Non-HLA markers of transplant rejection

Abstract

A composition comprising a collection of solid phase substrates coated with one or more homogeneous populations of binding agents, wherein each homogeneous population of binding agents specifically binds to an antibody directed against a single antigen selected from the group consisting of: dexamethasone-induced transcript (DEXI), CXC motif chemokine ligand 11 (CXCL11), cytokeratin 18 (KRT18), cytokeratin 8 (KRT8), tubulin, including tubulin alpha 1 b (also referred to as TUBa1b or TUBA1B), latrophilin 1 (LPHN1), colony stimulating factor 2 (CSF2), signal transducer and activator of transcription 6 (STAT6), lectin galactoside-binding soluble 3 (lectin galactoside-binding soluble 3“;LGALS3), SHC adaptor protein 3 (SHC3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutathione S-transferase theta-1 (GSTT1), phospholipase A2 receptor 1 (PLA2R1), interleukin 8 (IL-8), lectin galactoside-binding soluble 8 (LGALS8), small nuclear ribonucleoprotein polypeptide N (SNPRN), myosin, peroxisomal trans-2-enoyl-CoA reductase (PECR), vimentin (VIM), ATP synthase H+ transporting mitochondrial F1 complex beta polypeptide (ATP5B), collagen II, prelamin A / C (LMNA), small nuclear ribonucleoprotein polypeptide B (SNRPB2), fibronectin 1 (FN1), nephrosis 1, congenital Finnish type nephrosis 1 (NPHS1), thyroglobulin (TG) and vinculin (VCL).;

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 844,027, filed May 6, 2019, the entire contents of which are incorporated by reference into this application. RECOGNITION OF STATE SUPPORT

[0002] This invention was made with government support under Grant Numbers AI042819, AI135201, and DK104687, awarded by the National Institutes of Health. The government retains certain rights in this invention. FIELD OF THE INVENTION

[0003] The present invention relates generally to organ transplant rejection. More specifically, the present invention provides compositions, kits, and assays for determining whether an individual has allograft rejection or is at increased risk for developing post-transplant rejection. Furthermore, the present invention provides biomarkers, such as non-HLA antibodies, associated with transplant rejection. BACKGROUND

[0004] Organ transplantation from a donor to a host recipient is a feature of certain medical procedures and treatment regimens. After transplantation, the organ recipient is typically administered immunosuppressive therapy to maintain the viability of the donor organ and prevent graft rejection. When rejection occurs after organ transplantation, the reaction is usually classified as hyperacute rejection, acute rejection, or chronic rejection. Hyperacute rejection occurs within minutes to hours after organ transplantation, usually due to antibodies in the recipient's blood that react with the new organ, and is typically characterized by extensive glomerular capillary thrombosis and necrosis.Acute rejection (AR) typically occurs within the first 6 to 12 months after organ transplantation and is a complex immune response involving T cell recognition of the alloantigen in the graft and an inflammatory response within the graft itself. Chronic rejection is less well defined than hyperacute or acute rejection and is likely due to both antibodies and lymphocytes.

[0005] Despite advances in immunosuppressive therapies and transplant procedures, transplant rejection remains a common risk in organ transplant recipients. For example, despite improvements in immunosuppressive therapy over the years, approximately 30–40% of heart transplant recipients require treatment for AR in the first year after transplantation (see Taylor et al., J Heart Transplant, 2009, 28(10):1007–22). Furthermore, AR remains a risk factor for graft dysfunction, mortality, and the development of cardiac allograft vasculopathy (CAV), the leading cause of late graft failure (see Raichlin et al., J Heart Lung Transplant, 2009, 28(4):320–7).

[0006] Thanks to advances in human leukocyte antigen (HLA) antibody detection and improved immunosuppression, short-term allograft survival rates have increased over the past decade. However, long-term outcomes remain largely unchanged, and graft loss due to chronic rejection remains a significant problem.

[0007] HLA antibodies, particularly donor-specific antibodies (DSA), contribute to antibody-mediated rejection (AMR) and acute cellular rejection (ACR) after transplantation. However, a significant proportion of heart transplant patients have been found to have AMR even without HLA or DSA antibodies, suggesting that antibodies directed against non-HLA antigens are associated with an increased risk of AMR. Antibodies against non-HLA antigens such as vimenten, MHC class I polypeptide-related sequence A (MICA), and angiotensin II receptor type 1 (AT1R), as well as antibodies directed against endothelial cells, have been associated with AMR and chronic allograft vasculopathy after heart transplantation.In addition, antibodies against non-HLA antigens have been identified, which are associated with poor outcomes after transplantation of other organs.

