CN109633165B

CN109633165B - Application of anti-HSPA 4 autoantibody as breast cancer diagnosis or prognosis evaluation marker

Info

Publication number: CN109633165B
Application number: CN201811552785.8A
Authority: CN
Inventors: 曹雪涛; 顾炎; 刘艳芳
Original assignee: Institute of Basic Medical Sciences of CAMS
Current assignee: Institute of Basic Medical Sciences of CAMS
Priority date: 2018-12-18
Filing date: 2018-12-18
Publication date: 2021-03-09
Anticipated expiration: 2038-12-18
Also published as: CN109633165A

Abstract

The invention discloses and provides application of an anti-HSPA 4 autoantibody as a breast cancer diagnosis or prognosis evaluation marker. Specifically, the invention discloses an application of a substance for detecting a heat shock protein (HSPA4) autoantibody in preparing a product for breast cancer diagnosis or prognosis evaluation, an application of the heat shock protein HSPA4 autoantibody as a breast cancer diagnosis or prognosis evaluation marker, a related product and an application thereof. The technology has good clinical application prospect in the field of breast cancer medical diagnosis.

Description

Application of anti-HSPA 4 autoantibody as breast cancer diagnosis or prognosis evaluation marker

Technical Field

The invention relates to the field of biotechnology and medical diagnosis, in particular to application of a serum heat shock protein HSPA4 autoantibody as a diagnostic marker in preparation of a breast cancer diagnosis or patient prognosis evaluation kit.

Background

Along with population growth, aging and social and economic development, malignant tumors are becoming important global health problems, and have great influence on economic and social development and human health. Therefore, the prevention and control of tumors have become the focus of global health prevention and control. According to the latest data issued by the authoritative journal of oncology (CA Cancer J Clin), 1810 million new Cancer cases are expected to increase globally in 2018, 960 million people die due to Cancer, and the prevalence rate of Cancer in China is at a medium upper level internationally (Siegel RL et al, Cancer statistics,2018.CA Cancer J Clin.2018; 68(1): 7-30.).

Taking breast cancer as an example, it is the most common malignancy in women. In recent years, the incidence and mortality of breast Cancer in women have increased year by year in the world, and the incidence and mortality of malignant tumors in women have increased (Picon-Ruiz M et al, importance and additive breast Cancer and death: mechanical instruments and strategies for intervention. Ca Cancer J Clin,2017,67 (5)). According to the statistics of "journal of clinical tumors", breast cancer is the most common type of cancer in women (24.2%), which is also the number one killer (15%) of female cancer patients. In 2014, about 27.89 ten thousand new cases of breast cancer of women all over the country account for 16.51 percent of the onset of malignant tumors of the women and are positioned at the first of the onset of the malignant tumors of the women. Moreover, the disease burden of breast cancer in our country still shows a tendency to increase year by year (li he et al, 2014 analysis of breast cancer morbidity and mortality in chinese women, china journal of tumor, 2018,40 (3)).

Therefore, the early diagnosis and prognosis judgment of the tumor have important significance for the selection of cancer treatment modes and treatment curative effect.

Currently, tumor diagnosis mainly relies on imaging means (CT, MRI, etc.) and serum marker detection. However, for many types of cancer, good diagnostic indicators and screening techniques are still lacking. Although the popularity of medical imaging techniques has improved the ability to detect tumors early, some patients with tumors have already been diagnosed with advanced disease. Therefore, there is a need to develop strategies for blood-based detection and prognosis.

Autoantibodies refer to antibodies directed against tissues, organs, cells and Cell components of the body, which may have low titers in the blood of normal humans, and which cause dysfunction of tissues and organs, i.e., induce autoimmune diseases, etc., once they bind to a target antigen (Pagan J D et al, Cell, 2017; 172(3): 564-577). The tumor also induces the generation of antibodies due to the existence of specific antigens and related antigens, which is an important mechanism for resisting the tumor of the organism and is the most important means for clinical tumor hematological detection at present.

In one aspect, autoantibodies that detect a particular antigen can indirectly reflect the presence of a tumor associated antigen. Research shows that the autoantibody generated by the tumor antigen is a better index for early diagnosis of tumors, and can also be used for relapse of tumors, monitoring of curative effect, concomitant diagnosis of medication and the like. At present, the detection of more tumor autoantibodies is used for the diagnosis of tumors, such as autoantibodies of NY-SEO-1, p53, LAMR1 and the like used for the diagnosis of lung cancer; autoantibodies such as p62 and HCC1 (Caron M et al. Mol Cell Proteomics,2007,6(7):1115) for liver cancer diagnosis. However, the specificity and sensitivity of currently known autoantibody detection is not high. Therefore, a new tumor autoantibody is searched, the change trend of the tumor autoantibody in the occurrence and development of the tumor is detected, and the tumor autoantibody has important value for the diagnosis and prognosis judgment of the tumor.

On the other hand, autoantibodies present in tumor patients are also involved in the development of tumors. Clinical studies have shown that high concentrations of autoantibodies are present in the serum of human breast, genitourinary tract and head and neck cancers and are closely associated with tumor staging, metastasis and low survival rates. Further studies have shown that antibodies can activate the FcR gamma receptor on myeloid cells to promote the development of squamous Cell carcinoma (Andreu P et al Cancer Cell2010,17(2): 121-134.). Recent studies further reveal negative regulatory functions of plasma cells and antibodies in tumor immunity: human IgG 4-induced inflammatory responses of the Th2 type antagonize antibody-mediated anti-tumor immunity (Karagiannis P et al J Clin invest.2013,123(4): 1457-74.); IgA-expressing immunosuppressive plasma cells suppress T-cell dependent tumor chemotherapy responses (Shalapour S et al Nature 2015,521(7550): 94-98.). Therefore, the search of the autoantibody and the target antigen of the tumor patient is not only beneficial to searching an important target for disease diagnosis, but also beneficial to clarifying the occurrence and development mechanism of the disease, and can be used for predicting the prognosis of the tumor patient in time and selecting a proper tumor treatment scheme.

