TR201815921T4 - KIT FOR MONITORING, DETECTING AND STAGING GVHD - Google Patents

Abstract

The present invention is in the field of diagnostic and regulatory kits for the treatment of disorders associated with pathological cell proliferation. More specifically, the invention is adapted to measure and regulate activation and inactivation levels of glycogen synthase kinase 3 (GSK-3) as a marker of T-cell proliferation, particularly in cases such as Graft versus Host Disease (GVHD).

Description

DESCRIPTION KIT TO MONITOR, DETECT AND STAGE GVHD Background of the Invention 1. Field of Invention The present invention is diagnostic for the treatment of disorders associated with pathological cell proliferation. and regulatory kits. More specifically, the invention, specifically Graft versus Host Glycogen as a marker of T-cell proliferation in conditions such as GVHD Measuring and regulating the levels of activation and inactivation of synthase kinase 3 (GSK-3) adapted to. 2. Background of the Technique Glycogen synthase kinase 3 (GSK-3), multiple signaling initiated by various stimuli known to act as a downstream regulator that determines the output of the serine/threonine kinase. Through these pathways, GSK-3 is involved in cellular proliferation and apoptosis. plays a central role. The ability to detect the activation state of the GSK-3 and when necessary regulate it, treat disorders related to cell proliferation and apoptosis, and It allows for a better understanding and ability to diagnose.

Relating to tissue rejection, inflammation, infection, and immune response, including GVHD In many cases, an artista is present in cell proliferation, particularly in T-cell proliferation. there is a marked difference.

The incidence of GVHD has increased significantly in recent years. Allogeneic occurrence every year The number of hematopoietic stem cell transplants (HSCT) continues to increase each year.

GVHD can also sometimes occur as a complication of blood transfusion. Clinic As a result, the patient has one or more of the following observable symptoms: GVHD is suspected when it develops: dermatitis (skin rash), cutaneous blisters, diarrhea or crampy abdominal pain without diarrhea, persistent nausea and vomiting, hepatitis (bilirubin and/or liver enzymes) (Jacobsohn DA and Vogelsang GB 2007). clinical with current diagnostic techniques Observations are time consuming and obtained from the relevant organ to confirm and diagnose GVHD. Requires interventional tissue sampling including tissue biopsies taken. Today Although biopsies are the only way to accurately diagnose GVDH, the diagnosis is only T cells are already attacking host tissues and the GVHD process has reached phase 3 of the disorder. then detected. Biopsy procedures to obtain tissue are also invasive and go undiagnosed. causes delays of 24 to 48 hours beforehand. In addition, a definitive diagnosis with biopsies is always is not possible and other post-transplant conditions may exist in a similar way. Considering the clinical symptoms, the diagnosis is not reliable. specific for GVHD biomarkers have been described in the prior art. For example, in EP 2 163 896, GVHD of CCL8 shows that it is a marker for early diagnosis.

Therefore, it inhibits cell proliferation in the early stages of GVHD and the disease process. Rapid, reliable, relatively non-invasive for the detection of other pathological conditions including There has long been a need to provide a method.

Summary of the Invention The present invention relates to diagnostic and regulatory kits and more specifically to Graft versus Host Disease. It relates to a diagnostic and regulatory kit for

The present invention, according to appended independent claim 1 and dependent dependent claims 2-8, including a human It relates to a method for detecting GVHD in a mammal, such as

The present invention also provides GVHD according to the appended independent claim 9 and the appended dependent claim 10 of a kit. It is related to its use in diagnosis.

Brief Description of Figures The invention is described herein by way of example only, with reference to the accompanying drawings.

The details shown are for Exemplary purposes only and are used only as preferred expressions of the present invention. for an explanatory description of its regulations. The description, which is considered together with the figures, is in the art. it is clear to those with experience how various forms of invention can be embodied in practice. should reveal.

