KR102479643B1 - A method for diagnosing severe subarachnoid hemorrhage comprising analyzing the TCRB CDR3 repertoire - Google Patents

Abstract

The present invention provides a composition for diagnosing subarachnoid hemorrhage comprising an agent capable of measuring the expression levels of TCRBV28-01 and TCRBJ02-06, a kit for diagnosing subarachnoid hemorrhage comprising the composition, and diagnosis of subarachnoid hemorrhage using the composition and kit It is about how to provide 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, 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. can be used for prediction or diagnosis of

Description

A method for diagnosing severe subarachnoid hemorrhage comprising analyzing the TCRB CDR3 repertoire}

The present invention provides a composition for diagnosing subarachnoid hemorrhage comprising an agent capable of measuring the expression levels of TCRBV28-01 and TCRBJ02-06, a kit for diagnosing subarachnoid hemorrhage comprising the composition, and diagnosis of subarachnoid hemorrhage using the composition and kit It is about how to provide information for

Subarachnoid hemorrhage is a condition that occurs when arteries on the surface of the brain are damaged, and it is a type of stroke that can damage the brain. Blood from subarachnoid hemorrhage flows into the space between the brain and skull and mixes with cerebrospinal fluid, which acts as a buffer between the brain and brain and spinal cord, which increases the pressure around the brain and interferes with brain function.

Emergency CT and MRI examinations can be performed to diagnose subarachnoid hemorrhage. If the symptoms are strongly suspected but not found on CT or MRI, a lumbar puncture is performed to examine the spinal fluid. CT and MRI blood vessel examination and catheter cerebrovascular angiography can be performed to examine blood vessels in detail to detect aneurysms and cerebrovascular malformations. However, this diagnosis method requires expensive test equipment, and the process may also be complicated, so there is a disadvantage in that it is inconvenient to quickly diagnose the state of subarachnoid hemorrhage.

In the T-cell receptor (TCR), one α chain and one β chain are connected to each other by a disulfide bond. Like the B-cell receptor, the T-cell receptor also has a transmembrane region at the end of the constant region, which is fixed to the T cell, and the variable region (V) at the end of the α and β chains forms a single antigen attachment site.

With the recent development of diagnostic molecular genetic testing techniques, analysis of the variable region of the TCR β chain has become easier, making it possible to identify clones of specific immune cells. Recently, using these molecular genetic testing techniques, specific T-cell clones have been tested in diseases such as aplastic anemia, graft-versus-host disease, large granular lymphocytic leukemia, and paroxysmal nocturnal hemoglobinuria (PNH). Research on diagnostic tests and clinical usefulness is being actively conducted.

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

Republic of Korea Patent Publication No. 2002-0064396

A first aspect of the present application is to provide a composition for diagnosing subarachnoid hemorrhage, including an agent capable of measuring the expression levels of TCRBV28-01 and TCRBJ02-06.

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 TCRBV28-01 and TCRBJ02-06 in a biological sample isolated from a subject.

A fourth aspect of the present application is to provide a marker composition for diagnosing subarachnoid hemorrhage, including TCRBV28-01 and TCRBJ02-06.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed herein may also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of the present application is not to be construed as being limited by the specific descriptions described below.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed herein may also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of the present application is not to be construed as being limited by the specific descriptions described below.

A first aspect of the present application provides a composition for diagnosing subarachnoid hemorrhage, including an agent capable of measuring the expression levels of TCRBV28-01 and TCRBJ02-06.

The term “diagnosis” used throughout the present specification means determining whether subarachnoid hemorrhage has occurred in a specific individual, and more specifically, determining the severity of subarachnoid hemorrhage.

As used throughout the present specification, the term “subarachnoid hemorrhage (SAH)” is a type of stroke in which hemorrhage occurs as a result of damage to an artery on the surface of the brain. A subarachnoid hemorrhage causes blood to flow into the space between the brain and skull, which mixes with cerebrospinal fluid, which acts as a buffer between the brain and cerebrospinal fluid, increases the pressure around the brain (increased intracranial pressure), and interferes with brain function. , may cause brain damage. A subarachnoid hemorrhage is most often caused by a rupture of an arterial wall protruding from the surface of the brain. The protrusion of an artery is called an aneurysm, and usually this aneurysm is the cause of rupture, or blood leaks from an arteriovenous malformation made by entangling abnormal blood vessels, resulting in subarachnoid hemorrhage.