[0008] Endothelial cells (EC) are the first point of contact between the allograft and the transplant recipient's immune system and thus a potential source of non-HLA antigens that can trigger a humoral immune response. Endothelial cell crossmatch (ECXM) testing using primary human aortic endothelial cells (HAEC) and the XM-ONE ®Assay (Olerup SSP AB, Stockholm, Sweden), which uses EC precursors, have both been shown to be clinically relevant for identifying patient sera containing antibodies to EC. However, the utility of cell-based assays is limited because they do not identify the antigen that binds the non-HLA antibodies present in patient sera. Consequently, the inability to detect and characterize non-HLA antibodies limits the understanding of the range of non-HLA antigens that trigger a humoral response leading to poor allograft outcomes.

[0009] Early detection of AR is one of the most important clinical issues in the care of transplant recipients, including recipients of solid organs such as the heart, liver, lung, kidney, and intestine. Detecting AR before the onset of organ dysfunction allows for successful treatment of AR with aggressive immunosuppression. Equally important is reducing immunosuppression in patients who do not have AR to minimize drug toxicity. However, for most organs, rejection can only be definitively diagnosed by a biopsy of the affected organ.

[0010] For example, the current definitive diagnosis of cardiac allograft rejection relies on endomyocardial biopsy (EMB), an expensive, invasive, and uncomfortable procedure. Most heart transplant recipients undergo EMB up to 15 times in the first year, and even more frequently if rejection is detected. However, this procedure is limited by sampling error and interobserver variability (see Deng et al., Am J Transplant., 2006, 6(1):150-60; Wong et al., Cardiovasc Pathol., 2005, 14(4):176-80). Potential complications include arterial puncture, vasovagal reactions and prolonged bleeding during catheter insertion, arrhythmias and conduction disturbances, pneumothorax, biopsy-induced tricuspid regurgitation, and even cardiac perforation (see Baraldi-Junkins et al., J Heart Lung Transplant, 1993, 12(1 Part 1):63-7; Deckers et al., J Am Coll Cardiol., 1992, 19(1):43-7; Navia et al., J Heart Valve Dis., 2005, 14(2):264-7).

[0011] Although the diagnosis of acute rejection can be difficult, timely detection of immune-mediated damage is critical for graft health and long-term survival. A noninvasive biomarker panel for acute rejection that allows for frequent immunological monitoring of the graft would be of great value (see Evans et al., Am J Transplant., 2005, 5(6):1553-8; Mehra et al., Nat Clin Pract Cardiovasc Med., 2006, 3(3):136-43). Recently, a highly sensitive and specific gene-based biomarker panel for the diagnosis and prediction of biopsy-confirmed acute kidney transplant rejection was developed (see Li et al., Am J Transplant., 2012, 12(10):2710-8; Bromberg et al., Am J Transplant, 2012, 12(10):2573-4), which was independently validated in a randomized multicenter trial (see Chaudhuri et al., Pediatric Transplantation., 2012, 16(5):E183-7; Naesens et al., Am J Transplant., 2012, 12(10):2730-43). The diagnosis of acute rejection before the appearance of histopathological changes may allow optimization of immunosuppressive therapy to prevent progression of chronic allograft failure (see Kienzl et al., Transplantation, 2009, 88(4):553-60).

[0012] A noninvasive test that enables the detection of acute allograft rejection in various organs with high specificity (to reduce invasive protocol biopsies in patients at low risk for AR) and with high sensitivity (to improve clinical monitoring of patients at high risk for AR), earlier than currently possible, would lead to timely clinical intervention to mitigate AR and reduce immunosuppression protocols for quiescent and stable patients. Many tests are likely dependent on the recipient's age, comorbidities, the use of immunosuppression, and / or the cause of end-stage renal disease. Therefore, there remains a need for systems and methods to predict, diagnose, and monitor an AR response in a patient who has received an organ transplant.

[0013] Furthermore, rejection phenomena are not limited to cardiac allografts. Rejection phenomena (e.g., host-versus-graft disease) occur in all organ transplants, including kidney transplants. Furthermore, rejection-like events are associated with graft-versus-host disease (e.g., when transplanted leukocytes and lymphocytes attack host tissue) and autoimmune diseases (e.g., rheumatic fever, in which the heart is the target of autoantibodies and autoreactive lymphocytes). While aggressive immunosuppression is indicated in all cases to reverse or correct the immune response, the associated risk of promoting opportunistic infections limits its use.

[0014] Accordingly, there is a need in the field for highly accurate prognostic indicators and non-invasive tests for the likelihood of allograft rejection. Furthermore, there is a need in the field to identify markers that pathologically contribute to rejection or are exclusively prognostic indicators of rejection.

