CN115925924A - Antibody for detecting TSP-1 specific peptide fragment and application thereof - Google Patents

Abstract

The present invention relates to the field of cancer diagnosis. Specifically, the invention provides an antibody for detecting TSP-1 specific peptide fragment, and a method for detecting TSP-1 specific peptide fragment by using the antibody, wherein the TSP-1 specific peptide fragment can be used as a marker for diagnosing esophageal cancer, and therefore, the invention also provides application of the antibody in diagnosing esophageal cancer.

Description

Antibody for detecting TSP-1 specific peptide fragment and application thereof

Technical Field

The present invention relates to the field of cancer diagnosis. Specifically, the invention provides an antibody for detecting TSP-1 specific peptide fragment, and a method for detecting TSP-1 specific peptide fragment by using the antibody, wherein the TSP-1 specific peptide fragment can be used as a marker for diagnosing esophageal cancer, and therefore, the invention also provides application of the antibody in diagnosing esophageal cancer.

Background

Esophageal cancer is one of common digestive tract malignant tumors, in the treatment of esophageal cancer, many patients are already in an advanced metastatic stage at the time of diagnosis, the 5-year survival rate is less than 20%, and in patients diagnosed at an early stage, the five-year survival rate of early-stage esophageal cancer is close to 90%. A screening method for early detection of esophageal cancer is urgently needed in clinic.

Thrombospondin-1 (thrombospondin-1, TSP-1) was found in 1971 in platelet alpha granules and is a trimeric glycoprotein of 450kDa, belonging to the platelet coagulation protein family. TSP-1 has various biological functions, and can be used as an endogenous angiogenesis inhibitor to inhibit angiogenesis so as to inhibit tumor growth, while high expression of TSP-1 in gastric cancer can promote gastric cancer growth and lymph node metastasis, and different functions of TSP-1 may be related to different expression amounts of TSP-1 in a group and expression of auxiliary factors such as bFGF. Early studies have found that the expression of TSP1 protein specific N-terminal fragment in the serum of patients with esophageal cancer is increased, which indicates that the peptide fragment can be used for early screening of esophageal cancer.

Therefore, the development of a simple, accurate and high-sensitivity reagent for detecting the specific N-terminal fragment of the TSP1 protein has urgent practical significance in the early screening of esophageal cancer.

Disclosure of Invention

The present inventors have conducted extensive experimental studies to provide monoclonal antibodies particularly useful for detecting specific N-terminal fragments of TSP1 protein. On the basis, the inventor develops a novel detection kit and a detection method for diagnosing esophageal cancer, and the kit and the detection method have great clinical application values.

C-terminal antibodies

In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds a TSP-1 polypeptide, comprising: a heavy chain variable region (VH) comprising the following 3 Complementarity Determining Regions (CDRs): HCDR1 of sequence SEQ ID NO. 3, HCDR2 of sequence SEQ ID NO. 4, and HCDR3 of sequence SEQ ID NO. 5; and/or, a light chain variable region (VL) comprising the following 3 Complementarity Determining Regions (CDRs): LCDR1 with sequence SEQ ID NO. 6, LCDR2 with sequence SEQ ID NO. 7, and LCDR3 with sequence SEQ ID NO. 8.

In certain embodiments, the TSP-1 polypeptide is a specific N-terminal fragment of TSP1 protein having an amino acid sequence shown in SEQ ID NO. 17.

In certain embodiments, the antibody or antigen-binding fragment thereof does not bind the TSP-1 full-length protein. In certain embodiments, the antibody or antigen-binding fragment thereof does not bind to a polypeptide fragment that continues for one or more residues from the specific N-terminal fragment of the TSP1 protein.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID No. 1 or a variant thereof; and/or a VL comprising the sequence shown in SEQ ID NO. 2 or a variant thereof;

wherein the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) as compared to the sequence from which it is derived, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; preferably, the substitutions are conservative substitutions.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown as SEQ ID NO. 1 and a VL comprising the sequence shown as SEQ ID NO. 2.

N-terminal antibodies

In a second aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds a TSP-1 polypeptide, comprising: a heavy chain variable region (VH) comprising the following 3 Complementarity Determining Regions (CDRs): HCDR1 of sequence SEQ ID NO. 11, HCDR2 of sequence SEQ ID NO. 12, and HCDR3 of sequence SEQ ID NO. 13; and/or, a light chain variable region (VL) comprising the following 3 Complementarity Determining Regions (CDRs): LCDR1 with a sequence of SEQ ID NO. 14, LCDR2 with a sequence of SEQ ID NO. 15 and LCDR3 with a sequence of SEQ ID NO. 16.

In certain embodiments, the TSP-1 polypeptide is a specific N-terminal fragment of TSP1 protein having an amino acid sequence shown in SEQ ID NO. 17.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID No. 9 or a variant thereof; and/or, a VL comprising the sequence shown as SEQ ID NO. 10 or a variant thereof;

wherein the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; preferably, the substitutions are conservative substitutions.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown as SEQ ID NO. 9 and a VL comprising the sequence shown as SEQ ID NO. 10.

In certain embodiments of the first or second aspect, the antibody or antigen-binding fragment thereof comprises a constant region.

In certain embodiments of the first or second aspect, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from an immunoglobulin (e.g., an IgG, such as an IgG1, an IgG2, an IgG3, or an IgG 4) and the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from an immunoglobulin (e.g., κ or λ).

In certain embodiments of the first or second aspect, the antibody or antigen-binding fragment thereof comprises a mouse heavy chain constant region and a mouse light chain constant region. In certain embodiments, the antibody of the first aspect is an IgG2a, optionally the light chain is kappa. In certain embodiments, the antibody of the second aspect is an IgG1, optionally the light chain is lambda.