In patients with suspected subarachnoid hemorrhage, it is necessary to diagnose aneurysmal subarachnoid hemorrhage more quickly through accurate diagnostic techniques, to prevent rebleeding by removing the aneurysm, and to receive fluid therapy and intracranial pressure control treatment. In addition, specific complications (rebleeding, hydrocephalus, cerebrovascular spasm, epilepsy) and medical complications (neurogenic pulmonary edema, venous thrombosis, cardiovascular complications, electrolyte abnormalities, gastrointestinal complications) of subarachnoid hemorrhage at each stage of treatment should be evaluated and treated. Therefore, promptly diagnosing the exact 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 the present specification means that one α chain and one β chain are connected to each other by a single disulfide bond, and the transmembrane region at the end of the constant region It is fixed to T cells, and the variable region (V) at the ends of α and β chains forms a single antigen attachment site. In addition, the binding site at the tip of the T cell receptor binds to an antigen site called an epitope or an epitope. At this time, the binding method is similar to the binding between the enzyme and the substrate, and the binding occurs non-covalently. In addition, T-cell receptors, unlike B-cell receptors, bind only to manipulation of antigens presented on the host cell surface with the help of a protein called Major Histocompatibility Complex (MHC).

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

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

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: 3, wherein the amino acid sequence of SEQ ID NO: 3 is the TCRBV28-01 and It may contain TCRBJ02-06.

In one embodiment of the present application, the composition may further include an agent capable of measuring the expression levels 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.

In one embodiment of the present application, the composition may further include an agent capable of measuring the expression levels of TCRBV19-01 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: 1, wherein the amino acid sequence of SEQ ID NO: 1 is the TCRBV19- 01 and TCRBJ02-04.

In one embodiment of the present application, the composition may further include an agent capable of measuring the expression levels 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.

In one embodiment of the present application, the diagnosis of subarachnoid hemorrhage herein may be diagnosing the severity of subarachnoid hemorrhage, predicting the risk of occurrence, or diagnosing the prognosis of subarachnoid hemorrhage, and specifically, subarachnoid hemorrhage is mild or It may be to diagnose whether it is severe.

The term "Hunt-Hess grade or Hunt-Hess grade" used throughout the present specification is a representative evaluation method for patients with subarachnoid hemorrhage, and each grade can be classified according to the following criteria.

- Grade 0: unruptured aneurysm

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

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

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

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

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

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

In one embodiment of the present application, when the expression level of peptides including TCRBV28-01 and TCRBJ02-06 increases, or when the expression level of peptides including TCRBV30-01 and TCRBJ02-04 decreases, the subject is treated with severe arachnoid It can be judged that hemorrhage has occurred or is highly likely to occur.

In one embodiment of the present application, when the expression level of peptides including TCRBV 19-01 and TCRBJ02-04 is increased, or when the expression level of peptides including TCRBV28-01 and TCRBJ02-04 is decreased, the subject is mild It can be determined that subarachnoid hemorrhage has occurred or is highly likely to develop.

As used throughout the present specification, the term "measurement of expression level" refers to measuring the expression level or expression level of a specific protein (peptide) or a gene encoding the protein, specifically including TCRBV28-01 and TCRBJ02-06. It may be to measure the expression level of the peptide or mRNA or gene encoding the same, and may additionally measure the expression level of the peptide including TCRBV30-01 and TCRBJ02-04 or the 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 (Western blotting), ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA: radioimmunoassay), radioimmuno-diffusion method (radical immunodiffusion) , Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence ( It may be immunofluorescence, immunochromatography, fluorescence activated cell sorter analysis (FACS), 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" refers to a molecule that can be used to determine the expression level of a specific protein or gene encoding it, specifically detecting the protein or the gene And / or may include an agent capable of amplification, but is not limited thereto.

As used throughout the present specification, the term "an 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, "an 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 is specifically It may mean a primer capable of amplification or a probe capable of specifically binding, but is not limited thereto.

In one embodiment of the present application, the composition comprises peptides including TCRBV28-01 and TCRBJ02-06 or genes encoding the peptides, or primers, probes, nucleotides, antibodies or antigen-binding fragments thereof that specifically bind to mRNA , a ligand, a receptor, a protein, or a combination thereof.

