[go: up one dir, main page]

WO2022007283A1 - Kit et méthode de diagnostic d'une maladie associée à une expression de fap anormale, et support de stockage lisible par ordinateur - Google Patents

Kit et méthode de diagnostic d'une maladie associée à une expression de fap anormale, et support de stockage lisible par ordinateur Download PDF

Info

Publication number
WO2022007283A1
WO2022007283A1 PCT/CN2020/127378 CN2020127378W WO2022007283A1 WO 2022007283 A1 WO2022007283 A1 WO 2022007283A1 CN 2020127378 W CN2020127378 W CN 2020127378W WO 2022007283 A1 WO2022007283 A1 WO 2022007283A1
Authority
WO
WIPO (PCT)
Prior art keywords
fap
protein
polypeptide
cancer
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2020/127378
Other languages
English (en)
Chinese (zh)
Inventor
苏金
李军旗
杨鹏辉
彭晓敏
边寰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen International Institute For Biomedical Research
Original Assignee
Shenzhen International Institute For Biomedical Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen International Institute For Biomedical Research filed Critical Shenzhen International Institute For Biomedical Research
Publication of WO2022007283A1 publication Critical patent/WO2022007283A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/548—Carbohydrates, e.g. dextran
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/20—Dermatological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction

Definitions

  • the present invention relates to diagnostic kits, diagnostic methods and computer-readable media for diseases related to abnormal FAP expression, more particularly, to diagnosing diseases related to abnormal FAP expression by detecting the presence of FAP autoantibodies.
  • Fibroblast activation protein also known as Seprase, has a monomer of 97kDa and requires dimerization for enzymatic activity.
  • FAP is a membrane-integrated serine peptidase that, along with dipeptidyl peptidase IV (DPPIV, also known as CD26), a closely related cell surface enzyme, and other peptidases, belongs to the dipeptidyl peptidase IV family .
  • DPPIV dipeptidyl peptidase IV
  • CD26 dipeptidyl peptidase IV
  • FAP contains two N-glycosylated subunits with a large C-terminal extracellular domain, in which the catalytic domain of the enzyme is located. In its glycosylated form, FAP has dipeptidyl peptidase and endopeptidase activities and can cleave gelatin and type I collagen.
  • Human FAP exists on cells as homodimers containing 760 amino acid residues per monomer, including: a short cytoplasmic tail of only 6 amino acids, a single transmembrane domain of 20 amino acids, and Extracellular domain of 734 amino acids. Crystal structure determination revealed that the extracellular domain of FAP contains two domains: an alpha/beta hydrolase domain (residues 27-53 and 493-760) and an eight-lobed beta-propeller domain (residues 54-492) ), they enclose a diameter of approx. large cavity.
  • residues Ala657, Asn704, Arg123, Glu203 and Glu204 are all required for FAP catalytic activity. can be opened through the side Access to this cavity allows only elongated peptides or unfolded or partially unfolded protein fragments to reach the active site cavity.
  • FAP contains six potential N-linked glycosylation sites (motif Asn-X-Ser/Thr) at Asn residues 49, 92, 99, 227, 314 and 679. Most of these sites are located on the ⁇ -propeller surface, and only one is on the hydrolase domain, close to the cell surface. In baculovirus-expressed soluble human FAP, all amino acids except Asn99 are glycosylated.
  • FAP has a unique tissue distribution and is expressed on reactive stromal fibroblasts in more than 90% of all primary and metastatic epithelial tumors, including lung, colorectal, bladder, ovarian, and breast cancers, among others. Highly upregulated and not normally present in normal adult tissues. It has been reported that FAP is expressed not only in mesenchymal fibroblasts, but also in certain types of malignant cells of epithelial origin, and that FAP expression is directly related to the malignant phenotype.
  • Autoantibodies are antibodies directed against one's own tissues, organs, cells and cellular components. The growth, development and survival of the human body are maintained by a complete autoimmune tolerance mechanism. The normal immune response has a protective defense effect, that is, it does not react to its own tissues and components. Once the integrity of self-tolerance is destroyed, the body regards its own tissues and components as "foreign bodies", and an autoimmune reaction occurs and autoantibodies are produced. There can be low titers of autoantibodies in normal human blood, but no disease will occur, but if the titer of autoantibodies exceeds a certain level, it may cause damage to the body and induce disease.
  • the level of autoantibodies in the body is significantly increased, it indicates that there is an abnormal immune situation in the body, indicating that the level of related antigens in the body fluctuates, indicating the existence of a disease or an original disease. aggravated.
  • the main advantages of using autoantibodies as disease markers are: strong specificity, high sensitivity, simple operation, good repeatability, fast detection speed, and low false positive rate.
  • the level of autoantibodies in serum can be used to monitor different stages of the disease, and early diagnosis of the disease can be made, which is beneficial to the early treatment of the disease and improves the prognosis of patients.
  • the inventors of the present application have unexpectedly found that FAP autoantibodies exist in samples from patients with diseases related to abnormal FAP expression, while FAP autoantibodies cannot be detected in normal samples.
  • the inventors detected the presence of autoantibodies using proteins or polypeptides that can bind to FAP antibodies, thereby diagnosing diseases related to abnormal FAP expression.
  • one aspect of the present invention provides a kit for diagnosing a disease associated with abnormal FAP expression, the kit comprising a protein or polypeptide capable of binding to a fibroblast activating protein (FAP) antibody.
  • FAP fibroblast activating protein
  • Another aspect of the present invention provides the use of a protein or polypeptide capable of binding to a fibroblast activating protein (FAP) antibody in the preparation of a kit for diagnosing diseases related to abnormal expression of FAP.
  • FAP fibroblast activating protein
  • Another aspect of the present invention provides a protein or polypeptide capable of binding to fibroblast activating protein (FAP) antibody for diagnosing diseases associated with abnormal expression of FAP.
  • FAP fibroblast activating protein
  • Another aspect of the present invention provides a method for diagnosing a disease associated with abnormal FAP expression in a subject, the method comprising contacting the protein or polypeptide capable of binding to FAP autoantibodies of the present invention with a sample from the subject; detecting the presence of a complex of the protein or polypeptide and the FAP autoantibody in the contacted sample; and determining that the subject has or is at risk of having a disease associated with abnormal FAP expression based on the presence of the complex.