In the figures: Figure 1, GSK30L and GSK3ß subunits and their known activation and inactivation a pictorial representation of its regions; Figure 2 is a flowchart connecting GSK3ß to GVHD; Figure 3 is a pictorial representation of the disease process in GVHD; Figure 4A, ser 9-phosphorylated GSK-3ß (p-GSK-3ß) protein in spleen Samples a graphical representation of expression levels; Figure 43 is a graphical representation of total GSK-36 protein expression levels in spleen samples. is a representation; Figure 5, phosphorylated PKC (p-PKC) protein expression in spleen samples is a graphical representation of the levels; Figure 6A, ser 9-phosphorylated GSK-3ß (p-GSK-3ß) protein in blood samples a graphical representation of expression levels; and, Figure GB is a graphical representation of total GSK-3ß protein expression levels in blood samples. is a representation.

Detailed Description of the Invention The present invention is associated with pathological cell proliferation and pathological cell apoptosis. current method for monitoring patients suffering from conditions, particularly GVHD, and It solves many problems of systems.

Therefore, a preferred embodiment of the present invention is glycogen synthase kinase 3ß (GSK-3ß) is a description of a diagnostic kit that can detect expression level. In the current arrangement The level of GSK-SB expression is indicative of GVHD in the patient.

GSK-3ß outputs multiple signaling pathways initiated by different stimuli. It is known to act as a downstream regulating switch (Frame et al. Activation status of GSK-3ß, expression levels of GSK-3ß or on GSK-3ß The area responsible for activation or inactivation is used to detect and monitor GVHD.

Two subunits of the Figures, GSK-3, GSK-3d (10) and GSK-3ß (15) and their phosphorylation Reference is made to Figure 1 showing their locations. Serine 9 (S9) (16) and tyrosine Specific to the GSK-3ß subunit, which shows known phosphorylation sites, including has been referenced. In this embodiment, the normal physiological state of GSK-3ß is in an active state that is either unphosphorylated or dephosphorylated. Cool 9 in this arrangement phosphorylation can be identified and quantified according to the methods described in the specification. It inactivates GSK-3ß, which can be synthesized. Serine 9 phosphorylation is not limited to these, but cyclic adenosine monophosphate (cAMP), Phosphoinositide 3-kinase (PI3K), phosphatidylinositol 4,5-biphosphate (PIPZ), protein kinase A (PKA), and various other proteins including Akt (serine/threonine protein kinase family). associated with proteins.

In another embodiment of the present invention, GSK-SB is via the Wnt signaling pathway. inactivation may occur. Wnt activation is independent of serine 9 phosphorylation. inactivates GSK-3ß through its inactivation domain, which is unphosphorylated ß-catenin It is defined and quantified by measuring the changes in the levels. another one Activation of GSK-3ß in regulation, dephosphorylation or tyrosine 216 (Y216) occurs through phosphorylation. GSK-3ß phosphorylation methods described in definition It can be identified and quantified by changes in the molecular weight of GSK-3ß using can be done. In one aspect of this embodiment, the diagnostic kit of the present invention, GSK-SB expression It uses antibodies to identify and quantify levels.

A preferred embodiment of the present invention is the detection of GSK-Sß, GVHD, and a diagnostic and/or therapeutic kit included in a diagnostic and/or therapeutic kit for monitoring It is a therapeutic molecule.

A preferred embodiment of the diagnostic kit of the present invention is that GSK3ß activity is at ser 9 Detection and quantification of total GSK3ß and GSK3ß protein levels that are phosphorylated characterized by determination. Also in this embodiment, the diagnostic kit, GSK3ß inactivation of activated protein kinase C (PKC) via protein level and GSK3ß It can detect its inactivation via activated Wnt and quantify it.

Therefore, a preferred embodiment of the present invention is to improve the accuracy of GVHD detection. GSK-SB-associated markers, for example, affected by the activated Wnt pathway or is the identification of proteins induced by activated PKC. The domain on GSK-3ß is activated. It is affected by the Wnt pathway and the domain on GSK-SB is stimulated by the activated PKC pathway.

