or its antigen-binding portion or other binder has a mouse constant region. An immunoglobulin constant region refers to a heavy chain or light chain constant region. The amino acid sequences of human heavy chain and light chain constant regions are known in the art. The constant region can be of any suitable type, selected from the classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM. Several immunoglobulin classes can be further subdivided into isotypes, such as IgG1, IgG2 (e.g., IgG2a, IgG2b, IgG2c), IgG3, IgG4, or IgA1 and IgA2. The heavy chain constant regions (Fc) corresponding to different classes of immunoglobulins can be α, δ, ε, γ, and μ, respectively. The light chain can be either kappa (or κ) or lambda (or λ).

[0162] In some embodiments, the constant region may have an IgG1 isotype. In some embodiments, the constant region may have an IgG2 isotype. In some embodiments, the constant region may have an IgG1 isotype. In some embodiments, the constant region may have an IgG2a isotype. In some embodiments, the constant region may have an IgG1 isotype. In some embodiments, the constant region may have an IgG2b isotype. In some embodiments, the constant region may have an IgG1 isotype. In some embodiments, the constant region may have an IgG2c isotype. In some embodiments, the constant region may have an IgG3 isotype. In some embodiments, the constant region may have an IgG4 isotype. In some embodiments, the Fc domain may have a heterozygous form comprising constant regions from two or more isotypes. In some embodiments, the immunoglobulin constant region may be an IgG1 or IgG4 constant region.

[0163] In some embodiments, the PTK7 antibody heavy chain has a constant region having the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26. In some embodiments, the PTK7 antibody light chain has a constant region having the amino acid sequence shown in SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27.

[0164] In some embodiments, the PTK7 antibody comprises a heavy chain and a light chain, the heavy chain having a constant region having the amino acid sequence shown in SEQ ID NO. 22, and the light chain having a constant region having the amino acid sequence shown in SEQ ID NO: 23.

[0165] In some embodiments, the PTK7 antibody comprises a heavy chain and a light chain, the heavy chain having a constant region having the amino acid sequence shown in SEQ ID NO. 24, and the light chain having a constant region having the amino acid sequence shown in SEQ ID NO: 25.

[0166] In some embodiments, the PTK7 antibody comprises a heavy chain and a light chain, the heavy chain having a constant region having the amino acid sequence shown in SEQ ID NO. 26, and the light chain having a constant region having the amino acid sequence shown in SEQ ID NO: 27.

[0167] In some embodiments, the PTK7 antibody comprises a heavy chain and a light chain, the heavy chain having the amino acid sequence shown in SEQ ID NO. 28 and the light chain having the amino acid sequence shown in SEQ ID NO: 29.

[0168] In some embodiments, the PTK7 antibody comprises a heavy chain and a light chain, the heavy chain having the amino acid sequence shown in SEQ ID NO: 30 and the light chain having the amino acid sequence shown in SEQ ID NO: 31.

[0169] In some embodiments, the PTK7 antibody comprises a heavy chain and a light chain, the heavy chain having the amino acid sequence shown in SEQ ID NO. 32 and the light chain having the amino acid sequence shown in SEQ ID NO: 33.

[0170] Furthermore, the PTK7 antibody or its antigen-binding moiety or other binding agent may be part of a larger binding agent formed by covalent or non-covalent association of the antibody or antigen-binding moiety with one or more other proteins or peptides. Related to such binding agents are the use of, for example, the streptavidin core region to prepare tetrameric scFv molecules (Kipriyanov, SM et al., (1995), Human Antibodies and Hybridomas 6:93-101) and the use of cysteine ​​residues, marker peptides, and C-terminal multihistidine peptides, such as a hexahistidine tag (“hexahistidine tag” disclosed as SEQ ID NO: 59) to prepare divalent and biotinylated scFv molecules (Kipriyanov, SM et al., (1994) Mol. Immunol. 31:10471058).

[0171] In some implementations, when the binder is a bispecific antibody, the constant region of the heavy chain may have "knobs-into-holes" modifications to promote heterodimer formation and thus promote bispecific antibody production.

[0172] Fc domain modification to alter effector function

[0173] In some embodiments, the Fc region or Fc domain of the PTK7 antibody or its antigen-binding moiety or other binding agent is substantially non-binding to at least one Fc receptor selected from FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). In some embodiments, the Fc region or domain exhibits substantially non-binding to any Fc receptor selected from FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). As used herein, “substantially non-binding” means weak to no binding with one or more selected Fc γ receptors. In some embodiments, “substantially non-binding” means a decrease in binding affinity to the Fc γ receptor (i.e., an increase in Kd) of at least 1000-fold. In some embodiments, the Fc domain or region is Fc null. As used herein, “Fc null” refers to an Fc region or Fc domain that exhibits weak to no binding to any Fc γ receptor. In some embodiments, the Fc null region or Fc domain exhibits a binding affinity for the Fc γ receptor that is reduced (i.e., Kd is increased) by at least 1000-fold.

[0174] In some embodiments, the Fc domain has reduced or substantially no effector functional activity. As used herein, “effector functional activity” refers to antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). In some embodiments, the Fc domain exhibits reduced ADCC, ADCP, or CDC activity compared to the wild-type Fc domain. In some embodiments, the Fc domain exhibits a reduction in ADCC, ADCP, and CDC compared to the wild-type Fc domain. In some embodiments, the Fc domain exhibits substantially no effector function (i.e., the ability to stimulate or affect ADCC, ADCP, or CDC). As used herein, “substantially no effector function” means an effector functional activity reduced by at least 1000-fold compared to the wild-type or reference Fc domain.

[0175] In some implementations, the Fc domain has reduced ADCC activity or no ADCC activity. As used herein, reduced ADCC activity or no ADCC activity means that the ADCC activity of the Fc domain is reduced by at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, or at least 500-fold.

[0176] In some implementations, the Fc domain has reduced CDC activity or no CDC activity. As used herein, reduced CDC activity or no CDC activity means a reduction in CDC activity of the Fc domain of at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, or at least 500-fold.

[0177] In vitro and / or in vivo cytotoxicity assays can be performed to confirm a decrease / depletion of ADCC and / or CDC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks Fcγ receptor binding (and therefore may lack ADCC activity). Primary NK cells, used to mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of molecules of interest are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, for example, the ACTITM non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, Calif.; and the CytoTox 96TM non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, such as in animal models, as disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).

[0178] C1q binding assays can also be performed to confirm that the antibody or Fc domain or region cannot bind C1q and therefore lacks or has reduced CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)).

[0179] In some implementations, the Fc domain has reduced ADCP activity or no ADCP activity. As used herein, reduced ADCP activity or no ADCP activity means that the ADCP activity of the Fc domain is reduced by at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500 times.

[0180] ADCP binding assays can also be performed to confirm the absence or reduced ADCP activity of the antibody or its Fc domain or region. See, for example, US20190079077 and US20190048078 and the references published therein.

[0181] PTK7 antibodies or their antigen-binding moieties or other binders with reduced effector activity include those with substitutions of one or more Fc region residues, such as, for example, substitutions according to Kabat EU numbers 238, 265, 269, 270, 297, 327, and 329 (see, for example, U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of the amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant where residues 265 and 297 are substituted for alanine, according to Kabat EU numbers (see, for example, U.S. Patent No. 7,332,581). Certain antibody variants with reduced binding to the FcR are also known. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312 and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).) PTK7 antibodies containing such amino acid modifications that have weakened binding to FcR, or their antigen-binding moieties or other binding agents, can be prepared.

[0182] In some embodiments, the PTK7 antibody or its antigen-binding portion or other binding agent comprises an Fc domain or region having one or more amino acid substitutions that weaken Fc γR binding, such as substitutions at positions 234 and 235 of the Fc region (EU numbers of the residues). In some embodiments, the substitutions are L234A and L235A (LALA), according to the Kabat EU number. In some embodiments, the Fc domain contains D265A and / or P329G in an Fc region derived from the human IgG1 Fc region, according to the Kabat EU number. In some embodiments, the substitutions are L234A, L235A, and P329G in an Fc region derived from the human IgG1 Fc region (LALA-PG), according to the Kabat EU number. (See, for example, WO 2012 / 130831). In some embodiments, the substitutions are L234A, L235A, and D265A in an Fc region derived from the human IgG1 Fc region (LALA-DA), according to the Kabat EU number.

[0183] In some implementations, alterations are made in the Fc region that result in altered (i.e., reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), for example, as in U.S. Patent Nos. 6,194,551, WO 99 / 51642 and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0184] Methods for preparing antibodies, antigen-binding moieties, and other binding agents

[0185] In various implementations, PTK7 antibodies, their antigen-binding moieties, and other binders can be produced in cell lines derived from humans, mice, or other animals. Recombinant DNA expression can be used to produce PTK7 antibodies, their antigen-binding moieties, and other binders. This allows for the production of PTK7 antibodies, as well as a range of PTK7 antigen-binding moieties and other binders (including fusion proteins), in selected host species. For cell-based production systems, the production of PTK7 antibodies, their antigen-binding moieties, and other binders in bacteria, yeast, transgenic animals, and eggs is also an alternative. A major advantage of transgenic animals is the potential for high yields from renewable resources.

[0186] In some embodiments, the PTK7 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 8, or 15 is encoded by a nucleic acid. In some embodiments, the PTK7 VL polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 9, or 16 is encoded by a nucleic acid. In some embodiments, the nucleic acid encodes the PTK7VH polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 8, or 15. In some embodiments, the nucleic acid encodes the PTK7 VL polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 9, or 16. In some embodiments, the nucleic acid encodes the PTK7 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the nucleic acid encodes the PTK7 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the nucleic acid encodes the PTK7 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the nucleic acid encodes the PTK7 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the nucleic acid encodes the PTK7 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 9. In some implementations, the nucleic acid encodes a PTK7 VH polypeptide having the amino acid sequence shown in SEQ ID NO: 16.

[0187] In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences shown in SEQ ID NO: 1 and 2. In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences shown in SEQ ID NO: 8 and 9. In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences shown in SEQ ID NO: 15 and 16.

[0188] As used herein, the terms "nucleic acid," "nucleic acid sequence," "polynucleotide sequence," or "nucleotide" refer to a polymer molecule incorporating units of ribonucleic acid, deoxyribonucleic acid, or similar substances. Nucleic acids can be single-stranded or double-stranded. A single-stranded nucleic acid can be one strand of denatured double-stranded DNA. In some embodiments, the nucleic acid can be cDNA, such as a nucleic acid lacking introns. In one embodiment, a single nucleic acid is provided encoding both a light chain and a heavy chain of an antibody. In another embodiment, a pair of nucleic acids is provided, one encoding the light chain of an antibody and the other encoding the heavy chain.

[0189] Nucleic acid molecules encoding the amino acid sequence of a PTK7 antibody, its antigen-binding moiety, and other binding agents can be prepared by a variety of methods known in the art. These methods include, but are not limited to, the preparation of synthetic nucleotide sequences encoding PTK7 antibodies, antigen-binding moieties, or other binding agents. Furthermore, oligonucleotide-mediated (or site-directed) mutagenesis, PCR-mediated mutagenesis, and cassette mutagenesis can be used to prepare nucleotide sequences encoding PTK7 antibodies or antigen-binding moieties and other binding agents. As described herein, nucleic acid sequences encoding at least a PTK7 antibody, its antigen-binding moiety, a binding agent, or a polypeptide thereof can be recombined with vector DNA using conventional techniques, such as blunt or staggered ends for ligation, restriction enzyme digestion to provide suitable ends, appropriate filling of sticky ends, alkaline phosphatase treatment to avoid undesirable ligation, and ligation with a suitable ligase or other techniques known in the art. Techniques for such operations are disclosed, for example, in Maniatis et al., Molecular Cloning, Lab. Manual (Cold Spring Harbor Lab. Press, NY, 1982 and 1989) and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons), 1987–1993, and can be used to construct nucleic acid sequences and vectors encoding PTK7 antibodies or their antigen-binding moieties or their VH or VL peptides or other binding agents.

[0190] A nucleic acid molecule (such as DNA) is said to "express" a polypeptide if it contains nucleotide sequences that contain information regulating transcription and translation, and these sequences are "operably linked" to nucleotide sequences encoding a polypeptide. An operable link is a link in which the regulating DNA sequence and the DNA sequence seeking expression (e.g., a PTK7 antibody or its antigen-binding moiety or other binding agent) are linked in a manner that allows the polypeptide or antigen-binding moiety to be expressed in a recoverable amount of the gene. The exact nature of the regulatory regions required for gene expression can vary from organism to organism, as is well known in similar fields. See, for example, Sambrook et al., 1989; Ausubel et al., 1987–1993.