  • Another aspect of the present invention provides a computer-readable storage medium having stored thereon computer instructions for reading and execution by a computer, the computer instructions being executed to perform a method for detecting the presence or absence of FAP autoantibodies in a sample of a subject,
  • the method comprises: (a) contacting a sample of the subject with a protein or polypeptide capable of binding to a fibroblast activating protein (FAP) antibody; (b) detecting and reading a signal from the contacted sample to detect the protein and (c) determining whether the signal exceeds a predetermined threshold, and when the signal exceeds a predetermined threshold, determining the presence of FAP autoantibodies in the sample.
  • FAP fibroblast activating protein
  • Figure 1 Human FAP full-length sequence (SEQ ID NO: 1).
  • Figure 2 Human FAP ectodomain sequence (SEQ ID NO: 2).
  • Figure 3 Exemplary ectodomain sequence (SEQ ID NO: 22) according to the present invention.
  • Figure 4 Detection of recombinant proteins from the extracellular segment of eukaryotic expression of FAP. 293i eukaryotic expression of FAP extracellular segment recombinant protein was detected by SDS-PAGE (A) and WB (B). In A, 1: Marker, 2: 100ng recombinant protein, 3: 500ng recombinant protein. In B, 1: Marker, 2: 500ng recombinant protein.
  • Figure 5 Detection of serum FAP autoantibodies by ELISA. Using FAP extracellular segment recombinant protein as antigen, ELISA was used to detect autoantibodies in serum, and FAP commercial antibody was used as positive control, irrelevant antibody was used as negative control, and blank control was set. *, P ⁇ 0.05, ***, P ⁇ 0.001, ****, P ⁇ 0.0001.
  • FIG. 6 Immunofluorescence detection of serum FAP autoantibodies. Using FAP-overexpressing A549 cells as material, immunofluorescence was used to detect FAP autoantibodies in the serum to be tested, and commercial FAP antibodies were used as positive controls, and blank controls were set.
  • sequence eg, terms such as “amino acid sequence”, “antibody sequence”, “variable domain sequence”, or “protein sequence”
  • amino acid sequence eg., terms such as “amino acid sequence”, “antibody sequence”, “variable domain sequence”, or “protein sequence”
  • amino acid sequence is interpreted to mean a single amino acid or an unbranched sequence of two or more amino acids, depending on the context.
  • Nucleotide sequences are interpreted to mean unbranched sequences of 3 or more nucleotides.
  • protein/protein protein/protein
  • peptide polypeptide
  • polypeptide polypeptide
  • Each term refers to an organic compound consisting of a linear chain of two or more amino acids.
  • Compounds can have ten or more amino acids, twenty-five or more amino acids, fifty or more amino acids, one hundred or more amino acids, two hundred or more amino acids, and even three amino acids. Hundred or more amino acids.
  • polypeptides generally contain fewer amino acids than proteins; polypeptides can be prepared by chemical synthesis or recombinant methods; It is prepared in vitro or in vivo by known recombinant methods.
  • order of amide bonds in the primary structure of a polypeptide follows the amino acids as written, with the amino-terminus (N-terminus) of the polypeptide always on the left and the carboxy-terminus (C-terminus) on the right.
  • amino acid substitutions, deletions or insertions can be made to make conservative substitutions or changes in "non-essential" regions.
  • a polypeptide or amino acid sequence derived from a specified protein except for one or more individual amino acid substitutions, insertions, or deletions (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 The remainder may be identical to the starting sequence except for one or more single amino acid substitutions, insertions or deletions).
  • the polypeptide or amino acid sequence derived from a given protein has 1 to 5, 1 to 10, 1 to 15, or 1 to 20 individual amino acid substitutions, insertions or deletions relative to the starting sequence.
  • antibody refers to any form of antibody that exhibits a desired biological activity (eg, inhibition of binding of a ligand to its receptor or by inhibition of ligand-induced receptor signaling).
  • Antibody fragment and “antigen-binding fragment” refer to antigen-binding fragments of antibodies and antibody analogs, which typically include at least a portion of the antigen-binding or variable regions (eg, one or more CDRs) of the parent antibody.
  • Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, antibody fragments retain at least 10% of the parent binding activity when the activity is expressed on a molar basis.
  • the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody for the target.
  • antibody fragments include, but are not limited to: Fab, Fab', F(ab') 2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies; Fragmented multispecific antibodies.
  • FAP antibody refers to an antibody that specifically binds to FAP, including autoantibodies and engineered antibodies, and any form of antibody, such as antibody fragments and antigen-binding fragments as defined above.
  • autoantibody refers to an antibody that specifically binds to structures from the organism from which it is produced.
  • FAP autoantibody refers to an autoantibody that specifically binds to the FAP protein from which it is produced. Particularly preferably, it is a mammalian autoantibody, more preferably a human autoantibody, even more preferably a human autoantibody of the IgG class. Its variable domains are capable of specifically binding to self-antigens (eg FAP proteins).
  • FAP autoantibodies are polyclonal antibodies having distinct variable or complementarity determining regions that recognize one or more epitopes of FAP.
  • Antibodies are generally produced by the immune system after foreign proteins or other substances enter the body, and are used for immune responses to eliminate foreign harmful substances. Normally, the immune system recognizes and ignores the body's own cells without producing antibodies to them; at the same time, the immune system does not overreact to non-threatening substances in the environment. However, under certain circumstances, the immune system fails to recognize the body's own substances, and regards them as foreign invaders, thereby producing antibodies against these substances (ie, autoantibodies), triggering autoimmunity. There are many autoantibodies in autoimmune diseases.
  • epitope refers to a portion or segment of a molecule that is recognized by a binding agent.
  • epitopes are discrete three-dimensional sites on a molecule that are recognized by a binding agent.
  • Epitopes typically consist of chemically active surface groups of molecules (eg, amino acids or sugar side chains), and typically have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • Epitopes of a protein preferably comprise contiguous or discontinuous stretches of the protein, and are preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, most preferably 10 to 25 amino acids in length.
