Recombinant antibody, kit comprising same, and use thereof in diagnosing cancer

Abstract

Disclosed herein is a recombinant antibody or fragment thereof comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain. In accordance with some embodiments of the present disclosure, the VH domain and the VL domain of the recombinant antibody or the antibody fragment comprise amino acid sequences of at least 85% sequence similarity to sequence numbers: 4 and 8, respectively. The invention also discloses a method for diagnosing whether a subject has cancer or not by using the recombinant antibody or the fragment thereof. And methods of treating a subject diagnosed with cancer.

Description

1. Technical Field

[0002] The present disclosure generally relates to the field of disease diagnosis. More specifically, the present disclosure relates to recombinant antibodies and their use in diagnosing cancer.

Background Art

[0003] 2. Description of Related Art

[0004] Cancer refers to a group of diseases characterized by abnormal cell growth, which grow uncontrollably and exhibit the ability to invade and destroy normal tissues. It is a major cause of death worldwide, causing approximately 10 million deaths each year, that is, nearly one in every six deaths. According to statistics from the World Health Organization (WHO), cancer mortality can be reduced when cases are detected and treated early, and the WHO has proposed two early detection strategies: early diagnosis and early screening. Early diagnosis of cancer or pre-cancerous lesions enables early intervention to slow down or even prevent the development, invasion, and / or metastasis of cancer, thereby improving the survival rate and quality of life of cancer patients and also significantly reducing the cost and complexity of cancer treatment. The purpose of screening is to identify subjects with cancer or pre-cancerous lesions before they develop symptoms.

[0005] For cancer screening, tumor-associated antigen (TAA) is a useful target. In practice, a biomarker test is performed on a biological sample (e.g., blood or biopsy sample) isolated from a subject suspected of having cancer to identify the expression of one or more TAAs in the biological sample. Then, a person of ordinary skill in the art or a clinical operator can initially identify a cancer case and may selectively perform further tests to establish a definite diagnosis. During tumor growth, angiogenesis, invasion, and metastasis, cancer cells release plasminogen activators, which mediate the cleavage of plasminogen into plasmin, which degrades fibrin into fibrin-fibrinogen degradation products (FDPs). It is well known that all major cancers produce FDPs, including lung cancer, breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, tongue cancer, and hepatocellular carcinoma (HCC). DR-70 is a marker used to measure FDPs and can be used as a new target for cancer screening. However, to date, there has been no monoclonal antibody available for detecting DR-70. In view of this, there is a continuous hope in the related art to develop a novel anti-DR-70 monoclonal antibody.

Summary of the Invention

[0006] A simplified summary of the present disclosure is presented below to provide a basic understanding to the reader. This summary is not an extensive overview of the present disclosure, and it does not point out the key / important components of the invention or depict the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description presented later.

[0007] As implemented and broadly described herein, a first aspect of the present disclosure is directed to a recombinant antibody and its fragments (e.g., single-chain variable fragment, scFv) called "14G12-5". Structurally, each recombinant antibody and its fragments comprise a heavy chain variable (VH) domain and a light chain variable (VL) domain. According to an embodiment of the present disclosure, the VH domain comprises a first complementarity determining region (CDR-H1) of SEQ ID NO: 1, a second complementarity determining region (CDR-H2) of SEQ ID NO: 2, and a third complementarity determining region (CDR-H3) of SEQ ID NO: 3; and the VL domain comprises a first complementarity determining region (CDR-L1) of SEQ ID NO: 5, a second complementarity determining region (CDR-L2) of SEQ ID NO: 6, and a third complementarity determining region (CDR-L3) of SEQ ID NO: 7.

[0008] According to some exemplary embodiments, the fragment of the VH domain of the present recombinant antibody has an amino acid sequence of SEQ ID NO: 9, 10, 11, or 12; while the fragment of the VL domain of the present recombinant antibody has an amino acid sequence of SEQ ID NO: 13, 14, 15, or 16.