As shown in Figure 2, abnormal cell proliferation and apoptosis, Graft versus host T- disturbances related to other immune responses involving proliferation of cells is a basic feature. In this embodiment and other preferred embodiments, the diagnostic molecule and the kit is used in GVHD and its applications are available. Another of the present invention regulation of expression levels and activation Inactivation status of GSK-3ß GSK-3ß activation status by identification and quantification of regulation and associated pathways to regulate and monitor GVHD has been adapted. In this embodiment, the expression level of GSK-3ß is determined by the diagnostic kit of the present invention. determined and quantified.

By identifying and quantifying GSK-3ß expression and activation state A diagnostic kit that can identify GVHD in at least phase 2 of the disease is a part of the present invention. is another arrangement. In this embodiment, the diagnostic kit GSK-3ß activation states and their associated measures a number of parameters associated with pathways and to determine the extent of GVHD progression and with deviations from standard measurements taken from healthy subjects to make the appropriate diagnosis of GVHD. compares.

The diagnostic kit of the present invention can be used by measuring the expression levels of GSK-3ß and related proteins or early detection of GVHD by examining changes in GSK-313 activity by detecting It is adapted to detect the beginning. Also, in the current arrangement, the diagnostic kit and methods of the present invention, the above-mentioned proliferation and apoptosis of GSK-3ß by determining how it regulates T cell proliferation and apoptosis in relation to It will determine the cause of the GVHD. In this aspect of the preferred embodiment, the diagnostic kit The information provided by the GSK-3B indicates the activation and/or inactivation status of the GSK-3B. It is used to determine the most effective treatment for GVHD through regulation of

Another preferred embodiment of the present invention is that the diagnostic kit, GVHD, is accurate and precise. in a specific and unique way for GVHD to enable it to be properly diagnosed. characteristic is its adaptation to detect protein profiles. In this arrangement, The unique protein profile prevents false positive GVHD diagnosis.

Thus, a preferred embodiment of the present invention is GSK-Sß, GVHD and tissue rejection. in a therapeutic or pharmaceutical kit in applications that include, but are not limited to It acts as a regulatory and therapeutic molecule.

Reference is made to Figure 3, which shows the 3 phases of progression of GVHD disease. In phase 1 (400) receiver conditioning regime ( activated, host tissue (, Interleukin-l (IL-1), Interleukin-6 (IL-6) and Lipopolysaccharide (LPS) immune complexes. causes the release of cytokines (404). Increased levels of these cytokines It leads to the activation of APCs. II of GVHD (410). donor T cell activation occurs, host APCs (411) activate donor T-cells (412); this is regulatory T cells (Treg) (413). II of GVHD. increased survival following the phase (ie, apoptosis reduction), proliferation, migration (416) and differentiation of activated T-cells, more, including cytotoxic T Lymphocytes, Natural Killer (NK) cells, TNFoi and IL-1 produces additional effectors that mediate tissue damage. Leakage from damaged intestinal mucosa (415) LPS triggers additional TNFα production. TNFα causes necrosis and apoptosis in the skin and gastrointestinal tract. It can damage tissue directly by inducing TNF receptors or via the Fas pathway. TNFoi, gut plays a direct role in GVHD damage, which in a 'cytokine storm' causes skin, further increases the damage to the liver and lungs. In addition, type 1 T helper cells (Th1) (414) by releasing cytokines and aiding the presentation of antigens to T-cells II to facilitate the immune response. is released in phase. III of GVHD (420). phase T- targets host cells for destruction with a range of cellular and inflammatory effectors.

Target cells ( and CTL expressing T-cells. To macrophages (424), LPS and LPS in phase 3 of the GVHD disease course mediated by interferon-y (IFN-y). Once activated, macrophages tissue damage by affecting tissues and organs that are constantly proliferating, such as skin and skin initiates a cascade of inflammatory responses, including the release of mediators.

One embodiment of the present invention is GSK-3ß (GSK-3ß activation levels and GSK-BB inactivation levels) can detect and quantify its expression and activation status. The diagnostic kit that can determine the disease, II. T cell proliferation and migration during phase is that it can diagnose GVHD at the stage it occurs. New diagnostic kit, fast and relatively non-invasively, comparatively treat GVHD before extensive tissue damage occurs. can be diagnosed at an early stage.

