KR102415817B1 - A composition for diagnosing subarachnoid hemorrhage comprising a formulation capable of measuring the expression level of TCRBV 19-01 and TCRBJ02-04 - Google Patents

Abstract

The present invention provides a composition for diagnosing subarachnoid hemorrhage comprising an agent capable of measuring the expression level of TCRBV 19-01 and TCRBJ02-04, a kit for diagnosing subarachnoid hemorrhage comprising the composition, and diagnosis of subarachnoid hemorrhage using the composition and kit It relates to a method of providing information for
According to the composition for diagnosing subarachnoid hemorrhage, the kit for diagnosing subarachnoid hemorrhage, and the method for providing information for diagnosing subarachnoid hemorrhage of the present invention, the expression level of VJ recombination in the TCRB CDR3 repertoire is measured to determine whether the severity of subarachnoid hemorrhage is mild or severe. It can be used for prediction or diagnosis of

Description

A composition for diagnosing subarachnoid hemorrhage comprising a formulation capable of measuring the expression level of TCRBV 19-01 and TCRBJ02-04 }

The present invention provides a composition for diagnosing subarachnoid hemorrhage comprising an agent capable of measuring the expression level of TCRBV 19-01 and TCRBJ02-04, a kit for diagnosing subarachnoid hemorrhage comprising the composition, and diagnosis of subarachnoid hemorrhage using the composition and kit It relates to a method of providing information for

Subarachnoid hemorrhage (SAH) is a subtype of intracranial hemorrhage. As blood in the blood vessels flows out (extravasation), it enters the subarachnoid space and is a brain tissue located in the periphery of the brain parenchyma. It is a disease that causes various inflammatory responses and damage to brain cells.

Subarachnoid hemorrhage is a hemorrhage that occurs in the subarachnoid space surrounding the brain and causes damage throughout the brain, resulting in immediate death or serious permanent brain damage. Causes of subarachnoid hemorrhage include trauma caused by an external impact on the head, and a cerebral aneurysm caused by changes in the blood vessel wall of the brain. In addition, in terms of prognosis, subarachnoid hemorrhage is still a disease with high mortality and poor prognosis even in modern medicine. 10-15% of patients with subarachnoid hemorrhage die before they even arrive at the hospital, and 25% of patients die within 24 hours of bleeding. In addition, an average of 40% of patients die within 1 month even if they do not die at the beginning of the onset and are hospitalized, and 50% of patients with subarachnoid hemorrhage die within 6 months, and overall, 51-80% of patients eventually die. will do Even if they survive, they cause neurological complications due to rebleeding, hydrocephalus, and vasospasm within 2 weeks. life, or they become paralyzed and unable to walk.

For the diagnosis of subarachnoid hemorrhage, ct-angiography and cerebral angiography are performed for ruptured cerebral aneurysms. To diagnose subarachnoid hemorrhage due to rupture of a cerebral aneurysm, brain computed tomography (brain ct) is the first test method performed. If computed tomography is performed within 48 hours of subarachnoid hemorrhage, the diagnosis can be made in more than 95% of cases. However, this diagnostic method requires expensive test equipment, and the process may also be complicated, so it is inconvenient to quickly diagnose the condition of subarachnoid hemorrhage.

Accordingly, the present inventors tried to develop a method for diagnosing subarachnoid hemorrhage, specifically diagnosing the severity of subarachnoid hemorrhage, and predicting the prognosis efficiently and easily. As a result, the expression level of the V-J recombinant form of the T cell receptor beta chain was measured. And by comparison, the present invention was completed by developing a diagnostic composition capable of diagnosing or predicting the severity of subarachnoid hemorrhage.

Republic of Korea Patent Publication No. 2002-0064396

A first aspect of the present application is to provide a composition for diagnosing subarachnoid hemorrhage, comprising an agent capable of measuring the expression level of TCRBV19-01 and TCRBJ02-04.

A second aspect of the present application is to provide a kit for diagnosing subarachnoid hemorrhage comprising the composition for diagnosing subarachnoid hemorrhage.

A third aspect of the present application is to provide an information providing method for diagnosing subarachnoid hemorrhage, comprising measuring the expression levels of TCRBV19-01 and TCRBJ02-04 from a biological sample isolated from a subject.

A fourth aspect of the present application is to provide a marker composition for diagnosis of subarachnoid hemorrhage, comprising TCRBV19-01 and TCRBJ02-04.

This will be described in detail as follows. On the other hand, each description and embodiment disclosed herein may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present application fall within the scope of the present application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.

This will be described in detail as follows. On the other hand, each description and embodiment disclosed herein may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present application fall within the scope of the present application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.

A first aspect of the present application provides a composition for diagnosing subarachnoid hemorrhage, comprising an agent capable of measuring the expression level of TCRBV 19-01 and TCRBJ02-04.

As used throughout this specification, the term “diagnosis” refers to determining whether a subarachnoid hemorrhage occurs in a specific individual, and more specifically, the severity of the subarachnoid hemorrhage.

