Tissue factor binding antibodies and uses thereof

Abstract

The invention relates to a tissue factor binding antibody and application thereof, in particular to an antibody specifically binding to TF or an antigen binding fragment thereof and a kit containing the antibody or the antigen binding fragment, the antibody or the antigen binding fragment comprises (i) an LCDR1 sequence, an LCDR2 sequence and an LCDR3 sequence of a light chain variable region as shown in SEQ ID NO: 1 and (ii) an HCDR1 sequence, an HCDR2 sequence and an HCDR3 sequence of a heavy chain variable region as shown in SEQ ID NO: 2, the CDR is defined according to the IMGT, the Kabat, the Chothia, the Contact or any combination of the IMGT, the Kabat, the Chothia and the Contact. The tissue factor binding antibody provided by the invention has high sensitivity, high accuracy and high precision, and can meet the requirement of accurate detection of tissue factors.

Description

Technical Field

[0001] This invention relates to the field of antibody medicine. Specifically, this invention relates to tissue factor-binding antibodies and their uses, particularly to antibodies that specifically bind to TF or their antigen-binding fragments, and kits containing said antibodies or antigen-binding fragments. Furthermore, this invention relates to nucleic acids encoding said antibodies and host cells containing said nucleic acids, as well as methods for preparing said antibodies. This invention also relates to the detection and diagnostic uses of said antibodies. Background Technology

[0002] Tissue factor (TF), also known as prothrombin kinase, CD142, or coagulation factor 3, is a transmembrane glycoprotein composed of an extracellular domain, a transmembrane domain, and an intracellular domain.

[0003] TF is an essential molecule for initiating extrinsic coagulation events and is expressed functionally on the cell surface. Under normal physiological conditions, TF is located on the adventitia of the blood vessel wall and on the fibroblasts surrounding the blood vessel, but it is scarce in the media or intima. Only when the integrity of the blood vessel wall is compromised is TF exposed to the circulating blood, where it exerts its hemostatic effect by activating the coagulation cascade.

[0004] In contrast to its limited expression in normal tissues and cells, TF has been confirmed to be overexpressed in a variety of malignant tumors, including cervical cancer, pancreatic cancer, lung cancer, prostate cancer, bladder cancer, ovarian cancer, breast cancer, and colorectal cancer. Therefore, TF can serve as a target for the development of antibody drugs and ADC (antibody-drug conjugate) drugs, as seen in published literature such as WO2023 / 160651.

[0005] With the development of such targeted drugs, there is an urgent need in the field for detection and diagnostic methods that can accurately and reliably detect the expression and distribution of TF target antigens in the pathological tissues of patients or suspected patients, in order to help make effective treatment decisions and monitor treatment progress. Summary of the Invention

[0006] To meet the above requirements, the inventors have provided TF-binding antibodies suitable for clinical pathological detection. As shown in the examples, the antibodies of the present invention possess high specificity, high sensitivity, and high robustness. Using the antibodies of the present invention, in immunohistochemical (IHC) detection, the expression, distribution, and abundance of TF on various types of cancer cells can be effectively detected, thereby allowing for the diagnosis of TF-related cancers, the identification of TF-positive cancer patients suitable for anti-TF drug therapy, and the prediction of patient responsiveness to anti-TF drug therapy and prognosis assessment.

[0007] In a first aspect, this disclosure provides antibodies that bind tissue factor (TF) (also referred to herein as "TF-binding antibodies") or antigen-binding fragments thereof. In some embodiments, the TF-binding antibody of the present invention comprises the LCDR1, LCDR2, and LCDR3 sequences of the light chain variable region as shown in SEQ ID NO:1, and the HCDR1, HCDR2, and HCDR3 sequences of the heavy chain variable region as shown in SEQ ID NO:2, wherein the CDRs are defined according to IMGT, Kabat, Chothia, or Contact, or any combination thereof.

[0008] In a second aspect, this disclosure provides a nucleic acid encoding an antibody or antigen-binding fragment of a TF-binding agent according to the first aspect of this disclosure, a vector containing the nucleic acid (preferably an expression vector), a host cell containing the nucleic acid or the vector, and a method for preparing the antibody or antigen-binding fragment.

[0009] In a third aspect, this disclosure provides a method for detecting TF in a biological sample. In some embodiments, the method includes: contacting the biological sample with an antibody or antigen-binding fragment thereof that binds to TF according to a first aspect of this disclosure; and detecting a complex formed between the antibody or antigen-binding fragment thereof and the antigen TF. In some embodiments, the complex is detected by immunohistochemistry (IHC) or immunofluorescence (IF) staining.

[0010] In a fourth aspect, this disclosure provides a kit for TF detection. In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof that binds to TF according to a first aspect of this disclosure. In some embodiments, the kit further comprises an antigen retrieval buffer and / or a secondary antibody detection system.

[0011] In a fifth aspect, this disclosure provides uses and methods of using antibodies binding to TF or antigen-binding fragments thereof according to the first aspect of the present invention, and kits according to the fourth aspect of the present disclosure. In some embodiments, the use is for the preparation of products for the diagnosis / adjunctive diagnosis or prognosis of TF-positive tumors in subjects. In other embodiments, the use is for the preparation of products for determining whether a subject is suitable for anti-TF drug treatment, and in some embodiments, the use is for the preparation of products for predicting the responsiveness of a subject to anti-TF drug treatment. In some embodiments, this disclosure also provides methods for diagnosing or prognosing TF-positive tumors in subjects and methods for determining whether a subject is suitable for anti-TF drug treatment or predicting the responsiveness of a subject to anti-TF drug treatment, including detecting the presence or expression level of antigen TF in a biological sample from the subject using the TF-binding antibody or antigen-binding fragment of the present invention. In some embodiments, the tumor is selected from cervical cancer, pancreatic cancer, lung cancer, prostate cancer, bladder cancer, ovarian cancer, breast cancer, colorectal cancer, esophageal cancer, head and neck cancer, and gastric cancer. In some embodiments, the tumor includes primary, advanced, and metastatic cancers. In some embodiments, the treatment is administered using an anti-TF antibody drug or an anti-TF antibody-based ADC drug, such as the anti-TF antibody and its camptothecin drug conjugate disclosed in this disclosure (e.g., WO2023 / 160651, which is hereby incorporated herein by reference in its entirety), particularly "antibody-drug conjugate A" as defined herein. Attached Figure Description

[0012] Refer to the instruction manual. Figure 1 Reading this description will provide a better understanding of the preferred embodiments of the invention as detailed below. For illustrative purposes, the figures show presently preferred embodiments. However, it should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the figures.

[0013] Figure 1 This is a schematic diagram showing the immunohistochemical staining results of the TF-binding antibody of the present invention with the human skin cancer cell line A431; Figure 2 This is a schematic diagram showing the immunohistochemical staining results of the TF-binding antibody of the present invention with human breast ductal carcinoma cells BT474; Figure 3 This is a schematic diagram showing the immunohistochemical staining results of the TF-binding antibody of the present invention with normal human kidney tissue; Figure 4The diagram shows the immunohistochemical staining results of different concentrations (1 μg / ml, 5 μg / ml) of TF-binding antibody, mouse IgG antibody, commercial TF-binding antibody (Abcam), TF-binding antibody (CST), and rabbit IgG antibody of the present invention with sample numbers MDA-MB-468 and HCC1954, respectively. Figure 5 The diagram shows the immunohistochemical staining results of TF-binding antibody, mouse IgG antibody, commercial TF-binding antibody (Abcam), TF-binding antibody (CST), and rabbit IgG antibody at different concentrations (1 μg / ml, 5 μg / ml) of the present invention with sample numbers Caski and SW756, respectively. Figure 6 The diagram shows the immunohistochemical staining results of samples OIU11-030 and OIU11-031 with different concentrations (1 μg / ml, 5 μg / ml) of TF-binding antibody, mouse IgG antibody, commercial TF-binding antibody (Abcam), TF-binding antibody (CST), and rabbit IgG antibody of the present invention, respectively. Figure 7 The diagram shows the immunohistochemical staining results of samples OIU14-029 and OIU03-022 with different concentrations (1 μg / ml, 5 μg / ml) of TF-binding antibody, mouse IgG antibody, commercial TF-binding antibody (Abcam), and rabbit IgG antibody of the present invention, respectively. Figure 8 The diagram shows the immunohistochemical staining results of samples OIU17-015 and OIU17-016 with different concentrations (1 μg / ml, 5 μg / ml) of TF-binding antibody, mouse IgG antibody, commercial TF-binding antibody (Abcam), and rabbit IgG antibody of the present invention, respectively. Figure 9 The diagram shows the immunohistochemical staining results of different concentrations (2.5 μg / ml, 3.5 μg / ml, 5 μg / ml) of the TF-binding antibody of the present invention and the control positive antibody (commercial TF-binding antibody, Abcam) with TF-positive cell line A431, TF-negative cell line BT474 and normal kidney tissue, respectively. Figure 10 The diagram shows the immunohistochemical staining results of the TF-binding antibody of the present invention and the control positive antibody (commercial TF-binding antibody, Abcam) at different concentrations (2.5 μg / ml, 3.5 μg / ml, 5 μg / ml) with sample numbers OIU03-022, OIU11-005, and OIU11-036, respectively. Figure 11 This is a schematic diagram of the immunohistochemical staining results of the TF-binding antibody of the present invention in CDX and PDX animal model samples in Example 3 of the present invention. Detailed Implementation

[0014] definition 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. For the purposes of this invention, the following terms are defined.

[0015] As used herein, the term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​having a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value. The term is also intended to cover values ​​within the range of ±4%, ±3%, ±2%, ±1%, ±0.5%, or ±0.1% of the specified numeric value.

[0016] As used herein, the term “and / or” when used in conjunction with multiple options should be understood to mean any one of the options or any combination of two or more of the options.