In another embodiment of the present invention, the diagnostic kit GSK-SB expression for GVHD can determine the status of the measured GSK-3ß by examining its levels. Diagnostic of the present invention kit, prevent misdiagnosis with other conditions involving T-cell proliferation and activation for the further detection of unique RNA or protein profiles for GVHD. adapted to accurate and differential diagnosis.

In another embodiment of the invention, different proteins associated with GVHD in a given sample bind to a glass slide or equivalent substrate to determine expression levels. A protein chip using a set of antibodies, biomarkers or antigens is used.

In another embodiment of the invention, GVHD is preceded by clinical symptoms. A diagnostic kit based on a protein chip is used to diagnose it. In some arrangements The kit includes the activation status and/or expression level of GSK-3ß, related pathways, apoptosis. markers, proliferation markers, and other biological and biochemical molecules It contains a custom-made protein chip containing antibodies adapted to monitor This kind molecule candidates can be detected beforehand and can be detected by western blot, FACS, and other fingerprinting techniques. can be analysed.

In other preferred embodiments of the invention, the installation and efficacy of the GVHD diagnostic kit It is tested with the following steps: 0 GVHD mouse model - to determine the sequence of antibodies loaded on the protein chip Samples from mice are used. The aforementioned chip, then the mouse tested on samples. i Clinical trials - to determine the efficacy of the protein chip (diagnostic kit prototype) and to It is used to predict the development of GVHD in transplant patients.

To understand the invention and to see how it can be applied in practice, refer to the following examples. numerous preferred embodiments, by reference only by non-limiting illustration will be described.

Now, the role played by GSK-BB and other proteins from related pathways and their to identify their potential use for the diagnosis of GVHD at an early disease stage. experiments in a mouse model are mentioned.

Levels of GSK-SB and proteins from related pathways may be associated with an early disease To demonstrate that they can be used as markers for the diagnosis of GVHD during the period.

GVHD experimental mouse model: Samples were collected from blood and spleen at 0, 1, 3, 4, 5 and 6 days from the following 3 groups (group at least 3 mice per): 1. Untreated: Injection with plain medium (no irradiation). Expected result: healthy mice. 2. Genetically identical (sygeneic): donor matched to Balb/c mice after irradiation Injection is done with a splenocyte transplant from Balb/c mice. Expected result: short-term effects of irradiation and full recovery, no GVHD. 3. Allogeneic: To BaIb/c mice, mismatched donor C57Bl/6 after irradiation injected with a splenocyte transplant from mice. Expected result: warming up short-term effects and subsequent development of GVHD.

Summary of results: i p-GSK-3ß (ser-9 phosphorylated GSK-3ß) and total GSK-3ß levels in GVHD mice than both genetically identical and untreated control mice. is low. 0 The above-mentioned effect occurs 3-6 days after transplantation. seen per day.

GVHD mouse model: Spleen results Now, untreated (control), genetically identical and derived from allogeneic mice in spleen samples (phosphorylated at the ser 9 domain) p-GSK-3ß (Figure 4A) and total Figure 4, which shows a graphical representation of the protein expression levels of GSK-Bß (Figure 48). reference is made.

The results were normalized such that the values of the control mice were set to 100%.

Each parameter was compared with the values of untreated mice.

As shown in Figure 4, an extended stretch of total GSK-3ß and p-GSK-3ß over time decrease was determined exclusively in the spleen of allogeneic group mice.

In the genetically identical group, collect cells by day 5 of the splenocyte population. It should be noted that he has not recovered sufficiently for

Earlier in the allogeneic group as a result of the immune response characteristic of GVHD An increase in splenocytes was detected on days. . When symptoms appeared in mice on days 6 and 6, the total and The levels of phosphorylated GSK-3ß were maintained at a lower level than the other groups. However, in genetically identical mice, levels were nearly normal. has been seen.

Therefore, the results obtained suggest that in the allogeneic group, the clinical significance of p-GSK-3ß and total GSK-3ß decrease before symptoms appear (symptoms begin on day 4) and throughout the illness remained at a lower level than untreated and genetically identical groups. shows.