[0009] According to some exemplary embodiments, the VH and VL domains of the recombinant antibody / antibody fragment each comprise an amino acid sequence having at least 85% sequence similarity to that shown in SEQ ID NOs: 4 and 8; preferably, at least 90% sequence similarity to that shown in SEQ ID NOs: 4 and 8; more preferably, at least 95% sequence similarity to that shown in SEQ ID NOs: 4 and 8. In an exemplary example of the present disclosure, the VH and VL domains of the recombinant antibody / antibody fragment each comprise the amino acid sequences of SEQ ID NOs: 4 and 8 (i.e., the VH and VL domains respectively comprise amino acid sequences having 100% sequence similarity to that shown in SEQ ID NOs: 4 and 8).

[0010] A second aspect of the present disclosure relates to the use of the recombinant antibody or antibody fragment in the preparation of a kit for detecting DR-70 (FDP). According to some embodiments of the present disclosure, the kit comprises: a first container containing the recombinant antibody 14G12-5 or a fragment thereof (such as the single-chain variable region fragment of 14G12-5); and a second container containing a pharmaceutically acceptable carrier.

[0011] Also disclosed herein is a method for treating a subject suffering from cancer, which first diagnoses the subject by using a biological sample isolated from the subject; and treats the subject according to the diagnosis thus made. The method comprises: (i) obtaining a biological sample from the subject; (ii) mixing a biological sample with the recombinant antibody or a fragment thereof described in the present disclosure so that DR-70 in the biological sample reacts with the recombinant antibody or a fragment thereof described in the present disclosure, thereby forming a complex in an immunoassay, wherein the formation of the complex indicates that the subject suffers from the cancer; and (iii) administering an anti-cancer treatment to the subject to remove the cancer or inhibit the growth of the cancer.

[0012] Alternatively or optionally, the method further comprises adding a secondary antibody to detect the complex formed in step (ii), wherein the secondary antibody comprises a VH domain of SEQ ID NO: 20 and a VL domain of SEQ ID NO: 24.

[0013] According to embodiments of the present disclosure, the cancer is lung cancer, breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, tongue cancer or HCC.

[0014] According to embodiments of the present disclosure, the subject is a mammal; preferably, a human.

[0015] According to embodiments of the present disclosure, the anti-cancer treatment can be any one of surgery, chemotherapy, radiotherapy, hormone therapy, targeted therapy, immunotherapy or anti-angiogenesis therapy.

[0016] Many accompanying features and advantages of the present disclosure will be more easily understood by referring to the following detailed description.

Detailed Description

[0017] The following detailed description is intended as a description of these examples and is not intended as the only form for constructing or utilizing the examples. The description sets forth the functions of the examples and the sequence of steps for constructing and operating the examples. However, the same or equivalent functions and sequences can be achieved by different examples.

[0018] I. Definitions

[0019] For convenience, certain terms used in this specification, the examples, and the appended claims are consolidated and explained herein. Unless otherwise defined herein, scientific and technical terms used in the present disclosure shall have the meanings commonly understood and used by one of ordinary skill in the art. Additionally, unless the context otherwise requires, it should be understood that singular terms shall include the same plural forms, and plural terms shall include the singular. Specifically, as used herein and in the claims, the singular forms "a" and "an" include plural references unless the context clearly dictates otherwise. Further, as used herein and in the claims, the terms "at least one" and "one or more" have the same meaning and include one, two, three, or more.

[0020] Although the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Additionally, as used herein, the term "about" generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within the standard error of the mean as acceptable to one of ordinary skill in the art when considered. Except in the operating / work examples, or unless otherwise expressly stated, all numerical ranges, amounts, values, and percentages, such as amounts of materials, time durations, temperatures, operating conditions, ratios of amounts, etc., disclosed herein are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and the appended claims are approximations that may vary as desired. At the very least, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0021] The term "antibody" (Ab) is used in its broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific or multivalent antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, and antibody fragments, so long as they exhibit the desired biological activity. The term "antibody fragment" or "fragment of an antibody" refers to a portion of a full-length antibody, typically the antigen-binding domain or variable domain of a full-length antibody (i.e., the VH and VL domains). Examples of antibody fragments include the fragment antigen-binding (Fab), Fab', F(ab') 2, single-chain variable fragment (scFv), diabody, linear antibody, single-chain antibody molecule, and multispecific antibody formed by antibody fragments. Immunoglobulins can be classified into different classes according to the amino acid sequence of the antibody in their heavy-chain conserved domains, including immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE), and some of these classes can be further divided into subtypes (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to different classes of immunoglobulins are respectively called alpha (α), gamma (γ), delta (δ), epsilon (ε), and mu (μ). The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art, such as in "Celular and Molecular Immunology" by Abbas et al., 4th edition (2000). An antibody can be part of a larger fusion molecule, which is formed by covalent or non-covalent binding of the antibody to one or more other proteins or peptides.

