TWI702400B - Method for detecting ovarian cancer, method for monitoring progression of ovarian cancer in an ovarian cancer patient, and use of a reagent that recognizes a cleaved c3 polypeptide - Google Patents

Abstract

The present invention relates to biomarkers, methods and assay kits for the identification and screening for ovarian cancer and monitoring treatment of ovarian cancer.

Description

Methods for detecting ovarian cancer, methods for monitoring the development of ovarian cancer in patients with ovarian cancer, and Use of reagent capable of identifying cleavage C3 multipeptide

The present invention relates to biomarkers, methods and test kits for identifying and screening ovarian cancer and monitoring the treatment of ovarian cancer.

Ovarian cancer is one of the most common cancers among women in the world. It has a high prevalence rate in developed countries, and its incidence and mortality are still rising. Undoubtedly, if ovarian cancer can be detected early, it can provide the best cure opportunity and improve the survival rate of patients. Although ultrasound can see images of suspected tumors, it cannot distinguish whether it is ovarian cancer, and the sensitivity is not high, and it is also limited by equipment, which makes it difficult to promote large-scale screening. The currently reported ovarian cancer markers include CA-125 (cancer antigen 125, cancer antigen 125) and HE4 (paratestin 4, epididymis protein 4), but they are mainly used as prognostic indicators. As for ovarian cancer screening, especially Detection of early ovarian cancer, the effect is still not good.

CA-125 is the most widely used tumor indicator in obstetrics and gynecology, but only about 50-60% of patients with stage I ovarian cancer will have abnormal expression of CA-125. In addition, other malignant tumors, such as cervical cancer and breast cancer, or during pregnancy, endometriosis, pelvic inflammatory disease, etc., will also increase the level of CA-125. Therefore, CA-125 is not very specific for ovarian cancer. Furthermore, about 20% of patients with ovarian cancer can only detect a small amount, or even not detect CA-125 at all (Moore RG. et al ., Curr Opin.Oncol.Sep.22(5):492 -7.2010).

The HE4 gene is located on chromosome 20, and the protein structure consists of four disulfide bonds. It was first identified from male paratestines (Hellström I. et al ., CANCER RESEARCH 63, 3695-3700, July 1, 2003). Normal tissues also have the expression of this protein, for example, the gene expression is highest in the trachea and salivary glands. Although the function of HE4 is still unclear, in recent years, HE4 has been used to detect ovarian cancer, and it has been found that there are fewer false positives than CA-125, and it can better distinguish endometriosis and malignant ovarian tumors, and In some patients with ovarian cancer who did not show CA-125 indicators, increased levels of HE4 in the blood can be detected (Rosen DG. et al ., Gynecologic Oncology, November 2005 Volume 99, Issue 2, p255-526). However, even so, studies have pointed out that the content of HE4 in the blood will only increase significantly in patients whose lesions are obviously diagnosed as ovarian cancer after surgery. Therefore, HE4 is mainly used as a prognostic indicator, and it is difficult to be used for ovarian cancer screening and early stage Effective marker for diagnosis.

The types of ovarian cancer are complex, including at least endometrioid ovarian cancer, clear cell ovarian cancer, serous ovarian cancer, mucinous ovarian cancer, and other types of ovarian cancer, such as , Ovarian carcinosarcoma (ovarian carcinosarcoma), ovarian immature teratoma (Immature teratoma), ovarian mucinous and granulosa tumor (ovarian mucinous and granulosa tumor), and metastatic ovarian cancer (Krukenberg, ovarian Krukenberg tumor) Wait. So far, there is no effective biomarker that can be used to widely detect different types of ovarian cancer, and there is no effective biomarker for early detection of ovarian cancer.

Therefore, there is still a need to propose a biomarker for ovarian cancer, which can be used for extensive ovarian cancer screening, especially for early detection of ovarian cancer.

The present invention has unexpectedly discovered that in patients with ovarian cancer, the plasma protein complement component 3 (C3) is activated to produce a peptide fragment with a size of about 40kDa, which can be specific to different types It can be detected in the plasma of ovarian cancer patients, especially in the early stage, but it is impossible or almost impossible to detect in individuals without ovarian cancer. In addition, the content of this fragment increased in patients with worsening ovarian cancer. Therefore, the cleavage type of C3 protein can be used as a specific molecular marker for the diagnosis of ovarian cancer, especially for early diagnosis. It can also be used to monitor the progress of ovarian cancer in patients with ovarian cancer, or to evaluate ovarian cancer therapy The effectiveness of treatment.

In one aspect, the present invention provides a method for detecting ovarian cancer, comprising: (a) providing a biological sample from an individual to be tested; (b) detecting the cleaved complement component 3 (C3) of the biological sample from the individual to be tested The content of the multipeptide to obtain the detection amount of the cleaved C3 multipeptide; (c) compare the detection amount of the cleaved C3 multipeptide with the normal amount of the cleaved C3 multipeptide from a normal individual to obtain a comparison result; and (d ) Determine the possibility of ovarian cancer of the individual to be tested according to the comparison result, wherein if the comparison result shows that the detected amount of cleaved C3 multipeptide is higher than the normal amount of cleaved C3 multipeptide, then it is determined that the test individual has Ovarian cancer may be at risk of ovarian cancer.

In another aspect, the present invention provides a method for monitoring the development of ovarian cancer in a patient with ovarian cancer, the method comprising: (a) at a first time point, providing a first biological sample derived from the patient; (b) in At a second time point, a second biological sample derived from the patient is provided, wherein the second time point is later than the first time point; (c) Detect the content of cleaved C3 multipeptide in the first biological sample to obtain the first detection amount of cleaved C3 multipeptide, and detect the content of cleaved C3 multipeptide in the second biological sample to obtain the cut The second detection amount of type C3 multipeptide; (d) compare the first detection amount of cleaved C3 multipeptide with the second detection amount of cleaved C3 multipeptide to obtain a comparison result; and (e) judge based on the comparison result For the progression of ovarian cancer in the patient, if the comparison result shows that the second detected amount of cleaved C3 multipeptide is higher than the first detected amount of cleaved C3 multipeptide, it means that ovarian cancer is progressing.

In some specific embodiments, according to the method of the present invention, the patient is receiving treatment for ovarian cancer, and the first biological sample and the second biological sample are taken before or after treatment, or during treatment.

In some specific embodiments, the method of the present invention further comprises evaluating the therapeutic effect of the ovarian cancer treatment on the patient, wherein if the content of the cleaved C3 polypeptide shows a decreasing trend after treatment or during treatment, It means that the treatment is effective for the patient.

In some specific embodiments, the cleaved C3 multipeptide is C3 alpha ( α ) chain fragment 2.

In some specific embodiments, the cleaved C3 polypeptide has an amino acid sequence corresponding to position 1321 to position 1663 of the C3 protein of SEQ ID NO:1.

In some specific embodiments, the cleaved C3 multipeptide is a peptide fragment having the amino acid sequence of SEQ ID NO: 2.

In some specific embodiments, the detection step of the present invention is performed by mass spectrometry or immunoassay.

In some specific embodiments, the detection step of the present invention is performed by an immunoassay method that uses an antibody that specifically binds to the cleaved C3 polypeptide.

In some specific embodiments, the biological sample used in the method of the present invention is a body fluid sample, a tissue sample, or a biological specimen sample.

In some specific embodiments, the biological sample and body fluid sample used in the method of the present invention is selected from blood samples (such as plasma samples, heparinized plasma samples, EDTA-plasma samples, serum samples), urine samples, and ascites samples. Formed group.