[0015] The present invention develops and validates, for the first time, a large, high-throughput multiplex bead array to test for the presence of novel and known non-HLA antibodies associated with rejection.

[0016] All patents, patent applications, publications, documents and articles referred to herein are incorporated in their entirety unless otherwise indicated. SHORT SUMMARY

[0017] The present invention provides compositions, tests and kits for determining whether an individual has allograft rejection or subclinical allograft rejection or is at increased risk of suffering from rejection following transplantation.

[0018] In some embodiments, the present invention provides a composition comprising a collection of solid phase substrates coated with one or more homogeneous populations of binding agents, wherein each homogeneous population of binding agents specifically binds to an antibody directed against a single antigen selected from the group consisting of: dexamethasone-induced transcript (DEXI), CXC motif chemokine ligand 11 (CXCL11), cytokeratin 18 (KRT18), cytokeratin 8 (KRT8), tubulin, including tubulin alpha 1 b (also referred to as TUBα1 b or TUBA1 B), latrophilin 1 (LPHN1), colony stimulating factor 2 (CSF2), signal transducer and activator of transcription 6 (STAT6), lectin galactoside-binding soluble 3 (“lectin galactoside-binding soluble 3”;LGALS3), SHC adaptor protein 3 (SHC3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutathione S-transferase theta-1 (GSTT1), phospholipase A2 receptor 1 (PLA2R1), interleukin 8 (IL-8), lectin galactoside-binding soluble 8 (“lectin galactoside-binding soluble 8”;LGALS8), small nuclear ribonucleoprotein polypeptide N (SNPRN), myosin, peroxisomal trans-2-enoyl-CoA reductase (PECR), vimentin (VIM), ATP synthase H+ transporting mitochondrial F1 complex beta polypeptide (ATP5B), collagen II, prelamin A / C (LMNA), small nuclear ribonucleoprotein polypeptide B (SNRPB2), fibronectin 1 (FN1), vinculin (VCL), thyroglobulin (TG), and nephrosis 1 (NPHS1). In some embodiments, the collection of solid phase substrates further comprises one or more additional homogeneous populations of binding agents, wherein each additional homogeneous population of binding agents specifically binds to an antibody directed against a single additional antigen selected from the group consisting of: alpha-enolase (ENO1), agrin (AGRN), endomucin (EMCN), Sjogren's syndrome antigen B (SSB), actin, fms-related tyrosine kinase 3 ligand (FLT3LG), protein kinase C eta (PRKCH), and interleukin 21 (IL-21).;

[0019] In another embodiment, the compositions of the present invention comprise a collection of one or more different homogeneous populations of binding agents, wherein each different homogeneous population of binding agents is only capable of specifically binding an antibody directed against tubulin, LPHN1, SNRPN, KRT18, KRT8, LGALS3, SNRPB2, DEXI, collagen II, PLA2R1, GSTT1, VCL, CSF2, LGALS8, STAT6, TG, IL-8 and SHC3.

[0020] In another embodiment, the compositions of the present invention comprise a collection of distinct homogeneous populations of binding agents, wherein each distinct homogeneous population of binding agents is only capable of specifically binding an antibody directed against tubulin, LPHN1, SNRPN, KRT18, KRT8, LGALS3, SNRPB2, DEXI, collagen II, PLA2R1, GSTT1, VCL, CSF2, LGALS8, and STAT6.

[0021] In another embodiment, the compositions of the present invention comprise a collection of distinct homogeneous populations of binding agents, wherein each distinct homogeneous population of binding agents is only capable of specifically binding an antibody directed against tubulin, LPHN1, SNRPN, KRT18, KRT8, LGALS3, SNRPB2, DEXI, collagen II, PLA2R1, GSTT1, VCL, CSF2, LGALS8, STAT6, IL-8, and SHC3.

[0022] In another embodiment, the compositions of the present invention comprise a collection of distinct homogeneous populations of binding agents, wherein each distinct homogeneous population of binding agents is only capable of specifically binding an antibody directed against tubulin, LPHN1, SNRPN, KRT18, KRT8, LGALS3, SNRPB2, DEXI, collagen II, PLA2R1, and GSTT1.

[0023] In another embodiment, the compositions of the present invention comprise a collection of distinct homogeneous populations of binding agents, wherein each distinct homogeneous population of binding agents is only capable of specifically binding an antibody directed against tubulin, LPHN1, TG, GAPDH, FN1, NPHS1, VIM, myosin, VCL, and PECR.

[0024] In some embodiments, the compositions of the present invention comprise a collection of distinct homogeneous populations of binding agents, wherein each distinct homogeneous population of binding agents is only capable of specifically binding an antibody directed against DEXI, LGALS3, SNPRN, CSF2, IL-8, STAT6, LGALS8, KRT18, KRT8, GSTT1, LMNA, Collagen II, ATP5B, SNRPB2, and PLA2R1.