As used throughout this specification, the term “subarachnoid hemorrhage (SAH)” is a type of stroke that occurs due to damage to an artery on the brain surface. A subarachnoid hemorrhage causes blood to flow into the space between the brain and the skull, where it mixes with the cerebrospinal fluid, which acts as a buffer between the brain and the cerebrospinal fluid, increases the pressure around the brain (increased intracranial pressure), and interferes with brain function. , may cause brain damage. Subarachnoid hemorrhage is most often caused by rupture of a protruding arterial wall on the brain surface. The protruding part of the artery is called an aneurysm, and this aneurysm usually ruptures, or subarachnoid hemorrhage may occur due to blood leakage from arteriovenous malformations made by entangled abnormal blood vessels.

In patients with suspected subarachnoid hemorrhage, a quicker diagnosis of aneurysmal subarachnoid hemorrhage through an accurate diagnostic technique is required, rebleeding should be prevented by removing the aneurysm, and fluid therapy and intracranial pressure control treatment should be performed. In addition, at each stage of treatment, specific complications of subarachnoid hemorrhage (rebleeding, hydrocephalus, cerebrovascular spasm, epilepsy) and medical complications (neurogenic pulmonary edema, venous thrombosis, cardiovascular complications, electrolyte abnormalities, gastrointestinal complications) should be evaluated and treated. Therefore, promptly diagnosing an accurate condition such as the severity of subarachnoid hemorrhage is an important part in treating it and preventing complications.

The term "T-Cell Receptor (TCR)" used throughout this specification means that one α chain and one β chain are connected to each other by one disulfide bond, and the transmembrane region is located at the end of the constant part. Thereby, it is fixed to T cells, and the mutation region (V) at the ends of the α and β chains forms a single antigen attachment site. In addition, the binding site at the end of the T cell receptor binds to an antigenic site called an epitope or epitope. In this case, the binding method is similar to the binding between the enzyme and the substrate, and the binding occurs non-covalently. In addition, unlike the B-cell receptor, the T-cell receptor binds only to the manipulation of the antigen presented on the surface of the host cell with the help of a protein called the Major Histocompatibility Complex (MHC).

The T cells generate a predetermined DSB at the developmental stage, and specifically, through a reaction called V(D)J recombination of a functional antigen receptor gene, the constituent gene fragments V (Variable), D (Diversity), and J (Joining). ) is known to be completed by rearranging genes in order, and the α chain and β chain of the T cell receptor can acquire diversity through the above process. Specifically, the CDR3 of the T-cell receptor β chain can have a diverse repertoire through V-J recombination, and the V-J recombination can generate a combination of various types of TCR beta variable genes (TCRBV) and TCR beta joining genes (TCRBJ). have.

Various types of TCRBV and TCRBJ and amino acid sequence information thereof can be found in known literature, and specifically, as TCRBV, there may be TCRBV19-01, TCRBV28-01, or TCRBV30-01, and as TCRBJ, TCRBJ02-04 or TCRBJ02 -06, etc. In addition, the V-J recombinant form of the T-cell receptor β chain can be expressed by various combinations of TCRBV and TCRBJ, which can be found in known literature, specifically TCRBV19-01/TCRBJ02-04, TCRBV28-01/TCRBJ02-04, TCRBV28-01/TCRBJ02-06 or TCRBV30-01/TCRBJ02-04 and the like.

In one embodiment of the present application, the composition may include an agent capable of measuring the expression level of a peptide comprising the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence of SEQ ID NO: 1 is the TCRBV 19-01 and TCRBJ02-04.

In one embodiment of the present application, the composition may further include an agent capable of measuring the expression level of TCRBV28-01 and TCRBJ02-04.

In one embodiment of the present application, the composition may further include an agent capable of measuring the expression level of a peptide comprising the amino acid sequence of SEQ ID NO: 2, wherein the amino acid sequence of SEQ ID NO: 2 is the TCRBV28- 01 and TCRBJ02-04 may be included.

In one embodiment of the present application, the composition may further include an agent capable of measuring the expression level of TCRBV28-01 and TCRBJ02-06.

In one embodiment of the present application, the composition may further include an agent capable of measuring the expression level of a peptide comprising the amino acid sequence of SEQ ID NO: 3, wherein the amino acid sequence of SEQ ID NO: 3 is the TCRBV28- 01 and TCRBJ02-06 may be included.

In one embodiment of the present application, the composition may further include an agent capable of measuring the expression level of TCRBV30-01 and TCRBJ02-04.

In one embodiment of the present application, the composition may further include an agent capable of measuring the expression level of a peptide comprising the amino acid sequence of SEQ ID NO: 4, wherein the amino acid sequence of SEQ ID NO: 4 is the TCRBV30- 01 and TCRBJ02-04 may be included.

In one embodiment of the present application, the diagnosis of subarachnoid hemorrhage of the present application may be diagnosing the severity of subarachnoid hemorrhage, predicting the risk of onset, or diagnosing the prognosis of subarachnoid hemorrhage, specifically, if subarachnoid hemorrhage is mild or It may be a diagnosis of whether it is serious.

The term "Hunt-Hess classification or Hunt-Hess grade" used throughout this specification is a representative evaluation method for a subarachnoid hemorrhage patient, and each grade may be classified according to the following criteria.