In some specific embodiments, the ovarian cancer detectable by the method of the present invention can be endometrioid ovarian cancer, clear cell ovarian cancer, serous ovarian cancer, mucinous ovarian cancer ) Ovarian cancer, and other types of ovarian cancer, for example, ovarian carcinosarcoma (ovarian carcinosarcoma), ovarian immature teratoma (Immature teratoma), ovarian mucinous and granulosa tumor (ovarian mucinous and granulosa tumor), and metastatic ovarian cancer (Krukenberg, Krugenberg tumor of the ovary) and so on.

In some specific embodiments, the method of the present invention can detect stage I ovarian cancer, stage II ovarian cancer, stage III ovarian cancer, and stage IV ovarian cancer.

In another aspect, the present invention provides the use of a reagent that can identify cleavage-type C3 polypeptides, which is used for diagnosing or monitoring the development of ovarian cancer, or for preparing reagents for diagnosing or monitoring the development of ovarian cancer Or set.

The detailed description of one or more specific embodiments of the present invention is described in the following description. Other features or advantages of the present invention will be clarified by the following detailed description of various specific embodiments and the scope of the attached patent application.

Figure 1 shows Western blot analysis of plasma samples derived from ovarian cancer patients (P, patient) and normal individuals (N, normal), using antibodies against cleavage-type complement component 3 (C3) protein. The protein in the plasma sample derived from the subject is subjected to electrophoresis in a reduced state. The number on the left represents the molecular weight (kDa) and is the position of the molecular weight marker. The arrow indicates the approximately 40kDa peptide fragment of the complement component 3 protein.

Figure 2 shows the Western blot analysis of plasma samples of patients with ovarian cancer at different time points. Number 1 is the result of plasma sample analysis of patients with ovarian cancer at the first time point, and number 2 is the same patient after receiving chemotherapy The analysis results of plasma samples returned to the clinic for about one year showed that the content of cleaved C3 protein increased. The patient was further confirmed that his condition had deteriorated and cancer metastasis had occurred; number 3 is the analysis result of plasma samples of normal individuals, showing that cleaved C3 was not detected protein.

In order to provide a clear and easy way to understand the present invention, some terms are first defined. Other definitions are also explained in the detailed description. Unless otherwise defined, all technical and scientific terms in this article have the same meaning as those known to those skilled in the field of the invention.

The terms "一(a)" and "一(an)" as used herein refer to one or more (ie at least one) objects that conform to the context. For example, "a component" means one type of component or more than one type of component.

As used herein, the term "multipeptide" or "protein" refers to a polymer composed of amino acid residues connected by peptide bonds. The term ``protein'' generally refers to polymers with relatively large molecular weight (more amino acid residues) (e.g., containing more than 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 amino acid residues). The term "multipeptide" or "peptide" generally refers to polymers with relatively small molecular weight (fewer amino acid residues) (e.g., containing up to 500, 400, 300, or less amino acid residues). ). Amino acids can be represented by three letters or one letter as known in the art.

The term "about" or "approximately" mentioned in this article refers to the acceptable degree of variation for those with ordinary skills in the field, and it may vary to different degrees depending on the content. Generally speaking, "about" or "approximately" may mean that a value has a range of ±10% of the stated value.

The terms "subject", "individual", and "patient" mentioned herein are used interchangeably herein and refer to any mammalian individual, which is suitable for diagnosis, prognosis, treatment or course of treatment, specifically refers to Humanity. Other individuals may include cows, dogs, cats, guinea pigs, rabbits, rats, mice, horses, etc.

The second word "diagnosis" mentioned in this article generally includes determining whether an individual is likely to suffer from a disease, illness or loss of function. Those skilled in the field usually make a diagnosis based on one or more indicators (such as a marker, presence or absence, or its content, which can be used as an indicator of the presence or severity of a disease, disease, or loss of function). It can be understood in the art that diagnosis does not refer to determining the presence or absence of a specific disease with 100% accuracy, but refers to an increase in the possibility of a certain disease in the subject.

The term "treatment" as used herein refers to the purpose of curing, healing, alleviating, alleviating, changing, correcting, improving, improving or affecting the disease, the symptoms of the disease, the disability caused by the disease, or the tendency to suffer from the disease , While administering or administering one or more active ingredients to individuals suffering from the disease, symptoms of the disease, or prone to suffering from the disease.

As used herein, "normal individuals" and "control individuals" can be used interchangeably, and refer to individuals who are healthy and do not suffer from the specified disease (such as ovarian cancer), and can refer to a single normal individual or a group of normal individuals.

The term "abnormal amount" mentioned herein means that an indicator has an increased amount compared to an individual without a target disease (such as ovarian cancer) or a reference amount or a control amount. Specifically, for the examiner, the measured abnormal amount is higher than the reference amount or the control amount by 10%, 20%, 30%, 40%, 50%, 60%, 70% %, 80%, 90% or 100%. The reference amount or the control amount can be the amount measured by a biomarker from a normal individual or a control sample (such as a tissue or cell that does not contain the target disease). In this field, conventional detection and statistical methods can be used to obtain the numerical range of the normal amount by analyzing the labeled detection amount of samples of normal individual populations.

As used herein, a biomarker is a characteristic (such as protein, gene, gene expression), which can be measured and evaluated objectively as a normal or abnormal biological response, disease, treatment process, or response to treatment , Or indicators of interventional therapy. The marker can include the feature, or the pattern of the feature, or the presence or absence of the set, and the feature can be an indicator of a specific biological response. The measurement of the biomarker can be increased or decreased to represent a specific biological event or process. Markers are mainly used for diagnosis and prognosis purposes. However, it can also be used for treatment, monitoring, drug screening, and other purposes described herein, including evaluating the effectiveness of cancer therapy.

The biological samples described herein can be analyzed by any of the methods described herein, and the type of sample analyzed can be any sample obtained from a diagnosed individual, including samples derived from blood, urine, or ascites. Typically, the blood sample may be whole blood or a portion thereof, such as serum or plasma, heparinized or EDTA processed sample to avoid blood clotting. In addition, the biological sample may be a tissue sample or a biological specimen.

The present invention mainly (at least part of it) discovers a novel and reliable biomarker for ovarian cancer, namely the cleaved C3 polypeptide. The following examples confirm that the biomarker is specifically and significantly present in blood samples of ovarian cancer patients of various stages and various cell types, but blood samples of individuals with other cancer types or other diseases are not detected This biomarker. Therefore, the eggs described in this article The nest cancer detection method can identify individuals with, suspected, or risk of ovarian cancer. The detection methods described in this article can be used by any individual (such as female human individuals), especially for initial and routine screening, to screen patients with ovarian cancer, or those who may have ovarian cancer in progress. Patients at risk.

I. Cleaved C3 multipeptides can be used as novel biomarkers for ovarian cancer

Complement component 3 (C3) is a plasma glycoprotein, a member of the immune system, with a molecular weight of about 187kDa, and is the most abundant component in the complement system. When the complement system is activated, C3 can be cleaved by invertase into three fragments, including a larger C3 beta (β) fragment, and two smaller C3 alpha ( α ) fragments, including C3 α chain fragment 1 (C3 α chain fragment 1) and C3 α chain fragment 2 (C3 α chain fragment 2). Specifically, the human C3 protein contains a full-length amino acid sequence as shown in SEQ ID NO:1 (1663 amino acid), and the cleaved C3β fragment is a multi-peptide fragment of the amino acid sequence from position 23 to position 667. C3 α chain fragment 1 is a multipeptide fragment of the amino acid sequence from position 749 to position 954, and C3 α chain fragment 2 is a multipeptide fragment of the amino acid sequence from position 1321 to position 1663 (about C3 full length C A peptide fragment with about 342 amino acids at the end).