In certain embodiments of the first or second aspect, the antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, fab ', (Fab') ₂ Fv, disulfide-linked Fv, scFv, diabody (diabody), murine, chimeric, humanized, bispecific, or multispecific antibody.

In certain embodiments of the first or second aspect, the antibody or antigen-binding fragment thereof is detectably labeled. Preferably, the detectable label is selected from the group consisting of an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters), a fluorescent dye, biotin, colloidal gold, or a latex particle.

Preparation of antibodies

The antibody of the first aspect or the antibody of the second aspect may be prepared by various methods known in the art, for example, by genetic engineering recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. The transfected host cells are then cultured under specific conditions and the antibodies of the invention are expressed.

The antigen-binding fragment of the first aspect or the antigen-binding fragment of the second aspect may be obtained by hydrolysis of an intact antibody molecule. Alternatively, these antigen binding fragments may be produced directly by the recombinant host cell. For example, fab' fragments can be derived directly from the host cellObtaining; fab 'fragments can be chemically coupled to form F (ab') ₂ And (3) fragment. In addition, fv, fab or F (ab') ₂ The fragments may also be isolated directly from the culture medium of the recombinant host cell. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.

In another aspect, the invention provides an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or encoding an antibody or antigen-binding fragment thereof according to the second aspect of the invention.

In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding a light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecules of the invention comprise a first nucleic acid molecule comprising the first nucleotide sequence and a second nucleic acid molecule comprising the second nucleotide sequence.

In another aspect, the invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule as described above. In certain embodiments, the vectors of the invention are, e.g., plasmids, cosmids, phages, and the like.

In certain embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding a light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

In certain embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of the invention, and/or a second nucleotide sequence encoding the light chain variable region of the antibody or antigen-binding fragment thereof of the invention; wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.

In certain embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain of the antibody or antigen-binding fragment thereof of the present invention, and/or a second nucleotide sequence encoding a light chain of the antibody or antigen-binding fragment thereof of the present invention; wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.

In another aspect, the invention provides a host cell comprising an isolated nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E.coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.).

In another aspect, there is provided a method of making an antibody or antigen-binding fragment thereof of the invention comprising culturing a host cell as described above under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the culture of the cultured host cell.

Reagent kit

In a third aspect, the present invention provides a kit comprising:

(i) A first antibody which is an antibody of the first aspect or an antigen-binding fragment thereof; and (c) and (d),

(ii) A second antibody which is the antibody of the second aspect or an antigen-binding fragment thereof.

In certain embodiments, the first and second antibodies comprise constant regions. In certain embodiments, the heavy chains of the first and second antibodies comprise heavy chain constant regions derived from an immunoglobulin (e.g., igG, e.g., igG1, igG2, igG3, or IgG 4) and the light chains of the first and second antibodies comprise light chain constant regions derived from an immunoglobulin (e.g., κ or λ).

In certain embodiments, the second antibody is detectably labeled, or the kit further comprises a third antibody capable of specifically binding the second antibody, the third antibody being detectably labeled.

Herein, the detectable label may be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. It is particularly preferred that such labels be capable of being adapted for immunological detection (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, immunochromatographic assays, and the like). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., ³ H、 ¹²⁵ I、 ³⁵ S、 ¹⁴ c or ³² P), fluorescent dyes (e.g., fluorescein Isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., cy7, alexa 750)), chemiluminescent substances (e.g., acridinium compounds), colloidal gold, latex particles, and the like, and biotin for binding the above-described label-modified avidin (e.g., streptavidin). The markers encompassed by the present invention can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or scintillation calculators, and fluorescent labels can be detected using photodetectors to detect the emitted light. Enzyme labels are typically detected by providing a substrate for the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate. Calorimetric markers are detected by simply visualizing a colored marker. Chemiluminescent substances, such as acridinium compounds, are commonly used to detect emission by providing the substance with an excitation liquid and/or catalystOf (2) is detected. Biotin is generally detected by providing biotin with a label-modified avidin (e.g., streptavidin) as described above and detecting the label carried by the avidin linked to the biotin. In certain embodiments, a detectable label as described above can be attached to an antibody or antigen-binding fragment thereof of the invention via linkers of varying lengths to reduce potential steric hindrance.

In certain embodiments, the detectable label is selected from the group consisting of an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters), a fluorescent dye, biotin, colloidal gold, a latex particle.

In certain embodiments, the kit may further comprise reagents for allowing the detection of the corresponding detectable label. For example, when the detectable label is an enzyme, the kit may further comprise a chromogenic substrate for the corresponding enzyme, such as o-phenylenediamine (OPD), tetramethylbenzidine (TMB), ABTS or luminol-like compounds for horseradish peroxidase, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase. For example, when the detectable label is a chemiluminescent reagent (e.g., an acridinium ester compound), the kit may further comprise a pre-excitation liquid and/or an excitation liquid for chemiluminescence.

In certain embodiments, the third antibody is specific for an antibody of the species (e.g., mouse) from which the constant region comprised by the second antibody is derived. In certain embodiments, the third antibody is an anti-immunoglobulin antibody, such as an anti-IgG antibody. In certain embodiments, the third antibody is an anti-murine IgG1 antibody.

In certain embodiments, the kit further comprises a solid support. In certain embodiments, the solid support comprises a welled plate, tube, bead (e.g., latex particle) or film (e.g., nitrocellulose membrane) made of or coated with a polymeric material (e.g., polyvinyl chloride, polystyrene, polyacrylamide, or cellulose), or magnetic beads pre-coated with a functional group (e.g., amino, carboxyl, biotin, or avidin). In certain embodiments, the solid support is selected from the group consisting of magnetic beads, microtiter plates (e.g., microwell plates or microtiter plates), chromatographic membranes.