[0017] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations consisting of the mentioned elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0018] As used herein, the terms “tissue factor” or “TF” are used interchangeably and, unless otherwise stated, include any variant of human tissue factor, including sequence variants, especially naturally occurring variants, allele variants, and post-translational modification variants and conformational variants, and encompassing its species homologs. Furthermore, it should be understood that the term covers TF expressed in natural or recombinant cells. An example of tissue factor is the human TF protein containing the amino acid sequence UniProtKB-P13726. In this document, unless otherwise specified, the terms “tissue factor” or “TF” refer to tissue factor derived from humans.

[0019] As used herein, the term "TF-positive" cells / tissue refers to cells / tissues that express TF on their cell surface, such as tumor cells / tissues. The TF expression level on the cell / tissue surface can be determined using a TF-binding antibody (e.g., the TF-binding antibody of this invention) and any conventional method known in the art for determining cell surface antigen expression levels, such as immunohistochemical staining, FACS detection, or immunofluorescence staining, and compared with a preset threshold to determine whether the cells / tissue are TF-positive or TF-negative. For example, in the case of applying immunohistochemistry (IHC) to test samples, an IHC score can be used to assess the TF expression status of sample cells. IHC scores may include, but are not limited to, cell staining intensity scores, percentage of positive cells scores, and combinations thereof. In some embodiments of this disclosure, preferably, a four-level rating system of 0, 1+, 2+, and 3+ is used to assess the IHC staining intensity of cells, where "0" indicates that the cell has a staining intensity no higher than the background (i.e., negative cells); "1+" indicates that the cell is clearly visible under a 20x objective (i.e., weakly positive cells); "2+" indicates that the cell is clearly visible under a 10x objective (i.e., positive cells); and "3+" indicates that the cell is clearly visible under a 4x objective (i.e., strongly positive cells). Optionally, the IHC background is determined using isotype control antibody staining. In some further embodiments, the test sample can be identified as TF-positive or TF-negative based on the percentage of TF-positive cells with different staining intensities. In some preferred embodiments, a TF-positive sample is defined as a sample in which the total percentage of positive cells with cell membrane staining intensities of 1+, 2+, and 3+, as determined by IHC, exceeds (≥) 1%; a TF-negative sample is defined as a sample in which the total percentage of positive cells with cell membrane staining intensities of 1+, 2+, and 3+, as determined by IHC, is less than (<) 1%. In other embodiments, the test samples may be further categorized according to a histochemical score (H-score), where the H-score is defined as [1 × (percentage of positive cells with intensity 1+) + 2 × (percentage of positive cells with intensity 2+) + 3 × (percentage of positive cells with intensity 3+)]. In some embodiments, the H-score may be used to assess or assist in assessing the responsiveness of a subject's tumor to anti-TF drug immunotherapy (including, for example, anti-TF antibodies or anti-TF antibody-based ADC drugs).

[0020] As used herein, the term "antibody" refers to a polypeptide containing at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen. This term encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, single-chain or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), murine antibodies, rabbit antibodies, chimeric or humanized antibodies, full-length antibodies, and antibody fragments, provided they exhibit the desired antigen-binding activity.

[0021] As used herein, the term "whole antibody" (which may be used interchangeably with "full-length antibody," "complete antibody," and "intact antibody") comprises at least two heavy chains (H) and two light chains (L). Each heavy chain consists of a heavy chain variable region (abbreviated VH herein) and a heavy chain constant region. The heavy chain constant region consists of domains CH1, CH2, and CH3, and optionally CH4. Each light chain consists of a light chain variable region (abbreviated VL herein) and a light chain constant region. The light chain constant region consists of a domain CL. The variable region is a domain in the heavy or light chain of the antibody that participates in the binding of the antibody to its antigen. The constant region does not directly participate in the binding of the antibody to the antigen but exhibits various effector functions. The light chain of an antibody can be classified into one of two types (called kappa (κ) and lambda (λ)) based on the amino acid sequence of its constant domain. Antibody heavy chains can be classified into five main types based on the amino acid sequence of their heavy chain constant regions: IgA, IgD, IgE, IgG, and IgM. Several of these types can be further subdivided into subclasses, such as IgG1, IgG2, IgG3 and IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different antibody types are designated α, δ, ε, γ, and µ, respectively.

[0022] As used herein, the term "isotype" refers to the antibody type determined by the antibody heavy chain constant region. For example, antibodies according to the invention may be IgA (e.g., IgA1 or IgA2), IgG1, IgG2 (e.g., IgG2a or IgG2b), IgG3, IgG4, IgE, IgM, and IgD antibodies. In some embodiments, antibodies according to the invention have a non-human (e.g., murine) constant region, such as a murine IgG constant region.

[0023] As used herein, the term "antigen-binding fragment" of an antibody (which may be used interchangeably with "antibody fragment") refers to a molecule that is not a complete antibody but contains the portion of the complete antibody used to bind the antigen bound by that complete antibody. As will be understood by those skilled in the art, the antigen-binding portion of an antibody typically contains amino acid residues from a "complementarity-determining region" or "CDR". Antigen-binding fragments can be prepared by recombinant DNA technology or by enzymatic or chemical cleavage of a complete antibody. Antigen-binding fragments include, but are not limited to, Fab, scFab (single-chain Fab), disulfide-linked scFab, Fab', F(ab')2, Fab'-SH, Fv, scFv (single-chain Fv), disulfide-linked scFv, diabody, triabody, tetrabody, minibody, dAb, and sdAb. In this disclosure, unless the context clearly indicates otherwise, reference to "antibody" is equivalent to reference to "antibody and its antigen-binding fragment".

[0024] As used herein, the term "mouse antibody" refers to an antibody having a variable region derived from a murine immunoglobulin sequence. Furthermore, if the antibody contains a constant region, that constant region is also derived from a murine immunoglobulin sequence. However, it should be understood that murine antibodies may include amino acids not encoded by a murine immunoglobulin sequence (e.g., mutations introduced through random or point-specific mutagenesis in vitro or somatic mutations in vivo).

[0025] As used herein, the term "chimeric antibody" refers to an antibody whose sequence (e.g., the variable region sequence) is derived from one species, while another part of the antibody's sequence (e.g., the constant region sequence) is derived from another species.

[0026] As used herein, the term "isolated" antibody or antigen-binding fragment thereof refers to a recombinantly produced, artificially synthesized antibody or antigen-binding fragment thereof that has been isolated from its components in its natural environment. In some embodiments, the antibody or antigen-binding fragment thereof is purified to a purity greater than 90%, 95%, or 99%, which purity can be determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0027] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, the IgG heavy chain Fc region extends from Cys226 or Pro230 of the heavy chain to the C-terminus. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (MD), 1991, NIH Publication No. 91-3242.

[0028] As used herein, the antibody-related term "variant" refers to an antibody that, compared to a given antibody, contains a target antibody region having an amino acid alteration by substitution, deletion, and / or insertion of at least one, for example, 1-30, or 1-20, or 1-10, for example, 1, or 2, or 3, or 4, or 5 amino acids, wherein the variant substantially retains at least one biological property (e.g., antigen-binding capacity) of the antibody molecule before the alteration. The target antibody region can be the full length of the antibody, or a heavy chain variable region or a light chain variable region or a combination thereof, or one or more heavy chain CDR regions or one or more light chain CDR regions or a combination thereof. Such variants of specific exemplary antibodies of the present invention are considered in this disclosure.

[0029] As used herein, the term "sequence identity" refers to the degree of sequence similarity on a nucleotide-by-nucleotide or amino acid-by-amino acid basis within a comparison window. The "sequence identity percentage" can be calculated by comparing two optimally aligned sequences within a comparison window, determining the number of positions in both sequences containing the same amino acid residue to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the sequence identity percentage. Optimal alignment for determining the sequence identity percentage can be performed in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full-length sequence being compared or within the target sequence region.

[0030] In this disclosure, the percentage of amino acid sequence identity, with respect to the antibody sequence, is determined by optimally aligning a candidate antibody sequence to a given antibody sequence, preferably according to the Kabat numbering rules. In this document, without specifying a comparison window (i.e., the target antibody region to be compared), alignment across the entire length of the given antibody sequence will be applicable. In some embodiments, sequence identity, with respect to the antibody, may be distributed across the entire heavy chain variable region and / or the entire light chain variable region, or the percentage of sequence identity may be limited only to the framework region, while the sequence of the corresponding CDR region remains 100% identical.

[0031] As used herein, the term "conservative substitution" refers to an amino acid change that results in the replacement of an amino acid with a chemically similar amino acid. Amino acid modifications such as substitutions can be introduced into the antibodies of this invention using standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A table of conserved substitutions for functionally similar amino acids is well known in the art. The following eight groups contain amino acids that are conserved substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, *Proteins* (1984)). In some implementations, the term "conservative amino acid modification" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of antibodies containing amino acid sequences.

[0032] As used herein, the term "vector" refers to an artificial construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing said genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, and DNA or RNA expression vectors encapsulated in liposomes.

[0033] As used in this article, the term "host cell" refers to a cell in which exogenous nucleic acids have been introduced, including the progeny of these cells. Host cells include "transformed cells," which include primary transformed cells and their progeny, regardless of passage number.

[0034] As used herein, the terms “individual,” “subject,” or “patient” are used interchangeably and refer to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the subject is a human.

[0035] As used herein, the terms “tumor” and “cancer” are used interchangeably and refer to a physiological disorder in mammals characterized by unregulated cell growth. The term encompasses both primary and metastatic forms of tumors. It also covers all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0036] As used herein, the term "anti-TF drugs" refers to drugs based on anti-TF antibodies, including but not limited to anti-TF antibodies that specifically bind to TF and anti-TF antibody-drug conjugates.