  • the epitope may preferably be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length.
  • Epitopes define the minimal binding site of immunoglobulins and thus represent specific targets of immunoglobulins.
  • a single antigenic molecule can have one or more different epitopes. Generally speaking, only epitopes located on the surface of antigenic substances are easily bound to antigen-recognition receptors or antibodies, which are called functional epitopes.
  • the epitopes located inside the molecule have no immunogenicity and are called hidden epitopes.
  • the epitope of the autoantigen in the present invention is located in the extracellular domain of FAP.
  • the term "binding” preferably relates to specific binding.
  • “Specifically binds” means that an agent binds more strongly to a specific target than to another target. If the agent binds to the first target with a dissociation constant (K D ) that is less than the dissociation constant for the second target, it binds more strongly to the first target than to the second target.
  • the agent does not specifically bind a target with a dissociation constant (K D) compared to the target specific binding reagent with a dissociation constant (K D) more than 102-fold, 103-fold, 104-fold, 10 5 times, 10 6 times, 10 7 times, 10 8 times, 10 9 times or 10 10 times less.
  • an agent eg, a protein or polypeptide
  • an agent is an all-purpose target if it is capable of binding to a predetermined target but not other targets, ie does not have significant affinity for and does not bind significantly to other targets in standard assays specific to the predetermined target.
  • an agent is specific for FAP or FAP autoantibody if it is able to bind to FAP or FAP autoantibody but is (substantially) unable to bind to other targets (eg DPP IV).
  • the agent binding to a predetermined target of K D is at least 102 times their non-specific target binding K D of 103 times, 104 times, 105 times, 106 times, 107 times, 108 times , 109-fold or 1010-fold lower, the reagent is specific for said target.
  • Binding of an agent to a target can be determined experimentally using any suitable method, which is within the purview of those skilled in the art. Affinity can be readily determined using conventional techniques, such as by equilibrium dialysis; by surface plasmon resonance analysis using general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigens; or by methods known to the skilled artisan Other methods. Affinity data can be analyzed, for example, by methods known in the art. The measured affinity of a particular interaction can vary if measured under different conditions (eg, salt concentration, pH). Thus, the binding affinity and other parameters (e.g., K D, IC 50) is preferably measured and standardized solution standardized buffer and binding agent for a target.
  • the binding affinity and other parameters e.g., K D, IC 50
  • enzyme-linked immunosorbent assay or ELISA refers to a method for quantitative or semi-quantitative determination of protein concentration in a sample (eg plasma, serum or cell/tissue extract) in a multi-well plate format (usually 96 wells per plate) . Briefly, proteins in solution were adsorbed onto ELISA plates. Plates can be probed with antibodies specific for the protein of interest. Background was minimized by optimizing blocking and washing methods, and specificity was ensured by the presence of positive and negative controls. Detection methods are usually based on colorimetry or chemiluminescence.
  • domain refers to a folded protein structure that has the ability to retain its tertiary structure independently of the rest of the protein. Typically, domains are responsible for discrete functional properties of proteins, and in many cases, can be added, removed, or transferred to other proteins without loss of function of the rest of the protein and/or domains.
  • extracellular domain/extracellular domain/extracellular segment refers to a segment of a molecule (eg a protein) which faces the extracellular space of a cell and is preferably accessible from the outside of said cell, eg Accessed by binding agents (eg, autoantibodies) located outside the cell.
  • binding agents eg, autoantibodies
  • the term refers to one or more extracellular loops, extracellular domains or fragments thereof.
  • the terms "subject” or "patient”, “individual” refer to any subject for which diagnosis, prognosis or treatment is desired, particularly mammalian subjects. Mammals include humans, domestic animals, farm animals, zoo animals, sports animals or pets such as dogs, cats, pigs, rabbits, rats, mice, horses, cows, cows, and the like. The subject referred to herein is preferably a human.
  • “about” means that the index value is within an acceptable error range of the particular value determined by one of ordinary skill in the art, which value depends in part on how the measurement or determination is made (ie, the limits of the measurement system). For example, “about” can mean within 1 or more than 1 standard deviation in every practice in the art. Alternatively, “about” or “substantially comprising” can mean a range of up to 20%. Also, with respect to a biological system or process, the term can mean up to an order of magnitude or up to 5 times the value. Unless stated otherwise, when a specific value appears in this application and in the claims, the meaning of "about” or “substantially comprising” should be assumed to be within an acceptable error range for the specific value.
  • Proteins or polypeptides capable of binding to FAP antibodies are Proteins or polypeptides capable of binding to FAP antibodies
  • the term "protein or polypeptide capable of binding to FAP antibody” refers to a protein or polypeptide capable of forming a detectable complex with FAP autoantibodies or artificially designed antibodies. Compared with the technical solution of detecting the existence of FAP protein by designing antibodies in the prior art, in the present invention, the structure and characteristics of the FAP protein itself can be used to simply and efficiently design and obtain a protein that can bind to FAP autoantibodies. or peptides.
  • the protein or polypeptide may be a protein or polypeptide having one or more epitopes of the FAP protein.
  • the protein or polypeptide is capable of binding to the FAP autoantibody through the binding of the epitope to the FAP autoantibody.
  • Suitable proteins or polypeptides for binding to FAP antibodies as disclosed herein include full-length FAP proteins, extracellular segments thereof, or polypeptides comprising epitopes of FAP.
  • FAP includes FAP full-length protein, its extracellular segment and polypeptides comprising FAP epitopes.
  • FAP antibodies may be FAP autoantibodies or artificially designed antibodies. Proteins or polypeptides suitable for the purpose of the present invention can be combined with FAP autoantibodies or artificially designed anti-FAP antibodies to form chemically, physically or biologically detectable antigen-antibody complexes. In some embodiments, proteins or polypeptides suitable for the purposes of the present invention are capable of binding to one or more artificially designed anti-FAP antibodies, such as BMS168 (BMS), orb402355 (Biorbyt), 6D394 (Santa Cruz Biotechnology), F11-24 (Santa Cruz Biotechnology), 983802 (Novus Biologicals) or a combination thereof.