As described herein, the present invention relates to the presence and amount of the cleaved C3 polypeptide related to the appearance and development of ovarian cancer. The term "cleaved C3 polypeptide" as used herein or similar terms (such as "cleaved C3") refers to the C3 α chain fragment 2 after the full-length cleavage of C3. Specifically, the cleaved C3 polypeptide used herein refers to human C3 α chain fragment 2, which is a peptide fragment with about 342 amino acids at the C-terminus of the human C3 polypeptide, and has a molecular weight of about 40kDa. Specifically, it is a multi-peptide fragment having an amino acid sequence corresponding to the amino acid sequence from position 1321 to position 1663 of the C3 protein of SEQ ID NO:1, which is the Arg( at position 1320 of the C3 protein of SEQ ID NO:1 R) and Ser(S) at position 1321 are produced by hydrolytic cleavage. More specifically, the cleaved C3 multipeptide used herein is a multipeptide fragment having the amino acid sequence of SEQ ID NO:2. It is understandable that the polypeptide can have a limited number of changes or modifications, which can be carried out within a certain part of the polypeptide that is not related to its activity or function, and still result in an acceptable degree of equivalent or similar biological activity or Features.

The cleaved C3 multipeptide according to the present invention can be prepared by traditional methods, such as recombinant technology, using suitable host cells (such as E. coli, yeast, mammalian, insect or cell-free host systems) or synthetic methods. In some examples, the cleaved C3 multipeptide is different from the naturally occurring peptide in at least post-translational modification (such as glycation). The cleaved C3 polypeptide can be mixed with a pharmaceutically acceptable reagent (such as a carrier that does not co-exist with the cleaved C3 polypeptide in nature, or a non-naturally occurring carrier, or an immune adjuvant), To form a composite, for medical purposes or for inducing antibody production.

Ⅱ. Method for detecting cleaved C3 multipeptide and measuring its amount

Any conventional technique can be used to determine the presence and amount of cleaved C3 polypeptide in a biological sample. In some embodiments, the presence and/or amount of the cleaved C3 polypeptide can be determined by mass spectrometry, which can directly measure the analyte with high sensitivity and reproducibility. Many different mass spectrometry methods can be used. Examples of mass spectrometry include, but are not limited to, media-assisted laser desorption ionization/time difference ranging (MALDI-TOF), surface enhanced laser desorption ionization/time difference ranging (SELDI-TOF), liquid chromatography- Mass spectrometry (LC-MS), liquid chromatography tandem mass spectrometer (LC-MS-MS), and electrospray ionization mass spectrometry (ESI-MS). An example of a part of this method is a tandem mass spectrometer (MS/MS), which involves multiple steps for mass selection or analysis, usually separated by partial fragmentation patterns.

In other specific embodiments, the presence and/or amount of the cleaved C3 polypeptide can be determined by immunoassay methods. Examples of immunoassay methods include, but are not limited to, Western blot analysis (reduced or denaturing electrophoresis), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunoassay The precipitation method (RIPA), immunofluorescence method (IFA) and electrochemical fluorescence method (ECL).

In some examples, the presence and/or amount of the cleaved C3 polypeptide can be detected by using a reagent that can specifically identify the cleaved C3 polypeptide, for example, an antibody.

(i) Antibodies that specifically bind to the cleaved C3 multipeptide

The term "antibody" as used herein refers to a complete immunoglobulin, or a fragment thereof, and a multipeptide containing an antigen-binding domain or antigen-binding fragment, which can specifically bind to a specific antigen. The term includes, but is not limited to, single-strain, single-specific, multi-strain, multi-specific, humanized, human, single-stranded, chimeric, synthetic, recombinant, mutant, and heterozygous Of antibodies. Applicable antibodies can be prepared according to methods known in the field. The antibody may not exist in nature (if not artificially, it will not be produced in nature).

An intact or complete antibody contains two heavy chains and two light chains. Each heavy chain variable region by a line (V _H) and a first, second, and third constant region (C _H 1, C _H 2 and C _H 3) consisting, and each line consists of a light chain variable It consists of a region (V _L ) and a constant region (C _L ).

The term "antigen-binding domain" or "antigen-binding fragment" refers to the part or region of the entire antibody molecule that is responsible for binding to the antigen. The part of an antigen that can be specifically bound or recognized by an antibody is called an "antigenic determinant (or epitope, epitope)". Antigen-binding domain may comprise a heavy chain variable region (V _H) and light chain variable region (V _L); however, it is not necessary to include both. The variable regions in the two strands typically contain three highly variable regions, which are called complementarity determining regions (CDRs). The three CDRs are separated by framework regions (FRs), which are more highly conserved than CDRs. The constant regions of the heavy chain and light chain have nothing to do with antigen binding, but have different functions. The classification of antibodies is based on the amino acid sequence of the constant region of the heavy chain. The five main antibody types or isotypes are IgG, IgM, IgA, IgD, and IgE, which are defined by the constant regions of the heavy chain γ, μ, α , δ, and ε, respectively. Antibody antigen-binding example fragment, comprising: (1) a Fab fragment, having a V _L, a monovalent fragment V _H, C _L and C _H 1 domain of the; (2) a F (ab ') ₂ fragments, having a bivalent fragment comprising two Fab fragments, the lines were bridged through disulfide bonds to the coupling region, i.e., the Fab diabodies; (3) having a single-stranded V _L and V _H domains of the Fv fragment of the antibody; (4) a single complementarity determining region isolated (CDR); (5) a single-stranded Fv (scFv), a single strand of multiple peptide chains, Department of the V _H domain and a V _L domain, whose link through peptide linker; and (6) A (scFv) ₂ comprises three peptide chains; two V _H domains by linker peptides and a disulfide bond and two V _L domains.

The term "human antibody" includes antibodies with variable and constant regions that essentially correspond to, or are derived from, the immunoglobulin sequence of the human germline. The human antibody of the present invention, however, may contain amino acid residues that do not encode human germline immunoglobulin sequences (such as mutations induced by random or in vitro mutations at specific locations, or by body mutations in vivo), for example In CDRs. Specifically, the human antibody may have at least 1, 2, 3, 4, 5 or more positions replaced with amino acid residues, which are non-coding human germline immunoglobulin sequences.

In some specific embodiments, the antibody used in any of the methods described herein is an antibody that can specifically bind to the cleavage-type C3 multipeptide, for example, can specifically bind to the sequence of SEQ ID NO: 2 Cleaved C3 multi-peptide.

An antibody that can "specifically" identify a target (such as cleavage-type C3 polypeptide) is a term well known in the field, and the method used to determine the specific identification is also well-known in the field. If a molecule reacts or associates with a specific target antigen (but not with other targets) more frequently, faster, longer, and/or with higher affinity, the molecule is said to have "specific identification" Ability. Specifically, if an antibody binds to its target antigen (compared to other substances) with a higher affinity, tendency, faster, and/or longer time, then the antibody has a "specific recognition of the target antigen" " Ability. For example, an antibody can specifically (or preferably) bind to the cleaved C3 multipeptide or its epitope, which means that the antibody and its target antigen (compared to its binding to other antigens, such as full-length C3 , Or other epitopes in the same antigen) have higher affinity, tendency, faster, and/or longer binding capacity. It should be understood that "specific combination" or "preferred combination" does not necessarily require exclusive combination (although it may include exclusive combination). In general, but not necessarily, reference to bonding refers to preferably bonding.

