WO2021153649A1

WO2021153649A1 - Method for detecting risk of complications after transplantation of hematopoietic stem cells, predictive diagnostic agent, and kit

Info

Publication number: WO2021153649A1
Application number: PCT/JP2021/002963
Authority: WO
Inventors: 岡村　浩史; 博久中前; 岳郎進藤; 泰史大塚
Original assignee: 公立大学法人大阪; 国立大学法人佐賀大学
Priority date: 2020-01-31
Filing date: 2021-01-28
Publication date: 2021-08-05
Also published as: JP7347767B2; JPWO2021153649A1

Abstract

The purpose of the present invention is to provide a method capable of broadly predicting complications that would occur after transplantation of hematopoietic stem cells, including NRM, and predicting complications at a very early stage within 20 days after transplantation of hematopoietic stem cells. A method for detecting the risk of complications that would occur after transplantation of hematopoietic stem cells, the method comprising: (i) a step for measuring Ba in a biological sample derived from a subject; and (ii) a step for detecting the presence or absence of the risk of complications that would occur after transplantation of hematopoietic stem cells, on the basis of the result of step (i). The method for detecting the risk of complications that would occur after transplantation of hematopoietic stem cells can predict the risk of complications that would occur after transplantation of hematopoietic stem cells at a very early stage within 20 days after transplantation of hematopoietic stem cells, and can further predict NRM at a very early stage.

Description

Methods for detecting complication risk after hematopoietic stem cell transplantation, predictive diagnostic agents and kits

The present invention relates to a method for detecting the risk of complications after hematopoietic stem cell transplantation. More specifically, the present invention provides a method capable of early prediction of the onset of thrombotic microangiopathy (TA-TMA) after hematopoietic stem cell transplantation and the risk of non-recurrence mortality (NRM) after hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation is the only treatment method for diseases such as hematological cancer and immunodeficiency that are difficult to treat with ordinary chemotherapy and immunosuppressive therapy, and is the only treatment method for the purpose of completely curing Japan. In, about 3,500 cases are carried out annually. On the other hand, after allogeneic hematopoietic stem cell transplantation, TA-TMA was observed as a complication with a relatively high frequency (10 to 30%), and in severe cases, the median case fatality rate within 3 months after diagnosis was 75. It is so serious that it is reported as%.

The reason why complications after allogeneic hematopoietic stem cell transplantation cause such a poor prognosis is that once the disease develops, it becomes severe and difficult to treat because there are many unclear points regarding the pathophysiology and there is no means for early diagnosis. it is conceivable that. Therefore, establishment of a means for predicting complications after allogeneic hematopoietic stem cell transplantation is one of the unmet needs in clinical practice.

Assuming that sC5b-9, which is a complement complex of the terminal pathway in the complement activation pathway, is associated with the diagnosis and prognosis prediction of TA-TMA, sC5b-9 can be used as a marker for predicting the onset of TA-TMA. Sex has been reported. Specifically, in children, sC5b-9 measured 28 days after allogeneic hematopoietic stem cell transplantation has been shown to be useful as a marker for predicting the occurrence of TA-TMA (Non-Patent Document 1), and is used by adults. In sC5b-9, which develops graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation and is measured 2 to 6 weeks after the onset of GVHD, its usefulness as a marker for predicting the occurrence of TA-TMA has been shown. (Non-Patent Document 2).

The usefulness of sC5b-9 as a predictive marker for the onset of TA-TMA has been reported, but the timing at which this predictive marker can be effectively used is 28 days after allogeneic hematopoietic stem cell transplantation in children and allogeneic hematopoiesis in adults. It is 2 to 6 weeks after the onset of graft-versus-host disease (GVHD) after stem cell transplantation. However, in view of the poor prognosis of TA-TMA, it is extremely important to start the preventive measures as soon as possible after transplantation in controlling the onset of TA-TMA. Then, it must be said that the prediction of TA-TMA onset at the timing reported so far will still be lost late. At the latest, within 20 days after hematopoietic stem cell transplantation, the risk of complications should be determined. In addition, there has been no report that NRM could be predicted even in TA-TMA, and in fact, it is clearly stated in the above-mentioned Non-Patent Document 1 that NRM could not be predicted by sC5b-9. However, if it is possible to predict even the most serious NRM among TA-TMAs, it has great clinical significance.

Therefore, an object of the present invention is to provide a method that can widely predict complications after hematopoietic stem cell transplantation, including NRM, and can predict them very early within 20 days after hematopoietic stem cell transplantation.

The present inventors focused on using a protein associated with a pathway upstream of sC5b-9 in the complement activation pathway as a predictive marker in order to carry out complications after hematopoietic stem cell transplantation even earlier. However, such proteins are associated with complement activator C5a occurring upstream of sC5b-9 of the terminal pathway, complement component C3 mediating the common pathway upstream of the terminal pathway, and the function of the classical pathway. Although CH50, the complement component C4 that mediates the classical and lectin pathways, the complement regulators CFI and CFH that control the activation of C3, which is an upstream pathway from the terminal pathway, and autoantibodies to CFH, these proteins were investigated. Did not function as a predictable marker of TA-TMA very early within 20 days after transplantation. Under such circumstances, it was found that TA-TMA functions as a predictable marker at an extremely early stage within 20 days after transplantation for Ba, which is a complement activated product of the second pathway. Not only that, we also found that Ba also functions as a predictor of NRM at a similar very early stage. The present invention has been completed by further studies based on these findings.