  • BMS168 BMS
  • orb402355 Biorbyt
  • 6D394 Sura Cruz Biotechnology
  • F11-24 Sura Cruz Biotechnology
  • 983802 Novus Biologicals
  • proteins or polypeptides suitable for the purpose of the present invention are capable of binding to multiple (eg, 5 or more, 10 or more, 20 or more) artificially designed anti-FAP antibodies.
  • the FAP antibody is a monoclonal antibody or a polyclonal antibody.
  • the FAP antibody is any form of antibody or antibody fragment as defined herein.
  • FAP can be of any origin, such as human FAP (GenBank Accession: AAB49652.1), rodent FAP (GenBank Accession: CAA71116.1; Accession: EDL79010.1; Accession: AAH19190.1), mammalian FAP (GenBank Accession: CAA71116.1; Accession: EDL79010.1; Accession: AAH19190.1) : DAA32677.1), primate FAP (GenBank Accession:JAA37566.1; Accession:JAA21430.1).
  • the protein or polypeptide suitable for binding as an antibody to FAP of the present disclosure is FAP of human origin.
  • proteins or polypeptides suitable for binding as FAP antibodies of the present disclosure may be FAP full-length proteins, such as human FAP (GenBank Accession: AAB49652.1), rodent FAP (GenBank Accession: CAA71116.1; Accession : EDL79010.1; Accession: AAH19190.1), mammalian FAP (GenBank Accession: DAA32677.1), primate FAP (GenBank Accession: JAA37566.1; Accession: JAA21430.1).
  • the protein or polypeptide suitable for binding to FAP antibody as the present disclosure comprises the full-length sequence of human FAP as shown in SEQ ID NO: 1 of FIG. 1 .
  • one or more amino acid changes can be made to the full-length FAP sequence.
  • one or more conservative substitutions are contained in a polypeptide variant.
  • Constant substitution refers to a substitution in which one amino acid is substituted for another amino acid having similar properties such that those skilled in the art of peptide chemistry would expect that the The secondary structure and hydrophilic properties of the polypeptides have not changed substantially.
  • amino acid substitutions are therefore generally based on the relative similarity of the amino acid side chain substituents in question, eg, their hydrophilicity, hydrophobicity, charge, size, and the like nature.
  • Exemplary substitutions that take into account the various features described above are well known to those skilled in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine glutamine and asparagine; and valine, leucine and isoleucine.
  • Amino acid substitutions may be further based on the similarity of the residues in the following aspects: polarity, charge, solubility, hydrophilicity, hydrophobicity and/or the amphiphilic nature of the residues .
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • those with uncharged apical groups have similar hydrophobicity
  • Numerical amino acids include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine, and tyrosine.
  • amino acids that may represent conservative changes include: (1) Alanine, Proline, Glycine, Glutamic acid, Aspartic acid, Glutamine, Asparagine, Serine, Threonine; (2) ) cysteine, serine, tyrosine, threonine; (3) valine, isoleucine, leucine, methionine, alanine, phenylalanine; (4) lysine amino acid, arginine, histidine; and (5) phenylalanine, tyrosine, tryptophan, histidine.
  • a variant may also, or alternatively, contain non-conservative changes.
  • the variant polypeptide differs from a native sequence by substitution, deletion or addition of five or fewer amino acids.
  • Variants may also (or alternatively) be modified by, for example, deletions or additions of amino acids that are immunogenic, Secondary structure and hydrophilic properties will have the least effect.
  • protein and polypeptide are intended to include the protein or polypeptide itself as well as variants derived from the protein or polypeptide.
  • a “variant” derived from the protein or polypeptide refers to a protein or polypeptide that generally differs from a specifically disclosed protein or polypeptide by having one or Various substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be produced synthetically, for example, by modifying one or more of the protein or polypeptide sequences described in the present invention, and as described in the present invention and/or Or one or more biological activities possessed by the polypeptide can be assessed using any of a variety of techniques well known in the art.
  • a protein or polypeptide suitable for binding as a FAP antibody of the present disclosure is the extracellular domain of a full-length FAP protein.
  • the extracellular domain of the FAP full-length protein can be of any origin, eg, human, rodent, mammalian, or primate.
  • a FAP ectodomain sequence comprised as a protein or polypeptide suitable for binding to a FAP antibody of the present disclosure may be an amino-terminal (N-terminal) precursor to a native FAP ectodomain sequence (eg, SEQ ID NO: 2).
  • a native FAP ectodomain sequence eg, SEQ ID NO: 2.
  • One or more (e.g. top 1-100, top 1-90, top 1-80, top 1-70, top 1-60, top 1-50, top 1-40, top 1 -30, the first 1-20, the first 1-10, etc.) amino acids are deleted or replaced.
  • An exemplary ectodomain deleted for the first 12 amino acids of the amino terminus (N-terminus) of SEQ ID NO:2 is shown in SEQ ID NO:22 of Figure 3.
  • the FAP ectodomain sequence included as a protein or polypeptide suitable for binding to a FAP antibody of the present disclosure may be the carboxy-terminus (C-terminus) of the native FAP ectodomain sequence (eg, SEQ ID NO: 2).
  • C-terminus the carboxy-terminus of the native FAP ectodomain sequence
  • One or more amino acids are deleted or replaced.
  • the protein or polypeptide suitable for binding to FAP antibodies as the present disclosure comprises the sequence of the extracellular segment of human FAP as shown in SEQ ID NO:2 of FIG. Amino acids 27 to 760.
  • a protein or polypeptide suitable for binding as a FAP antibody of the present disclosure comprises amino acids 25 to 760 of SEQ ID NO:1.
  • a protein or polypeptide suitable for binding as a FAP antibody of the present disclosure comprises amino acids 26 to 760 of SEQ ID NO:1.
  • the present invention expects that FAP extracellular segments derived from different species may have slightly different amino acid starting and ending positions relative to the corresponding full-length FAP, but all are expected to be used for the purpose of the present invention, and all fall within the scope of the present invention.
  • extracellular segments of full-length FAP sequences from other species can be determined extracellular segments of full-length FAP sequences from other species, and will appreciate that extracellular segments of full-length FAP sequences from other species may also be suitable for use in the present invention.
  • one or more amino acid changes such as insertions, deletions or substitutions, can be made in the extracellular segment of the full-length FAP sequence.