[0191] Therefore, the expression of PTK7 antibodies or their antigen-binding moieties, as described herein, can occur in prokaryotic or eukaryotic cells. Suitable hosts include bacteria or eukaryotic hosts, including in vivo or in situ yeast, insect, fungal, avian, and mammalian cells, or host cells derived from mammals, insects, birds, or yeast. Mammalian cells or tissues can be derived from humans, primates, hamsters, rabbits, rodents, cattle, pigs, sheep, horses, goats, dogs, or cats, but any other mammalian cells may also be used. Furthermore, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be achieved by using, for example, a yeast ubiquitin hydrolase system. The resulting fusion proteins can be processed in vivo or purified and processed in vitro, allowing the synthesis of PTK7 antibodies or their antigen-binding moieties, or other binding agents, with specific amino-terminal sequences as described herein. Furthermore, problems associated with the retention of start codon-derived methionine residues in direct yeast (or bacterial) expression can be avoided. (See, for example, Sabin et al., 7 Bio / Technol. 705 (1989); Miller et al., 7 Bio / Technol. 698 (1989).). A range of yeast gene expression systems incorporating promoters and terminators from active gene encoding glycolytic enzymes produced in large quantities when yeast is grown in glucose-rich media can be used to obtain recombinant PTK7 antibodies or their antigen-binding moieties or other binding agents. Known glycolytic genes can also provide very efficient transcriptional control signals. For example, promoter and terminator signals from phosphoglycerate kinase genes can be utilized.

[0192] The production of PTK7 antibodies or their antigen-binding moieties or other binders in insects can be achieved, for example, by infecting insect hosts with baculoviruses engineered to express peptides, using methods known to those skilled in the art. See Ausubel et al., 1987–1993.

[0193] In some implementations, an introduced nucleic acid sequence (encoding a PTK7 antibody or its antigen-binding moiety or other binding agent or polypeptide thereof) is incorporated into a plasmid or viral vector capable of autonomous replication in recipient host cells. Any of a variety of vectors can be used for this purpose and are known and available to those skilled in the art. See, for example, Ausubel et al., 1987–1993. Important factors in selecting a particular plasmid or viral vector include: ease of identification and selection of vector-containing recipient cells from those that do not contain the vector; the desired vector copy number in a particular host; and whether it is desired that the vector can “shuttle” between host cells of different species.

[0194] Exemplary prokaryotic vectors known in the art include plasmids, such as those capable of replicating in *Escherichia coli*. Other gene expression elements that can be used to express DNA encoding a PTK7 antibody or its antigen-binding moiety or other binding agents include, but are not limited to, (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983)), Rous sarcoma virus LTR (Gorman et al., 79 PNAS 6777 (1982)), and Moloney murine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985)); (b) splice regions and polyadenylation sites, such as those derived from the late SV40 region (Okayarea et al., 1983); and (c) polyadenylation sites, such as those in SV40 (Okayama et al., 1983). Immunoglobulin-encoding DNA genes can be expressed as described by Liu et al., hereinafter and Weidle et al., 51 Gene 21 (1987), using the SV40 early promoter and its enhancer, mouse immunoglobulin H chain promoter enhancer, SV40 late region mRNA splicing, rabbit S-globulin insertion sequence, immunoglobulin and rabbit S-globulin polyadenylation sites and SV40 polyadenylation elements as expression elements.

[0195] For the nucleotide sequence encoding immunoglobulin, the transcription promoter can be, for example, human cytomegalovirus, and the promoter enhancer can be cytomegalovirus and mouse / human immunoglobulin.

[0196] In some embodiments, to express the DNA coding region in rodent cells, the transcription promoter may be a viral LTR sequence, the transcription promoter enhancer may be either or both of a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, and a polyadenylation and transcription termination region. In other embodiments, DNA sequences encoding other proteins are combined with the above-described expression elements to achieve protein expression in mammalian cells.

[0197] Each coding region or gene fusion is assembled into or inserted into an expression vector. Recipient cells capable of expressing the PTK7 variable region or its antigen-binding moiety or other binders are then transfected individually with nucleotides encoding PTK7 antibodies or antibody peptides or their antigen-binding moiety or other binders, or co-transfected with polynucleotides encoding the VH and VL chain coding regions or other binders. Transfected recipient cells are cultured under conditions allowing expression of the incorporated coding regions, and the expressed antibody chains or intact antibodies or antigen-binding moieties or other binders are recovered from the culture.

[0198] In some embodiments, nucleic acids containing coding regions encoding PTK7 antibodies or their antigen-binding moieties or other binding agents are assembled into separate expression vectors and then co-transfected into recipient host cells. Each vector may contain one or more selectable genes. For example, in some embodiments, two selectable genes are used, a first selectable gene designed for selection in a bacterial system and a second selectable gene designed for selection in a eukaryotic system, wherein each vector has a set of coding regions. This strategy produces vectors that first direct the production of nucleotide sequences in a bacterial system and allow their amplification. DNA vectors thus produced and amplified in a bacterial host are then used for co-transfection into eukaryotic cells, allowing selection of co-transfected cells carrying the desired transfected nucleic acid (e.g., containing the heavy and light chains of the PTK7 antibody). Non-limiting examples of selectable genes for bacterial systems are genes conferring resistance to ampicillin and genes conferring resistance to chloramphenicol. Selectable genes for eukaryotic transfectants include the xanthine-guanine phosphoribosyltransferase gene (named gpt) and the phosphotransferase gene from Tn5 (named neo). Alternatively, the fusion nucleotide sequences encoding the VH and VL chains can be assembled on the same expression vector.

[0199] For the transfection of the expression vector and the generation of the PTK7 antibody or its antigen-binding moiety or other binding agents, the recipient cell line can be a Chinese hamster ovary cell line (e.g., DG44) or myeloma cells. Myeloma cells can synthesize, assemble, and secrete immunoglobulins encoded by the transfected immunoglobulin gene and possess the mechanism of immunoglobulin glycosylation. For example, in some embodiments, the recipient cells are SP2 / 0 myeloma cells that produce recombinant Ig. SP2 / 0 cells produce only immunoglobulins encoded by the transfected gene. Myeloma cells can be grown in cultures or in the peritoneal cavity of mice (where the secreted immunoglobulins can be obtained from ascites fluid).

[0200] Expression vectors encoding PTK7 antibodies or their antigen-binding moiety or other binding agents can be introduced into suitable host cells by any of a variety of suitable methods, including biochemical methods such as transformation, transfection, protoplast fusion, calcium phosphate precipitation, and application of polycationic substances such as diethylaminoethyl (DEAE) dextran, and mechanical methods such as electroporation, direct microinjection, and microparticle bombardment. Johnston et al., 240 Science 1538 (1988), as is known to those skilled in the art.

[0201] Yeast offers certain advantages over bacteria in the production of both heavy and light chains of immunoglobulins. The production, secretion, and stability levels of antibodies and assembled PTK7 antibodies from yeast gene expression systems, along with their antigen-binding moieties and other binding agents, can be routinely assessed.

[0202] Bacterial strains (e.g., Escherichia coli K12 strain) can also be used as hosts for producing the antibody molecules described herein or their antigen-binding portions or other binding agents.

[0203] Host mammalian cells can grow in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin molecules, including leader peptide removal, folding and assembly of VH and VL chains, glycosylation of antibody molecules, and secretion of functional antibodies and / or their antigen-binding moieties or other binders.

[0204] In addition to the lymphoid-derived cells mentioned above, mammalian cells that can be used as hosts for antibody protein production include fibroblast-derived cells, such as Vero or CHO-K1 cells. Exemplary eukaryotic cells that can be used to express immunoglobulin peptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; PERC6™ cells (Crucell); and NSO cells. In some embodiments, specific eukaryotic host cells are selected based on their ability to perform desired post-translational modifications to the heavy and / or light chains. For example, in some embodiments, peptides produced by CHO cells have a higher level of sialylation than the same peptides produced by 293 cells.

[0205] In some implementations, one or more PTK7 antibodies or their antigen-binding moieties or other binders may be produced in vivo in animals that have been engineered or transfected with one or more nucleic acid molecules encoding polypeptides, according to any suitable method.

[0206] In some embodiments, the antibody or its antigen-binding portion is generated in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); and Endo et al., Biotechnol. Adv. 21:695-713 (2003).

[0207] Many vector systems can be used to express VH and VL chains in mammalian cells (see Glover, 1985). Various methods can be followed to obtain complete antibodies. Furthermore, plants have become a convenient, safe, and economical alternative expression system for large-scale culture production of recombinant antibodies based on microorganisms or animal cells.

[0208] For intact antibodies, the variable regions (VH and VL regions) of a PTK7 antibody are typically linked to at least a portion of an immunoglobulin constant region (Fc) or domain (typically the constant region (Fc) or domain of human immunoglobulins). Human constant region DNA sequences can be isolated from various human cells, such as immortalized B cells, using well-known methods (WO 87 / 02671). PTK7 binding antibodies may contain both light and heavy chain constant regions. The heavy chain constant region may include CH1, hinge, CH2, CH3, and optionally CH4 regions. In some embodiments, the CH2 domain may be omitted or delimited.

[0209] Techniques for producing single-chain antibodies are described (see, for example, U.S. Patent No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) that are adapted to produce single-chain antibodies that specifically bind to PTK7. Single-chain antibodies are formed by linking the heavy and light chain variable regions of the Fv region via amino acid bridges to produce single-chain polypeptides. Techniques for assembling functional Fv moieties in E. coli can also be used (see, for example, Skerra et al., Science 242:1038-1041 (1988); which is incorporated herein by reference in its entirety).

[0210] In some embodiments, the antigen-binding moiety or other binding agent comprises one or more scFvs. In some embodiments, the antigen-binding moiety or other binding agent is a single-domain antibody, which is an antibody moiety composed of a single monomeric variable antibody domain. Techniques for generating single-domain antibodies (DABs or VHHs) are known in the art, such as those disclosed, for example, in Cossins et al. (2006, Prot Express Purif 51:253-259) and Li et al. (Immunol. Lett. 188:89-95, 2017).

[0211] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of heavy-light chain pairs of two immunoglobulins with different specificities (see, for example, Milstein and Cuello, Nature 305:537 (1983)), WO93 / 08829 and Traunecker et al., EMBO J. 10:3655 (1991)), and mortar and pestle engineering (see, for example, U.S. Patent No. 5,731,168; Carter (2001), J Immunol Methods 248, 7-15).

[0212] Engineered antibodies with three or more functional antigen-binding sites (including “octopus antibodies”) can also be binding agents (see, for example, US 2006 / 0025576A1).

[0213] The conjugates (e.g., antibody or antigen-binding moiety) described in this article also include “dual-acting FAbs” or “DAFs” that contain antigen-binding sites that bind to two different antigens (see, for example, US 2008 / 0069820 and Bostrom et al., 2009, Science 323:1610-14). This article also includes “Crossmab” antibodies (see, for example, WO 2009 / 080251, WO 2009 / 080252, WO2009 / 080253, WO2009 / 080254, and WO2013 / 026833).

[0214] In some embodiments, the binder comprises a different antigen-binding site fused to one or the other of the two subunits of the Fc domain; thus, the two subunits of the Fc domain can be contained in two distinct polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization result in several possible combinations of the two polypeptides. To improve the yield and purity of bispecific molecules in recombinant production, it would be advantageous to introduce modifications into the Fc domain of the binder that promote the association of the desired polypeptide.

[0215] Generally, this method involves replacing one or more amino acid residues at the interface of two Fc domains with charged amino acid residues, making homodimer formation electrostatically unfavorable and heterodimerization electrostatically favorable.

[0216] In some embodiments, the binder is a “bispecific T-cell connector” or BiTE (see, for example, WO2004 / 106381, WO2005 / 061547, WO2007 / 042261, and WO2008 / 119567). This method utilizes two antibody variable domains arranged on a single polypeptide. For example, the single polypeptide chain may include two single-chain Fv (scFv) moieties, each having a variable heavy chain (VH) and a variable light chain (VL) domain separated by a polypeptide linker of sufficient length to allow intramolecular association between the two domains. The single polypeptide further includes a polypeptide spacer sequence between the two scFvs. Each scFv recognizes a different epitope, and these epitopes can be specific to different proteins, such that both proteins are bound by BiTE.

[0217] In some implementations, the binder is multispecific, such as IgG-scFV. IgG-scFv forms include IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG, and IgG-2scFv. These and other bispecific antibody forms and methods for preparing them have been described, for example, in Brinkmann and Kontermann, MAbs 9(2):182-212 (2017); Wang et al., Antibodies, 2019, 8, 43; Dong et al., 2011, MAbs 3:273-88; Natsume et al., J. Biochem. 140(3):359-368, 2006; Cheal et al., Mol. Cancer Ther. 13(7):1803-1812, 2014; and Bates and Power, Antibodies, 2019, 8, 28.

[0218] IgG-like dual variable domain antibodies (DVD-Ig) have been described in Wu et al., 2007, Nat Biotechnol 25:1290-97; Hasler et al., Mol. Immunol. 75:28-37, 2016 and WO 08 / 024188 and WO 07 / 024715. TriomAb has been described by Chelius et al., MAbs 2(3):309-319, 2010. Dual-IgG has been described by Kontermann et al., Drug Discovery Today 20(7):838-847, 2015. Tanden antibodies or TandAbs have been described by Kontermann et al., id. ScFv-HSA-scFv antibodies have also been described by Kontermann et al. (id).