That is, the present invention provides the inventions of the following aspects.
Item 1. (I) A step of detecting complement activator Ba in a biological sample derived from a test subject, and
(Ii) A method for detecting a risk of complications after hematopoietic stem cell transplantation, which comprises a step of detecting the presence or absence of a risk of complications after hematopoietic stem cell transplantation based on the result of the step (i).
Item 2. The amount of the complement activator Ba is increased by comparing the result of the step (i) obtained for the test subject with the result of the step (i) obtained for the control by the step (ii). Item 2. The detection method according to Item 1, which is carried out using the above as an index.
Item 3. Item 3. The detection method according to Item 1 or 2, wherein the complication after hematopoietic stem cell transplantation is thrombotic microangiopathy after hematopoietic stem cell transplantation.
Item 4. Item 8. The detection method according to any one of Items 1 to 3, wherein the complication after hematopoietic stem cell transplantation is a fatal complication that causes non-recurrence death.
Item 5. Item 4. The detection method according to any one of Items 1 to 4, wherein the test subject is a test subject within 20 days after hematopoietic stem cell transplantation.
Item 6. Item 2. The detection method according to any one of Items 1 to 5, wherein the step (i) is performed by a detection method based on a biospecific affinity.
Item 7. Predictive diagnostic agent for complication risk after hematopoietic stem cell transplantation, including anti-Ba antibody.
Item 8. A predictive diagnostic kit for complication risk after hematopoietic stem cell transplantation, which comprises the predictive diagnostic agent according to Item 7.
Item 9. A companion diagnostic for selecting treatments, including anti-Ba antibody, to reduce the risk of developing complications after hematopoietic stem cell transplantation.
Item 10. (I) A step of measuring the amount of complement activator Ba in a biological sample derived from a test subject, and
(Ii) A step indicating that there is a risk of complications after hematopoietic stem cell transplantation when the amount of the complement activator Ba is increased as compared with the result of the step (i) obtained for the control. Methods for diagnosing the risk of complications after hematopoietic stem cell transplantation, including.
Item 11. (I) To identify patients at risk of complications after hematopoietic stem cell transplantation, the steps of measuring complement activator Ba in biological samples of patients who received hematopoietic stem cell transplantation and (II) were identified. A method of treating a patient at risk of complications after hematopoietic stem cell transplantation, comprising the step of administering ecrizumab to a patient at risk of complications after hematopoietic stem cell transplantation.
Item 12. Item 2. The therapeutic method according to Item 11, wherein the step (I) and the step (II) are performed on a patient within 20 days after hematopoietic stem cell transplantation.
Item 13. Use of anti-Ba antibody for the manufacture of predictive diagnostic agents for the risk of complications after hematopoietic stem cell transplantation.
Item 14. Use of anti-Ba antibody for the manufacture of companion diagnostics for selecting treatments to reduce the risk of developing complications.

According to the present invention, by using Ba in a biological sample derived from a test subject as a biomarker for predicting the risk of complications after hematopoietic stem cell transplantation, the risk of complications after hematopoietic stem cell transplantation can be measured within 20 after transplantation, for example, transplantation. It is possible to make a very early prediction of the 7th day after that, and further, it is possible to make a very early prediction of NRM.

The selection flow of the patient (TA-TMA patient or non-TA-TMA patient) to be analyzed is shown. The contents of various complement activity pathway-related factors (Ba, CH50) contained in plasma on the day of transplantation and the 7th day after transplantation of TA-TMA patients (1) or non-TA-TMA patients (0) are shown. The contents of various complement activity pathway-related factors (C3, CFH) contained in plasma on the day of transplantation and the 7th day after transplantation of TA-TMA patients (1) or non-TA-TMA patients (0) are shown. The content of various complement activity pathway-related factors (CFI, CFH-IgG) contained in plasma on the day of transplantation and 7 days after transplantation in TA-TMA patients (1) or non-TA-TMA patients (0). show. The contents of various complement activity pathway-related factors (C4, C5b9) contained in plasma on the day of transplantation and the 7th day after transplantation in TA-TMA patients (1) or non-TA-TMA patients (0) are shown. The content of various complement activity pathway-related factors (C5a) contained in plasma on the day of transplantation and the 7th day after transplantation in TA-TMA patients (1) or non-TA-TMA patients (0) is shown. The ROC curve of the Ba value at 7 days after transplantation for the onset of TA-TMA is shown. The ROC curve of sC5b-9 at 28 days after transplantation for the onset of TA-TMA is shown. The time course of the cumulative onset of TA-TMA in the low value group (0) and the high value group (1) based on the cutoff of the Ba value on the 7th day after transplantation derived from FIG. 3 is shown. The time course of renal function based on the low value group (day7_Blow) and the high value group (day7_Ba high) based on the cutoff of the Ba value on the 7th day after transplantation is shown. The time course of the cumulative onset of non-recurrence mortality (NRM) based on the low value group (0) and the high value group (1) based on the cutoff of the Ba value on the 7th day after transplantation is shown. The time course of the cumulative onset of recurrence (Relapse) based on the low value group (0) and the high value group (1) based on the cutoff of the Ba value on the 7th day after transplantation is shown.

1. 1. Method for detecting complication risk after hematopoietic stem cell transplantation The method for detecting complication risk after hematopoietic stem cell transplantation according to the present invention includes (i) a step of detecting a complement activator Ba in a biological sample derived from a test subject. , (Ii) The step of detecting the presence or absence of the risk of complications after hematopoietic stem cell transplantation based on the result of the step (i). Hereinafter, the detection method of the present invention will be described in detail.

1-1. Complication risk prediction marker after hematopoietic stem cell transplantation In the detection method of the present invention, the complement activator Ba is used as a complication risk prediction marker after hematopoietic stem cell transplantation. The content of Ba in a biological sample derived from a subject who had complications after hematopoietic stem cell transplantation is significantly increased as compared with a hematopoietic stem cell transplantation patient who did not have the complications. Therefore, Ba makes it possible to distinguish between those who are at high risk of complications after hematopoietic stem cell transplantation and those who are not, in patients after hematopoietic stem cell transplantation.

Ba is known as a factor B degradation product produced by the degradation of complement B factor by complement D factor in the second pathway of the complement activation pathway. Regarding the amino acid sequence of Ba, for example, in the case of the human Ba amino acid sequence, the N-terminal side of factor B (26th to 764th amino acid sequence) of CFAB_HUMAN registered with the acceptance number P00751 in Uniprot / Swissprot. It is composed of the 26th to 259th amino acid sequences that occupy about 1/3 of the above.

Regarding the human Ba amino acid sequence, the specific amino acid sequence of the protein is shown in SEQ ID NO: 1.