In certain embodiments, the kit further comprises a coating reagent, such as a coating buffer (e.g., a carbonate buffer, a phosphate buffer, a Tris-HCl buffer, or a borate buffer), for coating the first antibody onto the solid support. Methods for coating proteins or polypeptides onto solid phase supports are well known in the art, e.g., physical adsorption, covalent coupling via aminated or carboxylated surfaces, or mediated binding via avidin-biotin systems, polylysine pre-coated surfaces, protein A or protein G pre-coated surfaces.

In certain embodiments, the first antibody is coated on the surface of a solid support.

In certain embodiments, the kit comprises at least the above-described solid support, and the above-described first antibody, in separate containers or in separate compartments of a single container unit.

In certain exemplary embodiments, the kit comprises: a primary antibody, and a secondary antibody with a detectable label. In certain exemplary embodiments, the kit comprises: a first antibody coated on the surface of the solid phase carrier, and a second antibody with a detectable label.

Detection method and application

In a fourth aspect, the present invention provides a method of detecting the presence or level of a TSP-1 polypeptide in a sample, comprising the steps of:

(1) Contacting the sample with a first antibody to form an antibody-antigen complex;

(2) Contacting the antibody-antigen complex with a second antibody to form an antibody-antigen-antibody complex; and

(3) Determining the amount of the antibody-antigen-antibody complex;

wherein the first antibody is the antibody of the first aspect or an antigen-binding fragment thereof and the second antibody is the antibody of the second aspect or an antigen-binding fragment thereof; alternatively, the first antibody is the antibody or antigen-binding fragment thereof of the second aspect, and the second antibody is the antibody or antigen-binding fragment thereof of the first aspect.

In certain embodiments, the TSP-1 polypeptide is a specific N-terminal fragment of TSP1 protein having an amino acid sequence shown in SEQ ID NO. 17.

The method may be used for diagnostic purposes, or for non-diagnostic purposes. In certain embodiments, the methods of the invention are used for non-diagnostic purposes. For example, since the sample to be tested is known to contain the TSP-1 polypeptide, that is, the same subject of the sample has had a diagnostic result prior to being tested using the method of the invention; thus, the method of the invention does not contribute to the diagnostic procedure of the sample. It follows that the direct aim of the method of the invention is not to obtain a diagnostic result of the same subject of the sample, but to carry out a further accurate quantitative determination of the sample for which diagnostic information is known.

In some embodiments, the second antibody is detectably labeled. In certain embodiments, the assay described in step (3) comprises the steps of: (3 a) detecting the amount of detectable label; (3b) Comparing the amount of detectable label obtained in step (3 a) with a standard curve representing the relationship between the known amount of TSP-1 polypeptide fragment and the amount of detectable label, and obtaining the amount of TSP-1 polypeptide. In certain embodiments, the assay described in step (3) comprises the steps of: (3 a) detecting the amount of the detectable label (e.g., a luminescence value); (3b) Comparing the amount of detectable label (e.g., luminescence) obtained in step (3 a) to the Cut off value, thereby determining whether the sample is positive for the TSP-1 polypeptide.

In other embodiments, the second antibody is not detectably labeled. In a class of embodiments, the assay described in step (3) comprises detecting the antibody-antigen-antibody complex using a third antibody with a detectable label. In certain embodiments, the third antibody is capable of specifically binding the second antibody (e.g., capable of specifically binding a constant region of the second antibody). In certain embodiments, the third antibody is specific for an antibody of the species (e.g., mouse) from which the constant region comprised by the second antibody is derived. In certain embodiments, the third antibody is an anti-immunoglobulin antibody, such as an anti-IgG antibody. In certain embodiments, the third antibody is an anti-murine IgG1 antibody. In certain embodiments, the assay described in step (3) may comprise the steps of: (3a) Contacting the antibody-antigen-antibody complex with a third antibody carrying a detectable label; (3 b) detecting the amount of detectable label; (3c) Comparing the amount of detectable label obtained in step (3 b) with a standard curve representing the relationship between the known amount of TSP-1 polypeptide and the amount of detectable label, and obtaining the amount of TSP-1 polypeptide. In certain embodiments, the assay described in step (3) comprises the steps of: (3a) Contacting the antibody-antigen-antibody complex with a third antibody bearing a detectable label; (3 b) detecting the amount (e.g., luminescence value) of the detectable label; (3c) Comparing the amount (e.g., luminescence) of detectable label obtained in step (3 b) to the Cut off value, thereby determining whether the sample is positive for the TSP-1 polypeptide.

In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters), a fluorescent dye, biotin, colloidal gold, or a latex particle.

In certain embodiments, in step (3), the assay is selected from an enzyme immunoassay, a chemiluminescent immunoassay, or an immunochromatography (e.g., immune colloidal gold or latex immunochromatography).

In certain embodiments, the first antibody is coated on the surface of a solid support. In certain embodiments, the solid support is selected from the group consisting of magnetic beads, microtiter plates (e.g., microwell plates or microtiter plates), chromatographic membranes.

In certain embodiments, a washing step is further included prior to step (2) and/or step (3). The washing step may remove unreacted materials.

In certain embodiments, the sample is a blood sample (e.g., whole blood, plasma, or serum), a tissue sample (e.g., a tumor tissue sample), or urine.

In another aspect, the invention also relates to the use of an antibody or antigen-binding fragment thereof of the first aspect, an antibody or antigen-binding fragment thereof of the second aspect, or a kit of the third aspect, in the preparation of a detection reagent for detecting the presence or level of a TSP-1 polypeptide in a sample.

