Method for early diagnosis, early prediction, monitoring or severity prediction of reduced graft function in kidney transplantation patients

Abstract

Subject of the present invention is a method for the early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of reduced graft function in a kidney transplantation patient, comprising the steps of: determining the level of pro-enkephalin or a fragment thereof in a bodily fluid sample obtained from said patient; and correlating the level of the pro-enkephalin or fragment thereof in the sample with the diagnosis and / or risk and / or severity of a reduced graft function, wherein the reduced graft function is a reduced graft function (SGF) or delayed graft function (DGF) of the kidney.

Description

Technical Field

[0001] The subject matter of this invention is a method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of decreased graft function in kidney transplant patients, the method comprising: ● Determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and ● Associate the levels of the proenkephalin or fragments thereof in the sample with the diagnosis and / or risk and / or severity of graft dysfunction. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

[0002] The subject matter of this invention is a method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft function decline in kidney transplant patients, the method comprising: ● Determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and ● The levels of the proenkephalin or fragments thereof in the samples were correlated with the risk and / or severity of graft function and / or reduced graft function. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

[0003] Another subject matter of the present invention is a method for patient stratification and / or patient selection for early treatment of graft dysfunction in kidney transplant patients, the method comprising: ● Determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and ● Patients can be stratified and / or selected for early treatment of graft-versus-graft dysfunction by correlating the levels of the proenkephalin or its fragments in the sample. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

[0004] Another subject matter of the present invention is a method according to the invention for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft function decline in kidney transplant patients, wherein the method is used for patient stratification and / or patient selection for early treatment of graft function decline in kidney transplant patients.

[0005] Another subject matter of the present invention is a method for the early diagnosis and / or early prediction of graft function and / or recovery from graft function decline in kidney transplant patients, the method comprising: ● Determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and ● The levels of the proenkephalin or fragments thereof in the samples were correlated with recovery from graft dysfunction. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

[0006] Another subject matter of the present invention is a method for early diagnosis and / or early prediction of immediate graft function (IGF) in kidney transplant patients, the method comprising: ● Determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and ● The levels of the proenkephalin or fragments thereof in the sample were correlated with graft function. The graft function mentioned above is immediate graft function (IGF) of the kidney. Background Technology

[0007] Delayed graft function (DGF) is a term used to describe the failure of a transplanted kidney to function immediately after transplantation. It can be considered a form of acute kidney injury after transplantation and is a significant complication of kidney transplantation.

[0008] The definition of DGF varies from transplant center to transplant center, although most transplant centers define it as acute kidney injury (AKI) that occurs in the first week after transplantation and requires dialysis treatment. Yarlagadda et al., 2008. Nephrol Dial Transplant 23: 2995–3003 DGF is a common complication, with an incidence rate between 25% and 30%. Mannon et al. 2018. Nephron Exp. Nephrol. 140: 94–98 DGF remains a major clinical challenge in kidney transplant recipient management. In fact, DGF is associated with higher rejection rates and poorer short- and long-term outcomes. Bahl et al., 2019. Curr. Opin. Organ Transplant. 24: 82–86 ).

[0009] The main risk factors involved in the development of DGF are ischemia-reperfusion injury (IRI), the source of the donated kidney (deceased donors vs. living donors), the quality of the donated kidney, and the recipient's clinical condition. Ponticelli et al., 2022. J. Pers. Med. 12, 1557 In addition to the well-known complications of acute kidney injury and dialysis, DGF also makes grafts susceptible to acute and chronic rejection. Boom et al., 2000. Kidney Int 58: 859–866 ), and increases the risk of chronic allogeneic graft-versus-nephropathy and premature graft failure. Giral-Classe et al., 1998. Kidney Int 54: 972–978 ).

[0010] Immediate postoperative functional impairment necessitates dialysis, which can last from a few days to several months, thus prolonging hospital stays and increasing healthcare costs. Buchanan et al., 2011. J Nephrol Therapeutic. S4: 001 Furthermore, DGF complicates post-transplant management for outpatients and increases morbidity.

[0011] Kidneys for transplantation can be retrieved from living donors (recipient relatives or unrelated donors) or from deceased donors with irreversible brain injury and continuous circulation maintained through supportive measures (brain-dead donors) or from donors who have undergone post-circulation death. The risk of DGF is higher with deceased donors. Yasseri et al., 2021. Urol. J. 88: 185–189 Although it also exists in living donors ( Narayanan et al., 2010. Am. J. Kidney Dis. 56: 961–970 Brain-dead donors aged 60 years or older, or 50-59 years old, with two of the following abnormalities (history of hypertension, final serum creatinine greater than 1.5 mg / dL (133 mmol / L) or cause of death being cerebrovascular) are defined as extended standard donors (ECD). Metzger et al., 2003. Am. J. Transplant. 3 (Suppl. S4): 114–125 ECDs are typically excluded from donation. When a kidney is received from an ECD, it usually develops DGF (degenerative factor aging). Kidney allografts from ECDs have a twice the risk of DGF, more frequent acute rejection, and lower long-term graft function. Port et al., 2002. Transplantation 74: 1281– 1286 ).

[0012] Post-transplant renal function has historically been categorized solely based on the presence or absence of DGF (diuretic factor). Therefore, many patients may have significant impairment, but are considered by default to have "sufficient" graft function if they avoid dialysis. Akkina et al., 2009. Am J Transplant 9: 1460–66 An intermediate phenotype known as slowed graft function (SGF) can be characterized by a slower initial postoperative decline in serum creatinine (Cr) compared to immediate graft function (IGF), but without the need for dialysis. Moore et al., 2010. Transplantation 90: 1113–1116 However, SGF may influence early treatment decisions, such as optimizing volume status, reducing exposure to calcineurin inhibitors, avoiding nephrotoxicity, and using calcium channel blockers. Peeters and Vanholder 2008. Transplantation 85: S31–37 SGF (Severe Fibroblast Growth Factor) is generally defined as not requiring renal replacement therapy and having a serum creatinine (SCr) quotient (i.e., the difference in SCr between day 0 and day 7 post-surgery divided by the SCr on day 7) < 0.7. However, other slightly different definitions may also be applied, such as the absolute level of SCr on a given post-operative day, such as SCr ≥ 3 mg / ld on day 5. Humar et al., 1997. Clin Transplant 11: 623–27 ) or Cr ≥2.5 on day 7 ( Zeraati et al., 2009. Transplant Proc 41: 2777–80 ), or insufficient percentage reduction of SCr within a given time period, such as a creatinine reduction rate (CRR) <30% between day 1 and day 2 ( Rodrigo et al., 2004. Am J Transplant 4: 1163–69 ).

[0013] Proenkephalin A is a precursor to the endogenous opioid enkephalin family. It is a pro-hormone that undergoes proteolytic processing to form several bioactive pentapeptides, such as methionine-enkephalin (Met-Enk) and leucine-enkephalin (Leu-Enk), as well as several other peptide fragments (enkelytin and C-terminal elongated Met-Enk peptides). In addition to mature enkephalins, other peptides are produced, one of which is the stable proenkephalin peptide 119-159 (PENK 119-159). The level of this peptide fragment in plasma / serum can be used as a surrogate indicator of systemic enkephalin synthesis, as proenkephalin A is the primary source of mature enkephalins. Ernst et al., 2006. Peptides 27: 1835-1840 Enkephalins are widely secreted and act on locally expressed opioid receptors, particularly delta opioid receptors. These opioid receptors are also widely expressed, with the highest density in the kidneys. Denning et al., 2008. Peptides 29 （1）: 83–921 After receptor binding, the biological effects of enkephalins include pain perception, anesthetic effects, and cardiovascular regulation. Holaday 1983. Annu. Rev. Pharmacol. Toxicol. 23: 541–594 These delta-opioid agonists stimulate urinary sodium excretion and polyuria ( ). Sezen et al., 1998. J. Pharmacol. Exp. Ther. 287(1): 238–245 Although several studies have demonstrated an association between elevated concentrations and adverse consequences, this association is generally proportional to changes in kidney function. In fact, in several populations, including sepsis, elevated concentrations are associated with decreased kidney function. Marino et al., 2015. J Nephrol 28:717–724 ), heart failure ( Ng et al., 2017. J. Am. Coll. Cardiol. 69(1): 56–69; Matsue et al., 2017. J. Card. Fail. 23(3): 231–239 ), heart surgery ( Shah et al., 2015. Clin. Nephrol. 83(1):29–35 ) and myocardial infarction ( Ng et al., 2014. J. Am. Coll. Cardiol. 63(3) (2014) 280–289 PENK 119-159 is strongly correlated with renal function and GFR measurement. Beunders et al., 2020 54(3): 308–314 Therefore, it has been proposed as a biomarker for assessing renal function in critically ill patients. Beunders et al. 2017. Appl Lab Med 2(3): 400-412; Donato et al., 2018. Clin Biochem 58: 72-77; Beunders et al., 2020 54(3): 308–314; Khorashadi et al., 2020. Nephron 144(12):655-661 ).

[0014] Plasma PENK has been shown to be associated with renal function, as reflected by its correlation with measured GFR in both kidney transplant recipients and donors one year post-transplantation. Furthermore, using PENK levels measured at least one year post-transplantation, PENK has been shown to be independently associated with an increased risk of late graft failure in kidney transplant recipients. Kienecker et al., 2017. Transplantation Direct 3: e190 ).

[0015] However, nothing is known about PENK levels measured in newly transplanted patients (e.g., hours or days after kidney transplantation). The surprising finding of this invention is that the levels of proenkephalins and their fragments (particularly proenkephalin 119-159 (MR-PENK, SEQ ID No. 6)) are suitable for the early prediction and monitoring of graft dysfunction in kidney transplant patients. Summary of the Invention

[0016] The subject matter of this invention is a method for early diagnosis and / or early prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients, the method comprising: ● Determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and ● Associate the levels of the proenkephalin or fragments thereof in the sample with the diagnosis and / or risk and / or severity of graft dysfunction. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

[0017] The subject matter of this invention is a method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft function decline in kidney transplant patients, the method comprising: ● Determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and ● The levels of the proenkephalin or fragments thereof in the samples were correlated with the risk and / or severity of graft function and / or reduced graft function. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

[0018] Throughout this specification, the term "early diagnosis" always refers to "early diagnosis of graft function".

[0019] Another subject matter of the present invention is a method for patient stratification and / or patient selection for early treatment of graft dysfunction in kidney transplant patients, the method comprising: ● Determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and ● Patients can be stratified and / or selected for early treatment of graft-versus-graft dysfunction by correlating the levels of the proenkephalin or its fragments in the sample. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

[0020] Another subject matter of the present invention is a method according to the invention for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft function decline in kidney transplant patients, wherein the method is used for patient stratification and / or patient selection for early treatment of graft function decline in kidney transplant patients.

[0021] Another subject matter of the present invention is a method for early diagnosis and / or early prediction of recovery from graft dysfunction in kidney transplant patients, the method comprising: ● Determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and ● The levels of the proenkephalin or fragments thereof in the samples were correlated with recovery from graft dysfunction. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

[0022] Another subject matter of the present invention is a method for early diagnosis and / or early prediction of immediate graft function (IGF) in kidney transplant patients, the method comprising: ● Determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and ● The levels of the proenkephalin or fragments thereof in the sample were correlated with graft function. The graft function mentioned above is immediate graft function (IGF) of the kidney.

[0023] Another subject matter of the present invention is a method for early treatment of graft dysfunction in kidney transplant patients, wherein the treatment is selected from renal replacement therapy and / or administration of drugs and / or adjustment of immunosuppressive therapy and / or adjustment of nephrotoxic drugs, wherein the patient is selected by a diagnostic method comprising the following steps: ● Determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and ● Associate the levels of the proenkephalin or fragments thereof in the sample with the diagnosis and / or risk and / or severity of graft dysfunction. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

[0024] Another subject matter of the present invention is a medicament for the early treatment of impaired graft function in kidney transplant patients, wherein the patients are selected by a diagnostic method comprising the following steps: ● Determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and ● The levels of the proenkephalin or fragments thereof in the samples were associated with the diagnosis of graft-deficient function and / or the risk of graft-deficient function. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

[0025] Surprisingly, proenkephalin (PENK) or fragments thereof have been shown to be a powerful and highly important biomarker for the early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft function decline in kidney transplant patients. Furthermore, it has been shown to be particularly useful for the early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of delayed graft function (DGF) in kidney transplant patients. Another surprising finding is that the levels of PENK or fragments thereof can be used to predict short-term kidney function in kidney transplant patients, particularly for kidney function decline (e.g., decreased GFR) up to 30 days post-transplantation.

[0026] The term "associated" as used in this article when referring to the use of diagnostic and prognostic biomarkers (such as proenkephalin or fragments thereof) means comparing the presence or level of a biomarker in a patient to its presence or amount in a person known to have a given condition (e.g., DGF or SGF or decreased renal function) or known to be at risk for a given condition. Biomarker levels in a patient sample can be compared to levels known to be associated with a specific diagnosis. The biomarker level in a sample is said to be associated with a diagnosis; that is, a skilled professional can use the biomarker level to determine whether the patient has a specific type of disease and respond accordingly. Furthermore, biomarker levels in a sample can be compared to biomarker levels known to be predictively associated with disease or disease outcomes (e.g., the development of a disease or condition (e.g., DGF or SGF), the severity of a disease or condition, or the improvement or worsening of a disease or condition (e.g., renal function)).

[0027] As used herein, the term "patient" refers to a living human or non-human organism. Preferably, the patient in this document is a human kidney transplant patient.

[0028] The term "kidney transplant patient" refers to a patient who is planning to undergo a kidney transplant, is currently undergoing a kidney transplant, or has already received a kidney transplant.

[0029] As used herein, the term "child" refers to a subject who is 18 years of age or younger, more preferably 14 years of age or younger, even more preferably 12 years of age or younger, even more preferably 8 years of age or younger, even more preferably 5 years of age or younger, even more preferably 2 years of age or younger, and most preferably 1 year of age or younger.

[0030] The term "elevated level" means a level above a certain (predetermined) threshold level. The term "elevated" level may mean a level above a value considered as a reference and / or threshold level.

[0031] In the context of this invention, the term "diagnosis" refers to the identification and (early) detection of a disease or clinical condition in a subject, and may also include differential diagnosis.

[0032] In the context of this invention, the term "early diagnosis" refers to the time point at which a disease or clinical condition is diagnosed in a subject before the usual time required for diagnosis by the gold standard method. In the context of this invention, "early diagnosis" means within 96 hours, preferably within 72 hours, more preferably within 48 hours, even more preferably within 24 hours, and most preferably within 12 hours after kidney transplantation.

[0033] In a preferred embodiment of the invention, the early diagnosis means within 12 to 48 hours after kidney transplantation.

[0034] In another preferred embodiment of the invention, the early diagnosis means within 12 to 24 hours after kidney transplantation.

[0035] In one embodiment of the invention, the early diagnosis means within 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or 12 hours.

[0036] In one implementation, "early diagnosis" refers to "early diagnosis" of the graft function of the kidney in kidney transplant patients.

[0037] In one particular implementation, "early diagnosis" refers to the early diagnosis of decreased graft function in a kidney transplant patient. In the context of this invention, the term "prediction" refers to a prediction of how a patient's medical condition will progress. This may include an estimate of the patient's chances of recovery or the risk of adverse outcomes (e.g., SGF or DGF, decreased kidney function).

[0038] In the context of this invention, the term "early prediction" refers to the point in time at which a patient's medical condition will progress, prior to the usual occurrence of a specific event (e.g., the onset of the initial symptoms of the disease). In the context of this invention, "early prediction" means within 96 hours after a kidney transplant, preferably within 72 hours, more preferably within 48 hours, even more preferably within 24 hours, and most preferably within 12 hours.

[0039] In a preferred embodiment of the invention, the early prediction means within 12 to 48 hours after kidney transplantation.

[0040] In another preferred embodiment of the invention, the early prediction means within 12 to 24 hours after kidney transplantation.

[0041] In one embodiment of the invention, the early prediction means within 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or 12 hours.

[0042] In one embodiment of the invention, in the context of diagnosis and / or prediction, the term "early" means the point in time when graft dysfunction is diagnosed and / or the risk and / or severity of graft dysfunction are predicted using proenkephalin or a fragment thereof, and the biomarker serum creatinine (SCr) in kidney transplant patients is not used to diagnose graft dysfunction and / or the risk and / or severity of graft dysfunction are not predicted.

[0043] In a particular embodiment of the invention, in the context of diagnosis and / or prediction, the term “early” means the time point at which graft-versus-graft function is diagnosed and / or the risk and / or severity of graft-versus-graft function is predicted using proenkephalin or a fragment thereof, (i) serum creatinine (SCr) levels are still above a predetermined threshold level and / or (ii) the relative change in SCr is a decrease of less than 50%.

[0044] In another specific embodiment of the invention, the predetermined threshold level of SCr is in the range of 1.5 to 4 mg / dL, more preferably in the range of 1.5 to 3 mg / dL, even more preferably in the range of 1.5 to 2.5 mg / dL, and most preferably the predetermined threshold is 2 mg / dL.

[0045] In one implementation, in the context of diagnosis and / or prediction, the term “early” means within 96 hours, preferably within 72 hours, more preferably within 48 hours, even more preferably within 24 hours, most preferably within 12 hours, and wherein in the kidney transplant patient (i) the SCr level is above a predetermined threshold level and / or (ii) the relative change in SCr is a reduction of less than 50%.

[0046] In one embodiment of the invention, in the context of diagnosis and / or prediction, the term "early" means that at a specific point in time, the level or relative change of the proenkephalin or fragment thereof diagnoses or predicts that the kidney transplant patient does not have or will not have graft function impairment, but the level or relative change of the SCr is still above a predetermined threshold or reduced by less than 50%.

[0047] If a physician uses only the level or relative change of SCr, and it remains above a predetermined threshold or decreases by less than 50%, the kidney transplant patient may be incorrectly diagnosed with reduced graft function if the level or relative change of proenkephalin or its fragments is not considered in the first few days after kidney transplantation.

[0048] In one embodiment of the invention, the prediction of renal function in kidney transplant patients means a prediction within 12 months, more preferably within 9 months, more preferably within 6 months, more preferably within 3 months, more preferably within 1 month, and most preferably within 14 days.

[0049] In a more specific embodiment of the invention, the prediction of renal function in kidney transplant patients means a short-term prediction within 1 month, more preferably within 28 days, even more preferably within 21 days, and most preferably within 14 days.

[0050] The term “monitoring” refers to controlling the development of a patient’s disease and / or pathophysiological condition (e.g., the risk or severity of the disease or symptom or the response to therapy) (detecting any changes in it).

[0051] The patient monitoring or follow-up will continue for 28 days after kidney transplantation, or until the transplanted kidney recovers its graft function.

[0052] The follow-up measurements can be performed for up to 7 days, preferably up to 14 days, more preferably up to 21 days, and most preferably up to 28 days. In one embodiment, the follow-up measurements can be performed until kidney function is preserved.

[0053] In the context of this invention, the term "monitoring the success of a therapy or intervention" refers to controlling and / or adjusting the therapeutic treatment of the patient.

[0054] Predicting or monitoring the success of a therapy or intervention can, for example, be done in patients diagnosed after kidney transplantation and / or at risk of reduced graft function by measuring proenkephalin (PENK) or fragments thereof to predict or monitor the success of renal replacement therapy and / or administration of medications and / or adjustment of immunosuppressive drugs and / or adjustment of nephrotoxic drugs.

[0055] Predicting or monitoring the success of a therapy or intervention can, for example, be done in patients at risk of reduced graft function after kidney transplantation by using measurements of PENK or fragments thereof before and after renal replacement therapy and / or administration of medications and / or adjustment of immunosuppressive drugs and / or adjustment of nephrotoxic drugs to predict or monitor the recovery of renal function.

