CN112540176B - Kit, method and computer-readable storage medium for diagnosing diseases associated with FAP expression abnormality - Google Patents

Abstract

The invention discloses application of a protein or polypeptide capable of being combined with a Fibroblast Activation Protein (FAP) antibody in preparing a kit for diagnosing diseases related to FAP abnormal expression. Diagnosing a disease associated with abnormal expression of FAP by detecting the presence of FAP autoantibodies using a protein or polypeptide capable of binding to the FAP antibody. The invention provides a more efficient and convenient method for diagnosing FAP abnormal expression related diseases.

Description

Kit, method and computer-readable storage medium for diagnosing diseases associated with FAP expression abnormality

Technical Field

The present invention relates to a diagnostic kit, a diagnostic method and a computer readable medium for diseases associated with abnormal expression of FAP, and more particularly, to diagnosing diseases associated with abnormal expression of FAP by detecting the presence of FAP autoantibodies.

Background

Fibroblast Activation Protein (FAP), also known as Seprase, is a 97kDa monomer that requires dimerization to exert enzymatic activity. FAP is a membrane-integrated serine peptidase, belonging to the dipeptidyl peptidase IV family, along with dipeptidyl peptidase IV (DPPIV, also known as CD26), a closely related cell surface enzyme, and other peptidases. FAP contains two N-glycosylated subunits with a large C-terminal extracellular domain in which the catalytic domain of the enzyme is located. FAP has dipeptidyl peptidase and endopeptidase activities in its glycosylated form, and cleaves gelatin and type I collagen. Human FAP exists on cells as a homodimer, each monomer of which comprises 760 amino acid residues, comprising: a short cytoplasmic tail of only 6 amino acids, a single transmembrane domain of 20 amino acids, and an extracellular domain of 734 amino acids. Crystal structure determination indicates that the extracellular domain of FAP comprises two domains: an alpha/beta hydrolase domain (residues 27-53 and 493-760) and an eight-leaf beta propeller domain (residues 54-492) that enclose a diameter of about

Is provided with a large cavity. In a pocket in the cavity, at the interface of the α/β hydrolase and the β -propeller domain, a catalytic triad is comprised of residues Ser624, Asp702 and His 734. In addition to the catalytic triad, residues Ala657, Asn704, Arg123, Glu203 and Glu204 are all essential for FAP catalytic activity. Can pass through the side opening

Into this cavity, only the elongated peptide or unfolded or partially unfolded protein fragment is allowed to reach the active site cavity. FAP contains six potential N-linked glycosylation sites at Asn residues 49, 92, 99, 227, 314, and 679 (the motif Asn-X-Ser/Thr). Most of these sites are located on the surface of the beta-propeller, only one on the hydrolase domain, near the cell surface. In baculovirus-expressed soluble human FAP, all amino acids except Asn99 are glycosylated.

FAP has a unique tissue distribution with high up-regulation of expression on the reactive stromal fibroblasts of more than 90% of all primary and metastatic epithelial tumors (including lung, colorectal, bladder, ovarian, and breast cancers, etc.) that are 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 expression of FAP is directly correlated with the malignant phenotype.

Autoantibodies refer to antibodies directed against self tissues, organs, cells and cellular components. The growth, development and survival of human body have the maintenance of complete autoimmune tolerance mechanism, and the normal immune reaction has protective defense function, i.e. no reaction to self tissues and components. Once the integrity of self tolerance is destroyed, the body can generate autoimmune reaction to generate autoantibody according to the fact that self tissues and components are 'foreign bodies'. There may be low titers of autoantibodies in normal human blood, but no disease will occur, but if the titer of autoantibodies exceeds a certain level, damage may occur to the body, inducing disease.

Numerous studies have shown that disease-associated antigens in serum can induce the body to produce autoantibodies, both antigens and autoantibodies to the antigens being present in the patient's serum. Therefore, it is possible to detect either an antigen or an autoantibody using an antibody, but the specificity and sensitivity of disease detection using an autoantibody are much higher than those of disease detection using an antigen. Many disease-associated antigens exist not only in patients but also in normal humans, and therefore detection of disease-associated antigens as diagnostic markers is not reliable. The autoantibody is low in content in normal human bodies and cannot be detected or does not exist at all, if the level of the autoantibody in the human bodies is obviously increased, the situation of abnormal immunity exists in the human bodies, and the fluctuation of the level of the related antigen in the human bodies is indicated, so that the existence of diseases or the aggravation of the original diseases is indicated. The main advantages of using autoantibodies as disease markers are: strong specificity, high sensitivity, simple operation, good repeatability, high detection speed and low false positive rate. The level change of the autoantibody in the serum can be used for monitoring different stages of diseases, and the early diagnosis of the diseases can be realized, thereby being beneficial to the early treatment of the diseases and improving the prognosis of patients.

At present, no report about FAP autoantibodies exists, and even no attempt is made to diagnose diseases related to abnormal expression of FAP through FAP autoantibodies.

Disclosure of Invention

The inventors of the present application unexpectedly found that FAP autoantibodies are present in patient samples with diseases associated with abnormal expression of FAP, whereas FAP autoantibodies are not detected in normal samples. The inventors use a protein or polypeptide capable of binding to the FAP antibody to detect the presence of autoantibodies, thereby diagnosing a disease associated with abnormal FAP expression.

In view of the above findings, one aspect of the present invention provides a kit for diagnosing a disease associated with abnormal expression of FAP, the kit comprising a protein or polypeptide capable of binding to a Fibroblast Activation Protein (FAP) antibody.

Another aspect of the invention provides a protein or polypeptide capable of binding to a Fibroblast Activation Protein (FAP) antibody for use in the preparation of a kit for diagnosing a disease associated with abnormal expression of FAP.

Another aspect of the invention provides a protein or polypeptide capable of binding to a Fibroblast Activation Protein (FAP) antibody for use in diagnosing a disease associated with abnormal expression of FAP.

Another aspect of the invention provides a method for diagnosing a disease associated with abnormal expression of FAP in a subject, the method comprising contacting a protein or polypeptide capable of binding to an FAP autoantibody of the invention with a sample from the subject; detecting the presence of a complex of said protein or polypeptide and said FAP autoantibody in the contacted sample; and determining that the subject has or is at risk of having a disease associated with aberrant expression of FAP based on the presence of the complex.

Another aspect of the invention provides a computer readable storage medium having stored thereon computer instructions for reading and execution by a computer, the computer instructions being executable to perform a method of detecting the presence of FAP autoantibodies in a sample of a subject, the method comprising: (a) contacting a sample of a subject with a protein or polypeptide capable of binding to a Fibroblast Activation Protein (FAP) antibody; (b) detecting and reading the signal of the contacted sample to detect whether the protein and the polypeptide form a complex with FAP autoantibody in the sample; and (c) determining whether the signal exceeds a predetermined threshold, and determining that an FAP autoantibody is present in the sample when the signal exceeds a predetermined threshold.

Additional aspects and advantages of the invention will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the invention as described herein.

Drawings

FIG. 1: the full-length sequence of human FAP (SEQ ID NO: 1).

FIG. 2: the sequence of human FAP ectodomain (SEQ ID NO: 2).

FIG. 3: exemplary ectodomain sequences according to the invention (SEQ ID NO: 22).

FIG. 4: and (3) detecting the recombinant protein of the eukaryotic expression FAP extracellular segment. 293i eukaryotic expression of FAP extracellular segment recombinant protein, and detection by SDS-PAGE (A) and WB (B). In A, 1: marker, 2: 100ng of recombinant protein, 3: 500ng of recombinant protein. 1 in B: marker, 2: 500ng of recombinant protein.

FIG. 5: ELISA detects serum FAP autoantibodies. And (3) detecting an autoantibody in serum by ELISA (enzyme-Linked immunosorbent assay) by taking FAP extracellular recombinant protein as an antigen, taking an FAP commercially available antibody as a positive control, taking an irrelevant antibody as a negative control, and setting a blank control. P <0.05, P <0.001, P < 0.0001.

FIG. 6: and (3) detecting serum FAP autoantibodies by immunofluorescence. An A549 cell over expressing FAP is used as a material, an immunofluorescence method is used for detecting FAP autoantibodies in serum to be detected, FAP commercial antibody is used as a positive control, and a blank control is set.

Detailed Description

Definition of

Throughout the specification and claims, unless the context indicates otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer, step or component but not the exclusion of any other integer, step or component. The term "comprising" as used herein may be substituted with the term "comprising" or "containing" or sometimes with the term "having" as used herein.

As used herein, the term "sequence" (e.g., terms such as "amino acid sequence," "antibody sequence," "variable domain sequence," or "protein sequence") is generally understood to include related amino acid sequences as well as nucleic acids or nucleotide sequences encoding the amino acid sequences, unless the context requires a more stringent interpretation. Depending on the context, an amino acid sequence is to be interpreted as meaning a single amino acid or an unbranched sequence of two or more amino acids. The nucleotide sequence is interpreted to mean an unbranched sequence of 3 or more nucleotides.

The terms "protein", "peptide" and "polypeptide" are used interchangeably throughout this disclosure and each has the same meaning for the present disclosure. Each term refers to an organic compound consisting of a linear chain of two or more amino acids. The compound may 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 more amino acids, and even three hundred or more amino acids. The skilled artisan will appreciate that polypeptides typically comprise fewer amino acids than proteins, although there is no art-recognized criticality in distinguishing between the number of amino acids of a polypeptide and a protein; the polypeptide can be prepared by chemical synthesis or recombinant methods; and proteins are typically prepared in vitro or in vivo by recombinant methods known in the art. By convention, the order of the amide bonds in the primary structure of a polypeptide follows the amino acids written, with the amino terminus (N-terminus) of the polypeptide always being on the left and the carboxy terminus (C-terminus) on the right.

It will also be appreciated by those of ordinary skill in the art that amino acid substitutions, deletions or insertions may be made to provide conservative substitutions or alterations in "non-essential" regions. For example, a polypeptide or amino acid sequence derived from a given protein may be identical to the starting sequence except for one or more individual amino acid substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more single amino acid substitutions, insertions, or deletions). In certain embodiments, 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.

As used herein, the term "antibody" refers to any form of antibody that exhibits a desired biological activity (e.g., inhibits binding of a ligand to its receptor or by inhibiting ligand-induced receptor signaling). "antibody fragments" and "antigen-binding fragments" refer to antigen-binding fragments and antibody analogs of an antibody, which typically include at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of the parent antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, an antibody fragment retains at least 10% of the parent binding activity when expressed as activity on a molar basis. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parent antibody to the target. Examples of antibody fragments include, but are not limited to: fab, Fab ', F (ab')₂And Fv fragments; a diabody; a linear antibody; single chain antibody molecules, such as sc-Fv; a nanobody; a domain antibody; and multispecific antibodies formed from antibody fragments. The term "FAP antibody" refers to an antibody that specifically binds to FAP, including autoantibodies and artificially designed antibodies, as well as any form of antibody, such as antibody fragments and antigen binding fragments as defined above.

The term "autoantibody" as used herein refers to an antibody that specifically binds to a structure from the organism from which it was produced. "FAP autoantibody" refers to an autoantibody that specifically binds to the FAP protein from which it was produced. Particularly preferably, it is a mammalian autoantibody, more preferably a human autoantibody of the IgG class. The variable domain of which is capable of specifically binding to a self-antigen (e.g. a FAP protein). The invention contemplates that the FAP autoantibodies are polyclonal antibodies having distinct variable regions 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 have entered the body, and are used in immune reactions to destroy foreign harmful substances. Generally, the immune system is able to recognize and ignore the body's own cells and does not produce antibodies to them; meanwhile, the immune system can not generate excessive reaction to substances without threat in the environment. However, in certain cases, the immune system fails to recognize the body's own substances and recognizes them as foreign invaders, thereby generating antibodies (i.e., autoantibodies) against these substances and inducing autoimmunity. There are many autoantibodies in autoimmune diseases.