The term "primer" used throughout this specification is a nucleic acid sequence having a free 3' hydroxyl group, capable of forming base pairs with a template complementary to a specific base sequence, and Refers to a nucleic acid sequence that serves as a starting point for strand copying. Primers can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., 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 7 to 50 nucleotide sequences as specific primers for mRNA of genes encoding TCRBV28-01 and TCRBJ02-06 to measure the amount of production of the desired product Through this, it is possible to predict the resistance of the object to the cancer treatment. PCR conditions and lengths of sense and antisense primers can be appropriately selected according to techniques known in the art. The primer 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.

The term "probe" used throughout this specification refers to a target nucleic acid, for example, a nucleic acid fragment such as RNA or DNA that can specifically bind to mRNA, and the presence, content and expression level of a specific mRNA. It may be labeled so that it can be identified. The probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe, or the like. For example, by performing hybridization using a probe having a nucleic acid sequence complementary to the mRNA of the gene encoding TCRBV28-01 and TCRBJ02-06, and measuring the expression level of the mRNA through the degree of hybridization, resistance to cancer treatment of the individual can predict Selection of an appropriate probe and hybridization conditions can be appropriately selected according to techniques known in the art. 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, such nucleic acid sequences can be modified through various methods known in the art. Examples of such modifications are methylation, capping, substitution of one or more homologs of the natural nucleotide, or modifications between nucleotides, e.g., uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates). etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.). Primers or probes can also be modified with labels that can directly or indirectly provide a detectable signal. Examples of the label may include a radioactive isotope, a fluorescent molecule, or biotin.

As used throughout this specification, the term "antibody" is an art-recognized term and refers to a specific immunoglobulin directed against an antigenic site. The antibody may specifically bind to proteins or fragments thereof including TCRBV28-01 and TCRBJ02-06. Fragments of proteins comprising TCRBV28-01 and TCRBJ02-06 may be, for example, immunogenic fragments. The fragment refers to a protein fragment having at least one epitope that can be recognized by antibodies against proteins including TCRBV28-01 and TCRBJ02-06. Genes encoding proteins including TCRBV28-01 and TCRBJ02-06 were cloned into an expression vector, proteins containing TCRBV28-01 and TCRBJ02-06 encoded by the genes were obtained, and from the obtained proteins, Antibodies can be prepared according to conventional methods.

Types of the antibody include polyclonal antibodies, monoclonal antibodies, or recombinant antibodies, and all immunoglobulin antibodies are included. The antibody is meant in its complete form with two full-length light chains and two full-length heavy chains. In addition, the antibody also includes special antibodies such as humanized antibodies. A polyclonal antibody can be produced by injecting a biomarker protein or a fragment thereof, which is an immunogen, 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 may be used. When the immunogen is injected intramuscularly, intraperitoneally or subcutaneously, it may be administered together with an adjuvant to increase antigenicity. Thereafter, blood from the external host is periodically collected to collect serum showing improved titer and antigen specificity, or antibodies can be separated and purified therefrom.

Monoclonal antibodies can be produced by a technique known to those skilled in the art for generating an immortalized cell line by fusion. Briefly about the method for producing a monoclonal antibody, 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, the antigen-producing lymphocytes isolated from the mouse are fused with human or mouse myeloma to generate an immortalized hybridoma, and only hybridoma cells producing the desired monoclonal antibody are selected and proliferated using an ELISA method After that, monoclonal antibodies can be isolated and purified from the culture. In addition, as monoclonal antibodies, commercially available antibodies against proteins including TCRBV28-01 and TCRBJ02-06 may be obtained and used.

Using these antibodies, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, western blotting or immunoblotting on polyacrylic gels known in the art. It is possible to confirm whether the protein is expressed in a biological sample by methods such as the above.

As used throughout this specification, the term "fragment" refers to a polypeptide that does not have, for example, the structure of an intact antibody, peptide or protein, but has a specific antigen-binding site or binding domain directed to an antigenic site. Such fragments include functional fragments of antibody molecules that are not complete 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 include at least 7 or more amino acids, for example, 9 or more amino acids or 12 or more amino acids.

A second aspect of the present application provides a kit for diagnosing subarachnoid hemorrhage, including the composition for diagnosing subarachnoid hemorrhage. Content overlapping with the first aspect is also 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 TCRBV28-01 and TCRBJ02-06. The kit may further include antisense oligonucleotides, primer pairs, probes, etc. for diagnosis of subarachnoid hemorrhage, as well as one or more other component compositions, solutions, or devices suitable for analysis methods, and PCR kits, RT- PCR kits, qRT-PCR kits, methylation-specific PCR kits, DNA chip kits, Western blot kits, or ELISA kits, and the like.