[0037] As used in this article, the term "anti-TF antibody-drug conjugate (ADC)" refers to a compound obtained by linking an anti-TF antibody or its antigen-binding fragment to a drug (small molecule) via a linker.

[0038] As used herein, a “biological sample” from a subject refers to a collection of cells, tissues, or body fluids obtained from an individual or subject. The source of a tissue or cell sample can be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; or cells from any stage of pregnancy or development in the subject. Tissue samples may contain compounds that are naturally occurring and do not contaminate with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. Examples of tumor samples include, but are not limited to, tumor biopsies, fine needle aspirates, bronchoalveolar lavage fluid, pleural fluid, sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like characteristics, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor tissue sections or frozen tumor samples.

[0039] As used herein, the terms “reference sample,” “reference cell,” “reference tissue,” “control sample,” “control cell,” or “control tissue” refer to a sample, cell, tissue, or standard used for comparative purposes. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or disease-free portion of the same subject or individual's body, and is healthy and / or disease-free tissue or cells. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from healthy tissue or cells of an individual who is not a subject.

[0040] As used herein, the term "primary antibody" refers to a TF antibody that specifically binds to a biological sample; the term "post-primary antibody reagent" refers to a reagent that can directly and specifically bind to the primary antibody, thereby forming a bridge between the primary antibody and subsequent reagents; and the term "secondary antibody" refers to an antibody that can bind to the primary antibody via the post-primary antibody reagent or directly (if the post-primary antibody reagent is not present). In TF antigen detection, the combined use of the primary antibody, post-primary antibody reagent, and secondary antibody can amplify the detection signal.

[0041] As used in this article, the term "detection antibody" refers to an antibody that has been directly labeled as detectable. If a primary antibody is directly labeled as a detectable marker, then that primary antibody is a detection antibody. If the primary antibody is indirectly labeled as a detectable marker through a secondary antibody, then that secondary antibody is a detection antibody.

[0042] As used herein, the term "nuclear counterstaining reagent" refers to a reagent used to stain the nuclei of nucleated cells in immunostained samples (e.g., samples stained by immunohistochemistry (IHC) or immunofluorescence (IF)). Staining the nucleated cells in a sample reveals the background outline of cells that have been specifically immunostained. Generally, the color of the nuclear counterstain should be as distinct as possible from the color of the detecting antibody. Therefore, the choice of nuclear counterstaining reagent will depend on the staining method of the sample: a chromogenic agent (a dye visible under a standard optical microscope) or fluorescent staining. Hematoxylin is the most commonly used nuclear counterstaining reagent in horseradish peroxidase (HRP) or alkaline phosphatase (AP) chromogenic systems. In hematoxylin-eosin (H&E) staining, hematoxylin and eosin are stained separately; this is a rapid staining process that does not require antibodies and is particularly useful for pathologists. DAPI is an abbreviation for 4',6-diamidinyl-2-phenylindole, which is the most commonly used nuclear fluorescent dye in immunohistochemistry (IHC) and immunocytochemistry (ICC) fluorescent staining.

[0043] Various aspects of the invention will be further described in the following subsections.

[0044] I. The antibody of the present invention Immunohistochemistry (IHC) is a technique that uses colorimetric antibodies to detect, locate, identify, and quantify target antigens in tissue cells. IHC has been widely used in tumor pathology diagnosis. By examining the distribution, location, and abundance of tumor biomarkers in clinicopathological samples, IHC can provide information on tumor staging and grading, benign or malignant nature, metastatic origin, and localization to the primary tumor, and can be used to predict a patient's clinical responsiveness to biomarker-targeted therapy. A key element in this type of pathological diagnosis is the availability of validated and reliable IHC antibodies for detecting biomarker-positive cases.

[0045] In principle, both monoclonal and polyclonal antibodies can be used for IHC. Polyclonal antibodies can recognize multiple epitopes of a target, but they are also more likely to cross-react, leading to nonspecific detection. One advantage of using monoclonal antibodies is that they are generally more specific, but this also means that the epitopes they recognize are more likely to be masked. In particular, for antigens expressed on membrane surfaces, different antibodies can exhibit significantly different detection performances due to factors such as the location of the recognized epitopes and the membrane surface binding activity. Furthermore, the complexity of clinical samples also poses challenges to the detection performance of monoclonal antibodies. Therefore, monoclonal antibodies used for IHC often require specialized screening and validation.

[0046] After extensive screening, the inventors proposed the TF-binding antibody of this invention and validated its detection performance on various cell lines and different types of tumor tissue samples. Clinical pathological applications also demonstrate that the TF-binding antibody of this invention possesses high specificity, high accuracy, and high sensitivity, meeting the desired detection / diagnostic objectives. The antibody of this invention can be reliably used by pathologists to detect TF expression in human cancer tissues.

[0047] Therefore, in a first aspect, this disclosure provides an antibody or antigen-binding fragment thereof that binds to TF.

[0048] I-1 antibody CDR region In some embodiments, the TF-binding antibody of the present invention comprises three complementary determinant regions of the light chain variable region as shown in SEQ ID NO:1 and three complementary determinant regions of the heavy chain variable region as shown in SEQ ID NO:2.

[0049] The "complementarity-determining region" (CDR), "CDR region," or "hypervariant region" is a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are sequentially numbered starting from the N-terminus and are commonly referred to as CDR1, CDR2, and CDR3. CDRs located within the antibody heavy chain variable domain are also called HCDR1, HCDR2, and HCDR3, while those located within the antibody light chain variable domain are called LCDR1, LCDR2, and LCDR3. Within a given amino acid sequence of a light chain variable region or heavy chain variable region, the CDR sequence can be determined using various schemes known in the art. Such schemes include, but are not limited to, the Kabat, AbM, Chothia, Contact, and IMGT definition schemes.

[0050] Table 1 shows exemplary CDR area ranges under some different CDR definition schemes using the Kabat and Chothia numbering systems.

[0051] Table 1

[0052] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the foregoing methods or any combination thereof.

[0053] CDRs can also be determined based on having the same Kabat numbering position as the reference CDR sequence. Unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it means the position according to the Kabat numbering system (e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (MD), 1991, NIH Publication No. 91-3242).

[0054] In some preferred embodiments, the CDR sequence of the TF-binding antibody according to the present invention is defined according to IMGT, Kabat, Chothia, or Contact, or any combination thereof.

[0055] Therefore, in one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that binds to TF, comprising three complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region and three complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region, wherein: (i) According to the CDR defined by IMGT, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 3, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 4, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 5, and HCDR1 contains the amino acid sequence shown in SEQ ID NO: 6, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 7, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 8; or (ii) According to the CDR defined by Kabat, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 10, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 11, and HCDR1 contains the amino acid sequence shown in SEQ ID NO: 12, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 13, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 14; or (iii) According to the CDR defined by Chothia, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 15, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 16, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 17, and HCDR1 contains the amino acid sequence shown in SEQ ID NO: 18, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 19, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 20; or (iv) According to the CDR defined by Contact, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 21, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 22, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 23, and HCDR1 contains the amino acid sequence shown in SEQ ID NO: 24, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 25, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 26.

[0056] As is known in the art, although CDRs differ between antibodies, only a limited number of amino acid sites within a CDR directly participate in antigen binding. Minimal overlapping regions can be determined using at least two of the Kabat, Chothia, AbM, IMGT, and Contact methods, thus providing a “minimum binding unit” for antigen binding. Such a minimum binding unit can be a sub-part of a CDR. The remaining residues of the CDR sequence, as will be apparent to those skilled in the art, can be determined by the antibody’s structure and protein folding. Therefore, the present invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be substituted with conserved amino acid residues.

[0057] In some embodiments, the antibody or antigen-binding fragment of the TF-binding antibody comprises: (i) according to the CDR defined by IMGT, the amino acid sequence of LCDR1 as shown in SEQ ID NO: 3, the amino acid sequence of LCDR2 as shown in SEQ ID NO: 4, the amino acid sequence of LCDR3 as shown in SEQ ID NO: 5, and the amino acid sequence of HCDR1 as shown in SEQ ID NO: 6, the amino acid sequence of HCDR2 as shown in SEQ ID NO: 7, and the amino acid sequence of HCDR3 as shown in SEQ ID NO: 8; or (ii) According to the CDR defined by Kabat, the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 9, the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 10, the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 11, and the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 12, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 13, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 14; or (iii) According to the CDR defined by Chothia, the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 15, the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 16, the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 17, and the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 18, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 19, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 20; or (iv) According to the CDR defined by Contact, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 21, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 22, the amino acid sequence of LCDR3 is shown in SEQ ID NO: 23, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 24, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 25, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 26.

[0058] I-2 antibody variable region In some embodiments, this disclosure also provides antibodies or antigen-binding fragments thereof comprising heavy chain variable regions and light chain variable regions that bind to TFs. A “variable region” or “variable domain” is a domain in the heavy or light chain of an antibody that participates in the binding of the antibody to its antigen. The heavy chain variable region (VH) and light chain variable region (VL) each consist of a hypervariable region (HVR, also known as a complementarity-determining region (CDR)) and interspersed, more conserved regions (i.e., framework regions (FR)). Specifically, each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0059] Since the CDR sequence is responsible for most antibody-antigen interactions, the variable region of an antibody can be modified while still retaining its desired biological activity. As an example, in some aspects, one or more residues in the variable region of an antibody can be modified, for example, by modifying one or more CDR regions and / or one or more framework regions, particularly by substitution of conserved residues, to obtain antibody variants that still substantially retain at least one biological property of the parent antibody (e.g., antigen-binding ability). As another example, the variable region of an antibody can be modified via CDR transplantation. In such antibody variants, a CDR sequence from a known antibody is transplanted into the framework region of a different antibody with different properties, and one to several residue mutations can be made as needed, such as reversion mutations, to refine the desired properties of the antibody.