[0022] As used herein, the term "monoclonal antibody" (mAb) refers to an antibody obtained from a substantially homogeneous population of antibodies. In contrast to polyclonal antibodies, which can contain different antibodies directed against different epitopes, each monoclonal antibody is directed against a single determinant (i.e., epitope) on an antigen. Monoclonal antibodies can be produced by fusing normal short-lived antibody-producing B cells with rapidly growing cells, such as immortalized cells. The resulting fused cells or hybridomas multiply rapidly, generating a large number of antibody-producing plants. Alternatively, monoclonal antibodies can be produced by recombinant DNA methods, in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from one species or belonging to an antibody type or subtype, and the remaining portion of the chain is identical or homologous to the corresponding sequence in an antibody derived from one species or belonging to an antibody type or subtype, and fragments of such antibodies, provided that they exhibit the desired biological activity.

[0023] As used herein, the term "recombinant antibody" refers to an antibody expressed and isolated in a cell or cell line transfected with an expression vector (or possibly more than one expression vector, usually two expression vectors), which contains a sequence encoding the antibody, where the encoding sequence is not naturally associated with the cell.

[0024] The "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. These domains are generally the most variable parts of the antibody and contain the antigen-binding sites. The term "variable" refers to the fact that certain portions of the variable domains vary extensively in sequence among antibodies and are responsible for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domain of the antibody. It is concentrated in three segments in the variable domains of the light and heavy chains, called complementarity-determining regions (CDRs) or hypervariable regions. The highly conserved portions of the variable domains are called framework (FR). The variable domains of the native heavy and light chains each contain four FR regions, which are mainly connected by β-sheet structures and are linked by three CDRs to form loops connecting the β-sheet structures and, in some cases, form part of the β-sheet structure. The CDRs in each chain are closely connected by the FR regions and together with the CDRs in the other chain form the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).

[0025] As discussed herein, minor changes in the amino acid sequence of an antibody (particularly minor changes in the FR sequences of an antibody) are considered to be encompassed within the present disclosure and the claimed inventive concept, provided that the changes in the amino acid sequence maintain at least 85% sequence similarity, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence similarity. The antibodies of the present disclosure can be specifically modified to alter antibody properties that are not related to their physiological activity. For example, certain amino acid residues in the antibody framework (FR) region can be altered and / or deleted without affecting the physiological activity of the antibodies in this study (i.e., their ability to treat cancer). In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are replacements that occur within a family of amino acid residues that are related by their side chains. Genetically encoded amino acid residues are generally divided into the following categories: (1) acidic: aspartic acid, glutamic acid; (2) basic: lysine, arginine, histidine; (3) nonpolar: alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; (4) uncharged polar: glycine, asparagine, glutamine, cysteamine, serine, threonine, tyrosine. Preferred families are: serine and threonine are the aliphatic hydroxyl family; asparagine and glutamine are the amide-containing family; alanine, valine, leucine and isoleucine are the aliphatic family; phenylalanine, tryptophan and tyrosine are the aromatic family. For example, it can be reasonably expected that replacing leucine with isoleucine or valine alone, aspartic acid with glutamic acid, threonine with serine, or making similar replacements of amino acids with structurally related amino acids will not have a significant impact on the binding or properties of the resulting molecule, especially if the replacement does not involve amino acid residues within the antigen-binding site (i.e., the CDRs). Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the peptide derivative. Those of ordinary skill in the art can readily prepare fragments or analogs of proteins / peptides without difficulty. Preferred amino and carboxyl termini of the fragments or analogs occur near the boundaries of the functional domains.

[0026] "Percent sequence identity" is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in a specific peptide sequence, after aligning the sequences and introducing gaps as necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. The alignment for the purpose of determining percent sequence identity can be achieved in a variety of ways within the skill in the art, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One of ordinary skill in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared. For the purposes herein, the sequence comparison between two amino acid sequences is performed by the computer program Blastp (protein-protein BLAST) provided online by the National Center for Biotechnology Information (NCBI). The percent amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can also be expressed as a given amino acid sequence A having a certain percentage amino acid sequence similarity to a given amino acid sequence B) is calculated by the following formula: where X is the number of amino acid residues scored as identical matches by the sequence alignment program BLAST in the alignment of A and B, and where Y is the total number of amino acid residues in A or B, whichever is shorter.