Now, spleen derived from untreated (control), genetically identical and allogeneic mice A graphical representation of protein expression levels of p-PKC (active form) over time in samples Reference is made to Figure 5, which shows its representation.

Here, PKC is located upstream of GSK-3ß in many pathways and also GSK- It has been confirmed that it can phosphorylate 3ß directly.

As shown in Figure 5, levels of activated PKC were both genetically modified relative to control. was significantly lower in splenocytes of both identical and allogeneic mice.

The results obtained were set to set the values of control mice to 100%. normalization should be emphasized. Each parameter is independent of other parameters. compared with the values of untreated mice.

Implications of the spleen results: . Total GSK-3ß in the spleen of allogeneic mice compared to genetically identical mice p-GSK-3ß differences can be detected as early as day 3, so these proteins to identify the early onset of GVHD in mice and potentially humans, and can be used as markers for diagnosis.

- Based on the above results, a diagnostic kit using protein chip technology, GSK-38 to predict and detect early onset of GVHD in humans changes in expression levels and activation of GSK-3ß in the spleen. It can be used to monitor changes in status.

GVHD mouse model: blood results Now, blood derived from Untreated (control), genetically identical and allogeneic mice samples (phosphorylated at the ser 9 domain) p-GSK-3ß (Figure 6A) and total GSK- Figure 6, which shows a graphical representation of the protein expression levels of 3ß (Figure GB). Ima ktadir referred to.

As illustrated in Figure 6, extended periods of total GSK-3ß and p-GSK-3ß in blood over time A decrease was observed only in the allogeneic group.

In the genetically identical group, a reduction in total GSK-3ß and p-GSK-3ß was observed. however, it occurs at a later stage than in allogeneic mice.

In the allogeneic group, a decrease in total GSK-SB and p-GSK-3ß levels occurred over time. This is greater than the reduction observed in the genetically identical group.

The results were normalized such that the values of the control mice were set to 100%. should be specified. Each parameter was compared with the values of untreated mice.

Implications of blood results: - Reduction of total GSK-BB and p-GSK-3ß in blood seen in allogeneic mice and larger than genetically identical mice.

. The reduction is most significant on the 3rd and 4th days and these are the early signs of GVHD. phase.

- Based on the above results, a diagnostic kit using protein chip technology, predicting early onset of GVHD in mammals, including humans and changes in GSK-SB expression levels and blood It can be used to monitor changes in the activation state of GSK-3ß.

. Blood samples showing GVHD were obtained from mice and included in humans. demonstrates the ability to obtain usable blood samples from other mammals. i Without being bound by theory, here the blood detected in mice Confirmation that levels of GVHD-related proliferation occur in the spleen has been made. Thus, in genetically identical mice on days 3, 4, and perhaps 5 The observed cells are cells that were present in the environment prior to transplantation. can be assumed. This is due to the complete absence of cells observed in allogeneic mice at day 4. on the contrary, these are new cells from the spleen.

The above results were initially associated with the progression and onset of GSK-3ß in GVHD. indicates that it is directly related.

Fingerprinting of GVHD disease Another key feature of the present invention is the unique fingerprint of GVHD disease. is research. In this way, a fingerprint is one that is uniquely correlated with GVHD. Allows the establishment of a diagnostic kit as described among others containing the protein set.

For fingerprint detection of GVHD, mouse model examples use the following data. analyzed by mass spectrometry to identify: . Proteins present in the analyzed system . Changes in protein expression . Changes in protein phosphorylation Based on the analysis of mouse samples, a custom-made protein chip is prepared. Kit to GSK-3ß candidate molecules identified to be involved in GVHD, such as antibodies against or related pathways, apoptosis markers, proliferation markers and (west blot analysis, FACS and fingerprinting) contains proteins included in the others (obtained using techniques such as

The kit mentioned above is used for the prognosis and early-stage diagnosis of GVHD.