In a specific embodiment, the antibody used in the method described herein is an antibody that can specifically recognize the cleaved C3 polypeptide, for example, an antibody that can specifically recognize the polypeptide of SEQ ID NO: 2. More specifically, the antibodies described herein that can specifically identify the cleaved C3 multipeptide means that the antibody can specifically bind to the epitope (epitope) in the cleaved C3 multipeptide, so that it can be In the biological sample tested, the antibody can be distinguished by conventional immunoassay methods (eg, Western blotting method, for example, by the determination of molecular weight) whether there is a cleaved C3 polypeptide, or the cleavage can be measured Type C3 multi-peptide content. In some embodiments, the antibody system capable of specifically identifying the cleavage C3 multipeptide of the present invention can specifically identify the N-terminal single amino acid residue of the cleavage C3 multipeptide, for example, SEQ ID NO: 2 The N-terminal serine (S) of the antibody does not bind to the full-length C3 protein, and can be used to distinguish whether there is a cleaved C3 polypeptide, or to further measure the content of the cleaved C3 polypeptide.

Any antibody described herein belongs to the scope of the present invention.

(ii) Antibody preparation

The antibodies described herein can bind to cleaved C3, which can be prepared by any method known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

In some specific embodiments, the antibodies described herein can be multiple strains of antibodies, which can be routinely Obtained by multiple antibody preparation methods. Specifically, cleaved C3 purified from human serum or recombinant peptides prepared by genetic engineering as antigens can be added with carrier proteins, such as bovine serum albumin (BSA) or keyhole blood blue Protein (keyhole limpet hemocyanin, KLH) to increase its immunogenicity. The antigen (or mixed with the carrier protein) is injected into an animal (such as a rabbit) to induce an immune response, and additional immunization can be performed, for example, once every two weeks, for a total of about 2-5 times. Subsequent detection is performed by enzyme immunoassay to ensure that the serum titer after immunization is greater than 1:4000 before all blood sampling can be performed. After the blood is collected, after the blood is coagulated, the supernatant is centrifuged to obtain an antiserum containing multiple antibodies against cleavage C3. Preferably, the antiserum can be purified, for example, by affinity chromatography (protein A/G) for the purification of immunoglobulin (Ig), and further precipitation with ammonium sulfate to obtain antibodies containing target specificity It can also be combined with ion exchange column chromatography to remove impurities in the precipitate.

In some specific embodiments, the antibody specifically binds to a target antigen (such as the cleavage type C3, such as SEQ ID NO: 2), which can be prepared by conventional fusion tumor technology. The full-length target antigen or fragments thereof can optionally be combined with a carrier protein (such as KLH) to immunize host animals to produce antibodies that can bind to the antigen. The immune time course and route of the host animal are generally the same as established and conventional techniques used to stimulate antibody production and production (further explanation herein). The general techniques used to produce mouse, humanized, and human antibodies are all known in the art and are described herein. It should be considered that any mammalian individual, including human or antibody-producing cells, can be manipulated as the basis for the production of mammalian and human-containing fusionoma cell lines. Generally speaking, the host animal is vaccinated with a certain amount of immunogens, including those described herein, by intraperitoneal, intramuscular, oral, subcutaneous, plantar, and/or intradermal methods.

Use general somatic cell hybridization technique (Kohler, B. and Milstein, C. (1975) Nature 256: 495-497 or modified from Buck, DW, et al., In Vitro , 18: 377-381 (1982)), Fusion tumors can be prepared from lymphocytes and immortal myeloma cells. The available myeloma cell lines, including but not limited to X63-Ag8.653 and the cells derived from Salk Institute, Cell Distribution Center, San Diego, Calif., USA, can all be used for heterozygous. Generally speaking, this technique involves the use of a fusion agent to fuse myeloma cells and lymphocytes. The fusion agent, such as polyethylene glycol, or by electrical means, is well known to those skilled in the art. After fusion, the cells are separated from the fusion medium and cultured in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium to eliminate the non-fusion Parental cells. Any of the medium described herein, with or without serum, can be used to cultivate fusion tumors, which can secrete monoclonal antibodies. After these fusion tumors are proliferated and subcultured, if necessary, the supernatant can be analyzed for anti-immunogen activity by routine immunoassay steps (such as radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

Fusion tumors can be used as a source of antibodies, which include all derivatives. The progeny cells of parental fusion tumors can produce monoclonal antibodies, which can bind to cleaved C3. Fusion tumors that can produce these antibodies can be cultured in vitro or in vivo using conventional methods. The monoclonal antibody can be separated from the culture medium or body fluids by traditional immunoglobulin purification steps such as ammonium sulfate precipitation, colloidal electrophoresis, dialysis, chromatography, and ultrafiltration, if applicable. If undesired activity occurs, it can be removed, for example, by using the preparation with an absorbent prepared from an immunogen, the immunogen is attached to a solid phase, and the desired The antibody is eluted or released from the immunogen. Use the target antigen or a fragment containing the target amino acid sequence linked to a protein to immunize a host animal to obtain the produced antibody (such as a monoclonal antibody); wherein the antigen system is immunogenic to the immune species, such as keyhole Insect hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitors using bifunctional or derivative agents, such as maleiminobenzoic acid sulfosuccinimidyl ester (through and half Cystine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl, or R1N=C=NR, Where R and R1 are different alkyl groups.

If necessary, the antibody (such as a single strain or multiple strains) of interest (such as produced by a fusion tumor) can be sequenced, and the polynucleotide sequence can be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a host cell's vector, and the host cell can be propagated and frozen for future use. Alternatively, the polynucleotide sequence can be used to genetically transform into a "humanize" antibody or improve its affinity (affinity maturity) or other antibody characteristics. For example, if the antibody is used in clinical trials and humans are treated, the constant region can be modified to be more similar to human constant regions to avoid immune response. In addition, it is possible to genetically modify the antibody sequence to obtain a higher affinity for the target antigen and a higher efficiency in detecting cleavage of C3. For those skilled in the art, it should be understood that one or more polynucleotide changes can be made to the antibody and still maintain its binding specificity to the target antigen.

In other specific embodiments, intact human antibodies can be prepared using commercially available mice, which are modified to express specific human immunoglobulins. The transgenic animal line is designed to generate more desired (such as intact human antibodies) or stronger immune response, and can also be used to generate humanized or human antibodies. Examples of such technologies as Amgen, Inc. (Fremont, Calif .) The Xenomouse ^RTM, and Medarex, Inc. (Princeton, NJ ) and the HuMAb-MouseRTM TC Mouse ^TM. In another possibility, the antibodies can be prepared by phage expression or yeast technology. See, for example, US Patent Nos. 5,565,332; 5,580,717; 5,733,743 and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455. In addition, phage expression technology (McCafferty et al., (1990) Nature 348:552-553), derived from immunoglobulin variable region (V) gene groups from unimmunized contributors, was used to produce human antibodies, and Antibody fragments in vitro.

The antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by conventional methods. For example, F(ab') ₂ fragments can be prepared by pepsin hydrolyzing antibody molecules, and Fab fragments can be produced by reducing the disulfide bonds of F(ab') ₂ fragments.

Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single chain antibodies, and bispecific antibodies, can be prepared by, for example, traditional recombinant techniques. In one example, a DNA encoding a monoclonal antibody (specific to the target antigen) can be isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes, it can be specifically compared with the encoding monoclonal antibody). Gene combination of heavy and light chains of antibodies). The fusion tumor cell can be used as a better source of the DNA. Once isolated, the DNA can be placed in one or more expression vectors and then transfected into host cells, such as E. coli cells, ape COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells , It does not produce immunoglobulins to obtain synthetic monoclonal antibodies from these recombinant host cells. See, for example, PCT Patent Publication No. WO 87/04462. The DNA can then be modified, for example, by replacing the sequences encoding the human heavy and light chain constant regions with homologous murine sequences, Morrison et al., (1984) Proc. Nat. Acad. Sci .81:6851, or by covalently binding the immunoglobulin coding sequence, all or part of the coding sequence and a non-immunoglobulin polypeptide sequence. In this way, genetically engineered antibodies, such as "chimeric" or "fusion" antibodies, can be prepared and can specifically bind to the target antigen.

The techniques involved in the preparation of "chimeric antibodies" are all well-known in this field. See, for example, Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81,6851; Neuberger et al. (1984) Nature 312,604; and Takeda et al. (1984) Nature 314:452.

The methods used to construct humanized antibodies are also well known in the art. See, for example, Queen et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989). In one example, parental V _H and V _L variable regions of a non-human antibody-based three-dimensional molecular model analysis performed to methods well known in the art. Next, the framework amino acid residues are predicted to be important for forming the correct CDR structure, and the same molecular model analysis is used to identify the framework amino acid residues. Meanwhile, the human V _H and V _L chain having the amino acid sequence, with such a non-human parent antibody is homologous to the genus; to the human amino acid sequence of the parental lines V _H and V _L sequence as a search word, from Found in various antibody gene databases. Then the selection of human V _H and V _L receptor gene.

The CDR region located in the selected human receptor gene can be replaced with the CDR region of the parental non-human antibody or its functional variant. When necessary, residues located in the framework region of the parental chain are predicted to be important and are used to interact with the CDR region (see above) and can be used to replace the corresponding counterpart in the human receptor gene Residues.

A single-chain antibody can be prepared by recombinant technology by linking a nucleotide sequence encoding the variable region of the heavy chain to a nucleotide sequence encoding the variable region of the light chain. Preferably, a flexible linker can be added between the two variable regions. Alternatively, the single-stranded antibody production technology described in (US Patent Nos. 4,946,778 and 4,704,692) can also be used to generate a phage or yeast scFv library and scFv colonies, which are specific for cleaved C3 and can be used Find out from the database by conventional means. The positive colonies can then be further screened to identify those that bind to cleaved C3.

The properties of antibodies prepared from methods known in the field and the methods described herein can be confirmed by methods known in the field. For example, a method is used to identify the epitope that binds to the antigen, or "epitope map". There are many different known methods in this field that can be used to locate and analyze the position of epitopes on proteins, including analysis of the crystal structure of antibody-antigen complexes, competitive analysis, gene fragment expression analysis, and synthetic peptide analysis, such as , As described in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In another sample, the epitope map can be used to determine the sequence of antibody binding. The epitope can be a linear epitope, for example, contained in a single-stranded amino acid, or a conformational epitope formed by three-dimensional amino acid interaction Base, which may not need to be included in a single-strand extended branch (the main structure of a linear sequence). Peptides of different lengths (such as at least 4-6 amino acids in length) can be isolated or synthesized (eg recombinantly), and an antibody can be used for binding analysis. In another example, the antigenic determinant that can be bound by the antibody can be determined in a systematic screening by using overlapping peptides (derived from the target antigen sequence) to determine the antibody binding position. According to gene fragment performance analysis, the open reading frame encoding target antigen system is randomly fragmented, or fragmented by specific gene construction, and the reactivity of the expressed antigen fragment can be determined by testing using antibodies. The gene fragment may, for example, be prepared by PCR, and then transcribed and translated into protein in vitro in a radioactive amino acid environment. Whether the antibody binds to the radioactively labeled antigen fragment can be determined by immunoprecipitation and gel electrophoresis. Part of the epitope can also be identified in a large database (phage library) of random peptide sequences displayed on the surface of phage particles. In addition, an analyzed database of overlapping peptide fragments can be used to test the binding of the test antibody in a simple binding test. In another example, for the mutation of the antigen binding domain, functional region switching test and alanine screening mutation method can be performed to identify the required, sufficient, or necessary residues for epitope binding. For example, the functional region switching test can be performed using a mutation of the target antigen, in which various fragments of a cleaved C3 polypeptide are replaced (replaced) with a similar but non-antigenic protein sequence. By analyzing antibodies that bind to the mutant C3, the importance of antibodies that bind to specific antigen fragments can be evaluated. The antibody that binds to the desired epitope can be identified by the epitope mapping map.

Ⅲ. Application of novel ovarian cancer markers in diagnosis and prognosis

As described in this article, the cleaved C3 multipeptide after protease decomposition was found to be related to the appearance and progression of ovarian cancer. Therefore, the methods described herein for screening patients with ovarian cancer, monitoring the progress of ovarian cancer, and methods for evaluating the therapeutic effect of ovarian cancer can use the cleaved C3 multipeptide as a possible Biomarker of trust. As mentioned above, the detection method described herein can be used as a preliminary screening method for any female individual to detect whether they may have ovarian cancer (especially early stage ovarian cancer).

The individual analyzed by this method can be any female mammalian individual, such as a human individual. In some examples, the individual may have one or more symptoms associated with ovarian cancer. In other instances, the individual may be at risk of developing ovarian cancer. In another example, the individual is asymptomatic. In another example, the diagnostic method described herein can be used as a routine screening method for healthy female individuals, such as women over 20, 35, 40, 45, 50 or 55 years old, to detect potential ovaries Cancer risk or development.

In order to perform the method described herein, a biological sample taken from an individual in need (such as a human patient who does not have any symptoms of ovarian cancer, or a human patient who has, is suspected of, or is at risk of having ovarian cancer ) Detecting or measuring the cleaved C3 polypeptide, or the amount thereof, in the sample by any method known in the art, such as those described herein, such as mass spectrometry and immunoassay. The biological sample may be a biological liquid sample, such as a blood sample, such as a plasma or serum sample. The cut C3 can be detected quantitatively or qualitatively. Any effective method in this field to measure the presence, amount, or activity of the marker is included in the present invention. Those skilled in the field have the ability to determine which method is most suitable for measuring a particular mark.

In a specific embodiment, a sample obtained from an individual to be tested is tested for the presence or absence of the cleaved C3 polypeptide. If the cleaved C3 polypeptide can be detected in a sample derived from an individual to be tested, it means that the individual has been diagnosed with ovarian cancer or has a risk of ovarian cancer.

In some embodiments, the amount of cleaved C3 polypeptide in a sample derived from a candidate individual can be compared with a standard value to determine whether the candidate individual has or is at risk of ovarian cancer. The standard value represents the amount of cleaved C3 polypeptide in the control sample. The control sample can be taken from an individual (e.g. A female individual) who does not have ovarian cancer. In addition, the control sample can be taken from a mixture of multiple individuals. Optionally, the control group individuals match the candidate individuals, such as age, gender, and/or ethnic background. Preferably, the control sample and the biological sample of the candidate individual are samples of the same type. In some specific embodiments, if the cleaved C3 polypeptide is measured, or the amount of cleaved C3 polypeptide is higher than the standard value (for example, about 10% or more higher than the standard value), the candidate individual May be diagnosed with, suspected, or at risk of ovarian cancer. In some examples, using conventional methods, such as those described herein, the amount of cleaved C3 polypeptide in the control sample is undetectable in the control sample (the standard value is 0, which is lower than the detection Test limit). In this example, the same method is used to detect the presence of the cleaved C3 polypeptide in a biological sample derived from an individual, which means that the individual has, is suspected of having, or is at risk of ovarian cancer.