[0027] The term "pharmaceutically acceptable" refers to molecular entities and compositions that are "generally recognized as safe", e.g., physiologically tolerable and typically do not produce an allergic or similar untoward reaction, such as gastric upset, dizziness and nausea. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or State government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0028] As used herein, the term "detect", whether used as a verb or a noun, in addition to its recognized meaning in the art, also refers to any process of observing a marker in a biological sample, whether or not the marker is actually detected. In other words, the act of probing for a marker in a sample is "detecting" even if it is determined that the marker is absent or below the level of sensitivity. Detection can be a quantitative, semi-quantitative or non-quantitative observation.

[0029] As used herein, the term "diagnosis" refers to a method by which a person skilled in the art can estimate and / or determine the probability (i.e., "likelihood") that a patient has a given disease or disorder (e.g., cancer). In the context of the present invention, "diagnosis" involves using the test results of the present invention (i.e., the presence or absence of DR-70), optionally together with other clinical features, to obtain a diagnosis of cancer (i.e., occurrence or non-occurrence) from a sample obtained from a subject and analyzed. Such a diagnosis being "definitive" does not mean that the diagnosis is 100% accurate. Different biomarkers are indicative of a variety of conditions. A skilled clinician does not use the result of a single biomarker in the absence of information, but rather uses the test results together with other clinical metrics to arrive at a diagnosis. Thus, a measured biomarker level on one side of a predetermined diagnostic threshold indicates a greater likelihood of the disease occurring in the subject than a measured level on the other side of the predetermined diagnostic threshold.

[0030] The term "subject" as used herein refers to an animal. Preferably, the animal is a mammal, such as a human, cow, goat, sheep, horse, dog, cat, rabbit, monkey, rat, or mouse. The term "subject" means both male and female unless a particular gender is specified.

[0031] II. Summary of the Invention

[0032] (i) Recombinant antibody

[0033] The present invention aims to provide novel antibodies for detecting DR-70 (FDP), and a kit for this purpose.

[0034] Accordingly, a first aspect of the present disclosure provides a recombinant mAb designated "14G12-5". According to an embodiment of the present disclosure, mAb 14G12-5 comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain. The VH domain comprises a first complementarity determining region (CDR-H1) of SEQ ID NO: 1, a second complementarity determining region (CDR-H2) of SEQ ID NO: 2, and a third complementarity determining region (CDR-H3) of SEQ ID NO: 2. And the VL domain comprises a first complementarity determining region (CDR-L1) of SEQ ID NO: 5, a second complementarity determining region (CDR-L2) of SEQ ID NO: 6, and a third complementarity determining region (CDR-L3) of SEQ ID NO: 7.

[0035] According to some alternative embodiments, the VH domain of the monoclonal antibody 14G12-5 comprises at least 85% (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identical to SEQ ID NO: 4, and the VL domain of the monoclonal antibody 14G12-5 comprises at least 85% (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identical to SEQ ID NO: 8. According to some preferred embodiments, the VH and VL domains of the monoclonal antibody 14G12-5 each comprise an amino acid sequence that is at least 90% identical to SEQ ID NOs: 4 and 8, respectively. More preferably, the VH and VL domains of the monoclonal antibody 14G12-5 each comprise an amino acid sequence that is at least 95% identical to SEQ ID NOs: 4 and 8, respectively. Most preferably, the VH and VL domains of the monoclonal antibody 14G12-5 each comprise an amino acid sequence that is 100% identical to SEQ ID NOs: 4 and 8, respectively.

[0036] Since the binding affinity and specificity of an antibody are mainly determined by its CDR sequences, as can be appreciated, the framework (FR) sequences of the VH and VL domains can vary (e.g., be replaced by conservative or non-conservative sequences) without altering the binding affinity and / or specificity of the current antibody. Preferably, the FR sequences are conservatively substituted with one or more suitable amino acids having similar properties; for example, leucine (a non-polar amino acid residue) is replaced with isoleucine, alanine, valine, proline, phenylalanine or tryptophan (another non-polar amino acid residue); aspartic acid (an acidic amino acid residue) is replaced with glutamic acid (another acidic amino acid residue); or lysine (a basic amino acid residue) is replaced with arginine or histidine (another basic amino acid residue).