  • one or more conservative substitutions are contained in a polypeptide variant.
  • Constant substitution refers to a substitution in which one amino acid is substituted for another amino acid having similar properties such that those skilled in the art of peptide chemistry would expect that the The secondary structure possessed by the polypeptide is essentially unchanged.
  • the present invention is intended to include the FAP ectodomain polypeptide itself, as well as variants derived from the polypeptide, of any origin.
  • proteins or polypeptides suitable for binding as FAP antibodies of the present disclosure are polypeptides comprising FAP epitopes.
  • polypeptide comprising an epitope of FAP is a polypeptide comprising one or more parts or segments of FAP as an epitope.
  • FAP full-length protein and its ectodomain meet this definition, the term is intended to specifically refer to other polypeptides other than FAP full-length protein and its ectodomain comprising one or more FAP antigens or a combination thereof .
  • the antigenic epitope is located on the surface of the FAP full-length protein or its extracellular domain, thereby facilitating antigen-recognition receptor binding.
  • a polypeptide comprising a FAP epitope may comprise conformational epitopes consisting of amino acids or polysaccharides that are linked or unlinked in sequence but are spatially adjacent to each other.
  • the antigenic epitope is not located on the surface of the antigenic molecule, and the antigen must be processed by the antigen-presenting cell into a small molecule polypeptide and combined with the MHC molecule before it can be recognized by the TCR.
  • the FAP epitope is a linear epitope consisting of consecutive amino acids in sequence.
  • a polypeptide comprising a FAP epitope when used, a polypeptide comprising more than one FAP epitope is used, eg, the polypeptide comprises two or more, five or more, ten one or more, twenty or more epitopes of FAP. In other embodiments, when a polypeptide comprising a FAP epitope is used, two or more, five or more, ten or more, twenty or more FAP-containing epitopes are used A mixture or fusion protein of epitope-containing polypeptides, wherein each FAP epitope-containing polypeptide comprises one or more FAP epitopes. Fusion forms of polypeptides (eg, fusion proteins) can be achieved and tested by means known in the art (eg, genetic engineering methods).
  • FAP epitope sequences are shown in the table below (aa denotes amino acids).
  • FAP can be native or recombinant.
  • Native FAP can be obtained by purification procedures derived from organs, tissues or cells that highly express FAP.
  • Recombinant FAP is an artificial FAP prepared and purified by genetic engineering methods, and it has the exact same or highly similar conformation, function and properties of natural FAP. Methods to obtain FAP from natural sources or to prepare recombinant FAP using genetic engineering methods are known in the art.
  • An exemplary production system for FAP full-length recombinant proteins is described in: Nagase, Takahiro et al. "Exploration of human ORFeome: high-throughput preparation of ORF clones and efficient characterization of their protein products.” DNA research: an international journal for rapid publication of reports on genes and genes vol.
  • FAP recombinant proteins from different sources can also be purchased from Beijing Yiqiao Shenzhou Science and Technology Co., Ltd. (Sino Biological Inc.), such as human recombinant FAP full-length protein (10464-H07H), biotinylated human recombinant FAP full-length protein (10464 -H07H-B) or cynomolgus monkey recombinant FAP full-length protein (90879-C07H).
  • FAP is in monomeric or dimeric form. Although FAP requires dimerization for enzymatic activity, in some embodiments of the invention, FAP may exist in monomeric form.
  • a monomeric FAP includes a monomeric FAP full-length protein or a monomeric FAP ectodomain, preferably a monomeric FAP ectodomain.
  • a protein or polypeptide suitable for binding as an antibody to FAP of the present disclosure comprises the sequence of the extracellular segment of human FAP as set forth in SEQ ID NO:2.
  • the recombinant FAP full-length protein or its extracellular segment obtained by genetic engineering methods is in monomeric form. FAPs of natural origin are usually present in dimer form.
  • Proteins or polypeptides suitable for binding as FAP antibodies of the present disclosure may be in monomeric or homodimeric form.
  • FAP of natural origin may be in a heterodimeric form with DPP4 or ⁇ 1 integrin.
  • the present invention contemplates that all of the above forms of FAP can be used for the purposes of the present invention.
  • FAP plasma concentrations measured by ELISA in healthy individuals are approximately 100 ng/mL or 0.6 nmol/L (median concentrations reported in various studies range from 15-500 ng/mL, depending on the materials and methods used Inside).
  • FAP is upregulated by multiple stimuli accompanying tissue remodeling in several states. For example, FAP expression is increased in healed wounds, keloids, scleroderma, pulmonary fibrosis, cirrhosis, arthritis, scar tissue after myocardial infarction, advanced atherosclerotic lesions, and stenotic areas in Crohn's disease . In these states, FAP is predominantly upregulated in activated mesenchymal cells such as fibroblasts.
  • FAP is also upregulated in various tumor types, and its expression is associated with an immunosuppressive tumor microenvironment, higher tumor grade, increased lymph node metastasis, and poorer overall survival.
  • FAP is an important hallmark of cancer-associated fibroblasts (CAFs) and appears to contribute to some of its tumor-promoting activities by regulating the structure and composition of the extracellular matrix and affecting the CAF secretome.
  • CAFs cancer-associated fibroblasts
  • endothelial cells and macrophages has also been reported.
  • FAP is also expressed in cancer cells and enhances its tumorigenicity and proliferative capacity in pancreatic, esophageal, and breast cancers, among others.
  • FAP inhibition in oral cancer reduces the activity of the PI3K/AKT and Ras-ERK pathways, resulting in reduced cancer cell proliferation, migration, and invasion.
  • disease associated with abnormal FAP expression refers to a disease in which the expression of FAP protein in individuals with the disease is compared to the level of FAP protein expression in individuals without the disease level increased significantly.
  • disease associated with abnormal FAP expression preferably refers to a disease in which significant FAP expression is detected in an individual suffering from the disease.
  • disease associated with abnormal FAP expression refers to a FAP plasma concentration in an individual with the disease of at least about 1 ⁇ g/mL, at least about 2 ⁇ g/mL, at least about 3 ⁇ g/mL, at least about 4 ⁇ g/mL, at least about about 5 ⁇ g/mL, at least about 6 ⁇ g/mL, at least about 7 ⁇ g/mL, at least about 8 ⁇ g/mL, at least about 9 ⁇ g/mL, at least about 10 ⁇ g/mL, at least about 15 ⁇ g/mL, at least about 20 ⁇ g/mL, at least about 25 ⁇ g /mL, at least about 50 ⁇ g/mL, at least about 100 ⁇ g/mL or higher.