The use of autoantibodies as diagnostic markers for tumors has the following advantages: firstly, the generation of the autoantibody can be induced by extremely low tumor antigen, so the autoantibody can be detected when the expression of the tumor antigen is very low, and the sensitivity is very good; the autoantibody can be detected before clinical symptoms appear in a tumor patient, so that the autoantibody can be used as a tumor marker for early diagnosis; the autoantibody reaction is closely related to the malignant transformation process of the tumor cells, and can also be used for evaluating the tumor progression and the malignant transformation of the cells; and fourthly, the autoantibody has good stability, is long in time existing in serum, and is convenient for long-term storage and large-scale screening. Therefore, the use of autoantibodies induced by tumors to reflect the disease progression of tumor generators in patients is becoming an important direction for finding new targets for early diagnosis and prognosis of tumors.

However, the search for target antigens that induce autoantibody production is a difficult point in their research and application, as it directly determines the detection of autoantibodies. The generation of autoantibodies is easily influenced by the internal and external environment of the body, especially by inflammatory factors, thereby influencing the specificity and sensitivity of detection. Furthermore, high levels of antibodies are also present in human peripheral blood itself. Therefore, finding out a tumor-specific target antigen and an autoantibody induced by the tumor-specific target antigen is very important for the application of the autoantibody to the detection of tumors, and is also a key point and a difficulty point of research in the field.

Heat Shock Proteins (HSPs) are a group of proteins expressed by cells under stress, can repair or degrade damaged proteins, have chaperone activity, assist correct folding of proteins, protect cells from external environmental stimulation, and participate in processes such as growth, metabolism, signal transduction and the like of cells. HSPs can be divided into six families, namely the HSP27 family, the HSP40 family, the HSP60 family, the HSP70 family, the HSP90 family and other HSP families (HSP110 and GRP170) according to their relative molecular size (Chatterjee S et al, Targeting Heat Shock Proteins in Cancer: A formulating Therapeutic approach. int J Mol Sci.2017; 18 (9)).

Researches show that HSP is highly expressed in various tumor tissues and plays an important role, and the HSP can promote the occurrence and development of tumors by promoting the proliferation and invasion of tumor cells and inhibiting the apoptosis of the tumor cells. For example, HSP70 is involved in the development of various neoplasms, and is closely related to poor prognosis of biliary tract Cancer, chondrosarcoma, melanoma, colon, bladder, and other tumors (Court ka et al, HSP70inhibition synergy industries the effects of magnetic fluidized in vitro Cancer. mol Cancer ther.,2017,16 (5)). HSP90 is highly expressed in a variety of tumor types, with high expression associated with poor prognosis in lung, esophageal, bladder, melanoma and leukemia, and various HSP90 inhibitors are currently used in clinical tumor studies (Isaacs J S. Hsp90as a "Chaperone" of the epidenemic: instruments and Opportunities for Cancer therapy. adv Cancer Res.,2016,129: 107-.

However, no report has been made that HSP as a tumor antigen induces autoantibody production to participate in the process of tumorigenesis and development. At present, no research report about the application of the anti-HSPA 4 autoantibody in tumor diagnosis and patient prognosis evaluation exists at home and abroad.

There is an urgent need in the art to find and use indices that can be effectively used for tumor diagnosis, tumor treatment protocol selection, and tumor prognosis evaluation in these applications.

Disclosure of Invention

One of the objectives of the present application is to find autoantibodies and their applications that can be effectively used for breast cancer diagnosis and patient prognosis evaluation.

In a first aspect of the present disclosure there is provided the use of a substance for detecting the level of autoantibodies to HSPA4 in the manufacture of a product for use in the diagnosis or prognostic assessment of breast cancer in a subject.

In some embodiments of the present application, the detection is performed on a mammal or a sample obtained from the mammal that has or is suspected of having breast cancer or is at risk of having breast cancer or has been at risk of having breast cancer but is cured.

In some embodiments, the mammal is selected from the group consisting of: primates, rodents, farm mammals, mammalian pets, and the like, e.g., humans, apes, orangutans, monkeys, cows, sheep, horses, camels, pigs, dogs, cats, rabbits, mice, and the like.

In some embodiments, the mammal has been identified as having breast cancer or being cured of breast cancer in a manner known in the art.

In some embodiments of the present application, the sample is selected from the group consisting of: blood samples, e.g., plasma, serum, whole blood; a tissue or cell sample, such as a breast tissue or cell sample, a cancer tissue or cell sample, a tissue or cell sample adjacent to a cancer, or a cell sample.

In some embodiments, the sample is a fresh sample, a cryopreserved sample, a fixed sample (e.g., a formalin fixed sample), an embedded sample (e.g., a paraffin embedded sample).

In some embodiments of the present application, the breast cancer is selected from the following classifications: invasive ductal carcinoma, non-invasive ductal carcinoma; alternatively, luminal a, luminal B, luminal-HER 2, HER2 overexpressing, basal cell-like, TNP-non-basal; alternatively, the hormone receptor positive type, the HER2/neu receptor positive type, the triple negative type.

In some embodiments of the present application, the substance for detecting the level of anti-HSPA 4 autoantibodies is a substance for detecting anti-HSPA 4 autoantibodies at the gene level and/or protein level; for example, the substance is a substance used in one or more detection techniques or methods selected from the group consisting of: immunohistochemistry (e.g., immunofluorescence analysis, reverse enzyme-linked immunosorbent assay, immunogold immunoassay), Western blotting, Northern blotting, PCR, and biochip.