[0037] Based on conservative substitutions, one of ordinary skill in the art can substitute the amino acid residues of the FR sequences of the VH and VL domains of the monoclonal antibody 14G12-5 without affecting its activity and / or efficacy (i.e., binding to DR-70 and / or diagnosing cancer). Accordingly, antibodies comprising substituted amino acids in the FR sequences of their VH and VL domains are included within the scope of the present disclosure. Accordingly, fragments of the VH or VL domain of the monoclonal antibody 14G12-5 are created. According to some embodiments of the present disclosure, the fragment of the VH domain has the amino acid sequence of SEQ ID NO: 9, 10, 11 or 12. According to other embodiments of the present disclosure, the fragment of the VL domain has the amino acid sequence of SEQ ID NO: 13, 14, 15 or 16.

[0038] For the intended purpose, the monoclonal antibody 14G12-5 of the present invention can be produced in the form of IgG, IgA, IgM, IgD or IgE. According to some exemplary embodiments, the monoclonal antibody of the present invention is produced in the form of IgG.

[0039] Fragments of the monoclonal antibody of the present invention are also disclosed herein, including scFv, Fab, Fab', F(ab')2 and diabodies. According to some embodiments, the antibody fragment is scFv.

[0040] The monoclonal antibodies and antibody fragments of the present invention can be produced by any method known in the art, such as phage display libraries, traditional immunization methods (i.e., immunizing animals with specific peptides to induce peptide-specific Abs in the animals), or recombinant DNA technology (also known as DNA cloning technology; i.e., constructing recombinant DNA encoding a specific Ab and transducing it into a host cell to express the Ab). Preferably, the mAb14G12-5 of the present invention is produced by recombinant DNA technology, in which nucleic acids encoding CDR sequences and / or VH and VL sequences are cloned into an expression vector (such as an IgG expression vector), and then the expression vector is transfected. It is introduced into a suitable host cell (preferably a mammalian cell, such as HEK293 cells) so that the monoclonal antibody of the present invention can be produced from the host cell. The procedures for recombinant DNA technology are known to those of ordinary skill in the art; therefore, for the sake of brevity, detailed descriptions are omitted here.

[0041] (ii) A kit containing the monoclonal antibody or antibody fragment of the present invention

[0042] According to a specific embodiment of the present disclosure, the monoclonal antibody 14G12-5 of the present invention exhibits binding affinity for DR-70. Therefore, a second aspect of the present disclosure relates to a kit for detecting DR-70. According to some embodiments, the kit of the present invention comprises a first container containing the monoclonal antibody 14G12-5 or a fragment thereof (e.g., 14G12-5 scFv) as described in section (i) of the present disclosure, and a second container containing a pharmaceutically acceptable carrier.

[0043] The pharmaceutically acceptable carrier can be a solvent, a dispersant, an antibacterial agent, an antifungal agent, an isotonic agent or other physiologically compatible reagents. Examples of pharmaceutically acceptable carriers suitable for this kit include, but are not limited to, water, saline, phosphate buffered saline (PBS), tris-buffered saline (TBS), glycerol, ethanol and combinations thereof. The pharmaceutically acceptable carrier may also contain a small amount of auxiliary substances, such as wetting agents or emulsifiers, preservatives or buffers, which enhance the stability or effectiveness of the antibody or its antigen-binding portion.