[0025] In some embodiments, the compositions of the present invention comprise a collection of distinct homogeneous populations of binding agents, wherein each distinct homogeneous population of binding agents is only capable of specifically binding an antibody directed against tubulin, SHC3, and CXCL11.

[0026] In certain embodiments, the solid phase substrates are porous or non-porous. In certain embodiments, the solid phase substrates comprise particles, nanoparticles, beads, nanospheres, or microspheres. In certain embodiments, the beads are polystyrene beads. In certain embodiments, the collection of solid phase substrates comprises those that are plate- or membrane-bound, or a microarray. In certain embodiments, the solid phase substrates are fluorescently labeled, magnetically labeled, chemiluminescent, or radiolabeled. In certain embodiments, the solid phase substrates are labeled with a small molecule. In certain embodiments, the one or more homogeneous populations of binders are conjugated to the surface of the solid phase substrates. In certain embodiments, the conjugation is covalent.In certain embodiments, the one or more homogeneous populations of binding agents are bound by affinity to the surface of the solid phase substrates. In certain embodiments, the binding agent is a protein, peptide, or polypeptide. In certain embodiments, the solid phase substrates are coated with one or more different homogeneous populations of binding agents that bind to one or more different antigens, and each of the solid phase substrates is detectably distinguishable from the other solid phase substrates.

[0027] In some cases, a method for determining the presence of one or more antibodies in a biological sample obtained from an individual may comprise contacting the biological sample with the composition disclosed herein and detecting binding of the one or more homogeneous populations of binding agents to the one or more antibodies. In certain embodiments, the individual is a mammal. In certain embodiments, the individual is a human. In certain embodiments, the individual has received or will receive an organ transplant, such as a heart or kidney transplant. In certain embodiments, the heart or kidney transplant is an allogeneic heart or kidney transplant.In certain embodiments, the biological sample is blood, plasma, serum, urine, spinal fluid, lymph fluid, synovial fluid, cerebrospinal fluid, tears, saliva, milk, mucosal secretions, effusion, sweat, biopsy aspirates, ascites, or fluid extracts. In certain embodiments, detection is performed by measuring fluorescence intensity or by immunological analysis.

[0028] In some cases, a method for diagnosing transplant rejection in an individual who has undergone a heart or kidney transplant may comprise contacting a biological sample obtained from the individual with the composition disclosed herein and measuring levels of the one or more antibodies in the sample. In certain embodiments, elevated levels of the one or more antibodies compared to reference levels indicate that the individual has developed transplant rejection in response to the heart or kidney transplant.

[0029] In some cases, a method for predicting the likelihood of transplant rejection in an individual requiring a heart or kidney transplant may comprise contacting a biological sample from the individual with the composition disclosed herein and measuring the levels of the one or more antibodies in the sample. In certain embodiments, elevated levels of the one or more antibodies, compared to reference levels, indicate that the individual has an increased likelihood of developing transplant rejection following a heart or kidney transplant.

[0030] In some embodiments, the present invention provides a therapeutic agent for use in treating an individual in need of treatment for transplant rejection after receiving a heart or kidney transplant. In some embodiments, the treatment comprises the individual having elevated levels of one or more antibodies in a biological sample from the individual compared to reference levels, and wherein the levels were determined by contacting a biological sample obtained from the individual with the composition disclosed herein.