In some embodiments of the present application, the substance is selected from the group consisting of: substances specific for anti-HSPA 4 autoantibodies, such as anti-antibodies (preferably monoclonal antibodies) thereof; an HSPA4 antigen or fragment or analog thereof having binding specificity for an anti-HSPA 4 autoantibody, e.g., an epitope, a fragment comprising an epitope thereof, or an analog thereof; specific probes, gene chips, PCR primers, gRNA and the like for the anti-HSPA 4 autoantibody.

In some embodiments, the substance carries a detectable label, e.g., the detectable label is selected from the group consisting of: a radioisotope, a fluorophore, a chemiluminescent moiety, an enzyme substrate, an enzyme cofactor, an enzyme inhibitor, a colloidal chromogenic label, a dye, a metal ion, or a ligand (e.g., biotin or hapten).

In some embodiments, the substance is immobilized, e.g., on a solid phase matrix.

In some embodiments, the solid phase matrix is a planar or curved solid phase matrix.

In some embodiments, the solid substrate comprises a substrate comprising a microplate, a microsphere, and a porous membrane.

In some embodiments of the present application, an increased level of anti-HSPA 4 autoantibodies in the subject or in a sample obtained from the subject, as compared to a normal control value, indicates that the subject is susceptible to or has undergone cancer metastasis, or indicates that the subject has a poor prognosis of cancer, or indicates that the subject has suffered cancer.

In some embodiments, the level of anti-HSPA 4 autoantibodies is used to assess disease-free survival and/or overall survival of the patient.

In some embodiments, a level of anti-HSPA 4 autoantibodies that is higher than a control is indicative of a decreased disease-free survival and/or overall survival of the subject. For example, levels of anti-HSPA 4 autoantibodies that are higher than controls may indicate that the disease-free survival time of the subject may be less than 40 months, e.g. less than 24 months, less than 12 months, less than 9.8 months, and the overall survival time may be less than 40 months, e.g. less than 24 months, less than 12 months, less than 9.8 months.

In some embodiments, a level of anti-HSPA 4 autoantibodies that is lower than a control is indicative of disease-free survival and/or long overall survival of the subject. For example, a level of anti-HSPA 4 autoantibodies that is lower than a control may indicate that the disease free survival of the subject is at least 40 months, such as at least 50 months, at least 60 months, and the overall survival may be at least 40 months, such as at least 50 months, at least 60 months.

In some embodiments, the normal control value is obtained from a sample from a subject not suffering from breast cancer, a sample from normal tissue of the subject, a normal subject's level of anti-HSPA 4 autoantibodies.

In some embodiments, the normal control value is: anti-HSPA 4 autoantibody levels measured in a normal biological sample other than breast cancer (e.g., a sample obtained from a healthy human or normal tissue of a subject to be tested), a population standard level determined by statistics, or a normalized level.

In some embodiments of the present application, the product is a test kit.

In some embodiments, the test kit further comprises one or more substances selected from the group consisting of: container, buffer, auxiliary agent, solvent, negative control substance, positive control substance and instruction for use.

In one embodiment, the test kit is a reverse enzyme-linked immunosorbent assay based kit for detecting anti-HSPA 4 autoantibodies in a biological sample.

In another aspect of the present application, there is also provided a method for predicting breast cancer metastasis, prognostically evaluating breast cancer, or diagnosing breast cancer in a subject, the method comprising: a step of detecting the level of anti-HSPA 4 autoantibodies in the subject or in a sample obtained from the subject.

In some embodiments, an increased level of anti-HSPA 4 autoantibodies in the subject, or in a sample obtained from the subject, as compared to a normal control, is indicative of the subject being susceptible to or having metastasized breast cancer, or is indicative of a poor prognosis for the subject's breast cancer patient, or is indicative of the subject having had cancer.

In some embodiments, the various features to which the methods of the present application relate are as defined or set forth in the foregoing applications.

In another aspect of the present application, there is also provided a product (e.g., a kit) for breast cancer diagnosis or prognosis evaluation, comprising: (ii) a substance for detecting the level of anti-HSPA 4 autoantibodies; and optionally other substances for the diagnosis or prognosis of breast cancer, for example detection substances for existing breast cancer markers, such as detection substances for serological CA15-3, CEA detection, pathological immunohistochemical detection (e.g. Ki-67, ER, PR, CerbB 2). In some embodiments, the various features of the products of the present application may be as defined or set forth above.

In another aspect of the present application, there is also provided a method of screening a candidate drug for treating breast cancer, ameliorating or preventing breast cancer metastasis and/or improving the prognosis of breast cancer, the method comprising detecting the effect of the candidate drug on the level of anti-HSPA 4 autoantibodies in a subject or a sample obtained from a subject, wherein a decrease in the level of anti-HSPA 4 autoantibodies following administration of the candidate drug indicates that the candidate drug has the effect of treating breast cancer, ameliorating or preventing breast cancer metastasis and/or improving the prognosis of breast cancer.

In some embodiments of the present application, the detection of anti-HSPA 4 autoantibody levels is performed using a product as described herein.

In some embodiments, the reduction is relative to the level of anti-HSPA 4 autoantibodies in a sample obtained from a subject not suffering from breast cancer, a sample obtained from normal tissue of the subject, a normal subject. In some embodiments, the decrease is relative to a normal control value that is: a level of an anti-HSPA 4 autoantibody molecule measured in a normal biological sample other than breast cancer (e.g., a sample obtained from a healthy human or normal tissue of a subject to be tested), a population standard level determined by statistics, or a normalized level.

In some embodiments, each feature involved in the drug candidate screening methods of the present application is as defined or set forth in the aforementioned application.