Earlier studies have found that the expression level of the specific N-terminal fragment of TSP1 protein is positively correlated with the risk of esophageal cancer (see Chinese patent application CN 201510947274.6), therefore, the reagent and the method for detecting TSP-1 polypeptide provided by the invention can be used for evaluating the risk of esophageal cancer or the disease of esophageal atypical hyperplasia or diagnosing esophageal cancer. Thus, the invention also provides the use of an antibody according to the first or second aspect or a kit according to the third aspect for the diagnosis of esophageal cancer or esophageal dysplasia.

In a fifth aspect, the present invention also relates to a method of assessing the risk of or diagnosing esophageal cancer or esophageal dysplasia, comprising detecting the presence or level of TSP-1 polypeptide in a sample from a subject using the method of the fourth aspect, and optionally assessing the risk of or diagnosing esophageal cancer or esophageal dysplasia based on the detection result.

In certain embodiments, the test result is compared to a reference value for a healthy population or a reference value for a patient with benign esophageal disease (e.g., esophagitis), and when the test result for the test sample is up-regulated, the subject is an esophageal cancer or esophageal dysplasia patient or at high risk of having esophageal cancer or esophageal dysplasia.

In certain embodiments, the test result is compared to a reference value for a patient with esophageal dysplasia, and when the test result for the test sample is up-regulated, the subject is indicated as a patient with esophageal cancer or as having esophageal cancer at high risk.

In certain embodiments, the esophageal cancer is Esophageal Squamous Cell Carcinoma (ESCC).

In certain embodiments, the esophageal dysplasia comprises mild dysplasia, moderate dysplasia and/or severe dysplasia. In certain embodiments, the esophageal dysplasia is severe dysplasia.

In certain embodiments, the subject is a human.

In another aspect, the present invention also relates to the use of the antibody or antigen-binding fragment thereof according to the first aspect, the antibody or antigen-binding fragment thereof according to the second aspect, or the kit according to the third aspect, in the preparation of a detection reagent for assessing the risk of or diagnosing esophageal cancer or esophageal dysplasia.

Definition of terms

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, virological, biochemical, immunological laboratory procedures used herein are all routine procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, the terms "TSP-1 specific peptide" and "TSP1 protein specific N-terminal fragment" have the same meaning and are used interchangeably, and refer to the amino acid sequence shown in SEQ ID NO. 17, which peptide can be used as a biomarker for esophageal cancer, as described in Chinese patent application CN201510947274.6.

As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one Light Chain (LC) and one Heavy Chain (HC). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, igD, igG, igA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains are not directly involved in the binding of antibodies to antigensAnd exhibit a variety of effector functions, such as mediating binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The VH and VL regions may also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each V _H And V _L By the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antigen-binding sites, respectively. The distribution of amino acids in each region or domain may follow Kabat, sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda, md. (1987 and 1991)), or Chothia&Lesk (1987) J.mol.biol.196:901-917; chothia et al (1989) Nature 342, 878-883.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs, designated CDR1, CDR2 and CDR3, in the variable regions of the heavy and light chains. The precise boundaries of these CDRs may be defined according to various numbering systems known in the art, such as those defined in the Kabat numbering system (Kabat et al, sequences of Proteins of Immunological Interest,5th Ed. Public Health service, national Institutes of Health, bethesda, md., 1991), chothia numbering system (Chothia & Lesk (1987) J.mol.biol.196:901-917, chothia et al (1989) Nature 342. For a given antibody, one skilled in the art will readily identify the CDRs as defined by each numbering system. Also, the correspondence between the different numbering systems is well known to those skilled in the art (see, e.g., lefranc et al, dev. Complex. Immunol.27:55-77, 2003).

In the present invention, the CDRs contained in the antibodies of the present invention or antigen binding fragments thereof can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained by the antibodies or antigen binding fragments thereof of the present invention are preferably determined by the Kabat, chothia, or IMGT numbering system. In certain embodiments, the CDRs contained by the antibodies or antigen-binding fragments thereof of the present invention are preferably determined by the IMGT numbering system.

The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies can be of different isotypes, e.g., igG (e.g., igG1, igG2, igG3, or IgG4 subtypes), igA1, igA2, igD, igE, or IgM antibodies.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, fab ', F (ab') ₂ Fd, fv, complementarity Determining Region (CDR) fragments, scFv, diabodies (diabodies), single domain antibodies (single domain antibodies), chimeric antibodies, linear antibodies (linear antibodies), nanobodies (technical from Domantis), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding capability on the polypeptide.

As used herein, the term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al, nature 341 544 546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F (ab') ₂ Fragment "means an antibody fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; the term "Fab 'fragment" means a reductively linked F (ab') ₂ The fragment obtained after disulfide bonding of the two heavy chain fragments in the fragment consists of one complete Fd fragment of the light and heavy chains, consisting of VH and CH1 domains.

As used herein, the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that form an entire antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity on the antibody. However, even one variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) is capable of recognizing and binding an antigen, although its affinity may be lower than the entire binding site.

As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker (linker). Such scFv molecules can have the general structure: NH ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a peptide having an amino acid sequence (GGGGS) ₄ But variants thereof may also be used. In some cases, a disulfide bond may also be present between the VH and VL of the scFv.

As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but that a linker is used that is too short to allow pairing between the two domains of the same chain, thereby forcing the domains to pair with the complementary domains of the other chain and create two antigen binding sites (see, e.g., holliger p. Et al, proc.natl.acad.sci.usa.90: 6444-6448 (1993), and Poljak r.j. Et al, strucure 2 1121-1123 (1994)).