Ba, which is a complication risk prediction marker after hematopoietic stem cell transplantation used in the present invention, may be human Ba shown in SEQ ID NO: 1 and other mutants thereof. That is, as the complication risk prediction marker Ba after hematopoietic stem cell transplantation used in the present invention, in addition to the human Ba shown in SEQ ID NO: 1, a mutant of human Ba, a non-human species Ba, and a non-human organism Examples include variants of Ba of the species.

More specifically, Ba, which is a complication risk prediction marker after hematopoietic stem cell transplantation used in the present invention, includes the following proteins (1) and (2), preferably the protein (1). Can be mentioned.
(1) Protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (2) Amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 1. A protein consisting of and having complement activity as Ba

The phrase "one or several amino acids have been substituted, deleted, inserted, and / or added" is not particularly limited, but is a known mutation such as a site-specific mutagenesis method. Depending on the protein production method and polymorphism, the number of substitutions, deletions, insertions, and / or additions (preferably 5 or less, more preferably 4 or less, still more preferably 3 or less, still more preferably 2). Hereinafter, particularly preferably one) amino acid is intended to be substituted, deleted, inserted, and / or added. In addition, Ba is composed of amino acid residues corresponding to about 1/3 of the amino group side of factor B, and the activity of factor B binding to C3b bound to the cell membrane in the second pathway of the complement activation pathway. And because it has the property of being degraded by factor D, the protein of (2) is degraded by factor D and its binding activity to C3b similar to that of human factor B in the second pathway of the complement activation pathway. It suffices to be composed of an amino acid sequence occupying about 1/3 of the N-terminal side of factor B having the above-mentioned characteristics.

1-2. Complications after hematopoietic stem cell transplantation Specific examples of complications after hematopoietic stem cell transplantation include thrombotic microangiopathy (TA-TMA) that develops after hematopoietic stem cell transplantation. TA-TMA is characterized by microangiopathic hemolytic anemia (MAHA), consumable thrombocytopenia, and organ dysfunction due to microcirculatory insufficiency. TA-TMA is a known TA-TMA diagnostic criterion (eg, (1) LDH normal upper limit or higher, (2) 50% or higher thrombocytopenia or platelet transfusion dependence, (3) progressive anemia or red blood cell transfusion dependence, (4) Appearance of crushed red blood cells of 1% or more in peripheral blood or histological evidence of microangiopathy, (5) No abnormal coagulation, and (6) Haptoglobin below normal lower limit, and (7) Coombs test negative Is diagnosed by (satisfying all). TA-TMA is classified as a secondary TMA, and its representative diseases are thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS). Can be mentioned. TA-TMA develops when vascular endothelial cells are damaged due to various causes after hematopoietic stem cell transplantation, the formation of platelet thrombocytosis is promoted, and as a result, the living body suffers from organ dysfunction due to circulatory insufficiency.

The complication risk prediction marker after hematopoietic stem cell transplantation used in the present invention also predicts serious complications after hematopoietic stem cell transplantation that result in transplant-related non-relapse mortality (NRM). be able to. From this point of view, a preferred example of the complications after hematopoietic stem cell transplantation to be predicted in the present invention is fatal complications resulting in NRM.

1-3. Subject The test subject may be any animal as long as it has undergone hematopoietic stem cell transplantation, and specifically, rodents such as mice, rats, hamsters and guinea pigs, and experimental animals such as rabbits; Livestock such as pigs, cows, goats, horses and sheep; pets such as dogs and cats; primates such as humans, monkeys, orangoutans and chimpanzees. The test subject in the present invention is preferably a primate, more preferably a human, and particularly preferably a human adult. The lower limit of the adult age is 16 years or older, preferably 30 years or older, more preferably 35 years or older, and the upper limit of the adult age is not limited as long as it is an indication for hematopoietic stem cell transplantation, for example. 80 years or younger, preferably 75 years or younger, more preferably 70 years or younger.

In addition, since Ba, which is a marker for predicting the risk of complications after hematopoietic stem cell transplantation, can predict the complications at an early timing within 20 days after transplantation, a preferable example of the test subject is the date of hematopoietic stem cell transplantation. As 0 days, the test subjects are within 20 days after transplantation. Further, from the viewpoint of accurately predicting the complications, as a more preferable example of the test subject, 1 to 19 days, preferably 2 to 17 days, more preferably 3 to 15 days, still more preferably 4 to 13 days after transplantation. Subject examples include days, more preferably 5 to 11 days, and particularly preferably 6 to 9 days.

1-4. Step of detecting Ba in a biological sample derived from a test subject In step (i), Ba in a biological sample derived from a test subject is detected. The detection target sample in the step (i) is a biological sample, and the detection target substance is the complication risk prediction marker (that is, Ba) after the hematopoietic stem cell transplantation.

The biological sample may be derived from the test subject, and specific examples thereof include a blood sample, a tissue sample, a cell sample, and the like collected from the test subject.

From the viewpoint of minimal invasiveness, a blood sample is preferable as a biological sample. Examples of the blood sample include whole blood, serum, plasma and the like. From the viewpoint of diagnostic accuracy, plasma is preferable. The collection and preparation of various blood samples can be performed according to a known method. For example, in the case of plasma, the collected blood (whole blood) is placed in a container containing an anticoagulant (EDTA, sodium citrate, heparin, etc.) and centrifuged to remove cell components (erythrocytes, white blood cells, platelets). Can be prepared. Blood samples are used, if necessary, diluted to an appropriate concentration.

From the viewpoint of diagnostic accuracy, biological samples preferably include tissue samples and cell samples. Examples of tissue samples and cell samples include samples obtained by biopsy, surgical pathological diagnosis, or cytodiagnosis. Specifically, the tissue samples and cell samples include fresh tissues and cells, frozen tissues and cells, and histopathologically treated tissues and cells (for example, formalin-fixed or paraffin-embedded). It may be there. These samples may be used as sections or as protein-soluble fractions prepared from tissues or cells.

Ba can be detected by detecting the presence or absence of Ba in a biological sample derived from a test subject. Preferably, Ba is detected by measuring the amount of Ba in the biological sample.