[0219] Intact (e.g., whole) antibodies, their dimers, individual light and heavy chains, or their antigen-binding moieties and other binders can be recovered and purified using known techniques, such as immunoadsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high-performance liquid chromatography), ammonium sulfate precipitation, gel electrophoresis, or any combination thereof. See generally Scopes, Protein Purification (Springer-Verlag, NY, 1982). Substantially pure PTK7-binding antibodies, their antigen-binding moieties, or other binders with at least about 90% to 95% homogeneity are advantageous, as are those with 98% to 99% or higher homogeneity, particularly for pharmaceutical use. Once partially purified or purified to homogeneity as required, intact PTK7 antibodies, their antigen-binding moieties, or other binders can be used for therapeutic purposes or for developing and performing assays, immunofluorescence staining, etc. See generally Vols. I & II Immunol. Meth. (edited. Lefkovits and Pernis, Acad. Press, NY, 1979 and 1981).

[0220] Conjugate

[0221] The conjugates of the present invention, particularly antibodies, can be provided in the form of conjugates. In one embodiment, the conjugates (particularly antibodies) of the present invention can be provided in the form of conjugates (particularly antibodies) conjugated with a label. The label can be any of those mentioned herein, for example. In one embodiment, the conjugates (particularly antibodies) of the present invention can be provided in the form of conjugates (particularly antibodies) conjugated with a drug. The drug can be, for example, a chemotherapeutic agent. In one embodiment, the conjugates (particularly antibodies) of the present invention can be provided in the form of conjugates (particularly antibodies) conjugated with a portion that allows for easier purification of the conjugate. This portion can be, for example, biotin.

[0222] IHC method

[0223] This invention provides the use of the binding agent of this invention in the detection and / or quantification of PTK7 protein. In one particular embodiment, this invention provides the use of the binding agent of this invention in immunohistochemistry (IHC).

[0224] This disclosure also provides an immunohistochemical (IHC) method for detecting PTK7-expressing cells in a sample using the binding agent of the present invention. In some embodiments, the method includes contacting the sample with a detection agent comprising any one of an antibody of the present disclosure, its antigen-binding portion, and other binding agents, and detecting the formation of a binding complex between the detection agent and PTK7. In some embodiments, the detection of the binding complex indicates the presence of PTK7-expressing cells in the sample.

[0225] According to this disclosure, the term "sample" from a subject can be a sample of the subject's tissue or any bodily fluid that may contain PTK7. Exemplary bodily fluids include, for example, blood, saliva, nasal mucus, or lymph. In some embodiments, the sample is a blood sample, such as those selected from the group consisting of whole blood, serum, plasma, capillary blood, arterial blood, venous blood, or any mixture thereof. Exemplary tissue samples include, but are not limited to, tissues of the tonsils, appendix, breast, ovary, colon, prostate, skin, lung, uterus, cervix, kidney, pancreas, bladder, brain, thyroid, ear, nose, larynx, esophagus, and liver. In one embodiment, the tissue is ovarian tissue. In another embodiment, the tissue is breast cancer tissue. In one embodiment, the tissue is tissue from a breast cancer biopsy. Those skilled in the art are aware of various means and methods that can be applied to obtain samples from a subject and in the required quantities suitable for the purposes of the methods, products, and uses disclosed herein. In some embodiments, the sample may be obtained from the subject by a physician, while in other cases, the sample may be obtained by the subject themselves. In some embodiments, the sample may be derived from a single subject, i.e., a single individual. In some embodiments, the sample may comprise tissue or bodily fluids from more than one subject. In some embodiments, the sample may be a tissue section. In embodiments utilizing tissue samples, the method is therefore an in vitro method. The binder of the present invention can also be used for in vivo imaging.

[0226] According to this disclosure, "subject" or, interchangeably herein, "patient" refers to a mammal, preferably a human, but may alternatively refer to different mammals, such as non-human primates or other mammals, or even non-mammals, any of which produce cells expressing PTK7. In some embodiments, the subject is a human patient. In some embodiments, the subject is a human cancer patient.

[0227] In some embodiments, the detection reagent further comprises a detectable biomarker that labels the binder. Definitions and examples of detectable biomarkers and detection reagents can be found elsewhere in this disclosure.

[0228] As used herein, the term "labeled" or "labeled" in relation to a binding agent is intended to include embodiments in which a detectable substance (such as a radioactive agent or another molecule providing a detectable signal) is labeled by coupling (e.g., by physical coupling or covalent binding), for example, but not intended to be limited to fluorescent tags (i.e., fluorophores, such as small organic chemical fluorophores or fluorescent proteins), binding molecules (e.g., biotin, which may bind avidin or streptavidin with high affinity), or enzyme-catalyzed activity labels ("enzyme labels"), i.e., enzymes, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), whose presence can be assessed and optionally quantified based on their reaction and / or transformation with a substrate. Various suitable detectable biomarkers are known in the art, and some of these are also described herein.

[0229] In some implementations, the detectable biomarker includes biotin, which can bind avidin or streptavidin with high affinity. Such duals (biotin-avidin, biotin-streptavidin) have been used quite extensively for labeling and detecting target molecules (e.g., antigens).

[0230] In some embodiments, the detectable biomarkers include enzyme biomarkers. In some embodiments, enzyme biomarkers include horseradish peroxidase (HRP) or alkaline phosphatase (AP). Such biomarkers catalyze reactions that can produce detectable signals indicating the formation of a complex between the binder and PTK7. Common substrates for HRP include, but are not limited to, 3,3',5,5'-tetramethylbenzidine (TMB), 3,3'-diaminobenzidine (DAB), 2,2'-azidobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3-amino-9-ethylcarbazole (AEC), o-phenylenediamine dihydrochloride (OPD), homovanillic acid, AmplexRed, and luminol.

[0231] In some embodiments, detecting the formation of a binding complex between the detectant and PTK7 includes: adding a mating agent of the detectable marker; and detecting the reaction between the detectable marker and the mating agent as an indication of the formation of the binding complex.

[0232] Definitions and examples of conjugate agents can be found elsewhere in this disclosure. Essentially, a detectable biomarker and a conjugate agent react to produce a detectable signal (e.g., staining of tissue) via microscopy and imaging techniques. In some embodiments, the conjugate agent is a conjugate of biotin (e.g., avidin or streptavidin). In some embodiments, the conjugate agent is a substrate of an enzyme biomarker (e.g., a substrate of HRP or AP).

[0233] An example of a pair of detectable biomarkers and their matching agents is biotin and DAB. By contacting a sample with a biotinylated binding agent (i.e., the detection agent) and adding DAB, PTK7-positive cells will be stained and can be visualized, imaged, and quantified if necessary, according to a predetermined protocol.

[0234] In some embodiments, the detectable marker includes a fluorescent tag, which provides a means of generating a detectable signal without the need for the addition of a coupler agent. In some embodiments, detecting the formation of a binding complex between the detectant and PTK7 includes detecting a signal from the fluorescent tag as an indication of the formation of the binding complex. In some embodiments, the fluorescent tag includes fluorescein (FITC), phycoerythrin (PE), preferably R-PE or allophycocyanin (APC).

[0235] In some embodiments, detecting the formation of a binding complex between the detection agent and PTK7 includes: contacting the sample with a labeled secondary antibody configured to bind the binding complex; and detecting a signal from the secondary antibody as an indication of the formation of the binding complex.

[0236] According to this disclosure, a "secondary antibody" refers to any type of "binding moiety," such as a binding protein, capable of specifically binding to IgA, IgG, and / or IgM antibodies or fragments thereof. Non-limiting examples of binding moieties include antibodies, such as antibodies, antibody fragments, domains, or portions thereof (e.g., Fab, Fab', F(ab')2, scFab, Fv, scFv, VH, VHH, VL, VLR, etc.) that are immunely or genetically derived from any species (e.g., human, chicken, camel, llama, lamprey, shark, goat, rodent, cattle, dog, rabbit, etc.), biantibodies, monoclonal antibodies (mAbs), and polyclonal antibodies (pAbs).

[0237] In some implementations, the secondary antibody is an immunoglobulin (Ig) (e.g., IgG produced in a non-human species), wherein the secondary antibody specifically binds to one or more specific Ig classes or fragments thereof (e.g., constant domains of specific Ig classes) of another selected species (e.g., mice or humans).

[0238] In some embodiments, the secondary antibody is labeled with a detectable biomarker, such that a detectable signal can be generated directly by the secondary antibody or after the labeled secondary antibody reacts with another molecule. Means and methods for developing, screening, and identifying suitable binding molecules for various scaffolds against desired target structures (such as IgA and / or IgG antibodies), including but not limited to those described above, are known and employed in the art. Exemplary methods currently routinely employed include, but are not intended to be limited to, high-throughput (HT) combinatorial library-based display and selection methods, such as phage display, ribosome display, mRNA display, and cell surface display (e.g., yeast display).

[0239] In some embodiments, detecting the formation of a binding complex between the detectant and PTK7 includes: adding a mating agent of the detectable marker; and detecting the reaction between the detectable marker and the mating agent as an indication of the formation of the binding complex.

[0240] In some embodiments, the detectable biomarkers include biotin or enzyme biomarkers. The descriptions of the detectable biomarkers, biotin, enzyme biomarkers, and conjugate agents of the secondary antibody are substantially the same as those of such molecules or portions thereof provided elsewhere for use as binding agents.

[0241] In some embodiments, the detectable marker includes a fluorescent tag. In some embodiments, detecting the formation of a binding complex between the detector and PTK7 includes detecting a signal from the fluorescent tag as an indication of the formation of the binding complex. In some embodiments, the fluorescent tag includes fluorescein (FITC), phycoerythrin (PE), preferably R-PE, or allophycocyanin (APC).

[0242] In some embodiments, the sample is derived from the subject's tissue. In some embodiments, the tissue is selected from the group consisting of tonsils, appendix, breast, ovary, colon, prostate, skin, lung, uterus, cervix, kidney, pancreas, bladder, brain, thyroid, ear, nose, larynx, and esophagus. In some embodiments, the tissue is selected from the group consisting of breast, ovary, colon, prostate, skin, lung, uterus, esophagus, and bladder. In some embodiments, the tissue is breast. In some embodiments, the tissue is ovary. In some embodiments, the tissue is prostate. In addition to the methods described herein, the present invention also provides tissue samples stained using the binding agent of the present invention.

[0243] In some implementations, the sample is taken from the subject, and the presence of cells expressing PTK7 indicates that the subject has a disease.

[0244] In some implementations, the sample is from a subject, and the method further includes: quantifying the binding of PTK-expressing cells in the sample to obtain a quantitative result, wherein a quantitative result exceeding a predetermined threshold indicates that the subject has a disease.

[0245] In some embodiments, disease occurrence is associated with the presence of PTK7 expression. In some embodiments, disease occurrence is associated with the presence of cells expressing PTK7. In some embodiments, disease occurrence is associated with the number of PTK7-expressing cells in a tissue section exceeding a predetermined threshold. In some embodiments, disease occurrence is associated with the percentage of PTK7-expressing cells in a tissue section relative to the total number of cells exceeding a predetermined percentage threshold.

[0246] In some implementations, the disease is cancer. In some implementations, the disease is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, melanoma, lung cancer, esophageal cancer, stomach cancer, endometrial cancer, head and neck cancer, and bladder cancer.

[0247] In some implementations, the disease is breast cancer. In some implementations, the disease is ovarian cancer. In some implementations, the disease is squamous cell carcinoma of the lung or adenocarcinoma of the lung.

[0248] In some embodiments, the disease is colon cancer. In some embodiments, the disease is prostate cancer. In some embodiments, the disease is melanoma. In some embodiments, the disease is lung cancer. In some embodiments, the disease is esophageal cancer. In some embodiments, the disease is stomach cancer. In some embodiments, the disease is endometrial cancer. In some embodiments, the disease is head and neck cancer. In some embodiments, the disease is bladder cancer.

[0249] Disease detection and treatment

[0250] In some embodiments, this disclosure provides a method for determining whether a subject has a disease, comprising: obtaining a sample from the subject; contacting the sample with a detection agent comprising a binder of this disclosure; determining whether the subject has the disease by detecting and / or quantifying the formation of a binding complex between the detection agent and PTK7, wherein the presence of the binding complex or a level of the binding complex exceeding a predetermined threshold indicates that the subject has the disease. In some embodiments, the method is performed on a sample already obtained from the subject, and therefore the method does not include a sample collection step.

[0251] In some embodiments, disease occurrence is associated with the presence of PTK7 expression. In some embodiments, disease occurrence is associated with the presence of cells expressing PTK7. In some embodiments, disease occurrence is associated with the number of PTK7-expressing cells in a tissue section exceeding a predetermined threshold. In some embodiments, disease occurrence is associated with the percentage of PTK7-expressing cells in a tissue section relative to the total number of cells exceeding a predetermined percentage threshold.

[0252] In some implementations, the disease is cancer. In some implementations, the disease is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, melanoma, lung cancer, esophageal cancer, stomach cancer, endometrial cancer, head and neck cancer, and bladder cancer.

[0253] In some implementations, the disease is breast cancer. In some implementations, the disease is ovarian cancer. In some implementations, the disease is squamous cell carcinoma of the lung or adenocarcinoma of the lung.