The term "epitope" refers to the portion or segment of a molecule that is recognized by a binding agent. For example, an epitope is a discrete three-dimensional site on a molecule that is recognized by a binding agent. Epitopes usually consist of chemically active surface groupings of molecules (e.g., amino acids or sugar side chains) and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. The epitope of a protein preferably comprises a continuous or discontinuous segment of said protein and is preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, most preferably 10 to 25 amino acids in length. For example, 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. An epitope defines the minimum binding site of an immunoglobulin and therefore represents a specific target for an immunoglobulin. A single antigenic molecule may have one or more different epitopes. In general, only an epitope on the surface of an antigenic substance is easy to bind to an antigen-recognizing receptor or antibody, and is called a functional epitope. While the epitope located inside the molecule is not immunogenic and is called a cryptic epitope. There are two types of epitope structures, conformational epitopes and linear epitopes. Preferably, the epitope of the autoantigen in the present invention is located in the extracellular domain of FAP.

According to the present invention, the term "binding" preferably relates to specific binding. By "specifically binds" is meant that the agent binds more strongly to a specific target than to another target. Dissociation constant (K) if the agent binds to the first target_D) Less than the dissociation constant for the second target, it binds more strongly to the first target than to the second target. Preferably, the dissociation constant (K) of the target that does not specifically bind to the agent_D) In contrast, the dissociation constant (K) of the target to which the agent specifically binds_D) Is in excess of 10²10 times of³10 times of⁴10 times of⁵10 times of⁶10 times of⁷10 times of⁸10 times of⁹Multiple or 10 times¹⁰The times are lower.

Preferably, an agent (e.g. a protein or polypeptide) is specific for a predetermined target if it is capable of binding to the target but it is not capable of binding to other targets, i.e. does not have significant affinity for and does not significantly bind to other targets in a standard assay. According to the invention, an agent is FAP-specific or FAP autoantibody-specific if it is capable of binding to FAP or FAP autoantibody but (substantially) incapable of binding to other targets (e.g. DPP IV). Preferably, K if the agent binds to the predetermined target_DIs K bound to its non-specific target_DAt least 10²10 times of³10 times of⁴10 times of⁵10 times of⁶10 times of⁷10 times of⁸10 times of⁹Multiple or 10 times¹⁰Lower fold, the agent is specific for the target.

The binding of the agent to the target can be determined experimentally using any suitable method, which is within the scope of the skilled person. Affinity can be readily determined using conventional techniques, e.g., by equilibrium dialysis; surface plasmon resonance analysis was used by using the general procedure outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by other methods known to the skilled person. Affinity data can be analyzed, for example, by methods known in the art. The measured affinity of a particular interaction may vary if measured under different conditions (e.g., salt concentration, pH). Thus, affinity and other binding parameters (e.g., K)_D、IC₅₀) The measurement of (b) is preferably performed with a standardized solution of the binding agent and the target and a standardized buffer.

The term "enzyme-linked immunosorbent assay or ELISA" as used herein relates to a method for quantitative or semi-quantitative determination of the protein concentration of a sample (e.g. plasma, serum or cell/tissue extract) in a multi-well plate format (typically 96 wells per plate). Briefly, proteins in solution were adsorbed onto ELISA plates. The plate can be probed using an antibody specific for the protein of interest. The background was minimized by optimizing the blocking and washing methods, and specificity was ensured by the presence of positive and negative controls. Detection methods are typically based on colorimetric or chemiluminescent detection.

As used herein, the term "domain" (of a polypeptide or protein) refers to a folded protein structure that has the ability to retain its tertiary structure independent of the rest of the protein. In general, domains are responsible for discrete functional properties of proteins, and in many cases can be added, removed, or transferred to other proteins without losing the function of the rest of the protein and/or domain.

In the present invention, the term "extracellular domain/ectodomain/extracellular segment" refers to a segment of a molecule (e.g. a protein) which is directed towards the extracellular space of a cell and which is preferably accessible from the outside of said cell, e.g. by a binding agent (e.g. an autoantibody) located outside said cell. Preferably, the term refers to one or more extracellular loops, extracellular domains or fragments thereof.

As used herein, the term "subject" or "patient" or "individual" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis or treatment is desired. Mammals include humans, domestic animals, farm animals, zoo animals, sports animals, or pets, such as dogs, cats, pigs, rabbits, rats, mice, horses, cows, etc. The subject referred to herein is preferably a human.

In the present invention, "about" means that the numerical value is within an acceptable error range for the specific value determined by one of ordinary skill in the art, which numerical value depends in part on how the value is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean within 1 or over 1 standard deviation per practice in the art. Alternatively, "about" or "substantially comprising" may mean a range of up to 20%. Furthermore, for biological systems or processes, the term may mean up to an order of magnitude or up to 5 times the value. Unless otherwise indicated, when a particular value appears in the application and claims, the meaning of "about" or "consisting essentially of" should be assumed to be within an acceptable error range for that particular value.

Protein or polypeptide capable of binding to FAP antibody

As used herein, the term "protein or polypeptide capable of binding to an FAP antibody" refers to a protein or polypeptide capable of forming a detectable complex with an FAP autoantibody or an artificially designed antibody. Compared with the technical scheme of detecting the existence of the FAP protein by designing the antibody in the prior art, the protein or polypeptide capable of being combined with the FAP autoantibody can be simply and efficiently designed and obtained by the structure and the characteristics of the FAP protein.

In one embodiment, the protein or polypeptide may be a protein or polypeptide having one or more epitopes of the FAP protein. The protein or polypeptide can bind to the FAP autoantibody through the binding of the epitope and the FAP autoantibody.

Proteins or polypeptides suitable for binding to FAP antibodies as disclosed herein include FAP full-length proteins, extracellular segments thereof, or polypeptides comprising an epitope of FAP. In the present invention, unless otherwise indicated, the term "FAP" includes FAP full-length proteins, extracellular segments thereof, and polypeptides comprising epitopes of FAP.

As defined above, a "FAP antibody" may be a FAP autoantibody or an artificially designed antibody. Proteins or polypeptides suitable as means for achieving the objects of the invention are capable of binding to FAP autoantibodies or artificially designed anti-FAP antibodies to form chemically, physically or biologically detectable antigen-antibody complexes. In some embodiments, a protein or polypeptide suitable as a means of achieving the objects of the invention can be conjugated 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. Preferably, a protein or polypeptide suitable for achieving the objects of the invention is capable of binding to a plurality (e.g. 5 or more, 10 or more, 20 or more) of artificially designed anti-FAP antibodies. In some embodiments, the FAP antibody is a monoclonal antibody or a polyclonal antibody. More FAP antibodies are seen in https:// www.antibodypedia.com/gene/33750/FAP (last visit date 2020, 7, 5). In some embodiments, the FAP antibody is any form of antibody or antibody fragment as defined herein.

FAP can be any source, such as human FAP (GenBank Accession: AAB49652.1), rodent FAP (GenBank Accession: CAA 71116.1; Accession: EDL 79010.1; Accession: AAH19190.1), mammal FAP (GenBank Accession: DAA32677.1), primate FAP (GenBank Accession: JAA 37566.1; Accession: JAA 21430.1). Preferably, the protein or polypeptide suitable for binding to FAP antibodies of the present disclosure is FAP of human origin.

In some embodiments, a protein or polypeptide that binds to FAP antibodies suitable as disclosed herein can be a FAP full-length protein, such as human FAP (GenBank Accession: AAB49652.1), rodent FAP (GenBank Accession: CAA 71116.1; Accession: EDL 79010.1; Accession: AAH19190.1), mammalian FAP (GenBank Accession: DAA32677.1), primate FAP (GenBank Accession: JAA 37566.1; Accession: JAA 21430.1). In a preferred embodiment, a protein or polypeptide suitable for binding to FAP antibodies of the present disclosure comprises the full-length sequence of human FAP as shown in SEQ ID No.1 of fig. 1.

As previously described, changes in one or more amino acids, such as insertions, deletions or substitutions, may be made to the full-length sequence of FAP. In many cases, one or more conservative substitutions are included in a polypeptide variant. "conservative substitution" refers to a substitution in which one amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the polypeptide to have a secondary structure and hydrophilic properties that are not substantially changed.

As outlined above, amino acid substitutions are therefore generally based on the relative similarity that the amino acid side-chain substituents possess, for example, their hydrophilicity, hydrophobicity, charge, size, and similar properties. Exemplary substitutions that take into account the various features previously described 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 made further based on the similarity of the residues in question: polarity, charge, solubility, hydrophilicity, hydrophobicity of the residue and/or the amphipathic nature. For example, amino acids with negative charges include aspartic acid and glutamic acid; amino acids with positive charges include lysine and arginine; and amino acids with similar hydrophobicity numbers with uncharged top groups include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine, and tyrosine. Other groups of 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, arginine, histidine; and (5) phenylalanine, tyrosine, tryptophan, histidine. A variant may also, or alternatively, contain non-conservative changes. In a preferred embodiment, the difference between the variant polypeptide and a native sequence is the occurrence of a substitution, deletion or addition of five amino acids or less. Also (or alternatively) the variant may be modified, for example by the deletion or addition of certain amino acids, which have minimal effect on the immunogenic, secondary structure and hydrophilic properties of the polypeptide.

In the present invention, the terms "protein", "polypeptide" are intended to include the protein or polypeptide itself as well as variants derived from the protein or polypeptide. "variant" derived from the protein or polypeptide refers to a protein or polypeptide that differs from a specifically disclosed protein or polypeptide, typically by having one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be produced synthetically, for example, by modification of one or more of the protein or polypeptide sequences described herein, and evaluation of one or more biological activities possessed by the polypeptide in accordance with the teachings of the present invention and/or using any of a variety of techniques well known in the art.

When it is desired to make changes to the amino acid sequence of a polypeptide to produce an equivalent, or even an improved, variant or portion of the polypeptide of the invention, one skilled in the art will typically make changes to one or more codons in the coding DNA sequence.

In some embodiments, a protein or polypeptide suitable for binding to FAP antibodies of the present disclosure is the extracellular domain of a FAP full-length protein. As previously described, the extracellular domain of the FAP full-length protein may be of any origin, e.g., human, rodent, mammalian or primate origin.

In some embodiments, a FAP ectodomain sequence suitable for inclusion in a protein or polypeptide that binds to a FAP antibody of the present disclosure can be formed by deletion or substitution of the first one or more (e.g., first 1-100, first 1-90, first 1-80, first 1-70, first 1-60, first 1-50, first 1-40, first 1-30, first 1-20, first 1-10, etc.) amino acids from the amino terminus (N-terminus) of a native FAP ectodomain sequence (e.g., SEQ ID NO: 2). An exemplary extracellular domain deleted of the first 12 amino acids at the amino terminus (N-terminus) of SEQ ID NO. 2 is shown in FIG. 3 as SEQ ID NO. 22.

In some embodiments, a FAP ectodomain sequence suitable for inclusion in a protein or polypeptide that binds to a FAP antibody of the present disclosure can be formed by deletion or substitution of one or more (e.g., the last 1-100, the last 1-90, the last 1-80, the last 1-70, the last 1-60, the last 1-50, the last 1-40, the last 1-30, the last 1-20, the last 1-10, etc.) amino acids from the carboxy-terminus (C-terminus) of a native FAP ectodomain sequence (e.g., SEQ ID NO: 2).