As the method for detecting Ba, a known protein detection method can be used without particular limitation, and examples thereof include a detection method based on biospecific affinity and a detection method by mass spectrometry.

In the detection method based on the biospecific affinity, an anti-Ba antibody capable of specifically recognizing Ba present in the biological sample collected from the test subject as an antigen (however, factor B is not recognized) is used. Such anti-Ba antibodies are known. The anti-Ba antibody may be either a monoclonal antibody or a polyclonal antibody as long as it specifically recognizes Ba as an antigen and does not recognize factor B, but a monoclonal antibody is preferable.

The anti-Ba antibody used to detect Ba in a biological sample is not particularly limited as long as it can be detected, and may be any isotype such as IgG, IgD, IgE, IgA, sIgA, and IgM. good. IgG is exemplified as a preferred antibody isotype in the present invention. Further, the anti-Ba antibody may have at least a complementarity determining region (CDR) for specifically recognizing and binding to Ba, and specifically, (ab') ₂ , Fab', Fab, etc. It may be a binding fragment of an antibody such as Fv, sFv, dsFv, sdAb). The anti-Ba antibody can be genetically engineered according to a conventional method.

As a specific detection method of Ba using the detection method based on the biospecific affinity, the anti-Ba antibody and the biological sample are brought into contact with each other, and the anti-Ba antibody and the complication risk prediction marker Ba after hematopoietic stem cell transplantation are used. There is a method of directly or indirectly detecting the specific binding of. Specific examples of such a detection method include immunoassays such as ELISA method, Western blotting method, immunoprecipitation method, radioimmunoassay (RIA) method, and fluorescence immunoassay method. When Ba is detected by these immunoassays, it can be carried out by binding a label such as an enzyme label, a color-developing label, a radiolabel or a luminescent label to another antibody that binds to Ba, and detecting or measuring this label. can. The other antibody may or may not have at least the ability to bind to Ba, and may or may not have the ability to bind to factor B. Further, the other antibody may be either a monoclonal antibody or a polyclonal antibody as long as it has at least the ability to bind to Ba.

The conditions for carrying out the immunoassay are not particularly limited as long as the specific binding between Ba and the anti-Ba antibody in the biological sample can be detected, and are set based on conventionally known conditions.

For example, when Ba is detected by the ELISA method, a biological sample collected from a test subject is added to each well of a multi-well plate on which an anti-Ba antibody is immobilized, and the anti-Ba antibody in the well and Ba in the biological sample are separated. React. Then, a labeled antibody that binds to Ba is added to each well for reaction, and then the reaction product obtained by adding the enzyme substrate is detected and / or quantified to obtain the biomarker in the biological sample. It can be detected and / or quantified. Here, the labeled antibody can be appropriately selected based on the animal to be tested from which a biological sample is collected, but when the test subject is a human, a non-human labeled antibody that specifically binds to human Ba. (For example, rabbit-derived anti-human Ba antibody) and the like.

Further, the enzyme used for labeling the labeled antibody can also be appropriately selected from those usually used, and for example, peroxidase, alkaline phosphatase, luciferase, esterase, glucose oxidase, β-D-galactosidase, etc. Examples thereof include β-D-glucuronidase. The enzyme substrate can be appropriately selected from known substrates depending on the type of enzyme. For example, when the enzyme is peroxidase, 3,3', 5,5'-tetramethylbenzidine (TMB) Can be used as a substrate.

Detection and / or quantification of the reaction product produced by the reaction of the enzyme with the substrate can be performed by measuring the absorbance of the reaction product, for example, 3,3', 5,5'-tetramethylbenzidine ( When TMB) is used as the enzyme substrate, it can be carried out by measuring the absorbance at 450 nm.

In a radioimmunoassay (RIA), an anti-Ba antibody is labeled with a radioisotope and reacted with Ba in a biological sample to form an immune complex, which is detected based on the radioactivity released from the radioisotope. Can be done.

In the case of a fluorescent immunoassay, an anti-Ba antibody is immobilized on a plate or the like, a biological sample is added thereto and reacted, and then a labeled antibody that binds to Ba present in the biological sample of the test subject is further reacted. , It can be done by detecting fluorescent color development. As the labeled antibody that specifically binds to Ba derived from the test subject, as described in the ELISA method, an antibody labeled with a fluorescent dye is used. Examples of the fluorescent dye include FITC, PE, APC, Cy-3, Cy-5 and the like.

In the immunoprecipitation method, it is possible to detect by reacting an anti-Ba antibody with a biological sample to form an immune complex and precipitating it as an insolubilizer using an active adsorbent such as protein A or protein G. can. Furthermore, immunoprecipitation and Western blotting can be combined for detection. More specifically, an anti-Ba antibody to which a tag such as FLAG is linked is reacted with a biological sample, and if Ba is present in the sample, an immune complex is formed, so that the antibody is precipitated by the above-mentioned active adsorbent. .. Then, the obtained precipitate is subjected to Western blotting. That is, Ba was present in the biological sample by separating and developing the precipitate by SDS-PAGE, transferring it to a nitrocellulose membrane, PVDF membrane, etc., and then performing an antigen-antibody reaction with an antibody against the tag on the transfer membrane. In some cases, it can be detected as a band.

In the detection method by mass spectrometry, a known mass spectrometer can be used without particular limitation. For example, as a method for introducing a sample into an instrument, a method of connecting to a separation device such as high performance liquid chromatography, dropping the sample on a stainless steel plate, immersing the probe in the sample, and the like can be mentioned. Examples of the method for ionizing the introduced sample include an electrospray ionization (ESI) method and a matrix-assisted laser desorption / ionization (MALDI) method. As a type of device for measuring the mass of ions, a quadrupole type, an ion trap type, a time-of-flight (TOF) type, a Fourier transform ion cyclotron resonance (FTICR) type, or the like is used alone or in combination. Those skilled in the art can perform mass spectrometry by arbitrarily selecting the optimum combination from these various options.