As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen to which the full-length antibody binds. Single domain antibodies are also known as nanobodies (nanobodies).

Each of the above antibody fragments retains the ability to specifically bind to the same antigen to which the full length antibody binds, and/or competes with the full length antibody for specific binding to the antigen.

Antigen-binding fragments of antibodies (e.g., antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of antibodies are specifically screened for specificity in the same manner as for intact antibodies.

Herein, when the term "antibody" is referred to, it includes not only intact antibodies, but also antigen-binding fragments of antibodies, unless the context clearly indicates otherwise.

As used herein, the term "Chimeric antibody" refers to an antibody in which a portion of the light chain or/and heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass) and another portion of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which nevertheless retains binding activity to an antigen of interest.

As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered to have an amino acid sequence modified to increase homology to the sequence of a human antibody. Generally, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FR and/or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies generally retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, and the like. The donor antibody can be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody having a desired property (e.g., antigen specificity, affinity, reactivity, etc.).

The chimeric antibody or humanized antibody of the present invention can be prepared based on the sequence of a murine monoclonal antibody. DNA encoding the heavy and light chains can be obtained from a murine hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques.

As used herein, the term "immunological detection" refers to an assay that utilizes specific antigen-antibody interactions/binding affinities, which are generally useful for detecting the presence or level of a particular antigen or antibody in a sample. Such immunological assays are well known to those skilled in the art and include, but are not limited to, enzyme Immunoassay (EIA), chemiluminescence immunoassay (CLIA), immunochromatography, radioimmunoassay (RIA), fluorescence Immunoassay (FIA), western blotting, immunoturbidimetry, surface plasmon resonance, and the like.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection such that the genetic material element it carries is expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs), or artificial chromosomes of P1 origin (PACs); bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), pox viruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication origin.

As used herein, the term "host cell" refers to a cell that can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells, or human cells.

As used herein, the term "identity" is used to refer to the match of sequence between two polypeptides or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of the total 6 positions match). Typically, the comparison is made when the two sequences are aligned to yield maximum identity. Such alignments can be performed by using, for example, needleman et al (1970) j.mol.biol.48: 443-453. The algorithm of e.meyers and w.miller (comput.appl biosci.,4, 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0) can also be used to determine percent identity between two amino acid sequences using a PAM120 weight residue table (weight residue table), a gap length penalty of 12, and a gap penalty of 4. In addition, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48:444-453 (1970)) algorithms that have been incorporated into the GAP program of the GCG software package (available at www. GCG. Com), using either the Blossum 62 matrix or the PAM250 matrix, and GAP weights (GAP weights) of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., of similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family.

The twenty conventional amino acids referred to herein are written following conventional usage. Amino acids are generally indicated by the single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "subject" includes, but is not limited to, various animals, particularly mammals, such as humans.

Advantageous effects

The invention provides a monoclonal antibody pair particularly suitable for detecting a specific fragment of TSP1 protein and an established double-antibody sandwich method based on the antibody pair. The technical scheme of the invention has good detection sensitivity, can realize rapid high-throughput detection of the specific N-terminal fragment of the TSP1 protein in the sample, thereby realizing early screening of esophageal cancer and having great clinical application value.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Drawings

FIG. 1 shows a standard curve for a double antibody sandwich ELISA detection system.

FIG. 2 shows a standard curve for a chemiluminescent detection system.

FIG. 3 shows the results of plasma samples from esophageal cancer patients and healthy controls using a double antibody sandwich ELISA assay system.

FIG. 4 shows the results of a chemiluminescence system blind assay of plasma samples from healthy controls, benign esophageal disease, precancerous esophageal lesions (atypical hyperplasia), and patients with esophageal cancer.

FIG. 5A shows ROC curves for TSP-1 specific polypeptides between esophageal cancer patients and healthy controls in a chemiluminescence detection system.

FIG. 5B shows ROC curves for TSP-1 specific polypeptides between esophageal precancerous patients and healthy controls under a chemiluminescence detection system.

Sequence information

Information on the sequences to which the present application relates is described in the following table.

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, the molecular biological experimental methods and immunoassay methods used in the present invention are essentially described by reference to j.sambrook et al, molecular cloning: a laboratory manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, eds. Molecular biology laboratory Manual, 3 rd edition, john Wiley & Sons, inc., 1995; the use of restriction enzymes follows the conditions recommended by the product manufacturer. Reagents of which sources are not indicated in the examples are conventional in the art or commercially available. It will be appreciated by those skilled in the art that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.

Example 1: preparation of TSP-1 peptide N-terminal and C-terminal monoclonal antibody

1.1 immunization of mice

The C-terminal fragment and N-terminal fragment of TSP-1 specific polypeptide (SEQ ID NO: 17) were chemically synthesized and linked to carrier proteins KLH, BSA as immunogens. Balb/C mice were immunized and purchased from Beijing Huafukang Biotech GmbH. Mice were immunized with complete adjuvant and appropriate amount of antigen for the first immunization, and then were immunized with incomplete adjuvant and appropriate amount of antigen every other week. After the 3 rd and 4 th immunizations, the eye sockets of the mice are bled, antiserum is obtained through centrifugation, the antibody titer of the serum of the mice is determined through an ELISA method, and the mice with the optimal titer are selected to perform a cell fusion experiment to prepare the monoclonal antibody.