In a preferred embodiment, the protein or polypeptide suitable for binding to FAP antibodies of the present disclosure comprises the extracellular segment sequence of human FAP as shown in SEQ ID No. 2 of fig. 2, having amino acids 27 to 760 of SEQ ID No. 1. In another preferred embodiment, proteins or polypeptides suitable for binding to FAP antibodies of the present disclosure comprise amino acids 25 to 760 of SEQ ID No. 1. In another preferred embodiment, proteins or polypeptides suitable for binding to FAP antibodies of the present disclosure comprise amino acids 26 to 760 of SEQ ID No. 1. The present invention contemplates FAP extracellular segments of different species origin, which may differ slightly in their start and stop positions relative to the amino acid corresponding to full-length FAP, but are contemplated as being useful for the purposes of the present invention and are within the scope of the present invention.

One skilled in the art can determine extracellular segments of FAP full-length sequences from other species, and it will be appreciated that extracellular segments of FAP full-length sequences from other species may also be suitable for use in the present invention. As previously described, one or more amino acid changes, such as insertions, deletions or substitutions, may be made in the extracellular segment of the FAP full-length sequence. In many cases, one or more conservative substitutions are included in a polypeptide variant. "conservative substitution" refers to a substitution in which one amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure of the polypeptide to be essentially unchanged. The invention is intended to include FAP ectodomain polypeptides per se of any origin as well as variants derived from such polypeptides.

In other embodiments, a protein or polypeptide suitable for binding to FAP antibodies of the present disclosure is a polypeptide comprising an epitope of FAP. Herein, the term "polypeptide comprising an epitope of FAP" is a polypeptide comprising one or more of FAP as part or segment of an epitope. In the present invention, while the FAP full-length protein and its extracellular domain conform to this definition, the term is intended to refer specifically to other polypeptides comprising one or more FAP antigens or combinations thereof in addition to the FAP full-length protein and its extracellular domain. In some embodiments, the epitope is on the surface of the FAP full-length protein or the extracellular domain thereof, thereby facilitating antigen recognition receptor binding. In some embodiments, a polypeptide comprising an epitope of FAP may comprise conformational epitopes consisting of amino acids or polysaccharides that are linked or unlinked in sequence, but spatially adjacent to each other. In some embodiments, the epitope is not on the surface of an antigenic molecule and is processed by an antigen presenting cell into a small polypeptide that binds to an MHC molecule before it can be recognized by the TCR. In some embodiments, the FAP epitope is a linear epitope consisting of amino acids linked in sequence.

In some embodiments, when a polypeptide comprising an epitope of FAP is used, a polypeptide comprising more than one epitope of FAP is used, e.g., the polypeptide comprises two or more, five or more, ten or more, twenty or more epitopes of FAP. In other embodiments, when a polypeptide comprising an epitope of FAP is used, a mixture or fusion protein of two or more, five or more, ten or more, twenty or more polypeptides comprising an epitope of FAP is used, wherein each polypeptide comprising an epitope of FAP comprises one or more epitopes of FAP. Fusion forms of the polypeptides (e.g., fusion proteins) can be achieved and tested by means known in the art (e.g., genetic engineering methods).

Exemplary FAP epitope sequences are shown in the table below (aa denotes amino acids).

FAP may be natural or recombinant. Native FAP can be obtained from purification procedures of organs, tissues or cells that highly express FAP. The recombinant FAP is artificial FAP prepared and purified by using a genetic engineering method, and has the conformation, the function and the property which are completely the same or similar to those of natural FAP. Methods for obtaining FAP from natural sources or for making 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, "expression of human ORFeome: high-throughput prediction of ORF clones and efficiency characterization of the same protein products," DNA research: an international project for rapid publication of genes and genes vol.15,3(2008):137-49.doi:10.1093/dnares/dsn004(GenBank: BAI 47344.1). Recombinant proteins of FAP from different sources are also available from Beijing-sense Qianzhou science and technology Inc. (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. While FAP needs to dimerize to exert enzymatic activity, in some embodiments of the invention FAP may be present in monomeric form. The monomeric FAP comprises a monomeric FAP full-length protein or a monomeric FAP ectodomain, preferably a monomeric FAP ectodomain. In one embodiment, a protein or polypeptide suitable for binding to FAP antibodies of the present disclosure comprises the extracellular domain sequence of human FAP as set forth in SEQ ID No. 2. Generally, the recombinant FAP full-length protein obtained by genetic engineering methods or its extracellular segment is in monomeric form. FAP, which is naturally derived, is usually present as a dimer. Proteins or polypeptides suitable for binding to FAP antibodies of the present disclosure may be in monomeric or homodimeric forms. In some embodiments, FAP from a natural source may be in the form of a heterodimer with DPP4 or a β 1 integrin. The present invention contemplates that the above various forms of FAP may be used for the purposes of the present invention.

Diseases associated with abnormal FAP expression

Soluble forms of FAP are present in the plasma of various species, although most normal adult tissues show little or no detectable expression of FAP. In humans, FAP plasma concentrations of approximately 100ng/mL or 0.6nmol/L were measured by ELISA in healthy individuals (median concentrations reported for various studies ranged from 15 to 500ng/mL depending on the materials and methods used).

FAP is upregulated by multiple stimuli in several states with tissue remodeling, in contrast to low expression in most quiescent stromal cells. For example, FAP expression is increased in narrow areas of healed wounds, keloids, scleroderma, pulmonary fibrosis, cirrhosis, arthritis, scar tissue following myocardial infarction, advanced atherosclerotic lesions, and crohn's disease. Under these conditions, FAP is primarily upregulated in activated mesenchymal cells (e.g., fibroblasts).

FAP is also upregulated in various tumor types, with expression associated with immunosuppressive tumor microenvironment, higher tumor grade, increased lymph node metastasis, and poor overall survival. FAP is an important hallmark of cancer-associated fibroblasts (CAF) and appears to promote certain of their tumor-promoting activities by modulating the structure and composition of the extracellular matrix and affecting the CAF secretory component. Expression of FAP in other types of tumor-associated stromal cells (e.g., endothelial cells and macrophages) has also been reported. FAP is also expressed in cancer cells and increases its tumorigenicity and proliferative capacity in pancreatic, esophageal, and breast cancers, among others. One study has shown that inhibition of FAP in oral cancer decreases the activity of the PI3K/AKT and Ras-ERK pathways, resulting in decreased proliferation, migration, and invasion of cancer cells.

As used herein, the term "a disease associated with aberrant expression of FAP" refers to a disease in which the level of expression of FAP protein is significantly increased in an individual having the disease as compared to the level of expression of FAP protein in an individual not having the disease.

As described above, most normal adult tissues show little or no detectable expression of FAP, and the plasma concentration of FAP in humans as measured by ELISA in healthy individuals is approximately 100ng/mL or 0.6 nmol/L. Thus, a "disease associated with aberrant FAP expression" preferably refers to a disease in which significant FAP expression is detected in an individual having the disease. More preferably, a "disease associated with aberrant expression of FAP" refers to a disease in which the FAP plasma concentration in an individual having the disease is at least about 1 μ g/mL, at least about 2 μ g/mL, at least about 3 μ g/mL, at least about 4 μ g/mL, at least about 5 μ g/mL, at least about 6 μ g/mL, at least about 7 μ g/mL, at least about 8 μ g/mL, at least about 9 μ g/mL, at least about 10 μ g/mL, at least about 15 μ g/mL, at least about 20 μ g/mL, at least about 25 μ g/mL, at least about 50 μ g/mL, at least about 100 μ g/mL, or more.

In embodiments of the invention, examples of diseases associated with aberrant expression of FAP include, but are not limited to: keloids, scleroderma, pulmonary fibrosis (in particular idiopathic pulmonary fibrosis), cirrhosis, arthritis (in particular osteoarthritis and rheumatoid arthritis), atherosclerosis, crohn's disease, basal cell carcinoma, squamous cell carcinoma, skin cancer, oral cancer (in particular oral squamous cell carcinoma), melanoma, esophageal cancer, adenocarcinoma, squamous cell carcinoma, gastric cancer (in particular intestinal gastric cancer), colorectal cancer, rectal cancer, pancreatic cancer, hepatocellular carcinoma, lung cancer (in particular non-small cell lung cancer), mesothelioma, cholangiocarcinoma, liver cancer, bladder cancer, breast cancer (e.g. ductal adenocarcinoma, lobular carcinoma, ductal carcinoma), kidney cancer (in particular renal clear cell carcinoma), prostate cancer, cervical cancer, ovarian cancer, glioma (in particular glioblastoma, interstitial glioblastoma, glioma), thyroid cancer (in particular papillary thyroid cancer, papillary carcinoma, and squamous cell carcinoma, Medullary thyroid carcinoma), parathyroid tumor, sarcoma (e.g., fibrosarcoma, leiomyosarcoma, malignant fibrous histiocytoma, low-grade myofibrosarcoma, osteosarcoma, osteoid tumor, and osteosarcoma), myeloma, astrocytoma.

In one embodiment of the present invention, the disease associated with abnormal FAP expression is idiopathic pulmonary fibrosis. In other embodiments of the invention, the disease associated with abnormal FAP expression is lung cancer, cholangiocarcinoma, liver cancer, glioma, ovarian cancer, breast cancer or pancreatic cancer.

Reagent kit

In one aspect of the invention, the invention provides a kit for diagnosing a disease associated with abnormal expression of FAP, the kit comprising a protein or polypeptide capable of binding to an antibody to FAP as defined herein.

In some embodiments of the invention, the protein or polypeptide capable of binding to the FAP antibody is the FAP full-length sequence, the extracellular domain of FAP, and/or a polypeptide comprising an epitope of FAP. Thus, in some embodiments, the kit comprises: (i) a FAP full-length sequence; (ii) the extracellular domain of FAP; and/or (iii) a polypeptide comprising an epitope of FAP.

In some embodiments, the kit further comprises additional reagents for detecting binding of the protein or polypeptide to FAP autoantibodies. Such additional agents include, but are not limited to, buffers (e.g., Tris, phosphate, and carbonate), stabilizers, excipients, biocides, and/or inert proteins (e.g., bovine serum albumin). For example, the protein or polypeptide and the additional agent may be provided as a lyophilized mixture, or the additional agent may be provided separately to the user for combined use. These additional agents are typically 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 concentration of protein/polypeptide.

In some embodiments, in the kit, the protein or polypeptide capable of binding to the FAP antibody is bound to (e.g., immobilized to) a solid support. Suitable solid supports are selected from the group consisting of enzyme plates, magnetic beads, polyvinylidene fluoride membranes, agarose beads, polystyrene and nitrocellulose membranes.

In some embodiments, the kit further comprises a secondary antibody with a probe label or unlabeled that is capable of recognizing the FAP autoantibody. In one embodiment, the secondary antibody with a probe label in the kit may be fluorescently modified or non-fluorescently modified.

In other embodiments, the kit further comprises a FAP antibody control at different concentrations. The FAP antibody control may be selected, for example, from artificially designed monoclonal antibodies as described above. .

In some embodiments, the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

In some embodiments, the protein or polypeptide capable of binding to the FAP antibody is labeled with a detectable label. In some embodiments, the label is selected from the group consisting of an enzymatic label, an isotopic label, a chemiluminescent label, a fluorescent label, and a dye.

Detection or diagnosis method and computer-readable storage medium

One aspect of the present invention provides a method for diagnosing a disease associated with abnormal expression of FAP in a subject, the method comprising determining the presence and/or amount of FAP autoantibodies in a sample obtained from the subject using a protein or polypeptide of the invention as described above.

Various immunoassays may be used in the diagnostic methods. In some embodiments, such immunoassays comprise the use of competitive and non-competitive detection systems such as radioimmunoassays, immunochromatography, ELISA, "sandwich" immunoassays, precipitation reactions, immunoblot analysis, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and the like. Both in vitro and in vivo assays can be used.