Any combination of the above-described solutions and features may be made by those skilled in the art without departing from the spirit and scope of the present invention. Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

The present invention will now be further described with reference to the accompanying drawings, wherein the showings are for the purpose of illustrating embodiments of the invention only and not for the purpose of limiting the scope of the invention.

FIG. 1: screening of specific binding partners for serum autoantibodies from breast cancer tumor-bearing mice:

a: the amount of antibodies (IgG1, IgG2a, IgG2b, and IgG3) in the sera of normal and tumor mice, P-values were calculated using Unpaired Student's t-tests in SPSS 17.0;

b: immunoblotting (Western blot) detects binding of serum antibodies to HSPA4 (protein NCL and ITGB3 are controls).

FIG. 2: SDS-PAGE analysis of the expression of human target protein (HSPA4) in the eluate:

marker: labeling with molecular weight; lane 1: buffer control; lane 2: eluent 1; lane 3: eluent 2.

FIG. 3: correlation of serum heat shock protein HSPA4 autoantibody levels with patient disease-free survival and overall survival:

a: a Kaplan-Meier survival curve of the level of serum heat shock protein HSPA4 autoantibodies and the disease-free survival time of a patient;

b: Kaplan-Meier survival curves of serum heat shock protein HSPA4 autoantibody levels versus overall patient survival time.

FIG. 4: correlation of serum heat shock protein HSPA4 autoantibody levels with patient lymph node metastasis:

normal serum (from normal human donations, n 60); lymph node negative (LN-, n ═ 52); lymph nodes are positive (LN +, n ═ 56). P values were calculated using unpaired two-tailed students's t-tests in SPSS 17.0.

Detailed Description

The invention relates to application of an autoantibody of a heat shock protein HSPA4 as a molecular marker for prognosis, diagnosis, drug screening and the like of breast cancer, and a related product and application thereof.

In particular, the inventors have applied proteomics techniques to identify autoantibodies against HSPA4 present in breast cancer patient samples. The expression level of the anti-HSPA 4 autoantibody in serum is detected by purifying HSPA4 protein and adopting reverse enzyme-linked immunosorbent assay analysis, and the result shows that the anti-HSPA 4 autoantibody in the serum of a breast cancer patient is obviously higher than that of a normal person and is related to lymph node metastasis of the breast cancer patient, the high expression of the antibody is related to the short total survival time and the difference in prognosis of the patient and can be used as an independent prognosis judgment factor. Based on the molecular marker, the invention provides a molecular marker for breast cancer diagnosis, prognosis evaluation and drug screening by using the level of the autoantibody of the heat shock protein HSPA4, provides a corresponding product, and has certain clinical and pharmaceutical application prospects.

All numerical ranges provided herein are intended to expressly include all numbers between the end points of the ranges and numerical ranges there between. The features mentioned with reference to the invention or the features mentioned with reference to the embodiments can be combined. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

As used herein, "comprising," having, "or" including "includes" comprising, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …", and "consisting of … …" are subordinate concepts of "comprising", "having", or "including".

anti-HSPA 4 autoantibody and detection substance thereof

As used herein, the terms "anti-HSPA 4 autoantibody" and "protein or polypeptide encoded by an anti-HSPA 4 autoantibody gene" are used interchangeably and all refer to a protein or polypeptide encoded by an anti-HSPA 4 autoantibody gene, a conservatively variant polypeptide thereof, or a homologous protein or polypeptide thereof, or an active fragment thereof.

As used herein, the terms "detection substance", "detection reagent" or "reagent for detecting an anti-HSPA 4 autoantibody molecule" or "reagent for detecting the expression of an anti-HSPA 4 autoantibody" in relation to an anti-HSPA 4 autoantibody, used interchangeably, refer to a substance that is specific for an anti-HSPA 4 autoantibody molecule and that can be used to directly or indirectly detect the presence and/or amount of an anti-HSPA 4 autoantibody molecule. These detection substances can detect the anti-HSPA 4 autoantibodies at the gene level or at the protein level.

Detection substances for anti-HSPA 4 autoantibodies may include, but are not limited to: substances specific for anti-HSPA 4 autoantibodies, such as anti-antibodies (preferably monoclonal antibodies) thereof; an HSPA4 antigen or fragment or analog thereof having binding specificity for an anti-HSPA 4 autoantibody, e.g., an epitope, a fragment comprising an epitope thereof, or an analog thereof; specific probes, gene chips, PCR primers, gRNA and the like for the anti-HSPA 4 autoantibody.

As used herein, the term "autoantibody specific binding partner" refers to a partner capable of specifically binding to an anti-HSPA 4 antibody, such as HSPA4 (including its full length sequence) or a fragment or analogue thereof, such as an epitope, a fragment comprising an epitope thereof or an analogue thereof, having binding specificity for an anti-HSPA 4 autoantibody.

Specific binding partners for HSPA4 autoantibodies for use herein can be synthesized using genetic engineering methods known to those skilled in the art. For example, the synthesis is performed by a genetic engineering method, and a prokaryotic expression system or an eukaryotic expression system can be selected according to requirements, wherein the prokaryotic expression system is mainly represented by an escherichia coli expression system, and the eukaryotic expression system is represented by a yeast, insect cell, and mammalian expression system.

As used herein, the terms "HSPA 4 antigen", "HSPA 4 protein or polypeptide" and "protein or polypeptide encoded by the HSPA4 gene" are used interchangeably and all refer to a protein or polypeptide encoded by the HSPA4 gene, conservative variants thereof, or homologous proteins or polypeptides thereof, or active fragments thereof. The HSPA4 protein is a member of the heat shock protein family known in the art, for example the sequence of human HSPA4 can be as shown in Gene ID: 3308; the mouse HSPA4 sequence can be shown as Gene ID: 15525. Since the sequence of the antigen HSPA4 is known in the art, autoantibodies specific for HSPA4 can be made by one of ordinary skill in the art based on routine means or obtained commercially.