[0044] In practical applications, the anti-DR-70 antibody of the present kit (e.g., monoclonal antibody 14G12-5 or 14G12-5 scFv) is used as a detection or capture antibody to detect DR-70 expression. Depending on the desired purpose, the present kit may further comprise a second anti-DR-70 antibody, a blocker (i.e., a reagent for blocking or minimizing non-specific binding between an antigen and an antibody), and / or a reporter molecule-conjugated secondary antibody (e.g., anti-mouse, anti-rat, anti-rabbit, or anti-human antibody depending on the intended use), such as a fluorescent molecule or horseradish peroxidase (HRP). For example, when measuring the expression level of DR-70 in a sample by Western blotting, flow cytometry, immunochemistry, or immunofluorescence assay using the kit of the present invention, the kit of the present invention may comprise the anti-DR-70 antibody of the present invention (e.g., monoclonal antibody 14G12-5 or 14G12-5 scFv) as the first antibody for detecting DR-70, a reporter molecule-conjugated anti-mouse antibody as the second antibody for detecting the first antibody (i.e., the anti-DR-70 antibody of the present invention), and optionally a blocker. Alternatively, when using the present kit to detect the expression level of DR-70 by enzyme-linked immunosorbent assay (ELISA), it may comprise the anti-DR-70 antibody of the present invention (e.g., monoclonal antibody 14G12-5 or 14G12-5 scFv) as a capture antibody or a detection antibody, a second anti-DR-70 antibody as a detection antibody (in the case of using the anti-DR-70 antibody of the present invention as a capture antibody) or a capture antibody (in the case of using the anti-DR-70 antibody of the present invention as a detection antibody), and optionally a blocker. In this case, preferably for detection purposes, the detection antibody is conjugated to a reporter molecule.

[0045] (iii) Use of the antibody or kit of the present invention for diagnosing cancer

[0046] A third aspect of the present disclosure relates to a method for diagnosing cancer by using the antibody or kit of the present invention. This method comprises: (i) isolating a biological sample from a subject in need (e.g., a subject suspected of having cancer); (ii) detecting the presence or absence of DR-70 in the biological sample by mixing the biological sample with the recombinant antibody or fragment thereof of the present invention so that DR-70 in the biological sample reacts with the recombinant antibody or fragment thereof of the present invention, by forming a complex in an immunological assay, wherein the formation of the complex indicates that the subject has cancer.

[0047] Alternatively or optionally, the method further comprises adding a primary and a secondary antibody to detect the complex formed in step (ii), wherein the secondary antibody comprises the VH domain of SEQ ID NO: 20 and the VL domain of SEQ ID NO: 24.

[0048] Preferably, the subject suitable for diagnosis by this method is a mammal, such as a human, cow, goat, sheep, horse, dog, cat, rabbit, monkey, rat or mouse. More preferably, the subject is a human.

[0049] In step (i), a biological sample is isolated from the subject. Depending on the intended use, the biological sample can be a whole blood sample, a plasma sample, a serum sample or a biopsy sample. According to some preferred embodiments, the biological sample is the serum sample.

[0050] In step (ii), the recombinant antibody of the present invention (such as monoclonal antibody 14G12-5 or 14G12-5 scFv) is used to detect whether there is DR-70 in the biological sample, wherein the complex of DR-70 and the recombinant antibody of the present invention is formed in an immunoassay, such as ELISA, flow cytometry, Western blot assay, immunochemical assay, etc.

[0051] According to an embodiment of the present disclosure, if the DR-70 level in the biological sample is higher than a control sample or a reference sample isolated from a healthy subject (for example, the reference data obtained from a gene / protein expression database is normalized and can be directly quantitatively compared with the data of the test sample), then the subject has cancer. In contrast, when the DR-70 level in the biological sample is equal to or lower than the DR-70 level in a control sample or a reference sample isolated from a healthy subject, then the subject does not have cancer.

[0052] Alternatively, the presence of DR-70 in the biological sample can be regarded as a positive screening result, and a technician or practitioner can preliminarily identify cancer cases and selectively perform further tests to establish a definitive diagnosis.

[0053] According to a specific alternative embodiment, the presence of DR-70 in the biological sample indicates that the subject is at high risk of having cancer and may require further tests to make a definitive diagnosis, such as computed tomography (CT) scan, bone scan, magnetic resonance imaging (MRI), positron emission tomography (PET) scan, ultrasound, X-ray, biopsy, urine and blood tests and / or physical examination. Conversely, the absence of DR-70 in the biological sample indicates that the subject has a lower risk of developing cancer.

[0054] According to some alternative embodiments, when the expression level of DR-70 in the biological sample is higher than that in a control sample or reference sample isolated from a healthy subject, then the subject has a high risk of suffering from cancer and may require further tests for a definitive diagnosis, such as CT scan, bone scan, MRI, PET scan, ultrasound, X-ray, biopsy, urine and blood tests, and / or physical examination. In contrast, when the expression level of DR-70 in the biological sample is equal to or lower than that in a control sample or reference sample isolated from a healthy subject, then the subject has a lower risk of suffering from cancer.