[0060] Therefore, this disclosure considers antibodies that bind to TF and have the CDR sequence characteristics according to the invention but different variable region sequences. Preferably, the variable region sequence according to the invention has structural similarity to the specific exemplary variable region sequence described herein, for example, having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0061] In one embodiment, this disclosure provides an antibody or antigen-binding fragment thereof that binds to TF, comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In another embodiment, this disclosure provides an antibody or antigen-binding fragment thereof that binds to TF, comprising a heavy chain variable region and a light chain variable region, wherein: the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0062] In some preferred embodiments, this disclosure provides an antibody or antigen-binding fragment thereof that binds to TF, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith, and wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. Preferably, the antibody or antigen-binding fragment comprises the heavy chain variable region of SEQ ID NO: 2 and the light chain variable region of SEQ ID NO: 1.

[0063] I-3 antibody heavy and light chains In some embodiments, the antibody according to the invention may comprise a heavy chain constant region and / or a light chain constant region. The heavy chain constant region contained in the antibody of the invention may be any isotype or subtype, such as the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4 isotypes. The light chain constant region contained in the antibody of the invention may be a κ light chain constant region or a λ light chain constant region.

[0064] In some embodiments, the antibody according to the invention is a full-length antibody composed of two heavy chains and two light chains. In some embodiments, the antibody according to the invention is a murine antibody.

[0065] In some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:32, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it, or is composed of SEQ ID NO:32; and the light chain comprises the amino acid sequence shown in SEQ ID NO:31, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it, or is composed of SEQ ID NO:31.

[0066] In some embodiments, the amino acid sequence of the heavy chain is shown in SEQ ID NO:32, and the amino acid sequence of the light chain is shown in SEQ ID NO:31.

[0067] II. Polynucleotides, vectors, hosts, and antibody preparation methods In a second aspect, this disclosure provides a nucleic acid encoding an antibody or a fragment thereof that binds to a TF according to a first aspect of this disclosure; a vector comprising the nucleic acid; a host cell comprising the nucleic acid or the vector; and a method for preparing the antibody or a fragment thereof.

[0068] In some embodiments, this disclosure provides nucleic acid molecules comprising polynucleotides encoding at least one CDR region and typically all three CDR regions of a heavy chain VH or light chain VL sequence of an antibody binding TF as described above. In some further embodiments, the invention provides nucleic acid molecules comprising polynucleotides encoding a complete or substantially complete variable region sequence of a heavy chain and / or light chain of an antibody binding TF as described above. As will be apparent to those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences.

[0069] In one embodiment, this disclosure provides one or more vectors comprising the nucleic acids of the present invention, including cloning vectors and expression vectors. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors that can be used in the present invention include, but are not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YAC).

[0070] In one embodiment, this disclosure provides a host cell comprising the vector of the present invention. Suitable host cells for cloning or expressing the vector encoding an antibody include prokaryotic or eukaryotic cells. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. After expression in bacteria such as *Escherichia coli*, the antibody can be separated from the bacterial cell paste in a soluble fraction and can be further purified. In yet another embodiment, the host cell is a eukaryotic cell. In yet another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments thereof. Examples of useful mammalian host cell lines include monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293HEK or 293 cells); Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines such as Y0, NSO, and Sp2 / 0.

[0071] In one embodiment, this disclosure provides a method for preparing the TF-binding antibody or antigen-binding fragment thereof of the present invention. In one embodiment, the method of the present invention includes culturing a host cell containing a nucleic acid encoding the antibody of the present invention under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0072] In another embodiment, the present invention also provides an antibody or antigen-binding fragment thereof that binds to TF having one or more of the following properties, and a method thereof for preparation thereof: (i) Epitopes that bind to the same or overlapping antibodies according to the invention; (ii) Competing with the antibody according to the invention to bind human TF; (iii) Inhibition (e.g., competitive inhibition) of the binding of the antibody according to the invention to cells expressing human TF on the cell surface, wherein the antibody according to the invention comprises the heavy chain variable region of SEQ ID NO: 2 and the light chain variable region of SEQ ID NO: 1.

[0073] In one embodiment, the preparation method includes: using an antibody according to the invention as a reference, screening for antibodies having the above-described properties from an antibody mixture in a binding affinity assay. The antibody mixture that can be used herein includes, but is not limited to, antiserum from TF-immunized animals, or a yeast or mammalian display antibody library.

[0074] The antibodies of the present invention prepared as described herein can be purified using known prior art techniques such as high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art. The purity of the antibodies of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, and HPLC.

[0075] III. Detection and Diagnostic Methods, Applications, and Reagent Kits In the third to fifth aspects, this disclosure provides the application of the antibodies of the present invention in detection and diagnostic methods and methods of using them, as well as kits containing the antibodies of the present invention and their use in the preparation of products for said detection and diagnostic.

[0076] III-1 Detection Method In some embodiments, this disclosure provides methods for detecting TF in a sample. In this document, the term "detection" includes quantitative, semi-quantitative, or qualitative detection. Exemplary detection methods include, but are not limited to, immunohistochemistry (IHC), immunocytochemistry (ICC), flow cytometry (e.g., FACS), ELISA assays, Western blotting, immunofluorescence (IF) assays, and / or co-immunoprecipitation (Co-IP) assays.

[0077] In some embodiments, the detection method of the present invention includes: (a) contacting a biological sample with an antibody or an antigen-binding fragment thereof according to the first aspect of the invention; and (b) Detect the antibody or its antigen-binding fragment and the complex formed by TF to determine whether the biological sample contains TF; optionally, the antibody is directly or indirectly detectably labeled.

[0078] There are no particular limitations on the biological samples suitable for use in the methods of this invention, including but not limited to cell samples (e.g., cultured cells, cell lines, cell samples from a subject), tissue samples (e.g., tissue sections, pathological sections, and tissue microarrays), and body fluid samples (e.g., blood, serum, urine, saliva, and tissue fluid). In some embodiments, the biological sample comprises cells or tissue. In other embodiments, the biological sample is derived from hyperplastic or cancerous lesions. In some preferred embodiments, the biological sample is a tissue section (such as a paraffin section or frozen section), and more preferably, the biological sample is a formaldehyde-fixed paraffin-embedded (FFPE) section.

[0079] Depending on the chosen detection method, the antibody binding to TF used in the method of this invention can be directly or indirectly detectably labeled. In this disclosure, "directly labeled" means that the antibody is directly linked to the label; correspondingly, "indirectly labeled" means that the antibody is linked to the label via one or more bridging reagents. For example, a primary antibody can be indirectly detectably labeled via a secondary antibody or a combination of a primary antibody followed by a reagent and a secondary antibody. Labels that can be used in this invention include, but are not limited to, fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, radioactive labels, and enzymes or ligands. Some specific examples of labels include: radioisotopes. 32 P, 14 C 125 I, 3 H and 131 I. Fluoresceins such as rare earth chelates or luciferin and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase (e.g., firefly luciferase and bacterial luciferase), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidases (e.g., glucose oxidase, galactose oxidase, glucose-6-phosphate dehydrogenase, heterocyclic oxidases (e.g., uricase and xanthine oxidase)), and enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, microperoxidase, biotin / avidin, spin labeling, phage labeling, stable free radicals, etc.

[0080] The TF-binding antibodies of the present invention are applicable not only to different types of samples (e.g., cell lines, tissue sections, and tissue microarrays (TMA)), but also to different types of protein expression analysis (e.g., IHC and IF) and quantitative / qualitative assessment methods (e.g., image analysis and microscopic observation).

[0081] In some embodiments, the method according to the invention detects the complex in step (b) by immunohistochemistry (IHC). Immunohistochemistry (IHC) is one of the marker-based protein detection techniques. The principle of this technique is based on the in situ specific binding of a specific antibody to a matching specific antigen in a sample (tissue or cell). The bound antigen-antibody complex can then be visualized using a series of detection techniques, allowing for the display and recording of the high-resolution distribution and localization of specific cellular components within the cell and appropriate histological background. Markers that can be used for IHC visualization include, for example, enzyme markers (such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) and their corresponding chromogenic substrates) and fluorescent markers (such as fluorescent dyes or fluorophores). Depending on the markers and the sample, the detection of the complex can be performed by image analysis, microscopic observation, etc., and can be qualitative, semi-qualitative, and quantitative assessments. There are no particular limitations on the biological samples used for IHC, including, for example, paraffin sections and frozen sections.

[0082] In some embodiments of the method according to the invention, IHC is used for the detection of antigen-antibody complexes. Depending on the biological sample used, those skilled in the art can select a suitable known IHC procedure. As a specific example of an IHC procedure applied to paraffin sections, the IHC includes: performing antigen retrieval on paraffin sections (e.g., FFPE sections) after dewaxing and hydration using an antigen retrieval buffer; adding a primary antibody diluted to a working concentration (an antibody binding to TF according to the invention; a negative control antibody; or a positive control antibody); and visualizing the antigen bound to the antibody. In some embodiments, the visualization includes directly or indirectly labeling the antibody according to the invention using a marker, such as an enzyme marker or a fluorescent marker.

[0083] In some embodiments, this disclosure provides a method for IHC detection on paraffin sections.

[0084] In some specific implementations, the method includes: (i) Obtain paraffin sections; (ii) Dewaxing and hydration; (iii) Antigen retrieval; (iv) Contact the slice with the antibody that binds TF; and (v) Detect the TF antigen that binds to the antibody.

[0085] In some embodiments of the IHC detection method according to the present invention, the antigen retrieval is heat-induced antigen retrieval. In some embodiments, the antigen retrieval step includes treating the slide with an antigen retrieval buffer at a temperature of approximately 95 to 100°C for 5-30 minutes (e.g., approximately 5, 10, 15, 20, 25, and 30 minutes), preferably approximately 20 minutes. In some embodiments, the antigen retrieval buffer is selected from citrate buffer (pH 6.0 ± 0.5) or EDTA buffer (pH 8.0 ± 0.5), and preferably citrate buffer (pH 6.0). In some preferred embodiments, the antigen retrieval step includes treating the slide in citrate buffer (pH 6.0) at a temperature of 95 to 100°C for approximately 20 minutes.