In one embodiment of the method of the invention, the assay is performed on a biological sample isolated from the subject.

Typically, the level of FAP autoantibody in the biological sample is compared to a reference level, wherein a deviation from the reference level is indicative of the presence and/or stage of the associated 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 the reference level indicates any significant change, e.g. 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 as compared to a reference level, e.g., as compared to a subject not suffering from a disease, can indicate the presence or risk of development of the associated disease in the subject.

The method for diagnosis allows quantitative and/or qualitative assessment, such as absolute and/or relative measurement of the target molecule, for example measuring the content of FAP autoantibodies.

In some embodiments of the methods of the invention, the presence of FAP autoantibodies or a higher amount of FAP autoantibodies as compared to a reference that does not suffer from a disease indicates that the subject suffers from a related disease.

In some embodiments of the methods of the invention, 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 an FAP autoantibody, and (ii) detecting the formation of a complex between the protein or polypeptide and the FAP autoantibody and/or determining the amount of the complex.

In some embodiments of the methods of the invention, the biological sample is pretreated, e.g., subjected to an enrichment procedure, prior to contacting with the protein or polypeptide of the invention to remove components that may interfere with antigen-antibody binding and/or to enrich for FAP autoantibodies that may be present in the biological sample.

In some embodiments, the sample is a bodily fluid sample. The term "body fluid sample" as used herein refers to any fluid sample from the body of a patient. The body fluid sample may be a blood sample, a urine sample, a sputum sample, a breast milk sample, a cerebrospinal fluid sample, an earwax (earwax) sample, an endolymph sample, an perilymph sample, a gastric fluid sample, a mucus sample, a peritoneal fluid sample, a pleural fluid sample, a saliva sample, a sebum (skin oil) sample, a semen sample, a sweat sample, a tear sample, a vaginal secretion sample, or a vomit sample, including components or fractions thereof. The bodily fluid samples may be mixed or combined. Thus, the body fluid sample may be a mixture of blood and urine samples or a mixture of blood and cerebrospinal fluid samples. The body fluid sample may be provided by removing body fluid from the patient, but may also be provided by using a previously isolated body fluid sample material. In a preferred embodiment, the sample is a whole blood sample or a blood fraction sample, such as a blood cell fraction, serum or plasma sample.

In some embodiments, the detection/diagnosis methods of the invention may be used in combination with other methods for detecting/diagnosing diseases associated with aberrant expression of FAP. For example, the detection/diagnostic methods of the present invention may be used in conjunction with the detection of other biomarkers of pulmonary fibrosis. For another example, the detection/diagnostic methods of the present invention can be used in conjunction with the detection of other biomarkers of cancer.

In some embodiments of the invention, a computer-readable storage medium having stored thereon computer instructions for reading and execution by a computer, the computer instructions being executable to perform a method of detecting the presence of FAP autoantibodies in a biological sample of a subject, the method comprising: (a) contacting a sample of a subject with a protein or polypeptide of the invention; (b) detecting and reading the signal of the contacted sample to detect whether the protein and the polypeptide form a complex with FAP autoantibody in the sample; and (c) determining whether the signal exceeds a predetermined threshold, and determining that an FAP autoantibody is present in the sample when the signal exceeds a predetermined threshold.

In some embodiments, 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 associated 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 the reference level indicates any significant change, e.g. 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 as compared to a reference level, e.g., as compared to a subject not suffering from a disease, can indicate the presence or risk of development of the associated disease in the subject.

Examples

Example 1 purification of FAP extracellular domain protein expression

Eukaryotic expression vector construction

Designing a primer:

search for the mRNA sequence of the human FAP gene (NM-004460.5) from the Gene Bank (GeneBank) primers containing the CDS sequence of the FAP gene were designed with a Primer blast plug-in the NCBI website to allow efficient amplification. Adding a proper restriction enzyme cutting site at the 5' end of the upstream and downstream primers of the target gene according to the requirement, and adding a corresponding protective base before the enzyme cutting site.

PCR amplification of FAP target gene:

the FAP gene was amplified by PCR using a plasmid containing the FAP gene (FAP-pUC19) as a template.

The PCR reaction was prepared according to the following table:

PCR amplification procedure:

1% agarose gel detection of PCR amplification products: mix 2 μ L PCR amplification product with 0.5 μ L DNA loading buffer, add to 1% agarose gel containing 1:10000EB substitute, and electrophoresis at 90V for 15 min.

Gel imaging: and (3) taking out the gel after the electrophoresis is finished, and placing the gel in a gel imaging system to observe whether the amplified band is single or not and whether the band size is correct or not. And if the band is single and the size of the band is consistent with the expected size, performing gel cutting and recovery on the target nucleic acid band.

And (3) carrying out enzyme digestion, connection and detection on the PCR product and a target vector:

(1) and (3) carrying out enzyme digestion and reaction system on the PCR product and the target plasmid:

the mixture is subjected to thermostatic water bath for 3 hours at 37 ℃. Detecting whether the product is completely digested by 1% agarose gel in a trace manner, and performing gel cutting recovery or directly purifying the target fragment according to the requirement.

(2) Ligation of DNA to the vector of interest

The ligation system was formulated as follows:

the reaction was carried out at 16 ℃ overnight.

Transformation and identification of ligation products:

(1) DH 5. alpha. was taken out from a-80 ℃ ultra-low temperature freezer and dissolved on ice, after complete dissolution 10. mu.L of ligation product was added and ice-cooled for 30 min.

(2) The ice-bath competent cells were placed in a preset thermostatic water bath at 42 ℃ for 90s by heat shock, and then rapidly placed on ice and allowed to stand for 2 min.

(3) Add 500. mu.L of liquid LB medium and incubate the competent cells at 37 ℃ on a shaker at 200rpm for 1 h.

(4) Taking a proper amount of culture product, uniformly coating the culture product on LB solid plates with corresponding resistance (plates with different resistances are selected according to the resistance of the target vector), and placing the plates in a constant temperature incubator at 37 ℃ until a monoclonal antibody grows out.

(5) Selecting a single clone in the plate, preparing a colony PCR identification reaction system by referring to the PCR reaction system, carrying out PCR amplification, detecting whether the single clone to be detected carries a target gene fragment by using 1% agarose gel, selecting a corresponding positive clone for sequencing, and determining the correctness of the fragment sequence, wherein the sequencing result is shown as SEQ ID NO. 2.

Protein expression and purification

Preparation of transfected cells:

(1) approximately 14-16 10cm dishes of 293T cells were prepared prior to transfection, and 1 dish of cells was passaged 1/3, which required 3 days to transfer to 16 dishes.

(2) On day 4, i.e., the day before transfection, 293T cells from 14-16 (depending on cell growth density) dishes were seeded into 8T 175 cell flasks to reach a 90% density at the growth density at the next day of transfection.

Transfection:

(1) the melted plasmid was mixed by shaking and vortexed to bring the liquid to the bottom of the EP tube (to avoid contamination, the plasmid was filtered through a 0.22 μm filter before transfection).

(2) The PEI was left to melt at room temperature.

(3) Two 50ml centrifuge tubes were placed in a biosafety cabinet (transfection mix prepared for 8 flasks of cells) and 7.2 × 4 ═ 28.8ml serum-free medium was added to each tube.

(4) Add 27. mu.g of plasmid 4 to each 50ml tube and gently rotate the tube to mix well.

(5) PEI was centrifuged by pipetting and 150. mu.l of PEI (1. mu.g/. mu.l) was added to each 50ml tube, the lid was quickly closed, vortexed for 20s and mixed well and incubated at room temperature for 15 min.

(6) When 5min had left the end of the incubation (time adjusted according to the number of transfected cell flasks), the supernatant of 293T cells was aspirated off in vacuo.

(7) Carefully add 7.4ml of transfection mixture gently along the side wall of the vial to the T175 cell vial, carefully invert the cell vial to completely cover the 293T cell surface with liquid at 37 ℃ and 5% CO₂An incubator.

(8) After 3-6 hours, each flask was supplemented with 25ml serum-free DMEM medium and the culture was continued at 37 ℃ with 5% CO 2.

(9) The supernatant was collected after 72-96 hours.

Protein purification:

(1) the transfection supernatant from 8 flasks 175 was collected aseptically and added to the conical flask and centrifuged at 4000rpm for 1 hour at 4 ℃.

(2) And when 10 minutes are left after the centrifugation is finished, adding 1mL of nickel column filler balanced by Binding buffer in advance into each tube, and combining the tubes by a decoloring shaker at 4 ℃ for 2-3 hours.

(3)3000g, centrifuged at 4 ℃ for 5min, the packing was collected and carefully transferred to a gravity column.

(4) After all the liquid flows out, the Beads are washed by 1 × Native Wash Buffer, the elution times are determined according to the impurity content in the eluent, and the elution is carried out for 7 times in the experiment.

(5) After the Elution solution was drained from the gravity column, the target protein was eluted with 20mL of 1 × Native Elution Buffer, and the eluate was collected with a 30KD ultrafiltration tube.

(6)3000g, centrifuging at 4 ℃ to obtain target protein about 1mL, subpackaging the target protein and freezing and storing in a refrigerator at-80 ℃ to avoid repeated freezing and thawing.

Results

FIG. 4 shows the results of detection of eukaryotic recombinant proteins expressing the extracellular domain of FAP by SDS-PAGE (A) and Western Blot (B). In A, 1: marker, 2: 100ng of recombinant protein, 3: 500ng of recombinant protein. 1 in B: marker, 2: 500ng of recombinant protein. As can be seen, the expressed recombinant protein of the extracellular domain has a molecular weight of about 97kDa, which is consistent with the expected molecular weight.

Example 2ELISA detection of FAP autoantibodies in peripheral blood

Serum separation

(1) The yellow head separation gel-coagulant tube draws 4-5ml of whole blood, immediately turns upside down and uniformly mixes for several times, stands for several minutes at room temperature (blood is coagulated, serum is separated out), then centrifugalizes, and can be placed in a refrigerator at 4 ℃ for a short time if not processed in time.

(2) Precooling at 4 ℃ by using a centrifuge.

(3) Centrifuging at 2500rpm at 4 deg.C for 10 min, carefully sucking the supernatant, transferring to new EP tube, and freezing at-80 deg.C.

(4) The method comprises the following steps of collecting 65 serum samples in total, wherein the serum samples comprise 20 normal human serum samples, 5 serum samples of Idiopathic Pulmonary Fibrosis (IPF) patients, and 40 tumor samples (16 lung cancer samples, 3 breast cancer samples, 4 ovarian cancer samples, 5 liver cancer samples, 3 bile duct cancer samples, 4 pancreatic cancer samples and 5 glioma samples).

ELISA detection

(1) The FAP extracellular domain antigen protein obtained in example 1 was diluted with coating buffer to a concentration of 1. mu.g/ml, 100. mu.l per well, and coated overnight at 4 ℃.

(2) And (3) sealing: the plate was washed once, 300. mu.l of blocking solution was added to each well, and blocked at 37 ℃ for 2 h.

(3) Adding a primary antibody: the plate was washed once and the primary antibody was diluted in a gradient of blocking solution (the serum from the patient collected above, starting concentration 1:30, diluted in a gradient of 3 times; supernatant of the culture medium, starting concentration stock solution, diluted in a gradient of 3 times), 100. mu.l per well and incubated at 37 ℃ for 1 hour. The positive control was FAP positive antibody (CST: 66562S), the negative control was irrelevant antibody, and the initial concentration was 1: 400.