[0056] "Pre-transplant" is defined as any time up to 7 days before the start of the transplant procedure.

[0057] In one specific embodiment of the invention, the sample obtained before transplantation is obtained within 7 days, preferably within 6 days, more preferably within 5 days, even more preferably within 4 days, even more preferably within 3 days, even more preferably within 48 hours, more preferably within 24 hours, preferably within 12 hours, and most preferably within 6 hours before the start of the transplantation procedure.

[0058] The standard hospital procedure is to obtain the sample within 24 hours before the transplant procedure begins.

[0059] One embodiment of the present invention is a method for predicting or monitoring the success of a therapy or intervention in patients identified as having reduced graft function and / or at risk of reduced graft function after kidney transplantation, wherein the prediction or monitoring is conducted before and / or after the therapy or intervention to assess the deterioration or recovery of kidney function.

[0060] The term "delayed graft function" (DGF) is defined as the need for at least one dialysis session within the first 7 days after a kidney transplant.

[0061] The term “slowed graft function” (SGF) is defined as no RRT within 7 days post-transplantation and a creatinine quotient on day 7 / day 0 < 0.7. Immediate graft function (IGF) is defined as no RRT within 7 days post-transplantation and a creatinine quotient on day 7 / day 0 > 0.7.

[0062] Reduced graft function is defined as graft function slowdown (SGF) or graft function delay (DGF).

[0063] A key indicator of kidney function is the glomerular filtration rate (GFR). GFR equals the total filtration rate of functional nephrons in the kidney and is considered the most useful indicator of kidney function in both health and disease. GFR is usually recorded as volume per unit time (e.g., milliliters per minute (mL / min)).

[0064] Glomerular filtration cannot be directly measured in the human body; therefore, the “true” GFR cannot be known definitively. However, GFR can be assessed from clearance measurements (measuring GFR [mGFR]) or serum levels of endogenous filtration markers (estimating GFR [eGFR]).

[0065] GFR can be measured by injecting inulin or an inulin analogue, scallion sugar, into the bloodstream (“measuring GFR”). Measuring renal function using inulin is currently the “gold standard” for comparison with other methods of estimating glomerular filtration rate. Contrast agents iohexol and iodophthalate have become more popular alternatives for measuring GFR and are considered to have shown sufficient accuracy in determining GFR. Soveri et al., 2014. Am J Kidney Dis. 64(3):411-24 ).

[0066] Creatinine clearance (CCr or CrCl) is the plasma volume of creatinine removed per unit time and is a useful indicator for approximating GFR. Creatinine clearance exceeding GFR is due to creatinine secretion, which can be blocked by cimetidine. Both GFR and CCr can be accurately calculated through comparative measurements of substances in blood and urine, or estimated using formulas based solely on blood test results (eGFR and eCCr). The results of these tests are used to assess renal excretory function. Currently, clinical practice guidelines and regulatory agencies recommend using estimated GFR (eGFR) for routine GFR assessment, while when a more accurate assessment is required, measuring GFR (mGFR) is recommended as a confirmatory test. Levey et al., 2020. Nat Rev Nephrol. 16(1): 51–64 ).

[0067] Several methods for determining eGFR are known in the art. For example, GFR can be estimated using the CKD-EPI creatinine equation, the CKD-EPI cystatin C equation or the CKD-EPI creatinine-cystatin C equation, the diet-modified renal disease (MDRD) study equation, and the Cockcroft-Gault equation. Santos and Martins 2015. World J Nephrol 4(3): 345- 353 ).

[0068] The severity of chronic kidney disease (CKD) is described by six stages; the three most severe stages are defined by MDRD-eGFR values, while the first three stages also depend on the presence of evidence of other kidney disease (such as proteinuria). 0) Normal renal function – GFR greater than 90 (mL / min) / (1.73 m 2 No proteinuria 1) CKD1—GFR higher than 90 (mL / min) / (1.73 m 2 There is evidence of kidney damage. 2) CKD2 (mild) – GFR 60 to 89 (mL / min) / (1.73 m 2 There is evidence of kidney damage. 3) CKD3 (moderate) – GFR 30 to 59 (mL / min) / (1.73 m 2 ) 4) CKD4 (severe) – GFR 15 to 29 (mL / min) / (1.73 m 2 ) 5) CKD5 renal failure – GFR less than 15 (mL / min) / (1.73 m 2 ).

[0069] Therefore, in the context of this invention, renal function can be determined by glomerular filtration rate (GFR), creatinine clearance (CCr), serum creatinine (SCr), serum cystatin C (CyC), urinalysis, blood urea nitrogen, or urine output. GFR can be selected from estimated GFR (eGFR), true GFR, or measured GFR (mGFR).

[0070] In one embodiment of the invention, a decline in renal function is predicted in the patient. In a particular embodiment of the invention, the decline in renal function is determined by GFR. In a very particular embodiment of the invention, a GFR decline to below 60 is predicted, preferably below 45, preferably below 30, and most preferably below 15.

[0071] In one embodiment of the invention, proenkephalin or a fragment thereof can diagnose and / or predict whether a patient has or will experience graft function impairment or graft function delay. In other words, the level of proenkephalin or a fragment thereof can distinguish between a patient with or likely to develop SGF and a patient with or likely to develop DGF. In another embodiment of the invention, the level of proenkephalin or a fragment thereof can distinguish between a patient with or likely to develop SGF and a patient with or likely to develop DGF, wherein (i) if the level of proenkephalin or a fragment thereof in a bodily fluid sample obtained from the patient is elevated above a predetermined threshold level, the patient has DGF or is at risk of having DGF, and (ii) if the level of proenkephalin or a fragment thereof in a bodily fluid sample obtained from the patient is below a predetermined threshold level, the patient has SGF or is at risk of having SGF.

[0072] In one particular embodiment of the invention, the predetermined threshold level for distinguishing between patients with or about to develop SGF and patients with or about to develop DGF is in the range of 100 to 500 pmol / L, more preferably in the range of 150 to 400 pmol / L, and most preferably in the range of 200 to 350 pmol / L.

[0073] In a more specific embodiment of the invention, if graft function delay is diagnosed and / or predicted, the patient will receive renal replacement therapy, and if graft function slowdown is diagnosed or predicted, renal replacement therapy will not be received. The severity of graft function delay is defined as the number of days the patient requires renal replacement therapy. Graft function delay can be categorized into severity groups as defined below: 0 / 1 (RRT only in the first 24 hours of day 0 / day 1) indicates low severity DGF; 2-7 (RRT ends between day 2 and day 7) indicates moderate severity; and >7 (RRT continues beyond day 7) indicates high severity.

[0074] The body fluid may be selected from blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva. In one embodiment of the invention, the body fluid is selected from whole blood, plasma, and serum.

[0075] Enkephalin or fragments thereof can diagnose and / or predict the severity of graft-versus-graft function decline in kidney transplant patients, wherein samples from said patients (a) are obtained at least once before and after kidney transplantation, or (b) are obtained at least twice after kidney transplantation, and wherein (i) an increase in level above a predetermined threshold or (ii) a relative change in the level of enkephalin or fragments thereof between samples obtained before and after kidney transplantation or between samples obtained after kidney transplantation is a decrease of less than 50% or an increase (when the level of the earlier sample is set to 100%) can diagnose and / or predict the severity of graft-versus-graft function decline in said patients.

[0076] Enkephalin or fragments thereof can diagnose and / or predict graft function delay and / or the severity of graft function delay in kidney transplant patients, wherein fluid samples from said patients are obtained at least once before and after kidney transplantation or at least twice after kidney transplantation, and wherein (i) the level increases to above a predetermined threshold or (ii) the relative change between the levels of enkephalin or fragments thereof in samples obtained before and after kidney transplantation or in two samples obtained after kidney transplantation is a decrease of less than 50% or an increase (when the level of the earlier sample is set to 100%), can diagnose and / or predict graft function delay and / or the severity of graft function delay in said patients.

[0077] During follow-up measurements after kidney transplantation, (i) the level of enkephalin or a fragment thereof or (ii) the relative change of enkephalin or a fragment thereof was associated with improvement or deterioration of renal function. In patients who were diagnosed and / or predicted to be at risk of reduced graft function (particularly slowed or delayed graft function), a decrease in (i) the level of enkephalin or a fragment thereof or a relative change of enkephalin or a fragment thereof of more than 50% was associated with improvement of renal function, and an increase in (i) the level of enkephalin or a fragment thereof or a relative change of enkephalin or a fragment thereof of less than 50% or an increase was associated with deterioration of renal function.

[0078] During follow-up measurements after kidney transplantation, (i) the level of enkephalin or a fragment thereof or (ii) the relative change in enkephalin or a fragment thereof was associated with the success of the therapy or intervention. In patients who were diagnosed and / or predicted to be at risk of reduced graft function (particularly slowed or delayed graft function), a decrease in (i) the level of enkephalin or a fragment thereof or a relative change in enkephalin or a fragment thereof of more than 50% was associated with a successful therapy or intervention, and an increase in (i) the level of enkephalin or a fragment thereof or a relative change in enkephalin or a fragment thereof of less than 50% or an increase was associated with an unsuccessful therapy or intervention.

[0079] In one embodiment of the invention, the therapy or intervention is renal replacement therapy, which is stopped and / or suspended if the level of the enkephalin or a fragment thereof is below a predetermined threshold or if the relative change in the enkephalin or a fragment thereof is a decrease of more than 50%.

[0080] The relative change in the level of a biomarker (e.g., proenkephalin or a fragment thereof, or serum creatinine) is calculated between the level of a biomarker in a sample obtained at a specific time point and the level of the same biomarker in another sample obtained after the first sample, and when the level of the earlier sample is set to 100%.

[0081] Compared to other biomarkers (e.g., serum creatinine, creatinine clearance), proenkephalin or fragments thereof are superior for the early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients. Superiority implies higher specificity, higher sensitivity, better correlation with clinical endpoints, and earlier timing. Kidney function can be measured by GFR, creatinine clearance, SCr, cystatin C, urinalysis, blood urea nitrogen, or urine output.

[0082] In one embodiment of the invention, if a risk of reduced graft function is diagnosed and / or predicted in the patient, the patient requires renal replacement therapy and / or administration of medications and / or adjustment of immunosuppressive therapy and / or adjustment of nephrotoxic drugs.

[0083] Renal replacement therapy (RRT) replaces the kidneys' normal blood filtration function and refers to a therapy used to replace the kidneys' normal blood filtration function. Renal replacement therapy can refer to dialysis (such as hemodialysis or peritoneal dialysis), hemofiltration, and hemodiafiltration. These techniques involve diverting blood into a machine, cleaning it, and then returning it to the body in various different ways. Hemodialysis, hemofiltration, and hemodiafiltration can be continuous or intermittent and can use either an arteriovenous route (where blood leaves from an artery and returns via a vein) or a venous-venous route (where blood leaves from a vein and returns via a vein). This results in various types of RRT. For example, renal replacement therapy may be selected from, but is not limited to, continuous renal replacement therapy (CRRT), continuous hemodialysis (CHD), continuous arterial-venous hemodialysis (CAVHD), continuous venous-venous hemodialysis (CVVHD), continuous hemofiltration (CHF), continuous arterial-venous hemofiltration (CAVH or CAVHF), continuous venous-venous hemofiltration (CVVH or CVVHF), continuous hemodiafiltration (CHDF), continuous arterial-venous hemodiafiltration (CAVHDF), continuous venous-venous hemodiafiltration (CVVHDF), intermittent renal replacement therapy (IRRT), intermittent hemodialysis (IHD), intermittent venous-venous hemodialysis (IVVHD), intermittent hemofiltration (IHF), intermittent venous-venous hemofiltration (IVVH or IVVHF), intermittent hemodiafiltration (IHDF), and intermittent venous-venous hemodiafiltration (IVVHDF).

[0084] In one particular embodiment of the invention, the renal replacement therapy is selected from dialysis (hemodialysis or peritoneal dialysis), hemofiltration, and hemodiafiltration.

[0085] In one particular embodiment of the invention, patients at risk of reduced graft function after kidney transplantation may be given a drug selected from recombinant alkaline phosphatase, pegylated carboxyhemoglobin, relaxin, hepatocyte growth factor, mirocept, and C1 esterase inhibitors.

[0086] Immunosuppressive drugs are selected from interleukin-2 receptor antagonists, calcineurin inhibitors (such as cyclosporine A and tacrolimus), mammalian rapamycin inhibitors, corticosteroids (such as prednisolone), mycophenolate mofetil, sirolimus, and azathioprine.

[0087] Nephrotoxic drugs can be selected from calcineurin inhibitors used for immunosuppression (such as cyclosporine A and tacrolimus), analgesics (such as nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen and aspirin), antimicrobial agents (such as aminoglycosides, cephalosporins, penicillins, quinolones, rifampin, and vancomycin), cholesterol-lowering statins, angiotensin-converting enzyme inhibitors (ACE inhibitors), angiotensin receptor blockers (ARBs), diuretics, and chemotherapeutic agents (such as cisplatin).

[0088] In the context of this invention, the adjustment of nephrotoxic drugs and / or immunosuppressive therapy for the patient may involve the initiation and / or alteration and / or discontinuation of the nephrotoxic drugs and / or immunosuppressive therapies. The adjustment of nephrotoxic drugs and / or immunosuppressive therapies may be a change in the dosage, route of administration, regimen, or other parameters of the nephrotoxic drug treatment. Furthermore, adjustments in the treatment with nephrotoxic drugs and / or immunosuppressive therapies may also, and potentially additionally, involve changes to one or more nephrotoxic agents and / or immunosuppressive therapies used to treat the patient. Therefore, in some embodiments, the change may involve replacing one or more nephrotoxic drugs and / or immunosuppressive therapies with one or more other agents. In a very specific embodiment of this invention, the nephrotoxic drugs and / or immunosuppressive therapies are discontinued in the patient.

[0089] Throughout this specification, the terms proenkephalinogen and PENK are used synonymously.

[0090] Proenkephalin has the following sequence: SEQ ID NO. 1 (Proenaphthol 1-243) ECSQDCATCSYRLVRPADINFLACVMECEGKLPSLKIWETCKELLQLSKPELPQDGTSTLRENSKPEESHLLAKRYGGFMKRYGGFMKKMDELYPMEPEEEANGSEILAKRYGGFMKKDAE EDDSLANSSDLLKELLETGDNRERSHHQDGSDNEEEVSKRYGGFMRGLKRSPQLEDEAKELQKRYGGFMRRVGRPEWWMDYQKRYGGFLKRFAEALPSDEEGESYSKEVPEMEKRYGGFMRF Proenkephalin fragments that can be measured in body fluids may be selected from, for example, the following fragments: SEQ ID NO. 2 (Synenkephalin, proenkephalin 1-73) ECSQDCATCSYRLVRPADINFLACVMECEGKLPSLKIWETCKELLQLSKPELPQDGTSTLRENSKPEESHLLA SEQ ID NO. 3 (Met-Enkephalin) YGGFM SEQ ID NO. 4 (Leu-enkephalin) YGGFL SEQ ID NO. 5 (Proenaphthol 90-109) MDELYPMEPEEEANGSEILA SEQ ID NO. 6 (Proenaphthol 119-159, mid-fragment of proenaphthol, MR-PENK) DAEEDDSLANSSDLLKELLETGDNRERSHHQDGSDNEEEVS SEQ ID NO. 7 (Met-Enkephalin-Arg-Gly-Leu) YGGFMRGL SEQ ID NO. 8 (Proenaphthol 172-183) SPQLEDEAKELQ SEQ ID NO. 9 (Proenaphthol 193-203) VGRPEWWMDYQ SEQ ID NO. 10 (Proenaphthol 213-234) FAEALPSDEEGESYSKEVPEME SEQ ID NO. 11 (Proenaphthol 213-241) FAEALPSDEEGESYSKEVPEMEKRYGGFM SEQ ID NO. 12 (Met-Enkephalin-Arg-Phe) YGGFMRF In one embodiment of the present invention, it should be understood that the term proenkephalin fragment also includes Leu-enkephalin and Met-enkephalin.

[0091] Determining the levels of proenkephalins or fragments thereof, including Leu-enkephalin and Met-enkephalin, may imply the determination of immunoreactivity to proenkephalins or fragments thereof, including Leu-enkephalin and Met-enkephalin. The binding agents used to determine proenkephalins or fragments thereof, including Leu-enkephalin and Met-enkephalin, depending on the binding region, may bind to more than one of the molecules shown above. This will be clear to those skilled in the art.

[0092] This means that if a binding agent that binds to a region within the amino acid sequence of proenkephalin (PENK) in bodily fluids is used in the method of the present invention, the term "determining the level of proenkephalin (PENK) or a fragment thereof in bodily fluids obtained from said patient" is equivalent to "determining the level of an immunoreactive analyte using at least one binding agent that binds to a region within the amino acid sequence of proenkephalin (PENK) in bodily fluids obtained from said patient." In one particular embodiment, a binding agent that binds to a region within the amino acid sequence of proenkephalin (PENK) in bodily fluids is used in the method of the present invention. In one particular embodiment, the binding agent used in the method of the present invention does indeed bind to a region within the amino acid sequence of leu-enkephalin or met-enkephalin in bodily fluids. In another particular embodiment of the present invention, said at least one binding agent binds to the mid-proenkephalin (MR-PENK) or a fragment thereof.

[0093] Therefore, according to the present invention, the level of an immunoreactive analyte in bodily fluids obtained from the subject was determined by using at least one binding agent that binds to a region within the amino acid sequence of any of the aforementioned peptides and peptide fragments (i.e., proenkephalinogen (PENK) and fragments according to any of sequences 1 to 12); and in connection with specific embodiments relevant to clinical practice.

[0094] In a more specific embodiment of the method according to the invention, the level of MR-PENK (SEQ ID NO. 6: proenkephalin 119-159, proenkephalin mid-fraction, MR-PENK) was determined. In a more specific embodiment, the level of the immunoreactive analyte was determined by using at least one binding agent that binds to MR-PENK and correlated with the above-described embodiments according to the invention.

[0095] Therefore, according to the method of the present invention, the immunoreactivity level of the above-mentioned binder was determined in bodily fluids obtained from the patient. Immunoreactivity level refers to the concentration of the analyte, quantitatively, semi-quantitatively, or qualitatively determined by the binding reaction of the binder with the analyte, wherein preferably the affinity constant of the binder for the analyte is at least 10. 8 M -1The binding agent may be an antibody or antibody fragment or a non-Ig scaffold, and the binding reaction is an immunoassay.

[0096] In one specific embodiment, the level of enkephalin or a fragment thereof was determined by using at least one binding agent that binds to a region within the amino acid sequence of a peptide selected from proenkephalin or a fragment of at least five amino acids thereof. In one specific embodiment, the at least one binding agent binds to a region having a sequence selected from SEQ ID No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In one specific embodiment, the binding agent does not bind to the proenkephalin peptides Met-enkephalin SEQ ID No: 3 and Leu-enkephalin SEQ ID No: 4. In one specific embodiment, the at least one binding agent binds to a region having a sequence selected from SEQ ID No. 1, 2, 5, 6, 8, 9, 10, and 11. In another specific embodiment, the at least one binding agent binds to a region having a sequence selected from SEQ ID No. 1, 2, 5, 6, 8, and 9. In yet another very specific embodiment, the binding agent binds to proenkephalin 119-159, the proenkephalin mid-fraction, and MR-PENK (SEQ ID No. 6).

[0097] The aforementioned binder binds to the peptide in bodily fluids obtained from the subject.