The method of the present invention specifically and extensively identifies various types of ovarian cancer and different stages of ovarian cancer, but blood samples of individuals suffering from other cancer types or other diseases do not detect this biomarker; and blood The sample is tested without complicated equipment, and it is highly acceptable to the subjects. It is suitable for large-scale screening, which is helpful for early diagnosis and continuous tracking of the disease.

Ovarian cancer as described herein includes but is not limited to endometrioid ovarian cancer, clear cell ovarian cancer, serous ovarian cancer, mucinous ovarian, and other types of ovarian cancer , For example, ovarian carcinosarcoma (ovarian carcinosarcoma), ovarian immature teratoma (Immature teratoma), ovarian mucinous and granulosa tumor (ovarian mucinous and granulosa tumor) and metastatic ovarian cancer (Krukenberg, ovarian Krukenberg tumor ).

The ovarian cancer described herein includes stage I ovarian cancer, stage II ovarian cancer, stage III ovarian cancer, and stage IV ovarian cancer. The symptoms of each stage of ovarian cancer are shown in Table 1.

Table 1: Symptoms of various stages of ovarian cancer.

In some embodiments, the amount of cleaved C3 polypeptide in multiple samples from candidate individuals (such as ovarian cancer patients) can be measured to determine the progression of the disease. For example, at least two biological samples (such as serum samples or plasma samples) can be obtained from candidate individuals at different time points. The amount of cleaved C3 polypeptide in the at least two biological samples can be measured as described herein. If the trend of the cleaved C3 polypeptide is observed, it will increase with time (for example, the amount of cleaved C3 polypeptide in the sample obtained later is higher than that of the sample obtained earlier) , The system is diagnosed with, suspected or at risk of ovarian cancer. If the individual is a patient with ovarian cancer, when the amount of cleaved C3 polypeptide shows an increasing trend, it means that ovarian cancer is progressing (deteriorating).

When an individual, such as a human patient, is diagnosed as having, suspected, or at risk of ovarian cancer, the individual can undergo further tests (such as routine physical testing, including imaging methods, such as X-ray photography, magnetic resonance imaging (MRI) ), or ultrasound or biopsy with surgery) to confirm the occurrence of the disease and/or determine the time and type of ovarian cancer.

In some embodiments, the method described herein may further comprise treating the ovarian cancer patient to at least relieve the symptoms related to the disease. The treatment can be any traditional anti-ovarian cancer treatment Methods include radiotherapy, chemotherapy and surgery. Exemplary anti-ovarian cancer chemotherapy agents include, but are not limited to, cytarabine (ARA-C), bevacizumab, bleomycin, carboplatinum, cisplatin, Jiaplatin Carboplatin, cyclophosphamide, etoposide (VP-16), docetaxel, doxorubicin, gemcitabine, 5-fluorouracil (5-FU) ), ifosfamide, niraparib, olaparib, paclitaxel, rucaparib phosphate, Tamoxifene, cancer Topotecan, vinblastine and vincristine.

In another embodiment, the method provided herein is used to evaluate the efficacy of ovarian cancer treatment administered to patients with ovarian cancer. For example, multiple biological samples can be collected from ovarian cancer patients undergoing ovarian cancer treatment during the treatment period. If during the treatment period, the amount of the cleaved C3 multi-peptide shows a decreasing trend, that is, the amount of cleaved C3 multi-peptide in the biological sample obtained later is compared with the amount of the biological sample obtained earlier. If the amount of type C3 polypeptide is low, it means that the treatment is effective for ovarian cancer patients. On the contrary, if the amount of the cleaved C3 multipeptide remains maintained or even increased during the treatment period, it means that the treatment is ineffective for the patient or the condition deteriorates.

The "lower", "reduced", "reduced" amount or similar terms used here refer to the amount lower than the desired comparison (such as the amount of label detection before treatment), and the degree of reduction is generally The statistical significance recognized by the field. In certain embodiments, the degree of reduction may be a reduction of 20% or more, a reduction of 30% or more, a reduction of 40% or more, and a reduction of 50% or more compared to the amount of label detected before treatment. 60% or more reduction, 70% or more reduction, 80% or more reduction, or 90% or more reduction.

The amount of "higher", "increased", "increased" or similar used here The term refers to an amount higher than the desired comparison (such as the amount of label detection before treatment), and the degree of improvement has a statistical significance generally recognized in this field. In a specific embodiment, the degree of improvement can be an increase of 20% or more, an increase of 30% or more, an increase of 40% or more, an increase of 50% or more, compared to the amount to be compared. % Or more increase, 70% or more increase, 80% or more increase, or 90% or more increase.

If an ovarian cancer therapy is deemed to be ineffective for an ovarian cancer patient, the treatment strategy can be adjusted, such as increasing the dose of the drug, the frequency of treatment, or changing a more suitable therapy.

Ⅳ. In Vitro Diagnostic Kit

The present invention also provides a kit for performing the method, which includes the reagent (such as an antibody) capable of specifically identifying the cleaved C3 polypeptide described herein. The set may further include instructions for using the set to detect the presence or content of the cleaved C3 polypeptide described herein, thereby detecting ovarian cancer, and can also be used to monitor the progress of ovarian cancer, or To evaluate the effects of treatments administered to patients with ovarian cancer.

V. Use of antibodies against cleavage type C3 multipeptides in the detection of ovarian cancer

Any of the antibodies described herein can specifically recognize the cleavage pattern C3 polypeptide for detecting ovarian cancer. For example, the antibody can be conjugated to a detectable label (such as an in vivo imaging agent known in the art) and used for diagnostic purposes. Preferably, the antibody system is used for in vitro analysis of samples from individuals to be tested by immunoassay methods, such as Western blot analysis, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunoassay (RIPA), immunofluorescence method (IFA) and electrochemical fluorescence method (ECL) to determine whether there is a cleaved C3 multipeptide in the sample or determine its content.

The subject to be treated by the diagnostic method or the treatment method described herein may be a female mammal, preferably a human female subject. Mammals include, but are not limited to farm animals, sports animals, pets, primates, horses, dogs, cats, mice, and rats. A human individual in need of this treatment, It may be a human patient with ovarian cancer, a human patient suspected of having ovarian cancer, or a human patient at risk of ovarian cancer. The patient can be identified by conventional medical treatment or the diagnostic methods described herein.

As used herein, "effective amount" refers to the amount of each active substance that can impart a therapeutic effect on the individual, whether used alone or in combination with one or more other active substances. The effective amount may vary and must be judged by those skilled in the field, based on the specific conditions at the time of administration, the severity of the situation, individual patient parameters, including age, physical condition, size, gender and weight, and treatment time The process, (if any) the nature of concurrent therapy, the specific delivery route, and the possible factors of the knowledge and professional judgment of other medical personnel. These factors are well known to those with ordinary skills in the field, and can be introduced without further routine experimentation. It is generally preferable to use the maximum dose of the individual composition or its composition, which is the highest safe dose based on wise medical judgment. It should be understood that those with ordinary skills in this field, however, a patient may insist on using a lower dose or a tolerable dose based on medical reasons, physiological reactions or other possible reasons.

Based on empirical considerations, such as half-life, it is generally believed that it affects the dose decision. For example, antibodies compatible with the human immune system, such as humanized antibodies or fully human antibodies, can extend the half-life of the antibody and prevent the antibody from being attacked by the host immune system. The frequency of administration can be determined and adjusted according to the treatment period. Generally speaking, if not necessary, the frequency of administration can be adjusted according to treatment and/or suppression, and/or improvement of ovarian cancer. In addition, the continuous release of anti-cutting C3 formula can also be applied. Various formulations and devices for sustained release are all known in the field.