[0086] In some embodiments of the IHC detection method according to the present invention, step (iv) includes incubating the antibody at a concentration of 1-10 μg / ml at approximately 10-35°C for 20-100 minutes. In some preferred embodiments, the antibody concentration is 1-5 μg / ml, for example, approximately 1 μg / ml, 1.5 μg / ml, 2 μg / ml, 2.5 μg / ml, 3 μg / ml, 3.5 μg / ml, 4 μg / ml, 4.5 μg / ml, or 5 μg / ml, or a range consisting of any two of the foregoing values ​​(e.g., 3-4 μg / ml) or any value therein (e.g., 3.3 μg / ml, 3.35 μg / ml, 3.4 μg / ml, 3.45 μg / ml, 3.5 μg / ml, 3.55 μg / ml, 3.6 μg / ml, 3.65 μg / ml, etc.); and the incubation is performed at room temperature (e.g., 20-26°C) for approximately 30-60 minutes. In some more preferred embodiments, step (iv) includes incubation at 20-26°C for approximately 60 minutes at an antibody concentration of approximately 3.5 μg / ml.

[0087] In some embodiments of the IHC detection method according to the present invention, the method includes: blocking endogenous cellular peroxidase with a peroxidase blocking agent before the sample is contacted with the antibody of the present invention. In some embodiments, the peroxidase blocking agent is 3%-4% hydrogen peroxide by mass fraction.

[0088] In some embodiments of the IHC detection method according to the present invention, in step (iv), the TF antigen bound to the antibody or its antigen-binding fragment is detected visually. In some embodiments, the method includes: performing the visualization with a detectable labeled secondary antibody. In some embodiments, the detectable labeled secondary antibody is an enzyme-labeled secondary antibody, and the method includes color development using a chromogenic substrate of the enzyme. In some embodiments, the enzyme is horseradish peroxidase (HRP) or alkaline phosphatase (AP), and preferably, the enzyme-labeled secondary antibody is a secondary antibody conjugated with polyhortradish peroxidase (polyHRP) or alkaline phosphatase (AP).

[0089] In some embodiments of the IHC detection method according to the present invention, the method includes: visualization using a secondary antibody detection system comprising a secondary antibody containing a detectable marker. In some embodiments, the secondary antibody detection system comprises the following components: (i) Optionally, a peroxidase blocking agent; (ii) Optionally, reagents used after primary antibody to amplify the detection signal; (iii) Enzyme-labeled secondary antibodies; (iv) The chromogenic substrate of the enzyme; and (v) Optional, nuclear counterstaining reagent.

[0090] In some embodiments, the enzyme is horseradish peroxidase (HRP), and the chromogenic substrate is DAB (3,3'-Diaminobenzidine) reagent. In some cases, advantageously, the IHC further includes: after staining with the chromogenic substrate, counterstaining the sections with hematoxylin. The IHC staining procedure according to the method of the present invention can be performed on a variety of commercially available automated staining systems, such as the Leica Bond Max (from Leica Biosystems, Nussloch GmbH).

[0091] In some specific embodiments, the IHC method includes: dewaxing for 0.5 min, incubation with antigen retrieval buffer at 100°C for 20 min, peroxidase blocking for 5 min, primary antibody incubation for 60 min, incubation with primary antibody and post-primary antibody reagent for 8 min, incubation with poly-HRP conjugated secondary antibody for 8 min, DAB staining for 10 min, and hematoxylin staining for 5 min.

[0092] III-2 Reagent Kit In some embodiments, this disclosure provides kits that can be used in the detection methods of the present invention. In some embodiments, the kit according to the present invention comprises the antibody binding to TF of the present invention, and optionally a packaging insert instructing the use of the kit.

[0093] In some embodiments, the kit according to the invention further comprises an antigen retrieval buffer. In some embodiments, the antigen retrieval buffer is selected from citrate buffer (pH 6.0±0.5) or EDTA buffer (pH 8.0±0.5), and preferably citrate buffer (pH 6.0).

[0094] In some embodiments, the kit according to the invention further comprises a secondary antibody detection system. In some embodiments, the secondary antibody detection system comprises a detectable labeled secondary antibody. In other embodiments, the secondary antibody detection system comprises the following components: (i) Optionally, a peroxidase blocking agent; (ii) Optionally, reagents used after primary antibody to amplify the detection signal; (iii) Enzyme-labeled secondary antibodies; (iv) The chromogenic substrate of the enzyme; and (v) Optional, nuclear counterstaining reagent.

[0095] In some embodiments, the peroxidase blocking agent is 3-4% hydrogen peroxide by mass. In some embodiments, the post-primary antibody reagent is an anti-IgG antibody bound to the primary antibody. In some embodiments, the enzyme is HRP, and the chromogenic substrate is DAB reagent. In some embodiments, the nuclear counterstaining reagent includes hematoxylin.

[0096] III-3 Diagnostic Uses Studies have shown that in cancer, TF promotes primary tumor growth, angiogenesis, tumor invasion, and metastasis; therefore, TF expression is associated with poor prognosis in various solid tumors. Simultaneously, TF expression in solid tumors makes it a meaningful target for cancer therapy, and preclinical proof-of-concept studies of TF-targeting antibodies, antibody-drug conjugates, immunoconjugates, and TF pathway inhibitors have been described.

[0097] Therefore, in clinicopathological diagnosis, accurate and sensitive immunohistochemical (IHC) analysis of target TF antigens using routinely processed tissue sections is of great significance for reliably identifying true target antigen-positive cases and determining whether patients are suitable for immunotherapy targeting the target antigen. Furthermore, validated IHC analysis on paraffin-embedded (e.g., FFPE) samples can help predict a patient's clinical responsiveness to immunotherapy and assist in monitoring treatment response by quantitatively detecting the tissue expression of the target.

[0098] As demonstrated in the examples, the TF-binding antibody of the present invention, which has been functionally verified by IHC, can be applied to clinical pathological samples to count positive tumor cells with different TF expression levels with high sensitivity and high specificity, and is therefore suitable for the above-mentioned pathological diagnosis and detection applications.

[0099] In some embodiments, this disclosure provides the use of the antibody or antigen-binding fragment thereof described in any of the foregoing claims, or the kit described in any of the foregoing claims, in the preparation of medicaments for the diagnosis, auxiliary diagnosis, and / or monitoring of the occurrence or progression of TF-related diseases.

[0100] In some implementations, TF-related diseases include tumors associated with abnormal TF expression (TF-positive tumors).

[0101] In some embodiments, this disclosure provides the use of the antibody or antigen-binding fragment thereof described in any of the foregoing claims, or the kit described in any of the foregoing claims, in the preparation of a product for determining whether a subject is suitable for treatment with an anti-TF drug.

[0102] In some embodiments, this disclosure provides the use of any of the antibodies or antigen-binding fragments thereof described above, or any of the kits described above, in the preparation of products for predicting a subject's responsiveness to TF drug treatment.

[0103] In some embodiments, this disclosure provides a method for diagnosing patients with TF-positive tumors, comprising: applying the detection method according to the invention to detect the presence and / or expression level of TF in a biological sample from the patient, and optionally comparing the detected presence or expression level of TF with the presence or expression level of TF in a reference sample. In some embodiments, elevated presence and / or expression levels of TF in the patient's biological sample, relative to the presence or expression level of TF in a reference sample from a healthy individual or from healthy tissue from the patient, indicate the presence of a TF-positive tumor.

[0104] In some embodiments, this disclosure provides a method for prognosticating TF-positive tumor patients, comprising: applying the detection method according to the invention to detect the presence and / or expression level of TF in a biological sample from the patient, and optionally comparing the detected presence or expression level of TF with the presence or expression level of TF in a reference sample. In some embodiments, the patient is a patient who has been treated with an antitumor agent, said antitumor agent including but not limited to metabolic inhibitors, antibiotic anticancer agents, plant alkaloid anticancer agents, topoisomerase inhibitors, antitumor alkylating agents, monoclonal antibodies, ADCs, etc. In some embodiments, a decrease in the presence and / or expression level of TF in a post-treatment biological sample compared to a pre-treatment patient biological sample indicates a good prognosis.

[0105] In some embodiments, this disclosure provides a method for determining whether a patient is suitable for treatment with an anti-TF drug, comprising: detecting the presence and / or expression level of TF on a biological sample from the patient using a detection method according to the invention, and optionally comparing the detected presence or expression level of TF with the presence or expression level of TF in a reference sample. In some embodiments, the anti-TF drug is an anti-TF antibody or an anti-TF antibody-drug conjugate (ADC). Examples of such TF drugs may be the anti-TF antibody and anti-TF antibody-drug conjugate (ADC) disclosed in WO2023 / 160651. In some embodiments, the anti-TF drug is an antibody-drug conjugate (ADC) having the structure of formula (I): Formula (I), Ab represents anti-TF antibody. Where q is the antibody-drug ratio (i.e., the ratio of the drug portion coupled to the Ab portion to the Ab portion).

[0106] In some implementations, q is an integer from 1 to 8; in specific and preferred implementations, q is 2, 3, 4, 5, 6, 7, or 8. In some of the most preferred implementations, the ADC has an average DAR value of approximately 8.

[0107] In some embodiments, the Ab comprises three CDRs of the heavy chain variable region (VH) sequence of SEQ ID NO: 27 and three CDRs of the light chain variable region (VL) sequence of SEQ ID NO: 28; or more preferably comprises the heavy chain variable region of SEQ ID NO: 27 and the light chain variable region of SEQ ID NO: 28, and even more preferably comprises the heavy chain of SEQ ID NO: 29 and the light chain of SEQ ID NO: 30.