[0098] Therefore, the subject matter of this invention is a method for early risk prediction, risk monitoring, or severity prediction of graft dysfunction in kidney transplant patients, the method comprising: ● The levels of proenkephalin or fragments thereof in bodily fluids obtained from the patient were determined by using at least one binding agent that binds to a region within the amino acid sequence of a peptide selected from SEQ ID No. 1 to 12; and ● The levels of the proenkephalin or fragments thereof are correlated with the risk and / or severity of graft dysfunction. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

[0099] In one embodiment of the invention, the binding agent is selected from antibodies, antibody fragments, or non-Ig scaffolds that bind to proenkephalin or fragments of at least 5 amino acids thereof.

[0100] In one particular embodiment, the level of the enkephalin or a fragment thereof is measured by an immunoassay using an antibody or antibody fragment that binds to the enkephalin or a fragment thereof. An immunoassay that can be used to determine the level of the enkephalin or a fragment of at least five amino acids thereof may include the steps outlined in Example 1. All thresholds and values ​​must be viewed in relation to the test and calibration used according to Example 1. Those skilled in the art will recognize that the absolute values ​​of the thresholds may be affected by the calibration used. This means that all values ​​and thresholds given herein should be understood in the context of the calibration used herein (Example 1).

[0101] The thresholds mentioned above may differ in other assays if these thresholds are calibrated differently from the assay system used in this invention. Therefore, taking into account calibration differences, the above thresholds should be applicable to such differently calibrated assays. One possibility for quantifying calibration differences is to perform a method comparison analysis (correlation) of the two methods by measuring the corresponding biomarker (e.g., proenkephalin or fragment thereof) in the sample using the assay discussed (e.g., an assay for measuring proenkephalin or fragment thereof) and the corresponding biomarker assay used in this invention. Another possibility is to determine the median level of the biomarker in a representative normal population using the assay discussed (given the sufficient analytical sensitivity of the test), and compare the results with the median levels of the biomarker described in the literature (e.g., ...). Donato et al., 2018. Clin Biochem. 58: 72-77 The calibration was recalculated based on the differences obtained through this comparison. Using the calibration used in this invention, samples from normal (healthy) subjects (n=100) were measured: the median plasma penKid (SEQ ID NO. 6) was 48.1 pmol / L (interquartile range Q1-Q3 41.7-55.7 pmol / L), and the central 95% reference limit was 36-83 pmol / L. Donato et al., 2018. Clin Biochem. 58: 72-77 Therefore, in one embodiment of the present invention, the predetermined threshold level of the proenkephalin or a fragment thereof is in the range of 50 to 750 pmol / L, more preferably in the range of 100 to 500 pmol / L, even more preferably in the range of 150 to 400 pmol / L, and most preferably in the range of 200 to 300 pmol / L.

[0102] Therefore, in one embodiment of the present invention, the predetermined threshold level of the proenkephalin or its fragment is x times the median level of proenkephalin or its fragment in healthy individuals. In a specific embodiment of the present invention, the threshold level of the proenkephalin or its fragment is in the range of 1.0 to 15.6 times the median level of proenkephalin or its fragment in healthy individuals, more preferably in the range of 1.7 to 10.4 times, even more preferably in the range of 2.1 to 8.3 times, and most preferably in the range of 3.1 to 6.2 times.

[0103] The prerequisite for using x times, for example, the median (or a specific percentile) level of proenkephalin or its fragments in healthy individuals as a threshold level is that the assays used in this invention and the assays with different calibrations (as described above) are performed in a linear manner.

[0104] Alternatively, the levels of any of the above analytes can be determined by other analytical methods, such as mass spectrometry. Mass spectrometry (MS) methods can include matrix-assisted laser desorption / ionization MS (MALDI-MS), liquid chromatography-mass spectrometry (LC-MS), and liquid chromatography-electrospray ionization MS (LC-ESI-MS).

[0105] According to the present invention, the binding agent against enkephalinogen is selected from antibodies (e.g., IgG, a typical full-length immunoglobulin) or antibody fragments containing at least a heavy chain and / or a light chain F variable domain, such as chemically conjugated antibodies (antigen-binding fragments), including but not limited to Fab fragments, including Fab mini-antibodies, single-chain Fab antibodies, monovalent Fab antibodies with epitope tags, such as Fab-V5Sx2; divalent Fab (mini-antibodies) dimerized with a CH3 domain; divalent or multivalent Fab, such as those formed by heterodomain-assisted polymerization (e.g., by dimerization of the dHLX domain), such as Fab-dHLX-FSx2; F(ab')2-fragments, scFv fragments, polymerized multivalent and / or multispecific scFv fragments, divalent and / or bispecific bisomatic antibodies, BITE® (bispecific T-cell conjugate), trifunctional antibodies, multivalent antibodies, such as those from a class different from G; and single-domain antibodies, such as nanobodies derived from camel or fish immunoglobulins.

[0106] In one particular embodiment, the level of the enkephalin or a fragment thereof is measured using a method for determining the binding of an aptamer, non-Ig scaffold, selected from those described in more detail below, to bind to the enkephalin or a fragment thereof.

[0107] The affinity constant of the binder for enkephalinogen or its fragments to enkephalinogen is at least 10. 7 M -1 Preferably 10 8 M-1 The preferred affinity constant is greater than 10. 9 M -1 The most preferred value is greater than 10. 10 M -1 Those skilled in the art will understand that lower affinity can be compensated for by applying higher doses of the compound, and such measures do not fall outside the scope of the invention. Binding affinity can be determined using the Biacore method, which is provided as a service analysis, for example, at Biaffin, Kassel, Germany (http: / / www.biaffin.com / de / ).

[0108] Besides antibodies, other biopolymer scaffolds are known in the art to be capable of recombining target molecules and have been used to generate highly target-specific biopolymers. Examples include aptamers, spiegelmers, anticalins, and cone snail toxins. Non-Ig scaffolds can be protein scaffolds and can be used as antibody mimics because they are able to bind to ligands or antigens. Non-Ig scaffolds can be selected from tetraconnectin-based non-Ig scaffolds (e.g., described in US 2010 / 0028995), fibronectin scaffolds (e.g., described in EP 1266 025), lipocalcin-based scaffolds (e.g., described in WO 2011 / 154420), ubiquitin scaffolds (e.g., described in WO 2011 / 073214), transferrin scaffolds (e.g., described in US2004 / 0023334), protein A scaffolds (e.g., described in EP 2231860), ankyrin repeat-based scaffolds (e.g., described in WO2010 / 060748), microprotein (preferably microproteins that form cystine knots) scaffolds (e.g., described in EP 2314308), Fyn SH3 domain-based scaffolds (e.g., described in WO 2011 / 023685), and EGFR-A domain-based scaffolds (e.g., described in WO 2010 / 0028995). (as described in 2005 / 040229) and scaffolds based on Kunitz structural domains (e.g., as described in EP 1941867).

[0109] A threshold level is a level that allows patients to be assigned to a group of patients diagnosed with adverse events (e.g., reduced graft function) and / or at risk of adverse events, or to a group of patients not yet diagnosed with adverse events and / or at risk of adverse events, or to a group of patients with a certain degree of severity (e.g., the severity of graft function delay). Therefore, a threshold level should allow for the differentiation between patients diagnosed with adverse events and / or at risk of adverse events and those not diagnosed with adverse events and / or at risk of adverse events. Threshold levels can be determined in the art in various ways. A threshold level is a predetermined value and is set to meet conventional requirements, such as specificity and / or sensitivity. These requirements may vary. For example, sensitivity or specificity may have to be set to certain limits, such as 80%, 90%, 95%, or 98%, respectively.

[0110] The sensitivity and specificity of diagnostic and / or prognostic tests depend not only on the analytical "quality" of the test but also on the definition of abnormal results. In practice, receiver operating characteristic (ROC) curves are typically calculated by plotting the values ​​of variables against their relative frequencies in a "reference group" (i.e., patients who do not experience graft dysfunction after kidney transplantation) and a "disease" population (i.e., patients with graft dysfunction after kidney transplantation). For any given biomarker, the distribution of biomarker levels may overlap between patients with and without graft dysfunction. In such cases, the test cannot absolutely distinguish between patients with and without graft dysfunction (e.g., slowed or delayed graft function) with 100% accuracy; the overlapping area indicates where the test fails to differentiate between normal and diseased individuals. A threshold is chosen above which the test is considered abnormal (or below which, depending on how the biomarker changes with disease), and below which it is considered normal. The area under the ROC curve is a measure of the probability that the assumed measurement allows for the correct identification of the disease. ROC curves can be used even when test results do not necessarily provide precise numbers. ROC curves can be created whenever the results can be sorted. For example, test results for "disease" samples might be sorted by severity (e.g., 1=low, 2=normal, 3=high). This sorting can then be correlated with results in a "reference" group, and an ROC curve can be created. These methods are well known in the art (see, for example...). Hanley et al., 1982. Radiology 143: 29-36 Preferably, the ROC curve produces an AUC greater than about 0.5, more preferably greater than about 0.7, even more preferably greater than about 0.8, and even more preferably greater than about 0.85, and most preferably greater than about 0.9. In this case, the term "about" means + / - 5% for a given measurement.

[0111] The reference group can be a healthy population, such as those without signs and symptoms of disease. In another aspect of the invention, the reference group can be a group of kidney transplant patients, particularly kidney transplant patients who have not experienced graft dysfunction (e.g., patients with immediate graft function). The reference group may consist of more than one reference subject.

[0112] The horizontal axis of the ROC curve represents (1-specificity), which increases with the false positive rate. The vertical axis of the curve represents sensitivity, which increases with the true positive rate. Therefore, for a specific cutoff threshold, a value for (1-specificity) can be determined, and the corresponding sensitivity can be obtained. The area under the ROC curve is a measure of the probability that the measured biomarker level allows for the correct identification of a disease or condition. Therefore, the area under the ROC curve can be used to determine the validity of the test.

[0113] For event occurrence time data, as in mortality risk prediction, the threshold level can be further obtained, for example, from Kaplan-Meier analysis, where the occurrence of the disease is correlated with, for example, the ternary, quartile, and quintile of biomarkers in the population (e.g., kidney biomarkers, cardiovascular biomarkers). There are also methods equivalent to the previously described ROC approach, namely, methods based on time-dependent ROC analysis or generalizations of the area under the ROC curve (C-index).

[0114] Other preferred thresholds are, for example, the 90th, 95th, or 99th percentile in the normal population. Using a percentile higher than the 75th percentile can reduce the number of false positives identified, but may miss identifying subjects with moderate risk (although the risk is still increased). Therefore, the threshold adopted may depend on whether it is considered more appropriate to identify the majority of at-risk subjects at the cost of identifying "false positives," or to primarily identify high-risk subjects at the cost of missing a few subjects with moderate risk.

[0115] For example, the value of the 75th percentile, more preferably the 90th percentile, even more preferably the 95th percentile, and most preferably the 99th percentile can be used as the upper limit of the normal range.

[0116] In addition to the normal range, other methods may be used to determine thresholds for specific indications, depending on the intended use and application / clinical context. These methods include, for example, the Youden optimum, thresholds that maximize overall accuracy, odds ratios, or positive or negative predictive values. In some cases, thresholds that achieve pre-defined levels of sensitivity or specificity (e.g., 80%, 90%, 95%, or 99%) may be appropriate for clinical application. The choice of method depends on the clinical application, weighing the costs of false positives and false negatives based on the impact of test results on patients and the healthcare system, as well as clinical needs. Finally, multiple methods can be combined to define consensus thresholds.

[0117] Threshold levels may vary with various physiological parameters (such as age, sex, or subgroup) and the methods mentioned in this article for determining proenkephalins and their fragments.

[0118] In one particular embodiment of the invention, the threshold level is age-dependent. The MR-PENK (proenkephalin 119-159; SEQ ID No: 6) values ​​revealed that they depended on the subject's age, using more than one threshold. The threshold decreased as the patient aged.

[0119] In one embodiment of the invention, the predetermined threshold level of the proenkephalin or its fragment is in the range of 50 to 750 pmol / L, more preferably in the range of 100 to 500 pmol / L, even more preferably in the range of 150 to 400 pmol / L, and most preferably in the range of 200 to 300 pmol / L.

[0120] In one particular embodiment, the proenkephalin fragment is MR-PENK (PENK 119-159, SEQ ID NO. 6).

[0121] In another specific embodiment of the invention, the predetermined threshold level of the MR-PENK (PENK 119-159, SEQ ID NO. 6) is in the range of 50 to 750 pmol / L, more preferably in the range of 100 to 500 pmol / L, even more preferably in the range of 150 to 400 pmol / L, and most preferably in the range of 200 to 300 pmol / L.

[0122] For other proenkephalin fragments derived from the proenkephalin precursor in a 1:1 molar ratio, the threshold levels are likely similar. Since mature Met-enkephalin is encoded by the proenkephalin precursor in a 6:1 molar ratio, meaning six Met-enkephalin molecules are derived from one precursor, the threshold level of the Met-enkephalin is six times higher than the MR-PENK threshold level.

[0123] In a very specific embodiment of the invention, if measured within 24 hours after kidney transplantation, the predetermined threshold level of the proenkephalin or fragments thereof (particularly MR-PENK (PENK 119-159, SEQ ID NO. 6)) is 300 pmol / L.

[0124] In another very specific embodiment of the invention, if measured more than 24 to 48 hours after kidney transplantation, the predetermined threshold level of the proenkephalin or fragments thereof (particularly MR-PENK (PENK 119-159, SEQ ID NO. 6)) is 200 pmol / L.

[0125] In another embodiment of the invention, the levels of proenkephalins or fragments thereof in bodily fluid samples obtained from the patient are measured before and after kidney transplantation. The samples obtained after kidney transplantation are obtained within the range of 3 to 96 hours post-transplantation, more preferably 6 to 72 hours, even more preferably 9 to 48 hours, and most preferably 12 to 24 hours post-transplantation.

[0126] In a more specific embodiment of the invention, the sample obtained after kidney transplantation is obtained within 96 hours, more preferably within 72 hours, even more preferably within 48 hours, even more preferably within 24 hours, and most preferably within 12 hours after kidney transplantation.

[0127] In one embodiment of the invention, the level of the proenkephalin or a fragment thereof can be determined as a follow-up assay more than 96 hours after kidney transplantation.

[0128] In one embodiment of the invention, the relative changes in the levels of proenkephalin or fragments thereof in body fluid samples obtained (i) at least once before and after kidney transplantation or (ii) at least twice after kidney transplantation are calculated and correlated with the diagnosis and / or risk and / or severity of reduced graft function in the patient's kidney.

[0129] In one particular embodiment of the invention, the relative change in the level of the proenkephalin or a fragment thereof is either a decrease or even an increase.

[0130] In another specific implementation, when the level of an earlier sample is set to 100%, if the reduction is less than 35%, preferably less than 40%, even more preferably less than 45%, and most preferably less than 50%, then a reduction in kidney graft function is diagnosed and / or predicted in the patient.

[0131] In a particular embodiment of the invention, when the level of an earlier sample is set to 100%, if the relative change between the levels of proenkephalin or fragments thereof in the samples obtained at least once before and after kidney transplantation or at least twice after kidney transplantation is a decrease of less than 50% or an increase, then a risk of graft dysfunction is diagnosed and / or predicted in the patient.

[0132] If the sample is obtained, for example, once before kidney transplantation (sample 1) and once after kidney transplantation (e.g., 24 hours later) (sample 2), and the relative change in the level of proenkephalin or a fragment thereof, calculated between sample 1 and sample 2 (set to 100%), is less than 50% reduced or increased, then the patient is diagnosed and / or predicted to have a reduced graft function. A third sample may then be obtained after sample 2 (e.g., 48 hours after kidney transplantation) for monitoring of the patient. Similarly, the relative change in the level of proenkephalin or a fragment thereof, now set to 100%, is calculated between sample 2 and sample 3.

[0133] The subject matter of this invention is also a medicine for the early treatment of impaired graft function in kidney transplant patients, wherein the patients are selected through a diagnostic method including the following steps: - To determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and - Associate the level of the proenkephalin or fragment thereof in the sample with the diagnosis of graft-deficient function and / or the risk of graft-deficient function. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

[0134] In one embodiment, the drug is selected from recombinant alkaline phosphatase, PEGylated carboxyhemoglobin, relaxin, hepatocyte growth factor, mirocept, and C1 esterase inhibitors.

[0135] In one particular embodiment, an immunoassay is used to measure the level of proenkephalin or a fragment thereof, and the binding agent is an antibody or antibody fragment that binds to proenkephalin or a fragment thereof.

[0136] In one particular embodiment, the assay used comprises two binding agents that bind to two distinct regions within the proenkephalin region, namely amino acids 133-140 (LKELLETG, SEQ ID No. 13) and 152-159 (SDNEEEVS, SEQ ID No. 14), wherein each region contains at least 4 or 5 amino acids.

[0137] In one embodiment of the present invention, the sensitivity of the assay for determining proenkephalin or fragments in a sample is < 15 pmol / L, preferably < 10 pmol / L, and most preferably < 6 pmol / L.

[0138] The subject matter of this invention is the use of at least one binder that binds to a region within the amino acid sequence of a peptide selected from SEQ ID No. 1 to 12 and fragments thereof from bodily fluids obtained from the subject, in a method for early risk prediction, risk monitoring, or severity prediction of graft dysfunction in kidney transplant patients.

[0139] In one embodiment of the invention, the binding agent is selected from antibodies, antibody fragments, or non-Ig scaffolds that bind to proenkephalin or fragments of at least five amino acids thereof. In a particular embodiment, the at least one binding agent binds to a region having a sequence selected from SEQ ID No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In a particular embodiment, the binding agent binds to proenkephalin peptides met-enkephalin (SEQ ID No: 3) and leu-enkephalin (SEQ ID No: 4). In a particular embodiment, the at least one binding agent binds to a region having a sequence selected from SEQ ID No. 1, 2, 5, 6, 8, 9, 10, and 11. In another particular embodiment, the at least one binding agent binds to a region having a sequence selected from SEQ ID No. 1, 2, 5, 6, 8, and 9. In yet another very particular embodiment, the binding agent binds to proenkephalin 119-159, a proenkephalin mid-fraction, and MR-PENK (SEQ ID No. 6).

[0140] In a more specific embodiment, the at least one binding agent binds to regions within the amino acid sequence of proenkephalin 119-159, the middle segment of proenkephalin, and MR-PENK (SEQ ID No. 6) in bodily fluids obtained from the subject, more specifically to amino acids 133-140 (LKELLETG, SEQ ID No. 13) and / or amino acids 152-159 (SDNEEEVS, SEQ ID No. 14), wherein each of the regions contains at least 4 or 5 amino acids.

[0141] In addition, the at least one measurable clinical parameter is selected from alanine aminopeptidase, alkaline phosphatase, gamma-glutamyl transferase, calprotectin, CC motif chemokine ligand 14, chitinase 3-like protein 1, hepatocyte growth factor, hepcidin, IL-18, β-trace protein (BTP), cystatin C, KIM-1, TIMP-2, IGFBP-7, blood urea nitrogen (BUN), NGAL, liver-type fatty acid-binding protein, monocyte chemoattractant peptide-1, creatinine clearance, serum creatinine (SCr), urea, metrin-1, osteopontin, retinol-binding protein, tumor necrosis factor, and Apache score.

[0142] In one embodiment of the invention, the method is performed more than once to monitor the patient's risk or to monitor the treatment progress of the kidney transplant patient. In a particular embodiment, the monitoring is performed to assess the patient's response to preventive and / or treatment measures taken.

[0143] In one embodiment of the invention, the method is used to stratify the patient into risk groups. The patient may be stratified into a low, intermediate, or high risk group for reduced graft function. The patient may also be stratified into a low, intermediate, or high risk group for slowed graft function and / or delayed graft function.

[0144] Furthermore, the subject matter of this invention is a method for determining proenkephalin and proenkephalin fragments in a sample, comprising two binding agents that bind to two different regions within the proenkephalin region (i.e., amino acids at positions 133-140 (LKELLETG, SEQ ID NO. 13) and amino acids at positions 152-159 (SDNEEEVS, SEQ ID NO. 14)), wherein each said region contains at least 4 or 5 amino acids.