- Grade 0: Unruptured aneurysm

- Grade Ⅰ: Asymptomatic or minimal headache and slight neck stiffness

- Grade Ⅱ: No neurological deficit other than moderate to severe headache, stiff neck, cranial nerve palsy

- Grade III: drowsiness, confusion or mild lack of focus

- Grade IV: Stupor, moderate to severe hemiparesis, premature cerebral spasm and disorders of the vegetative state

- Grade V: Appearance of deep coma, cerebral spasm, and moribund state

In one embodiment of the present application, the mild subarachnoid hemorrhage may have a Hunt-Hess grade of less than or equal to grade III, specifically grade I, grade II, or grade III.

In one embodiment of the present application, the severe subarachnoid hemorrhage may be a Hunt-Hess grade or higher, specifically grade IV or V.

In one embodiment of the present application, when the expression level of a peptide comprising TCRBV 19-01 and TCRBJ02-04 is increased, or when the expression level of a peptide comprising TCRBV28-01 and TCRBJ02-04 is decreased, the subject is mild It can be judged that subarachnoid hemorrhage develops or is highly likely to develop.

In one embodiment of the present application, when the expression level of a peptide comprising TCRBV28-01 and TCRBJ02-06 is increased, or when the expression level of a peptide comprising TCRBV30-01 and TCRBJ02-04 is decreased, the subject subject is subjected to severe arachnoid It can be judged that bleeding has occurred or is highly likely to occur.

As used throughout this specification, the term "measurement of expression level" refers to measuring the expression or level of expression of a specific protein (peptide) or a gene encoding the protein, specifically including TCRBV 19-01 and TCRBJ02-04 It may be to measure the expression level of a peptide or mRNA or gene encoding the same, and additionally to measure the expression level of a peptide comprising TCRBV28-01 and TCRBJ02-04 or mRNA or gene encoding the same.

According to one embodiment of the present application, the method for measuring the expression level of the protein is Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA: radioimmunoassay), radioimmunodiffusion (radical immunodiffusion) , Ouchterlony immunodiffusion, rocket immunoeletrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence ( It may be immunofluorescence, immunochromatography, fluorescenceactivated cell sorter analysis, or protein chip technology.

According to one embodiment of the present application, the method for measuring the expression level of the mRNA or gene is reverse transcriptase polymerization (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real-time reverse transcriptase polymerase reaction ( It may be real time quantitative RT-PCR, quantitative RT-PCR, RNase protection method, Northern blotting, or DNA chip technology.

According to one embodiment of the present application, "agent capable of measuring the expression level" means a molecule that can be used to check the expression level of a specific protein or gene encoding the same, specifically detecting the protein or the gene and/or an agent capable of amplifying, but is not limited thereto.

As used throughout this specification, the term "agent capable of detecting a specific protein or a gene encoding the same" refers to an agent capable of specifically binding to and recognizing the specific gene or protein, or detecting and amplifying the specific gene or protein. And, "agent capable of amplifying a specific gene or protein" means an agent capable of increasing the number by repeating the replication of the specific gene or protein, for example, a polynucleotide containing the gene It may mean a primer that can be amplified or a probe that can specifically bind, but is not limited thereto.

In one embodiment of the present application, the composition comprises a peptide comprising TCRBV 19-01 and TCRBJ02-04 or a gene encoding the peptide, or a primer, probe, nucleotide, antibody or antigen binding thereof that specifically binds to mRNA It may include fragments, ligands, receptors, proteins, or a combination thereof.

As used throughout this specification, the term "primer" is a nucleic acid sequence having a free 3-terminal hydroxyl group, which can form a base pair with a template complementary to a specific base sequence and form a template Refers to a nucleic acid sequence that serves as a starting point for strand copying. The primer is capable of initiating DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. For example, PCR amplification is performed using sense and antisense primers having a sequence of 7 to 50 nucleotides as specific primers for the mRNA of the genes encoding TCRBV 19-01 and TCRBJ02-04 to measure the amount of production of the desired product Through this, it is possible to predict the resistance of an individual to a cancer treatment. PCR conditions and lengths of sense and antisense primers may be appropriately selected according to techniques known in the art. The primers are 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 10 to 20, 15 to 80, 15 to 50, 15 to 30, 15 to 20, 20 to 100, 20 to 80 , 20 to 50, or 20 to 30 nt.

The term "probe" as used throughout this specification refers to a nucleic acid fragment such as RNA or DNA capable of specifically binding to a target nucleic acid, for example, mRNA, and the presence, absence, content and expression level of a specific mRNA. It may be labeled so that it can be confirmed. The probe may be manufactured in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or the like. For example, hybridization is performed using a probe having a nucleic acid sequence complementary to the mRNA of the gene encoding TCRBV 19-01 and TCRBJ02-04, and the expression level of mRNA is measured through the degree of hybridization. resistance can be predicted. Suitable probe selection and hybridization conditions can be appropriately selected according to techniques known in the art. The probe is 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 10 to 20, 15 to 80, 15 to 50, 15 to 30, 15 to 20, 20 to 100, 20 to 80 , 20 to 50, or 20 to 30 nt.

In addition, the primer or probe may be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods. In addition, these nucleic acid sequences can be modified through various methods known in the art. Examples of such modifications include methylation, encapsulation, substitution of one or more homologues of natural nucleotides, or modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates. etc.) or charged linkages (eg, phosphorothioate, phosphorodithioate, etc.). Primers or probes may also be modified with labels capable of directly or indirectly providing a detectable signal. Examples of such labels may include radioactive isotopes, fluorescent molecules, or biotin.