[0254] In some embodiments, the disease is colon cancer. In some embodiments, the disease is prostate cancer. In some embodiments, the disease is melanoma. In some embodiments, the disease is lung cancer. In some embodiments, the disease is esophageal cancer. In some embodiments, the disease is stomach cancer. In some embodiments, the disease is endometrial cancer. In some embodiments, the disease is head and neck cancer. In some embodiments, the disease is bladder cancer.

[0255] In some embodiments, this disclosure provides a method for treating a disease in a patient who has been diagnosed with the disease by the detection methods described herein. In one embodiment, the detection method includes obtaining a sample from a subject; contacting the sample with a detection agent comprising a binder of this disclosure; determining whether the subject has the disease by detecting and / or quantifying the formation of a binding complex between the detection agent and PTK7, wherein the presence of the binding complex or a level of the binding complex exceeding a predetermined threshold indicates that the subject has the disease; and treating the disease. In some embodiments, the binder comprises VH and VL regions containing the amino acid sequences shown below: SEQ ID NO: 1 and SEQ ID NO: 2, respectively; SEQ ID NO: 8 and SEQ ID NO: 9, respectively; or SEQ ID NO: 15 and SEQ ID NO: 16, respectively. Anticancer agents for such methods are also provided.

[0256] In some embodiments, disease occurrence is associated with the presence of PTK7 expression. In some embodiments, disease occurrence is associated with the presence of cells expressing PTK7. In some embodiments, disease occurrence is associated with the number of PTK7-expressing cells in a tissue section exceeding a predetermined threshold. In some embodiments, disease occurrence is associated with the percentage of PTK7-expressing cells in a tissue section relative to the total number of cells exceeding a predetermined percentage threshold.

[0257] In some implementations, the disease is cancer. In some implementations, the disease is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, melanoma, lung cancer, esophageal cancer, stomach cancer, endometrial cancer, head and neck cancer, and bladder cancer.

[0258] In some implementations, the disease is breast cancer. In some implementations, the disease is ovarian cancer. In some implementations, the disease is squamous cell carcinoma of the lung or adenocarcinoma of the lung.

[0259] In some embodiments, the disease is colon cancer. In some embodiments, the disease is prostate cancer. In some embodiments, the disease is melanoma. In some embodiments, the disease is lung cancer. In some embodiments, the disease is esophageal cancer. In some embodiments, the disease is stomach cancer. In some embodiments, the disease is endometrial cancer. In some embodiments, the disease is head and neck cancer. In some embodiments, the disease is bladder cancer.

[0260] In some implementations, the treatment includes administering chemotherapy to the subject.

[0261] In some implementations, the treatment includes administering hormone therapy to the subject.

[0262] In some implementations, the treatment includes administering radiation therapy to the subject.

[0263] In some implementations, the treatment includes administering immunotherapy to the subject.

[0264] In some implementations, the treatment includes administering stem cell therapy to the subject.

[0265] In some implementations, treatment includes administering a targeted therapy to the subject.

[0266] In some implementations, the treatment includes performing surgery on the subject.

[0267] In some implementations, the disease is breast cancer, and the treatment includes removal of the breast tumor or mastectomy.

[0268] In some implementations, the disease is ovarian cancer, and the treatment includes hysterectomy or salpingo-oophorectomy.

[0269] In some embodiments, this disclosure provides a method for monitoring the progression of disease in a subject, comprising the steps of: obtaining a sample from the subject; contacting the sample with a detection agent containing a binder of this disclosure; assessing the formation of a binding complex between the detection agent and PTK7 to obtain a first assessment result; repeating steps (a), (b), and (c) using samples from the subject at subsequent time points to obtain a second assessment result; and comparing the second assessment result with the first assessment result to determine the progression of disease in the subject.

[0270] In some embodiments, disease occurrence is associated with the presence of PTK7 expression. In some embodiments, disease occurrence is associated with the presence of cells expressing PTK7. In some embodiments, disease occurrence is associated with the number of PTK7-expressing cells in a tissue section exceeding a predetermined threshold. In some embodiments, disease occurrence is associated with the percentage of PTK7-expressing cells in a tissue section relative to the total number of cells exceeding a predetermined percentage threshold.

[0271] In some implementations, the disease is cancer. In some implementations, the disease is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, melanoma, lung cancer, esophageal cancer, stomach cancer, endometrial cancer, head and neck cancer, and bladder cancer.

[0272] In some implementations, the disease is breast cancer. In some implementations, the disease is ovarian cancer. In some implementations, the disease is squamous cell carcinoma of the lung or adenocarcinoma of the lung.

[0273] In some embodiments, the disease is colon cancer. In some embodiments, the disease is prostate cancer. In some embodiments, the disease is melanoma. In some embodiments, the disease is lung cancer. In some embodiments, the disease is esophageal cancer. In some embodiments, the disease is stomach cancer. In some embodiments, the disease is endometrial cancer. In some embodiments, the disease is head and neck cancer. In some embodiments, the disease is bladder cancer.

[0274] Reagent test kit

[0275] This disclosure also provides kits for detecting cells expressing PTK7 in samples. In some embodiments, such kits typically contain one or more components necessary for performing the IHC method (i.e., assay) described herein. Components may be compounds, reagents, containers, and / or devices.

[0276] In some embodiments, the kit includes a detection agent that comprises any one of the antibodies, antibody fragments, and other detection agents (collectively, “binding agents”) of this disclosure.

[0277] In some implementations, the reagent kit's binder is tagged with a detectable biomarker.

[0278] In some embodiments, the kit further comprises a secondary antibody that is labeled and configured to bind to a binding complex that can be formed by the detection agent and PTK7. In some embodiments, the secondary antibody is labeled with a detectable biomarker.

[0279] In some embodiments, the kit also includes a partner agent. Descriptions of the detectable biomarkers and partner agents are provided elsewhere in this disclosure.

[0280] In some implementations, the kit also includes a buffer for assaying.

[0281] In some implementations, the kit also includes instructions for carrying out the methods of the present invention.

[0282] In some embodiments, the kit contains all the reagents mentioned herein with respect to the methods of the invention. In some embodiments, the kit also contains instructions for performing the methods.

[0283] use

[0284] This disclosure also provides the use of the binder in cells expressing PTK7 in a test sample, the use of the binder in the preparation of a kit for detecting cells expressing PTK7 in a sample, and / or the use for determining whether a subject has a disease.

[0285] In some implementations, the disease is cancer. In some implementations, the disease is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, melanoma, lung cancer, esophageal cancer, stomach cancer, endometrial cancer, head and neck cancer, and bladder cancer.

[0286] In some implementations, the disease is breast cancer. In some implementations, the disease is ovarian cancer. In some implementations, the disease is squamous cell carcinoma of the lung or adenocarcinoma of the lung.

[0287] In some embodiments, the disease is colon cancer. In some embodiments, the disease is prostate cancer. In some embodiments, the disease is melanoma. In some embodiments, the disease is lung cancer. In some embodiments, the disease is esophageal cancer. In some embodiments, the disease is stomach cancer. In some embodiments, the disease is endometrial cancer. In some embodiments, the disease is head and neck cancer. In some embodiments, the disease is bladder cancer.

[0288] The use of the binder of the present invention in the detection and / or quantification of PTK7 is also provided.

[0289] The invention also provides the use of the binding agent for staining tissue samples. Furthermore, the invention provides the use of the binding agent in flow cytometry.

[0290] Further implementation plan

[0291] Any of the binding agents described herein, particularly antibodies, can be used to detect cancers expressing PTK7. In one embodiment, the binding agents (particularly antibodies) of the present invention can be selected for use in the methods or applications described herein based on the staining pattern exhibited by the binding agents of the present invention, particularly antibodies (e.g., staining patterns as described in the forms of this application). In one embodiment, they can be selected, for example, because they exhibit a stronger binding to the target cancer tissue than other antibodies described herein. For example, they can be selected because they stain membrane PTK7. For example, they can be selected because they stain intracellular PTK7.

[0292] In one embodiment, an antibody comprising six CDRs of the M10-5F7 antibody, or an antigen-binding fragment thereof, can be used to detect ovarian cancer (HCDR1 of SEQ ID NO: 17, HCDR2 of SEQ ID NO: 18, HCDR3 of SEQ ID NO: 19, LCDR1 of SEQ ID NO: 20, LCDR2 of RVS, and LCDR3 of SEQ ID NO: 21). In one embodiment, the antibody or its antigen-binding fragment comprises the VH and VL sequences of the M10-5F7 antibody (SEQ ID NO: 15 and 16, respectively). Variant antibodies may also be used.

[0293] In one embodiment, an antibody comprising the six CDRs of the M10-5F7 antibody, or an antigen-binding fragment thereof, can be used to detect tonsil cancer. In one embodiment, the antibody or its antigen-binding fragment comprises the VH and VL sequences of the M10-5F7 antibody. Variant antibodies may also be used.

[0294] In one embodiment, an antibody comprising the six CDRs of the M10-5F7 antibody, or an antigen-binding fragment thereof, can be used to detect renal cell carcinoma. In one embodiment, the antibody or its antigen-binding fragment comprises the VH and VL sequences of the M10-5F7 antibody. Variant antibodies may also be used.

[0295] In one embodiment, an antibody comprising the six CDRs of the M10-5F7 antibody, or an antigen-binding fragment thereof, can be used to detect uterine cancer. In one embodiment, the antibody or its antigen-binding fragment comprises the VH and VL sequences of the M10-5F7 antibody. Variant antibodies may also be used.

[0296] In one embodiment, an antibody comprising six CDRs of the M6-8G5 antibody (HCDR1 of SEQ ID NO: 10, HCDR2 of SEQ ID NO: 11, HCDR3 of SEQ ID NO: 12, LCDR1 of SEQ ID NO: 13, LCDR2 of QMS, and LCDR3 of SEQ ID NO: 14), or an antigen-binding fragment thereof, can be used to detect ovarian cancer. In one embodiment, the antibody or its antigen-binding fragment comprises the VH and VL sequences of the M6-8G5 antibody (SEQ ID NO: 8 and 9, respectively). Variant antibodies may also be used.

[0297] In one embodiment, an antibody comprising the six CDRs of an M6-8G5 antibody, or an antigen-binding fragment thereof, can be used to detect breast cancer. In one embodiment, the antibody or its antigen-binding fragment comprises the VH and VL sequences of the M6-8G5 antibody. Variant antibodies may also be used.

[0298] In one embodiment, an antibody comprising the six CDRs of an M6-8G5 antibody, or an antigen-binding fragment thereof, can be used to detect pancreatic cancer. In one embodiment, the antibody or its antigen-binding fragment comprises the VH and VL sequences of the M6-8G5 antibody. Variant antibodies may also be used.

[0299] In one embodiment, an antibody comprising the six CDRs of an M6-8G5 antibody, or an antigen-binding fragment thereof, can be used to detect lung cancer. In one embodiment, the antibody or its antigen-binding fragment comprises the VH and VL sequences of the M6-8G5 antibody. Variant antibodies may also be used.

[0300] In one embodiment, an antibody comprising the six CDRs of an M6-8G5 antibody, or an antigen-binding fragment thereof, can be used to detect uterine cancer. In one embodiment, the antibody or its antigen-binding fragment comprises the VH and VL sequences of the M6-8G5 antibody. Variant antibodies may also be used.

[0301] In one embodiment, an antibody comprising the six CDRs of an M6-8G5 antibody, or an antigen-binding fragment thereof, can be used to detect renal cell carcinoma. In one embodiment, the antibody or its antigen-binding fragment comprises the VH and VL sequences of the M6-8G5 antibody. Variant antibodies may also be used.

[0302] In one embodiment, an antibody comprising the six CDRs of an M6-8G5 antibody, or an antigen-binding fragment thereof, can be used to detect pancreatic cancer. In one embodiment, the antibody or its antigen-binding fragment comprises the VH and VL sequences of the M6-8G5 antibody. Variant antibodies may also be used.

[0303] In one embodiment, an antibody comprising the six CDRs of an M6-8G5 antibody, or an antigen-binding fragment thereof, can be used to detect prostate cancer. In one embodiment, the antibody or its antigen-binding fragment comprises the VH and VL sequences of the M6-8G5 antibody. Variant antibodies may also be used.

[0304] In one embodiment, an antibody comprising six CDRs (HCDR1 of SEQ ID NO: 3, HCDR2 of SEQ ID NO: 4, HCDR3 of SEQ ID NO: 5, LCDR1 of SEQ ID NO: 6, LCDR2 of LVS, and LCDR3 of SEQ ID NO: 7) of the M6-11E2 antibody, or an antigen-binding fragment thereof, may be used to detect breast cancer. In one embodiment, the antibody or its antigen-binding fragment comprises the VH and VL sequences of the M6-11E2 antibody (SEQ ID NO: 1 and 2, respectively). Variant antibodies may also be used.

[0305] In one embodiment, an antibody comprising the six CDRs of an M6-11E2 antibody, or an antigen-binding fragment thereof, can be used to detect ovarian cancer. In one embodiment, the antibody or its antigen-binding fragment comprises the VH and VL sequences of the M6-11E2 antibody. Variant antibodies may also be used.