Also, to facilitate detection, the detection reagents of the invention may also carry detectable labels including, but not limited to: radioisotopes, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, colloidal chromophoric labels, metal ions, ligands (e.g., biotin or haptens), and the like. For example, a chemical moiety that can be detected by a medical device to obtain a quantitative optical, electrical, or radiological signal corresponding to the detectable substance can be used to characterize the presence/amount of autoantibodies in the sample as a function of the quantitative signal obtained. The enzyme label can adopt horseradish peroxidase, alkaline phosphatase, etc. and substrate solution thereof, and the detection instrument adopts visible light enzyme labeling instrument, etc. The fluorescent label can be various fluorescent dyes or time-resolved fluorescent dyes known to those skilled in the art, such as FITC, PE, Cy3, Cy5, rare earth metals, and the like. The colloidal color-developing label can be a colloidal gold label, and the result can be observed by naked eyes.

The detection reagents described herein may be present in solution, immobilized on a support (e.g., substrate, adsorbate), or in other ways conventional in the art, so long as the presence is suitable for the detection of anti-HSPA 4 autoantibodies in biological samples. For example, when the detection reagent described herein is a nucleotide probe, it may be present in the form of a biochip (or "microarray").

In one embodiment herein, a kit for detecting anti-HSPA 4 autoantibodies in serum/plasma is provided that consists essentially of a solid phase matrix, a specific binding partner for said autoantibody, a second specific binding partner, and a detectable group; wherein the solid phase matrix is bound to a specific binding partner and the second specific binding partner is bound to a detectable group.

In some embodiments, the detection reagents described herein can be immobilized, e.g., on a solid phase matrix. Solid phase matrices suitable for use herein may be planar or curved solid phase matrices, including but not limited to: microplates, microspheres, and porous membranes. The solid phase matrix is a plane or curved surface solid phase matrix. Common solid phase matrixes are the bottom surface of polystyrene of a microporous plate, the surface of a nitrocellulose membrane, a nylon membrane, a PVDF membrane, the surface of a glass matrix, the surfaces of various organic polymers or inorganic microspheres and the like, and can be used as the solid phase matrix of the kit. The binding of the specific binding partner to the solid phase substrate can be by various binding modalities such as physical adsorption, electrostatic adsorption or covalent binding, and such binding/conjugation methods are applicable methods known to those skilled in the art.

Breast cancer diagnosis and/or prognosis evaluation product

Provided herein is a product (e.g., a kit) for breast cancer diagnosis and/or prognosis evaluation, comprising: (ii) a substance for detecting anti-HSPA 4 autoantibody levels (e.g. as not described); and optionally other substances for the diagnosis or prognosis of breast cancer, for example detection substances for existing breast cancer markers, such as detection substances for serological CA15-3, CEA detection, pathological immunohistochemical detection (e.g. Ki-67, ER, PR, CerbB 2).

Depending on the requirements of the detection method used, an appropriate detection substance for the autoantibodies against HSPA4 may be selected and formulated into a kit suitable for the detection method used. The detection mode and the reagents contained in the kit can be adjusted and changed by those skilled in the art according to actual conditions and needs.

Thus, also provided herein is a detection kit comprising: (i) detecting an effective amount of one or more reagents for detecting anti-HSPA 4 autoantibodies; (ii) optionally, one or more selected from the group consisting of: containers, instructions for use, positive controls, negative controls, buffers, adjuvants or solvents, such as solutions for suspending or immobilizing cells, detectable labels or labels, solutions for facilitating hybridization of nucleic acids, solutions for lysing cells, or solutions for nucleic acid purification.

In one example, provided herein is a test kit suitable for detecting expression of anti-HSPA 4 autoantibodies in a biological sample by a reverse enzyme-linked immunosorbent assay. The detection kit may comprise: coating buffer solution; an autoantibody specific binding partner, e.g. or a fragment or analogue thereof having binding specificity for an anti-HSPA 4 autoantibody, e.g. an epitope, a fragment comprising an epitope thereof or an analogue thereof; sealing liquid; secondary antibodies, such as anti-human IgG-HRP labeled secondary antibodies; substrate buffers, such as DAB substrate buffer; a color developing solution; and optionally a container containing the above reagents and instructions for use.

The kit may further comprise instructions for using the kit, wherein the instructions describe how to use the kit to perform the detection, and how to use the detection result to judge the metastasis and prognosis of the breast cancer and select a treatment scheme.

Of course, the kit may also comprise other reagents that are clinically useful in the judgment of breast cancer development, selection of treatment regimens and/or prognostic assessment in a subject to aid or validate the results obtained by detecting anti-HSPA 4 autoantibodies. One of ordinary skill in the art can routinely select the desired compound according to particular needs.

Application and phase of anti-HSPA 4 autoantibody in breast cancer metastasis prediction, prognosis evaluation, diagnosis and drug screening Method for detecting and evaluating

According to the disclosure in the present application, the level of the anti-HSPA 4 autoantibody is closely related to metastasis, prognosis and diagnosis of breast cancer, and thus can be used as an indicator for the prediction, prognosis evaluation and diagnosis of breast cancer metastasis.

As used herein, the term "prognosis" refers to the prediction of the likely course and outcome of a disease, which includes the judgment of the specific outcome of the disease (e.g., recovery, the appearance or disappearance of other abnormalities, such as certain symptoms, signs, and complications, and death). Poor prognosis as described herein includes, but is not limited to: the survival period is shortened, the breast cancer is easy to be transferred, the number of the breast cancer is increased, the breast cancer is enlarged, the TNM grade is increased, and the like. After predicting the patient's prognosis, the patient's prognosis may be improved in combination with a treatment that reduces the amount of the anti-HSPA 4 autoantibody molecule.