According to one embodiment of the present application, the kit may include not only the polynucleotide, cDNA, mRNA, peptide, etc. of the present application, but also 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 that includes essential elements required to perform RT-PCR, and the RT-PCR kit is a kit for each specific gene or methylation region. In addition to primers, a test tube or other suitable container, reaction buffer (with varying pH and magnesium concentration), 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 required to perform DNA chip, and the DNA chip kit may include polynucleotides, primers or probes specific for the genes or methylation regions. and 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 containing essential elements required to perform a microarray, and the DNA microarray kit may contain cDNA corresponding to a gene or fragment thereof as a probe. The substrate may include a quantitative control gene or a cDNA corresponding to a fragment thereof, and may 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 method is used to immobilize the searched markers as probe DNA molecules on a substrate of a DNA chip. A method using a spotter or the like may 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 TCRBV28-01 and TCRBJ02-06 in a biological sample isolated from a subject. Contents overlapping with the first and second aspects are also applied to the information provision method of the third aspect.

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

As used throughout the present specification, the term "sample" means a material derived from an individual with or likely to have subarachnoid hemorrhage, and specifically, tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, It may include urine and the like, but is not limited thereto. In addition, gene and/or protein samples may be obtained from these samples, and the genetic samples may include nucleic acids, for example, DNA, mRNA, or cDNA synthesized from mRNA, from which expression of specific genes/proteins may be obtained. 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 TCRBV28-01 and TCRBJ02-06 from a biological sample separated from a control group; and comparing the expression levels of TCRBV28-01 and TCRBJ02-06 in the individual and the control group.

As used throughout the present specification, the term "control group" may refer to a general subject, a non-subarachnoid hemorrhage patient group, a non-patient group, and the like without subarachnoid hemorrhage.

In one embodiment of the present application, the method, when the expression levels of TCRBV28-01 and TCRBJ02-06 in the subject are higher than the expression levels of TCRBV28-01 and TCRBJ02-06 in the control group, the subject has severe subarachnoid hemorrhage It may further include the step of determining that the disease has occurred or predicting the risk of disease at a high level.

The term "prediction" used throughout the present specification refers to whether a specific individual is likely to develop mild or severe subarachnoid hemorrhage, and if there is a possibility of developing mild or severe subarachnoid hemorrhage, the likelihood of developing mild or severe subarachnoid hemorrhage 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 include measuring the expression levels of TCRBV30-01 and TCRBJ02-04 from a biological sample isolated from the subject.

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

In one embodiment of the present application, the method is, when the expression levels of TCRBV30-01 and TCRBJ02-04 in the subject are lower than the expression levels of TCRBV30-01 and TCRBJ02-04 in the control group, the subject is treated with severe arachnoid It may further include the step of determining that bleeding has occurred or predicting the risk of occurrence at a high level.

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

In one embodiment of the present application, the method is, when the expression levels of TCRBV19-01 and TCRBJ02-04 in the subject are higher than the expression levels of TCRBV28-01 and TCRBJ02-06 in the control group, the subject is subjected to mild arachnoid It may further include the step of determining that bleeding has occurred or predicting 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 TCRBJ02-04 from a biological sample separated from a control group; and comparing the expression levels of TCRBV28-01 and TCRBJ02-04 in the individual and the control group.

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

A fourth aspect of the present application provides a marker composition for diagnosing subarachnoid hemorrhage, including TCRBV28-01 and TCRBJ02-06. The contents overlapping with the first to third aspects are also applied to the composition of the fourth aspect.

As used throughout the present specification, the term "marker" refers to a biomarker and can detect changes in living organisms, thereby objectively measuring the normal or pathological state of living organisms, the degree of response to drugs, and the like.

In one embodiment of the present application, the marker composition can diagnose the severity of subarachnoid hemorrhage, and can specifically determine whether the subarachnoid hemorrhage is mild or severe.

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, a marker composition comprising TCRBV28-01 and TCRBJ02-06 and/or TCRBV30-01 and TCRBJ02-04 can diagnose or predict severe subarachnoid hemorrhage.

In one embodiment of the present application, the marker composition may further include TCRBV19-01 and TCRBJ02-04.