In the specific detection method of Ba using the detection method by mass spectrometry, a known protein measurement method using mass spectrometry can be used. For example, the presence of Ba can be detected by fragmenting Ba to be measured by trypsin digestion or the like to prepare a peptide sample and detecting a product ion having a sequence specific to Ba. Product ions having a sequence specific to the Ba are set within the measurement range (m / z) of the mass spectrometer to be used. In addition, using a stable isotope-labeled internal standard peptide having a sequence specific to the Ba, the relative intensity of the peak derived from a product ion having a sequence specific to the Ba is derived with respect to the intensity of the peak derived from the internal standard peptide. By doing so, Ba can be quantified.

1-5. In the step step (ii) of detecting the presence or absence of complication risk after hematopoietic stem cell transplantation, the presence or absence of complication risk after hematopoietic stem cell transplantation is detected based on the result of the step (i).

Regarding the presence or absence of complications after hematopoietic stem cell transplantation, the content of Ba in biological samples derived from subjects who had complications after hematopoietic stem cell transplantation was significantly higher than that in hematopoietic stem cell transplantation patients who did not have the complications. Judgment is based on the characteristics that increase in. Preferably, the determination of the presence or absence of complication risk after hematopoietic stem cell transplantation describes the result of step (i) obtained for the test subject as the result of step (i) obtained for the control (hereinafter, also referred to as the reference Ba amount). ), The increase in the amount of Ba can be used as an index.

Here, examples of the control used in step (ii) include animals of the same species as the test subject, animals after hematopoietic stem cell transplantation, and animals that do not develop post-transplantation complications, and are preferably tested. Examples include animals of the same species as the subject, animals after hematopoietic stem cell transplantation, animals that do not develop complications, and animals that have the same elapsed period after transplantation as the test subject.

As an example of the reference Ba amount, the range from the mean value of the Ba measurement values in the control population minus the standard deviation to the value obtained by adding the standard deviation to the mean value (the value obtained by subtracting the standard deviation and the standard deviation are added). (Including both the lower limit value), the range from the lower limit value to the upper limit value of the average value (including both the lower limit value and the upper limit value), and the like. When such a standard Ba amount is used, if the Ba measurement value in the subject exceeds the upper limit of the standard Ba amount, the subject is predicted to have a high risk of developing complications after hematopoietic stem cell transplantation (complication risk "complication risk". Yes ”can be detected).

As another example of the reference Ba amount, in the target population in which the presence or absence of complications after hematopoietic stem cell transplantation is known, the subjects who have complications after hematopoietic stem cell transplantation are included in a predetermined ratio below the cutoff value. The cutoff value determined in is mentioned. In determining such a cutoff value, it is possible to obtain a value that gives the best chi-square value for discrimination. When such a standard Ba amount is used, if the Ba measurement value in the subject exceeds the standard Ba amount, it is predicted that the subject has a high risk of developing complications after hematopoietic stem cell transplantation (complication risk "presence"). Can be detected).

A further example of the reference Ba amount is a cutoff value set based on a receiver operating characteristic (ROC) curve in a target population in which the presence or absence of complications after hematopoietic stem cell transplantation is known. In this case, the point on the ROC curve that is the closest to the point (coordinates (0,1)) whose sensitivity and singularity are both 1, or the origin coordinates (0,0) from the coordinates (1,1). The cutoff value can be determined based on the point on the ROC curve that is farthest from the straight line connecting the. When such a standard Ba amount is used, if the Ba measurement value in the subject exceeds the standard Ba amount, it is predicted that the subject has a high risk of developing complications after hematopoietic stem cell transplantation (complication risk "presence"). Can be detected).

For subjects who are predicted to have a high risk of developing complications after hematopoietic stem cell transplantation, as a measure to further reduce the risk of developing complications, adjustment of drugs at risk of developing TA-TMA and infection Complication control, prophylactic eculizumab administration, etc. can be examined and treated.

In addition, the complication risk prediction marker Ba after hematopoietic stem cell transplantation does not show a significant difference in the content in the biological sample between the case where the disease that caused the transplantation indication recurs and the case where the disease does not recur. Therefore, according to the present invention, complications and recurrence can be predicted separately. Furthermore, the complication risk prediction marker Ba after hematopoietic stem cell transplantation can also predict NRM, which is particularly serious among TA-TMAs. Moreover, the complication risk prediction marker Ba after hematopoietic stem cell transplantation can make these predictions very early within 20 days. Therefore, it is possible to appropriately select and start the treatment that should be taken for a subject who is predicted to have a high risk of developing complications after hematopoietic stem cell transplantation at an extremely early stage. Can be secured for a long time. Therefore, it can be expected that the risk of complications after hematopoietic stem cell transplantation can be reduced, and the fatality rate due to complications can be reduced more effectively.

2. Predictive Diagnostic Agents and Predictive Diagnostic Kits for Complication Risk After Hematopoietic Stem Cell Transplantation The present invention provides predictive diagnostic agents for complication risk after hematopoietic stem cell transplantation, including anti-Ba antibodies. The predictive diagnostic agent of the present invention can be used to perform the method for detecting the risk of complications after hematopoietic stem cell transplantation described in item 1 above.

The anti-Ba antibody used in the predictive diagnostic agent of the present invention is as described in item 1-4 above.

In the predictive diagnostic agent of the present invention, the anti-Ba antibody may be provided in a state of being immobilized on an insolubilized carrier. The material of the insolubilized carrier is not particularly limited as long as it does not interfere with the detection of the complication risk prediction marker Ba after hematopoietic stem cell transplantation, and is, for example, polystyrene, polyethylene, polypropylene, polyester, polyacrylic nitrile, polyvinyl chloride, fluororesin, crosslinked dextran. , Paper, silicon, glass, metal, agarose and the like can be exemplified. Moreover, you may use these materials in combination of 2 or more types. Examples of the shape of the insolubilized carrier include a substrate shape, a particle shape, and any other shape, and specific examples of the insolubilized carrier include microplates, trays, particles, fibers, rods, plates, containers, cells, test tubes, and the like. Can be mentioned.

Immobilization of the anti-Ba antibody on the insolubilized carrier can be performed according to a conventionally known method.