Depending on the route of administration, the bound antibody can be mixed with a suitable pharmaceutically acceptable carrier to form a suitable formulation. The injectable composition may contain different carriers, such as vegetable oil, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and multiple Alcohol (glycerin, propylene glycol, liquid polyethylene glycol and the like). For intravenous injection, you can use The water-soluble antibody is administered by drip method by injecting a pharmaceutical formula containing the antibody and physiologically acceptable excipients. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% physiological saline, Ringer's solution or other suitable excipients. For intramuscular formulations, such as a sterile formulation, the antibody is a suitable soluble salt, which can be dissolved by administering pharmaceutical excipients (such as water for injection, 0.9% saline, or 5% glucose solution).

Without further elaboration, we believe that those with ordinary skills in this field can apply the present invention to its most complete extension based on the above description. Therefore, the following specific embodiments are only for illustration, and are not intended to limit the applicable scope of the present invention in any way. All documents cited in this article are cited in this article for the purpose of citation or by reference.

Example: Specific proteolytic fragments of complement component 3 in patients with ovarian cancer

First, through a modified two-dimensional colloidal electrophoresis system, we found that certain fragments of complement component 3 (C3 protein) were detected in the plasma of some patients with ovarian cancer. By LC-MS/MS analysis of its trypsin hydrolysate, we found that the fragment has a molecular weight of about 40kDa. These data indicate that the specific fragment of the C3 protein may be the product of the proteolysis process. Further analysis of the amino acid sequence revealed that the specific fragment of the C3 protein is human C3 α chain fragment 2, which is a C-terminal fragment cut between positions 1320 and 1321 relative to the full-length human C3 protein SEQ ID NO:1 , Has the amino acid sequence of SEQ ID NO: 2.

Then, perform the Western blot method to further understand the characteristics of these protein fragments.

Antibody preparation

The preparation method of the multi-strain antibody of the present invention includes the following steps: the purified human cleavage C3 multi-peptide fragment or recombinant protein is used as the antigen, and the antigen purity is more than 95%. To increase the immunogenicity of the antigen, the peptide can be cross-linked to an appropriate carrier protein, usually bovine serum albumin (bovine serum albumin). albumin, BSA) or keyhole limpet hemocyanin (KLH). Injecting antigen into a rabbit body induces an immune response, and different epitopes (epitopes) induce B cells to secrete many different immunoglobulins (antibodies). Immunization is once every two weeks for a total of 5 immunizations. Subsequent detection is performed by enzyme immunoassay to ensure that the serum titer after immunization is greater than 1:4000 before all blood sampling can be performed. After the blood is coagulated, the supernatant is centrifuged to obtain the antiserum. The subsequent purification of the correct antibody molecule is the purification of immunoglobulin (Ig) by affinity chromatography (protein A/G) and precipitation with ammonium sulfate to obtain the antibody containing the target specificity. It can be combined with ion exchange column chromatography to remove impurities in the precipitate.

Western ink dot analysis

The protein in the plasma sample collected from a human individual was mixed with 6X Laemmli sample buffer containing 120mM Tris-HCl, pH 6.8, 2% SDS ( w/v ) and 10% sucrose ( w/v ). Add 1% 2-mercaptoethanol to some samples. After electrophoretic separation, the protein was transferred to Immobilon PVDF membrane (Millipore Corp.) using standard techniques. The membrane after spotting was blocked with a blocking buffer (containing 1% goat serum), and allowed to act at room temperature for 1 hour, followed by rabbit anti-complement component 3 antibody. After three times of washing with Tris buffered saline (containing 0.1% Tween 20), the membrane was treated with peroxidase-conjugated goat anti-rabbit IgG (Sigma-Aldrich). The chemical fluorescence system uses LAS-4000 (Fujifilm, Japan) for detection.

result

Western blot analysis showed that a specific complement component 3 fragment (cleaved C3 multipeptide fragment) was detected in samples from patients with ovarian cancer, about 40kDa in size. As shown in Figure 1, the cleaved C3 multipeptide fragments can be detected in samples from ovarian cancer patients, but not in samples from normal individuals. Therefore, the cleaved C3 multi-peptide fragment can be used as a diagnostic indicator for ovarian cancer (specifically appear in egg It is not detected in the plasma samples of patients with nest cancer, but not in the plasma of normal individuals).

In order to confirm the specificity of this cleaved C3 multi-peptide fragment for ovarian cancer, plasma samples from human individuals with other cancer types or other diseases were taken for Western blot analysis. The results are shown in Table 2.

The results showed that plasma samples of human individuals with other cancer types or other diseases did not detect the presence of cleaved C3 multipeptide fragments (the detection rate was zero).

In contrast, plasma samples from human individuals with different stages of ovarian cancer were taken for Western blot analysis. The results are shown in Table 3.

The results show that the cleaved C3 multi-peptide fragment is significantly associated with ovarian cancer, Among the patients classified by stage of ovarian cancer, 22 of the 28 patients were detected with cleaved C3 multipeptide fragments, the positive percentage was as high as 78.6%, and the detection rate was nearly 80%, so it can be used as an indicator of ovarian cancer ; Especially in the initial stage (Phase I) the positive percentage is as high as 86.7%, and the detection rate is close to 90%. This is a high detection rate that cannot be achieved by existing detection technologies. It is of great help to the early diagnosis and screening of ovarian cancer.

We further analyzed the types of ovarian cancer patients, and analyzed whether the biomarkers of cleaved C3 multipeptide fragments are widely present in different types of ovarian cancer patients. The results are shown in Table 4.

The results show that the cleaved C3 multi-peptide fragments are significantly related to different types of ovarian cancer, which means that the detection of cleaved C3 multi-peptide fragments can be used to screen for multiple types of ovarian cancer, especially for the current test The extremely low rate of bright cell ovarian cancer, with a detection rate of 100.0%, is of great help to the early diagnosis and screening of ovarian cancer.

In addition, Figure 2 shows the Western blot analysis of plasma samples of patients with ovarian cancer at different time points. Number 1 is the analysis result of plasma samples from patients with ovarian cancer at the first time point, and Number 2 is the same patient receiving The analysis of plasma samples returned to the clinic about one year after chemotherapy showed that the content of cleaved C3 protein increased. The patient was further confirmed that his condition had deteriorated and cancer metastasis had occurred; number 3 is the analysis result of plasma samples of normal individuals, showing that no cleave was detected Type C3 protein. The results show that the cleaved C3 multi-peptide fragment can be used as an indicator to monitor the therapeutic effect of ovarian cancer, and the increase in content indicates that ovarian cancer may worsen.

We have confirmed that the cleaved C3 multi-peptide fragment of about 40kDa specifically exists in patients with ovarian cancer, and can be used as a biomarker for ovarian cancer screening and as an indicator for monitoring the therapeutic effect of ovarian cancer. Therefore, the present invention proposes for the first time an ovarian cancer screening technology, which can specifically identify patients with ovarian cancer, suspected ovarian cancer, or who may be at risk of ovarian cancer, and can be used as an early stage (that is, in any symptoms). Prognostic indicators before occurrence) or after treatment. The technology of the present invention can develop a test reagent for ovarian cancer, using cleaved C3 protein as a biomarker for ovarian cancer screening and tracking the condition after treatment. Compared with the prior art, the technology of the present invention greatly improves the detection rate of ovarian cancer. It can not only identify various types of ovarian cancer, but also identify various stages of ovarian cancer, especially the early detection rate is quite high (approximately 90%) and low false positives (the presence of cleaved C3 multi-peptide fragments are not detected in patient samples from other types of cancers or diseases). This is a major breakthrough in screening for ovarian cancer. The simple protein measurement method completes the test, which is a non-invasive test, which is highly accepted by patients and is suitable for the development of large-scale early screening; on the other hand, it can also be used as an indicator to monitor the effect of ovarian cancer treatment and continuously track the patient's condition , So that the treatment plan can be adjusted in time for patients with poor treatment effect to avoid or delay the deterioration.