[0145] In one embodiment of the invention, it can be a so-called POC (point-of-care) test, a testing technique that allows testing to be performed near the patient in less than one hour without requiring a fully automated assay system. An example of such a technique is immunochromatographic assay.

[0146] In one embodiment of the invention, this assay is a sandwich immunoassay using any type of detection technology, including but not limited to enzyme labeling, chemiluminescent labeling, and electrochemiluminescent labeling, preferably a fully automated assay. In one embodiment of the invention, this assay is an enzyme-labeled sandwich assay. Examples of automated or fully automated assays include assays that can be used in one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, and Alere Triage®.

[0147] Various immunoassays are known and can be used in the assays and methods of the present invention, including: radioimmunoassay (“RIA”), homogeneous enzyme multiplication immunoassay (“EMIT”), enzyme-linked immunosorbent assay (“ELISA”), apoenzyme reactivation immunoassay (“ARIS”), test strip immunoassay, and immunochromatographic assay.

[0148] In one embodiment of the invention, at least one of the two binders is labeled for detection.

[0149] Preferred detection methods include various forms of immunoassays, such as radioimmunoassay (RIA), chemiluminescence and fluorescence immunoassays, enzyme-linked immunosorbent assay (ELISA), Luminex-based bead arrays, protein microarray assays, and rapid assays such as immunochromatographic strip assays.

[0150] In a preferred embodiment, the marker is selected from chemiluminescent markers, enzyme markers, fluorescent markers, and radioactive iodine markers.

[0151] The assay can be a homogeneous or heterogeneous assay, or a competitive or non-competitive assay. In one embodiment, the assay takes the form of a sandwich assay, a non-competitive immunoassay in which the molecule to be detected and / or quantified is bound to a first antibody and to a second antibody. The first antibody may be bound to a solid phase, such as the surface of beads, pores, or other containers, a chip, or a strip, and the second antibody is an antibody labeled, for example, with a dye, a radioisotope, or a reactive or catalytically active moiety. The amount of labeled antibody bound to the analyte is then measured by an appropriate method. The general composition and procedures involved in the "sandwich assay" are well-established and known to those skilled in the art.

[0152] In another embodiment, the assay includes two capture molecules, preferably antibodies, both present as dispersions in a liquid reaction mixture, wherein a first labeled component is attached to the first capture molecule, wherein the first labeled component is part of a labeling system based on fluorescence or chemiluminescence quenching or amplification, and a second labeled component of the labeling system is attached to the second capture molecule, such that after the two capture molecules bind to the analyte, a measurable signal is generated, allowing the detection of the formed sandwich complex in a solution containing the sample.

[0153] In another embodiment, the labeling system comprises rare earth cavities or rare earth chelates in combination with fluorescent dyes or chemiluminescent dyes (especially anthocyanin-type dyes).

[0154] In the context of this invention, the fluorescence-based assay includes the use of a dye, such as FAM (5- or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700 / 800, cyanine dyes such as CY3, CY5, CY3.5, CY5.5, Cy7, sulfadiazine, 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), T... ET, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), N,N,N',N'-tetramethyl-6-carboxy-rhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6), rhodamine, rhodamine green, rhodamine red, rhodamine 110, BODIPY dyes such as BODIPY TMR, Oregon Green, coumarins such as umbelliferone, benzoimides such as Hoechst 33258; phenanthridines such as Texas Red, Ikima Yellow, Alexa Fluor, PET, ethidium bromide, acridine dyes, carbazole dyes, phenanthrene Azine dyes, porphyrin dyes, polymethyst dyes, etc.

[0155] In the context of this invention, the chemiluminescence-based determination method includes the use of a dye based on the physical principles described for chemiluminescent materials in the following documents. Kirk-Othmer, *Encyclopedia of Chemical Technology* Chemical Technology, 4th Edition, Executive Editor: J.I. Kroschwitz; Editors: M. Howe-Grant, John Wiley & Sons, 1993, vol.15, pp. 518-562, incorporated herein by reference, including citations on pp. 551-562. arts The chemiluminescent label can be an acridine ester label, a steroidal label involving isoluminol labeling, etc. Acridine esters are preferred chemiluminescent dyes.

[0156] As described herein, the term "assay" or "diagnostic assay" can be of any type used in the diagnostic field. Such an assay can be based on the binding of the analyte to be detected to one or more capture probes with a certain affinity. Regarding the interaction between the capture molecule and the target molecule or molecule of interest, the affinity constant is preferably greater than 10. 8 M -1 .

[0157] In the context of this invention, a "binding molecule" is a molecule that can be used to bind a target molecule or molecule of interest, i.e., an analyte (i.e., PENK and fragments thereof in the context of this invention), from a sample. Therefore, the binding molecule must be sufficiently shaped in terms of spatial and surface characteristics (e.g., surface charge, hydrophobicity, hydrophilicity, presence or absence of Lewis donors and / or acceptors) to specifically bind the target molecule or molecule of interest. Thus, binding can be mediated, for example, by ionic, van der Waals, π-π, σ-π, hydrophobic, or hydrogen bonding interactions, or combinations of two or more of the above interactions, between the capturing molecule and the target molecule or molecule of interest. In the context of this invention, the binding molecule can be, for example, selected from nucleic acid molecules, carbohydrate molecules, PNA molecules, proteins, antibodies, peptides, or glycoproteins. Preferably, the binding molecule is an antibody, comprising a fragment having sufficient affinity for the target or molecule of interest, and comprising recombinant antibodies or recombinant antibody fragments, as well as chemically and / or biochemically modified derivatives of the antibody or fragments derived from variant chains of at least 12 amino acids in length.

[0158] Chemiluminescent markers can be acridine ester markers, steroid markers involving isoluminol markers, etc.

[0159] Enzyme markers can include lactate dehydrogenase (LDH), creatine kinase (CPK), alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), acid phosphatase, glucose-6-phosphate dehydrogenase, horseradish peroxidase (HRP), etc.

[0160] In one embodiment of the invention, at least one of the two binders is bound to the solid phase, which serves as both magnetic particles and the polystyrene surface.

[0161] In one embodiment of the assay for determining proenkephalin or proenkephalin fragments in a sample according to the present invention, such an assay is a sandwich assay, preferably a fully automated assay. It can be a fully automated or manual ELISA. It can be a so-called point-of-care (POC) test. Examples of automated or fully automated assays include assays that can be used in one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, BrahmsKryptor®, Biomerieux Vidas®, Alere Triage®, and Ortho Vitros®. Examples of test formats are provided above.

[0162] In one embodiment of the method for determining proenkephalin or proenkephalin fragments in a sample according to the present invention, at least one of the two binding agents is labeled for detection. Examples of the labels are provided above.

[0163] In one embodiment of the method for determining proenkephalin or proenkephalin fragments in a sample according to the present invention, at least one of the two binding agents is bound to a solid phase. Examples of solid phases are provided above.

[0164] In one embodiment of the assay method according to the present invention for determining proenkephalin or proenkephalin fragments in a sample, the label is selected from chemiluminescent labels, enzyme labels, fluorescent labels, and radioactive iodine labels. Another aspect of the present invention is a kit comprising the assay method according to the present invention, wherein the components of the assay method can be contained in one or more containers.

[0165] In one embodiment, the subject matter of the invention is a point-of-care device for performing the method according to the invention, wherein the point-of-care device comprises at least one antibody or antibody fragment targeting amino acids 133-140 (LKELLETG, SEQ ID No. 13) or 152-159 (SDNEEEVS, SEQ ID No. 14), wherein each region comprises at least 4 or 5 amino acids.

[0166] In one embodiment, the subject matter of the invention is a point-of-care device for performing the method according to the invention, wherein the point-of-care device comprises at least two antibodies or antibody fragments targeting amino acids 133-140 (LKELLETG, SEQ ID No. 13) and 152-159 (SDNEEEVS, SEQ ID No. 14), wherein each region comprises at least 4 or 5 amino acids.

[0167] In one embodiment, the subject matter of the invention is a kit for performing the method according to the invention, wherein the point of care device comprises at least one antibody or antibody fragment targeting amino acids 133-140 (LKELLETG, SEQ ID No. 13) or 152-159 (SDNEEEVS, SEQ ID No. 14), wherein each said region comprises at least 4 or 5 amino acids.

[0168] In one embodiment, the subject matter of the invention is a kit for performing the method according to the invention, wherein the point of care device comprises at least two antibodies or antibody fragments targeting amino acids 133-140 (LKELLETG, SEQ ID No. 13) and 152-159 (SDNEEEVS, SEQ ID No. 14), wherein each region comprises at least 4 or 5 amino acids.

[0169] The term "antibody" generally includes monoclonal and polyclonal antibodies and their binding fragments, particularly Fc-fragments, as well as so-called "single-chain antibodies" (Bird et al., 1988), chimeric, humanized, particularly CDR-grafted antibodies, and dimeric or tetrameric antibodies (Holliger et al., 1993). It also includes immunoglobulin-like proteins selected by techniques including, for example, phage display, that specifically bind to molecules of interest contained in a sample. In this context, the term "specific binding" refers to an antibody produced against a molecule of interest or a fragment thereof. An antibody is considered specific if its affinity for the molecule of interest or its aforementioned fragment is at least, preferably, 50 times, more preferably 100 times, and most preferably at least 1000 times, higher than its affinity for other molecules contained in a sample containing said molecule of interest. How to manufacture antibodies and select antibodies with given specificity is well known in the art.

[0170] The antibodies or fragments according to the invention are proteins that specifically bind to antigens, comprising one or more polypeptides essentially encoded by immunoglobulin genes. Recognized immunoglobulin genes include constant region genes for κ, λ, α (IgA), γ (IgG1, IgG2, IgG3, IgG4), δ (IgD), ε (IgE), and μ (IgM), as well as numerous variable region genes for immunoglobulins. Full-length immunoglobulin light chains are typically about 25 kDa or 214 amino acids in length.

[0171] The full-length immunoglobulin heavy chain is typically about 50 kDa or 446 amino acids in length. The light chain is encoded by a variable region gene (about 110 amino acids long) located at the NH2-terminus and a κ or λ constant region gene located at the COOH-terminus. The heavy chain is also encoded by a variable region gene (about 116 amino acids long) and one of the remaining constant region genes.

[0172] The basic structural unit of an antibody is typically a tetramer composed of two identical pairs of immunoglobulin chains, each pair consisting of one light chain and one heavy chain. In each pair, the variable regions of the light and heavy chains bind to the antigen, while the constant regions mediate effector function. Immunoglobulins also exist in a variety of other forms, including, for example, Fv, Fab, and (Fab')2, as well as bifunctional hybrid antibodies and single-chain antibodies (e.g., ...). Lanzavecchia et al., 1987. Eur. J. Immunol. 17:105; Huston et al., 1988. Proc. Natl. Acad. Sci. USA, 85:5879-5883; Bird et al., 1988. Science 242:423-426; Hood et al., 1984, Immunology, Benjamin, NY, 2nd ed.; Hunkapiller and Hood 1986. Nature 323:15-16 The variable regions of the immunoglobulin light or heavy chains include structural regions interrupted by three hypervariable regions, also known as complementarity-determining regions (CDRs) (see [link to relevant documentation]). Sequences of Proteins of Immunological Interest, E. Kabat et al., 1983, US Department of Health and Human Services As noted above, CDRs are primarily responsible for binding to epitopes of antigens. Immune complexes are antibodies specifically bound to antigens, such as monoclonal antibodies, chimeric antibodies, humanized antibodies, or human antibody or functional antibody fragments.

[0173] Chimeric antibodies are antibodies whose light and heavy chain genes are typically constructed by genetic engineering from variable and constant region genes of immunoglobulins from different species. For example, a variable region from a mouse monoclonal antibody gene can be linked to a human constant region such as κ and γ1 or γ3. Thus, in one instance, a therapeutic chimeric antibody is a hybrid protein consisting of a variable or antigen-binding domain from a mouse antibody and a constant or effector domain from a human antibody, although other mammalian species can also be used, or the variable region can be generated by molecular techniques. Methods for manufacturing chimeric antibodies are well known in the art, see, for example, U.S. Patent No. 5,807,715. A “humanized” immunoglobulin is an immunoglobulin comprising a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDR is referred to as the “donor,” and the human immunoglobulin providing the framework is referred to as the “recipient.” In one embodiment, in a humanized immunoglobulin, all CDRs are derived from the donor immunoglobulin. Constant regions may not necessarily exist, but if they do, they must be substantially identical to the constant regions of human immunoglobulins, i.e., with an identity of at least about 85-90%, such as about 95% or higher. Therefore, it is possible that all parts of a humanized immunoglobulin, except for the CDR, are substantially identical to their corresponding parts of the native human immunoglobulin sequence. A “humanized antibody” is an antibody comprising humanized light chains and humanized heavy chains of immunoglobulin. Humanized antibodies bind to the same antigen as the donor antibody that provides the CDR. The receptor framework of a humanized immunoglobulin or antibody may have a limited number of amino acid substitutions derived from the donor framework. Humanized or other monoclonal antibodies may have other conserved amino acid substitutions that substantially do not affect antigen binding or the function of other immunoglobulins. Exemplary conserved substitutions are, for example: gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed using genetic engineering (see, for example, U.S. Patent No. 5,585,089). Human antibodies are antibodies in which the light chain and heavy chain genes are of human origin. Human antibodies can be produced using methods known in the art. Human antibodies can be produced by immortalizing human B cells that secrete antibodies of interest. Immortification can be achieved, for example, through EBV infection or by fusing human B cells with myeloma or hybridoma cells to produce trivalent hybridoma cells. Human antibodies can also be produced by phage display methods (see, for example, WO91 / 17271, WO92 / 001047, WO92 / 20791), or by selection from human combinatorial monoclonal antibody libraries (see the Morphosys website). Human antibodies can also be prepared using transgenic animals carrying human immunoglobulin genes (see, for example, WO93 / 12227, WO 91 / 10741).

[0174] Humanization of mouse antibodies can be performed according to the following procedure: To humanize mouse-derived antibodies, the antibody sequences were analyzed to understand the structural interactions between the framework region (FR), complementarity-determining region (CDR), and antigen. Based on structural modeling, a suitable human-derived FR was selected, and the mouse CDR sequence was transplanted into the human FR. Mutations can be introduced into the amino acid sequences of the CDR or FR to restore structural interactions that were abolished by species switching of the FR sequence. This restoration of structural interactions can be achieved using phage display libraries via a randomized method or via a directed method guided by molecular modeling. Almagro and Fransson 2008. Anti- Humanization of antibodies, Front Biosci. 2008 Jan 1;13:1619-33 ).

[0175] Methods for obtaining monoclonal antibodies

[0176] In all the following embodiments, the term monoclonal antibody means including monoclonal antibodies as well as fragments of monoclonal antibodies, such as the fragments detailed herein, and more specifically, monoclonal antibodies.

[0177] Hybridoma

[0178] In another aspect, the antibody according to the invention is a monoclonal antibody that can be obtained by a method comprising the steps described below: i) Fusing antibody-secreting cells from animals previously immunized with antigens with myeloma cells to obtain a large number of hybridomas. ii) Isolate hybridomas from the large number of hybridomas that produce the desired monoclonal antibody.

[0179] In some embodiments, the antibody according to the invention is a monoclonal antibody that can be obtained by isolating a hybridoma from a large number of hybridomas that produce the desired monoclonal antibody, wherein the large number of hybridomas is produced by fusing antibody-secreting cells from an animal previously immunized with an antigen with myeloma cells to obtain a large number of hybridomas.

[0180] The required monoclonal antibody, in particular a monoclonal antibody that binds to the said antigen, especially one with a binding affinity of at least 10. 7 M -1 Preferably 10 8 M -1 More preferably, the affinity is greater than 10. 9 M -1 The most preferred value is greater than 10. 10 M -1 .

[0181] In some embodiments of the method for obtaining antibodies, in step i), the animal is a mammal, particularly a rabbit, mouse, or rat, more particularly a mouse, and more particularly a Balb / c mouse.

[0182] In some embodiments of the method for obtaining antibodies, in step i), the antibody-secreting cells are spleen cells, more particularly activated B cells.

[0183] In some embodiments of the method for obtaining antibodies, in step i), fusion involves the use of polyethylene glycol.

[0184] In some embodiments of the method for obtaining antibodies, in step i), the myeloma is derived from a mammal, and in some embodiments from a mammal of the same species from which the large number of antibody-secreting cells are obtained. In some specific embodiments of the method for obtaining antibodies, in step i), the myeloma cells are the cell line SP2 / 0.

[0185] In some embodiments of the method for obtaining antibodies, the fusion in step i) includes PEG-assisted fusion, Sendai virus-assisted fusion, or current-assisted fusion.

[0186] In some embodiments of the method for obtaining antibodies, the separation in step ii) includes performing antibody capture assays, antigen capture assays, and / or functional screening.

[0187] In some embodiments of the method for obtaining antibodies, in step ii), isolating the hybridoma that produces the desired monoclonal antibody may include cloning and recloning the hybridoma using limiting dilution techniques.

[0188] In one embodiment, the antigen capture assay includes: a) Binding the generated antibody to a substrate, particularly a solid substrate. b) Allow the antigen to bind to the antibody. c) Remove unbound antigens by washing. d) Detect the bound antigen; Alternatively, the antigen capture assay may include: a) Allowing the antigen to bind to the generated antibody to form an antibody-antigen complex. b) Binding the antibody-antigen complex to a substrate, particularly a solid substrate. c) Remove unbound antigens by washing. d) Detect the bound antigen.

[0189] In one embodiment, the separation in step ii) includes performing enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting, cell staining, immunoprecipitation, and / or Western blot.

[0190] In one embodiment, the detection of the antibody or antigen is performed using an immunoassay.

[0191] In one embodiment, the animal is a transgenic animal, particularly a transgenic mouse (wherein, specifically within the genome of the transgenic animal, the mouse immunoglobulin (Ig) locus has been replaced by a human locus), such as HuMabMouse or XenoMouse.

[0192] In one embodiment, the antigen comprises a peptide as described in Table 1 herein, which in some embodiments (particularly for immunization) may be coupled to a protein, particularly a serum protein, more particularly serum albumin, and even more particularly BSA.

[0193] In a preferred embodiment, the antibody according to the invention is a monoclonal antibody obtainable by a method comprising the steps described below: i) Using polyethylene glycol, spleen cells from Balb / c mice previously immunized with the peptides described in Table 1 of this document were fused with SP2 / 0 myeloma cells to obtain a large number of hybridomas. ii) Isolate hybridomas from the large number of hybridomas that produce the desired monoclonal antibody; More preferably, the method includes: 1) Grow the hybridomas in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement] for the first time period (especially 2 weeks). 2) Subsequently, use HT medium instead of HAT medium for multiple subcultures (especially 3 times). 3) The cells were then returned to normal cell culture medium for the second time period, particularly until three weeks after fusion. 4) Preliminary screening of antigen-specific IgG antibodies in cell culture supernatant 5) Propagate microcultures of cells that tested positive in step 4). 6) Retest for antigen-specific IgG antibodies in the cell culture supernatant of microcultures. 7) Cloning and recloning cultures that tested positive in step 6) using limiting dilution techniques. 8) Optionally determine the isotype of the clone obtained from 7). 9) Optionally purify the antibody via protein A.

[0194] Phage display

[0195] On the other hand, the antibody according to the invention is a monoclonal antibody that can be obtained by a method comprising the following steps: i) Isolate at least one antibody with affinity for the antigen from an antibody gene library; ii) Producing at least one cell line expressing at least one of the antibodies; iii) Isolate the at least one antibody from the culture of the at least one cell line obtained in step ii).