Further, the predictive diagnostic agent of the present invention may be formulated containing a buffer solution, a stabilizer, a preservative, etc. in addition to the anti-Ba antibody.

The predictive diagnostic agent of the present invention may be kitted in combination with other items that may be required to detect anti-Ba antibodies. That is, the present invention also provides a predictive diagnostic kit for complication risk after hematopoietic stem cell transplantation, which includes the predictive diagnostic agent.

Other items are not particularly limited as long as they can be required to detect an anti-Ba antibody, and include, for example, a labeled antibody, a labeling substance detection agent, a solubilizer, a detergent, a reaction terminator, and the like. Control samples and / or predictive diagnostic protocols and the like can be mentioned. The predictive diagnostic protocol includes information such as operations and procedures for carrying out the method for detecting the risk of complications after hematopoietic stem cell transplantation described above.

3. 3. Method for diagnosing complication risk after hematopoietic stem cell transplantation As described in item 1-5 above, the presence or absence of complication risk after hematopoietic stem cell transplantation is determined by the amount of Ba obtained for the test subject and the standard Ba obtained for the control. It can be done by using the fact that it is increasing in comparison with the amount as an index. That is, the present invention compares the results of (i) measuring the amount of complement activator Ba in a biological sample derived from a test subject with (ii) the results of step (i) obtained for a control. Also provided is a method for diagnosing the risk of complications after hematopoietic stem cell transplantation, which comprises a step of indicating that there is a risk of complications after hematopoietic stem cell transplantation when the amount of the complement activator Ba is increased.

The details of the steps (i) and (ii) in the diagnostic method of the present invention are the same as the steps (i) and (ii) of the method for detecting the risk of complications after hematopoietic stem cell transplantation described in item 1 above. ..

3. 3. Companion diagnostics for selecting treatments to reduce the risk of developing complications As described in item 1-1 above, Ba in biological samples is a risk of complications after hematopoietic stem cell transplantation in patients after hematopoietic stem cell transplantation. It makes it possible to distinguish between those who have a high risk and those who do not, and as described in item 1-5 above, for subjects who are predicted to have a high risk of developing complications after hematopoietic stem cell transplantation, further complications As measures for reducing the risk of developing TA-TMA, adjustment of drugs that are at risk of developing TA-TMA, countermeasures against infectious diseases, prophylactic administration of ecrizumab, etc. can be examined and treated. That is, the present invention also provides a companion diagnostic agent for selecting a treatment for reducing the risk of developing complications, including an anti-Ba antibody. The companion diagnostic agent of the present invention can be used in the method for detecting the risk of complications after hematopoietic stem cell transplantation described in item 1 above.

The anti-Ba antibody used in the companion diagnostic agent of the present invention is as described in item 1-4 above, and an example of a specific form of the anti-Ba antibody is the same as in item 2 above, and the anti-Ba antibody However, it is the same as the above item 2 in that it may be made into a kit in combination with other items that may be required for detecting the anti-Ba antibody.

4. Treatment method for patients at risk of complications after hematopoietic stem cell transplantation As described in item 1-5 above, by using Ba in a biological sample as a biomarker for predicting complication risk after hematopoietic stem cell transplantation, after hematopoietic stem cell transplantation. For subjects who can diagnose the risk of developing complications and are predicted to have a high risk of developing complications after hematopoietic stem cell transplantation (detection of complication risk "presence"), further complications As a treatment for reducing the risk of developing the disease, prophylactic administration of ecrizumab or the like can be examined and treated. Eculizumab is an anti-C5 chimeric antibody preparation that suppresses the production of the pro-inflammatory factor C5a and the terminal complement complex C5b-9 by specifically binding to complement C5. That is, the present invention is specified as (I) a step of measuring Ba in a biological sample of a patient who has undergone hematopoietic stem cell transplantation in order to identify a patient who is at risk of complications after hematopoietic stem cell transplantation, and (II). Also provided are methods of treating patients at risk of post-hematopoietic stem cell transplantation, including the step of administering ecrizumab to patients at risk of complications after hematopoietic stem cell transplantation.

Furthermore, as described in items 1-3 and 1-5 above, the complication risk prediction marker Ba after hematopoietic stem cell transplantation can be predicted very early within 20 days, and therefore, complications after hematopoietic stem cell transplantation. Actions that should be taken for subjects who are predicted to be at high risk of developing the disease can be appropriately selected and initiated very early. That is, in the therapeutic method of the present invention, the step (I) and the step (II) are performed within 20 days after the hematopoietic stem cell transplantation (preferably 1 to 19 days after the transplantation, more preferably 2 to 17 days, still more preferably. Can be performed on patients for 3 to 15 days, more preferably 4 to 13 days, even more preferably 5 to 11 days, particularly preferably 6 to 9 days).

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

<< Patients and methods >>
<Study design and target patient selection>
This test was performed by retrospective observation. Patient selection was made in the order shown in FIG. Specifically, patients who underwent allogeneic hematopoietic stem cell transplantation at Osaka City University between December 2012 and December 2016 (excluding patients who did not obtain consent and patients who could not use samples). Of the cases, TA-TMA difficult-to-diagnose cases (uncertain TA-TMA) were excluded. uncertain TA-TMA is a case in which TA-TMA is suspected but the test items such as haptoglobin, Coombs test, and crushed red blood cells necessary for TA-TMA diagnosis are not performed, and other factors that cause thrombocytopenia or hemolysis ( Includes cases in which primary blood disease, drugs, etc.) are observed. After selecting the target TA-TMA patients in this way, a propensity score was calculated for each case, and the same number of non-TA-TMA patients were matched. Finally, non-TA-TMA patients matched with TA-TMA onset patients were analyzed.

Nine items of complement activity pathway-related factors were measured using frozen plasma samples collected from TA-TMA patients and non-TA-TMA patients before and within 20 days after transplantation (specifically, on the 7th day). We compared and examined. Among the factors related to the complement activity pathway, sC5b-9, which was reported to be useful as a marker for predicting the occurrence of TA-TMA on the 28th day after transplantation in Non-Patent Document 1, was frozen on the 28th day after transplantation. Plasma samples were also measured. Table 1 shows the factors related to the complement activity pathway measured in this test.