In some aspects of the disclosure, levels of anti-HSPA 4 autoantibodies are used to assess disease-free survival and/or overall survival of a patient.

In some embodiments, a level of anti-HSPA 4 autoantibodies that is higher than a control is indicative of an increased risk of disease-free survival and/or decreased overall survival of the subject. For example, disease-free survival and/or overall survival is reduced in subjects with levels of anti-HSPA 4 autoantibodies greater than controls compared to subjects with levels of anti-HSPA 4 autoantibodies less than controls. For example, the proportion of disease-free survival time and/or reduced overall survival time is increased for a population of subjects having levels of anti-HSPA 4 autoantibodies greater than the control, as compared to a population of subjects having levels of anti-HSPA 4 autoantibodies less than the control.

Generally, the following methods can be used for breast cancer metastasis prediction, prognostic assessment and/or diagnosis: detecting the level of an anti-HSPA 4 autoantibody molecule in a test subject or in a sample obtained from said subject and comparing said level to a control level; if the comparison shows that the level of the anti-HSPA 4 autoantibody molecule in the subject is higher than the control level, it is an indication that the subject is predisposed to developing breast cancer metastasis, a poor prognosis, or has suffered from breast cancer. In some embodiments, the methods of the present application further optionally comprise: obtaining a test sample from a subject; contacting the sample to be tested with a reagent or kit for detecting the level of autoantibodies to HSPA 4.

As used herein, the term "normal control" refers to the level of anti-HSPA 4 autoantibody molecules used as a reference, including but not limited to: a level of an anti-HSPA 4 autoantibody molecule measured in a non-breast cancer normal biological sample from the same subject (e.g., a sample obtained from non-breast cancer paracancerous or normal tissue of the subject), a population standard level determined by statistics, or a normalized level.

The test kit of the present application can be used for tumor diagnosis and patient prognosis evaluation, for example, by the following steps: (a) obtaining a test sample from a subject; (b) contacting a sample to be detected with a detection reagent in the detection kit of the invention; (c) detecting the level of anti-HSPA 4 autoantibodies in the test sample and comparing the level to a control level; (d) and (3) carrying out tumor diagnosis and patient prognosis evaluation according to the detection result: if the test result shows that the level of the anti-HSPA 4 autoantibody in the tissue of the subject is higher than the level of the normal control, the subject is suggested to have the possibility of breast cancer or tumor metastasis or poor prognosis of the patient.

Also provided herein is a method of screening for a candidate drug for treating, ameliorating or preventing breast cancer metastasis and/or improving the prognosis of breast cancer, the method comprising testing the effect of the candidate drug on the level of heat shock protein anti-HSPA 4 autoantibodies in a subject or a sample obtained from a subject, wherein a decrease in the level of heat shock protein anti-HSPA 4 autoantibodies following administration of the candidate drug indicates that the candidate drug has the effect of treating, ameliorating or preventing breast cancer metastasis and/or improving the prognosis of breast cancer. The various features involved in the candidate drug screening methods of the present application may be as defined or set forth herein.

The products of the present application may be used to screen or evaluate candidate drugs for treating breast cancer, alleviating or preventing breast cancer metastasis and/or improving breast cancer prognosis, for example, by:

(a) administering the drug candidate to a subject;

(b) detecting the effect of the candidate drug on the level of anti-HSPA 4 autoantibodies in the subject or in a sample obtained from the subject;

(c) judging whether the candidate drug has the effect of treating the breast cancer, relieving or preventing the breast cancer metastasis and/or improving the breast cancer prognosis according to the detection result,

wherein a decrease in the level of heat shock protein anti-HSPA 4 autoantibodies after administration of the drug candidate indicates that the drug candidate has the effect of treating breast cancer, ameliorating or preventing breast cancer metastasis and/or improving the prognosis of breast cancer.

In addition, the method has the characteristics of high sensitivity and high accuracy in the diagnosis and/or prognosis evaluation of the breast cancer of a subject by detecting the level of the anti-HSPA 4 autoantibody, and is even superior to the conventional breast cancer diagnosis and prognosis evaluation means. Moreover, the products and methods of the present application can also be used in combination with existing conventional breast cancer diagnosis and prognosis evaluation means, thereby more sensitively and accurately diagnosing and/or prognostically evaluating breast cancer. The combined use can produce certain superposition and even additive effect. Existing conventional means for breast cancer diagnosis and prognosis evaluation include, but are not limited to: serological CA15-3 and CEA detection, imaging diagnosis (such as molybdenum target), ultrasonic diagnosis, cytological diagnosis, and pathological immunohistochemical detection (such as Ki-67, ER, PR, Cerbb 2).

Examples

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art can make appropriate modifications and alterations to the present invention, which fall within the scope of the invention.

The experimental procedures for the conditions not specified in the examples below can be carried out by methods conventional in the art, for example, by referring to the molecular cloning, A Laboratory Manual, New York, Cold Spring Harbor Laboratory Press, 1989 or according to the conditions recommended by the supplier. Methods for sequencing DNA are conventional in the art and tests are also available from commercial companies.

Unless otherwise indicated, percentages and parts are by weight. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1: screening specific binding partner of breast cancer tumor-bearing mouse serum autoantibody by protein mass spectrometry technology

Mouse breast cancer cell line 4T1 was purchased from ATCC (DMEM medium + 10% fetal bovine serum) in the united states. Balb/c mice were purchased from Shanghai Sphall-BiKa laboratory animals Co., Ltd and were raised in an SPF-grade environment.