(4) Adding a secondary antibody: washing the plate twice, adding 100 μ l goat anti-human IgG-HRP to each well, diluting with blocking solution at a ratio of 1:10000, adding 15% goat serum, and incubating at 37 deg.C for 1 h.

(5) Color development and termination: the plate was washed twice with 100. mu.l of TMB per well, left at room temperature for about 6min, and immediately washed with 50. mu.l of 2M H₂SO₄And (6) terminating.

(6) Reading by a microplate reader, and detecting the OD value by using the dual wavelength of 450-.

Results

The results are shown in FIG. 5. FAP autoantibodies were significantly higher in tumor and IPF patient sera than normal controls (n-20) at the 1:900 dilution. 5 IPF samples were screened, 3 of which were positive for FAP autoantibodies with a positive rate of 60%. Screening 40 tumor samples, wherein 32 FAP autoantibodies are positive, and the positive rate is 80%; wherein, the lung cancer accounts for 16 parts, and the positive rate is 87.5 percent; 3 parts of bile duct cancer, wherein the positive rate is 100%; 5 parts of liver cancer, the positive rate is 100%; 5 parts of glioma, and the positive rate is 60%; 4 parts of ovarian cancer, wherein the positive rate is 100%; 3 parts of breast cancer, wherein the positive rate is 66.7%; pancreatic cancer 4 portions, positive rate 75%.

Example 3 immunofluorescence detection of FAP autoantibodies in peripheral blood

Construction of stably expressing FAP cell line

(1) Finding and confirming the full-length sequence (shown as SEQ ID NO. 1) of the FAP gene according to NCBI, constructing an FAP lentivirus over-expression vector, and packaging the virus in vitro after sequencing verification.

(2) Taking A549 cells in logarithmic growth phase, counting cells, adjusting cell density, and transmitting to 2 × 10⁶Individual cells were transferred to new petri dishes for the next day of viral infection.

(3) And (3) replacing 6ml of complete culture medium for the cells in the step (2), sucking 1ml of complete culture medium to dilute the over-expressed FAP lentivirus obtained in the step (1), slowly and uniformly dripping a virus diluent into the cell culture medium, culturing in a constant-temperature cell culture box at 37 ℃ for 48 hours, and then replacing with the new complete culture medium.

(4) And (3) carrying out cell passage after the cells grow to be full, adding 2 mu g/ml puromycin into a new culture medium for drug screening, and determining that the A549 cells stably express FAP through continuous drug screening of 3-4 generations.

Cellular immunofluorescence detection of FAP autoantibodies in peripheral blood

(1) Taking A549 and A549: FAP cells in logarithmic growth phase, counting the cells, adjusting the cell density, paving a 24-hole plate according to 50000/hole, and culturing overnight in a constant-temperature incubator at 37 ℃ for immunofluorescence detection.

(2) Cell fixation: the medium in 24-well plates was discarded, and 500. mu.l of pre-cooled 4% paraformaldehyde was added to each well to fix the cells for 15 minutes at 4 ℃.

(3) Cell permeation: the fixed solution was washed with PBS, and 500. mu.l of a 0.5% Triton-X100 permeation solution prepared in advance was added to each well, followed by permeation for 15 minutes at room temperature.

(4) And (3) sealing: the permeate was washed with PBS, 500. mu.l of pre-prepared 15% blocking goat serum was added to each well and blocked for 1 hour at room temperature.

(5) Incubation of serum to be tested (taking serum of IPF patient, positive sample of ELISA test result (IPF-1) and normal human serum, and negative sample of ELISA test result (D4) in example 2 as an example): diluting the serum to be detected by PBS according to the proportion of 1:10, dripping 200 mul of serum diluent to be detected into each hole, taking the complete covering of cells as the standard, and incubating the cell culture plate at 4 ℃ overnight. The positive control was FAP positive antibody (CST: 66562S) and was assayed at 1:400 starting concentration.

(6) And (3) secondary antibody incubation: the incubation serum was washed with PBS, and a pre-prepared FITC-labeled fluorescent secondary antibody of goat anti-human IgG was added dropwise, and the positive antibody was a FITC-labeled fluorescent secondary antibody of goat anti-rabbit IgG, and incubated at room temperature for 30 minutes.

(7) Nuclear counterstaining: and (3) washing the fluorescent secondary antibody by PBS, dripping a proper amount of DAPI nuclear dye containing an anti-fluorescence quencher for nuclear counterstaining, and performing data acquisition by a fluorescence microscope after the counterstaining is carried out for 15 minutes.

Results

The results are shown in fig. 6, and the sera to be tested all have a weak fluorescence signal in a549 cells without FAP expression at a dilution of 1: 10. In the A549 cells over-expressing FAP, IPF-1(ELISA detection shows positive) has a remarkable fluorescence signal compared with D4 (normal human serum sample, ELISA detection shows negative), and the fluorescence signal of IPF-1 in the A549 cells over-expressing FAP is remarkably higher than that of A549 blank cells.

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Val Thr Asp Glu Arg Val Cys Leu Gln Trp Leu Lys Arg Val Gln Asn

275 280 285

Val Ser Val Leu Ser Ile Cys Asp Phe Arg Glu Asp Trp Gln Thr Trp

290 295 300

Asp Cys Pro Lys Thr Gln Glu His Ile Glu Glu Ser Arg Thr Gly Trp

305 310 315 320

Ala Gly Gly Phe Phe Val Ser Thr Pro Val Phe Ser Tyr Asp Ala Ile

325 330 335

Ser Tyr Tyr Lys Ile Phe Ser Asp Lys Asp Gly Tyr Lys His Ile His

340 345 350

Tyr Ile Lys Asp Thr Val Glu Asn Ala Ile Gln Ile Thr Ser Gly Lys

355 360 365

Trp Glu Ala Ile Asn Ile Phe Arg Val Thr Gln Asp Ser Leu Phe Tyr

370 375 380

Ser Ser Asn Glu Phe Glu Glu Tyr Pro Gly Arg Arg Asn Ile Tyr Arg

385 390 395 400

Ile Ser Ile Gly Ser Tyr Pro Pro Ser Lys Lys Cys Val Thr Cys His

405 410 415

Leu Arg Lys Glu Arg Cys Gln Tyr Tyr Thr Ala Ser Phe Ser Asp Tyr

420 425 430

Ala Lys Tyr Tyr Ala Leu Val Cys Tyr Gly Pro Gly Ile Pro Ile Ser

435 440 445

Thr Leu His Asp Gly Arg Thr Asp Gln Glu Ile Lys Ile Leu Glu Glu

450 455 460

Asn Lys Glu Leu Glu Asn Ala Leu Lys Asn Ile Gln Leu Pro Lys Glu

465 470 475 480

Glu Ile Lys Lys Leu Glu Val Asp Glu Ile Thr Leu Trp Tyr Lys Met

485 490 495

Ile Leu Pro Pro Gln Phe Asp Arg Ser Lys Lys Tyr Pro Leu Leu Ile

500 505 510

Gln Val Tyr Gly Gly Pro Cys Ser Gln Ser Val Arg Ser Val Phe Ala

515 520 525

Val Asn Trp Ile Ser Tyr Leu Ala Ser Lys Glu Gly Met Val Ile Ala

530 535 540

Leu Val Asp Gly Arg Gly Thr Ala Phe Gln Gly Asp Lys Leu Leu Tyr

545 550 555 560

Ala Val Tyr Arg Lys Leu Gly Val Tyr Glu Val Glu Asp Gln Ile Thr

565 570 575

Ala Val Arg Lys Phe Ile Glu Met Gly Phe Ile Asp Glu Lys Arg Ile

580 585 590

Ala Ile Trp Gly Trp Ser Tyr Gly Gly Tyr Val Ser Ser Leu Ala Leu

595 600 605

Ala Ser Gly Thr Gly Leu Phe Lys Cys Gly Ile Ala Val Ala Pro Val

610 615 620

Ser Ser Trp Glu Tyr Tyr Ala Ser Val Tyr Thr Glu Arg Phe Met Gly

625 630 635 640

Leu Pro Thr Lys Asp Asp Asn Leu Glu His Tyr Lys Asn Ser Thr Val

645 650 655

Met Ala Arg Ala Glu Tyr Phe Arg Asn Val Asp Tyr Leu Leu Ile His

660 665 670

Gly Thr Ala Asp Asp Asn Val His Phe Gln Asn Ser Ala Gln Ile Ala

675 680 685

Lys Ala Leu Val Asn Ala Gln Val Asp Phe Gln Ala Met Trp Tyr Ser

690 695 700

Asp Gln Asn His Gly Leu Ser Gly Leu Ser Thr Asn His Leu Tyr Thr

705 710 715 720

His Met Thr His Phe Leu Lys Gln Cys Phe Ser Leu Ser Asp

725 730

<210> 3

<211> 77

<212> PRT

<213> Artificial sequence ()

<400> 3

Glu Arg Cys Gln Tyr Tyr Thr Ala Ser Phe Ser Asp Tyr Ala Lys Tyr

1 5 10 15

Tyr Ala Leu Val Cys Tyr Gly Pro Gly Ile Pro Ile Ser Thr Leu His

20 25 30

Asp Gly Arg Thr Asp Gln Glu Ile Lys Ile Leu Glu Glu Asn Lys Glu

35 40 45

Leu Glu Asn Ala Leu Lys Asn Ile Gln Leu Pro Lys Glu Glu Ile Lys

50 55 60

Lys Leu Glu Val Asp Glu Ile Thr Leu Trp Tyr Lys Met

65 70 75

<210> 4

<211> 17

<212> PRT

<213> Artificial sequence ()

<400> 4

Phe Phe Pro Asn Trp Ile Ser Gly Gln Glu Tyr Leu His Gln Ser Ala

1 5 10 15

Asp

<210> 5

<211> 100

<212> PRT

<213> Artificial sequence ()

<400> 5

Pro Pro Gln Phe Asp Arg Ser Lys Lys Tyr Pro Leu Leu Ile Gln Val

1 5 10 15

Tyr Gly Gly Pro Cys Ser Gln Ser Val Arg Ser Val Phe Ala Val Asn

20 25 30

Trp Ile Ser Tyr Leu Ala Ser Lys Glu Gly Met Val Ile Ala Leu Val

35 40 45

Asp Gly Arg Gly Thr Ala Phe Gln Gly Asp Lys Leu Leu Tyr Ala Val

50 55 60

Tyr Arg Lys Leu Gly Val Tyr Glu Val Glu Asp Gln Ile Thr Ala Val

65 70 75 80

Arg Lys Phe Ile Glu Met Gly Phe Ile Asp Glu Lys Arg Ile Ala Ile

85 90 95

Trp Gly Trp Ser

100

<210> 6

<211> 50

<212> PRT

<213> Artificial sequence ()

<400> 6

Ser Trp Glu Tyr Tyr Ala Ser Val Tyr Thr Glu Arg Phe Met Gly Leu

1 5 10 15

Pro Thr Lys Asp Asp Asn Leu Glu His Tyr Lys Asn Ser Thr Val Met

20 25 30

Ala Arg Ala Glu Tyr Phe Arg Asn Val Asp Tyr Leu Leu Ile His Gly

35 40 45

Thr Ala

50

<210> 7

<211> 238

<212> PRT

<213> Artificial sequence ()