[0306] In one embodiment, an antibody comprising the six CDRs of an M6-11E2 antibody, or an antigen-binding fragment thereof, can be used to detect pancreatic cancer. In one embodiment, the antibody or its antigen-binding fragment comprises the VH and VL sequences of the M6-11E2 antibody. Variant antibodies may also be used.

[0307] In one embodiment, an antibody comprising the six CDRs of an M6-11E2 antibody, or an antigen-binding fragment thereof, can be used to detect gastric cancer. In one embodiment, the antibody or its antigen-binding fragment comprises the VH and VL sequences of the M6-11E2 antibody. Variant antibodies may also be used.

[0308] other

[0309] The IHC methods and assays disclosed herein should be understood to encompass more than just the specific steps and reagents described herein. At a minimum, this document provides for the detection / quantification of PTK7 or its complexes and / or the detection / quantification of cells / tissues expressing PTK7. Such methods can be used for the prognosis and diagnosis of diseases (e.g., cancer) and for kits according to this disclosure for the same purposes. Methods / assays using the binders of this disclosure may include: immunodiffusion techniques, immunoelectrophoresis, light scattering immunoassays, agglutination techniques, labeled immunoassays (such as those selected from the group comprising radiolabeled immunoassays), enzyme immunoassays (such as colorimetric assays), chemiluminescent immunoassays, and immunofluorescence techniques. These methods are familiar to those skilled in the art and are also described in the prior art, for example in Zane, HD (2001): Immunology—Theoretical & Practical Concepts in Laboratory Medicine, WB Saunders Company, particularly in Chapter 14. Preferably, the assay is in the form of ELISA, and a microtiter plate containing wells is used as a diagnostically useful carrier.

[0310] sequence list

[0311] The table below provides a summary of the CDR, VL, VH, constant region, intact heavy chain, and intact light chain sequences of the antibody clones of this invention. This invention provides antibodies comprising the aforementioned six CDR sets, as well as variant forms. It also provides antibodies comprising one of the VL and VH sequence pairs from those clones, and variant sequences of those sequences. Furthermore, it provides antibodies having one of those antibodies, a pair of intact heavy chain and light chain sequences, and variants thereof.

[0312]

[0313]

[0314] Example

[0315] Materials and methods

[0316] 1. IHC method

[0317] Sample preparation: Bake FFPE glass slides at 60°C in a drying oven for 1 hour to improve the adhesion of the sample to the glass slide.

[0318] Dewaxing and rehydration: FFPE tissue was dewaxed by immersing it twice in fresh xylene for 10 minutes each time; it was then rehydrated for 5 minutes each time by a series of gradient EtOH washing solutions (100%, 95%, 85%, 75%).

[0319] Wash the sections in dH2O for 3 minutes and then in PBS twice for 3 minutes each time.

[0320] Epitope retrieval: Heat the slides in a pressure cooker with pH 9.0 EDTA antigen retrieval solution (1X) until it begins to boil, then perform epitope retrieval under high temperature and pressure for 3 minutes. Cool to room temperature. Wash the slides twice with PBS for 3 minutes each time.

[0321] Blocking: Remove the washing buffer and draw a hydrophobic zone around the tissue using a PAP pen. Block the sections with peroxidase blocking reagent at room temperature for 15 minutes. Wash the sections twice with PBS for 3 minutes each time.

[0322] Primary antibody incubation: Follow the product-specific protocol for antibody dilution. Remove the wash buffer and cover the slide with 100–400 μL of primary antibody diluted with the recommended antibody diluent. Incubate in a humidified chamber at 37°C for 1 hour. Wash the slide twice with PBS for 3 minutes each time.

[0323] Secondary antibody incubation: Remove the washing buffer and cover the sections with 1-3 drops of HRP-conjugated goat anti-rabbit and mouse reagent. Incubate in a humidified chamber at 37°C for 30 minutes. Wash the sections twice with PBS for 3 minutes each time.

[0324] DAB staining: Prepare a DAB-containing working solution by thoroughly mixing 1 drop of concentrated DAB solution into each 1 mL of DAB substrate buffer. Remove the wash buffer and allow the tissue sections to be covered with the 1X DAB working solution for 3 minutes at room temperature. Wash the sections twice in PBS for 3 minutes each time.

[0325] Counterstaining: Completely immerse the slide in hematoxylin and incubate for 1 minute. Immerse the slide in the bluing solution. Rinse the slide in tap water for 3 minutes.

[0326] Dehydration and clearing: Dehydrate with a series of gradient EtOH washing solutions (70%, 85%, 95%, 100%) for 3 minutes each. Immerse twice in fresh xylene for 10 minutes each time.

[0327] Mounting: Mount the slide with mounting medium.

[0328] 2. Preparation of FFPE blocks on cell lines

[0329] Cells were harvested during the logarithmic growth phase and counted using Count-star.

[0330] After centrifugation at 1000 rpm for 24 hours at 4°C, approximately 5 × 10⁻⁶ cells were collected. 6 1 x 10 cells 7 ).

[0331] Discard the supernatant, and then add 30 mL of 10% NBF to a 50 mL tube containing the cell pellet.

[0332] Cell suspensions were prepared by inverting the tubes several times. The tubes were then laid flat on the work surface, and the cells were fixed in 10% NBF for 30 minutes.

[0333] Centrifuge the sample at 1000 rpm for 5 minutes at 4°C, carefully discard all fixative without disturbing the cell pellet at the bottom of the tube, and preheat the sample in a 50°C incubator.

[0334] Heat HistoGel in a boiling water bath for 3-10 minutes, and place the liquid HistoGel in an incubator at 50°C.

[0335] Add one or two drops of HistoGel to the cell deposit and vortex for a few seconds to mix thoroughly. Then place the mixture on ice for 5-10 minutes to solidify the gel.

[0336] Place the cell gel in a long-well embedding cassette. Rinse the cell gel slowly with running water in a 250 mL beaker for 1 hour. Then, sequentially add 50%, 75%, 85%, and 95% ethanol to the cell block, immersing the cell gel in each ethanol solution for 1 hour. Finally, immerse the cell gel twice in 100% ethanol, 1 hour each time.

[0337] Remove the cell blocks from 100% ethanol and soak them twice in xylene for one hour each time.

[0338] Soak the cell blocks in liquid paraffin twice, for one hour each time, but overnight soaking is also acceptable.

[0339] FFPE blocks were prepared according to standard embedding procedures. The tissues were then embedded in paraffin at a paraffin embedding station.

[0340] Example 1 – Hybridoma Activity of Top Clones

[0341] The PTK7 ECD protein was prepared internally and used as an antigen. Monoclonal antibodies against PTK7 were developed by sequentially immunizing animals with the PTK7-his antigen protein and adjuvants, and the experimental animals were Balb / c mice.

[0342] For the first injection, animals were immunized with 100 μg antigen per animal. For the second and third injections, animals were immunized with 50 μg antigen per animal, and a booster immunization was performed with 25 μg. Freund's adjuvant (complete and incomplete) was used as the immunoadjuvant in the experiment. All animals were immunized via intraperitoneal injection. Mice with good immunohistochemical titers and positive serum immunohistochemistry (IHC) were selected for booster immunization. After booster immunization, mice were euthanized and immersed in 75% ethanol. The spleen was dissected, homogenized with a homogenizer, and filtered through a cell sieve to prepare a single-cell suspension. The spleen cell suspension was centrifuged at 1,500 rpm for 5 minutes, and the supernatant was discarded. 5 mL of erythrocyte lysate was added to lyse the erythrocytes at room temperature for 5 minutes, and PBS was added to bring the volume to 20 mL. After centrifugation at 1,500 rpm for 5 minutes, the supernatant was discarded. Viable cells were counted after resuspending. Sp2 / 0 cells were collected from culture flasks, and the supernatant was discarded after centrifugation at 1,500 rpm for 5 minutes. Count the viable cells after resuspending. Mix spleen cells with Sp2 / 0 cells at a ratio of 4:1 and centrifuge at 1,500 rpm for 5 minutes, discarding the supernatant. Resuspend the cells in 20 mL of electroporation buffer. After centrifugation at 1,500 rpm for 7 minutes, discard the supernatant and repeat this step once. Resuspend the cells with an appropriate amount of electroporation buffer to ensure a cell concentration of approximately 2 x 10⁻⁶ cells / mL. 7 Cells / mL. Add the cell suspension to a 9 mL electroporation well for fusion. After fusion, transfer the cell suspension to 20 mL of RPMI 1640 complete medium containing 20% ​​FBS and incubate at room temperature for 20 minutes. Resuspend the fused cells in RPMI 1640 medium containing 1xHAT, 1xBIOMYC3, and 20% FBS. Add the cell suspension at 100 μL / well to several 96-well cell culture plates to ensure approximately 5 x 10⁶ cells per well. 4 Cells were added to each well, and the plate was placed in a 37°C cell culture incubator. After 7 days, an additional 100 μL of RPMI 1640 complete medium containing 20% ​​FBS, 1xHAT, and 1xBIOMYC-3 was added to each well. Ten days after confluence, cell culture supernatant from the hybridoma parent clone was collected and used for screening by ELISA and IHC methods based on binding to human PTK7-his protein. Clones that were positive for both ELISA and IHC were then selected for further validation.

[0343] Example 2 - IHC Method - Mouse Serum Validation

[0344] FFPE (formalin-fixed paraffin-embedded) sections of ovarian cancer were baked in a drying oven at 60°C for 1 hour, then dewaxed in fresh xylene for 10 minutes, and rehydrated with a gradient of ethanol (100%, 95%, 85%, 75%) to distilled water. The sections were washed twice with PBS for 3 minutes each time. Epitope retrieval was performed by heating the sections in pH 9.0 EDTA antigen retrieval solution (1X) in a pressure cooker until it began to boil. The sections were cooled to room temperature. The sections were washed twice with distilled water for 3 minutes each time. Endogenous peroxidase activity was blocked by incubating the sections in a peroxidase blocking reagent at room temperature for 15 minutes, followed by washing twice with PBS for 3 minutes each time. Immunoassay for PTK7 was performed using primary antibodies (mouse serum) diluted 1:500 and 1:2000, respectively. The sections were covered with 100–400 μL of the primary antibody dilution and incubated at 37°C for 1 hour. The sections were washed twice with PBS for 3 minutes each time. The sections were then incubated with HRP-conjugated goat anti-rabbit and mouse reagents at 37°C for 30 minutes, and observed at room temperature for 3 minutes using a DAB-containing substrate working solution as the chromogen, resulting in brown staining. The sections were washed twice with PBS for 3 minutes each time. Finally, the sections were counterstained (with hematoxylin and incubated for 1 minute, then rinsed in tap water for 3 minutes), dehydrated (by passing through gradient concentrations of ethanol (70%, 85%, 95%, 100%) for 3 minutes each), and mounted in mounting medium.

[0345] Example 3 - IHC Method - Hybridoma Screening (Manual)

[0346] Formalin-fixed, paraffin-embedded tumor tissue sections were baked in a drying oven at 60°C for 1 hour, then dewaxed in fresh xylene for 10 minutes, and rehydrated with a gradient of ethanol (100%, 95%, 85%, 75%) to distilled water. The sections were then washed twice with PBS for 3 minutes each time. Epitope retrieval was performed by heating the slides in pH 9.0 EDTA antigen retrieval solution (1X) in a pressure cooker until boiling began. The sections were cooled to room temperature. The sections were washed twice with distilled water for 3 minutes each time. Endogenous peroxidase activity was blocked by incubating the sections in a peroxidase blocking reagent at room temperature for 15 minutes. The sections were then washed twice with PBS for 3 minutes each time. Immunoassay for PTK7 was performed using a primary antibody against PTK7 (hybridoma supernatant), and the sections were covered with 100–400 μL of primary antibody dilution and incubated at 37°C for 1 hour. The sections were washed twice with PBS for 3 minutes each time. The sections were then incubated with HRP-conjugated goat anti-rabbit and mouse reagents at 37°C for 30 minutes, and observed at room temperature for 3 minutes using a DAB-containing substrate working solution as the chromogen, resulting in brown staining. The sections were washed twice with PBS for 3 minutes each time. Finally, the sections were counterstained (with hematoxylin and incubated for 1 minute, then rinsed in tap water for 3 minutes), dehydrated (by passing through gradient concentrations of ethanol (70%, 85%, 95%, 100%) for 3 minutes each), and mounted in mounting medium. The results are shown below. Figure 1A-1D As shown.