As used throughout this specification, the term "antibody" is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. The antibody may specifically bind to a protein including TCRBV 19-01 and TCRBJ02-04 or a fragment thereof. Fragments of proteins comprising TCRBV 19-01 and TCRBJ02-04 may be, for example, immunogenic fragments. The fragment refers to a protein fragment having at least one epitope that can be recognized by an antibody against a protein including TCRBV 19-01 and TCRBJ02-04. A gene encoding a protein comprising TCRBV 19-01 and TCRBJ02-04 was cloned into an expression vector, and a protein comprising TCRBV 19-01 and TCRBJ02-04 encoded by the gene was obtained, and from the obtained protein, the technique Antibodies can be prepared according to conventional methods in the art.

The form of the antibody includes a polyclonal antibody, a monoclonal antibody, or a recombinant antibody, and all immunoglobulin antibodies are included. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. Moreover, the said antibody also includes special antibodies, such as a humanized antibody. Polyclonal antibodies can be prepared by injecting an immunogen biomarker protein or a fragment thereof into an external host according to a conventional method known to those skilled in the art. As the external host, mammals such as mice, rats, sheep, and rabbits can be used. When the immunogen is injected by an intramuscular, intraperitoneal or subcutaneous injection method, it may be administered together with an adjuvant to increase antigenicity in general. Thereafter, blood may be periodically collected from an external host to collect serum showing improved titer and antigen specificity, or antibodies may be isolated and purified therefrom.

Monoclonal antibodies can be prepared by techniques for generating immortalized cell lines by fusion known to those skilled in the art. Briefly describing the monoclonal antibody production method, the protein is purified and an appropriate amount of about 10 μg is immunized to Balb/C mice, or a polypeptide fragment of the protein is synthesized and combined with bovine serum albumin to immunize mice. Then, antigen-producing lymphocytes isolated from mice are fused with human or mouse myeloma to generate immortalized hybridomas, and ELISA is used to select and proliferate only hybridoma cells producing the desired monoclonal antibody. After this, monoclonal antibodies can be isolated and purified from the culture. In addition, monoclonal antibodies can be used by obtaining commercially available antibodies against proteins including TCRBV 19-01 and TCRBJ02-04.

Enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, Western blotting or immunoblotting on polyacrylic gels known in the art using these antibodies It can be confirmed whether the protein is expressed in the biological sample by such a method.

The term "fragment" as used throughout this specification refers to, for example, a polypeptide that does not have the structure of an intact antibody, peptide or protein, but has a specific antigen binding site or binding domain directed to the antigenic site. Such fragments include functional fragments of antibody molecules that are not intact antibodies having two light chains and two heavy chains. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab') ₂ or Fv. The binding fragment may comprise at least 7 or more amino acids, for example, 9 or more amino acids, 12 or more amino acids.

A second aspect of the present application provides a kit for diagnosing subarachnoid hemorrhage, comprising the composition for diagnosing subarachnoid hemorrhage. The content overlapping with the first aspect is equally applied to the kit of the second aspect.

In one embodiment of the present application, the kit has an effect of diagnosing or predicting the severity of subarachnoid hemorrhage by measuring the expression levels of TCRBV 19-01 and TCRBJ02-04. The kit may further include antisense oligonucleotides, primer pairs, probes, etc. for the diagnosis of subarachnoid hemorrhage, as well as one or more other component compositions, solutions, or devices suitable for the analysis method, PCR kit, RT- PCR kits, qRT-PCR kits, methylation-specific PCR kits, DNA chip kits, Western blot kits, ELISA kits, and the like.

According to one embodiment of the present application, the kit may include polynucleotides, cDNAs, mRNAs, peptides, etc. of the present application, as well as other component compositions, solutions or devices suitable for the analysis method.

According to one embodiment of the present application, the RT-PCR kit may be a kit including essential elements necessary for performing RT-PCR, and the RT-PCR kit is specific for the genes or methylation region. In addition to primers, test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), dideoxynucleotides (ddNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitors , DEPC-water, sterile water, and the like.

According to one embodiment of the present application, the DNA chip kit may be a kit including essential elements necessary for performing a DNA chip, and the DNA chip kit is a polynucleotide, primer or probe specific for the genes or methylation region. includes a substrate to which is attached, and the substrate may include a nucleic acid corresponding to a quantitative control gene or a fragment thereof.

According to one embodiment of the present application, the microarray kit may be a kit including essential elements necessary for performing a microarray, and the DNA microarray kit is a microarray kit, cDNA corresponding to a gene or a fragment thereof is probed. and a substrate attached thereto, and the substrate may contain cDNA corresponding to a quantitative control gene or a fragment thereof, and can be easily prepared by a manufacturing method commonly used in the art using the gene. In order to fabricate a microarray, a micropipetting method using a piezoelectric method or a pin type to immobilize the detected marker on a substrate of a DNA chip using the probe DNA molecule A method using a spotter or the like can be used. The substrate of the microarray chip may be coated with an active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde. In addition, the substrate may be one selected from the group consisting of slide glass, plastic, metal, silicon, nylon membrane, and nitrocellulose membrane.