[0031] In some embodiments, the present invention provides a kit. In certain embodiments, the kit comprises the compositions and reagents disclosed herein for detecting binding of the one or more homogeneous populations of binding agents to the antibodies. In other embodiments, the kit further comprises one or more reference samples. BRIEF DESCRIPTION OF THE DRAWINGS Fig.Figure 1 shows the non-HLA antibodies significantly associated with (A) pediatric and (B) adult renal allograft rejections. A. High-throughput multiplex bead analysis was used to identify non-HLA antibodies in sera from pediatric renal transplant patients (n=34 sera with rejection, n=95 sera without rejection). Antibodies against 15 non-HLA antigens (y-axis) were identified that were significantly associated with time to first rejection, with an odds ratio >1 (x-axis). Seven of these antibodies, DEXI, CSF2, IL-8, LGALS3, SNPRN, STAT6, and LGALS8, have been newly described in association with renal allograft rejection. The bars represent the 95% confidence interval (CI).(B) Antibodies against three non-HLA antigens (tubulin, CXCL11, SHC3) were significantly associated with renal transplant rejection in adult renal allograft recipients (n=70 rejection sera, n=90 non-rejection sera). Risk ratios for antibody binding to the remaining non-HLA antigens of the multiplex panel, which are not significantly different from one, are not shown. * denotes p<0.05; all other non-HLA antibodies shown are p<0.1. Fig.Figure 2 shows the results of a correlation matrix analysis demonstrating hierarchical clustering of non-HLA antibodies in independent studies of (A) adult cardiac, (B) pediatric renal, and (C) adult renal allograft rejection sera. (A) Non-HLA antibodies associated with cardiac allograft rejection are selectively clustered into four groups. Two non-HLA antibodies were newly identified in this study as being associated with cardiac allograft rejection (TG and LPHN1). (B) The matrix describes the correlation of non-HLA antibodies found in sera of pediatric renal transplant patients with rejection. Non-HLA antibodies associated with renal allograft rejection can be selectively clustered into six groups.Seven non-HLA antibodies were newly identified in this study as being associated with renal allograft rejection (DEXI, CSF2, IL-8, LGALS3, SNPRN, STAT6, and LGALS8). Four non-HLA antibodies cluster independently (PLA2R1, CSF2, GSTT1, and LGALS8). (C) The targets found to be significant in the pediatric renal cohort were used to develop a correlation matrix for the adult renal cohort. The antigens independently correlated with rejection are similar in pediatric and adult renal transplant patients with rejection. Fig.Figure 3 shows the results of a correlation matrix analysis demonstrating hierarchical clustering of non-HLA antibodies in a combined analysis of sera from pediatric kidney and adult kidney allograft rejection patients. The matrices describe the correlation of non-HLA antibodies found in sera from rejection patients in a combined analysis of pediatric and adult kidney transplant sera. Non-HLA antibodies associated with kidney allograft rejection can be selectively clustered into nine groups. Antibodies against eight non-HLA antigens were newly identified as associated with renal allograft rejection (LGALS8, SHC3, STAT6, DEXI, IL-8, LGALS3, SNPRN, and CSF2), and four non-HLA antigens cluster independently (PLA2R1, STAT6, GSTT1, and CSF2). Fig.Figure 4 shows a classification algorithm for identifying non-HLA antibodies that predict renal allograft rejection. The classification and regression tree (CART) analysis shows a binary decision tree that evaluates rejection classification based on the strength of non-HLA antibodies (MFI). A. CART analysis of sera isolated from adult cardiac allograft patients (n=67 sera). A cutoff of 1,000 MFI was used in the analysis. The root node, LPHN1, which contains all 67 sera, of which 49% are rejection samples, splits into child nodes at an MFI<1,000. As the algorithm proceeds to the terminal nodes, 65% of rejection samples are correctly identified (far right, dark boxes). The scale bar indicates the association with repulsion, with the lighter boxes of the terminal nodes correlating with non-repulsion. B.CART analysis of sera isolated from pediatric kidney allotransplantation patients (n=129 sera). A cutoff of 1,000 MFI was used in the analysis. The root node SNPRN, which contains all 129 sera, of which 26% are rejection samples, splits into subordinate nodes at an MFI <1,000. As the algorithm proceeds to the terminal nodes, 56% of the non-rejection samples are correctly identified (far left, light boxes). The scale bar indicates the association with rejection, with the lighter boxes of the terminal nodes correlating with non-rejection. Fig. Figure 5 shows non-HLA antibodies divided into 4 groups. The first group is the non-HLA antibodies found in several transplant cohorts (core: tubulin) and all those found in the CART analysis ( Fig.4) are predictive of rejection. Group 2 includes the non-HLA antibodies that are sorted independently in a correlation matrix and all newly identified ones (highlighted by shaded targets and including all such targets in all groups). Two non-HLA antibodies, PLA2R and GSTT1, are found in groups 1 and 2. Group 3 includes the non-HLA antibodies that are found together in correlation matrix analyses and are independent of groups 1 and 2. Group 4 includes all non-HLA antibodies that have been found to be associated with rejection (Table 4). Fig. Figure 6 shows the non-HLA antibodies that were divided into four groups after an extended analysis with cardiac allografts. The first group is the non-HLA antibodies that were found in several transplant cohorts (core: tubulin) and all those that were found in the CART analysis ( Fig.4) are predictive of rejection. Group 2 includes those non-HLA antibodies that are independently ranked in a correlation matrix, as well as all newly identified ones (highlighted by shaded markers, including all such targets in all groups). Group 3 includes those non-HLA antibodies that are found together in correlation matrix analyses and are independent of groups 1 and 2. Group 4 includes all non-HLA antibodies that have been found to be associated with rejection (Table 4). DETAILED DESCRIPTION

[0032] The present disclosure relates to the diagnosis, prognosis, and / or treatment of acute, chronic, or delayed rejection of heart or kidney transplants. In some cases, the disclosure relates to methods for determining whether an individual is at increased risk for developing rejection following transplantation. In some embodiments, the rejection is acute rejection.