[0347] Example 4 - IHC Method - Subclone Screening (Manual)

[0348] Formalin-fixed, paraffin-embedded tumor tissue sections were baked in a drying oven at 60°C for 1 hour, then dewaxed in fresh xylene for 10 minutes, and rehydrated with a gradient of ethanol concentrations (100%, 95%, 85%, 75%) to distilled water. Sections were washed twice with PBS for 3 minutes each time. Epitope retrieval was performed by heating slides in pH 9.0 EDTA antigen retrieval solution (1X) in a pressure cooker until boiling began. Sections were cooled to room temperature. Sections were washed twice with distilled water for 3 minutes each time. Endogenous peroxidase activity was blocked by incubating sections in a peroxidase blocking reagent at room temperature for 15 minutes, followed by washing sections twice with PBS for 3 minutes each time. Immunoassay for PTK7 was performed using a primary antibody against PTK7 (hybridoma supernatant), and sections were covered with 100–400 μL of primary antibody dilution and incubated at 37°C for 1 hour. Sections were washed twice with PBS for 3 minutes each time. The sections were then incubated with HRP-conjugated goat anti-rabbit and mouse reagents at 37°C for 30 minutes and observed at room temperature for 3 minutes using a DAB-containing substrate working solution as a chromogen, resulting in brown staining. The sections were washed twice with PBS for 3 minutes each time. Finally, the sections were counterstained (with hematoxylin and incubated for 1 minute, then rinsed in tap water for 3 minutes), dehydrated (by passing through gradient concentrations of ethanol (70%, 85%, 95%, 100%) for 3 minutes each) and mounted in mounting medium. Table 1 shows the IHC screening results of the top 8 subclones (underlined) in ovarian and breast cancer.

[0349] Table 1: Results of IHC screening of the top 8 subclones in ovarian and breast cancer

[0350]

[0351] Based on the good and unique staining results, 206-M6-9H5 (M10091-01) was selected (see...). Figure 2A-2B ), 222-M6-2C6 (M10091-04) (see also) Figures 3A-3B ), 235-M6-14H10 (M10091-05) (see) Figures 4A-4B ), 312-M10-5F7 (M10091-08) (see) Figures 5A-5B ), 58-M1-11E5 (M10091-06) (see also) Figures 6A-6B ), 134-M6-17F7 (M10091-03) (see) Figures 7A-7B ), 236-M6-8G5 (M10091-07) (see) Figures 8A-8B ), 259-M6-11E2 (M10091-02) (see also) Figures 9A-9B ).

[0352] Results of IHC for the first 3 subclones: Table 2 shows the recommended concentrations for the first 3 subclones used in the IHC test; Table 3 shows the ID and tissue origin of the tumor sections used in the titration assays for the first 3 subclones; Table 4 shows the tissue types used in the tissue microarray (TMA) for the first 3 subclones.

[0353] 11E2: Positive for ovarian cancer cell membranes, breast cancer cell membranes, and tumor stroma. (See also...) Figure 10 )

[0354] 8G5: Positive for ovarian cancer cell membranes, breast cancer cell membranes, and tumor stroma. (See also...) Figure 11 )

[0355] 5F7: Positive for ovarian cancer cell membranes, negative for breast cancer tumor cells, and positive for tumor stroma. (See also...) Figure 12 )

[0356] CST: Positive for ovarian cancer cell membranes, positive for breast cancer cell membranes, and positive for tumor stroma. (See also...) Figure 13 )

[0357] Table 2: Recommended IHC concentrations for the first three subclones

[0358]

[0359] Table 3: Samples

[0360]

[0361] Table 4: Organization Types

[0362]

[0363] Example 5 - IHC Method - Top-Level Cloning Optimization for TMA (Manual)

[0364] Formalin-fixed, paraffin-embedded tumor tissue sections were baked in a drying oven at 60°C for 1 hour, then dewaxed in fresh xylene for 10 minutes, and rehydrated with a gradient of ethanol concentrations (100%, 95%, 85%, 75%) to distilled water. Sections were washed twice with PBS for 3 minutes each time. Epitope retrieval was performed by heating the sections in pH 9.0 EDTA antigen retrieval solution (1X) in a pressure cooker until it began to boil. The sections were cooled to room temperature and then washed twice with distilled water for 3 minutes each time. Endogenous peroxidase activity was blocked by incubating the sections in a peroxidase blocking reagent at room temperature for 15 minutes, followed by washing twice with PBS for 3 minutes each time. PTK7 immunoassay was performed using primary antibodies against PTK7 at recommended concentrations (1, 2, 4, 8 μg / mL, respectively), and sections were covered with 100–400 μL of primary antibody dilution and incubated at 37°C for 1 hour. The sections were then washed twice with PBS for 3 minutes each time. The sections were then incubated with HRP-conjugated goat anti-rabbit and mouse reagents at 37°C for 30 minutes, and observed at room temperature for 3 minutes using a DAB-containing substrate working solution as the chromogen, resulting in brown staining. The sections were washed twice with PBS for 3 minutes each time. Finally, the sections were counterstained (with hematoxylin and incubated for 1 minute, then rinsed in tap water for 3 minutes), dehydrated (by passing through gradient concentrations of ethanol (70%, 85%, 95%, 100%) for 3 minutes each), and mounted in mounting medium.

[0365] The results of subclone 11E2 show Figures 14A-14D The results of subclone 8G5 show... Figures 15A-15D The results of subcloning 5F7 show... Figure 16A-16D middle.

[0366] Example 6 - IHC Method - TMA

[0367] A 1 mm core from a paraffin-embedded tumor block was used to generate TMA. Before staining, the microarrays were baked in a drying oven at 60°C for 1 hour. These sections were then dewaxed in fresh xylene for 10 minutes and rehydrated with a gradient of ethanol concentrations (100%, 95%, 85%, 75%) to distilled water, followed by washing twice with PBS for 3 minutes each time. Epitope retrieval was performed by heating the sections in a pH 9.0 EDTA antigen retrieval solution (1X) in a pressure cooker until it began to boil. The sections were cooled to room temperature and then washed twice with distilled water for 3 minutes each time. Endogenous peroxidase activity was blocked by incubating the sections in a peroxidase blocking reagent at room temperature for 15 minutes, followed by washing twice with PBS for 3 minutes each time. Immunoassay for PTK7 was performed using primary antibodies against PTK7 at recommended concentrations (1, 2, 2 μg / mL, respectively). The sections were covered with 100–400 μL of primary antibody dilution and incubated at 37°C for 1 hour. The sections were then washed twice with PBS for 3 minutes each time. The sections were then incubated with HRP-conjugated goat anti-rabbit and mouse reagents at 37°C for 30 minutes, and observed at room temperature for 3 minutes using a DAB-containing substrate working solution as the chromogen, resulting in brown staining. The sections were washed twice with PBS for 3 minutes each time. Finally, the sections were counterstained (with hematoxylin and incubated for 1 minute, then rinsed in tap water for 3 minutes), dehydrated (by passing gradient concentrations of ethanol (70%, 85%, 95%, 100%) for 3 minutes each), and mounted in mounting media. Table 5 shows TMA-Leica. Table 6 shows TMA-Manual.

[0368] Table 5: TMA - IHC via Leica Autostainer

[0369]

[0370]

[0371] Table 6: TMA - IHC by manual staining

[0372]

[0373]

[0374] Subclone 11E2 TMA results in Figures 17A-17B The results of subclone 8G5 TMA are shown in [the image / data]. Figures 18A-18B The results of subclone 5F7 TMA are shown in [the image / data]. Figures 19A-19B It is displayed in the middle.

[0375] Example 7 - IHC Method - Tumor Cell Block (Manual)

[0376] Formalin-fixed, paraffin-embedded tumor cell sections were baked in a drying oven at 60°C for 1 hour, then dewaxed in fresh xylene for 10 minutes, and rehydrated with a gradient of ethanol concentrations (100%, 95%, 85%, 75%) to distilled water. Sections were washed twice with PBS for 3 minutes each time. Epitope retrieval was performed by heating slides in pH 9.0 EDTA antigen retrieval solution (1X) in a pressure cooker until boiling began. Sections were cooled to room temperature. Sections were washed twice with distilled water for 3 minutes each time. Endogenous peroxidase activity was blocked by incubating sections in a peroxidase blocking reagent at room temperature for 15 minutes, followed by washing sections twice with PBS for 3 minutes each time. Immunoassay for PTK7 was performed using primary antibodies against PTK7 at recommended concentrations (1, 2, 2 μg / mL, respectively), and sections were covered with 100–400 μL of primary antibody dilution buffer and incubated at 37°C for 1 hour. Sections were washed twice with PBS for 3 minutes each time. The sections were then incubated with HRP-conjugated goat anti-rabbit and mouse reagents at 37°C for 30 minutes, and observed at room temperature for 3 minutes using a DAB-containing substrate working solution as the chromogen, resulting in brown staining. The sections were washed twice with PBS for 3 minutes each time. Finally, the sections were counterstained (with hematoxylin and incubated for 1 minute, then rinsed in tap water for 3 minutes), dehydrated (by passing through gradient concentrations of ethanol (70%, 85%, 95%, 100%) for 3 minutes each), and mounted in mounting medium.

[0377] The first three cell lines were IHC.

[0378] PTK7 antigen expression levels: PA-1 > MDA-MB-453 > MCF-7. Raji was a negative control. Subclone 11E2 results showed... Figure 20 The results of subclone 8G5 show... Figure 21 The results of subcloning 5F7 show... Figure 22 PA-1 is a cancer cell line originally isolated from an ovarian cancer patient. MDA-MB-453 is a cancer cell line originally isolated from a metastatic breast cancer patient. MCF-7 is a cancer cell line originally isolated from a breast adenocarcinoma patient.

[0379] Having described the basic concepts thus, it will be quite apparent to those skilled in the art, upon reading this detailed disclosure, that the foregoing detailed disclosure is intended to be presented by way of example only and not to be limiting. Even if not expressly stated herein, various changes, improvements, and modifications can be made or anticipated by those skilled in the art. Such changes, improvements, and modifications are intended to be set forth in this disclosure and are within the spirit and scope of the exemplary embodiments of this disclosure.

[0380] Furthermore, certain terms have been used to describe embodiments of this disclosure. For example, the terms "an embodiment," "an embodiment," and "some embodiments" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of this disclosure. Therefore, it should be emphasized and understood that in various parts of this specification, two or more references to "an embodiment" or "an embodiment" or "alternative embodiment" do not necessarily refer to the same embodiment. Moreover, in one or more embodiments of this disclosure, particular features, structures, or characteristics may be appropriately combined.

[0381] Furthermore, those skilled in the art will understand that aspects of this disclosure may be illustrated and described herein in any of a number of patentable classes or contexts, including any new and useful methods, machines, manufactures or compositions of matter, or any new and useful improvements thereof.

[0382] Furthermore, the order or sequence of the processing elements, or the use of numbers, letters, or other designations, is not intended to limit the claimed processing and methods to any order, unless otherwise specified in the claims. Although the foregoing disclosure has discussed various useful embodiments currently considered to be part of this disclosure by way of various examples, it should be understood that such details are for this purpose only, and the appended claims are not limited to the disclosed embodiments, but rather are intended to cover modifications and equivalent arrangements within the spirit and scope of the disclosed embodiments.

[0383] Similarly, it should be understood that in the embodiments described above in this disclosure, various features are sometimes combined together in a single embodiment, drawing, or description therein to simplify this disclosure and aid in understanding one or more of the various embodiments. However, the approach of this disclosure should not be construed as reflecting an intention that the claimed subject matter requires more features than those expressly recited in each claim. Rather, the subject matter of the claims covers fewer than all features of a single foregoing disclosed embodiment.

[0384] Further implementation plan - binder

[0385] 1. A binder for protein tyrosine kinase 7 (PTK7), comprising:

[0386] The heavy chain variable (VH) region and the light chain variable (VL) region, wherein the VH region contains complementarity-determining regions HCDR1, HCDR2, and HCDR3 disposed within the heavy chain variable region framework, and the VL region contains LCDR1, LCDR2, and LCDR3 disposed within the light chain variable region framework, wherein the VH and VL CDRs have amino acid sequences selected from the set of amino acid sequences shown below:

[0387] a. These are SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, LVS, and SEQ ID NO: 7, respectively;

[0388] b. SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, QMS and SEQ ID NO: 14 respectively; and

[0389] c. These are SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, RVS, and SEQ ID NO: 21, respectively.

[0390] 2. The binding agent according to claim 1, wherein the VH region and VL region have an amino acid sequence selected from the amino acid sequence pairs shown in the group consisting of:

[0391] a. SEQ ID NO: 1 and SEQ ID NO: 2, respectively;

[0392] b. SEQ ID NO: 8 and SEQ ID NO: 9 respectively; and

[0393] c. These are SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

[0394] 3. The binding agent according to claim 1, wherein the VH region and VL region have an amino acid sequence selected from the amino acid sequence pairs shown in the group consisting of:

[0395] a. SEQ ID NO: 1 and SEQ ID NO: 2, respectively;

[0396] b. SEQ ID NO: 8 and SEQ ID NO: 9 respectively; and

[0397] c. SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

[0398] The heavy and light chain framework regions are optionally modified by substitution, deletion, or insertion of 1 to 8 amino acids in the framework regions.

[0399] 4. The binder according to any one of claims 1-3, wherein the frame region is a human frame region.

[0400] 5. The binding agent according to any one of 1-3, wherein the frame region is a mouse frame region.

[0401] 6. The binding agent according to any one of 1-5, wherein the binding agent is an antibody.

[0402] 7. The binder according to any one of 1-6, wherein one of 1 to 6, wherein the binder further comprises a heavy chain constant region.