A third aspect of the present application provides an information providing method for diagnosing subarachnoid hemorrhage, comprising measuring the expression levels of TCRBV19-01 and TCRBJ02-04 from a biological sample isolated from a subject. The contents overlapping with the first aspect and the second aspect are equally applied to the information provision method of the third aspect.

As used throughout this specification, the term "individual" refers to any organism that has or is likely to develop subarachnoid hemorrhage, and specific examples include mice, monkeys, cattle, pigs, mini-pigs, livestock, humans, etc. It may include mammals, farmed fish, and the like, but is not limited thereto.

As used throughout this specification, the term "sample" refers to a substance derived from an individual who is or is likely to develop subarachnoid hemorrhage, and specifically, tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, It may include urine and the like, but is not limited thereto. In addition, a gene and/or protein sample may be obtained from these samples, and the genetic sample may include a nucleic acid, for example, DNA, mRNA, or cDNA synthesized from mRNA, and the expression of a specific gene/protein therefrom. As long as the level can be confirmed, the type is not limited thereto.

In one embodiment of the present application, the method comprises: measuring the expression levels of TCRBV19-01 and TCRBJ02-04 from a biological sample isolated from a control; and comparing the expression levels of TCRBV19-01 and TCRBJ02-04 of the subject and the control group.

As used throughout this specification, the term “control group” may refer to a general individual who has not developed subarachnoid hemorrhage, a non-subarachnoid hemorrhage patient group, a non-patient group, and the like.

In one embodiment of the present application, the method comprises treating the subject with mild subarachnoid hemorrhage when the expression level of TCRBV19-01 and TCRBJ02-04 of the subject is higher than the expression level of TCRBV19-01 and TCRBJ02-04 of the control group. It may be determined that the disease has occurred or further comprising the step of predicting the risk of developing a high level.

The term "prediction" used throughout the present specification means whether there is a possibility of developing mild or severe subarachnoid hemorrhage for a specific individual, and if there is a possibility of developing mild or severe subarachnoid hemorrhage, the likelihood of developing mild or severe subarachnoid hemorrhage is compared to an unspecified number of people. It means to determine whether it is relatively high.

In one embodiment of the present application, the method may further comprise measuring the expression levels of TCRBV28-01 and TCRBJ02-04 from the biological sample isolated from the subject.

In one embodiment of the present application, the method comprises: measuring the expression levels of TCRBV28-01 and TCRBJ02-04 from a biological sample isolated from a control; and comparing the expression levels of TCRBV28-01 and TCRBJ02-04 of the subject and the control group.

In one embodiment of the present application, the method is when the expression level of TCRBV28-01 and TCRBJ02-04 of the subject is lower than the expression level of TCRBV28-01 and TCRBJ02-04 of the control, the subject is treated with mild arachnoid It may further include determining that bleeding has occurred or estimating the risk of occurrence at a high level.

In one embodiment of the present application, the method comprises: measuring the expression levels of TCRBV28-01 and TCRBJ02-06 from a biological sample isolated from a control; and comparing the expression levels of TCRBV28-01 and TCRBJ02-06 of the subject and the control group.

In one embodiment of the present application, the method comprises treating the subject with severe arachnoidosis when the expression level of TCRBV28-01 and TCRBJ02-06 of the subject is higher than the expression level of TCRBV28-01 and TCRBJ02-06 of the control group. It may further include determining that bleeding has occurred or estimating the risk of occurrence at a high level.

In one embodiment of the present application, the method comprises the steps of: measuring the expression levels of TCRBV30-01 and TCRBJ02-04 from a biological sample isolated from a control; and comparing the expression levels of TCRBV30-01 and TCRBJ02-04 of the subject and the control group.

In one embodiment of the present application, the method comprises treating the subject with severe arachnoidosis when the expression level of TCRBV30-01 and TCRBJ02-04 of the subject is lower than the expression level of TCRBV30-01 and TCRBJ02-04 of the control group. It may further include determining that bleeding has occurred or estimating the risk of occurrence at a high level.

A fourth aspect of the present application provides a marker composition for diagnosis of subarachnoid hemorrhage, comprising TCRBV19-01 and TCRBJ02-04. The contents overlapping with the first to second aspects are equally applied to the composition of the fourth aspect.

As used throughout this specification, the term “marker” refers to a biomarker and can detect a change in a living organism, thereby objectively measuring a normal or pathological state of an organism, a degree of response to a drug, and the like.

In one embodiment of the present application, the marker composition is capable of diagnosing the severity of subarachnoid hemorrhage, and specifically, it may be used to determine whether subarachnoid hemorrhage is mild or severe.

In one embodiment of the present application, the marker composition may further include TCRBV28-01 and TCRBJ02-04.

In one embodiment of the present application, the marker composition comprising TCRBV19-01 and TCRBJ02-04 and/or TCRBV28-01 and TCRBJ02-04 can diagnose or predict mild subarachnoid hemorrhage.

In one embodiment of the present application, the marker composition may further include TCRBV28-01 and TCRBJ02-06.

In one embodiment of the present application, the marker composition may further include TCRBV30-01 and TCRBJ02-04.

In one embodiment of the present application, the marker composition comprising TCRBV28-01 and TCRBJ02-06 and/or comprising TCRBV30-01 and TCRBJ02-04 can diagnose or predict severe subarachnoid hemorrhage.