[0033] Furthermore, the present invention provides biomarkers (e.g., protein markers) associated with transplantation. The biomarkers (e.g., protein markers) described herein can be used in the prediction, diagnosis, prognosis, or treatment of heart or kidney transplant rejection.

[0034] The present disclosure further encompasses devices for analyzing one or more protein or antibody markers of an individual to determine the presence or absence, or level, of the one or more markers. The presence or absence, or level, of one or more markers is an indicator that the individual has an increased or decreased risk of developing organ transplant rejection compared to a control individual (e.g., a healthy individual who does not express the one or more markers).

[0035] The term "collection of solid phase substrates" refers to a group of substrates that are inherently solid or can be formed on a solid surface. The term "collection" means more than one solid substrate, and the number of substrates is determined by the number of different markers, such as an antibody, that are assayed according to the methods of the present disclosure.

[0036] The term "homogeneous population" refers to a population of molecules that is identical in terms of their molecular structure. In one embodiment, the homogeneous population is a collection of a single binding agent that specifically binds to an antibody in a sample, where the binding agent has the same amino acid sequence. In another embodiment, the homogeneous population is a collection of a single binding agent that specifically binds to an antibody in a sample, where the binding agents all have the same protein structure.

[0037] The term “transplantation” or “transfer” refers to the procedure by which donor tissue, such as a heart or kidney, is joined to the body of the transplant recipient.

[0038] The term "allogeneic" or "allograft" refers to the transplantation of an organ from the same animal species. However, "xenogeneic" transplants, i.e., the transplantation of organs from other animal species into a human, e.g., with hearts or kidneys harvested from transgenic pigs, are also conceivable within the scope of the present disclosure.

[0039] The term "rejection" or "graft rejection" is used herein to refer to rejection by the immune system of a tissue transplant recipient when the transplanted tissue is immunologically foreign. In certain embodiments, tissue rejection includes, but is not limited to, autoimmune organ rejection, e.g., pericarditis, and graft-versus-host rejection. Most commonly, organ rejection occurs following allotransplantation or xenotransplantation. In some examples, the rejection is acute rejection. In some examples, the rejection is chronic rejection.

[0040] As used in the field, “acute rejection” is the form of rejection that occurs within the first six months after transplantation and is often mediated by mononuclear cells that infiltrate the graft and cause acute damage to the graft parenchymal cells.

[0041] In this field, "chronic rejection" refers to the form of rejection that develops within months to years after transplantation. Chronic rejection is the main cause of long-term transplant loss.

[0042] The term "marker" or "biomarker" is used interchangeably herein and refers to a protein, such as an antibody, that has altered expression compared to normal levels prior to or during rejection of a heart or kidney transplant. In some embodiments, such proteins are antibodies directed against non-HLA proteins, "non-HLA antibodies," which are associated with immune or inflammatory responses. In other embodiments, a marker is a protein found in the tissue of the rejected organ before rejection begins. For example, a marker used herein is a non-HLA antibody disclosed herein, e.g., in Table 4. In a specific example, the markers of the disclosure are proteinaceous molecules that, as such, can be modified by the cells that express them.In some cases, partial sequence data confirm that spots exhibiting only minor variations in molecular weight, isoelectric point, or both represent differently modified forms of the same protein. Such modifications include, but are not limited to, differences in glycosylation, phosphorylation, N-terminal acetylation, C-terminal amidation, mRNA splicing variations, and the like.

[0043] The term "binding agent" refers to a molecule that specifically recognizes and binds to a target molecule of interest. Non-limiting examples of binding agents include any molecule capable of forming immune complexes with a target molecule. In a specific embodiment, the target molecule may be, for example, an antibody or antibody fragment, and the binding agent in that particular embodiment would be, but is not limited to, an antigenic molecule, such as a polypeptide, to which the antibody or fragment thereof specifically binds.

[0044] The term “antibody” refers to an immunoglobulin molecule or a fragment thereof that recognizes and specifically binds to a target, such as a protein, a polypeptide, a peptide, a carbohydrate, a polynucleotide, a lipid, or a combination of the foregoing, via at least one antigen recognition site in the variable region of the immunoglobulin molecule.