  • examples of diseases associated with abnormal FAP expression include, but are not limited to: keloids, scleroderma, pulmonary fibrosis (especially idiopathic pulmonary fibrosis), liver cirrhosis, arthritis (especially osteoarthritis and rheumatoid arthritis), atherosclerosis, Crohn's disease, basal cell carcinoma, squamous cell carcinoma, skin cancer, oral cancer (especially oral squamous cell carcinoma), melanoma, esophageal cancer, Adenocarcinoma, squamous cell carcinoma, gastric cancer (especially intestinal type gastric cancer), colorectal cancer, rectal cancer, pancreatic cancer, hepatocellular carcinoma, lung cancer (especially non-small cell lung cancer), mesothelioma, cholangiocarcinoma, liver cancer, bladder carcinoma, breast cancer (eg, ductal adenocarcinoma, lobular carcinoma, ductal carcinoma), kidney cancer (especially clear cell renal cell
  • the disease associated with abnormal FAP expression is idiopathic pulmonary fibrosis.
  • the disease associated with abnormal FAP expression is lung cancer, cholangiocarcinoma, liver cancer, glioma, ovarian cancer, breast cancer or pancreatic cancer.
  • the present invention provides a kit for diagnosing a disease associated with abnormal FAP expression, the kit comprising a protein or polypeptide capable of binding to FAP antibody as defined herein.
  • the protein or polypeptide capable of binding to the FAP antibody is the full-length sequence of FAP, the extracellular domain of FAP, and/or a polypeptide comprising an epitope of FAP.
  • the kit comprises: (i) the full-length sequence of FAP; (ii) the extracellular domain of FAP; and/or (iii) a polypeptide comprising an epitope of FAP.
  • the kit further includes additional reagents for detecting binding of the protein or polypeptide to FAP autoantibodies.
  • additional reagents include, but are not limited to, buffers (eg, Tris, phosphoric acid, and carbonic acid), stabilizers, excipients, biocides, and/or inert proteins (eg, bovine serum albumin).
  • buffers eg, Tris, phosphoric acid, and carbonic acid
  • stabilizers eg, excipients, biocides, and/or inert proteins (eg, bovine serum albumin).
  • the protein or polypeptide and the additional reagents may be provided as a lyophilized mixture, or the additional reagents may be provided separately to the user for use in combination.
  • These additional reagents are generally less than 5% by weight based on the amount of active protein/polypeptide, and the total amount is at least 0.001% by weight based on the protein/polypeptide concentration.
  • the protein or polypeptide capable of binding to the FAP antibody is bound to (eg, immobilized to) a solid support.
  • Suitable solid supports are selected from microtiter plates, magnetic beads, polyvinylidene fluoride membranes, agarose beads, polystyrene and nitrocellulose membranes.
  • the kit further comprises a probe-labeled or unlabeled secondary antibody capable of recognizing the FAP autoantibody.
  • the probe-labeled secondary antibody in the kit may be fluorescently modified or non-fluorescently modified.
  • the kit further includes FAP antibody controls at different concentrations.
  • the FAP antibody control substance for example, can be selected from the artificially designed monoclonal antibodies as described above. .
  • the kit is an enzyme-linked immunosorbent assay (ELISA) kit.
  • ELISA enzyme-linked immunosorbent assay
  • the protein or polypeptide capable of binding to the FAP antibody is labeled with a detectable label.
  • the label is selected from the group consisting of enzymatic labels, isotopic labels, chemiluminescent labels, fluorescent labels and dyes.
  • One aspect of the present invention provides a method for diagnosing a disease associated with abnormal FAP expression in a subject, the method comprising using the above-described protein or polypeptide of the present invention to determine the presence and/or presence of FAP autoantibodies in a sample obtained from the subject quantity.
  • immunoassays can be used in diagnostic methods.
  • such immunoassays include the use of, for example, radioimmunoassays, immunochromatography, ELISA, "sandwich” immunoassays, precipitation, immunoblot analysis, gel diffusion precipitation, immunodiffusion assays, agglutination Competitive and non-competitive detection systems such as detection, complement fixation detection, immunoradiometric detection, and fluorescence immunoassay. Both in vitro and in vivo assays can be used.
  • the assay is performed on a biological sample isolated from the subject.
  • the level of FAP autoantibodies in a biological sample is compared to a reference level, wherein the deviation from the reference level is indicative of the presence and/or stage of the relevant disease in the subject.
  • the reference level can be a level determined in a control sample (e.g., from a healthy tissue or subject) or a median level from a healthy subject.
  • a "deviation" from said reference level indicates any significant change, eg an increase or decrease of at least 10%, 20% or 30%, preferably at least 40% or 50%, or even higher.
  • the presence of FAP autoantibodies and/or an increased amount of FAP autoantibodies compared to a reference level, eg, compared to a subject without the disease can be indicative of the presence or risk of developing a related disease in the subject.
  • Methods for diagnosis allow quantitative and/or qualitative evaluation, eg absolute and/or relative measurement of target molecules, eg measuring the content of FAP autoantibodies.
  • the presence of FAP autoantibodies or a higher amount of FAP autoantibodies compared to a reference without the disease indicates that the subject has an associated disease.
  • determining the presence and/or amount of FAP autoantibodies comprises: (i) contacting a biological sample with a protein or polypeptide of the invention capable of binding to FAP autoantibodies, and (ii) detecting the formation and/or determination of the amount of a complex between the protein or polypeptide and the FAP autoantibody.
  • the biological sample is pre-treated, eg, subjected to an enrichment procedure, to remove components and/or enrichment that may interfere with antigen-antibody binding prior to contacting with the protein or polypeptide of the invention FAP autoantibodies that may be present in biological samples.
  • the sample is a body fluid sample.
  • body fluid sample refers to any fluid sample from the body of a patient.
  • the body fluid sample can be a blood sample, a urine sample, a sputum sample, a breast milk sample, a cerebrospinal fluid sample, an ear wax (ear wax) sample, an endolymph sample, a perilymph sample, a gastric juice sample, a mucus sample, a peritoneal fluid sample, a pleural fluid sample, Saliva samples, sebum (skin oil) samples, semen samples, sweat samples, tear samples, vaginal secretion samples or vomit samples, including components or fractions thereof.
  • the bodily fluid samples can be mixed or pooled.