[0055] Cancers suitable for diagnosis by the present method can be lung cancer, breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, tongue cancer, hepatocellular carcinoma (HCC), or any cancer that produces fibrin and fibrinogen degradation products (FDP) or is related to FDP, such as ovarian cancer, cervical cancer, lymphoma, nasopharyngeal carcinoma (NPC), and esophageal cancer.

[0056] Based on the diagnosis results, the subject can receive appropriate anti-cancer treatments, including but not limited to surgery, chemotherapy, radiotherapy, hormonal therapy, targeted therapy, immunotherapy, or anti-angiogenesis therapy, to remove the cancer or inhibit the growth of the cancer.

[0057] The following examples are provided to illustrate certain aspects of the present invention and to assist those skilled in the art in practicing the present invention. These examples should in no way be construed as limiting the scope of the present invention in any way. Without further detailed description, it is believed that those skilled in the art can make the most of the present invention based on the description herein. All publications cited herein are incorporated herein by reference in their entirety.

[0058] Examples

[0059] Materials and Methods

[0060] The antibody of the present invention is named "monoclonal antibody 14G12-5", and its amino acid sequence is summarized in Table 1 below.

[0061] Table 1 Amino acid sequences of monoclonal antibody 14G12-5 and its fragments of the present invention

[0062] The amino acid sequence of the detection antibody (or secondary antibody) named "monoclonal antibody 31H11-2" is summarized in Table 2 below.

[0063] Enzyme-linked immunosorbent assay (ELISA)

[0064] Coat a 96-well ELISA plate with monoclonal antibody 14G12-5 (48 ng / well) in sodium borate buffer (pH 8.5) and incubate overnight (at least 16 hours) at 20 - 25 °C, then wash 3 times with sodium borate buffer (pH 8.5) at room temperature. Add the blocking buffer (StabilCoat TM ) to the plate, then incubate overnight (at least 16 hours) at 20 - 25 °C. Remove the blocking buffer and wash 3 times with PBS-T (PBS containing 0.1% - 20). After that, add serum samples at different concentrations to the wells. Incubate the plate at 22 - 28 °C for 30 minutes, then wash 6 times with PBS-T. Add HRP-conjugated monoclonal antibody 31H11-2 (24 ng / well; it has the VH sequence of SEQ ID NO: 20 and the VL sequence of SEQ ID NO: 24) to the plate. Incubate the plate at 22 - 28 °C for 30 minutes. After washing three times with PBS-T, use 3,3',5,5'-tetramethylbenzidine (TMB) substrate to generate a signal. Terminate the reaction with TMB stop buffer and measure the absorbance at 450 nm by an ELISA reader.

[0065] Example 1 Characteristics of Monoclonal Antibody 14G12-5

[0066] First, examine the binding activity of the monoclonal antibody 14G12-5 of the present invention to human FDP. The results show that the monoclonal antibody 14G12-5 exhibits binding affinity to human FDP, and the EC 50 (half maximal effective concentration) is 30.1 ng / μl (about 2×10 -7 M) (data not shown). The kinetics and binding affinity of monoclonal antibody 14G12-5 to canine FDP or human FDP are summarized in Table 3 and Table 4 respectively.

[0067] Table 3 Kinetic Analysis of Monoclonal Antibody 14G12-5 Ka: Association rate constant. Kd: Dissociation rate constant. KD: Equilibrium dissociation constant. Rmax: Analyte binding capacity on the surface. Chi 2: Measure of the mean squared residuals (the difference between experimental data and the fitted curve).

[0068] Table 4 Binding affinity of monoclonal antibody 14G12-5 for canine FDP or human FDP KD: Equilibrium dissociation constant. Rmax: Analyte binding capacity of the surface. Chi 2 : Measure of the mean squared residuals (the difference between experimental data and the fitted curve).

[0069] The data in Table 3 and Table 4 demonstrate the binding affinity of monoclonal antibody 14G12-5 for FDP, where the binding ability of monoclonal antibody 14G12-5 for human FDP is 10 times higher than that of monoclonal antibody 14G12-5 for canine FDP.