Candidate antibodies used for the GVHD protein chip are antibodies that recognize the following biomarkers. 0 Total GSK-SB i GSK-3ß ser 9 phosphorylation - indicates GSK-3ß inactivation o PKC Activation - indicator of GSK-3ß inactivation o b-catenin - inactivation of GSK-3B via activated Wnt Pathway i A protein upstream of GSK-Sß in the Dvl - Wnt pathway and activation of the Wnt pathway an indicator . Akt - PI-3K signaling sequence protein that phosphorylates GSK-Sß at ser9.

- Erk - Map kinase signaling sequence protein that phosphorylates GSK-Sß at ser9 . Map kinase signaling sequence that directly phosphorylates GSK-3ß in p38 MAPK ser389 protein o GSK-3ß Tyrosine 216 phosphorylation - indicates GSK-3ß activation . Serum Albumin - potentially associated with the onset of GVHD shown. 0 Apoptosis markers (Morocco, Bcl family, Cytochrome C, caspases) o Immune activity markers (Nf-kB, CD25) - Proliferation markers (Cyclin D1, PCNA, p27) In other embodiments of the invention, the protein chip results also show the chip's efficacy, sensitivity and It was validated by comparison with western blot data to determine its accuracy.

The above-mentioned information indicates GSK-3ß in GVHD and other pathways involved in this disease. used to evaluate the regulating pathways.

One main aspect of the invention is the expression levels of GSK-3ß and the state of activation. where the changes are used as new markers for the diagnosis of GVHD.

Changes in GSK-3ß expression levels and activation of GSK-SB protein before observable clinical symptoms, by monitoring changes in Early onset of GVHD can be detected.

In another preferred embodiment of the invention, the use of GSK-3ß as a marker before the stage at which observable symptoms are shown by their patients and allows the diagnosis of GVHD before the phase in which T-cells attack in the body.

In other embodiments of the invention, Western blot analysis of mouse splenocyte strains for GVHD carried out using at least one of the following proteins as potential markers: i b-catenin - a for inactivation of GSK-3B via the activated Wnt Pathway . A protein upstream of GSK-3ß in the Dvl-Wnt pathway and activation of the Wnt pathway. an indicator . Akt - PI-3K signaling sequence protein that phosphorylates GSK-3ß at ser9.

. Erk - Map kinase signaling sequence protein that phosphorylates GSK-3ß at ser9 o Map kinase signaling sequence that directly phosphorylates GSK-3ß in P38 MAPK ser389 protein . GSK-3ß tyr216 phosphorylation - indicates GSK-3ß activation In other embodiments of the invention, Western blot analysis of mouse blood samples is used for GVHD. GSK-BB, p-GSK-3ß, PKC and most of the antibodies listed above as potential markers performed using at least one: In another embodiment of the invention, the livers from GVHD model mice are characterized by GSK-SB and analysis of proteins of interest using western blot and/or mass spectrometry techniques or protein used to perform the analysis using any other technique.

At the same time, we found that the regulation of GSK-BB expression is a model of an acute GVHD mouse model. Testing in time study is also within the scope of the present invention. That's why GSK- The onset of GVHD can be prevented with 3ß manipulation.

In another embodiment of the invention, substrates of GSK-3ß and associated with said substrates products are identified.

In another embodiment of the invention, the above-mentioned substrates and for GVHD specific products have been found. These substrates and products are what the present invention provides. used to increase the sensitivity of diagnostic technology.

In another embodiment of the invention, GSK-Sß associated with a specific related disease such as GVHD The specific pathway that stimulates the specific change in the activation state is identified. GSK- Such candidates that stimulate the activation state of 3ß may include: i Dsh - Wnt pathway (upstream of GSK-3ß) GSK- via activated Wnt pathway It is associated with the inactivation of 3ß.

- Akt - PI-3K/Akt pathway is associated with Ser 9 inactivation of the Wnt pathway.

. CAMP - Associated with Ser 9 inactivation of GSK-3ß The above information indicates that activation of GSK-3ß in the relevant specific condition such as GVHD. shows the ways that regulate its state. These results correlated with the activation state of GSK-3ß. used to fingerprint a specific disease. Fingerprint removal this way data are used to increase the sensitivity of the diagnostic kit for the relevant disease.