1.2 determination of antibody Titers

TSP-1 specific polypeptides were diluted to 2ug/ml with PBS, pipetted 100ul into 96-well plates using a sample gun, and incubated overnight at 4 ℃. After completion of incubation, excess PBS in the 96-well plate was discarded, 100ul of PBS containing 1% casein was added to the 96-well plate, and incubation was performed at 37 ℃ for 2 hours. 100ul of hybridoma cell culture supernatant, sp2/0 cell culture supernatant, (PBS) and 1000-fold diluted positive serum are sucked by a sample adding gun, respectively added into a 96-well plate coated with TSP1-BSA, and incubated for 2h at 37 ℃. After three washes, secondary antibody was added and incubated at 37 ℃ for 1h. Washing for three times, patting dry the ELISA plate, adding 100ul TMB developing solution, incubating for 15min at room temperature, adding stop solution to stop developing, and reading the absorbance value at 450nm by an enzyme-linked immunosorbent assay (ELISA) instrument.

1.3 Screening for antibody pairings

All monoclonal antibodies to be tested were diluted to 4ug/ml with PBS, pipetted 100ul into a 96-well plate using a sample gun, and the 96-well plate was placed in a 4 ℃ cold room for overnight incubation. After the incubation, excess PBS in the 96-well plate was discarded, 100ul PBS containing 1% casein was added to the 96-well plate, and the 96-well plate was incubated in a 37 ℃ incubator for 2 hours. Adding TSP-1 specific polypeptide standard substances with different concentrations, incubating at 37 ℃ for 1h, washing three times after reaction, adding an HRP (or ALP) labeled antibody (labeled by a Label Easy quick HRP or ALP coupling kit) for resisting the TSP-1 specific polypeptide, incubating at 37 ℃ for 1h, washing three times, adding TMB, incubating at room temperature for 15min, adding a stop solution, carrying out color comparison at 450-630nm, and selecting the optimal antibody pairing combination. The selected optimal pair is applied to an ELISA kit and a magnetic particle chemiluminescence kit.

Finally, optimally matched monoclonal antibodies specifically binding with the TSP-1 specific polypeptide N end (hereinafter referred to as N-end antibodies) and monoclonal antibodies specifically binding with the C end (hereinafter referred to as C-end antibodies) are obtained by screening. Furthermore, when a TSP-1 fragment extending one or more residues further on the C-terminus of SEQ ID NO:17 was used as the coating antigen, NO binding of the C-terminal antibody was detected, indicating the specificity of the C-terminal antibody for a TSP-1 specific polypeptide (SEQ ID NO: 17).

According to the IMGT numbering system, the CDR sequences of the N-terminal antibody are shown as SEQ ID NOs:11-16, and VH and VL are shown as SEQ ID NO:9 and 10, respectively; the CDR sequences of the C-terminal antibody are shown in SEQ ID NOs:3-8, and VH and VL are shown in SEQ ID NO:1 and 2, respectively.

Example 2: subtype identification

The Ig capture antibody was diluted to 0.5ug/ml using PBS, pipetted 100ul with a sample gun into a 96 well plate and incubated overnight at 4 ℃. After completion of incubation, excess PBS in the 96-well plate was discarded, 100ul of PBS containing 1% casein was added to the 96-well plate, and incubation was performed at 37 ℃ for 2 hours. 100ul of hybridoma culture supernatant was aspirated by a sample gun and incubated for 2h at 37 ℃ in an incubator. After the reaction, the 96-well plate was washed three times with a plate washer, 10000 times kappa,/lambda chain or 20000 HRP-labeled antibody diluted with PBS was added thereto, 100ul, and incubated at 37 ℃ for 1 hour. Washing for three times, adding 100ul TMB developing solution by using a sample adding gun, incubating for 15min at room temperature, adding 50ul stop solution to stop developing, and reading the absorbance value at 450nm by using an enzyme-linked immunosorbent assay (ELISA) instrument. The results show that the N-terminal antibody is IgG1, and the light chain is lambda; the C-terminal antibody is IgG2a, and the light chain is kappa.

Example 3: double-antibody sandwich ELISA detection system

Experimental reagent:

the buffer solution comprises: the coating buffer was 50mM PBS, pH 7.6; the blocking buffer solution is PBS buffer solution added with 10% blocking proteins such as BSA, OVA, casein and the like; the reaction buffer solution is a closed buffer solution, and a stable buffer solution is added into the closed buffer solution, and has a protection effect on a detection object in a sample; the washing buffer was TBS buffer.

Coating antibody: c-terminal monoclonal antibody obtained in example 1

Detecting an antibody: n-terminal monoclonal antibody obtained in example 1

The reaction steps are as follows:

1) A96-well ELISA plate was coated, 100. Mu.l of the coating antibody was added at a concentration of 6ug/ml, the plate was filled with a coating buffer, and the plate was sealed with a sealing plate and allowed to react overnight at 4 ℃. Washed three times with wash buffer.

2) Add 200. Mu.l/well blocking buffer and incubate for 3 hours at 37 ℃. Wash three times with wash buffer.

3) To each well was added 50. Mu.l of a sample to be tested (including: TSP-1 specific polypeptide standard, serum or plasma, cell culture supernatant, etc.). Then, 50. Mu.l/well of the reaction buffer was added thereto and mixed well. Incubate at 37 ℃ for 1 hour. Wash three times with wash buffer.

4) 100 μ l/well of HRP conjugated detection antibody was added at a concentration of 0.3ug/ml. Incubate at 37 ℃ for 1 hour. Washed three times with wash buffer.

5) 100. Mu.l/well of TMB developing solution was added thereto, and the mixture was developed in the dark at room temperature for 30 minutes. Then 100. Mu.l/well of stop buffer was added and mixed well.

6) OD (450-630 nm) was read with a microplate reader.

A typical standard curve for the detection of TSP-1-specific polypeptide standards according to the ELISA method described above is depicted in FIG. 1, and the assay can be used to quantitatively determine concentrations in plasma or serum of TSP-1-specific polypeptides above 30pg/ml (functional sensitivity).