[0070] To study whether monoclonal antibody 14G12-5 can be used to detect DR-70 in ELISA, monoclonal antibody 14G12-5 was added as a capture antibody to ELISA plates to capture analytes at specified concentrations (to provide DR-70 at 0, 0.625, 2.5, 5, and 10 μg / ml in AMDL-ELISA ), and HRP-conjugated monoclonal antibody 31H11-2 was used as a detection antibody to detect the analytes captured by monoclonal antibody 14G12-5. The results showed that the combination of monoclonal antibody 14G12-5 (48 ng / well) and monoclonal antibody 31H11-2 (24 ng / well) can be used to detect DR-70 in ELISA, where a test curve (Y-axis) of the mean absorbance was plotted against different DR-70 concentrations (X-axis) approaching the linear standard curve established by AMDL-ELISA (data not shown); the data indicate that monoclonal antibody 14G12-5 exhibits high accuracy in detecting DR-70.

[0071] Then, the combination of monoclonal antibody 14G12-5 and monoclonal antibody 31H11-2 (as the capture antibody and detection antibody in ELISA, respectively) was used to detect DR-70 in serum samples. Serum samples obtained from human subjects were serially diluted (0, 1, 2, or 3-fold dilutions) and subjected to ELISA analysis. The data indicate that monoclonal antibody 14G12-5 and monoclonal antibody 31H11-2 are capable of detecting DR-70 at different dilution levels (0, 1, 2, or 3-fold dilutions; data not shown). In addition, compared with the polyclonal antibody (pAb) against DR-70 provided in

[0072] In conclusion, the present invention provides a novel monoclonal antibody 14G12-5. According to the working examples of the present disclosure, the antibody 14G12-5 exhibits binding affinity for human FDP and DR-70, and thus can be used as an antibody (e.g., capture or detection antibody) in DR-70 immunoassays to detect DR-70 or diagnose cancer.

[0073] It should be understood that the above description of the embodiments is provided by way of example only, and those of ordinary skill in the art can make various modifications. The above specification, examples and data provide a complete description of the structure and use of the exemplary embodiments of the present invention. Although the various embodiments of the present invention have been described above with a certain degree of particularity or with reference to one or more individual embodiments, those of ordinary skill in the art can make various changes to the disclosed embodiments without departing from the spirit or scope of the present invention.

Claims

1. A recombinant antibody or fragment thereof, comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein The VH domain comprises a first complementarity determining region (CDR-H1) with a sequence number of 1, a second complementarity determining region (CDR-H2) with a sequence number of 2, and a third complementarity determining region (CDR-H3) with a sequence number of 3; and The VL domain comprises a first complementarity determining region (CDR-L1) with sequence number: 5, a second complementarity determining region (CDR-L2) with sequence number: 6, and a third complementarity determining region (CDR-L3) with sequence number:

7.

2. The recombinant antibody or fragment thereof according to claim 1, wherein the VH domain and the VL domain respectively have an amino acid sequence with at least 85% sequence similarity to SEQ ID NOs: 4 and 8. 3 . The recombinant antibody or fragment thereof according to claim 2 , wherein the VH domain and the VL domain respectively have an amino acid sequence with at least 100% sequence similarity to SEQ ID NOs: 4 and 8.

4. The recombinant antibody or fragment thereof according to claim 1, wherein the fragment of the VH domain has an amino acid sequence of SEQ ID NO: 9, 10, 11 or 12.

5. The recombinant antibody or fragment thereof according to claim 1, wherein the fragment of the VL domain has an amino acid sequence of SEQ ID NO: 13, 14, 15 or 16.

6. A kit for diagnosing cancer, comprising: a first container for containing the recombinant antibody or fragment thereof as claimed in claim 1; and A second container is used to hold a pharmaceutically acceptable carrier.

7. The kit of claim 6, wherein the VH domain and the VL domain each have an amino acid sequence having at least 85% sequence similarity to SEQ ID NOs: 4 and 8.

8. The kit of claim 6, wherein the VH domain and the VL domain each have an amino acid sequence with at least 100% sequence similarity to SEQ ID NOs: 4 and 8.

9. The kit of claim 6, wherein the fragment of the VH domain has an amino acid sequence with sequence number: 9, 10, 11 or 12.

10. The kit of claim 6, wherein the fragment of the VL domain has an amino acid sequence of SEQ ID NO: 13, 14, 15 or 16.

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