<Definition>
The diagnostic criteria for TA-TMA are in line with the diagnostic criteria proposed by Cho et al., And the following seven items are to be met at the same time. (1) LDH normal upper limit or higher, (2) 50% or higher thrombocytopenia or platelet transfusion dependence, (3) progressive anemia or erythrocyte transfusion dependence, (4) 1% or higher crushed erythrocyte appearance in peripheral blood or Histological evidence of microangiopathy, (5) no abnormal coagulation, and (6) below normal lower limit of haptoglobin, and (7) negative Coombs test.

According to previously reported, pretreatments containing any of more than 8 Gy of TBI, 7.2 mg / kg or more of intravenous busulfan, and 140 mg / m ² or more of melphalan should be myeloablative pretreatment, and the others should not be treated. Defined as myeloablative pretreatment. HLA concordance was defined by DNA typing at the HLA-A, HLA-B, HLA-C, and HLA-DR loci.

<Measurement method for factors related to complement activity pathway>
Plasma samples were prepared by collecting blood in a blood collection tube containing EDTA-2Na and immediately centrifuging at 3000 rpm / 20 minutes. The prepared plasma samples were controlled and cryopreserved at -80 ° C in the same type of ultra-low temperature freezer until the time of test measurement in the clinical laboratory department of Osaka City University.

Nine items related to the complement activity pathway (CH50, C3, C4, CFI, CFH, anti-CFH antibody C5a, sC5b-9, Ba) were quantified. One-point CH50 "Seiken" (Denka Seiken Co., Ltd.) was used to quantify CH50; N-assay TIA C3-SH Nittobo and N-assay TIA C4-SH Nittobo (both) were used to quantify C3 and C4, respectively. Nittobo Medical Co., Ltd.); CFI (Human) ELISA Kit, CFH (Human) ELISA Kit, and CFH IgG ELISA Kit (all from Abnova) were used to quantify CFI, CFH, and anti-CFH antibodies, respectively. MicroVue Ba EIA Kit, MicroVue sC5b-9 Plus EIA Kit, and MicroVue C5a EIA Kit (Quidel) were used for the quantification of Ba, sC5b-9, and C5a, respectively. Specifically, in the quantification of Ba using MicroVueBaEIAKit, a plasma sample was brought into contact with a Ba-specific (and factor B-unaware) monoclonal antibody coated on the substrate, and the monoclonal antibody was contacted. A horseradish peroxidase-labeled polyclonal antibody having a binding property to Ba is bound to Ba in a plasma sample bound to Ba, and the labeling is measured.

<Propensity score matching method>
The presence or absence of TA-TMA onset is the dependent variable, and the known pre-transplant risk factors for TA-TMA are patient age, gender, pre-transplant disease status, donor (related / unrelated), and HLA match (match / mismatch). , Graft type (bone marrow / peripheral blood / umbilical cord blood), pretreatment intensity (bone marrow destructive / non-myeloablative), GVHD prevention method (cyclosporin administration / tacrolimus administration), patient cytomegalovirus antibody (yes / no) Propensity scores for each patient were calculated using a logistic regression model as a variable. Patients who did not develop TA-TMA and survived without recurrence until the same time as the TA-TMA onset date of TA-TMA patients, and who had a propensity score closest to the propensity score of TA-TMA patients, were controlled 1: 1. Matched as a patient. Control patients were reconstructable and the matching calipers were within 0.25 of the standard deviation of the propensity score. Missing values were complemented by the multiple complement method.

<Statistical analysis method>
The Two way ANOVA method was used for comparison of the time course of the complement protein value and the time course of the Cre value between the two groups, and the sidek method was used for the correction of the multiple test. When a significant difference was obtained between the two groups, a time point with a significant difference was further obtained by Post hoc analysis. The optimum Cutoff value by Youden Index was obtained from the ROC curve, and the Gray method was used to compare the cumulative onset of TA-TMA and NRM. The onset of TA-TMA and the death of non-TA-TMA were considered competing events.

<< Result >>
<Patient background to be analyzed>
TA-TMA patients (n = 15) and control patients (n = 15) extracted by matching were analyzed. For these TA-TMA patients and control patients, the number of people in each group for each pre-transplant risk factor (however, the median and range (in parentheses) for age are shown, and the number of people for pre-transplant disease status and graft type. The ratio (in parentheses; unit%) is shown together with the p-value (there is no difference between the groups) by comparison between the groups, as shown in the table below. In this TA-TMA patient, the median date of onset of TA-TMA was 25 days after transplantation (interquartile range: 22.5-44), with the transplantation date as 0 day.

<Differences in complement activity pathway-related factors between the TA-TMA group and the non-TA-TMA group>
The measurement results of the complement activity pathway-related factors of Comparative Examples 1 to 8 and Example 1 between the TA-TMA group (1) and the non-TA-TMA group (0) are shown in FIGS. 2a to 2e. Of the nine complement activity pathway-related factors, except for Ba (Comparative Examples 1 to 8), no significant difference was shown between the two groups within 20 days after transplantation (specifically, 7 days after transplantation). Although not shown, sC5b-9, which was reported to be useful as a TA-TMA occurrence prediction marker 28 days after transplantation in Non-Patent Document 1, was reported on 28 days after transplantation (Comparative Example 9). The TA-TMA group (1) showed an abnormally high value. On the other hand, in Ba (Example 1), a significantly abnormally high value was shown in the TA-TMA group (1) within 20 days after transplantation (specifically, 7 days after transplantation) (mean ± standard error). , 1129 ± 109 ng / ml in the TA-TMA group (1), 584 ± 38 ng / ml in the non-TA-TMA group (0)).

<Ability to predict complications after hematopoietic stem cell transplantation based on Ba value on the 7th day after transplantation>
The ROC curve of the Ba value (Example 1) on the 7th day after transplantation for the TA-TMA onset group is shown in FIG. The AUC of the Ba value on the 7th day after transplantation for the TA-TMA onset group was 0.88 (95% confidence interval: 0.72-1.0), and the optimum cutoff value obtained from the ROC curve was 869 ng /. It was ml.