<400> 7

Ile Leu Pro Pro Gln Phe Asp Arg Ser Lys Lys Tyr Pro Leu Leu Ile

1 5 10 15

Gln Val Tyr Gly Gly Pro Cys Ser Gln Ser Val Arg Ser Val Phe Ala

20 25 30

Val Asn Trp Ile Ser Tyr Leu Ala Ser Lys Glu Gly Met Val Ile Ala

35 40 45

Leu Val Asp Gly Arg Gly Thr Ala Phe Gln Gly Asp Lys Leu Leu Tyr

50 55 60

Ala Val Tyr Arg Lys Leu Gly Val Tyr Glu Val Glu Asp Gln Ile Thr

65 70 75 80

Ala Val Arg Lys Phe Ile Glu Met Gly Phe Ile Asp Glu Lys Arg Ile

85 90 95

Ala Ile Trp Gly Trp Ser Tyr Gly Gly Tyr Val Ser Ser Leu Ala Leu

100 105 110

Ala Ser Gly Thr Gly Leu Phe Lys Cys Gly Ile Ala Val Ala Pro Val

115 120 125

Ser Ser Trp Glu Tyr Tyr Ala Ser Val Tyr Thr Glu Arg Phe Met Gly

130 135 140

Leu Pro Thr Lys Asp Asp Asn Leu Glu His Tyr Lys Asn Ser Thr Val

145 150 155 160

Met Ala Arg Ala Glu Tyr Phe Arg Asn Val Asp Tyr Leu Leu Ile His

165 170 175

Gly Thr Ala Asp Asp Asn Val His Phe Gln Asn Ser Ala Gln Ile Ala

180 185 190

Lys Ala Leu Val Asn Ala Gln Val Asp Phe Gln Ala Met Trp Tyr Ser

195 200 205

Asp Gln Asn His Gly Leu Ser Gly Leu Ser Thr Asn His Leu Tyr Thr

210 215 220

His Met Thr His Phe Leu Lys Gln Cys Phe Ser Leu Ser Asp

225 230 235

<210> 8

<211> 101

<212> PRT

<213> Artificial sequence ()

<400> 8

Pro Pro Gln Phe Asp Arg Ser Lys Lys Tyr Pro Leu Leu Ile Gln Val

1 5 10 15

Tyr Gly Gly Pro Cys Ser Gln Ser Val Arg Ser Val Phe Ala Val Asn

20 25 30

Trp Ile Ser Tyr Leu Ala Ser Lys Glu Gly Met Val Ile Ala Leu Val

35 40 45

Asp Gly Arg Gly Thr Ala Phe Gln Gly Asp Lys Leu Leu Tyr Ala Val

50 55 60

Tyr Arg Lys Leu Gly Val Tyr Glu Val Glu Asp Gln Ile Thr Ala Val

65 70 75 80

Arg Lys Phe Ile Glu Met Gly Phe Ile Asp Glu Lys Arg Ile Ala Ile

85 90 95

Trp Gly Trp Ser Tyr

100

<210> 9

<211> 253

<212> PRT

<213> Artificial sequence ()

<400> 9

Met Lys Ser Val Asn Ala Ser Asn Tyr Gly Leu Ser Pro Asp Arg Gln

1 5 10 15

Phe Val Tyr Leu Glu Ser Asp Tyr Ser Lys Leu Trp Arg Tyr Ser Tyr

20 25 30

Thr Ala Thr Tyr Tyr Ile Tyr Asp Leu Ser Asn Gly Glu Phe Val Arg

35 40 45

Gly Asn Glu Leu Pro Arg Pro Ile Gln Tyr Leu Cys Trp Ser Pro Val

50 55 60

Gly Ser Lys Leu Ala Tyr Val Tyr Gln Asn Asn Ile Tyr Leu Lys Gln

65 70 75 80

Arg Pro Gly Asp Pro Pro Phe Gln Ile Thr Phe Asn Gly Arg Glu Asn

85 90 95

Lys Ile Phe Asn Gly Ile Pro Asp Trp Val Tyr Glu Glu Glu Met Leu

100 105 110

Ala Thr Lys Tyr Ala Leu Trp Trp Ser Pro Asn Gly Lys Phe Leu Ala

115 120 125

Tyr Ala Glu Phe Asn Asp Thr Asp Ile Pro Val Ile Ala Tyr Ser Tyr

130 135 140

Tyr Gly Asp Glu Gln Tyr Pro Arg Thr Ile Asn Ile Pro Tyr Pro Lys

145 150 155 160

Ala Gly Ala Lys Asn Pro Val Val Arg Ile Phe Ile Ile Asp Thr Thr

165 170 175

Tyr Pro Ala Tyr Val Gly Pro Gln Glu Val Pro Val Pro Ala Met Ile

180 185 190

Ala Ser Ser Asp Tyr Tyr Phe Ser Trp Leu Thr Trp Val Thr Asp Glu

195 200 205

Arg Val Cys Leu Gln Trp Leu Lys Arg Val Gln Asn Val Ser Val Leu

210 215 220

Ser Ile Cys Asp Phe Arg Glu Asp Trp Gln Thr Trp Asp Cys Pro Lys

225 230 235 240

Thr Gln Glu His Ile Glu Glu Ser Arg Thr Gly Trp Ala

245 250

<210> 10

<211> 101

<212> PRT

<213> Artificial sequence ()

<400> 10

Thr Asp Glu Arg Val Cys Leu Gln Trp Leu Lys Arg Val Gln Asn Val

1 5 10 15

Ser Val Leu Ser Ile Cys Asp Phe Arg Glu Asp Trp Gln Thr Trp Asp

20 25 30

Cys Pro Lys Thr Gln Glu His Ile Glu Glu Ser Arg Thr Gly Trp Ala

35 40 45

Gly Gly Phe Phe Val Ser Thr Pro Val Phe Ser Tyr Asp Ala Ile Ser

50 55 60

Tyr Tyr Lys Ile Phe Ser Asp Lys Asp Gly Tyr Lys His Ile His Tyr

65 70 75 80

Ile Lys Asp Thr Val Glu Asn Ala Ile Gln Ile Thr Ser Gly Lys Trp

85 90 95

Glu Ala Ile Asn Ile

100

<210> 11

<211> 283

<212> PRT

<213> Artificial sequence ()

<400> 11

Met Lys Ser Val Asn Ala Ser Asn Tyr Gly Leu Ser Pro Asp Arg Gln

1 5 10 15

Phe Val Tyr Leu Glu Ser Asp Tyr Ser Lys Leu Trp Arg Tyr Ser Tyr

20 25 30

Thr Ala Thr Tyr Tyr Ile Tyr Asp Leu Ser Asn Gly Glu Phe Val Arg

35 40 45

Gly Asn Glu Leu Pro Arg Pro Ile Gln Tyr Leu Cys Trp Ser Pro Val

50 55 60

Gly Ser Lys Leu Ala Tyr Val Tyr Gln Asn Asn Ile Tyr Leu Lys Gln

65 70 75 80

Arg Pro Gly Asp Pro Pro Phe Gln Ile Thr Phe Asn Gly Arg Glu Asn

85 90 95

Lys Ile Phe Asn Gly Ile Pro Asp Trp Val Tyr Glu Glu Glu Met Leu

100 105 110

Ala Thr Lys Tyr Ala Leu Trp Trp Ser Pro Asn Gly Lys Phe Leu Ala

115 120 125

Tyr Ala Glu Phe Asn Asp Thr Asp Ile Pro Val Ile Ala Tyr Ser Tyr

130 135 140

Tyr Gly Asp Glu Gln Tyr Pro Arg Thr Ile Asn Ile Pro Tyr Pro Lys

145 150 155 160

Ala Gly Ala Lys Asn Pro Val Val Arg Ile Phe Ile Ile Asp Thr Thr

165 170 175

Tyr Pro Ala Tyr Val Gly Pro Gln Glu Val Pro Val Pro Ala Met Ile

180 185 190

Ala Ser Ser Asp Tyr Tyr Phe Ser Trp Leu Thr Trp Val Thr Asp Glu

195 200 205

Arg Val Cys Leu Gln Trp Leu Lys Arg Val Gln Asn Val Ser Val Leu

210 215 220

Ser Ile Cys Asp Phe Arg Glu Asp Trp Gln Thr Trp Asp Cys Pro Lys

225 230 235 240

Thr Gln Glu His Ile Glu Glu Ser Arg Thr Gly Trp Ala Gly Gly Phe

245 250 255

Phe Val Ser Thr Pro Val Phe Ser Tyr Asp Ala Ile Ser Tyr Tyr Lys

260 265 270

Ile Phe Ser Asp Lys Asp Gly Tyr Lys His Ile

275 280

<210> 12

<211> 81

<212> PRT

<213> Artificial sequence ()

<400> 12

Thr Asp Glu Arg Val Cys Leu Gln Trp Leu Lys Arg Val Gln Asn Val

1 5 10 15

Ser Val Leu Ser Ile Cys Asp Phe Arg Glu Asp Trp Gln Thr Trp Asp

20 25 30

Cys Pro Lys Thr Gln Glu His Ile Glu Glu Ser Arg Thr Gly Trp Ala

35 40 45

Gly Gly Phe Phe Val Ser Thr Pro Val Phe Ser Tyr Asp Ala Ile Ser

50 55 60

Tyr Tyr Lys Ile Phe Ser Asp Lys Asp Gly Tyr Lys His Ile His Tyr

65 70 75 80

Ile

<210> 13

<211> 51

<212> PRT

<213> Artificial sequence ()

<400> 13

Phe Phe Val Ser Thr Pro Val Phe Ser Tyr Asp Ala Ile Ser Tyr Tyr

1 5 10 15

Lys Ile Phe Ser Asp Lys Asp Gly Tyr Lys His Ile His Tyr Ile Lys

20 25 30

Asp Thr Val Glu Asn Ala Ile Gln Ile Thr Ser Gly Lys Trp Glu Ala

35 40 45

Ile Asn Ile

50

<210> 14

<211> 735

<212> PRT

<213> Artificial sequence ()