[0403] 8. The binding agent according to any one of claims 1-6, wherein the binding agent is a monoclonal antibody.

[0404] 9. The binder according to claim 8, wherein the heavy chain constant region is of the IgG isotype.

[0405] 10. The binder according to claim 8, wherein the heavy chain constant region is a human IgG1 constant region or a human IgG4 constant region.

[0406] 11. The binder according to claim 8, wherein the heavy chain constant region is a mouse IgG1 constant region, a mouse IgG2a constant region, a mouse IgG2c constant region, or a mouse IgG3 constant region.

[0407] 12. The binder according to claim 8, wherein the heavy chain constant region has the amino acid sequence shown in SEQ ID NO: 22, 24 or 26.

[0408] 13. The binder according to any one of 1 to 11, wherein the binder further comprises a light chain constant region.

[0409] 14. The binder according to claim 13, wherein the light chain constant region has the amino acid sequence shown in SEQ ID NO: 23, 25 or 27.

[0410] 15. The binding agent according to claim 1, wherein the binding agent is a monoclonal antibody, the monoclonal antibody comprising a VH region having the amino acid sequence shown in SEQ ID NO: 1, a VL region having the amino acid sequence shown in SEQ ID NO: 2, a heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 22, and a light chain constant region having the amino acid sequence shown in SEQ ID NO: 23.

[0411] 16. The binding agent according to claim 1, wherein the binding agent is a monoclonal antibody, the monoclonal antibody comprising a VH region having the amino acid sequence shown in SEQ ID NO: 8, a VL region having the amino acid sequence shown in SEQ ID NO: 9, a heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 24, and a light chain constant region having the amino acid sequence shown in SEQ ID NO: 25.

[0412] 17. The binding agent according to claim 1, wherein the binding agent is a monoclonal antibody comprising a VH region having the amino acid sequence shown in SEQ ID NO: 15, a VL region having the amino acid sequence shown in SEQ ID NO: 16, a heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 26, and a light chain constant region having the amino acid sequence shown in SEQ ID NO: 27.

[0413] Further implementation scheme - IHC method:

[0414] A1. A method for detecting cells expressing PTK7 in a sample, comprising:

[0415] Contact the sample with a detection agent comprising the binder of any one of embodiments 1-17, further numbered, and

[0416] The detection of the formation of a binding complex between the detection agent and PTK7 indicates the presence of cells expressing PTK7 in the sample.

[0417] A2. The method according to A1, wherein the detection agent further comprises a detectable marker that labels the binder.

[0418] A2.1. The method according to A2, wherein the detectable marker includes biotin or an enzyme marker.

[0419] A2.2. The method according to A2.1, wherein the enzyme marker includes horseradish peroxidase (HRP) or alkaline phosphatase (AP).

[0420] A2.3. The method according to any one of A2-A2.2, wherein detecting the formation of the binding complex between the detector and PTK7 comprises:

[0421] Add the mating agent of the detectable marker; and

[0422] The reaction between the detectable marker and the partner agent is detected as an indication of the formation of the binding complex.

[0423] A2.4. The method according to A2, wherein the detectable marker includes a fluorescent tag.

[0424] A2.5. The method according to A2.4, wherein detecting the formation of the binding complex between the detector and PTK7 comprises:

[0425] The signal from the fluorescent tag is detected as an indication of the formation of the binding complex.

[0426] A3. The method according to A1, wherein detecting the formation of the binding complex between the detector and PTK7 comprises:

[0427] The sample is brought into contact with a labeled secondary antibody configured to bind the binding complex; and

[0428] The signal from the secondary antibody is detected as an indication of the formation of the binding complex.

[0429] A3.1. The method according to A3, wherein the secondary antibody is labeled with a detectable marker.

[0430] A3.2. The method according to A3.1, wherein the detectable marker includes biotin or an enzyme marker.

[0431] A3.3. The method according to any one of A3.1-A3.2, wherein detecting the formation of the binding complex between the detector and PTK7 comprises:

[0432] Add the mating agent of the detectable marker; and

[0433] The reaction between the detectable marker and the partner agent is detected as an indication of the formation of the binding complex.

[0434] A3.4. The method of claim A3.1, wherein the detectable marker comprises a fluorescent tag.

[0435] A3.5. The method according to claim A3.4, wherein detecting the formation of the binding complex between the detector and PTK7 comprises:

[0436] The signal from the fluorescent tag is detected as an indication of the formation of the binding complex.

[0437] A3.6. The method according to A3.4, wherein the fluorescent tag comprises fluorescein (FITC), phycoerythrin (PE), preferably R-PE or allophycocyanin (APC).

[0438] A4. The method according to A1, wherein the sample is derived from the subject's tissue.

[0439] A4.1. The method according to A4, wherein the tissue is selected from the group consisting of tonsils, appendix, mammary gland, ovary, colon, prostate, skin, lung, uterus, cervix, kidney, pancreas, bladder, brain, thyroid gland, ear, nose, larynx and esophagus.

[0440] A4.2. The method according to any one of A4-A4.1, wherein the tissue is selected from the group consisting of mammary gland, ovary, colon, prostate, skin, lung, uterus, esophagus and bladder.

[0441] A4.3. The method according to any one of A4-A4.2, wherein the tissue is a mammary gland.

[0442] A4.4. The method according to any one of A4-A4.2, wherein the tissue is an ovary.

[0443] A5. The method according to A1, wherein the sample is from a subject, and the presence of cells expressing PTK7 indicates that the subject has a disease.

[0444] A5.1. The method according to A5.1, wherein the sample is from a subject, and the method further comprises: quantifying the binding of PTK-expressing cells in the sample to obtain a quantitative result, wherein the quantitative result exceeding a predetermined threshold indicates that the subject has a disease.

[0445] A5.2. The method described in A5 or A5.1, wherein the disease is cancer.

[0446] A5.3. The method according to A5 or A5.1, wherein the disease is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, melanoma, lung cancer, esophageal cancer, gastric cancer, endometrial cancer, head and neck cancer, and bladder cancer.

[0447] A5.4. The method according to claim A5 or A5.1, wherein the disease is breast cancer.

[0448] A5.5. The method according to claim A5 or A5.1, wherein the disease is ovarian cancer.

[0449] A5.6. The method according to claim A5 or A5.1, wherein the disease is squamous cell carcinoma of the lung or adenocarcinoma of the lung.

[0450] Further implementation plans - disease diagnosis, treatment and monitoring

[0451] B1. Methods for determining whether a subject is suffering from a disease, including:

[0452] Samples were obtained from the subjects;

[0453] Contact the sample with a detection agent comprising the binder of any one of embodiments 1-17, further numbered, and

[0454] Whether a subject has the disease is determined by detecting and / or quantifying the formation of a binding complex between the detection agent and PTK7, wherein the presence of the binding complex or the level of the binding complex exceeding a predetermined threshold indicates that the subject has the disease.

[0455] B1.1. According to the method described in B1, the disease is cancer.

[0456] B2. Methods of treating diseases, including:

[0457] Samples were obtained from the subjects;

[0458] The sample is brought into contact with a detection agent comprising any one of 1-17;

[0459] Whether a subject has the disease is determined by detecting and / or quantifying the formation of a binding complex between the detection agent and PTK7, wherein the presence of the binding complex or a level of the binding complex exceeding a predetermined threshold indicates that the subject has the disease; and

[0460] To treat the aforementioned disease.

[0461] B2.1. The disease described in B2 is cancer.

[0462] B2.2. The method according to B2.1, wherein the disease is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, melanoma, lung cancer, esophageal cancer, gastric cancer, endometrial cancer, head and neck cancer, and bladder cancer.

[0463] B2.3. The method according to any one of B2-B2.2, wherein the treatment comprises administering chemotherapy to the subject.

[0464] B2.4. The method according to any one of B2-B2.2, wherein the treatment comprises administering hormone therapy to the subject.

[0465] B2.5. The method according to any one of B2-B2.2, wherein the treatment comprises administering radiotherapy to the subject.

[0466] B2.6. The method according to any one of B2-B2.2, wherein the treatment comprises administering immunotherapy to the subject.

[0467] B2.7. The method according to any one of B2-B2.2, wherein the treatment comprises administering stem cell therapy to the subject.

[0468] B2.8. The method according to any one of B2-B2.2, wherein the treatment comprises administering a targeted therapy to the subject.

[0469] B2.9. The method according to any one of B2-B2.2, wherein the treatment comprises performing surgery on the subject.

[0470] B2.10. The method according to any one of B2-B2.2, wherein the disease is breast cancer, and the treatment includes mastectomy or lumpectomy.

[0471] B2.11. The method according to any one of B2-B2.2, wherein the disease is ovarian cancer, and the treatment includes hysterectomy or salpingo-oophorectomy.

[0472] B3. A method for monitoring disease progression in subjects, comprising the following steps:

[0473] (a) Obtaining samples from the subject;

[0474] (b) Contact the sample with a detection agent comprising the binder of any one of embodiments 1-17, further numbered;

[0475] (c) Assess the formation of a binding complex between the assay reagent and PTK7 to obtain a first assessment result;

[0476] (d) Repeat steps (a), (b), and (c) using samples from the subjects at subsequent time points to obtain a second assessment result; and

[0477] (e) Compare the second assessment result with the first assessment result to determine the progression of the disease in the subject.

[0478] B3.1. The disease described in B3 is cancer.

[0479] Further implementation plan - reagent kit

[0480] C1. A kit for detecting cells expressing PTK7 in a sample, the kit comprising a detection agent comprising a binding agent according to any one of embodiments 1-17, further numbered.

[0481] C2. The kit according to claim C1, further comprising a secondary antibody labeled and configured to bind to the binding complex formed by the detection agent and PTK7.

[0482] sequence list

[0483] SEQ ID NO:1QVTLKESGPGILQPSQTLSLTCSFSGFSLNTFGMGVSWIRQPSGNGLEWLAHIYWDDGKDYNPSLKSRLTISKDTSNNQVFLKITTVDTTDTATYYCAYGFAYWGQGTLVTVSA

[0484] SEQ ID NO:2 DVVMTQTPLTLSVTIGQPASISCKSSQSLLSINGKTYLNWLLQRPGQSPKRLIHLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPHTFGGGTKLEIK

[0485] SEQ ID NO:3 GFSLNTFGMG

[0486] SEQ ID NO:4 IYWDDGK

[0487] SEQ ID NO:5 AYGFAY

[0488] SEQ ID NO:6 QSLLSINGKTY

[0489] SEQ ID NO:7 VQGTHFPHT

[0490] SEQ ID NO:8 EVMLVESGGGSAKPGGSLKLSCEASGFTFSSYAMSWVRQTPGKRLEWVASISNGGSYTNYPDSVKGRFTISRDNAKNTLYLQMTSLRSEDTAIYYCSNTGTSYYGFEYWGQGTTLTVSS

[0491] SEQ ID NO:9 DIVMTQAAFSSPVTLGTSASISCRSSESLLYSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPYTFGGGTRLEIK

[0492] SEQ ID NO:10 GFTFSSYA

[0493] SEQ ID NO:11 ISNGGSYT

[0494] SEQ ID NO:12 SNTGTSYYGFEY

[0495] SEQ ID NO:13 ESLLYSNGITY

[0496] SEQ ID NO:14 AQNLELPYT

[0497] SEQ ID NO:15 EVQLQQSGPVLVKPGASVKISCKASGYTFTDYNMHWVRQSHGKSLEWIGYIYPYNGGTGYNQKFKSKATLTVDNSSNTAYLELRSLTSEDSAVYYCARGGWYFDVWGAGTTVTVSS

[0498] SEQ ID NO:16 DAVMTQTPLSLPVSLGDQASISCRSSQSLENSNGNTYLNWYLQKPGQSPQLLIYRVSNRFSGVLDRFSGSGSGTDFTLKISRVEAEDLGVYFCLQITHVPFTFGSGTKLEIK

[0499] SEQ ID NO:17 GYTFTDYN

[0500] SEQ ID NO:18 IYPYNGGT

[0501] SEQ ID NO:19 ARGGWYFDV

[0502] SEQ ID NO:20 QSLENSNGNTY

[0503] SEQ ID NO:21 LQITHVPFT

[0504] SEQ ID NO:22 AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0505] SEQ ID NO:23 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0506] SEQ ID NO:24 AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0507] SEQ ID NO:25 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0508] SEQ ID NO:26AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0509] SEQ ID NO:27 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0510] SEQ ID NO:28 QVTLKESGPGILQPSQTLSLTCSFSGFSLNTFGMGVSWIRQPSGNGLEWLAHIYWDDGKDYNPSLKSRLTISKDTSNNQVFLKITTVDTTDTATYYCAYGFAYWGQGTLVTVSAAKTTPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0511] SEQ ID NO:29DVVMTQTPLTLSVTIGQPASISCKSSQSLLSINGKTYLNWLLQRPGQSPKRLIHLVSKLDSGVPDRFTGSGSGTDFTLKISRVEADLGVYYVCQGTHFPHTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0512] SEQ ID NO:30 EVMLVESGGGSAKPGGSLKLSCEASGFTFSSYAMSWVRQTPGKRLEWVASISNGGSYTNYPDSVKGRFTISRDNAKNTLYLQMTSLRSEDTAIYYCSNTGTSYYGFEYWGQGTTLTVSSAKTTPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0513] SEQ ID NO:31 DIVMTQAAFSSPVTLGTSASISCRSSESLLYSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPYTFGGGTRLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0514] SEQ ID NO:32EVQLQQSGPVLVKPGASVKISCKASGYTFTDYNMHWVRQSHGKSLEWIGYIYPYNGGTGYNQKFKSKATLTVDNSSNTAYLELRSLTSEDSAVYYCARGGWYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0515] SEQ ID NO:33 DAVMTQTPLSLPVSLGDQASISCRSSQSLENSNGNTYLNWYLQKPGQSPQLLIYRVSNRFSGVLDRFSGSGSGTDFTLKISRVEAEDLGVYFCLQITHVPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