Establishing a mouse breast cancer model: mice (female, 8 weeks old) were injected 5X 10 under the right lower abdominal mammary fat pad⁵And detecting the antibody level in the serum of the mouse by an enzyme-linked immunosorbent assay (ELISA) method and observing the lymph node metastasis and the tumor growth condition of the mouse by using the breast cancer cell line/breast cancer cell line 4T1 cells.

Purification of mouse serum antibodies: antibodies were purified from mouse serum using an antibody Purification Kit (Melon Gel IgG Spin Purification Kit, Pierce, USA). The specific operation is carried out according to the instruction.

Protein mass spectrometry detection of specific binding partners after co-immunoprecipitation: tumor cells 4T1 were collected, cell lysis buffer (containing protease inhibitor) was added, and supernatants were removed after lysis. Purified serum antibody was added to the cell lysate and incubated overnight at 4 ℃ with slow shaking. The pretreated protein A agarose beads were added to the cell lysate incubated overnight with the antibody and incubated for 2-4h at 4 ℃ with slow shaking.

After the immunoprecipitation reaction, the agarose beads were centrifuged to the bottom of the tube; carefully aspirating the supernatant and washing the agarose beads 3-4 times with lysis buffer; finally, SDS loading buffer was added. The sample is sent to Beijing Huada protein research and development center, Inc. to be detected by protein mass spectrum, and the specific binding partner is screened.

The results show that: the amount of antibody in the serum of breast cancer tumor-bearing mice was significantly higher than that of normal mice (fig. 1A). Protein mass spectrometry detection of the specific binding partner bound by serum antibodies was performed and we screened for the tumor antigen HSPA 4. We further verified binding of serum antibodies to HSPA4 using an immunoblot (western blot) method (fig. 1B).

The above results suggest that the presence of an antibody targeting the tumor antigen HSPA4 in the serum of tumor-bearing mice suggests that this antibody may be a potential target for tumor detection. We further validated this finding in clinical samples.

Example 2: expression and production of human specific binding partner (HSPA4)

The specific binding partner of the selected autoantibody (HSPA4) was synthesized using genetic engineering methods known to those skilled in the art. Because of the different modifications of tumor antigens, the expression system of HSPA4 selects the commonly used human breast cancer cell line MBA-MD-231 (purchased from ATCC in usa) to obtain immunogenic antigenic proteins.

1. Construction of cloning plasmid: the full-length splicing primer (the sequence of human HSPA4 is shown as Gene ID: 3308) is designed by a PAS (PCR-based Accurate Synthesis) method and is connected into an expression vector pcDNA3.1; the obtained recombinant plasmid is transferred into a clone strain, and a positive clone is selected by a resistant plate. Performing plasmid light extraction on the positive clone, and performing double enzyme digestion and sequencing verification to ensure that the positive clone is correct; extracting positive clone plasmid, concentrating for use.

2. Transfection of human breast cancer cell line MBA-MD-231: after the MBA-MD-231 cells are cultured, the cells are plated and transfected with the density of 40-60%. A transfection solution containing the above plasmid was prepared using jetPRIME (purchased from Polyplus, France), slowly added to the culture solution, shaken well, left in an incubator at 37 ℃ for 6 hours, and replaced with a normal culture solution to continue the culture. After 48 hours of cell confluency, cellular proteins were extracted.

3. Protein column affinity purification of the target protein: the supernatant solution was applied to a Ni-IDA Binding-Buffer pre-equilibrated Ni-IDA-Sepharose CL-6B affinity chromatography column. And (4) washing with a cleaning solution. Eluting the target protein by using Elution-Buffer, and collecting effluent. The protein solution collected above was added to a dialysis bag and dialyzed overnight using 20mM Tris-HCl, 0.15M NaCl, pH 8.0.

4. Analysis by 12% SDS-PAGE showed bands stained with Coomassie Brilliant blue.

The results showed a single clear band at the target band (about 110kDa) by 12% SDS-PAGE analysis, confirming that the target protein (HSPA4) is predominantly present in the eluate (FIG. 2).

Example 3: binding coating of specific binding partners to solid phase matrices

Coating of the micro-porous plate: the specific binding partner was diluted with coating buffer (0.05mol/L carbonate buffer, pH9.6) and dispensed onto 96-well microtiter plates overnight at 4 ℃. The coating solution was removed and washed 2 times with PBS. Add blocking solution (5% nonfat dry milk-PBS) at room temperature for 2-4 hours. And (5) throwing off the confining liquid and washing with PBS.

Example 4: enzyme labeling method for detecting serum autoantibodies targeting antigen HSPA4

Patient serum or diluted serum 100 μ L was added to 96-well plates coated with specific binding partner and incubated at room temperature for 2 hours. PBST was washed 3 times. An anti-human IgG-HRP-labeled secondary antibody (purchased from Kyowa Kikuchi Biotech Co., Ltd., product No. ZDR-5301) was diluted with PBST 1:1000, added to a 96-well plate at 100. mu.L per well, incubated at room temperature for 1 hour, and then washed 3 times with PBST. Adding 100 μ L of TMB color developing solution, reacting for 20 min, and adding 2M H₂SO₄The reaction was terminated. Reading OD on microplate reader₄₅₀。

Example 5: correlation of serum heat shock protein HSPA4 autoantibody levels with patient prognosis

The correlation between the serum level of antibodies against the heat shock protein HSPA4 and the disease-free survival time and the overall survival time of the patients was analyzed.

2011-2012 women who had breast cancer (from Tianjin tumor hospital) were selected and received no chemotherapy or radiotherapy before surgery. All patients signed informed consent.

The coated microplate prepared in example 3 was used to test the levels of the 108 cases of serum heat shock proteins of breast cancer, HSPA4, autoantibodies, using the method of example 4, and to analyze the correlation between disease-free survival time and overall survival time of breast cancer patients. The P value was measured using Chi-square tests in SPSS 17.0.