[0196] Antibodies that have affinity for antigens are specifically those with a binding affinity of at least 10. 7 M -1 Preferably 10 8 M -1 The antibody, preferably with an affinity greater than 10. 9 M -1 The most preferred value is greater than 10. 10 M -1 .

[0197] In one embodiment, the antibody according to the invention is a monoclonal antibody that can be obtained by isolating at least one antibody from a culture derived from at least one cell line expressing at least one antibody with affinity for an antigen from an antibody gene library.

[0198] In one embodiment, the antigen comprises a peptide as described in Table 1 herein, which in some embodiments may be bound to a solid phase.

[0199] In some embodiments of the method for obtaining antibodies, in step i), the antibody gene library is a naive antibody gene library, particularly a human naive antibody gene library, and more specifically, in which the antibody is presented by phage display, i.e., presented on a phage containing a nucleotide sequence encoding such a corresponding antibody; more specifically, it is a library HAL 7, HAL 8, or HAL 9, and more specifically, a library containing human naive antibody gene libraries HAL7 / 8.

[0200] In some embodiments of the method for obtaining antibodies, in step i), screening includes using antigens, particularly antigens containing tags, more particularly biotin tags, attached to them via two different spacers. In a particular embodiment, this screening strategy involves a mixture of screening wheels using non-specifically binding antigens and antigens that specifically bind via tags (in the case of biotin tags, to streptavidin). In this way, the background of non-specific binders can be minimized.

[0201] In some embodiments of the method for obtaining antibodies, in step i), in embodiments where the library is a phage display library, the antibody is isolated by isolating the phage that presents the antibody (and contains a nucleotide sequence encoding the antibody).

[0202] In some embodiments of the method for obtaining antibodies, in step ii), the cell line is generated by introducing a nucleotide sequence encoding the antibody; in embodiments where the library in step i) is a phage display library, the phage isolated from step i) can be used to generate bacterial strains expressing the antibody, such as Escherichia coli (E. coli) strains.

[0203] In some embodiments of the method for obtaining antibodies, in step iv), where the library in step i) is a phage display library and in the embodiment where a bacterial strain is generated in step ii), antibodies can be isolated from the supernatant of the culture.

[0204] It should be understood that, when used in describing methods of obtaining antibodies, the term "an antibody" in the phrase "at least one antibody" specifically may include more than one antibody molecule having the same amino acid sequence. This understanding also applies, with necessary modifications, to the term "a cell line."

[0205] In some embodiments of the method for obtaining antibodies, more than one antibody (meaning multiple antibodies, each with a different amino acid sequence) is isolated in step i), thus generating more than one cell line in step ii). This method may involve selecting clones that are positive for binding to the antigen, for example by a binding assay, such as an ELISA assay involving the antigen, and cells that are positive for binding to the antigen may be isolated to generate monoclonal cell lines.

[0206] In a preferred embodiment, the antibody according to the invention is a monoclonal antibody obtainable by a method comprising the steps described below: i) Isolate at least one of the antibodies from the antibody gene library containing the human naive antibody gene library HAL7 / 8 by eluting bacteriophages carrying antibodies with affinity for the antigen; ii) Producing at least one Escherichia coli cell line expressing at least one of the antibodies; iii) Isolate the at least one antibody from the culture supernatant of at least one Escherichia coli cell line obtained in step ii).

[0207] In another aspect, the antibody fragments according to the invention are produced by a method involving enzymatic digestion of the antibody. In some embodiments, this method produces, for example, Fab or F(ab)2 antibody fragments. In some embodiments, this method involves digestion with pepsin or papain, the enzyme optionally immobilized on a surface.

[0208] In some implementations, antibodies can be humanized via CDR transplantation, particularly through methods involving the following steps: - Extract RNA from hybridomas expressing antibodies of interest (e.g., those obtained by the methods described herein); - The extracted RNA was amplified by RT-PCR, specifically using a set of primers specific to the heavy and light chains of the antibody of interest, to obtain the DNA product; - The DNA product was further amplified by PCR, particularly using a semi-nested primer set specific to the antibody variable region; - Determine the sequence of the DNA product; - Align the sequence with the homologous human framework sequence to determine the humanized sequences for the variable heavy chain and variable light chain sequences (of the desired antibody).

[0209] In some embodiments, antibody humanization can be achieved by comparing the sequence of the DNA product obtained by amplifying RNA extracted from a hybridoma expressing the antibody of interest via RT-PCR (particularly using a primer set specific to the heavy and light chains of the antibody of interest) and further amplifying the DNA obtained therefrom via PCR (particularly using a semi-nested primer set specific to the variable region of the antibody) with a homologous human scaffold sequence to determine the humanized sequences for the variable heavy and light chain sequences (of the desired antibody).

[0210] In some implementations, antibodies can be humanized in the following ways: - Identify the complementarity-determining region (CDR), which can be achieved by analyzing the structural interactions between the framework region (FR) and the CDR and the antigen; - The CDR sequence is transplanted into the human scaffold region.

[0211] In some embodiments, antibodies can be humanized by transplanting a CDR sequence into a human framework region, the CDR sequence being preferably determined by analyzing the structural interactions between the framework region (FR), the complementarity-determining region (CDR), and the antigen.

[0212] In some implementations, variations can be introduced into the amino acid sequence of the CDR or FR to maintain structural interactions with the antigen (which would otherwise be rendered invalid by introducing the human FR sequence), for example, by using a randomized method of phage display libraries or by a directed method guided by molecular modeling.

[0213] The DNA sequence encoding the antibody, as detailed herein, can be transferred into cells using known genetic engineering techniques and used to produce the antibody.

[0214] Antibody production

[0215] In another aspect, the antibody according to the invention is a monoclonal antibody obtainable by the method described herein, which is produced by a method comprising the steps of: - Cultivate cell lines containing nucleotide sequences encoding the antibodies; - Isolate the antibody from the culture.

[0216] In another particular aspect, the antibody according to the invention is a monoclonal antibody obtainable by the methods described herein, which is produced by isolating the antibody from a culture containing a cell line encoding a nucleotide sequence of the antibody.

[0217] In some embodiments of the method, the cell line is generated as described above and may include bacterial cells, such as Gram-negative bacteria like *Escherichia coli*, *Proteus mirabilis*, or *Pseudomonas putidas*; Gram-positive bacteria like *Bacillus brevis*, *Bacillus subtilis*, or *Bacillus megaterium*; lactobacilli like *Lactobacillus zeae / casei* or *Lactobacillus paracasei*; or *Streptomyces* like *Streptomyces lividans*; eukaryotic cells, such as yeasts like *Pichia pastoris*, *Saccharomyces cerevisiae*, *Hansenula polymorpha*, or *Schizosaccharomyces*. *Schwanniomyces occidentalis*, *Kluyveromyces lactis*, or *Yarrowia lipolytica*; fungi, such as filamentous fungi, such as *Trichoderma* or *Aspergillus* such as *A. niger* (e.g., *A. awamori* subgenus) and *Aspergillus oryzae*, *Trichoderma reesei*, *C. lucknowense*; protozoa, such as leishmania such as *L. tarentolae*; insect cells, such as insect cells transfected with baculoviruses (e.g., AcNPV), such as insect cell lines from *Spodoptera frugiperda* such as Sf-9 or Sf-21, *Drosophila melanogaster* such as DS2, or *Trichopulsia*. ni) such as High Five cells (BTI-TN-5B1-4); mammalian cells, such as hamster cells, such as Chinese hamster ovary cells such as K1-, DukX B11-, DG44, Lec13 or BHK cells; mouse cells, such as mouse myeloma cells such as NS0 cells; Homo sapiens cells, such as Per.C6, AGE1.HN, HEK293 cells.

[0218] In some embodiments of the method, the cell may be a hybridoma cell, such as the hybridoma cell described herein.

[0219] In some embodiments of the method, the culture can be carried out in static suspension culture, stirred suspension culture, membrane-based culture, matrix-based culture, or high cell density bioreactor; the vessel used for such culture can be selected from T-flasks, rolling culture, swirling culture, stirred tank bioreactors, airlift bioreactors, static membrane-based or matrix-based culture systems, suspension bioreactors, fluidized bed bioreactors, ceramic bioreactors, perfusion systems, and hollow fiber bioreactors.

[0220] In some embodiments of the method, the cells can be fixed to a matrix.

[0221] High-cell-density bioreactors are specifically designed to produce more than 10 8 A culture system with a cell density of 1 cell / ml.

[0222] In another aspect, the antibody according to the invention is a monoclonal antibody obtainable by the method described herein, which is produced by a method comprising the steps of: - To produce transgenic plants or animals containing nucleotide sequences encoding the antibodies; - Isolate the antibody from the plant or animal or from the secretions or products of the plant or animal.

[0223] In another particular aspect, the antibody according to the invention is a monoclonal antibody obtainable by the methods described herein, which is produced by isolating the antibody from a transgenic plant or animal having a nucleotide sequence encoding the antibody, or from the secretions or products of the transgenic plant or animal.

[0224] The animal may be selected, for example, from chickens, mice, rats, rabbits, cows, goats, sheep, or pigs; the secretion or product may be, for example, milk or eggs. The plant may be selected, for example, from tobacco (N. tabacum or N. benthamiana), duckweed (Lemna minor), Chlamydomonas reinhardtii, rice, Arabidopsis thaliana, alfalfa (Medicago sativa), lettuce, or corn.

[0225] In some embodiments, the antibodies can be separated by: physicochemical fractionation, such as size exclusion chromatography, precipitation (e.g., using ammonium sulfate), ion exchange chromatography, fixed metal chelate chromatography-gel filtration, and zone electrophoresis; classification based on their binding, such as to bacterial proteins A, G, or L, or jacalin; antigen-specific affinity purification by immobilized ligands / antigens; and, if necessary, removal of low molecular weight components by methods such as dialysis, desalting, and percolation.

[0226] In some embodiments, the antibody is encoded by a nucleotide sequence, wherein the nucleotide sequence is a reverse transcription of the amino acid sequence of an antibody produced by one of the methods described herein.

[0227] Against the backdrop described above, the following sequentially numbered embodiments provide other specific aspects of the invention: 1. A method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients, the method comprising: ● Determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and ● Associate the levels of the proenkephalin or fragments thereof in the sample with the diagnosis and / or risk and / or severity of graft dysfunction. Reduced graft function is defined as slowed graft function (SGF) or delayed graft function (DGF) in the kidneys.

[0228] 2. A method for patient stratification and / or patient selection for early treatment of graft dysfunction in kidney transplant patients, the method comprising: - To determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and - By correlating the levels of the proenkephalin or its fragments in the sample, patients can be stratified and / or selected for early treatment of graft-versus-graft dysfunction. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

[0229] 3. The method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiment 1 or 2, wherein if the level of the proenkephalin or fragment thereof in a body fluid sample obtained from the patient rises above a predetermined threshold level, the patient has graft dysfunction and / or is at risk of graft dysfunction and / or is stratified or selected for early treatment of graft dysfunction.

[0230] 4. The method for early diagnosis and / or early risk prediction and / or risk monitoring or severity prediction of graft dysfunction in kidney transplant patients according to Embodiment 3, wherein the predetermined threshold level is in the range of 50 to 750 pmol / L, more preferably in the range of 100 to 500 pmol / L, even more preferably in the range of 150 to 400 pmol / L, and most preferably in the range of 200 to 300 pmol / L.

[0231] 5. The method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 1 to 4, wherein the level of proenkephalin or fragments thereof in body fluid samples obtained from said patients is (i) measured at least once before and after kidney transplantation, or (ii) measured at least once after kidney transplantation.

[0232] 6. A method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of reduced graft function in kidney transplant patients according to embodiment 1, 2 or 5, wherein (i) body fluid samples are obtained from the patient at least once before and after kidney transplantation or (ii) at least twice after kidney transplantation, and the relative change in the level of the proenkephalin or a fragment thereof is calculated, wherein the relative change is related to the diagnosis and / or risk and / or severity of reduced kidney graft function.

[0233] 7. The method of claim 6 for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients, wherein when the level of an earlier sample is set to 100%, if the relative change between the levels of proenkephalin or fragments thereof in samples obtained at least once before and after kidney transplantation or at least twice after kidney transplantation is a decrease of less than 50% or an increase, then graft dysfunction is diagnosed and / or the risk of graft dysfunction is predicted in the patient.

[0234] 8. The method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 1 to 7, wherein...

[0235] (i) If the at least one body fluid sample is obtained after kidney transplantation, the sample is obtained within 96 hours, more preferably within 72 hours, even more preferably within 48 hours, even more preferably within 24 hours, and most preferably within 12 hours after kidney transplantation.

[0236] (ii) If the at least two body fluid samples are obtained before and after kidney transplantation, then the body fluid sample obtained after kidney transplantation is obtained within 96 hours, more preferably within 72 hours, even more preferably within 48 hours, even more preferably within 24 hours, and most preferably within 12 hours after kidney transplantation, or wherein...

[0237] (iii) If the at least two body fluid samples are obtained after kidney transplantation, the first sample after kidney transplantation is obtained within 96 hours, more preferably within 72 hours, even more preferably within 48 hours, even more preferably within 24 hours, and most preferably within 12 hours after kidney transplantation.

[0238] 9. The method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 1 to 8, wherein the determination of proenkephalin or fragments thereof is performed more than once as a follow-up measurement in the patients after kidney transplantation.

[0239] 10. The method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 1 to 9, wherein if graft dysfunction is diagnosed and / or the risk of graft dysfunction is predicted in the patient, the patient requires renal replacement therapy and / or administration of medication and / or adjustment of immunosuppressive therapy and / or adjustment of nephrotoxic drugs.

[0240] 11. The method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiment 10, wherein the renal replacement therapy is selected from dialysis (hemodialysis or peritoneal dialysis), hemofiltration, and hemodiafiltration.

[0241] 12. The method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiment 10, wherein the administered drug is a treatment with recombinant alkaline phosphatase, pegylated carboxyhemoglobin, relaxin, hepatocyte growth factor, mirocept, or a C1 esterase inhibitor.

[0242] 13. The method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 1 to 12, wherein the patient is receiving immunosuppressive therapy selected from interleukin-2 receptor antagonists, calcineurin inhibitors (cyclosporine A, tacrolimus), mammalian targets of rapamycin inhibitors, corticosteroids, mycophenolate mofetil, sirolimus, and azathioprine.

[0243] 14. A method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 1 to 13, wherein the nephrotoxic drug is selected from calcineurin inhibitors for immunosuppression (e.g., cyclosporine A, tacrolimus), analgesics (e.g., nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, aspirin), antimicrobial agents (e.g., aminoglycosides, cephalosporins, penicillins, quinolones, rifampin, and vancomycin), cholesterol-lowering statins, angiotensin-converting enzyme inhibitors (ACE inhibitors), angiotensin receptor blockers (ARBs), and diuretics, chemotherapeutic agents (e.g., cisplatin), and the nephrotoxic drug is modified and / or reduced and / or discontinued if graft dysfunction is diagnosed and / or the risk of graft dysfunction is predicted in the patient.

[0244] 15. The method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 1 to 14, wherein the enkephalinogen or fragment is selected from SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 and SEQ ID No. 12.

[0245] 16. The method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 1 to 15, wherein the patient is an adult or a child.

[0246] 17. The method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 1 to 16, wherein the body fluid sample may be selected from whole blood, serum, plasma, urine, cerebrospinal fluid (CSF) and saliva.

[0247] 18. The method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 1 to 17, wherein the body fluid sample may be selected from whole blood, serum, or plasma.

[0248] 19. The method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 1 to 18, wherein at least one additional clinical parameter is measured, selected from alanine aminopeptidase, alkaline phosphatase, gamma-glutamyl transferase, calprotectin, CC motif chemokine ligand 14, chitinase 3-like protein 1, hepatocyte growth factor, hepcidin, IL-18, β-trace protein (BTP), cystatin C, KIM-1, TIMP-2, IGFBP-7, blood urea nitrogen (BUN), NGAL, liver-type fatty acid-binding protein, monocyte chemoattractant peptide-1, creatinine clearance, serum creatinine (SCr), urea, metrin-1, osteopontin, retinol-binding protein, tumor necrosis factor, and Apache score.

[0249] 20. A method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 1 to 19, the method comprising determining the level of proenkephalin or a fragment thereof in a body fluid sample using at least one binding agent, wherein the at least one binding agent binds to a region within an amino acid sequence selected from SEQ ID No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, preferably the at least one binding agent binds to a region having a sequence selected from SEQ ID No. 1, 2, 5, 6, 8, 9, 10 and 11, preferably the at least one binding agent binds to a region having a sequence selected from SEQ ID No. 1, 2, 5, 6, 8 and 9, preferably the at least one binding agent binds to SEQ ID No. 6.

[0250] 21. The method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 1 to 20, wherein the level of proenkephalin or a fragment thereof is measured using an immunoassay, and the binding agent is an antibody or antibody fragment that binds to proenkephalin or a fragment thereof.

[0251] 22. The method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 1 to 21, wherein an immunoassay comprising two binding agents is used, said binding agents binding to two different regions within the proenkephalin region (i.e., amino acids 133-140 (LKELLETG, SEQ ID NO. 13) and amino acids 152-159 (SDNEEEVS, SEQ ID No. 14)), wherein each said region contains at least 4 or 5 amino acids.

[0252] 23. The method for early diagnosis and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 21 and 22, wherein the assay sensitivity is < 15 pmol / L, preferably < 10 pmol / L, and most preferably < 6 pmol / L.

[0253] 24. The method for early diagnosis and / or early risk prediction and / or risk monitoring of reduced graft function in kidney transplant patients according to embodiments 1 to 23, wherein the method is used to stratify the patients into risk groups.

[0254] 25. A method for early treatment of impaired graft function in kidney transplant patients, wherein the treatment is selected from renal replacement therapy and / or administration of medications and / or adjustment of immunosuppressive therapy and / or adjustment of nephrotoxic drugs, wherein the patient is selected by a diagnostic method comprising the following steps: - To determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and - Associate the level of the proenkephalin or fragment thereof in the sample with the diagnosis of graft-deficient function and / or the risk of graft-deficient function. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

[0255] 26. A medicine for the early treatment of impaired graft function in kidney transplant patients, wherein the patients are selected by a diagnostic method comprising the following steps: - To determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and - Associate the level of the proenkephalin or fragment thereof in the sample with the diagnosis of graft-deficient function and / or the risk of graft-deficient function. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

[0256] 27. A medicament for use according to embodiment 26, wherein the medicament is selected from recombinant alkaline phosphatase, PEGylated carboxyhemoglobin, relaxin, hepatocyte growth factor, mirocept, and C1 esterase inhibitors.

[0257] 28. Use of a point-of-care device for performing the method according to embodiments 1 to 27, wherein the point-of-care device comprises at least two antibodies or antibody fragments targeting amino acids 133-140 (LKELLETG, SEQ ID No. 13) and 152-159 (SDNEEEVS, SEQ ID No. 14).

[0258] 29. Use of a kit for performing the method according to embodiments 1 to 27, wherein the kit comprises at least two antibodies or antibody fragments targeting amino acids 133-140 (LKELLETG, SEQ ID No. 13) and 152-159 (SDNEEEVS, SEQ ID No. 14).

[0259] In the context described above, the following embodiments relating to aspect B of the present invention also constitute a part of the present invention: 1. A method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft function impairment in kidney transplant patients, the method comprising: ● Determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and ● The level of the proenkephalin or fragment thereof in the sample is associated with the risk and / or severity of graft function and / or reduced graft function.

[0260] Reduced graft function is defined as slowed graft function (SGF) or delayed graft function (DGF) in the kidneys.

[0261] 2. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiment 1 of aspect B, wherein the method is used for patient stratification and / or patient selection for early treatment of graft dysfunction in kidney transplant patients.