<400> 14

Leu Arg Pro Ser Arg Val His Asn Ser Glu Glu Asn Thr Met Arg Ala

1 5 10 15

Leu Thr Leu Lys Asp Ile Leu Asn Gly Thr Phe Ser Tyr Lys Thr Phe

20 25 30

Phe Pro Asn Trp Ile Ser Gly Gln Glu Tyr Leu His Gln Ser Ala Asp

35 40 45

Asn Asn Ile Val Leu Tyr Asn Ile Glu Thr Gly Gln Ser Tyr Thr Ile

50 55 60

Leu Ser Asn Arg Thr Met Lys Ser Val Asn Ala Ser Asn Tyr Gly Leu

65 70 75 80

Ser Pro Asp Arg Gln Phe Val Tyr Leu Glu Ser Asp Tyr Ser Lys Leu

85 90 95

Trp Arg Tyr Ser Tyr Thr Ala Thr Tyr Tyr Ile Tyr Asp Leu Ser Asn

100 105 110

Gly Glu Phe Val Arg Gly Asn Glu Leu Pro Arg Pro Ile Gln Tyr Leu

115 120 125

Cys Trp Ser Pro Val Gly Ser Lys Leu Ala Tyr Val Tyr Gln Asn Asn

130 135 140

Ile Tyr Leu Lys Gln Arg Pro Gly Asp Pro Pro Phe Gln Ile Thr Phe

145 150 155 160

Asn Gly Arg Glu Asn Lys Ile Phe Asn Gly Ile Pro Asp Trp Val Tyr

165 170 175

Glu Glu Glu Met Leu Ala Thr Lys Tyr Ala Leu Trp Trp Ser Pro Asn

180 185 190

Gly Lys Phe Leu Ala Tyr Ala Glu Phe Asn Asp Thr Asp Ile Pro Val

195 200 205

Ile Ala Tyr Ser Tyr Tyr Gly Asp Glu Gln Tyr Pro Arg Thr Ile Asn

210 215 220

Ile Pro Tyr Pro Lys Ala Gly Ala Lys Asn Pro Val Val Arg Ile Phe

225 230 235 240

Ile Ile Asp Thr Thr Tyr Pro Ala Tyr Val Gly Pro Gln Glu Val Pro

245 250 255

Val Pro Ala Met Ile Ala Ser Ser Asp Tyr Tyr Phe Ser Trp Leu Thr

260 265 270

Trp Val Thr Asp Glu Arg Val Cys Leu Gln Trp Leu Lys Arg Val Gln

275 280 285

Asn Val Ser Val Leu Ser Ile Cys Asp Phe Arg Glu Asp Trp Gln Thr

290 295 300

Trp Asp Cys Pro Lys Thr Gln Glu His Ile Glu Glu Ser Arg Thr Gly

305 310 315 320

Trp Ala Gly Gly Phe Phe Val Ser Thr Pro Val Phe Ser Tyr Asp Ala

325 330 335

Ile Ser Tyr Tyr Lys Ile Phe Ser Asp Lys Asp Gly Tyr Lys His Ile

340 345 350

His Tyr Ile Lys Asp Thr Val Glu Asn Ala Ile Gln Ile Thr Ser Gly

355 360 365

Lys Trp Glu Ala Ile Asn Ile Phe Arg Val Thr Gln Asp Ser Leu Phe

370 375 380

Tyr Ser Ser Asn Glu Phe Glu Glu Tyr Pro Gly Arg Arg Asn Ile Tyr

385 390 395 400

Arg Ile Ser Ile Gly Ser Tyr Pro Pro Ser Lys Lys Cys Val Thr Cys

405 410 415

His Leu Arg Lys Glu Arg Cys Gln Tyr Tyr Thr Ala Ser Phe Ser Asp

420 425 430

Tyr Ala Lys Tyr Tyr Ala Leu Val Cys Tyr Gly Pro Gly Ile Pro Ile

435 440 445

Ser Thr Leu His Asp Gly Arg Thr Asp Gln Glu Ile Lys Ile Leu Glu

450 455 460

Glu Asn Lys Glu Leu Glu Asn Ala Leu Lys Asn Ile Gln Leu Pro Lys

465 470 475 480

Glu Glu Ile Lys Lys Leu Glu Val Asp Glu Ile Thr Leu Trp Tyr Lys

485 490 495

Met Ile Leu Pro Pro Gln Phe Asp Arg Ser Lys Lys Tyr Pro Leu Leu

500 505 510

Ile Gln Val Tyr Gly Gly Pro Cys Ser Gln Ser Val Arg Ser Val Phe

515 520 525

Ala Val Asn Trp Ile Ser Tyr Leu Ala Ser Lys Glu Gly Met Val Ile

530 535 540

Ala Leu Val Asp Gly Arg Gly Thr Ala Phe Gln Gly Asp Lys Leu Leu

545 550 555 560

Tyr Ala Val Tyr Arg Lys Leu Gly Val Tyr Glu Val Glu Asp Gln Ile

565 570 575

Thr Ala Val Arg Lys Phe Ile Glu Met Gly Phe Ile Asp Glu Lys Arg

580 585 590

Ile Ala Ile Trp Gly Trp Ser Tyr Gly Gly Tyr Val Ser Ser Leu Ala

595 600 605

Leu Ala Ser Gly Thr Gly Leu Phe Lys Cys Gly Ile Ala Val Ala Pro

610 615 620

Val Ser Ser Trp Glu Tyr Tyr Ala Ser Val Tyr Thr Glu Arg Phe Met

625 630 635 640

Gly Leu Pro Thr Lys Asp Asp Asn Leu Glu His Tyr Lys Asn Ser Thr

645 650 655

Val Met Ala Arg Ala Glu Tyr Phe Arg Asn Val Asp Tyr Leu Leu Ile

660 665 670

His Gly Thr Ala Asp Asp Asn Val His Phe Gln Asn Ser Ala Gln Ile

675 680 685

Ala Lys Ala Leu Val Asn Ala Gln Val Asp Phe Gln Ala Met Trp Tyr

690 695 700

Ser Asp Gln Asn His Gly Leu Ser Gly Leu Ser Thr Asn His Leu Tyr

705 710 715 720

Thr His Met Thr His Phe Leu Lys Gln Cys Phe Ser Leu Ser Asp

725 730 735

<210> 15

<211> 255

<212> PRT

<213> Artificial sequence ()

<400> 15

Leu Arg Pro Ser Arg Val His Asn Ser Glu Glu Asn Thr Met Arg Ala

1 5 10 15

Leu Thr Leu Lys Asp Ile Leu Asn Gly Thr Phe Ser Tyr Lys Thr Phe

20 25 30

Phe Pro Asn Trp Ile Ser Gly Gln Glu Tyr Leu His Gln Ser Ala Asp

35 40 45

Asn Asn Ile Val Leu Tyr Asn Ile Glu Thr Gly Gln Ser Tyr Thr Ile

50 55 60

Leu Ser Asn Arg Thr Met Lys Ser Val Asn Ala Ser Asn Tyr Gly Leu

65 70 75 80

Ser Pro Asp Arg Gln Phe Val Tyr Leu Glu Ser Asp Tyr Ser Lys Leu

85 90 95

Trp Arg Tyr Ser Tyr Thr Ala Thr Tyr Tyr Ile Tyr Asp Leu Ser Asn

100 105 110

Gly Glu Phe Val Arg Gly Asn Glu Leu Pro Arg Pro Ile Gln Tyr Leu

115 120 125

Cys Trp Ser Pro Val Gly Ser Lys Leu Ala Tyr Val Tyr Gln Asn Asn

130 135 140

Ile Tyr Leu Lys Gln Arg Pro Gly Asp Pro Pro Phe Gln Ile Thr Phe

145 150 155 160

Asn Gly Arg Glu Asn Lys Ile Phe Asn Gly Ile Pro Asp Trp Val Tyr

165 170 175

Glu Glu Glu Met Leu Ala Thr Lys Tyr Ala Leu Trp Trp Ser Pro Asn

180 185 190

Gly Lys Phe Leu Ala Tyr Ala Glu Phe Asn Asp Thr Asp Ile Pro Val

195 200 205

Ile Ala Tyr Ser Tyr Tyr Gly Asp Glu Gln Tyr Pro Arg Thr Ile Asn

210 215 220

Ile Pro Tyr Pro Lys Ala Gly Ala Lys Asn Pro Val Val Arg Ile Phe

225 230 235 240

Ile Ile Asp Thr Thr Tyr Pro Ala Tyr Val Gly Pro Gln Glu Val

245 250 255

<210> 16

<211> 31

<212> PRT

<213> Artificial sequence ()

<400> 16

Glu Ala Ile Asn Ile Phe Arg Val Thr Gln Asp Ser Leu Phe Tyr Ser

1 5 10 15

Ser Asn Glu Phe Glu Glu Tyr Pro Gly Arg Arg Asn Ile Tyr Arg

20 25 30

<210> 17

<211> 51

<212> PRT

<213> Artificial sequence ()

<400> 17

Glu Glu Ser Arg Thr Gly Trp Ala Gly Gly Phe Phe Val Ser Thr Pro

1 5 10 15

Val Phe Ser Tyr Asp Ala Ile Ser Tyr Tyr Lys Ile Phe Ser Asp Lys

20 25 30

Asp Gly Tyr Lys His Ile His Tyr Ile Lys Asp Thr Val Glu Asn Ala

35 40 45

Ile Gln Ile

50

<210> 18

<211> 50

<212> PRT

<213> Artificial sequence ()

<400> 18

Asp Cys Pro Lys Thr Gln Glu His Ile Glu Glu Ser Arg Thr Gly Trp

1 5 10 15

Ala Gly Gly Phe Phe Val Ser Thr Pro Val Phe Ser Tyr Asp Ala Ile

20 25 30

Ser Tyr Tyr Lys Ile Phe Ser Asp Lys Asp Gly Tyr Lys His Ile His

35 40 45

Tyr Ile

50

<210> 19

<211> 260

<212> PRT

<213> Artificial sequence ()

<400> 19

Gln Trp Leu Lys Arg Val Gln Asn Val Ser Val Leu Ser Ile Cys Asp

1 5 10 15

Phe Arg Glu Asp Trp Gln Thr Trp Asp Cys Pro Lys Thr Gln Glu His

20 25 30

Ile Glu Glu Ser Arg Thr Gly Trp Ala Gly Gly Phe Phe Val Ser Thr

35 40 45

Pro Val Phe Ser Tyr Asp Ala Ile Ser Tyr Tyr Lys Ile Phe Ser Asp

50 55 60

Lys Asp Gly Tyr Lys His Ile His Tyr Ile Lys Asp Thr Val Glu Asn

65 70 75 80

Ala Ile Gln Ile Thr Ser Gly Lys Trp Glu Ala Ile Asn Ile Phe Arg

85 90 95

Val Thr Gln Asp Ser Leu Phe Tyr Ser Ser Asn Glu Phe Glu Glu Tyr

100 105 110

Pro Gly Arg Arg Asn Ile Tyr Arg Ile Ser Ile Gly Ser Tyr Pro Pro

115 120 125

Ser Lys Lys Cys Val Thr Cys His Leu Arg Lys Glu Arg Cys Gln Tyr

130 135 140

Tyr Thr Ala Ser Phe Ser Asp Tyr Ala Lys Tyr Tyr Ala Leu Val Cys

145 150 155 160

Tyr Gly Pro Gly Ile Pro Ile Ser Thr Leu His Asp Gly Arg Thr Asp

165 170 175

Gln Glu Ile Lys Ile Leu Glu Glu Asn Lys Glu Leu Glu Asn Ala Leu

180 185 190

Lys Asn Ile Gln Leu Pro Lys Glu Glu Ile Lys Lys Leu Glu Val Asp

195 200 205

Glu Ile Thr Leu Trp Tyr Lys Met Ile Leu Pro Pro Gln Phe Asp Arg

210 215 220

Ser Lys Lys Tyr Pro Leu Leu Ile Gln Val Tyr Gly Gly Pro Cys Ser

225 230 235 240

Gln Ser Val Arg Ser Val Phe Ala Val Asn Trp Ile Ser Tyr Leu Ala

245 250 255

Ser Lys Glu Gly

260

<210> 20

<211> 27

<212> PRT

<213> Artificial sequence ()

<400> 20

Ala Ala Leu Ala Leu Val Val Ile Cys Ile Val Leu Arg Pro Ser Arg

1 5 10 15

Val Tyr Lys Pro Glu Gly Asn Thr Lys Arg Ala

20 25

<210> 21

<211> 220

<212> PRT

<213> Artificial sequence ()

<400> 21

Gly Gly Pro Cys Ser Gln Ser Val Lys Ser Val Phe Ala Val Asn Trp

1 5 10 15

Ile Thr Tyr Leu Ala Ser Lys Glu Gly Ile Val Ile Ala Leu Val Asp

20 25 30

Gly Arg Gly Thr Ala Phe Gln Gly Asp Lys Phe Leu His Ala Val Tyr

35 40 45

Arg Lys Leu Gly Val Tyr Glu Val Glu Asp Gln Leu Thr Ala Val Arg

50 55 60

Lys Phe Ile Glu Met Gly Phe Ile Asp Glu Glu Arg Ile Ala Ile Trp

65 70 75 80

Gly Trp Ser Tyr Gly Gly Tyr Val Ser Ser Leu Ala Leu Ala Ser Gly

85 90 95

Thr Gly Leu Phe Lys Cys Gly Ile Ala Val Ala Pro Val Ser Ser Trp

100 105 110

Glu Tyr Tyr Ala Ser Ile Tyr Ser Glu Arg Phe Met Gly Leu Pro Thr

115 120 125

Lys Asp Asp Asn Leu Glu His Tyr Lys Asn Ser Thr Val Met Ala Arg

130 135 140

Ala Glu Tyr Phe Arg Asn Val Asp Tyr Leu Leu Ile His Gly Thr Ala

145 150 155 160

Asp Asp Asn Val His Phe Gln Asn Ser Ala Gln Ile Ala Lys Ala Leu

165 170 175

Val Asn Ala Gln Val Asp Phe Gln Ala Met Trp Tyr Ser Asp Gln Asn

180 185 190

His Gly Ile Ser Ser Gly Arg Ser Gln Asn His Leu Tyr Thr His Met

195 200 205

Thr His Phe Leu Lys Gln Cys Phe Ser Leu Ser Asp

210 215 220

<210> 22

<211> 722

<212> PRT

<213> Artificial sequence ()