According to the composition for diagnosing subarachnoid hemorrhage, the kit for diagnosing subarachnoid hemorrhage, and the method for providing information for diagnosing subarachnoid hemorrhage of the present invention, the expression level of V-J recombination of the TCRB CDR3 repertoire is measured to determine whether the severity of subarachnoid hemorrhage is mild or severe. It can be used for prediction or diagnosis of

1 is a diagram schematically showing the diagnosis of subarachnoid hemorrhage using VJ recombination of T cell receptor beta chain.
2 is a diagram showing the comparison results of TCRB clonogenic performance according to subarachnoid hemorrhage (SAH) severity, good grade vs bad grade. It was confirmed that patients with good grade SAH had higher clonogenic capacity than patients with poor grade SAH.
3 shows repertoire diversity at hospitalization and follow-up, where TCRB CDR3 clonal types are indicated by dots and ranked according to clonal frequency.
4 is a diagram showing the comparison results of CDR3 length repertoire according to subarachnoid hemorrhage (SAH) severity, good grade vs bad grade. It was found that patients with good grade SAH had shorter CDR lengths than patients with poor grade SAH.
5 is a diagram showing the results of quantification of the TCRB CDR3 sequence in each patient between the day of admission and 7-day follow-up. The ten most common sequences are marked in different colors. It was confirmed that patients with poor grade SAH tended to have a lower ratio among the top 10 most common sequences compared to patients with good grade SAH at admission.
6 is a diagram showing the distribution and change of unique clonal types of TCRBV and TCRBJ genes in SAH patients. Compared with TCRBJ, it was confirmed that TCRBV showed a different distribution between hospitalization and 7-day follow-up. Different colors were used to indicate the gene frequency of each patient.
7 is a view showing comparison results of average TCRB VJ gene utilization according to the severity of SAH. The V gene and J gene segments were arranged on the x-axis and the y-axis. The different colors indicate the difference in gene frequency in patients with poor grades of SAH compared to patients with good grades of SAH.
8 is a diagram showing the comparison results of average TCRB VJ gene utilization during hospitalization and 7-day follow-up according to good grade (A) and bad grade (B) according to the severity of SAH. The V gene and J gene segments were arranged on the x-axis and the y-axis. The different colors indicate the difference in gene frequency in patients with poor grades of SAH compared to patients with good grades of SAH.

Hereinafter, the present application will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes of the present invention, and the scope of the present application is not limited to these Examples and Experimental Examples.

Example 1: Cohort Collection

The cohort for carrying out the present invention was derived from the stroke database of participating hospitals, which is part of an ongoing prospective observation project at the regional medical center. All patients were admitted to the hospital from March 2016 to December 2019. The subarachnoid hemorrhage patients for the Examples and Experimental Examples herein met the following conditions: 1) an adult over 18 years old, 2) spontaneous subarachnoid hemorrhage due to aneurysm rupture associated with a saccular appearance. and 3) patients who underwent coil embolization. In addition, patients with the following conditions were excluded: 1) vertebral, anatomical, and infectious aneurysms, 2) angiogram-negative subarachnoid hemorrhage, and 3) arteriovenous malformation (AVM) or dural arteriovenous fistula. Patients with subarachnoid hemorrhage with other cerebrovascular disease including dural arteriovenous fistula.

The purpose of this study was to compare the composition and mutations of the TCRB (T cell receptor β-chain) CDR3 repertoire between the time of admission (admission) and follow-up after 7 days. In addition, patients were divided into two groups according to SAH severity, good and bad grades, and the TCRB repertoire between these groups was further compared. A good grade SAH was defined as an initial Hunt and Hess grade of III or lower. Clinical and radiological health information was reviewed, and clinical outcomes were assessed using modified Rankin scale (mRS) scores after 6 months of SAH. The above experiments were approved by the participating hospital institutional review committee (No. 2016-3, 2017-9, 2018-6), and informed consent was obtained from all patients or their relatives.

Example 2: DNA extraction and high-throughput sequencing

Peripheral blood samples were prepared on the day of admission and follow-up of the patients. Genomic DNA was extracted from whole blood using the QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany), quantified with DropSense96 and diluted for library preparation in buffer according to standards. Sample data were then generated using an immunoSEQ assay (Adaptive Biotechnologies, Seattle, WA). The somatic rearranged locus CDR3 region was amplified using bias-controlled multiplex polymerase chain (PCR). Specifically, the first PCR was performed for all V and J gene segments using forward and reverse amplification primers and hypervariable CDR3 was amplified. We then performed a proprietary barcode sequence and Illumina adapter sequence (https://support.illumina.com) in a second PCR. The CDR3 library was sequenced on an Illumina instrument according to the manufacturer's instructions.