Example 4: chemiluminescent detection system

Magnetic beads (Magnosphere MS160/Carboxyl from JSR) and a monoclonal antibody against FITC were coupled, this being reagent M; labeling FITC by using the C-terminal antibody of example 1 as a coating antibody, and after removing free FITC, determining the antibody labeling rate and the concentration of a labeled antibody, namely R1 reagent; after the N-terminal antibody of example 1 was labeled with acridinium ester or alkaline phosphatase as a detection antibody, the free acridinium ester or free alkaline phosphatase was removed, the concentration and labeling rate of the labeled antibody were measured, and a stabilizer, which was an R2 reagent, was added.

Mixing and incubating a sample to be detected, R1, R2 and an M reagent, and combining a Fluorescein (FITC) -labeled C-terminal antibody in the R1 and an alkaline phosphatase or acridinium ester-labeled N-terminal antibody in the R2 with an antigen molecule (TSP-1 specific peptide fragment) in the sample to form an immune complex. The immune complex is further captured by an anti-FITC antibody coupled on the surface of the magnetic particle and adsorbed on the surface of the magnetic particle. Washing, removing unbound material, adding luminescent substrate, and measuring relative luminescence intensity (RLU) with alkaline phosphatase enzyme substrate or acridinium ester. The RLU is in direct proportion to the concentration of the antigen molecules in a certain range, and the concentration of the antigen molecules of the sample to be detected can be read from the standard curve by an interpolation method.

A typical standard curve for the detection of TSP-1 specific polypeptide standards by the above described chemiluminescent method is depicted in FIG. 2, and the use of this assay allows the quantification of TSP-1 specific polypeptide immunoreactivity concentrations in plasma or serum above 1pg/ml (functional sensitivity).

Example 5: ELISA detection system and application of chemiluminescence detection system in esophageal cancer diagnosis

5.1ELISA detection System

The ELISA detection system of example 3 is used for detecting plasma samples of 15 esophageal cancer patients and 4 healthy controls, the detection result is shown in figure 3, the esophageal cancer and the healthy controls have significant difference, and the level of TSP-1 specific peptide fragment can be used for distinguishing the esophageal cancer from the healthy controls.

5.2 chemiluminescent detection System

The chemiluminescence detection system of example 4 was used to blindly detect TSP-1-specific peptides from plasma samples of 15 Healthy Controls (HC), 10 patients with Mild and Moderate esophagitis (esophagitis), 19 patients with esophageal squamous carcinoma and 30 patients with esophageal dysplasia (epithelial hyperplasia) (i.e., the samples were not known to the examiner), 10 cases with Mild dysplasia (milled epithelial hyperplasia), 10 cases with Moderate dysplasia (model epithelial hyperplasia) and 10 cases with Severe dysplasia (Severe epithelial hyperplasia). As shown in FIGS. 4-5, the median of the TSP-1 specific peptides in healthy control plasma was 404.89pg/ml, the median of the TSP-1 specific peptides in esophagitis patients plasma was 430.57pg/ml, the median of the TSP-1 specific peptides in light, medium and severe esophageal dysplasia patients plasma was 898.33pg/ml,1142.76pg/ml and 602.16ppg/ml, and the median of the TSP-1 specific peptides in esophageal squamous carcinoma patients plasma was 2475.37pg/ml. Compared with healthy control, the plasma TSP-1 specific peptide fragment level of the esophageal cancer patient is obviously increased (P < 0.0001). The plasma TSP-1 specific peptide fragment level of the esophageal cancer patient is obviously higher than that of benign esophagus diseases (esophagitis) and severe atypical hyperplasia (P < 0.05). The results show that the level of the TSP-1 specific peptide fragment can be used for distinguishing esophageal cancer or atypical hyperplasia from healthy control or esophageal benign diseases, and further indicate that the level of the TSP-1 specific peptide fragment is an early diagnosis marker of esophageal cancer.

The diagnostic efficacy of plasma TSP-1 on esophageal squamous carcinoma and esophageal dysplasia is analyzed by using a Receiver Operating Curve (ROC), and the result shows that the area under the ROC curve for diagnosing esophageal squamous carcinoma of the plasma TSP-1 specific peptide fragment level is 0.93 and the area under the ROC curve for diagnosing esophageal dysplasia is 0.73. 845.7pg/ml is selected as a Cut off value according to the John's index, the sensitivity and the specificity of the TSP-1 polypeptide fragment to the diagnosis of esophageal squamous carcinoma are respectively 86.7 percent and 89.5 percent, and the sensitivity and the specificity to the diagnosis of esophageal dysplasia are respectively 86.7 percent and 50.0 percent under the condition, and the results show that the plasma TSP-1 can be used as a biomarker of esophageal cancer and esophageal precancerous lesion (esophageal dysplasia) and is suitable for esophageal cancer screening and early diagnosis.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.

Claims (23)

1. An antibody or antigen-binding fragment thereof that specifically binds a thrombospondin-1 (TSP-1) polypeptide, comprising: a heavy chain variable region (VH) comprising the following 3 Complementarity Determining Regions (CDRs): HCDR1 of sequence SEQ ID NO. 3, HCDR2 of sequence SEQ ID NO. 4, and HCDR3 of sequence SEQ ID NO. 5; and/or, a light chain variable region (VL) comprising the following 3 Complementarity Determining Regions (CDRs): LCDR1 with a sequence of SEQ ID NO. 6, LCDR2 with a sequence of SEQ ID NO. 7and LCDR3 with a sequence of SEQ ID NO. 8.