As a result, it was found that: patients with high serum levels of anti-HSPA 4 autoantibodies (above mean OD value, n ═ 49) had shorter disease-free and overall survival times than those with low levels (below mean OD value, n ═ 59) (fig. 3A and 3B).

The results show that: serum anti-HSPA 4 autoantibody levels can be used as an indicator of prognosis in breast cancer patients, with high levels indicating poor prognosis.

Example 6: correlation of serum heat shock protein HSPA4 autoantibody levels with patient lymph node metastasis.

Lymph nodes are the most common metastasis sites of breast cancer patients and are also important indexes for prognosis judgment, and the correlation between the level of autoantibodies of serum heat shock protein HSPA4 and the lymph node metastasis of the patients is analyzed.

Using the coated plate of example 3, 60 normal sera (from blood donations from normal persons) and 108 breast cancers (52 lymph node negative (i.e., LN-) and 56 lymph node positive (i.e., LN +)) sera were tested for the level of heat shock protein HSPA4 autoantibodies by the method of example 4, and analyzed for association with lymph node metastasis in breast cancer patients.

As a result, it was found that: compared with normal people, the serum heat shock protein HSPA4 autoantibody level of two groups of breast cancer patients is increased; also, serum levels of autoantibodies to the heat shock protein HSPA4 were significantly higher in patients with lymph node metastases than in lymph node negative patients (fig. 4).

The above results show that: the serum anti-heat shock protein HSPA4 autoantibody level of a patient is obviously higher than the serum level of a normal human and is closely related to lymph node metastasis, and the high expression of the autoantibody level is obviously related to the poorer prognosis of the patient, and the result indicates that the serum anti-heat shock protein HSPA4 autoantibody level of the patient can be used as a marker for breast cancer diagnosis or auxiliary diagnosis, tumor metastasis prediction or patient prognosis evaluation.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. Use of a substance for detecting the level of autoantibodies against HSPA4 in the manufacture of a product for use in the diagnosis or prognosis of breast cancer in a subject.

2. Use according to claim 1, wherein the product is a kit.

3. The use of claim 1, wherein the detection is performed on a mammal or a sample obtained from the mammal that has or is suspected of having breast cancer or is at risk of having breast cancer or has been at risk of having breast cancer but has been cured.

4. The use of claim 3, wherein the sample is selected from the group consisting of: a blood sample; tissue or cell samples.

5. Use according to claim 4, wherein the blood sample is selected from: plasma, serum and whole blood.

6. Use according to claim 4, wherein the tissue or cell sample is selected from: breast tissue or cell samples, cancer tissue or cell samples, and paracancerous tissue or cell samples.

7. The use of claim 1, wherein the breast cancer is selected from the following group of types: invasive ductal carcinoma, non-invasive ductal carcinoma; alternatively, luminal a, luminal B, luminal-HER 2, HER2 overexpressing, basal cell-like, TNP-non-basal; alternatively, the hormone receptor positive type, the HER2/neu receptor positive type, the triple negative type.

8. The use according to claim 1, wherein the substance for detecting the level of anti-HSPA 4 autoantibodies is a substance for detecting anti-HSPA 4 autoantibodies at the gene level and/or protein level.

9. Use according to claim 1, wherein the substance is a substance used in one or more detection techniques or methods selected from the group consisting of: immunohistochemistry, Western blotting, Northern blotting, PCR, and biochip methods.

10. Use according to claim 9, wherein the immunohistochemistry is selected from the group consisting of: immunofluorescence analysis, reverse enzyme-linked immunosorbent assay and immunocolloidal gold method.

11. Use according to claim 1, wherein the substance is selected from: substances specific for anti-HSPA 4 autoantibodies; an HSPA4 antigen or fragment or analog thereof having binding specificity for an anti-HSPA 4 autoantibody.

12. Use according to claim 1, wherein the substance is selected from: an anti-antibody against an autoantibody to HSPA4, an epitope of the HSPA4 antigen or a fragment or analog thereof comprising said epitope.

13. The use of claim 12, wherein the anti-antibody is a monoclonal antibody.

14. Use according to claim 1, wherein the substance is selected from: specific probes, gene chips, PCR primers and gRNAs for anti-HSPA 4 autoantibodies.

15. The use according to claim 1, wherein an increased level of anti-HSPA 4 autoantibodies in the subject or in a sample obtained from the subject, compared to a normal control value, is indicative that the subject is susceptible to or has undergone cancer metastasis, or is indicative that the subject has a poor prognosis of cancer, or is indicative that the subject has suffered cancer.

16. A product for breast cancer diagnosis or prognosis evaluation, comprising:

(ii) a substance for detecting the level of anti-HSPA 4 autoantibodies; and

other substance for the diagnosis or prognostic evaluation of breast cancer, wherein said other substance is selected from the group consisting of: a detection substance against serological CA 15-3; breast cancer pathological immunohistochemical detection of agents against ER, PR and CerbB 2.

17. The product of claim 16, wherein the product is a kit.

18. A method of screening a candidate drug for treatment of, alleviation or prevention of metastasis to, and/or improvement in the prognosis of, breast cancer, the method comprising detecting the effect of the candidate drug on the level of anti-HSPA 4 autoantibodies in a subject or in a sample obtained from a subject, wherein a decrease in the level of anti-HSPA 4 autoantibodies following administration of the candidate drug indicates that the candidate drug has the effect of treating, alleviating or preventing metastasis to, and/or improving the prognosis of, breast cancer.

19. The method according to claim 18, wherein the detection of the level of autoantibodies against HSPA4 is performed using a product as mentioned in claims 1-17.