  • the body fluid sample may be a mixture of blood and urine samples or a mixture of blood and cerebrospinal fluid samples.
  • the bodily fluid sample can be provided by removing bodily fluid from the patient, but can also be provided by using previously isolated bodily fluid sample material.
  • the sample is a whole blood sample or a blood fraction sample, such as a blood cell fraction, serum or plasma sample.
  • the detection/diagnosis method of the present invention can be used in combination with other methods for detection/diagnosis of diseases associated with abnormal FAP expression.
  • the detection/diagnostic methods of the present invention can be used in conjunction with the detection of other biomarkers of pulmonary fibrosis.
  • the detection/diagnostic methods of the present invention can be used in conjunction with the detection of other biomarkers of cancer.
  • a computer-readable storage medium having stored thereon computer instructions for reading and execution by a computer, the computer instructions being executed to detect the presence of FAP in a biological sample of a subject
  • a method for an autoantibody comprising: (a) contacting a sample of a subject with the protein or polypeptide of the present invention; (b) detecting and reading the signal of the contacted sample to detect whether the protein or polypeptide is forming a complex with FAP autoantibodies in the sample; and (c) determining whether the signal exceeds a predetermined threshold, and when the signal exceeds a predetermined threshold, determining the presence of FAP autoantibodies in the sample.
  • the predetermined threshold may be a reference level as described above, wherein a deviation from the reference level is indicative of the presence and/or stage of the relevant disease in the subject.
  • the reference level can be a level determined in a control sample (eg, from a healthy tissue or subject) or a median level from a healthy subject.
  • a "deviation" from said reference level indicates any significant change, eg an increase or decrease of at least 10%, 20% or 30%, preferably at least 40% or 50%, or even higher.
  • the presence of FAP autoantibodies and/or an increased amount of FAP autoantibodies compared to a reference level, eg, compared to a subject without the disease can be indicative of the presence or risk of developing a related disease in the subject.
  • the mRNA sequence of human FAP gene (NM_004460.5) was searched from GeneBank (NM_004460.5) using the Primer blast plugin in the NCBI website to design primers that can efficiently amplify the CDS sequence of the FAP gene.
  • Appropriate restriction enzyme cleavage sites are added to the 5' ends of the upstream and downstream primers of the target gene as required, and corresponding protective bases are added before the enzyme cleavage sites.
  • the FAP gene was amplified by PCR using a plasmid containing the FAP gene (FAP-pUC19) as a template.
  • 1% agarose gel detection of PCR amplification products Take 2 ⁇ L of PCR amplification products and mix with 0.5 ⁇ L of DNA loading buffer, add to 1% agarose gel containing 1:10000EB substitute, and electrophoresis at 90V for 15min.
  • Gel imaging After electrophoresis, take out the gel and place it in a gel imaging system to observe whether the amplified band is single and the size of the band is correct. If the band is single and the size is the same as expected, perform gel cutting for the target nucleic acid band.
  • connection system is prepared according to the following system:
  • each bottle was supplemented with 25ml of serum-free DMEM medium, and the culture was continued at 37°C and 5% CO2.
  • Figure 4 shows the results of the detection of eukaryotic expression of FAP extracellular segment recombinant protein by SDS-PAGE (A) and Western Blot (B).
  • A 1: Marker
  • 2 100ng recombinant protein
  • 3 500ng recombinant protein
  • B 1: Marker, 2: 500ng recombinant protein. It can be seen from the figure that the molecular weight of the expressed ectodomain recombinant protein is about 97 kDa, which is consistent with the expected molecular weight.
  • Example 2 detects FAP autoantibodies in peripheral blood
  • IPF idiopathic pulmonary fibrosis
  • Example 1 The FAP extracellular segment antigen protein obtained in Example 1 was diluted with coating buffer to a concentration of 1 ⁇ g/ml, 100 ⁇ l per well, and coated overnight at 4°C.
  • Blocking Wash the plate once, add 300 ⁇ l of blocking solution to each well, and block at 37°C for 2 hours.
  • Color development and termination wash the plate twice, add 100 ⁇ l of TMB to each well, leave at room temperature for about 6 minutes, and immediately stop with 50 ⁇ l of 2M H 2 SO 4 .
  • the microplate reader reads, and the OD value is detected by the dual wavelength 450-630nm.
  • tumor samples were screened, of which 32 were positive for FAP autoantibodies, with a positive rate of 80%; 16 were for lung cancer, with a positive rate of 87.5%; 3 were for cholangiocarcinoma, with a positive rate of 100%; 5 were for liver cancer, with a positive rate of 100%; There were 5 tumors with a positive rate of 60%; 4 ovarian cancers with a positive rate of 100%; 3 breast cancers with a positive rate of 66.7%; and 4 pancreatic cancers with a positive rate of 75%.
  • step (3) Replace the cells in step (2) with a new 6ml complete medium, absorb the overexpressed FAP lentivirus obtained in 1ml of the complete medium and dilute (1), slowly and evenly drop the virus dilution into the cell culture medium, After culturing in a constant temperature cell incubator at 37°C for 48h, replace with a new complete medium.
  • Nuclear counterstaining The fluorescent secondary antibody was washed with PBS, and an appropriate amount of DAPI nuclear dye containing anti-fluorescence quencher was added dropwise for nuclear counterstaining. After 15 minutes of counterstaining, fluorescence microscopy was performed for data collection.
  • the serum to be tested has a weaker fluorescent signal in A549 cells without FAP expression.
  • IPF-1 positive by ELISA
  • D4 normal human serum sample, negative by ELISA
  • IPF-1 fluoresces in A549 cells overexpressing FAP The signal was significantly higher than that of A549 blank cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne une application d'une protéine ou d'un polypeptide, capable d'être lié à un anticorps de protéine d'activation des fibroblastes (FAP), dans la préparation d'un kit utilisé pour diagnostiquer une maladie associée à une expression de FAP anormale. La présente invention utilise la protéine ou le polypeptide capable d'être lié à l'anticorps de FAP pour diagnostiquer la maladie associée à l'expression de FAP anormale par détection de la présence de l'anticorps de FAP. La présente invention concerne une méthode de diagnostic plus efficace et plus commode pour la maladie associée à l'expression de FAP anormale.
PCT/CN2020/127378 2020-07-08 2020-11-07 Kit et méthode de diagnostic d'une maladie associée à une expression de fap anormale, et support de stockage lisible par ordinateur Ceased WO2022007283A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CNPCT/CN2020/100767 2020-07-08
CN2020100767 2020-07-08