2. The antibody or antigen-binding fragment thereof of claim 1, comprising: a VH comprising the sequence set forth in SEQ ID No. 1 or a variant thereof; and/or a VL comprising the sequence shown in SEQ ID NO. 2 or a variant thereof;

wherein the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; preferably, the substitutions are conservative substitutions;

preferably, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown as SEQ ID NO. 1 and a VL comprising the sequence shown as SEQ ID NO. 2.

3. An antibody or antigen-binding fragment thereof that specifically binds a thrombospondin-1 (TSP-1) polypeptide, comprising: a heavy chain variable region (VH) comprising the following 3 Complementarity Determining Regions (CDRs): HCDR1 of sequence SEQ ID NO. 11, HCDR2 of sequence SEQ ID NO. 12, and HCDR3 of sequence SEQ ID NO. 13; and/or, a light chain variable region (VL) comprising the following 3 Complementarity Determining Regions (CDRs): LCDR1 with sequence SEQ ID NO. 14, LCDR2 with sequence SEQ ID NO. 15, and LCDR3 with sequence SEQ ID NO. 16.

4. The antibody or antigen-binding fragment thereof of claim 3, comprising: a VH comprising the sequence set forth in SEQ ID No. 9 or a variant thereof; and/or, a VL comprising the sequence shown as SEQ ID NO. 10 or a variant thereof;

wherein the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; preferably, the substitutions are conservative substitutions;

preferably, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown as SEQ ID NO. 9 and a VL comprising the sequence shown as SEQ ID NO. 10.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof comprises a constant region;

preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from an immunoglobulin (e.g., an IgG, e.g., an IgG1, an IgG2, an IgG3, or an IgG 4) and the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from an immunoglobulin (e.g., a κ or λ).

6. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, fab ', (Fab') ₂ Fv, disulfide-linked Fv, scFv, diabody (diabody), murine, chimeric, humanized, bispecific, or multispecific antibody.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof is detectably labeled;

preferably, the detectable label is selected from the group consisting of an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters), a fluorescent dye, biotin, colloidal gold, or a latex particle.

8. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-7.

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

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

11. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-7, comprising culturing the host cell of claim 10 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

12. A kit, comprising:

(i) A first antibody selected from the antibody of claim 1 or 2, or an antigen-binding fragment thereof; and the combination of (a) and (b),

(ii) A second antibody selected from the antibodies of claims 3 or 4 or antigen binding fragments thereof.

13. The kit of claim 12, wherein the first and/or second antibody comprises a constant region;

preferably, the heavy chain of the first and/or second antibody comprises a heavy chain constant region derived from an immunoglobulin (e.g., an IgG, e.g., igG1, igG2, igG3 or IgG 4) and the light chain of the first and/or second antibody comprises a light chain constant region derived from an immunoglobulin (e.g., κ or λ).

14. The kit of claim 12 or 13, wherein the second antibody is detectably labeled, or further comprising a third antibody capable of specifically binding the second antibody, the third antibody being detectably labeled;

preferably, the detectable label is selected from the group consisting of an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters), a fluorescent dye, biotin, colloidal gold, or a latex particle.

15. The kit of any one of claims 12-14, wherein the kit further comprises a solid support;

preferably, the solid support is selected from a magnetic bead, a microtiter plate (e.g., a microplate or an elisa plate), or a chromatographic membrane;

preferably, the first antibody is coated on the surface of a solid support.

16. A method of detecting the presence or level of a TSP-1 polypeptide in a sample, comprising the steps of:

(1) Contacting the sample with a first antibody to form an antibody-antigen complex;

(2) Contacting the antibody-antigen complex with a second antibody to form an antibody-antigen-antibody complex; and

(3) Determining the amount of the antibody-antigen-antibody complex;

wherein the first antibody is the antibody or antigen-binding fragment thereof of claim 1 or 2 and the second antibody is the antibody or antigen-binding fragment thereof of claim 3 or 4; alternatively, the first antibody is the antibody or antigen-binding fragment thereof of claim 3 or 4, and the second antibody is the antibody or antigen-binding fragment thereof of claim 1 or 2;

preferably, the TSP-1 polypeptide is shown as SEQ ID NO. 17.

17. The method of claim 16, wherein the second antibody is detectably labeled; alternatively, the assay described in step (3) comprises the use of a third antibody with a detectable label;

preferably, the detectable label is selected from the group consisting of an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters), a fluorescent dye, biotin, colloidal gold, or a latex particle.

18. The method of claim 16 or 17, wherein in step (3) the assay is selected from an enzyme immunoassay, a chemiluminescent immunoassay, or an immunochromatography (such as immuno-colloidal gold or latex immunochromatography).

19. The method of any one of claims 16-18, wherein the first antibody is coated on the surface of a solid support;

preferably, the solid support is selected from the group consisting of magnetic beads, microtiter plates (e.g., microtiter plates or microtiter plates), or chromatographic membranes.

20. The method of any one of claims 16-19, wherein the first antibody is the antibody or antigen-binding fragment thereof of claim 1 or 2 and the second antibody is the antibody or antigen-binding fragment thereof of claim 3 or 4.

21. The method of any one of claims 16-20, wherein the sample is a blood sample (e.g., whole blood, plasma, or serum), a tissue sample (e.g., a tumor tissue sample), or urine.

22. Use of an antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 7 or a kit as claimed in any one of claims 12 to 15 in the manufacture of a test reagent for detecting the presence or level of a TSP-1 polypeptide in a sample and/or for assessing the risk of, or diagnosing, esophageal cancer or esophageal dysplasia.

23. The use of claim 22, wherein the sample is a blood sample (e.g., whole blood, plasma, or serum), a tissue sample (e.g., a tumor tissue sample), or urine from a subject (e.g., a human).

Images