Example 3: TCRB repertoire analysis

Raw Illumina sequence reads were demultiplexed and further processed with the following steps: removal of adapter and primer sequences, identification and correction of technical errors, removal of primer dimers, germline and other contaminating sequences. The data were then filtered and clustered using a relative frequency ratio and a modified nearest-neighbor algorithm to merge closely related sequences. For the variable region of an antibody chain, an antibody is produced by a combination of gene fragments, V, D and J genes. The V, D and J regions are separated and encoded with single or multiple exons. Accordingly, the V, D, and J genes were defined based on annotations according to the IMGT database (www.imgt.org). Biological CDR3 locus sequences were normalized and quantified. The immunoSEQ Analyzer toolset was used for data analysis. Comparison results were reported as the median (25th-75th percentile) using the Mann-Whitney U test. P values less than 0.05 were considered statistically significant, and the analysis was performed with SPSS V.19 (SPSS, Illinois, USA).

Experimental Example 1: Confirmation of basic characteristics of the cohort

The TCRB CDR3 repertoire of 6 patients with subarachnoid hemorrhage (SAH) in the cohort collected in Example 1 was analyzed and compared at two different time points (hospitalization and 7-day follow-up). Of the 6 patients, 3 patients had a good grade (Hunt-Hess grade Ⅲ or lower) SAH and the other 3 had a bad grade SAH (Hunt-Hess grade IV or higher). The mean age was 53.5 years (range, 41-77 years). Four were female (66.7%). Most of the aneurysms (n = 5, 83.3%) were located in the anterior circulation. Two patients experienced severe delayed cerebral ischemia and recovered well after chemical angioplasty. Three patients with good grade SAH recovered well without serious disability. However, 1 out of 3 patients (33.3%) showed neurologically poor results as indicated by a 3-point mRS (Modified Rankin Scale) score. The basic characteristics of the above-described cohorts are summarized in Table 1 below.

patient
No. age gender Hunt-Hess
Rating Fisher
Rating Aneurysm
part DCI 6-month mRS One 61 F II 2 MCA No 0 2 50 F II 3 MCA Yes 0 3 77 F III 3 A-com No 0 4 47 M IV 4 PICA No 3 5 57 M IV 4 A-com No 0 6 41 F IV 3 MCA Yes One

* A-com - anterior communicating artery; DCI—delayed cerebral ischemia; MCA - middle cerebral artery; mRS - modified Rankin scale; PICA - posterior inferior cerebellar artery

Experimental Example 2: TCRB sequence, clonogenic ability and CDR3 length analysis

In the cohort identified in Experimental Example 1, variables such as total and unique sequences, clonality, and frequency of the TCRB CDR3 repertoire were measured.

First, as a result of confirming clonogenic performance using the Shannon index during hospitalization and follow-up, the results were 0.059 (0.037-0.038) and 0.027 (0.014-0.082) (p = 0.699), respectively. The clonogenic performance of poor grade SAH [0.025 (0.011-0.038)] was compared to good grade SAH [0.095 (0.079-0.101); p = 0.026]. (Fig. 2). Also, a read frequency of 0.005% A < 0.05% predominated in clonal expansion, but the change between hospitalization and follow-up did not show a clear association (Table 2).

NO. One NO. 2 NO. 3 NO. 4 NO. 5 NO. 6 Read frequency (%) Adm F/U Adm F/U Adm F/U Adm F/U Adm F/U Adm F/U ≥5 One One One 0 One One 0 0 0 0 0 0 0.5≤- < 5 8 9 14 3 10 6 0 2 5 3 8 One 0.05≤- <0.5 65 45 56 217 1071 284 44 278 259 94 1372 265 0.005≤- <0.05 3995 4748 10787 2256 0 2000 3744 2557 1965 4646 0 3005

* Adm (admission) - time of admission; F/U (follow-up) - time of follow-up (7 days after hospitalization)

Next, the TCRB clonal frequencies were ranked and repertoire diversity was compared using the Simpson diversity index (D). Here, a score close to 1 indicates high diversity (Fig. 3). Overall, the variability index was 0.993 to 0.999 at admission and 0.987 to 0.999 at follow-up. Diversity scores of patients with poor grade SAH were 0.998 and 0.999, higher than those of patients with good grade SAH.

CDR3 length ranged from 15 to 78 nucleotides, peaking at 42 in patients with good grade SAH and at 45 in patients with poor grade SAH. The five most common CDR3 lengths were 36, 39, 42, 45 and 48 nucleotides. Results were calculated as the average of 5 participants. Good grade SAH cases tended to have shorter CDR lengths compared to low grade SAH ( FIG. 4 ).

Next, the 10 most extended sequences of these sequences are presented and compared in terms of the amino acid level ( FIG. 5 ). The distribution of the top ten most common sequences was different for each patient. Overall, it was confirmed that patients with poor grade SAH showed a lower proportion of the top 10 most common sequences than patients with good grade SAH.

Experimental Example 3: Confirmation of distribution of TCRBV and TCRBJ

In the cohort identified in Experimental Example 1, the distribution of unique clonal types in both TCRBV and TCRBJ gene segments was analyzed. Overall, the TCRBV gene segments from follow-up had a different clonal phenotype than that at the day of admission, but the distribution of TCRBJ gene segments was similar. It was confirmed that when compared with good grade SAH, bad grade SAH tends to exhibit a large number of different clonal types of the TCRBV gene segment ( FIG. 6 ).

In addition, the relative frequencies of V-J combinations were analyzed using different colors representing disease severity, and 754 annotated V-J pairs were identified. Of these, the top three V-J pairs most commonly increased in patients with poor grade SAH compared to patients with good grade SAH were TCRBV21-01/TCRBJ01-05, TCRBV21-01/TCRBJ01-04, and TCRBV21-01/TCRBJ02-04. was confirmed (FIG. 7).