[0262] 3. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiment 1 or 2 of aspect B, wherein if the level of the proenkephalin or fragment thereof in a bodily fluid sample obtained from the patient rises above a predetermined threshold level, the patient is considered to have graft dysfunction and / or is at risk of graft dysfunction and / or is stratified or selected for early treatment of graft dysfunction.

[0263] 4. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring or severity prediction of graft function reduction in kidney transplant patients according to embodiment 3 of aspect B, wherein the predetermined threshold level is in the range of 50 to 750 pmol / L, more preferably in the range of 100 to 500 pmol / L, even more preferably in the range of 150 to 400 pmol / L, and most preferably in the range of 200 to 300 pmol / L.

[0264] 5. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft function reduction in kidney transplant patients according to embodiments 1 to 4 of aspect B, wherein the level of proenkephalin or fragments thereof in body fluid samples obtained from said patients is (i) measured at least once before and after kidney transplantation, or (ii) measured at least once after kidney transplantation.

[0265] 6. A method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft function reduction in kidney transplant patients according to embodiment 1, 2 or 5 of aspect B, wherein (i) body fluid samples are obtained from the patient at least once before and after kidney transplantation or (ii) at least twice after kidney transplantation, and the relative change in the level of the proenkephalin or fragment thereof is calculated, wherein the relative change is related to the risk and / or severity of graft function and / or graft function reduction in the kidney.

[0266] 7. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft function reduction in kidney transplant patients according to embodiment 6 of aspect B, wherein when the level of an earlier sample is set to 100%, if the relative change between the levels of proenkephalin or fragments thereof in samples obtained at least once before and after kidney transplantation or at least twice after kidney transplantation is a decrease of less than 50% or an increase, then a risk of graft function reduction is diagnosed and / or predicted in the patient.

[0267] 8. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 1 to 7 of aspect B, wherein...

[0268] (iv) If the at least one body fluid sample is obtained after kidney transplantation, the sample is obtained within 96 hours, more preferably within 72 hours, even more preferably within 48 hours, even more preferably within 24 hours, and most preferably within 12 hours after kidney transplantation.

[0269] (v) If the at least two body fluid samples are obtained before and after kidney transplantation, then the body fluid sample obtained after kidney transplantation is obtained within 96 hours, more preferably within 72 hours, even more preferably within 48 hours, even more preferably within 24 hours, and most preferably within 12 hours after kidney transplantation, or wherein...

[0270] (vi) If the at least two body fluid samples are obtained after kidney transplantation, the first sample after kidney transplantation is obtained within 96 hours, more preferably within 72 hours, even more preferably within 48 hours, even more preferably within 24 hours, and most preferably within 12 hours after kidney transplantation.

[0271] 9. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft function reduction in kidney transplant patients according to embodiments 1 to 8 of aspect B, wherein the determination of proenkephalin or fragments thereof is performed more than once as a follow-up measurement in the patients after kidney transplantation.

[0272] 10. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft function reduction in kidney transplant patients according to embodiments 1 to 9 of aspect B, wherein if graft function reduction is diagnosed and / or the risk of graft function reduction is predicted in the patient, the patient requires renal replacement therapy and / or administration of medication and / or adjustment of immunosuppressive therapy and / or adjustment of nephrotoxic drugs.

[0273] 11. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft function decline in kidney transplant patients according to embodiment 10 of aspect B, wherein the renal replacement therapy is selected from dialysis (hemodialysis or peritoneal dialysis), hemofiltration and hemodiafiltration.

[0274] 12. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft function impairment in kidney transplant patients according to embodiment 10 of aspect B, wherein the administered drug is a treatment with recombinant alkaline phosphatase, pegylated carboxyhemoglobin, relaxin, hepatocyte growth factor, mirocept, or a C1 esterase inhibitor.

[0275] 13. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 1 to 12 of aspect B, wherein the patient is receiving immunosuppressive therapy selected from interleukin-2 receptor antagonists, calcineurin inhibitors (cyclosporine A, tacrolimus), mammalian targets of rapamycin inhibitors, corticosteroids, mycophenolate mofetil, sirolimus, and azathioprine.

[0276] 14. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiment 10 of aspect B, wherein the nephrotoxic drug is selected from calcineurin inhibitors for immunosuppression (e.g., cyclosporine A, tacrolimus), analgesics (e.g., nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, aspirin), antimicrobial agents (e.g., aminoglycosides, cephalosporins, penicillins, quinolones, rifampin, and vancomycin), cholesterol-lowering statins, angiotensin-converting enzyme inhibitors (ACE inhibitors), angiotensin receptor blockers (ARBs), and diuretics, chemotherapeutic agents (e.g., cisplatin), and the nephrotoxic drug is modified and / or reduced and / or discontinued if graft dysfunction is diagnosed and / or the risk of graft dysfunction is predicted in the patient.

[0277] 15. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 1 to 11 of aspect B, wherein the enkephalinogen or fragment is selected from SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 and SEQ ID No. 12.

[0278] 16. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to embodiments 1 to 12 of aspect B, wherein the body fluid sample may be selected from whole blood, serum, plasma, urine, cerebrospinal fluid (CSF) and saliva.

[0279] 17. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring of graft function decline in kidney transplant patients according to embodiments 1 to 13 of aspect B, wherein the body fluid sample may be selected from whole blood, serum, or plasma.

[0280] 18. A method for early treatment of graft dysfunction in kidney transplant patients, wherein the treatment is selected from renal replacement therapy and / or administration of medications and / or adjustment of immunosuppressive therapy and / or adjustment of nephrotoxic drugs, wherein the patient is selected by a diagnostic method comprising the following steps: - To determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and - The level of the proenkephalin or fragment thereof in the sample was associated with the risk of graft function and / or reduced graft function. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

[0281] 19. A medicine for the early treatment of impaired graft function in kidney transplant patients, wherein the patients are selected by a diagnostic method comprising the following steps: - To determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and - Associate the level of the proenkephalin or fragment thereof in the sample with the diagnosis of graft-deficient function and / or the risk of graft-deficient function. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

[0282] 20. The medicament for early treatment of graft dysfunction in kidney transplant patients according to embodiment 19 of aspect B, wherein the medicament is selected from recombinant alkaline phosphatase, pegylated carboxyhemoglobin, relaxin, hepatocyte growth factor, mirocept, and C1 esterase inhibitors.

[0283] 21. The medicament for early treatment of graft dysfunction in kidney transplant patients according to embodiments 19 and 20 of aspect B, wherein the patient is at risk of graft dysfunction and / or graft dysfunction if the level of the proenkephalin or a fragment thereof in a bodily fluid sample obtained from the patient rises above a predetermined threshold level.

[0284] 22. The medicament for early treatment of reduced graft function in kidney transplant patients according to embodiment 21 of aspect B, wherein the predetermined threshold level is in the range of 50 to 750 pmol / L, more preferably in the range of 100 to 500 pmol / L, even more preferably in the range of 150 to 400 pmol / L, and most preferably in the range of 200 to 300 pmol / L.

[0285] 23. The medicament for early treatment of graft dysfunction in kidney transplant patients according to embodiments 19 to 22 of aspect B, wherein the level of proenkephalin or fragments thereof in body fluid samples obtained from said patients is (i) measured at least once before and after kidney transplantation, or (ii) measured at least once after kidney transplantation.

[0286] 24. The medicament for early treatment of graft dysfunction in kidney transplant patients according to embodiments 19 to 23 of aspect B, wherein (i) at least once before and after kidney transplantation or (ii) at least twice after kidney transplantation, fluid samples are obtained from the patient, and the relative change in the level of the proenkephalin or a fragment thereof is calculated, wherein the relative change is related to the risk and / or severity of graft dysfunction and / or graft dysfunction.

[0287] 25. The medicament for early treatment of graft dysfunction in kidney transplant patients according to embodiment 24 of aspect B, wherein when the level of an earlier sample is set to 100%, if the relative change between the levels of proenkephalin or fragments thereof in samples obtained at least once before and after kidney transplantation or at least twice after kidney transplantation is a decrease of less than 50% or an increase, then graft dysfunction is diagnosed and / or the risk of graft dysfunction is predicted in the patient.

[0288] 26. The medicament for early treatment of graft dysfunction in kidney transplant patients according to embodiments 19 to 25 of aspect B, wherein...

[0289] (i) If the at least one body fluid sample is obtained after kidney transplantation, then the sample is obtained within 96 hours, more preferably within 72 hours, even more preferably within 48 hours, even more preferably within 24 hours, and most preferably within 12 hours after kidney transplantation, or wherein...

[0290] (ii) If the at least two body fluid samples are obtained before and after kidney transplantation, then the body fluid sample obtained after kidney transplantation is obtained within 96 hours, more preferably within 72 hours, even more preferably within 48 hours, even more preferably within 24 hours, and most preferably within 12 hours after kidney transplantation, or wherein...

[0291] (iii) If the at least two body fluid samples are obtained after kidney transplantation, the first sample after kidney transplantation is obtained within 96 hours, more preferably within 72 hours, even more preferably within 48 hours, even more preferably within 24 hours, and most preferably within 12 hours after kidney transplantation.

[0292] 27. The medicament for early treatment of graft dysfunction in kidney transplant patients according to embodiments 19 and 26 of aspect B, wherein the determination of proenkephalin or fragments thereof is performed more than once as a follow-up measurement in the patients after kidney transplantation.

[0293] 28. The medicament for early treatment of graft dysfunction in kidney transplant patients according to embodiments 19 to 27 of aspect B, wherein if graft dysfunction is diagnosed and / or the risk of graft dysfunction is predicted in the patient, the patient additionally requires renal replacement therapy and / or adjustment of immunosuppressive therapy and / or adjustment of nephrotoxic drugs.

[0294] 29. The medicine for early treatment of reduced graft function in kidney transplant patients according to embodiment 28 of aspect B, wherein the renal replacement therapy is selected from dialysis (hemodialysis or peritoneal dialysis), hemofiltration and hemodiafiltration.

[0295] 30. The nephrotoxic drug for early treatment of graft dysfunction in kidney transplant patients according to embodiment 28 of aspect B, wherein the nephrotoxic drug is selected from calcineurin inhibitors for immunosuppression (e.g., cyclosporine A, tacrolimus), analgesics (e.g., nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, aspirin), antimicrobial agents (e.g., aminoglycosides, cephalosporins, penicillins, quinolones, rifampin, and vancomycin), cholesterol-lowering statins, angiotensin-converting enzyme inhibitors (ACE inhibitors), angiotensin receptor blockers (ARBs), and diuretics, chemotherapeutic agents (e.g., cisplatin), and the nephrotoxic drug is modified and / or reduced and / or discontinued if graft dysfunction is diagnosed and / or the risk of graft dysfunction is predicted in the patient.

[0296] 31. The medicament for early treatment of graft dysfunction in kidney transplant patients according to embodiments 19 to 30 of aspect B, wherein the enkephalin or fragment is selected from SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 and SEQ ID No. 12.

[0297] 32. The medicament for early treatment of graft dysfunction in kidney transplant patients according to embodiments 19 to 31 of aspect B, wherein the fluid sample may be selected from whole blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.

[0298] 33. The medicament for early treatment of graft dysfunction in kidney transplant patients according to embodiment 32 of aspect B, wherein the body fluid sample may be selected from whole blood, serum, or plasma.

[0299] 34. Use of a point-of-care device for performing the method according to embodiments 1 to 16 of aspect B, wherein the point-of-care device comprises at least two antibodies or antibody fragments targeting amino acids 133-140 (LKELLETG, SEQ ID No. 13) and 152-159 (SDNEEEVS, SEQ ID No. 14).

[0300] 35. Use of a kit for performing the method according to embodiments 1 to 16 of aspect B, wherein the kit comprises at least two antibodies or antibody fragments targeting amino acids 133-140 (LKELLETG, SEQ ID No. 13) and 152-159 (SDNEEEVS, SEQ ID No. 14).

[0301] 36. A method for predicting renal function in kidney transplant patients, the method comprising: ● Determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and ● Correlate the levels of the proenkephalin or its fragments in the sample with the patient's renal function. The renal function mentioned therein is defined as glomerular filtration rate (GFR), creatinine clearance rate (CCr), serum creatinine (SCr), urinalysis, blood urea nitrogen, or urine output.

[0302] 37. The method for predicting renal function in a kidney transplant patient according to embodiment 36 of aspect B, wherein a decrease in renal function is predicted if the level of the proenkephalin or a fragment thereof in a body fluid sample obtained from the patient increases to above a predetermined threshold level.

[0303] 38. The method for predicting renal function in kidney transplant patients according to embodiments 36 and 37 of aspect B, wherein the prediction is made within 12 months, more preferably within 9 months, more preferably within 6 months, more preferably within 3 months, more preferably within 1 month, and most preferably within 14 days.

[0304] 39. The method for predicting renal function in kidney transplant patients according to embodiment 38 of aspect B, wherein the prediction is a short-term prediction within 3 months, preferably within 1 month, more preferably within 28 days, even more preferably within 21 days, and most preferably within 14 days.

[0305] 40. The method for predicting renal function in a kidney transplant patient according to embodiment 37 of aspect B, wherein the predetermined threshold level is in the range of 50 to 750 pmol / L, more preferably in the range of 100 to 500 pmol / L, even more preferably in the range of 150 to 400 pmol / L, and most preferably in the range of 200 to 300 pmol / L.

[0306] 41. The method for predicting renal function in a kidney transplant patient according to embodiments 36 to 40 of aspect B, wherein the level of proenkephalin or fragments thereof in a body fluid sample obtained from the patient is (i) measured at least once before and after kidney transplantation, or (ii) measured at least once after kidney transplantation.

[0307] 42. The method for predicting renal function in a kidney transplant patient according to embodiments 36 to 40 of aspect B, wherein (i) a body fluid sample is obtained from the patient at least once before and after kidney transplantation or (ii) at least twice after kidney transplantation, and a relative change in the level of the proenkephalin or a fragment thereof is calculated, wherein the relative change is related to the renal function.

[0308] 43. The method for predicting renal function in a kidney transplant patient according to embodiment 42 of aspect B, wherein when the level of an earlier sample is set to 100%, a decrease in renal function is predicted in the patient if the relative change between the levels of proenkephalin or fragments thereof in samples obtained at least once before and after kidney transplantation or at least twice after kidney transplantation is a decrease of less than 50% or an increase.

[0309] 44. The method for predicting renal function in a kidney transplant patient according to embodiments 36 to 43 of aspect B, wherein the enkephalin or fragment is selected from SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 and SEQ ID No. 12.

[0310] 45. The method for predicting renal function in kidney transplant patients according to embodiments 36 to 44 of aspect B, wherein the body fluid sample may be selected from whole blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.

[0311] 46. ​​The method for predicting renal function in kidney transplant patients according to embodiments 36 to 44 of aspect B, wherein the body fluid sample may be selected from whole blood, serum, and blood.

[0312] 47. The method for predicting renal function in kidney transplant patients according to embodiments 36 to 46 of aspect B, wherein the GFR is an estimated GFR (eGFR), a true GFR, or a measured GFR (mGFR).

[0313] 48. The method for predicting renal function in kidney transplant patients according to embodiments 36 to 47 of aspect B, wherein the predicted GFR decreases to below 60, preferably below 45, preferably below 30, and most preferably below 15.

[0314] Example

[0315] Example 1

[0316] Antibody development

[0317] peptides

[0318] Peptides are synthesized (JPT Technologies, Berlin, Germany).

[0319] Peptides / conjugates used for immunity: Peptides for immunization (Table 1) (JPT Technologies, Berlin, Germany) were synthesized with an additional N-terminal cysteine ​​residue for conjugating the peptide to bovine serum albumin (BSA). The peptide was covalently linked to BSA using Sulfo-SMCC (PerbioScience, Bonn, Germany). The conjugation procedure was performed according to the Perbio manual.

[0320] Table 1: Names of Immunopeptides and Antibodies

[0321] The antibody is produced according to the following method: BALB / c mice were immunized on days 0 and 14 with 100 µg of the peptide-BSA conjugate (emulsified in 100 µl of complete Freund's adjuvant), and on days 21 and 28 with 50 µg of the conjugate (in 100 µl of incomplete Freund's adjuvant). Three days prior to the fusion experiment, animals received 50 µg of the conjugate dissolved in 100 µl of saline, administered once intraperitoneally and once intravenously.

[0322] Using 1 ml of 50% polyethylene glycol, spleen cells from the immunized mice and myeloma cell line SP2 / 0 were fused at 37°C for 30 s. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growth in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement]. After two weeks, the HAT medium was replaced with HT medium, and the cells were passaged three times before returning to normal cell culture medium.

[0323] Three weeks after fusion, the cell culture supernatant was used for preliminary screening of antigen-specific IgG antibodies. Microcultures testing positive were transferred to 24-well plates for propagation. After retesting, selected cultures were cloned and re-cloned using limiting dilution techniques to determine the isotype. Lane, RD 1985 J. Immunol. Meth. 81: 223-228; Ziegler, B. et al., 1996. Horm. Metab. Res. 28: 11-15 ).

[0324] Monoclonal antibody production

[0325] Antibodies are produced using standard antibody production methods. Marx et al., 1997. ATLA 25, 121 The antibody was purified by protein A chromatography. Based on SDS gel electrophoresis analysis, the antibody purity was >95%.

[0326] Antibody labeling and coating

[0327] All antibodies were labeled with acridine esters according to the following procedure: Labeled compound (tracer): 100 µg (100 µl) of antibody (1 mg / ml, in PBS, pH 7.4) was mixed with 10 µl of acridine-NHS ester (1 mg / ml, in acetonitrile, InVent GmbH, Germany) (EP 0353971) and incubated at room temperature for 20 min. The labeled antibody was purified by gel filtration HPLC on a Bio-Sil SEC 400-5 (Bio-Rad Laboratories, Inc., USA). The purified labeled antibody was diluted in (300 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na-EDTA, 5 g / L bovine serum albumin, pH 7.0). The final concentration was approximately 800,000 relative optical units (RLU) of the labeled compound per 200 µl (approximately 20 ng of labeled antibody). The chemiluminescence of acridine esters was measured using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

[0328] Solid-phase antibody (coating antibody): Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated with antibody (1.5 µg antibody / 0.3 ml 100 mmol / L NaCl, 50 mmol / L Tris / HCl, pH 7.8) (at room temperature for 18 h). After blocking with 5% bovine serum albumin, the tubes were washed with PBS at pH 7.4 and vacuum dried.

[0329] antibody specificity

[0330] Antibody cross-reactivity was determined as follows: 1 µg of peptide in 300 µl of PBS (pH 7.4) was pipette into a polystyrene tube and incubated at room temperature for 1 h. After incubation, the tube was washed five times (1 ml each time) with 5% BSA solution in PBS (pH 7.4). Each labeled antibody (300 µl, in PBS, pH 7.4, 800,000 RLU / 300 µl) was added and incubated at room temperature for 2 h. After five washes (1 ml each time with 20 mmol / L PBS, pH 7.4, 0.1% Triton X 100), the remaining luminescence (labeled antibody) was quantified using an AutoLumat 953 luminometer. The synthesized MR-PENK peptide was used as a reference (100%).

[0331] Table 2 lists the cross-reactivity of different antibodies.

[0332] Table 2: Cross-reactivity of different PENK antibodies

[0333] All antibodies bound to the MR-PENK peptide, comparable to peptides used for immunization. Except for the NT-MR-PENK antibody (which showed 10% cross-reactivity with EEDDSLANSSDLLK), no antibodies showed cross-reactivity with MR-PENK fragments not used for individual antibody immunization.