[0108] In some preferred embodiments, the ADC is "antibody-drug conjugate A". In this disclosure, "antibody-drug conjugate A" refers to an ADC having the structure of formula (I) above, wherein the Ab is an anti-TF antibody composed of two heavy chains of SEQ ID NO: 29 and two light chains of SEQ ID NO: 30, and the ADC has an average DAR value of approximately 8.

[0109] In this disclosure, the heavy chain variable region sequence of the Ab antibody is shown in SEQ ID NO: 27.

[0110] SEQ ID NO: 27: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPGQGLEWMGMIDPSDSYTSYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCTRGSGPGLFAYWGQGTLVTVSS In this disclosure, the light chain variable region sequence of the Ab antibody is shown in SEQ ID NO: 28.

[0111] SEQ ID NO: 28: DIQETQSPSSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSVSGTDFTLTISSLQPEDFATYFCQQGNTLPYTFGQGTKVEIK In this disclosure, the heavy chain sequence of the Ab antibody is shown in SEQ ID NO: 29.

[0112] SEQ ID NO: 29: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPGQGLEWMGMIDPSDSYTSYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCTRGSGPGLFAYWGQGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK In this disclosure, the light chain sequence of the Ab antibody is shown in SEQ ID NO: 30.

[0113] SEQ ID NO: 30: DIQETQSPSSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSVSGTDFTLTISSLQPEDFATYFCQQGNTLPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC In some embodiments, this disclosure provides a method for predicting a subject's responsiveness to anti-TF drug treatment, comprising: detecting the presence and / or expression level of TF on a biological sample from a patient using a detection method according to the invention, and optionally comparing the detected presence or expression level of TF with the presence or expression level of TF in a reference sample. In some embodiments, the anti-TF drug is an anti-TF antibody or an anti-TF antibody-drug conjugate (ADC). Examples of such anti-TF drugs may be mentioned in WO2023 / 160651, which discloses anti-TF antibodies and anti-TF antibody-drug conjugates (ADCs). In some embodiments, the anti-TF drug is an ADC having the structure of formula (I) shown above. In some preferred embodiments, the ADC is "antibody-drug conjugate A" as defined herein.

[0114] In some embodiments of the diagnostic, prognostic, judgment, and / or predictive methods according to the present invention, the methods include: 1) Contact the biological sample from the subject with the antibody or antigen-binding fragment that binds to TF; 2) Detect the binding of the antibody or its antigen-binding fragment to the biological sample; and 3) Optionally, the presence or expression level of TF in the biological sample from the subject is compared with the presence or expression level of TF in a reference sample.

[0115] In some implementations, the detection is performed by immunohistochemical (IHC) staining.

[0116] In some embodiments, the biological sample is a tissue section (such as a paraffin section or frozen section), more preferably a formaldehyde-fixed-paraffin-embedded (FFPE) section. In some embodiments, the biological sample is a biopsy (such as a core biopsy), a surgical specimen (such as a specimen from a surgical excision), or a fine-needle aspirate.

[0117] In some embodiments, the biological sample is a precancerous sample, a primary cancer sample, or a sample obtained after the cancer has metastasized. In some embodiments, the biological sample is obtained prior to treatment with a therapeutic agent, such as an anti-TF antibody or an anti-TF antibody-drug conjugate. In some embodiments, the biological sample is a sample taken before treatment (e.g., before initiating treatment); or before a treatment at a later interval.

[0118] TF-positive tumors suitable for the methods and applications of this invention can be selected from: myeloma (such as multiple myeloma), acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia (AML), non-Hodgkin lymphoma, central nervous system tumors, gliomas, brain cancer, head and neck cancer (such as head and neck squamous cell carcinoma), gastrointestinal cancer, genitourinary cancer, lung cancer (such as non-small cell lung cancer, NSCLC), esophageal cancer, gastric cancer, hepatobiliary cancer, pancreatic cancer, colorectal cancer, rectal cancer, bladder cancer, kidney cancer, breast cancer (especially triple-negative breast cancer), prostate cancer, endometrial cancer, ovarian cancer, cervical cancer, melanoma, sarcoma, and skin cancer.

[0119] The TF-positive tumors suitable for the methods and applications of this invention can be early, intermediate, or late-stage cancers, or metastatic cancers. Furthermore, the TF-positive tumors suitable for the methods and applications of this invention can be tumors that have previously undergone treatment and have experienced immune escape.

[0120] In some embodiments, the TF-positive tumors used according to the method of the present invention are selected from: cervical cancer, pancreatic cancer, lung cancer, prostate cancer, bladder cancer, ovarian cancer, breast cancer, colorectal cancer, esophageal cancer, head and neck cancer, and gastric cancer, including primary, advanced, or metastatic cancers. In yet another embodiment, the TF-positive tumor is cervical cancer. In another embodiment, the TF-positive tumor is colon cancer. In yet another embodiment, the TF-positive tumor is ovarian cancer. In still another embodiment, the TF-positive tumor is pancreatic cancer.

[0121] In some embodiments, this disclosure also provides the use of the antibodies or kits of the present invention in the preparation of products for use in the methods described above.

[0122] Table 2 provides the abbreviations used in this disclosure.

[0123] Table 2

[0124] Example Example 1: Preparation of TF-binding antibody A murine monoclonal antibody that optimally binds to triglycerides (TF) was obtained through immunization of mice and screening of hybridomas, and named 1B6-10F12C3. Sequencing confirmed that this TF-binding antibody consists of two light chains and two heavy chains, wherein: The amino acid sequence of the light chain variable region is: DIQMTQTTSSLSASLGDRVTISCRASQDITNYLNWYQQKPDGTVKLLIY FTSKLHSGVPSRFSGRGSGTDYSLTISNLEPEDIATYYCQQYSRRPWTFGGGTKLEIK (SEQ ID NO:1).

[0125] The light chain amino acid sequence is: DIQMTQTTSSLSASLGDRVTISCRASQDITNYLNWYQQKPDGTVKLLIY FTSKLHSGVPSRFSGRGSGTDYSLTISNLEPEDIATYYCQQYSRRPWTTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 31).

[0126] The CDR region definition for light chains is shown in Table 3: Table 3

[0127] The amino acid sequence of the heavy chain variable region is: DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPG NKLEWMGYISYDGVNSHNPSLKNRISIIRDTSKNQFFLNLDSVTTEDTATYYCVRAMGYYGDWNFDVWGAGTTVTVSS (SEQ ID NO: 2).

[0128] The heavy chain amino acid sequence is: DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPG NKLEWMGYISYDGVNSHNPSLKNRISIIRDTSKNQFFLNLDSVTTEDTATYYCVRAMGYYGDWNFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMV TLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPK DVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVY TIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:32).

[0129] The heavy chain CDR region is defined as shown in Table 4: Table 4

[0130] Based on the above-mentioned amino acid sequence information of the antibody light and heavy chains, the amino acid sequence was converted into a DNA sequence using eukaryotic degenerate codons. While maintaining the post-translational amino acid sequence, the optimized light and heavy chain DNA expression sequence was obtained after optimization using mammalian cell preferred codons.

[0131] The optimized light and heavy chain DNA was integrated into the PTT5 expression vector by molecular cloning to construct a TF-binding antibody expression plasmid. The plasmid was then transiently transfected into 293E suspension cells or Chinese hamster ovary cells (CHO cell line) using liposomes. After transfection, the 293E cells were amplified in shake flasks and cultured in batches with feed, and antibodies that could bind to the TF target were obtained from the supernatant of the fermentation broth.

[0132] The TF-binding antibody harvested from the fermentation broth was purified and prepared by protein A affinity chromatography, anion exchange chromatography and cation exchange chromatography to obtain the final TF-binding antibody.

[0133] Example 2: Development of TF-binding antibody IHC Using a murine TF-binding antibody (1B6-10F12C3), the efficacy of the antibody prepared in Example 1 as an IHC detection antibody and the optimization of the IHC protocol were evaluated on an immunohistochemistry platform.

[0134] 2-1 The antibody to be tested used in this embodiment, as well as the reagents, equipment, and methods used in the experiment, are as follows: The information on the test antibodies and quality control antibodies used in this embodiment is shown in Table 5, and they were diluted to the working concentration using diluent (CST / 8112L).

[0135] Table 5

[0136] 2-2 The reagents and consumables used in this embodiment are shown in Table 6: Table 6

[0137] The secondary antibody detection system uses Leica Bond™ Polymer Refine Detection as its main component, including: a peroxidase blocking agent (3-4% hydrogen peroxide); and a primary antibody post-detection reagent (Tris buffered saline containing 10% animal serum, which contains rabbit anti-mouse IgG / 0.1% ProClin). TM 950); polymer (Tris-buffered saline solution containing 10% animal serum containing goat anti-rabbit poly-HRP-IgG / 0.1% ProClin) TM 950); DAB colorimetric reagent Part 1 (66mM 3,3-diaminobenzidine tetrahydrochloride hydrate, containing stabilizer) and DAB colorimetric reagent Part B (≤0.1% hydrogen peroxide solution, containing stabilizer); hematoxylin (<0.1% hematoxylin).

[0138] Antigen retrieval buffer 1 is citrate buffer (pH 6.0); Antigen retrieval buffer 2 is EDTA buffer (pH 8.0).

[0139] The experimental steps for IHC 2-3 are as follows: The paraffin blocks were sectioned on a paraffin microtome (Leica RM2235) to a thickness of 4µm. The sections were then placed on positively charged slides (Superfrost plus Slides). The sections were baked at 60°C for 60 ± 30 minutes. Automatic staining was then performed on an automated immunohistochemical staining system (Leica / Bond RX platform) according to the following procedure (as shown in Table 7).