The ROC curve of the sC5b-9 value (Comparative Example 9) 28 days after transplantation for the TA-TMA onset group is shown in FIG. The AUC of the sC5b-9 value on the 28th day after transplantation for the TA-TMA onset group was 0.69 (95% confidence interval: 0.49 to 0.89), and the optimum cutoff value obtained from the ROC curve was It was 197.2 ng / ml.

As shown in the comparison between FIGS. 3 and 4, the complication risk prediction marker Ba after hematopoietic stem cell transplantation of the present invention is useful as a TA-TMA prediction marker on the 28th day after transplantation in Non-Patent Document 1. Compared with the reported sC5b-9, not only is the prediction time of TA-TMA significantly earlier, but the accuracy is also significantly superior.

<Complication prediction ability after hematopoietic stem cell transplantation based on Ba value on the 7th day after transplantation-2>
Using the cutoff of the Ba value (Example 1) on the 7th day after transplantation derived from FIG. 3, the TA- The relationship between the cumulative incidence of TMA (vertical axis) and the elapsed months after transplantation (horizontal axis) is shown in FIG. Comparing the two groups in FIG. 5, the TA-TMA cumulative incidence rate was significantly higher in the high Ba group (1) on the 7th day after transplantation than in the low Ba group (0) on the 7th day after transplantation. (1 year cumulative incidence: 100% vs 17%; HR: 18.9 [95% confidence interval: 5.5-64.6]; p <0.001).

In addition, FIG. 6 shows the relationship between creatinine Cre (vertical axis), which similarly indicates renal function, and the elapsed day after transplantation (horizontal axis). As shown in FIG. 6, the Ba high value group (day7_Ba high) on the 7th day after transplantation showed a significantly higher creatinine level than the Ba low value group (day7_Ba low) on the 7th day after transplantation.

<Complication prediction ability after hematopoietic stem cell transplantation based on Ba value on the 7th day after transplantation-3>
Using the cutoff of the Ba value (Example 1) derived from FIG. 3 on the 7th day after transplantation, no recurrence occurred in the low value group (0) in which the Ba value was less than the cutoff and the high value group (1) in which the Ba value was greater than or equal to the cutoff. The relationship between the cumulative incidence of mortality (NRM) (vertical axis) and the elapsed months after transplantation (horizontal axis) is shown in FIG. Comparing the two groups in FIG. 7, the high Ba group (1) on the 7th day after transplantation showed a significantly higher cumulative incidence of NRM than the low Ba group (0) on the 7th day after transplantation. (P = 0.008).

Using the cutoff of the Ba value (Example 1) derived from FIG. 3 on the 7th day after transplantation, the low value group (0) having a Ba value less than the cutoff and the high value group (1) having a Ba value equal to or higher than the cutoff were used. The relationship between the cumulative incidence of recurrence (Relapse) (vertical axis) and the elapsed months after transplantation (horizontal axis) is shown in FIG. Even when the two groups were compared between the groups in the figure, there was no significant difference between the two groups.

As shown in the comparison between FIGS. 7 and 8, the complication risk prediction marker Ba after hematopoietic stem cell transplantation of the present invention can predict NRM separately from recurrence. On the other hand, Non-Patent Document 1 shows that sC5b-9 is useful as a TA-TMA prediction marker on the 28th day after transplantation, but NRM cannot be predicted. The complication risk prediction marker Ba after hematopoietic stem cell transplantation of the present invention has great clinical significance because it can predict the most serious case, NRM, at an extremely early stage.

Claims

(I) A step of detecting complement activator Ba in a biological sample derived from a test subject, and
(Ii) A method for detecting a risk of complications after hematopoietic stem cell transplantation, which comprises a step of detecting the presence or absence of a risk of complications after hematopoietic stem cell transplantation based on the result of the step (i).
The amount of the complement activator Ba is increased by comparing the result of the step (i) obtained for the test subject with the result of the step (i) obtained for the control by the step (ii). The detection method according to claim 1, which is carried out using the above as an index.
The detection method according to claim 1 or 2, wherein the complication after hematopoietic stem cell transplantation is thrombotic microangiopathy after hematopoietic stem cell transplantation.
The detection method according to any one of claims 1 to 3, wherein the complication after hematopoietic stem cell transplantation is a fatal complication that causes non-recurrence death.
The detection method according to any one of claims 1 to 4, wherein the test subject is a test subject within 20 days after hematopoietic stem cell transplantation.
The detection method according to any one of claims 1 to 5, wherein the step (i) is performed by a detection method based on a biospecific affinity.
Predictive diagnostic agent for complication risk after hematopoietic stem cell transplantation, including anti-Ba antibody.
A predictive diagnostic kit for complication risk after hematopoietic stem cell transplantation, which comprises the predictive diagnostic agent according to claim 7.
A companion diagnostic for selecting treatments, including anti-Ba antibody, to reduce the risk of developing complications after hematopoietic stem cell transplantation.
(I) A step of measuring the amount of complement activator Ba in a biological sample derived from a test subject, and
(Ii) A step indicating that there is a risk of complications after hematopoietic stem cell transplantation when the amount of the complement activator Ba is increased as compared with the result of the step (i) obtained for the control. Methods for diagnosing the risk of complications after hematopoietic stem cell transplantation, including.
(I) To identify patients at risk of complications after hematopoietic stem cell transplantation, the steps of measuring complement activator Ba in biological samples of patients who received hematopoietic stem cell transplantation and (II) were identified. A method of treating a patient at risk of complications after hematopoietic stem cell transplantation, comprising the step of administering ecrizumab to a patient at risk of complications after hematopoietic stem cell transplantation.
The treatment method according to claim 11, wherein the step (I) and the step (II) are performed on a patient within 20 days after hematopoietic stem cell transplantation.
Use of anti-Ba antibody for the manufacture of predictive diagnostic agents for the risk of complications after hematopoietic stem cell transplantation.
Use of anti-Ba antibody for the manufacture of companion diagnostics for selecting treatments to reduce the risk of developing complications.