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, a marker composition comprising TCRBV19-01 and TCRBJ02-04 and/or TCRBV28-01 and TCRBJ02-04 can diagnose or predict mild 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 in the TCRB CDR3 repertoire is measured to determine whether the severity of subarachnoid hemorrhage is mild or severe. can be used for prediction or diagnosis of

1 is a diagram schematically showing diagnosis of subarachnoid hemorrhage using VJ recombination of T cell receptor beta chain.
2 is a diagram showing the comparison results of TCRB clonogenicity according to subarachnoid hemorrhage (SAH) severity and good grade vs. bad grade. It was confirmed that patients with good grade SAH had higher clonogenicity than patients with bad grade SAH.
Figure 3 is a diagram showing repertoire diversity during hospitalization and follow-up. TCRB CDR3 clonotypes are indicated by dots and ranked according to clonotype frequency.
Figure 4 is a diagram showing the comparison results of CDR3 length repertoires according to subarachnoid hemorrhage (SAH) severity, good grade vs. bad grade. It was confirmed that patients with good grade SAH had shorter CDR lengths than those with poor grade SAH.
5 is a diagram showing the quantification results of TCRB CDR3 sequences in each patient between the day of hospitalization and the 7-day follow-up. The 10 most common sequences are shown in different colors. We found that patients with poor grade SAH tended to have a lower proportion of the top 10 most common sequences at admission compared to patients with good grade SAH.
Figure 6 is a diagram showing the distribution and change of unique clonotypes of TCRBV and TCRBJ genes in SAH patients. Compared to TCRBJ, it was confirmed that TCRBV showed a different distribution between the day of admission and the 7-day follow-up. Different colors were used to represent gene frequencies for each patient.
7 is a diagram showing the comparison results of average TCRB VJ gene utilization according to SAH severity. The V gene and J gene segments are arranged on the x and y axes. Different colors represent differences in gene frequencies in patients with poor grade SAH compared to patients with good grade SAH.
8 is a diagram showing comparison results of average TCRB VJ gene utilization at hospitalization and during 7-day follow-up according to good grade (A) and bad grade (B) according to SAH severity. The V gene and J gene segments are arranged on the x and y axes. Different colors represent differences in gene frequencies in patients with poor grade SAH compared to patients with good grade SAH.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are intended to illustrate 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 participating hospital's stroke database, which is part of an ongoing prospective observational project at a regional medical center. All patients were admitted to the hospital from March 2016 to December 2019. Subarachnoid hemorrhage patients for the present examples and experimental examples satisfied the following conditions: 1) an adult over 18 years of age, 2) spontaneous subarachnoid hemorrhage due to aneurysmal rupture associated with a saccular appearance. and 3) patients who underwent coil embolization. In addition, patients with the following conditions were excluded: 1) pendulum, anatomical, and infectious aneurysm, 2) angiogram-negative subarachnoid hemorrhage, and 3) arteriovenous malformation (AVM) or dural arteriovenous fistula. Subarachnoid hemorrhage with other cerebrovascular disease including dural arteriovenous fistula.

We aimed to compare the composition and mutation of the T cell receptor β-chain (TCRB) CDR3 repertoire between the time of admission (admission) and follow-up after 7 days. In addition, we divided patients into two groups according to SAH severity, good and bad grades, and further compared the TCRB repertoire between these groups. A good grade of SAH was defined as an initial Hunt and Hess grade of III or lower. Clinical and radiological medical information was reviewed, and clinical outcomes were evaluated using the modified Rankin scale (mRS) score 6 months after SAH. The experiment was approved by the Institutional Review Board of the participating hospitals (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. Then, sample data were generated using an immunoSEQ assay (Adaptive Biotechnologies, Seattle, WA). Somatic rearranged locus CDR3 regions were amplified using bias-controlled multiplex polymerase chain (PCR). Specifically, a first PCR was performed on all V and J gene segments using forward and reverse amplification primers and a hypervariable CDR3 was amplified. Then, in a second PCR, proprietary barcode sequences and Illumina adapter sequences (https://support.illumina.com) were performed. CDR3 libraries were 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 was then filtered and clustered using relative frequency ratio and a modified nearest-neighbor algorithm to merge closely related sequences. In the case of the variable regions of antibody chains, antibodies are produced by the combination of gene segments, V, D and J genes. The V, D and J regions are isolated and encoded as single or multiple exons. Accordingly, the V, D, and J genes were defined based on annotation 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. Comparative results were expressed as medians (25-75th percentile) using the Mann-Whitney U test. A P value of less than 0.05 was considered statistically significant, and analysis was performed with SPSS V.19 (SPSS, Illinois, USA).