[0140] Table 7

[0141] After completing the automated staining procedure as described above, remove the slides from the Bond Rx platform. In the Leica fully automated staining system, initiate the dehydration program using ethanol and xylene, and then mount the slides using a mounter or manually. Scan the stained slides using a full-field-of-view slide scanning analysis system (Leica, Aperio Versa8). Capture and visualize the images using a microscope (Zeiss / Axio LabA1) and imaging system and software (Nikon).

[0142] Example 2.1 IHC assay on tumor cell lines and quality control tissue samples TF immunohistochemical staining was performed on tumor cell lines (human skin cancer cell line A431 and human breast ductal carcinoma cell line BT474) and quality control samples (human normal kidney tissue).

[0143] Tumor cells were fixed in 10 mL of 4% PFA at room temperature for 30 minutes, then replaced with 70% ethanol for 30 minutes. The cells were then mixed with agarose to form agarose cell blocks, which were dehydrated and made into cell paraffin blocks for sectioning.

[0144] Freshly excised normal kidney tissue was directly fixed in standard formalin and embedded in paraffin to prepare paraffin blocks for sectioning. Immunohistochemical staining was then performed according to the IHC experimental procedures described above. Different concentrations of TF-binding antibody and different antigen retrieval solutions were used in the staining process, as shown in Table 8.

[0145] Table 8

[0146] Staining results as follows Figures 1 to 3 As shown. By Figures 1 to 3 It was found that, compared to the TF-negative cell line BT474 and normal kidney tissue (non-glomerular tissue), the TF-binding antibody of this invention obtained uniform and strong staining on the TF-positive cell line A431 and normal kidney tissue (glomeruli), indicating that the antibody has the IHC efficacy for detecting and distinguishing positive TF cells. The antibody was initially optimized using different concentrations and different retrieval solutions. Staining results showed that using antigen retrieval buffer 1 (ER1) resulted in a cleaner staining background; on the positive cell line A431, staining with antibody concentrations of 1 µg / ml and 5 µg / ml resulted in clearer membrane expression and no background. Therefore, in subsequent experiments, ER1 retrieval and antibody concentrations of 1 µg / ml and 5 µg / ml were selected for further validation and optimization of the IHC protocol.

[0147] Example 2.2 IHC assay on tumor tissue samples IHC assays were performed on CDX (tumor cell line xenograft) samples and human tumor tissue samples to evaluate the efficacy of the antibody of the present invention in detecting TF expression in tissue samples. A CDX tumor model was constructed by transplanting in vitro cultured xenogeneic (human) tumor cell lines into immunodeficient mice. Information on the CDX samples used in this embodiment is shown in Table 9 below. The human tumor tissue samples used in this embodiment were commercially available, and specific information is shown in Table 10 below.

[0148] Table 9

[0149] Table 10

[0150] Tumor tissue samples were collected and FFPE paraffin blocks were prepared. In this embodiment, a commercial rabbit anti-TF monoclonal antibody was used as a positive control, and mouse / rabbit IgG was used as a negative control. The FFPE sections were stained using the positive control antibody according to the manufacturer's instructions. Immunohistochemical staining was then performed according to the IHC experimental procedures described above. The antibody concentration and antigen retrieval solution information in the staining procedure are shown in Table 11.

[0151] Table 11

[0152] Staining results as follows Figures 4 to 8 As shown in the figure, the TF-binding antibody of this invention achieved similar staining effects on tissue samples as two commercial antibodies. Compared to a concentration of 1 µg / ml, the staining pattern and effect of the TF-binding antibody of this invention at a concentration of 5 µg / ml are closer to those of the commercial antibodies.

[0153] Example 2.3 Optimization of IHC antibody concentration In this study, in order to conserve antibody, the concentration gradient of the antibody of the present invention used for IHC was further refined, and the effects of three treatment groups of 2.5 µg / ml (incubation for 60 min), 3.5 µg / ml (incubation for 60 min), and 5 µg / ml (incubation for 30 min) on IHC staining were investigated.

[0154] The study was conducted on cell line-derived samples and quality control samples (FFPE paraffin blocks prepared according to Example 2.1) as well as human tumor tissue samples (commercially available). Specific sample information is provided in Table 12.

[0155] Table 12

[0156] Immunohistochemical staining was performed according to the above IHC experimental procedure. The antibody concentration and antibody incubation information in the staining procedure are shown in Table 13.

[0157] Table 13

[0158] Staining results as follows Figure 9 and Figure 10 As shown. The TF-binding antibody of this invention achieved staining effects on TF-positive cells and tumor tissue samples comparable to commercial antibodies under three IHC test conditions; among them, the staining pattern and effect obtained using 3.5 µg / ml antibody (incubation for 60 min) were more consistent with those of commercial antibodies. The following IHC operating conditions were determined to be the preferred IHC conditions for the TF-binding antibody of this invention (see Table 14).

[0159] Table 14

[0160] Example 3: Immunohistochemical staining of CDX and PDX animal model samples The antibody of the present invention was applied to tissue samples from CDX and PDX tumor-bearing animal models treated with anti-TF antibody-drug conjugates (ADCs) to detect their IHC staining performance. The CDX and PDX experimental groups used in this embodiment are shown in Table 15, where the ADC drug is "antibody-drug conjugate A" as defined above, and the ADC dosage for groups 1 and 8 is the effective dose for tumor-bearing animals determined in previous studies, while groups 4 and 7 received 1 / 10 and 1 / 3 of the effective dose of ADC, respectively, to examine the ability of the antibody of the present invention to monitor differential treatment effects.

[0161] The study included the following controls: (i) a control group that did not receive any treatment; and (ii) a control group that received an allotype IgG antibody (from Suzhou Yilian Biopharmaceutical Co., Ltd.). Intravenous administration was administered once weekly. The CV1664 cervical cancer PDX model was from Crown Bioscience, Inc. This patient-derived xenograft (PDX) model accurately reflects the human heterogeneity of this gynecological malignancy, providing an accurate method for predicting drug efficacy before clinical trials. The A2780 cell line does not express tissue factor TF, and a CDX model constructed from it was used as a negative control. The A2780 ovarian cancer CDX model was from WuXi AppTec Co., Ltd., Suzhou. The OVCAR8 ovarian cancer CDX model, the CaSki cervical cancer CDX model, and the KYSE520 esophageal cancer CDX model were from Pharmaron (Beijing) Co., Ltd.

[0162] Table 15

[0163] Immunohistochemical staining was performed according to the above IHC experimental procedure. The antibody concentration and antibody incubation information in the staining procedure are shown in Table 16.

[0164] Table 16

[0165] After staining, the pathologist performs immunohistochemical staining scoring on the sample slides according to the following evaluation method.

[0166] A. Definition of staining intensity: Pathologists assess only TF expression in tumor cells. Positive staining is defined as any intensity of staining above the background in the cytoplasmic membrane (completely circumferential and / or partially linear and / or polarized) and cytoplasm. The staining background is determined by comparing the staining of a sample with that of an isotype control.

[0167] B. The analytical criteria for tumor cell staining intensity are shown in Table 17.

[0168] Table 17

[0169] C. The results shall be reported in the following format: (1) Pathologists evaluate tumor cells that are TF-positive at any intensity higher than the background staining; (2) The tumor cell positivity rate was calculated using the following formula: Tumor cell positivity rate = (Number of TF-positive tumor cells / Total number of viable tumor cells) × 100%; (3) The TF staining score of the tissue is represented by the result of multiplying the positive rate of tumor cells by the staining intensity (0, 1+, 2+, 3+). Specifically, the positive rate of cells at each staining intensity level is first calculated, and then the H-score is calculated using the following formula: H-score = [1 × (percentage of positive cells with intensity 1+) + 2 × (percentage of positive cells with intensity 2+) + 3 × (percentage of positive cells with intensity 3+)]; (4) A positive sample is defined as a sample in which the total percentage of positive cells with cell membrane staining intensities of 1+, 2+, and 3+ exceeds (≥) 1%. A negative sample is defined as a sample in which the total percentage of positive cells with cell membrane staining intensities of 1+, 2+, and 3+ is less than (<) 1%. (5) Report the total H-score of the cell membrane and cytoplasm.

[0170] Staining results are shown Figure 11Table 18 shows the IHC staining scores of the tested samples, where “SiB” and “AB” in the sample number indicate that the sample was stained with the antibody of this invention and the commercial antibody ab228968, respectively.

[0171] Table 18

[0172] The results showed that the antibody of this invention was highly consistent with commercial TF-binding antibodies in terms of IHC staining scores. Compared to samples G3#88379 and blank-#88385 from the TF-negative A2780 tumor cell CDX model (positive rate 0%, H-score 0), the untreated TF-positive tumor CDX and PDX control samples (OVCAR-8 and 15521) exhibited significantly stronger TF staining (positive rate 100%, H-score 300); while the TF-positive tumor CDX and PDX model samples treated with antibody-drug conjugate A showed a dose-related reduction in TF staining (including positive rate and H-score). These results suggest that the antibody of this invention is suitable for monitoring the therapeutic response of subjects to TF-targeted drugs.

[0173] Example 4: IHC staining performance of the antibody of the present invention on clinical tissue samples To characterize the IHC application of the antibody of this invention, the staining performance of the antibody of this invention and commercial antibodies was detected and compared on clinical tissue samples. The staining conditions and IHC staining scoring method for sample sections described in Example 3 were used in the study on sections of FFPE samples (commercially obtained) from human tumor tissue. Specific sample information is shown in Table 19.

[0174] Table 19

[0175] Table 20 shows the IHC staining scores of the tested samples. In the sample number, “SiB” and “AB” indicate that the sample was stained with the antibody of this invention and the commercial antibody ab228968, respectively.