[0045] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. As used herein, the term polypeptide can refer to a natural or synthetic molecule comprising two or more amino acids joined by the carboxyl group of one amino acid to the alpha-amino group of another. In select cases, a polypeptide is the binding agent used in the methods and compositions of the present disclosure. The polypeptides used as binding agents in the methods and compositions of the present disclosure can be of various animal origins, including, but not limited to, human, monkey, mouse, pig, bovine, canine, equine, sheep, goat, and rabbit. In certain embodiments, the polypeptide used as a binding agent is a recombinant peptide.The polypeptide may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention, e.g., by the formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, or other manipulations or modifications such as conjugation with a labeling moiety. For example, the definition also includes polypeptides containing one or more analogues of an amino acid, or polypeptides having one or more conservative substitutions, as well as other modifications known in the art.

[0046] The definition of polypeptide as used in the present disclosure includes antigens or antibodies. For example, the polypeptide may be an antigenic binder that specifically binds to the non-HLA antibodies disclosed herein. In some embodiments, the antigenic binders comprise the full-length protein or a protein fragment thereof. In some embodiments, the antigenic binder comprises or consists of the antigenic determinant. In some embodiments, the antigenic binder is of human origin. In other embodiments, the antigenic binder is of non-human origin, such as from monkeys, mice, pigs, cattle, canines, horses, sheep, goats, and rabbits.

[0047] As used herein, the term "labeled" means that the moieties comprise a member of a signal-generating system and are thus detectable either directly or through the combined action of one or more additional members of a signal-generating system. Examples of directly detectable labels include isotopic and fluorescent moieties, which are often covalently bound to the solid-phase substrates, binding agents, and / or biological samples. The label may include, but is not limited to, a fluorescent label, an immunolabel, a magnetic label, a DNA label, a small molecule label, or a radiolabel.

[0048] The term "affinity," as used herein, means non-covalent bonding or association. The term "non-covalent" refers to interactions that do not involve the formation of covalent chemical bonds. Non-covalent bonds are associations between or among molecules in which one or more non-covalent forces, such as, but not limited to, hydrogen bonds, van der Waals forces, and electrostatic forces, may be at work. When a ligand has an affinity for a particular target, it means that there is a favorable tendency for the ligand to specifically and non-covalently associate with the target to form a complex or complexes.The affinity of a ligand for its target depends on several factors, including, but not limited to, the conformation of the ligand, the conformation of the target, and local environmental parameters such as temperature and ionic conditions, which can strongly influence binding without significantly altering the conformation. Non-limiting examples of affinity binding include the binding between biotin and streptavidin, histidine and nickel, or antibody and antigen.

[0049] As used herein, the term “detect” refers to the qualitative or quantitative measurement of undetectable, low, normal, or high concentrations of one or more biomarkers in the biological sample, such as antigens, antibodies, or other biological molecules.

[0050] The term “probability of organ rejection” refers to the probability of a rejection episode that can be predicted based on the expression level of one or more markers disclosed herein.

[0051] The term "increased expression" of a marker or markers in a test sample refers to increased expression levels of the marker(s) compared to the expression level of the corresponding marker(s) in a reference sample, or to the presence of the corresponding marker(s) in a test sample that are not expressed in a reference sample. In some embodiments, the level of a marker, as used herein, refers to the circulating amount of a marker. The term "circulating level" refers to the amount or concentration of a marker present in a circulating fluid. The circulating level can be expressed, for example, as an absolute amount, concentration, or amount per unit mass of the individual, as well as a relative amount. The level of a marker can also be a relative amount, for examplecompared to, but not limited to, an internal standard or baseline values, or it may be expressed as a range of levels, minimum and / or maximum levels, mean levels, median levels, or presence or absence of a marker. In one example, increased expression is measured by median fluorescence intensity (MFI). In other examples, increased expression is measured by, for example, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), or electrochemiluminescence ELISA. In one example, increased expression refers to the increased level or presence of a marker disclosed herein. In one example, increased expression refers to the level or presence of a collection of markers disclosed herein.

[0052] A "sample," "test sample," or "biological sample," as used interchangeably herein, in certain embodiments is of biological origin, e.g., from a mammal. In certain examples, the sample is a tissue or body fluid derived from an individual. In certain other examples, the sample is a human sample or an animal sample. Non-limiting sources of a sample include blood, plasma, serum, urine, spinal fluid, lymph fluid, synovial fluid, cerebrospinal fluid, tears, saliva, milk, mucosal secretions, effusion, sweat, biopsy aspirates, ascites, or fluid extracts. In a specific example, the sample is a liquid sample. In some embodiments, the samples are derived from an individual (e.g., a human) and include various sample sources described herein.