Clinical study using GVHD diagnostic kit The following clinical trial is a CRA with Helsinki approval and consultations with medical centers. It is carried out in association with (clinical research association): . Recruited patients for clinical trial (approval in Helsinki and negotiations with medical centers) included).

- Blood samples were taken from patients (year-round) who underwent bone marrow transplants.

. Protein chip to test its efficacy in predicting acute onset of GVHD The protein chip is used in the analysis of human samples. This is a retrospective study. Scarce analysis results are diagnostic for each patient (in accordance with current medical standards). compared to results.

If necessary, quantify human samples using kits western blot analysis. is calibrated.

Claims (1)

CLAIMS A method for detecting Graft versus Host Disease, GVHD, in a mammal, including a human, characterized in that; it includes the following steps: measuring the level of GSK-3ß in a sample obtained from said mammal, where a significant deviation from normal values is indicative of the presence of GVHD. It is a method according to claim 1, its feature is; It also includes a step of measuring the level of GSK-3ß by measuring at least one parameter selected from the group consisting of GSK-3ß expression levels, GSK-38 activation levels, GSK-SB inactivation levels, serine 9 phosphorylated GSK-3ß expression, and p-PKC expression. It is a method according to claim 1 and its feature is; further comprising a step of measuring GSK-SB expression and/or activation level by measuring at least one activation marker selected from the group consisting of overexpression of GSK-3ß, tyrosine 216 phosphorylation, and any combination thereof. It is a method according to claim 1, its feature is; further comprising the step of determining the expression and/or inactivation status of the GSK-SB level by measuring at least one inactivation indicator selected from the group consisting of: (i) serine 9 phosphorylation; (ii) Wnt activation detected by at least one of the following: a protein marker specific for inactivation of GSK-3ß via ß-catenin levels or the activated Wnt pathway, or where the activated Wnt pathway affects by stimulating inactivation of GSK-3ß area on GSK-SB; (iii) PKC expression levels (iv) a protein marker specific for inactivation of GSK-3ß by activated PKC, and any combination thereof. It is a method according to claim 1, its feature is; it also includes an additional step of taking a sample from the spleen, blood, another body fluid or tissue. It is a method according to claim 1, its feature is; one of the following multiples is correct: (a) the method also includes an additional step to compare the result of said GSK-3ß levels with a predetermined normal value from healthy subjects, and (b) an approximate 25% of the normal inactivation value. deviation indicates the presence of GVHD. It is a method according to claim 1, its feature is; It includes the following additional steps: a. From measuring ß-catenin levels in the absence of serine 9 phosphorylation of the GSK-38 inactivation level, measuring the area stimulated above GSK-3ß when stimulated via the activated Wnt pathway, measuring a protein marker unique for inactivation of GSK-3ß via the activated Wnt pathway. and measuring a protein marker unique for inactivation of GSK-3ß via activated PKC; and b. Comparing the result of said inactivation levels with the predetermined normal inactivation value obtained from healthy subjects, where a deviation of at least approximately 25% of the normal inactivation values indicates the presence of GVHD. It is a method according to claim 1, its feature is; It includes the following steps: a. Measurement of GSK-3ß inactivation indicators, b. Measurement of GSK-313 activation indicators, c. combining the measures of activation and inactivation indicators, to obtain the rate of inactivation with an activation characteristic of GVHD. 9. It is the use of a kit in the diagnosis of Graft versus Host Disease, GVHD, and its feature is; said kit contains a protein chip containing a sequence of bound antibodies that recognize biomarkers characteristic of GVHD, to detect the expression levels of different GVHD-related proteins in a given sample of said biomarkers Total GSK-SB, serine 9 phosphorylated GSK-3ß, GSK -SB serine 9 phosphorylation PKC activation is selected from the group consisting of GSK-3ß tyrosine 216 phosphorylation. 10. It is used according to claim 9, its feature is; said biomarkers can be detected prior to the onset of GVHD symptoms in a mammal, including a human.