[0176] Table 20

[0177] As the scoring results show, the antibody of this invention and the commercial TF-binding antibody have the same IHC staining score in the samples.

[0178] Example 5: IHC analysis of clinical tissue samples The clinical efficacy of the antibody of the present invention was tested on a series of clinical tumor tissue samples and control samples. IHC staining was performed essentially according to the method described in Example 2.

[0179] Specifically, FFPE paraffin blocks from patient pathological tissue were cut into 4 μm thick sections using a Leica RM2235 microtome. The sections were then baked in an oven at 60°C for 60 minutes. The TF-binding antibody (1B6-10F12C3) was diluted to 3.5 µg / ml using primary antibody dilution buffer (Leica, AR9352). The allotype mouse IgG control antibody (Abcam, ab18443) was diluted 1:100 using primary antibody dilution buffer (Leica, AR9352). Staining was performed using an automated immunohistochemical staining system (Leica / Bond RX platform). The IHC staining procedure included: dewaxing for 0.5 min, incubation with antigen retrieval buffer 1 at 100°C for 20 min, peroxidase blocking for 5 min, primary antibody incubation for 60 min, incubation with post-primary antibody reagent for 8 min, polymer incubation for 8 min, DAB staining for 10 min, and hematoxylin staining for 5 min.

[0180] After staining, the slides are removed and dehydrated and cleared in 70% ethanol, 100% ethanol, and xylene. The slides are then mounted using a mounting machine or manually, and the quality of the stained slides is checked. Pathologists evaluate the H&E-stained sections for each sample to ensure detection on tumor cells; and score the TF immunohistochemical staining for each sample according to the method described in Example 3. Furthermore, the interpretation of TF immunohistochemical results in clinical samples must be performed by at least two qualified pathologists. The report must include the staining intensity and positivity rate of TF.

[0181] On a set of 23 clinical tissue samples, slides were stained with the antibody of this invention and reviewed by two pathologists (Operator 1 and Operator 2). For accuracy assessment, the membrane staining H-scores given by the two pathologists on the same stained slides of the same sample were compared with a reference result for consistency evaluation. An H-score deviation within ±30 was considered accurate (marked "PASS"), otherwise inaccurate (marked "FAIL"). Table 21 shows the review comparison results, where the reference result shown in Table 21 is the IHC membrane staining H-score obtained by Operator 2 after reviewing slides from the same clinical tissue samples stained with the commercial antibody ab228968. For sensitivity and specificity assessment, correctly identified positive and negative samples were determined by re-examining the stained tissues. Sensitivity and specificity were calculated using the following formulas: Sensitivity = 1 - (Number of false negative samples / Total number of positive samples) * 100%; Specificity = 1 - (Number of false positive samples / Total number of negative samples) * 100%.

[0182] According to the calculation results, the antibody of this invention achieves slide reading accuracy, sensitivity, and specificity of 91.30%, 100%, and 100%, respectively. Through accuracy assessment, sensitivity assessment, and specificity assessment, the antibody of this invention possesses accuracy, sensitivity, and specificity that meet the requirements for diagnosis and detection.

[0183] Table 21

[0184] Furthermore, the accuracy of IHC staining for TF-binding antibodies provided by this invention was evaluated, including intra-batch and inter-batch repeatability. FFPE human specimens from three different patients were tested repeatedly in three batches. In each batch, three consecutive sections of each specimen were tested for antibody staining according to the above method, and the fourth consecutive section was tested using an isotype control. For accuracy assessment, the three batches were tested by two different physicians (Operator A and Operator B) on two different dates to cover as many variables as possible. The first and third batches of samples for ovarian cancer, cervical cancer, and pancreatic cancer were tested by Operator A; the second batch of samples for ovarian cancer, cervical cancer, and pancreatic cancer were tested by Operator B. The results are shown in Table 22. The accuracy assessment acceptance criteria are as follows: (1) For intra-batch accuracy: the H-score deviation of three consecutive sections from the same human tissue sample should be within ±30. (2) For inter-batch accuracy: the H-score deviation of all nine sections from the same human tissue should be within ±30.

[0185] Table 22

[0186] As shown in Table 22, the accuracy of IHC staining using the antibody of this invention reached 100%. The antibody of this invention passed the accuracy evaluation.

Claims

1. An antibody or antigen-binding fragment thereof that binds to tissue factor TF, comprising: (i) The LCDR1, LCDR2, and LCDR3 sequences of the light chain variable region as shown in SEQ ID NO:1, and (ii) The HCDR1, HCDR2, and HCDR3 sequences of the heavy chain variable region as shown in SEQ ID NO:2, in, CDR is defined according to IMGT, Kabat, Chothia, or Contact, or any combination thereof.

2. The antibody or antigen-binding fragment of tissue factor TF according to claim 1, comprising three complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region, and three complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region, wherein: (i) According to the CDR defined by IMGT, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 3, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 4, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 5, and HCDR1 contains the amino acid sequence shown in SEQ ID NO: 6, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 7, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 8; or (ii) According to the CDR defined by Kabat, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 10, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 11, and HCDR1 contains the amino acid sequence shown in SEQ ID NO: 12, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 13, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 14; or (iii) According to the CDR defined by Chothia, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 15, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 16, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 17, and HCDR1 contains the amino acid sequence shown in SEQ ID NO: 18, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 19, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 20; or (iv) According to the CDR defined by Contact, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 21, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 22, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 23, and HCDR1 contains the amino acid sequence shown in SEQ ID NO: 24, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 25, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:

26.

3. The antibody or antigen-binding fragment of tissue factor TF according to any one of claims 1-2, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

4. The antibody or antigen-binding fragment of tissue factor TF according to any one of claims 1-2, comprising a heavy chain variable region and a light chain variable region, wherein: The light chain variable region contains the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

5. The antibody or antigen-binding fragment thereof that binds to tissue factor TF according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:2, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

1.

6. The antibody or antigen-binding fragment thereof that binds to tissue factor TF according to claim 1, wherein, The antibody is a full-length antibody composed of two heavy chains and two light chains. The antigen-binding fragment is selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibody, scFv, scFab, disulfide-linked scFv, disulfide-linked scFab, (Fab')2 fragment, or linear antibody.

7. The antibody or antigen-binding fragment thereof that binds to tissue factor TF according to claim 1, wherein the antibody is a murine antibody.

8. An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof that binds to tissue factor TF as described in any one of claims 1-7.

9. A vector comprising the nucleic acid of claim 8.

10. A host cell comprising the nucleic acid of claim 8 or the vector of claim 9.

11. A method for preparing an antibody or antigen-binding fragment thereof that binds to tissue factor TF, the method comprising culturing a host cell containing the nucleic acid under conditions suitable for expressing a nucleic acid encoding an antibody or antigen-binding fragment thereof that binds to tissue factor TF as described in any one of claims 1-7, and isolating the antibody or antigen-binding fragment thereof.

12. A method for detecting TF in a biological sample, the method comprising: (a) Contacting a biological sample with the antibody or antigen-binding fragment thereof that binds tissue factor TF as described in any one of claims 1-7; and (b) Detect the complex formed by the antibody or its antigen-binding fragment and TF to determine whether the biological sample contains TF.

13. The method of claim 12, wherein the biological sample is selected from one or more of cell samples, tissue samples, and body fluid samples.

14. The method according to any one of claims 12-13, wherein the complex is detected by immunohistochemical staining.

15. The method of claim 14, wherein the method comprises: (i) Obtain paraffin sections; (ii) Dewaxing and hydration; (iii) Antigen retrieval; (iv) Contact the slide with the antibody that binds tissue factor TF; as well as (v) Detect TF antigens that bind to the antibody or its antigen-binding fragment.

16. The method of claim 15, wherein the antigen retrieval step comprises treating the slide at 95-100°C for 5-30 minutes using an antigen retrieval buffer; And / or, the antigen retrieval is performed using an antigen retrieval buffer containing citrate buffer or EDTA buffer.

17. The method according to claim 15 or 16, wherein step (iv) comprises incubating the antibody at a concentration of 1-10 μg / ml at 15-30°C for 30-90 minutes.

18. The method of claim 15 or 16, wherein step (v) comprises detection using a secondary antibody detection system, wherein the secondary antibody detection system comprises the following components: (i) Peroxidase blocking agent; (ii) Reagents after primary antibody used to amplify the detection signal; (iii) Enzyme-labeled secondary antibodies; (iv) The chromogenic substrate of the enzyme; and (v) Nuclear counterstaining reagent.

19. The method according to claim 18, wherein the enzyme-labeled secondary antibody is a polyhortradiction peroxidase or alkaline phosphatase-labeled secondary antibody.

20. A kit comprising an antibody or antigen-binding fragment thereof that binds to tissue factor TF as described in any one of claims 1-7.

21. The kit of claim 20, further comprising antigen retrieval buffer.

22. The kit according to any one of claims 20-21, further comprising a secondary antibody detection system, the secondary antibody detection system comprising the following components: (i) Peroxidase blocking agent; (ii) Reagents after primary antibody used to amplify the detection signal; (iii) Enzyme-labeled secondary antibodies; (iv) The chromogenic substrate of the enzyme; and (v) Nuclear counterstaining reagent.

23. Use of the antibody or antigen-binding fragment thereof that binds to tissue factor TF according to any one of claims 1-7, or the kit according to any one of claims 20-22, in the preparation of a medicament for the diagnosis, auxiliary diagnosis and / or monitoring of the occurrence or progression of TF-related diseases, or in the preparation of a product for determining whether a subject is suitable for receiving anti-TF drug treatment, or in the preparation of a product for predicting the responsiveness of a subject to anti-TF drug treatment.

24. The use according to claim 23, wherein TF-related diseases include tumors associated with abnormal TF expression; And / or, the anti-TF drug is an anti-TF antibody or an ADC drug based on an anti-TF antibody.

Images