Experimental Example 1: Confirmation of basic characteristics of cohort

The TCRB CDR3 repertoire of 6 patients with subarachnoid hemorrhage (SAH) from 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 SAH of good grade (Hunt-Hess grade III or lower) and the remaining 3 patients had SAH of poor grade (Hunt-Hess grade IV or higher). Mean age was 53.5 years (range, 41–77 years). 4 were female (66.7%). Most 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%) had a poor neurological outcome as indicated by a Modified Rankin Scale (mRS) score of 3 points. The basic characteristics of the cohort described above are summarized and described in Table 1 below.

patient
No. age gender Hunt-Hess
ranking Fisher
ranking 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, clonogenicity and CDR3 length analysis

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

First, the results of confirming the clonogenicity using the Shannon index during admission and follow-up were 0.059 (0.037-0.038) and 0.027 (0.014-0.082), respectively (p = 0.699). The clonogenicity of bad grade SAH [0.025 (0.011-0.038)] was significantly higher than that of good grade SAH [0.095 (0.079-0.101); p = 0.026]. (Fig. 2). In addition, a read frequency of 0.005% A < 0.05% was predominant for clonal expansion, but the change between admission 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 (after 7 days of hospitalization)

Next, we ranked TCRB clonotype frequencies and compared repertoire diversity using the Simpson diversity index (D). A score close to 1 indicates high diversity (Fig. 3). Overall, the diversity index ranged from 0.993 to 0.999 at admission and from 0.987 to 0.999 at follow-up. The diversity scores of patients with poor-grade SAH were 0.998 and 0.999, respectively, higher than those of patients with good-grade SAH.

CDR3 lengths ranged from 15 to 78 nucleotides, peaking at 42 in patients with good-grade SAH and 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 subjects. 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 were presented and compared at the amino acid level (FIG. 5). The distribution of the top 10 most common sequences was different for each patient. Overall, it was found that patients with poor grade SAH showed a lower proportion of the top 10 most common sequences than did patients with good grade SAH.

Experimental Example 3: Checking the Distribution of TCRBV and TCRBJ

In the cohort identified in Experimental Example 1, the distribution of unique clonotypes in both TCRBV and TCRBJ gene segments was analyzed. Overall, TCRBV gene segments from follow-up had different clonotypes than those on the day of admission, but the distribution of TCRBJ gene segments was similar. It was confirmed that compared to good-grade SAH, bad-grade SAH tended to represent a large number of different clonotypes of TCRBV gene segments (FIG. 6).

In addition, the relative frequency of V-J combinations was 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. confirmed (FIG. 7).

Next, we compared V–J combinations at two time points, admission and 7-day follow-up, in patients with good-grade and bad-grade SAH, respectively. As a result, it was confirmed that TCRBV19-01/TCRBJ02-04 was the most increased V-J pair and TCRBV28-01/TCRBJ02-04 was the most decreased V-J pair in SAH of good grade. In addition, it was confirmed that TCRBV28-01/TCRBJ02-06 was the V-J pair that increased the most and TCRBV30-01/TCRBJ02-04 was the V-J pair that decreased the most with regard to SAH patients with poor grade (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 sequence number 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 progress of immunotherapy in patients with mild and severe subarachnoid hemorrhage.

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

<110> Industry Academic Cooperation Foundation, Hallym University <120> A method for diagnosing severe subarachnoid hemorrhage comprising analyzing the TCRB CDR3 repertoire <130> P203030 <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)

A composition for diagnosing the prognosis of subarachnoid hemorrhage, comprising an agent capable of measuring the expression levels of TCRBV28-01 and TCRBJ02-06, and diagnosing whether or not severe subarachnoid hemorrhage with a Hunt-Hess grade of grade IV or higher .
The composition for 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: 3.
The method according to claim 1, wherein the composition further comprises an agent capable of measuring the expression level of TCRBV30-01 and TCRBJ02-04, the composition for prognosis of subarachnoid hemorrhage.
The method according to claim 3, wherein the composition further comprises an agent capable of measuring the expression level of the peptide comprising the amino acid sequence of SEQ ID NO: 4, the composition for prognosis of subarachnoid hemorrhage.
delete delete A kit for diagnosing the prognosis of subarachnoid hemorrhage, comprising the composition of any one of claims 1 to 4.
delete delete delete A marker composition for diagnosing the prognosis of subarachnoid hemorrhage, comprising TCRBV28-01 and TCRBJ02-06, and diagnosing whether or not severe subarachnoid hemorrhage with a Hunt-Hess grade of grade IV or higher.

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