Sequence Information

Complement component 3 (Homo sapiens)-full length (SEQ ID NO: 1)

Complement component 3 (Homo sapiens) C3α chain fragment 2 (1321-1663 aa) (SEQ ID NO: 2)

Complement component 3 (Homo sapiens) C3α chain fragment 1 (749-954 aa) (SEQ ID NO: 3)

Complement component 3 (Homo sapiens) C3β fragment (23-667 aa) (SEQ ID NO: 4)

Claims (26)

A method for detecting ovarian cancer, comprising: (a) providing a biological sample from an individual to be tested; (b) detecting the content of the cleaved complement component 3 (C3) multipeptide in the biological sample from the individual to be tested to obtain The detected amount of cleaved C3 multipeptide; (c) compare the detected amount of cleaved C3 multipeptide with the normal amount of cleaved C3 multipeptide from a normal individual to obtain a comparison result; and (d) judge the amount based on the comparison result The possibility of the individual to be tested suffering from ovarian cancer, where if the comparison result shows that the detected amount of cleaved C3 multipeptide is higher than the normal amount of cleaved C3 multipeptide, then it is determined that the subject has ovarian cancer or has ovarian cancer The risk of the cleavage type C3 multipeptide is human C3 α chain fragment 2. The method of claim 1, wherein the cleaved C3 polypeptide has an amino acid sequence corresponding to position 1321 to position 1663 of SEQ ID NO:1. The method of claim 1, wherein the cleavage type C3 polypeptide has the amino acid sequence of SEQ ID NO: 2. Such as the method of claim 1, wherein the detection in step (b) is performed by mass spectrometry or immunoassay. The method of claim 1, wherein the detection of step (b) is performed by an immunoassay method that uses an antibody that specifically binds to the cleavage C3 polypeptide of SEQ ID NO: 2. Such as the method of claim 1, wherein the biological sample is a body fluid sample, a tissue sample, or a biological specimen sample. Such as the method of claim 6, wherein the biological sample is a blood sample, a urine sample, or an ascites sample. The method according to any one of claims 1 to 7, wherein the ovarian cancer is selected from the group consisting of endometrioid ovarian cancer, clear cell ovarian cancer, serous ovarian cancer, and mucus Type (mucinous) ovarian cancer, or selected from ovarian carcinosarcoma (ovarian carcinosarcoma), ovarian immature teratoma (Immature teratoma), ovarian mucinous and granulosa tumor (ovarian mucinous and granulosa tumor), and metastasis Sexual ovarian cancer (Krukenberg, ovarian Krugenberg tumor). The method according to any one of claims 1 to 7, wherein the ovarian cancer is selected from the group consisting of stage I ovarian cancer, stage II ovarian cancer, stage III ovarian cancer, and stage IV ovarian cancer. The method of claim 9, wherein the ovarian cancer is selected from the group consisting of endometrioid ovarian cancer, clear cell ovarian cancer, serous ovarian cancer, and mucinous ovarian cancer Group consisting of ovarian carcinosarcoma (ovarian carcinosarcoma), ovarian immature teratoma (Immature teratoma), ovarian mucinous and granulosa tumor (ovarian mucinous and granulosa tumor), and metastatic ovarian cancer (Krukenberg, Klugenberg tumor of the ovary). A method for monitoring the development of ovarian cancer in patients with ovarian cancer, the method comprising: (a) at a first time point, providing a first biological sample derived from the patient; (b) at a second time point, providing a first biological sample derived from the patient A second biological sample of the patient, wherein the second time point is later than the first time point; (c) Detect the content of cleaved C3 multipeptide in the first biological sample to obtain the first detection amount of cleaved C3 multipeptide, and detect the content of cleaved C3 multipeptide in the second biological sample to obtain the cut The second detection amount of type C3 multipeptide; (d) compare the first detection amount of cleaved C3 multipeptide with the second detection amount of cleaved C3 multipeptide to obtain a comparison result; and (e) judge based on the comparison result For the progression of ovarian cancer in the patient, if the comparison result shows that the second detected amount of cleaved C3 multipeptide is higher than the first detected amount of cleaved C3 multipeptide, it means that ovarian cancer is developing, and the cleaved C3 The multipeptide is human C3 α chain fragment 2. The method of claim 11, wherein the patient is treated for ovarian cancer, and the first biological sample and the second biological sample are taken before or after treatment, or during treatment. The method of claim 11, which further comprises evaluating the therapeutic effect of the ovarian cancer treatment on the patient, wherein if the content of the cleaved C3 polypeptide shows a decreasing trend after treatment or during the course of treatment, it represents the treatment It is effective for this patient. The method of claim 11, wherein the cleaved C3 polypeptide has an amino acid sequence corresponding to position 1321 to position 1663 of SEQ ID NO:1. The method of claim 11, wherein the cleaved C3 polypeptide has an amino acid sequence of SEQ ID NO: 2. Such as the method of claim 11, wherein the detection in step (c) is performed by mass spectrometry or immunoassay. The method of claim 11, wherein the detection of step (c) is performed by an immunoassay method that uses an antibody that specifically binds to the cleavage C3 polypeptide of SEQ ID NO: 2. Such as the method of claim 11, wherein the biological sample is a body fluid sample, a tissue sample, or a biological specimen sample. Such as the method of claim 18, wherein the biological sample is a blood sample, a urine sample, or an ascites sample. The method according to any one of claims 11 to 19, wherein the ovarian cancer is selected from the group consisting of endometrioid ovarian cancer, clear cell ovarian cancer, serous ovarian cancer, and mucus Type (mucinous) ovarian cancer, or selected from ovarian carcinosarcoma (ovarian carcinosarcoma), ovarian immature teratoma (Immature teratoma), ovarian mucinous and granulosa tumor (ovarian mucinous and granulosa tumor), and metastasis Sexual ovarian cancer (Krukenberg, ovarian Krugenberg tumor). The method according to any one of claims 11 to 19, wherein the ovarian cancer is selected from the group consisting of stage I ovarian cancer, stage II ovarian cancer, stage III ovarian cancer, and stage IV ovarian cancer. The method of claim 21, wherein the ovarian cancer is selected from the group consisting of endometrioid ovarian cancer, clear cell ovarian cancer, serous ovarian cancer, and mucinous ovarian cancer Group consisting of ovarian carcinosarcoma (ovarian carcinosarcoma), ovarian immature teratoma (Immature teratoma), ovarian mucinous and granulosa tumor (ovarian mucinous and granulosa tumor), and metastatic ovarian cancer (Krukenberg, Klugenberg tumor of the ovary). The use of a reagent capable of identifying cleavage type C3 multipeptides, which is used to diagnose ovarian cancer or monitor the development of ovarian cancer, or to prepare reagents or kits for diagnosing or monitoring the development of ovarian cancer, wherein the cleavage type The C3 multipeptide is human C3 α chain fragment 2. Such as the use of claim 23, wherein the cleaved C3 polypeptide has an amino acid sequence corresponding to position 1321 to position 1663 of SEQ ID NO:1. Such as the use of claim 23, wherein the cleaved C3 polypeptide has the amino acid sequence of SEQ ID NO: 2. Such as the use of claim 23, wherein the reagent is an antibody.

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