Publications (1)

Publication Number Publication Date
WO2022007283A1 true WO2022007283A1 (fr) 2022-01-13

Family

ID=75013967

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/127378 Ceased WO2022007283A1 (fr) 2020-07-08 2020-11-07 Kit et méthode de diagnostic d'une maladie associée à une expression de fap anormale, et support de stockage lisible par ordinateur

Country Status (2)

Country Link
CN (1) CN112540176B (fr)
WO (1) WO2022007283A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024056075A1 (fr) * 2022-09-16 2024-03-21 菲创生物医学技术 （广州）有限公司 Utilisation du récepteur à domaine discoïdine 2 dans le diagnostic du gliome et support lisible par ordinateur associé
CN118914566A (zh) * 2022-12-09 2024-11-08 元典(天津)新技术有限公司 一种用于肝纤维化和肝硬化检测的试剂盒

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103154038A (zh) * 2010-08-13 2013-06-12 罗切格利卡特公司 抗成纤维细胞激活蛋白抗体及使用方法
WO2014193999A2 (fr) * 2013-05-28 2014-12-04 Caris Science, Inc. Méthodes et compositions d'identification de biomarqueurs

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0953639A1 (fr) * 1998-04-30 1999-11-03 Boehringer Ingelheim International GmbH Anticorps dirigé contre FAPalpha pouvant être produit avec une efficacité accrue
CA2696579C (fr) * 2007-08-17 2017-01-24 Boehringer Ingelheim International Gmbh Utilisation de derives de la purine pour le traitement de maladies liees au fab
BRPI0820793A2 (pt) * 2007-12-10 2015-06-16 Hoffmann La Roche Seprase como marcador de câncer
LT2990421T (lt) * 2009-04-30 2018-04-25 Tel Hashomer Medical Research Infrastructure And Services Ltd. Anti-ceacam1 antikūnai ir jų panaudojimo būdai
CN102414565B (zh) * 2009-05-04 2014-09-24 霍夫曼-拉罗奇有限公司 作为癌症的标记物的DPPIV/Seprase的用途
CN102532298B (zh) * 2012-03-01 2013-11-06 刘林林 与自身抗体特异性结合的abcc3抗原多肽及应用
CN103869086B (zh) * 2014-04-14 2016-02-17 杭州凯保罗生物科技有限公司 一种血清自身抗体检测试剂盒
CN105367626A (zh) * 2015-12-17 2016-03-02 苏州偲聚生物材料有限公司 用于检测宫颈癌的多肽、检测器件和检测试剂盒
CN106928364B (zh) * 2015-12-30 2020-09-25 广西医科大学 一种FAP纳米抗体Nb26
BR112019021411A2 (pt) * 2017-04-13 2020-05-05 Hoffmann La Roche métodos para tratar ou retardar a progressão do câncer e para melhorar a função, usos de um imunoconjugado, de um agonista, de um antagonista, composições, kit e invenção
US11571476B2 (en) * 2017-12-11 2023-02-07 Taipei Medical University Anti-tumor/anti-tumor associated fibroblast/anti-hapten trispecific antibodies and use thereof
CN110988346A (zh) * 2019-11-27 2020-04-10 上海端雅生物科技有限公司 一种辅助诊断肺癌的标记物以及检测方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103154038A (zh) * 2010-08-13 2013-06-12 罗切格利卡特公司 抗成纤维细胞激活蛋白抗体及使用方法
WO2014193999A2 (fr) * 2013-05-28 2014-12-04 Caris Science, Inc. Méthodes et compositions d'identification de biomarqueurs

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024056075A1 (fr) * 2022-09-16 2024-03-21 菲创生物医学技术 （广州）有限公司 Utilisation du récepteur à domaine discoïdine 2 dans le diagnostic du gliome et support lisible par ordinateur associé
CN118914566A (zh) * 2022-12-09 2024-11-08 元典(天津)新技术有限公司 一种用于肝纤维化和肝硬化检测的试剂盒

Also Published As

Publication number Publication date
CN112540176B (zh) 2021-09-28
CN112540176A (zh) 2021-03-23

Similar Documents

Publication Publication Date Title
RU2650778C2 (ru) Композиции и способы применения для определения не4а
JP6691060B2 (ja) 診断用の細胞表面前立腺癌抗原
CN112540176B (zh) 用于诊断fap表达异常相关疾病的试剂盒、方法及计算机可读存储介质
JP6276992B2 (ja) 胸膜中皮腫患者の早期発見のための分子マーカー及びその発現解析方法
JP2010502192A (ja) Ｂｏｒｉｓアイソフォーム並びに疾患を検出及び治療する方法
JP6122779B2 (ja) 抗ｗｔ１抗体の測定方法
JP5857334B2 (ja) 上皮性卵巣癌マーカー検出用抗体及び上皮性卵巣癌判定方法
US20220034890A1 (en) Use of bmmf1 rep protein as a biomarker for breast cancer
HK40039496B (en) Kits, methods and computer readable storage medium for diagnosing abnormal fap expression related diseases
HK40039496A (en) Kits, methods and computer readable storage medium for diagnosing abnormal fap expression related diseases
JP6729917B2 (ja) ＥｐｈＡ２ Ｎ末端フラグメント抗体
EP2957571B1 (fr) Anticorps anti-pvhl monoclonaux et leurs utilisations
JP2014532408A (ja) ＵＳＰ２ａペプチドおよび抗体
JP2000069963A (ja) アポリポプロテインｅ４特異モノクローナル抗体
JP5750646B2 (ja) Ｓｃｃａ２濃度測定によるアレルギー疾患の検査方法
JP4838436B2 (ja) 抗ヒト肝性トリグリセリドリパーゼ抗体
US20220120744A1 (en) Assessing and treating germ cell tumors and paraneoplastic autoimmunity
EP2650307A1 (fr) PROCÉDÉ DE MESURE IMMUNOLOGIQUE DE LA PROTÉINE cofiline-1
JP2024022080A (ja) ポリペプチド、抗ｅ型肝炎ウイルス抗体の検出方法、抗ｅ型肝炎ウイルス抗体及びｅ型肝炎ウイルスの同時検出方法、及びキット
JP5750645B2 (ja) アレルギー疾患の検査方法
CN103842504A (zh) 上皮性卵巢癌标志物检测用抗体和上皮性卵巢癌判定方法
JP2014115199A (ja) 胃癌又は食道癌の検出方法
JP2014167419A (ja) 胸膜中皮腫患者の早期発見のための分子マーカーの組合せとその発現解析方法
JP2011251969A (ja) 抗ヒト肝性トリグリセリドリパーゼ抗体
HK1191056B (en) Compositions and methods of use for determination of he4a

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20944237

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC

122 Ep: pct application non-entry in european phase

Ref document number: 20944237

Country of ref document: EP

Kind code of ref document: A1