Next, we compared V-J combinations at two time points, hospitalization and 7-day follow-up in patients with good-grade SAH and poor-grade SAH, respectively. As a result, it was confirmed that the most increased V-J pair in good grade SAH was TCRBV19-01/TCRBJ02-04, and the most decreased V-J pair was TCRBV28-01/TCRBJ02-04. In addition, with respect to patients with poor grade SAH, it was confirmed that the most increased V-J pair was TCRBV28-01/TCRBJ02-06, and the most decreased V-J pair was TCRBV30-01/TCRBJ02-04 (FIG. 8).

The specific amino acid sequence of the V-J combination is described in detail in Table 3 below.

V-J combination amino acid sequence SEQ ID NO: TCRBV19-01/TCRBJ02-04 CASSPGTGEFESKNIQYF One TCRBV28-01/TCRBJ02-04 CASSATGGGLNIQYF 2 TCRBV28-01/TCRBJ02-06 CASGRGTGANVLTF 3 TCRBV30-01/TCRBJ02-04 CAWSNPSEAKNIQYF 4

Therefore, based on the above results, the expression level of a specific V-J pair of TCRB, specifically TCRBV19-01/TCRBJ02-04, TCRBV28-01/TCRBJ02-04, TCRBV28-01/TCRBJ02-06 or TCRBV30-01/TCRBJ02-04 was confirmed By doing so, it is possible to confirm the diagnosis, prognosis, and whether immunotherapy is progressing in patients with mild or severe subarachnoid hemorrhage.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims to be described later and their equivalents.

<110> Industry Academic Cooperation Foundation, Hallym University <120> A composition for diagnosing subarachnoid hemorrhage comprising a formulation capable of measuring the expression level of TCRBV 19-01 and TCRBJ02-04 <130> P203010 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> TCRBV19-01/TCRBJ02-04 <400> 1 Cys Ala Ser Ser Pro Gly Thr Gly Glu Phe Glu Ser Lys Asn Ile Gln 1 5 10 15 Tyr Phe <210> 2 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> TCRBV28-01/TCRBJ02-04 <400> 2 Cys Ala Ser Ser Ala Thr Gly Gly Gly Leu Asn Ile Gln Tyr Phe 1 5 10 15 <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> TCRBV28-01/TCRBJ02-06 <400> 3 Cys Ala Ser Gly Arg Gly Thr Gly Ala Asn Val Leu Thr Phe 1 5 10 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> TCRBV30-01/TCRBJ02-04 <400> 4 Cys Ala Trp Ser Asn Pro Ser Glu Ala Lys Asn Ile Gln Tyr Phe 1 5 10 15

Claims (11)

For diagnosis of prognosis of subarachnoid hemorrhage, comprising an agent capable of measuring the expression level of TCRBV19-01 and TCRBJ02-04, and diagnosing whether or not the Hunt-Hess grade is mild subarachnoid hemorrhage of grade III or less composition.
The composition for diagnosis of prognosis of subarachnoid hemorrhage according to claim 1, wherein the composition comprises an agent capable of measuring the expression level of a peptide comprising the amino acid sequence of SEQ ID NO: 1.
The composition for diagnosis of prognosis of subarachnoid hemorrhage according to claim 1, wherein the composition further comprises an agent capable of measuring the expression level of TCRBV28-01 and TCRBJ02-04.
The composition for diagnosis of prognosis of subarachnoid hemorrhage according to claim 3, wherein the composition further comprises an agent capable of measuring the expression level of a peptide comprising the amino acid sequence of SEQ ID NO: 2.
delete delete A kit for diagnosing the prognosis of subarachnoid hemorrhage, comprising the composition of any one of claims 1 to 4.
A method for diagnosing whether or not a Hunt-Hess grade is a mild subarachnoid hemorrhage of grade III or less, comprising measuring the expression level of TCRBV19-01 and TCRBJ02-04 from a biological sample isolated from a subject , A method of providing information for the diagnosis of subarachnoid hemorrhage prognosis.
The method according to claim 8, wherein the method comprises: measuring the expression levels of TCRBV19-01 and TCRBJ02-04 from a biological sample isolated from a control; and
Comparing the expression levels of TCRBV19-01 and TCRBJ02-04 in the subject and the control group
Further comprising, Hunt-Hess grade (Hunt-Hess grade) of diagnosing whether or not mild subarachnoid hemorrhage of grade III or less, an information providing method for diagnosing the prognosis of subarachnoid hemorrhage.
The method according to claim 9, wherein when the expression level of TCRBV19-01 and TCRBJ02-04 of the subject is higher than the expression level of TCRBV19-01 and TCRBJ02-04 of the control, the subject is diagnosed as having mild subarachnoid hemorrhage. A method for providing information for diagnosing a prognosis of a subarachnoid hemorrhage, further comprising the step of determining or predicting the risk of developing at a high level.
A marker composition for prognosis diagnosis of subarachnoid hemorrhage, comprising TCRBV19-01 and TCRBJ02-04, and for diagnosing whether Hunt-Hess grade is mild subarachnoid hemorrhage of grade III or less.

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