[0334] Enkephalin immunoassay: Pipettes 50 µl of sample (or calibrator) into coated tubes, add 200 µl of labeled antibody, and incubate at 18–25 °C for 2 h. Unbound tracer is removed by washing five times (1 ml each time) with washing solution (20 mmol / L PBS, pH 7.4, 0.1% Triton X 100). The amount of labeled antibody bound to the tube is measured using a luminometer 953. A fixed concentration of 1000 pmol MR-PENK / L is used. Table 3 shows the signal-to-noise ratio (RLU) to noise (RLU without MR-PENK) for different antibody combinations. All antibodies form sandwich complexes with any other antibody. Surprisingly, the strongest signal-to-noise ratio (optimal sensitivity) is achieved by combining MR-MR-PENK and CT-MR-PENK antibodies. We subsequently used this antibody combination for MR-PENK immunoassays for further research. MR-MR-PENK antibody was used as the coating antibody, and CT-MR-PENK antibody was used as the labeling antibody.

[0335] Table 3: Signal-to-noise ratio of different antibody combinations

[0336] calibration: The assay was calibrated using a dilution of synthetic MR-PENK diluted in 20 mM K2PO4, 6 mM EDTA, 0.5% BSA, 50 μM aminopeptidase inhibitor, and 100 μM leucopeptide (pH 8.0). Figure 1 Typical proenkephalin dose / signal curves are shown. The assay sensitivity was 5.5 pmol / L for 20 measurements with calibrator zero (without MR-PENK) + 2 SD.

[0337] Example 2

[0338] The aim of this study was to evaluate the ability of the biomarker proenkephalin A 119-159 (PENK or penKid) to predict, compared with serum creatinine (SCr), kidney transplant recipients for reduced graft function, particularly slowed graft function (SGF) and delayed graft function (DGF).

[0339] Sample collection and analysis

[0340] EDTA plasma samples were obtained from 159 patients. Multiple blood samples were obtained from each patient: one sample each on the day of transplantation (day 0, pre-transplant, pre-Tx) until 35 days post-transplantation (every 24 hours). SCr in plasma samples was measured using a commercially available enzyme assay. (See Donato et al., 2018). Donato et al., 2018. Clin Biochem. 58: 72-77 As described in the paper, PENK was measured using a double monoclonal sandwich immunoassay.

[0341] Delayed graft function (DGF) was defined as any renal replacement therapy (RRT) within 7 days post-transplantation (n=53). Slowed graft function (SGF) was defined as no RRT therapy within 7 days post-transplantation and a creatinine quotient on day 7 / day 0 < 0.7. Immediate graft function (IGF) was defined as no RRT therapy within 7 days post-transplantation and a creatinine quotient on day 7 / day 0 > 0.7.

[0342] DGF severity was divided into three severity groups, defined as 0 / 1 (RRT performed only in the first 24 hours of day 0 / day 1), 2-7 (RRT ended between day 2 and day 7), and >7 (RRT lasted longer than day 7).

[0343] All patients underwent successful transplants and regained kidney function before discharge.

[0344] Statistical analysis

[0345] Values ​​are expressed as median and interquartile range (IQR) or count and percentage (as applicable). Group comparisons of continuous variables were performed using the Kruskal-Wallis test, and post-hoc tests were performed for variables with more than two categories. Logarithmic transformation was performed on biomarker data (SCr and PENK). Categorical data were compared using the Pearson chi-square test for count data. Logistic regression was used to assess and compare the predictive power of PENK and SCr for DGF. To demonstrate the independence of clinical variables, the added value of PENK in predicting DGF on top of a multivariate model with the most relevant clinical variables was assessed based on a likelihood ratio chi-square test of nested models. A concordance index (C-index or AUC) is given as an effect measure for both univariate and multivariate models. For multivariate models, a bootstrap-corrected version of the C-index / AUC is given. Receiver operating characteristic (ROC) curves were constructed and plotted to assess the sensitivity and specificity of PENK measurements obtained at different time points for predicting and diagnosing DGF. Changes in PENK and SCr were calculated as a percentage of pre-transplant biomarker concentrations.

[0346] All statistical tests were two-tailed, and a two-tailed p-value of 0.05 was considered significant. Statistical analyses were performed using R version 4.2.2 (http: / / www.r-project.org, libraries rms, Hmisc, ROCR) and the Social Sciences Statistics Package (SPSS) version 22.0 (SPSS Inc., Chicago, Illinois, USA).

[0347] result: This study included 159 kidney transplant recipients (n=109 of whom were cadaveric grafts). 53 patients developed disease-free grafting (DGF). Among the 106 non-DGF patients, n=45 developed severe disease-free grafting (SGF). Patients with DGF were grouped by severity: DGF 0 / 1 (n=19), DGF 2-7 (n=17), and DGF >7 (n=17). Patient characteristics are described in Table 4.

[0348] Table 4: Patient characteristics of the study cohort

[0349] like Figure 2A As shown, PENK can distinguish between DGF patients and non-DGF patients (including both IGF and SGF) as early as 24 hours post-transplantation. After the initial decrease in PENK after transplantation, PENK levels remain elevated in DGF patients until renal function is finally established in most patients in the group (after day 8). The separation between DGF and non-DGF patients is earlier and more pronounced compared to SCr. Figure 2B).

[0350] Patient levels before transplantation (pre-Tx) may vary, as can be seen from... Figure 2A This is evident from the distribution of the pre-transplantation bounding boxes in B. Therefore, the changes compared to the pre-transplantation values ​​were investigated. Figure 3A As shown in Figure B, the observed difference between PENK and SCr remains even after adjusting for pre-transplant concentrations. Figure 3A The results showed that, when assessing changes compared to pre-transplant concentrations, the PENK differences observed between DGF-positive and DGF-negative patients were present as early as 24 hours post-transplant. Although all patients experienced a decrease in PENK concentration, most DGF-negative patients saw their concentrations drop to below 50% of pre-transplant concentrations within 24 hours. Comparison with SCr showed that for most non-DGF-positive patients, a decrease to below 50% of pre-transplant concentrations took until day 3.

[0351] As shown in Figure 4A, PENK could distinguish DGF, SGF, and IGF as early as 24 hours after transplantation (day 1, p<0.0001), while SCr could not. Figure 4B (p=0.0657). Most notably, as early as day 1, PENK concentrations in SGF patients were significantly lower than those in DGF patients (post-hoc p=0.0002). SCr could not distinguish between SGF and DGF on day 1 (post-hoc p=0.3504). The trajectory up to day 21 indicated that changes in PENK preceded changes in SCr throughout the observation period.

[0352] As shown in Figures 5A and 5B, the differences between PENK and SCr observed remained after adjusting for pre-transplant concentrations. Changes in PENK as early as 24 hours post-transplantation (day 1, p < 0.0001) could distinguish DGF, SGF, and IGF, as could changes in SCr. Figure 5B (p<0.00001). However, most notably, PENK concentrations decreased more sharply in SGF patients as early as day 1 compared to DGF patients (post-hoc p<0.0001), and SGF and DGF patients could not be distinguished by changes in SCr, with the decrease in SGF patients being significantly smaller than that in DGF patients on day 1 (post-hoc p=0.0099). The trajectory up to day 21 indicated that changes in PENK preceded changes in SCr throughout the observation period, particularly for SGF patients.

[0353] While PENK can differentiate between SGF (which does not require post-transplant resuscitation therapy) and DGF (which requires post-transplant resuscitation therapy) as early as 24 hours post-transplantation, based on both absolute concentration and changes compared to pre-transplant levels, SCr cannot. This is a significant advantage of PENK, as it allows for earlier decision to treat patients with DGF who will develop DGF with resuscitation therapy, rather than those who will develop SGF, compared to the standard biomarker SCr.

[0354] Next, we performed logistic regression on the endpoints DGF and SGF (excluding IGF) (Table 5). This confirmed that PENK was superior to SCr on days 1, 2, and 3. PENK outperformed SCr on days 1, 2, and 3 (all p < 0.0001). For changes on days 1, 2, and 3, changes in PENK were also superior to changes in SCr (all p < 0.0001). The AUC of PENK increased from 0.73 on day 1 to 0.81 on day 2 and 0.84 on day 3. Notably, the change in PENK on day 1 already reached a similar AUC (0.79), suggesting a temporal benefit if the trajectory from before transplantation is taken into account.

[0355] Table 5: Logistic regression results of PENK, SCr, and pre-transplant changes in PENK and SCr on the first 3 days post-transplantation, with endpoints DGF and SGF (excluding IGF patients). Model Chi 2 Chi 2 Statistics; df: degrees of freedom; LR p-value: likelihood ratio p-value; C-index: consistency index or AUC; CI: confidence interval.

[0356]

[0357] Figure 6A The absolute values ​​and 6B (variation) illustrate the differences in DGF prediction between PENK and SCr on days 1, 2, and 3 post-transplantation.

[0358] Exemplary thresholds and corresponding sensitivity and specificity are as follows: Using an absolute PENK cutoff of 300 pmol / L on day 1 post-transplantation, DGF patients could be identified with 95% sensitivity and 45% specificity (odds ratio 16.2). Using an absolute PENK cutoff of 200 pmol / L on day 2 post-transplantation, DGF patients could be identified with 97% sensitivity and 40% specificity (odds ratio 22.7). Using an absolute PENK cutoff of 200 pmol / L on day 3 post-transplantation, DGF patients could be identified with 95% sensitivity and 45% specificity (odds ratio 16.2).

[0359] Using a relative change of less than 50% in PENK from pre-transplant to day 1 post-transplant, patients with diabetic leukemia (DGF) can be identified with 89% sensitivity and 41% specificity (odds ratio 5.8). Using a relative change of less than 50% in PENK from pre-transplant to day 2 post-transplant, patients with DGF can be identified with 71% sensitivity and 79% specificity (odds ratio 9.0). Using a relative change of less than 50% in PENK from pre-transplant to day 3 post-transplant, patients with DGF can be identified with 65% sensitivity and 93% specificity (odds ratio 24.0).

[0360] Next, we applied logistic regression to the DGF endpoint (Table 6). This confirmed the superiority of PENK over SCr on days 1, 2, and 3. PENK was superior to SCr on days 1, 2, and 3 (all p < 0.0001). Figure 7A For changes on days 1, 2, and 3, the change in PENK was also better than that in SCr (all p < 0.0001). Figure 7B The AUC of PENK increased from 0.81 on day 1 to 0.88 on day 2 and 0.91 on day 3. Notably, the change in PENK on day 1 already reached a similar AUC (0.87), suggesting a temporal benefit if the trajectory from pre-transplantation is taken into account. Table 7 shows that both PENK at day 1 and the change in PENK at day 1 provided added value on top of a multivariate model consisting of known clinical risk factors for DGF (both p < 0.0001). In both cases, either PENK or the change in PENK was the strongest predictor in the model (both Chi). 2 >15), and cold ischemia time is the second strongest factor in both models (Chi). 2 >5).

[0361] Table 6: Logistic regression results of DGF, the endpoint of changes in PENK and SCr before transplantation, in the first 3 days post-transplantation. Model Chi 2 Chi 2 Statistics; df: degrees of freedom; LR p-value: likelihood ratio p-value; C-index: consistency index or AUC; CI: confidence interval.

[0362]

[0363] Figure 7A (Absolute value) and 7B (variation) illustrate the differences in DGF prediction by PENK and SCr on days 1, 2 and 3 post-transplantation.

[0364] Table 7: Multivariate logistic regression results for endpoint DGF in models including donor age, donor SCr, living donor vs. deceased donor, cold ischemia time, and duration of RRT before transplantation in the patient (model "Multi"), and combinations of said models with PENK at d1 (model "Multi, PENK") or PENK change at d1 (model "Multi, chng PENK"). 2 Chi 2 Statistics; df: degrees of freedom; LR p-value: likelihood ratio p-value; C-index: bootstrap corrected consistency index or AUC.

[0365]

[0366] Figures 8A and 8B show that PENK also differentiated DGF severity earlier than SCr. Although SCr decreased only after two weeks in DGF severity 0–1 (days 12–15, post-hoc p-value 0.0010), PENK in this group decreased after week 1 (days 6–8, post-hoc p-value <0.0001 for PENK, 0.6113 for SCr, comparing severity groups 0–1 with >7). Similarly, PENK decreased in patients with DGF severity 2–7 in week 2 (days 12–15, post-hoc p-value 0.0374 for PENK, 0.6728 for SCr, comparing severity groups 2–7 with >7), while for SCr, only a trend was observed in week 3 (day 21 / last day, p=0.1226). This trend was also observed for changes compared to pre-transplant values; see [link to relevant documentation]. Figure 9A And B.

[0367] For example, using an absolute PENK cutoff of 300 pmol / L on day 1 post-transplantation, patients with DGF can be identified with 95% sensitivity and 57% specificity (odds ratio 25.4). Therefore, in patients with PENK values ​​below 300 pmol / L on day 1, 96% will not develop DGF (negative predictive value), while in patients with values ​​above 300 pmol / L, 49% have already developed or will develop DGF (positive predictive value).

[0368] As another example, using an absolute PENK cutoff of 200 pmol / L on day 2 post-transplantation, patients with DGF could be identified with 97% sensitivity and 58% specificity (odds ratio 46.2). Therefore, in patients with PENK values ​​below 200 pmol / L on day 1, 98% will not develop DGF (negative predictive value), while in patients with values ​​above 200 pmol / L, 57% have already developed or will develop DGF (positive predictive value).

[0369] Using a relative change of less than 50% in PENK levels from pre-transplant to day 1 post-transplant, patients with dilated factor fibrosis (DGF) can be identified with 89% sensitivity and 67% specificity (odds ratio 16.5). Therefore, among patients with a pre-transplant PENK concentration change of less than 50% on day 1, 58% have already developed or will develop DGF (positive predictive value), while among patients with a change greater than 50%, 92% have not developed or will not develop DGF (negative predictive value). Changes from pre-transplant to day 2 or 3 provide similar performance criteria.

[0370] These are examples of cutoff values, and other cutoff values ​​can be used, depending on whether it is more appropriate to identify the majority of patients at risk of developing DGF (or SGF) at the cost of also identifying "false positives," or to identify primarily high-risk patients at the cost of missing a few intermediate-risk patients.

[0371] In summary, the results indicate that PENK can differentiate between patients with delayed graft function and those with primary graft resorption earlier than SCr. Compared to SCr, PENK can distinguish between DGF and non-DGF as early as 24 hours post-transplantation, measured as a single value and compared with a threshold, or as a relative change. Furthermore, the serial data strongly suggest that PENK can differentiate between SGF and DGF and can predict the duration of DGF (as reflected by DGF severity) much earlier than SCr.

[0372] Example 3

[0373] In a single-center observational study, proenkephalin A 119-159 (PENK or penKid) was investigated and compared with serum creatinine (SCr) to predict delayed graft function (DGF) on day 7 and a decrease in estimated glomerular filtration rate (eGFR) on day 30 after kidney transplantation.

[0374] Sample collection and analysis: This study included patients aged 18 years and older who were planning to undergo kidney transplantation. Exclusion criteria were age under 18 years and pregnancy.

[0375] Ethylenediaminetetraacetic acid (EDTA) plasma samples were collected on the day of transplantation (0–12 hours before transplantation, day 0, or before transplantation) and up to one day after transplantation (12–24 hours after transplantation, day 1). Serum creatinine was measured using an enzymatic method on an automated chemiluminescence analyzer (Konelab20XT, Thermo Fisher Scientific, Waltham, MA, USA). For PENK measurement, a non-automated immunoluminescent assay as described in Donato et al., 2018 (Donato et al., 2018. Clin Biochem. 58: 72–77) was used.

[0376] Delayed graft function (DGF) was defined as requiring renal replacement therapy (RRT) within the first seven days post-transplantation, differentiated by DGF severity 0 / 1 (RRT performed only on day 0 or day 1 post-transplantation, i.e., up to 24 hours), DGF severity 2-7 (RRT completed between day 2 and day 7), and DGF severity >7 (RRT lasting beyond day 7). Slowed graft function (SGF) was defined as the absence of RRT within the first seven days post-transplantation, with a day 7 SCr / day 0 SCr quotient <0.7. Immediate graft function (IGF) was defined as the absence of RRT within the first seven days post-transplantation, with a day 7 SCr / day 0 SCr quotient >0.7.

[0377] Decreased eGFR was defined as eGFR < 30 ml / min / 1.73 m 2 eGFR was determined using the latest CKD-EPI equation (2021). (Inker et al., 2021. N Eng J Med. 385:1737-1749).

[0378] Statistical analysis: Depending on the context, median and interquartile range (IQR) or count and percentage were reported. Group comparisons of continuous variables were performed using the Kruskal-Wallis test, and post-hoc tests were conducted for variables with more than two categories. Categorical data were compared using the Pearson chi-square test for count data. All statistical tests were two-tailed, with a two-tailed p-value of 0.05 considered significant. Logarithmic transformations were performed on the PENK and SCr data, and logistic regression was used to predict DGF. Receiver operating characteristic (ROC) curves were constructed, and the area under the ROC curve (AUC) and concordance index (C-index) were calculated.

[0379] For all statistical analyses, use R version 4.2.2 (http: / / www.r-project.org, librariesrms, Hmisc, ROCR) or IBM SPSS Statistics version 22 (SPSS Inc., Chicago, Illinois, USA).

[0380] result: The study included 38 kidney transplant recipients. Baseline characteristics are presented in Table 5. DGF occurred in 15 patients (39.5%), with severity ranging from DGF 0 / 1 in 2 patients to DGF 2-7 in 5 patients and DGF >7 in 8 patients. Among the remaining patients, SGF occurred in 13 patients (34.2%), and IGF occurred in 10 patients (26.3%). eGFR decreased in 7 patients (18.4%) on day 30 post-transplantation.

[0381] Table 5: Baseline Characteristics

[0382] There were no differences in pre-transplant (pre-Tx) biomarker levels (PENK, SCr) between patients with and without DGF (Figure 10). At 12–24 hours post-transplant (Day 1), PENK levels were significantly elevated in patients with DGF compared to those without DGF (non-DGF, including both IGF and SGF) (573.5 [507.9–709.8] pmol / L vs. 234.4 [163.6–361] pmol / L, p < 0.0001) (Table 2). Figure 10A In contrast, for SCr, the separation between the two groups was not significant (p=0.062) (Table 5, Figure 10B This resulted in a C-index of 0.91 (0.81-1) for PENK on day 1 for predicting DGF, which was better than the C-index of 0.68 (0.51-0.86) for SCr on day 1.

[0383] Because patients exhibited varying baseline biomarker levels pre-transplant (pre-Tx), changes in biomarker levels (Δ) from pre-transplant to Day 1 were investigated. Both PENK and SCr decreased from pre-transplant to Day 1 (Table 2). This decrease was less pronounced in DGF patients compared to those without DGF, and was significant for PENK (-7.02% [-22.43–4.94]% pre-transplant vs. -61.23% [-76.26–36.15]% pre-transplant, p = 0.0005), but not for SCr (Table 2). This resulted in ΔPENK being superior to ΔSCr in predicting DGF, as shown by the ROC curves ( Figure 11 In logistic regression analysis, the C-index of ΔPENK (0.88 [0.75-1]) was better than that of ΔSCr (0.7 [0.51-0.88]).

[0384] In patients with IGF, SGF, and DGF, PENK and SCr levels gradually increased on day 1 (Figure 12). This difference was more pronounced and significant for PENK (p<0.0001). Figure 12A The result was not significant for SCr (p=0.1969). Figure 12B In the post-hoc analysis, significant differences were found between IGF and DGF (p<0.0001) and between SGF and DGF (p=0.01095) for PENK measurements taken on day 1. This indicates that PENK differentiated patients requiring post-transplant resuscitation (RRT) (DGF) from those not requiring RRT (IGF and SGF) as early as 12–24 hours post-transplant, allowing for timely treatment decisions, which is something that the most advanced biomarker SCr currently available cannot achieve.