<400> 22

Met Arg Ala Leu Thr Leu Lys Asp Ile Leu Asn Gly Thr Phe Ser Tyr

1 5 10 15

Lys Thr Phe Phe Pro Asn Trp Ile Ser Gly Gln Glu Tyr Leu His Gln

20 25 30

Ser Ala Asp Asn Asn Ile Val Leu Tyr Asn Ile Glu Thr Gly Gln Ser

35 40 45

Tyr Thr Ile Leu Ser Asn Arg Thr Met Lys Ser Val Asn Ala Ser Asn

50 55 60

Tyr Gly Leu Ser Pro Asp Arg Gln Phe Val Tyr Leu Glu Ser Asp Tyr

65 70 75 80

Ser Lys Leu Trp Arg Tyr Ser Tyr Thr Ala Thr Tyr Tyr Ile Tyr Asp

85 90 95

Leu Ser Asn Gly Glu Phe Val Arg Gly Asn Glu Leu Pro Arg Pro Ile

100 105 110

Gln Tyr Leu Cys Trp Ser Pro Val Gly Ser Lys Leu Ala Tyr Val Tyr

115 120 125

Gln Asn Asn Ile Tyr Leu Lys Gln Arg Pro Gly Asp Pro Pro Phe Gln

130 135 140

Ile Thr Phe Asn Gly Arg Glu Asn Lys Ile Phe Asn Gly Ile Pro Asp

145 150 155 160

Trp Val Tyr Glu Glu Glu Met Leu Ala Thr Lys Tyr Ala Leu Trp Trp

165 170 175

Ser Pro Asn Gly Lys Phe Leu Ala Tyr Ala Glu Phe Asn Asp Thr Asp

180 185 190

Ile Pro Val Ile Ala Tyr Ser Tyr Tyr Gly Asp Glu Gln Tyr Pro Arg

195 200 205

Thr Ile Asn Ile Pro Tyr Pro Lys Ala Gly Ala Lys Asn Pro Val Val

210 215 220

Arg Ile Phe Ile Ile Asp Thr Thr Tyr Pro Ala Tyr Val Gly Pro Gln

225 230 235 240

Glu Val Pro Val Pro Ala Met Ile Ala Ser Ser Asp Tyr Tyr Phe Ser

245 250 255

Trp Leu Thr Trp Val Thr Asp Glu Arg Val Cys Leu Gln Trp Leu Lys

260 265 270

Arg Val Gln Asn Val Ser Val Leu Ser Ile Cys Asp Phe Arg Glu Asp

275 280 285

Trp Gln Thr Trp Asp Cys Pro Lys Thr Gln Glu His Ile Glu Glu Ser

290 295 300

Arg Thr Gly Trp Ala Gly Gly Phe Phe Val Ser Thr Pro Val Phe Ser

305 310 315 320

Tyr Asp Ala Ile Ser Tyr Tyr Lys Ile Phe Ser Asp Lys Asp Gly Tyr

325 330 335

Lys His Ile His Tyr Ile Lys Asp Thr Val Glu Asn Ala Ile Gln Ile

340 345 350

Thr Ser Gly Lys Trp Glu Ala Ile Asn Ile Phe Arg Val Thr Gln Asp

355 360 365

Ser Leu Phe Tyr Ser Ser Asn Glu Phe Glu Glu Tyr Pro Gly Arg Arg

370 375 380

Asn Ile Tyr Arg Ile Ser Ile Gly Ser Tyr Pro Pro Ser Lys Lys Cys

385 390 395 400

Val Thr Cys His Leu Arg Lys Glu Arg Cys Gln Tyr Tyr Thr Ala Ser

405 410 415

Phe Ser Asp Tyr Ala Lys Tyr Tyr Ala Leu Val Cys Tyr Gly Pro Gly

420 425 430

Ile Pro Ile Ser Thr Leu His Asp Gly Arg Thr Asp Gln Glu Ile Lys

435 440 445

Ile Leu Glu Glu Asn Lys Glu Leu Glu Asn Ala Leu Lys Asn Ile Gln

450 455 460

Leu Pro Lys Glu Glu Ile Lys Lys Leu Glu Val Asp Glu Ile Thr Leu

465 470 475 480

Trp Tyr Lys Met Ile Leu Pro Pro Gln Phe Asp Arg Ser Lys Lys Tyr

485 490 495

Pro Leu Leu Ile Gln Val Tyr Gly Gly Pro Cys Ser Gln Ser Val Arg

500 505 510

Ser Val Phe Ala Val Asn Trp Ile Ser Tyr Leu Ala Ser Lys Glu Gly

515 520 525

Met Val Ile Ala Leu Val Asp Gly Arg Gly Thr Ala Phe Gln Gly Asp

530 535 540

Lys Leu Leu Tyr Ala Val Tyr Arg Lys Leu Gly Val Tyr Glu Val Glu

545 550 555 560

Asp Gln Ile Thr Ala Val Arg Lys Phe Ile Glu Met Gly Phe Ile Asp

565 570 575

Glu Lys Arg Ile Ala Ile Trp Gly Trp Ser Tyr Gly Gly Tyr Val Ser

580 585 590

Ser Leu Ala Leu Ala Ser Gly Thr Gly Leu Phe Lys Cys Gly Ile Ala

595 600 605

Val Ala Pro Val Ser Ser Trp Glu Tyr Tyr Ala Ser Val Tyr Thr Glu

610 615 620

Arg Phe Met Gly Leu Pro Thr Lys Asp Asp Asn Leu Glu His Tyr Lys

625 630 635 640

Asn Ser Thr Val Met Ala Arg Ala Glu Tyr Phe Arg Asn Val Asp Tyr

645 650 655

Leu Leu Ile His Gly Thr Ala Asp Asp Asn Val His Phe Gln Asn Ser

660 665 670

Ala Gln Ile Ala Lys Ala Leu Val Asn Ala Gln Val Asp Phe Gln Ala

675 680 685

Met Trp Tyr Ser Asp Gln Asn His Gly Leu Ser Gly Leu Ser Thr Asn

690 695 700

His Leu Tyr Thr His Met Thr His Phe Leu Lys Gln Cys Phe Ser Leu

705 710 715 720

Ser Asp

Claims (27)

1. Use of a protein or polypeptide capable of binding to a fibroblast activation protein FAP antibody in the preparation of a kit for diagnosing a disease associated with the abnormal expression of FAP in a subject, wherein the FAP antibody is a FAP autoantibody that is present in a blood sample isolated from the subject and is present in a higher amount as compared to a subject not suffering from the disease.

2. The use of claim 1, wherein the protein or polypeptide is selected from the group consisting of a FAP full-length protein, a FAP ectodomain polypeptide, and a polypeptide comprising an epitope of FAP.

3. The use of claim 1 or 2, wherein the FAP antibody is a monoclonal antibody, a polyclonal antibody, or a combination thereof.

4. The use according to claim 1 or 2, wherein the protein or polypeptide comprises the amino acid sequence shown in SEQ ID No.1 or 2 or a variant thereof.

5. The use of claim 1 or 2, wherein the kit further comprises additional reagents for detecting whether the protein or polypeptide binds to an FAP antibody.

6. The use of claim 5, wherein the additional agent is a secondary antibody.

7. The use of claim 1 or 2, wherein the disease associated with abnormal expression of FAP is selected from the group consisting of: keloids, scleroderma, pulmonary fibrosis, cirrhosis, arthritis, atherosclerosis, crohn's disease, basal cell carcinoma, squamous cell carcinoma, skin cancer, oral cancer, melanoma, esophageal cancer, adenocarcinoma, gastric cancer, colorectal cancer, rectal cancer, pancreatic cancer, hepatocellular cancer, lung cancer, mesothelioma, cholangiocarcinoma, bladder cancer, breast cancer, kidney cancer, prostate cancer, cervical cancer, ovarian cancer, glioma, thyroid cancer, parathyroid tumor, sarcoma, myeloma, astrocytoma.

8. The use of claim 7, wherein the disease associated with abnormal expression of FAP is idiopathic pulmonary fibrosis.

9. The use of claim 7, wherein the disease associated with abnormal expression of FAP is lung cancer, cholangiocarcinoma, liver cancer, glioma, ovarian cancer, breast cancer or pancreatic cancer.

10. The use of claim 1, wherein the protein or polypeptide is immobilized on a solid support.

11. The use of claim 10, wherein the solid support is selected from the group consisting of an enzyme label plate, a magnetic bead, a polyvinylidene fluoride membrane, an agarose bead, polystyrene, and a nitrocellulose membrane.

12. The use of claim 1, wherein the subject is a human.

13. The use of claim 1, wherein the blood sample is serum, plasma or whole blood.

14. The use of claim 2, wherein the FAP full-length protein is in monomeric form.

15. The use of claim 2, wherein the FAP extracellular domain polypeptide comprises a polypeptide consisting of FAP amino acids 27 to 760.

16. Use according to claim 1 or 2, wherein the protein or polypeptide is labelled with a label.

17. The use of claim 16, wherein the label is selected from the group consisting of an enzyme label, an isotope label, a chemiluminescent label, a fluorescent label, and a dye.

18. The use according to claim 1 or 2, wherein the protein or polypeptide is a natural or recombinant human protein or polypeptide.

19. The use of claim 1 or 2, wherein the kit further comprises a control of FAP recombinant antibody at different concentrations.

20. A computer readable storage medium having stored thereon computer instructions for reading and execution by a computer, the computer instructions being executable to perform a method of detecting the presence of FAP autoantibodies in a sample of a subject, the method comprising:

(a) contacting a sample of a subject with a protein or polypeptide capable of binding to an antibody to fibroblast activation protein, FAP;

(b) detecting and reading the signal of the contacted sample to detect whether the protein and the polypeptide form a complex with FAP autoantibody in the sample; and

(c) determining whether the signal exceeds a predetermined threshold and determining the presence of FAP autoantibodies in the sample when the signal exceeds a predetermined threshold, and wherein the threshold is the median level from healthy subjects.

21. A kit for diagnosing a disease associated with FAP aberrant expression in a subject, the kit comprising a protein or polypeptide capable of binding to a fibroblast activation protein FAP antibody, wherein the FAP antibody is a FAP autoantibody that is present in a blood sample isolated from the subject and is present in a higher amount as compared to a subject not suffering from the disease.

22. The kit of claim 21, wherein the protein or polypeptide is immobilized on a solid support.

23. The kit of claim 21, wherein the protein or polypeptide is selected from the group consisting of a FAP full-length protein, a FAP ectodomain polypeptide, and a polypeptide comprising an epitope of FAP.

24. The kit of claim 21, wherein the FAP full-length protein is in monomeric form.

25. The kit of claim 21, wherein the protein or polypeptide is labeled with a label.

26. The kit of claim 25, wherein the label is selected from the group consisting of an enzyme label, an isotope label, a chemiluminescent label, a fluorescent label, and a dye.

27. The kit of claim 21, further comprising a FAP recombinant antibody control at different concentrations.

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