Claims

1. A binding agent for protein tyrosine kinase 7 (PTK7), said binding agent comprising: The heavy chain variable (VH) region and the light chain variable (VL) region, wherein the VH region includes complementary determining regions HCDR1, HCDR2, and HCDR3 disposed in the heavy chain variable region framework region, and the VL region includes LCDR1, LCDR2, and LCDR3 disposed in the light chain variable region framework region, wherein the VH and VL are selected from one of the following VH and VL pairs shown in a. to c. below: a. VH and VL, wherein VH includes HCDR1 containing SEQ ID NO: 3, HCDR2 containing SEQ ID NO: 4 and HCDR3 containing SEQ ID NO: 5, and VL includes LCDR1 containing SEQ ID NO: 6, LCDR2 containing LVS and LCDR3 containing SEQ ID NO: 7; b. VH and VL, wherein the VH comprises HCDR1 comprising SEQ ID NO: 10, HCDR2 comprising SEQ ID NO: 11, and HCDR3 comprising SEQ ID NO: 12, and the VL comprises LCDR1 comprising SEQ ID NO: 13, LCDR2 comprising QMS, and LCDR3 comprising SEQ ID NO: 14; and c. VH and VL, wherein the VH includes HCDR1 comprising SEQ ID NO: 17, HCDR2 comprising SEQ ID NO: 18, and HCDR3 comprising SEQ ID NO: 19, and the VL includes LCDR1 comprising SEQ ID NO: 20, LCDR2 comprising RVS, and LCDR3 comprising SEQ ID NO:

21.

2. The binder according to claim 1, wherein the VH and VL regions are selected from one of the following VH and VL region pairs: a. VH containing SEQ ID NO: 1 and VL containing SEQ ID NO: 2; b. VH containing SEQ ID NO: 8 and VL containing SEQ ID NO: 9; and c. VH containing SEQ ID NO: 15 and VL containing SEQ ID NO:

16.

3. The binder according to claim 1, wherein the VH and VL regions have an amino acid sequence of one of the following VH and VL sequence pairs as shown below: a. VH containing SEQ ID NO: 1 and VL containing SEQ ID NO: 2; b. VH containing SEQ ID NO: 8 and VL containing SEQ ID NO: 9; and c. VH containing SEQ ID NO: 15 and VL containing SEQ ID NO: 6, The heavy and light chain framework regions are optionally modified by substitution, deletion, or insertion of 1 to 8 amino acids in the framework regions, and the antibody retains the ability to bind PTK7.

4. The binder according to any one of claims 1-3, wherein the frame region is a human frame region.

5. The binder according to any one of claims 1-3, wherein the frame region is a mouse frame region.

6. The binding agent according to any one of claims 1-5, wherein the binding agent is an antibody.

7. The binding agent according to any one of claims 1-6, wherein the binding agent is a monoclonal antibody.

8. The binder according to any one of the preceding claims, wherein the binder comprises a heavy chain, the heavy chain comprising a VH and a heavy chain constant region.

9. The binder according to claim 8, wherein the heavy chain constant region is of the IgG isotype.

10. The binder according to claim 8, wherein the heavy chain constant region is a human IgG1 constant region or a human IgG4 constant region.

11. The binder according to claim 8, wherein the heavy chain constant region is a mouse IgG1 constant region, a mouse IgG2a constant region, a mouse IgG2c constant region, or a mouse IgG3 constant region.

12. The binder according to claim 8, wherein the heavy chain constant region has the amino acid sequence shown in SEQ ID NO: 22, 24 or 26.

13. The binder according to any one of the preceding claims, wherein the binder comprises a light chain, the light chain comprising a VL and a light chain constant region.

14. The binder according to claim 13, wherein the light chain constant region has the amino acid sequence shown in SEQ ID NO: 23, 25 or 27.

15. The binding agent of claim 1, wherein the binding agent is an antibody, the antibody comprising: a. A heavy chain comprising a VH region having the amino acid sequence shown in SEQ ID NO: 1 and a heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 22; and b. A light chain comprising a VL region having the amino acid sequence shown in SEQ ID NO: 2 and a light chain constant region having the amino acid sequence shown in SEQ ID NO:

23.

16. The binding agent of claim 1, wherein the binding agent is an antibody, the antibody comprising: a. A heavy chain comprising a VH region having the amino acid sequence shown in SEQ ID NO: 8 and a heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 24; and b. A light chain comprising a VL region having the amino acid sequence shown in SEQ ID NO: 9 and a light chain constant region having the amino acid sequence shown in SEQ ID NO:

25.

17. The binding agent of claim 1, wherein the binding agent is an antibody, the antibody comprising: a. A heavy chain comprising a VH region having the amino acid sequence shown in SEQ ID NO: 15 and a heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 26; and b. A light chain comprising a VL region having the amino acid sequence shown in SEQ ID NO: 16 and a light chain constant region having the amino acid sequence shown in SEQ ID NO:

27.

18. The binder according to claim 1, comprising the heavy chain of SEQ ID NO: 28 and the light chain of SEQ ID NO:

29.

19. The binder according to claim 1, comprising the heavy chain of SEQ ID NO: 30 and the light chain of SEQ ID NO:

31.

20. The binder according to claim 1, comprising the heavy chain of SEQ ID NO: 32 and the light chain of SEQ ID NO:

33.

21. A method for detecting cells expressing PTK7 in a sample, comprising: Contact the sample with a detection agent comprising the binder according to any one of claims 1-20, and The detection of the formation of a binding complex between the detection agent and PTK7 indicates the presence of cells expressing PTK7 in the sample.

22. The method of claim 21, wherein the detection agent further comprises a detectable marker that labels the binder.

23. The method of claim 21, wherein the detectable marker comprises biotin or an enzyme marker.

24. The method of claim 23, wherein the enzyme marker comprises horseradish peroxidase (HRP) or alkaline phosphatase (AP).

25. The method according to any one of claims 21-24, wherein detecting the formation of the binding complex between the detector and PTK7 comprises: Add the coupler agent of the detectable marker; and The reaction between the detectable marker and the partner agent is detected as an indication of the formation of the binding complex.

26. The method of claim 22, wherein the detectable marker comprises a fluorescent tag.

27. The method of claim 21, wherein detecting the formation of the binding complex between the detector and PTK7 comprises: The signal from the fluorescent tag is detected as an indication of the formation of the binding complex.

28. The method of claim 21, wherein detecting the formation of the binding complex between the detector and PTK7 comprises: The sample is brought into contact with a labeled secondary antibody configured to bind the binding complex; and The signal from the secondary antibody is detected as an indication of the formation of the binding complex.

29. The method of claim 28, wherein the secondary antibody is labeled with a detectable marker.

30. The method of claim 29, wherein the detectable marker comprises biotin or an enzyme marker.

31. The method according to any one of claims 25 to 30, wherein detecting the formation of the binding complex between the detector and PTK7 comprises: Add the coupler agent of the detectable marker; and The reaction between the detectable marker and the partner agent is detected as an indication of the formation of the binding complex.

32. The method of claim 25, wherein the detectable marker comprises a fluorescent tag.

33. The method of claim 32, wherein detecting the formation of the binding complex between the detector and PTK7 comprises: The signal from the fluorescent tag is detected as an indication of the formation of the binding complex.

34. The method of claim 33, wherein the fluorescent tag comprises fluorescein (FITC), phycoerythrin (PE), preferably R-PE or allophycocyanin (APC).

35. The method of claim 21, wherein the sample is derived from the subject's tissue.

36. The method of claim 35, wherein the tissue is selected from the group consisting of tonsils, appendix, mammary gland, ovary, colon, prostate, skin, lung, uterus, cervix, kidney, pancreas, bladder, brain, thyroid gland, ear, nose, larynx and esophagus.

37. The method of claim 35, wherein the tissue is selected from the group consisting of mammary gland, ovary, colon, prostate, skin, lung, uterus, esophagus and bladder.

38. The method according to any one of claims 35 to 37, wherein the tissue is a mammary gland.

39. The method according to any one of claims 35 to 37, wherein the tissue is an ovary.

40. The method of claim 21, wherein the sample is derived from a subject, and the presence of cells expressing PTK7 indicates that the subject is suffering from a disease.

41. The method of claim 37, wherein the sample is derived from a subject, and the method further comprises: The binding of PTK-expressing cells in the sample is quantified to obtain a quantitative result, wherein a quantitative result exceeding a predetermined threshold indicates that the subject has a disease.

42. The method according to claim 40 or 41, wherein the disease is cancer.

43. The method according to claim 40 or 41, wherein the disease is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, melanoma, lung cancer, esophageal cancer, gastric cancer, endometrial cancer, head and neck cancer, and bladder cancer.

44. The method according to claim 42 or 43, wherein the disease is breast cancer.

45. The method according to claim 42 or 43, wherein the disease is ovarian cancer.

46. ​​The method according to claim 42 or 43, wherein the disease is squamous cell carcinoma of the lung or adenocarcinoma of the lung.

47. The method according to any one of claims 21 to 46, wherein the method is an immunohistochemical method.

48. Use of the binding agent according to any one of claims 1 to 20 in the detection of PTK7 protein.

49. Methods for determining whether a subject is suffering from a disease include: Samples were obtained from the subjects; Contact the sample with a detection agent comprising the binder according to any one of claims 1-20, and Whether a subject has the disease is determined by detecting and / or quantifying the formation of a binding complex between the detection agent and PTK7, wherein the presence of the binding complex or the level of the binding complex exceeding a predetermined threshold indicates that the subject has the disease.

50. The method of claim 49, wherein the disease is cancer.

51. A method for treating a subject's disease, wherein the subject has been determined to have the disease by the method of claim 40.

52. The method of claim 51, wherein the binder comprises VH and VL regions containing the amino acid sequences shown below: SEQ ID NO: 1 and SEQ ID NO: 2, respectively; SEQ ID NO: 8 and SEQ ID NO: 9, respectively; or SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

53. Methods of treating diseases include: Samples were obtained from the subjects; The sample is brought into contact with a detection agent comprising the binder according to any one of claims 1-20; Whether a subject has the disease is determined by detecting and / or quantifying the formation of a binding complex between the detection agent and PTK7, wherein the presence of the binding complex or a level of the binding complex exceeding a predetermined threshold indicates that the subject has the disease; and To treat the aforementioned disease.

54. The method of claim 53, wherein the disease is cancer.

55. The method according to claim 54, wherein the disease is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, melanoma, lung cancer, esophageal cancer, gastric cancer, endometrial cancer, head and neck cancer, and bladder cancer.

56. The method according to any one of claims 53 to 55, wherein the treatment comprises administering chemotherapy to the subject.

57. The method according to any one of claims 53 to 56, wherein the treatment comprises administering hormone therapy to the subject.

58. The method according to any one of claims 53 to 56, wherein the treatment comprises administering radiotherapy to the subject.

59. The method according to any one of claims 53 to 56, wherein the treatment comprises administering immunotherapy to the subject.

60. The method according to any one of claims 53 to 56, wherein the treatment comprises administering stem cell therapy to the subject.

61. The method according to any one of claims 53 to 56, wherein the treatment comprises administering a targeted therapy to the subject.

62. The method according to any one of claims 53 to 56, wherein the treatment comprises performing surgery on the subject.

63. The method according to any one of claims 53 to 56, wherein the disease is breast cancer, and the treatment comprises mastectomy or lumpectomy.

64. The method according to any one of claims 53 to 56, wherein the disease is ovarian cancer, and the treatment comprises hysterectomy or salpingo-oophorectomy.

65. A method for monitoring the progression of disease in subjects, comprising the following steps: (a) Contacting a sample from the subject with a detection agent comprising the binder of any one of claims 1-20; (b) Assess the formation of a binding complex between the assay reagent and PTK7 to obtain a first assessment result; (c) Repeat steps (a), (b) and (c) using samples from the subject at subsequent time points to obtain a second assessment result; and (d) Compare the second assessment result with the first assessment result to determine the progression of the disease in the subject.

66. The method of claim 65, wherein the disease is cancer.

67. A kit for detecting cells expressing PTK7 in a sample, the kit comprising a detection agent comprising a binding agent according to any one of claims 1-21.

68. The kit of claim 67, further comprising a secondary antibody labeled and configured to bind to the binding complex formed by the detection agent and PTK7.

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