[0385] PENK on day 1 can differentiate DGF severity, with PENK gradually decreasing from the highest (DGF 0-1) to the lowest (DGF>7) and non-DGF patients (ANOVA p>0.0001), while there was no significant difference for SCr on day 1 (p=0.10068) (Figure 13). In post-hoc analysis, compared with non-DGF patients, day 1 PENK was significantly improved in patients with DGF severity >7 (p=0.02167), DGF severity 2-7 (p=0.00993), and marginally significantly improved in patients with DGF severity 0-1 (p=0.07025).

[0386] Regarding the endpoint of decreased eGFR on day 30, there was no significant difference in pre-transplant biomarker levels (Figure 14). However, compared to day 30 with eGFR >30 ml / min / 1.73 m 2 Compared to patients who showed a decrease in eGFR on day 30, the PENK boundary on day 1 was significantly improved in patients with a decrease in eGFR on day 30 (p = 0.05247). In contrast, SCr could not distinguish between the two groups (p = 0.80663). Consistent with the performance of ΔPENK in predicting DGF, ΔPENK was able to predict a decrease in eGFR on day 30 (AUC 0.82), while SCr was not useful (AUC 0.55), as shown in the ROC curves. Figure 15In logistic regression analysis, the C-index of ΔPENK (0.82 [0.66-0.99]) was better than that of ΔSCr (0.54 [0.31-0.79]), and the C-index of PENK on day 1 (0.74 [0.59-0.89]) was also better than that of SCr on day 1 (0.53 [0.33-0.74]).

[0387] In summary, the study confirmed the results of Example 2 and further demonstrated that PENK can predict graft function delay as early as 12-24 hours after kidney transplantation, differentiate between SGF and DGF, grade the severity of DGF, and predict the decrease in eGFR on day 30, while SCr measured on day 1 after transplantation could not identify these high-risk patients. Attached Figure Description

[0388] Figure 1 – Typical proenkephalin dose / signal curve.

[0389] Figure 2A - Box-whisker diagrams of PENK based on DGF at the following time points: before transplantation (pre-Tx), days 1, 2, 3, one week later (days 6-8: day 7, or day 6 or 8 if day 7 is missing), two weeks later (days 12-15: day 14, or day 12, 13, or 15 if day 14 is missing), and three weeks later (day 21 / last day: day 21 or the last day of observation before discharge).

[0390] Figure 2B - Cassirhizograms of SCr were plotted against DGF at the following time points: pre-Tx, days 1, 2, 3, one week later (days 6–8: day 7, or day 6 or 8 if day 7 is missing), two weeks later (days 12–15: day 14, or day 12, 13, or 15 if day 14 is missing), and three weeks later (day 21 / last day: day 21 or the last day of observation before discharge). The cutoff was 1.2 mg / dL.

[0391] Figure 3A - Box-and-whisker plot of PENK changes (as a percentage compared to pre-transplant) in DGF and non-DGF patients. Time points are as follows: Figure 2A As defined in section B. The cutoff line is 50% compared to the pre-transplant concentration.

[0392] Figure 3B - Box-and-whisker plot of SCr changes (as a percentage compared to pre-transplant) in DGF and non-DGF patients. Time points are as follows. Figure 5A As defined in section B. The cutoff line is 50% compared to the pre-transplant concentration.

[0393] Figure 4A Box-and-whisker plots of PENK based on DGF, SGF, and IGF were generated at the following time points: pre-transplant (pre-Tx), days 1, 2, and 3; one week later (days 6–8: day 7, or day 6 or 8 if day 7 is missing); two weeks later (days 12–15: day 14, or day 12, 13, or 15 if day 14 is missing); and three weeks later (day 21 / last day: day 21 or the last day of observation before discharge). Cutoffs were 80 pmol / L (upper limit of normal) and 200 pmol / L.

[0394] Figure 4B - Cassette plots of SCr based on DGF, SGF, and IGF at the following time points: pre-transplant (pre-Tx), days 1, 2, 3, one week later (days 6–8: day 7, or day 6 or 8 if day 7 is missing), two weeks later (days 12–15: day 14, or day 12, 13, or 15 if day 14 is missing), and three weeks later (day 21 / last day: day 21 or the last day of observation before discharge). The cutoff was 1.2 mg / dL.

[0395] Figure 5A - Box-and-whisker plots of PENK changes (as a percentage compared to pre-transplant) in patients with IGF, SGF, and DGF. Time points are as follows. Figure 4A As defined in [the text]. The cutoff line is 50% compared to the pre-transplant concentration.

[0396] Figure 5B - Box-and-whisker plots of SCr changes (as a percentage compared to pre-transplant) in patients with IGF, SGF, and DGF. Time points are as follows. Figure 4B As defined in [the text]. The cutoff line is 50% compared to the pre-transplant concentration.

[0397] Figure 6A - ROC plot of endpoint DGF versus SGF (except for IGF patients), comparing PENK and SCr on days 1, 2, and 3 post-transplantation. AUC: Area under the receiver operating curve.

[0398] Figure 6B - The ROC plot of the endpoint DGF versus SGF (except for IGF patients) compared the changes in PENK and SCr on days 1, 2, and 3 post-transplantation with pre-transplantation levels. AUC: Area under the receiver operating curve.

[0399] Figure 7A – ROC plot of endpoint DGF, comparing PENK and SCr on days 1, 2, and 3 post-transplantation. AUC: Area under the receiver operating curve.

[0400] Figure 7B – ROC plot of endpoint DGF, comparing changes in PENK and SCr on days 1, 2, and 3 post-transplantation compared to pre-transplantation levels. AUC: Area under the receiver operating curve.

[0401] Figure 8A - Box-and-whisker plots of PENK based on DGF severity at the following time points: pre-Tx, days 1, 2, 3, one week later (days 6–8: day 7, or day 6 or 8 if day 7 is missing), two weeks later (days 12–15: day 14, or day 12, 13, or 15 if day 14 is missing), and three weeks later (day 21 / last day: day 21 or the last day of observation before discharge). DGF severity groups: non-DGF (no RRT after transplantation); 0–1 (RRT only on day 0 or day 1); 2–7 (RRT ends between day 2 and day 7); >7 (RRT lasts longer than day 7). Cutoffs were 80 pmol / L (upper limit of normal) and 200 pmol / L.

[0402] Figure 8B - Cassette plots of SCr based on DGF severity were drawn at the following time points: pre-transplant (pre-Tx), days 1, 2, 3, one week later (days 6–8: day 7, or day 6 or 8 if day 7 is missing), two weeks later (days 12–15: day 14, or day 12, 13, or 15 if day 14 is missing), and three weeks later (day 21 / last day: day 21 or the last day of observation before discharge). DGF severity groups: non-DGF (no RRT post-transplant); 0–1 (RRT only on day 0 or day 1); 2–7 (RRT ends between day 2 and day 7); >7 (RRT lasts longer than day 7). The cutoff was 1.2 mg / dL.

[0403] Figure 9A – A box-and-whisker plot showing changes in PENK (as a percentage compared to pre-transplantation levels) based on DGF severity. Groups and time points are defined in Figures 7A and 7B. The cutoff is 50% compared to pre-transplantation concentration.

[0404] Figure 9B – A box-and-whisker plot showing changes in SCr (as a percentage compared to pre-transplantation levels) based on DGF severity. Groups and time points are defined in Figures 7A and 7B. The cutoff is 50% compared to pre-transplantation concentrations.

[0405] Figure 10 A – Box-and-whisker plots of proenkephalin A 119-159 (PENK) at time points 0–12 hours before transplantation (pre-transplantation) and 12–24 hours after transplantation (Day 1), based on the defined delay in graft function (DGF) requiring RRT within the first seven days post-transplantation. The three gray lines represent cutoff values ​​of 300 pmol / L, 200 pmol / L (to distinguish between DGF and non-DGF), and 89 pmol / L (as a reference upper limit for healthy individuals). The x-axis is logarithmically transformed.

[0406] Figure 10 B - A box-and-whisker plot of serum creatinine (SCr) at time points 0–12 hours before transplantation (pre-transplantation) and 12–24 hours after transplantation (day 1), based on graft function delay (DGF) defined as requiring RRT within the first seven days post-transplantation. The gray line represents 2 mg / dL SCr used as a reference. The x-axis is logarithmically transformed.

[0407] Figure 11 – Receiver operating characteristic plots of graft function delay (DGF), defined as the endpoint requiring RRT within the first seven days post-transplantation, were used to compare changes (Δd0 / d1) in proenkephalin A119-159 (PENK) or serum creatinine (SCr) from 0–12 hours pre-transplantation to 12–24 hours post-transplantation. The area under the curve (AUC) for PENK and SCr is presented.

[0408] Figure 12 A – Box-and-whisker plots of proenkephalin A 119-159 (PENK) based on delayed (DGF), slowed (SGF), and immediate (IGF) graft function at time points 0–12 hours before transplantation (pre-transplantation) and 12–24 hours after transplantation (day 1). DGF was defined as the reduced response time (RRT) within the first seven days post-transplantation; SGF was defined as no RRT within the first seven days post-transplantation and a SCr / SCr ratio on day 7 < 0.7; IGF was defined as no RRT within the first seven days post-transplantation and a SCr / SCr ratio on day 7 > 0.7. The three gray lines represent cutoff values ​​of 300 pmol / L, 200 pmol / L, and 89 pmol / L (as upper reference values ​​for healthy individuals). The x-axis is logarithmically transformed.

[0409] Figure 12B - Box-and-whisker plot of serum creatinine (SCr) at time points 0–12 hours before transplantation (pre-transplantation) and 12–24 hours after transplantation (day 1), based on delayed (DGF), slowed (SGF), and immediate (IGF) graft function. DGF was defined as the receptive response time (RRT) within the first seven days post-transplantation; SGF was defined as no RRT within the first seven days post-transplantation and a quotient of SCr on day 7 / SCr on day 0 < 0.7; IGF was defined as no RRT within the first seven days post-transplantation and a quotient of SCr on day 7 / SCr on day 0 > 0.7. The gray line represents 2 mg / dL SCr used as a reference. The x-axis is logarithmically transformed.

[0410] Figure 13 A – Box-and-whisker plots of proenkephalin A 119-159 (PENK) at time points 0–12 hours before transplantation (pre-transplant) and 12–24 hours after transplantation (day 1), based on the severity of delayed graft function (DGF). The DGF severity groups are as follows: no DGF (no RRT post-transplant); 0–1 (RRT only on day 0 or day 1); 2–7 (RRT ends between day 2 and day 7); >7 (RRT lasts longer than day 7). The three gray lines represent cutoff values ​​of 300 pmol / L, 200 pmol / L, and 89 pmol / L (as upper reference values ​​for healthy individuals). The x-axis is logarithmically transformed.

[0411] Figure 13 B – Box-and-whisker plot of serum creatinine (SCr) at time points 0–12 hours before transplantation (pre-transplantation) and 12–24 hours after transplantation (day 1), based on the severity of delayed graft function (DGF). The DGF severity groups are as follows: no DGF (no RRT post-transplantation); 0–1 (RRT only on day 0 or day 1); 2–7 (RRT ends between day 2 and day 7); >7 (RRT lasts longer than day 7). The gray line represents 2 mg / dL SCr used as a reference. The x-axis is logarithmically transformed.

[0412] Figure 14 A - Decreased estimated glomerular filtration rate (eGFR) on day 30 at time points 0–12 hours before transplantation (pre-transplant) and 12–24 hours after transplantation (day 1) (defined as eGFR <30 ml / min / 1.73 m 2 Box-and-whisker plot of proenkephalin A 119-159 (PENK). The three gray lines represent cutoff values ​​of 300 pmol / L, 200 pmol / L, and 89 pmol / L (as upper reference values ​​for healthy individuals). The x-axis is logarithmically transformed.

[0413] Figure 14B - Decreased estimated glomerular filtration rate (eGFR) on day 30 at time points 0–12 hours before transplantation (pre-transplant) and 12–24 hours after transplantation (day 1) (defined as eGFR <30 ml / min / 1.73 m 2 A box-and-whisker plot of serum creatinine (SCr) was plotted. The gray line represents 2 mg / dL SCr used as a reference. The x-axis is logarithmically transformed.

[0414] Figure 15 – The estimated glomerular filtration rate (eGFR) decreased at the endpoint of day 30 (defined as eGFR <30 ml / min / 1.73 m). 2 The receiver operating characteristic (ROC) plots were used to compare changes (Δd0 / d1) in proenkephalin A119-159 (PENK) or serum creatinine (SCr) from 0–12 hours pre-transplant to 12–24 hours post-transplant. The area under the curve (AUC) for PENK and SCr is presented.

Claims

1. A method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients, the method comprising: ● Determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and ● The levels of the proenkephalin or fragments thereof in the samples were correlated with the risk and / or severity of graft function and / or reduced graft function. Reduced graft function is defined as slowed graft function (SGF) or delayed graft function (DGF) in the kidneys.

2. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to claim 1, wherein the method is used for patient stratification and / or patient selection for early treatment of graft dysfunction in kidney transplant patients.

3. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to claim 1 or 2, wherein if the level of the proenkephalin or a fragment thereof in a body fluid sample obtained from the patient rises above a predetermined threshold level, the patient is considered to have graft dysfunction and / or is at risk of graft dysfunction and / or is stratified or selected for early treatment of graft dysfunction.

4. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring or severity prediction of graft function reduction in kidney transplant patients according to claim 3, wherein the predetermined threshold level is in the range of 50 to 750 pmol / L, more preferably in the range of 100 to 500 pmol / L, even more preferably in the range of 150 to 400 pmol / L, and most preferably in the range of 200 to 300 pmol / L.

5. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to claims 1 to 4, wherein the level of proenkephalin or fragments thereof in body fluid samples obtained from said patient is (i) measured at least once before and after kidney transplantation, or (ii) measured at least once after kidney transplantation.

6. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to claims 1 to 5, wherein the determination of proenkephalin or fragments thereof is performed more than once as a follow-up measurement after the patient's kidney transplant.

7. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft function reduction in kidney transplant patients according to claims 1 to 6, wherein if graft function reduction is diagnosed and / or the risk of graft function reduction is predicted in the patient, the patient requires renal replacement therapy and / or administration of medication and / or adjustment of immunosuppressive therapy and / or adjustment of nephrotoxic drugs.

8. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to claim 7, wherein the administered drug is a treatment with recombinant alkaline phosphatase, pegylated carboxyhemoglobin, relaxin, hepatocyte growth factor, mirocept, or a C1 esterase inhibitor.

9. The method of claim 7 for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients, wherein the nephrotoxic drug is selected from calcineurin inhibitors for immunosuppression (e.g., cyclosporine A, tacrolimus), analgesics (e.g., nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, aspirin), antimicrobial agents (e.g., aminoglycosides, cephalosporins, penicillins, quinolones, rifampin, and vancomycin), cholesterol-lowering statins, angiotensin-converting enzyme inhibitors (ACE inhibitors), angiotensin receptor blockers (ARBs), and diuretics, chemotherapeutic agents (e.g., cisplatin), and the nephrotoxic drug is modified and / or reduced and / or discontinued if graft dysfunction is diagnosed and / or the risk of graft dysfunction is predicted in the patient.

10. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to claims 1 to 7, wherein the enkephalinogen or fragment is selected from SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 and SEQ ID No.

12.

11. The method for early diagnosis of graft function and / or early risk prediction and / or risk monitoring and / or severity prediction of graft dysfunction in kidney transplant patients according to claims 1 to 10, wherein the body fluid sample may be selected from whole blood, serum, plasma, urine, cerebrospinal fluid (CSF) and saliva.

12. A method for early treatment of reduced graft function in kidney transplant patients, wherein the treatment is selected from renal replacement therapy and / or administration of drugs and / or adjustment of immunosuppressive therapy and / or adjustment of nephrotoxic drugs, wherein the patient is selected by a diagnostic method comprising the following steps: - To determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient, and - The level of the proenkephalin or fragment thereof in the sample was associated with the risk of graft function and / or reduced graft function. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

13. A medicine for the early treatment of impaired graft function in kidney transplant patients, wherein the patients are selected by a diagnostic method comprising the following steps: - To determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient, and - Associate the level of the proenkephalin or fragment thereof in the sample with the diagnosis of graft-deficient function and / or the risk of graft-deficient function. The aforementioned reduced graft function refers to slowed graft function (SGF) or delayed graft function (DGF) in the kidney.

14. The medicament for early treatment of graft dysfunction in kidney transplant patients according to claim 13, wherein the medicament is selected from recombinant alkaline phosphatase, pegylated carboxyhemoglobin, relaxin, hepatocyte growth factor, mirocept, and C1 esterase inhibitors.

15. The medicament for early treatment of graft dysfunction in kidney transplant patients according to claims 13 and 14, wherein the patient is at risk of graft dysfunction and / or graft dysfunction if the level of the proenkephalin or a fragment thereof in a bodily fluid sample obtained from the patient rises above a predetermined threshold level.

16. The medicament for early treatment of graft dysfunction in kidney transplant patients according to claim 15, wherein the predetermined threshold level is in the range of 50 to 750 pmol / L, more preferably in the range of 100 to 500 pmol / L, even more preferably in the range of 150 to 400 pmol / L, and most preferably in the range of 200 to 300 pmol / L.

17. The medicament for early treatment of graft dysfunction in kidney transplant patients according to claims 13 to 16, wherein if graft dysfunction is diagnosed and / or the risk of graft dysfunction is predicted in the patient, the patient additionally requires renal replacement therapy and / or adjustment of immunosuppressive therapy and / or adjustment of nephrotoxic drugs.

18. The medicament for early treatment of graft dysfunction in kidney transplant patients according to claims 13 to 17, wherein the enkephalin or fragment is selected from SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 and SEQ ID No.

12.

19. The medicament for early treatment of graft dysfunction in kidney transplant patients according to claims 13 to 18, wherein the fluid sample may be selected from whole blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.

20. A method for predicting renal function in a kidney transplant patient, the method comprising: ● Determine the level of proenkephalin or fragments thereof in bodily fluid samples obtained from the patient; and ● Correlate the levels of the proenkephalin or its fragments in the sample with the patient's renal function. The renal function mentioned therein is defined as glomerular filtration rate (GFR), creatinine clearance rate (CCr), serum creatinine (SCr), urinalysis, blood urea nitrogen, or urine output.

21. The method of claim 20 for predicting renal function in a kidney transplant patient, wherein a decrease in renal function is predicted if the level of the proenkephalin or a fragment thereof in a body fluid sample obtained from the patient increases to above a predetermined threshold level.

22. The method for predicting renal function in kidney transplant patients according to claims 20 and 21, wherein the prediction is made within 12 months, more preferably within 9 months, more preferably within 6 months, more preferably within 3 months, more preferably within 1 month, and most preferably within 14 days.

23. The method for predicting renal function in a kidney transplant patient according to claim 21, wherein the predetermined threshold level is in the range of 50 to 750 pmol / L, more preferably in the range of 100 to 500 pmol / L, even more preferably in the range of 150 to 400 pmol / L, and most preferably in the range of 200 to 300 pmol / L.

24. The method for predicting renal function in a kidney transplant patient according to claims 20 to 23, wherein the level of proenkephalin or a fragment thereof in a body fluid sample obtained from the patient is (i) measured at least once before and after kidney transplantation, or (ii) measured at least once after kidney transplantation.

25. The method of claim 24 for predicting renal function in a kidney transplant patient, wherein the enkephalin or fragment is selected from SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 and SEQ ID No.

12.

26. The method for predicting renal function in kidney transplant patients according to claims 20 to 25, wherein the body fluid sample may be selected from whole blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.

27. The method for predicting renal function in kidney transplant patients according to claims 20 to 26, wherein the GFR is estimated GFR (eGFR), true GFR, or measured GFR (mGFR).

28. The method for predicting renal function in kidney transplant patients according to claims 20 to 27, wherein the predicted GFR decreases to below 60, preferably below 45, preferably below 30, and most preferably below 15.

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