[0053] The term "individual" refers to any animal, e.g., a mammal, including but not limited to humans and non-human primates, that is to be subjected to a particular treatment. Typically, the terms individual, patient, subject, and "test subject" are used interchangeably herein and refer to a mammal, particularly a human or a non-human primate. In some embodiments, the individual is an adult. In one embodiment, the adult individual is a post-pubertal human. In another embodiment, the individual is a pubescent human. The term "adult" does not include pre-pubertal children. An individual of interest is a person who is to be or has been tested for the evaluation (e.g., prediction, diagnosis, identification, etc.) of allograft rejection.The individual may have been previously screened or diagnosed using other methods as described in current clinical practice. In some embodiments, the individual belongs to a patient subpopulation. For example, any of the methods described herein may be used in the assessment of acute rejection in a patient subpopulation with cardiac or renal allograft rejection (e.g., acute rejection) with a score of Grade 0, Grade 1A, Grade 1B, Grade 2, Grade 3A, Grade 3B, or Grade 4. In some embodiments, the individual has an allograft rejection score of ≥ Grade 1B. In some embodiments, the individual has at least one histologically proven rejection episode.In some embodiments, acute cellular rejection (ACR) and antibody-mediated rejection (AMR) are assessed by endomyocardial biopsy (EMB) according to the International Society for Heart and Lung Transplantation (ISHLT) criteria. In some embodiments, the individual has ACR 1R. In some embodiments, the individual has ACR 2R. In some embodiments, the individual has ACR 3R. In some embodiments, the individual has AMR. In some embodiments, the individual has a mixture of ACR and AMR. In some embodiments, the individual may or may not have had a biopsy, such as a kidney or heart biopsy. Use of any of the methods, compositions, or kits described herein can non-invasively assess a rejection response in an individual who may have cardiac or renal allograft rejection.

[0054] A "reference sample" is used to correlate and compare the results obtained with a test sample. Reference samples can be any biological samples as used herein. The methods of the present disclosure involve a comparison between the levels of one or more markers, e.g., the non-HLA antibodies disclosed herein, in a test sample and a "reference level." A reference sample can be obtained from various subgroups of individuals, such as, but not limited to, healthy individuals, individuals who have never received an organ transplant, individuals who have received an organ transplant but never developed severe rejection of the transplant, and individuals of all ages. A reference sample can also be obtained from an individual before receiving a transplant or before the individual develops rejection of the transplant.The level of a marker in the reference sample is referred to as the "reference level." Non-limiting examples of reference samples or reference levels are listed in the "Examples" section below.

[0055] The term "immunological analysis" refers to the characterization of the markers disclosed herein based on immunospecific binding, i.e., reactivity with a specific binding partner of the marker. The paradigm of a specific binding partner is an antigen if the marker is an antibody. Accordingly, all techniques applicable to antigen-antibody binding also apply to the binding of any specific binding partner of a marker. Examples of immunological analysis techniques include, but are not limited to, immunoblotting, ELISA, radioimmunoassay (RIA), agglutination, immunofluorescence, immunochemiluminescence, immunochromatography, IHC, biosensor, optical sensor, and immunoprecipitation.

[0056] Reference herein to "about" a value or parameter includes (and describes) embodiments that relate to that value or parameter per se. For example, a description that refers to "about X" includes a description of "X." The term "about" is used to provide some flexibility to a numerical endpoint by providing that a particular value may be "a little above" or "a little below" the endpoint without affecting the desired result, but also includes a range of + / - 10% of the stated value. Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format.It should be understood that such a range format is used solely for convenience and brevity and is therefore to be flexibly interpreted to include not only the numeric values ​​expressly stated as range boundaries, but also all individual numeric values ​​or subranges contained within that range, as if each numeric value and subrange were expressly stated.

[0057] The singular forms “a”, “an” and “the” as used herein include plural forms unless the context clearly indicates otherwise.

[0058] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0059] In carrying out the present invention, unless otherwise indicated, conventional techniques of protein biology, protein chemistry, molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology are employed, which are well known in the art. Such techniques are fully explained in the literature, for example, in "Molecular Cloning: A Laboratory Manual," second edition (Sambrook et al., 1989); "Current Protocols in Molecular Biology" (Ausubel et al., eds., 1987, periodic updates); "PCR: The Polymerase Chain Reaction," (Mullis et al., eds., 1994); and Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd edition, J. Wiley & Sons (New York, NY 1994).

[0060] A biomarker within the meaning of the present invention comprises a protein, such as an antibody, that is expressed by cells of individuals who are or have been exposed to a rejection reaction following a heart or kidney transplant. The marker proteins are typically expressed in very small amounts or are not expressed in reference samples. In some embodiments, the expression of a marker protein increases in association with impending or onset of organ rejection, e.g., acute rejection.

[0061] The present invention provides homogeneous populations of binders for biomarkers, e.g., non-HLA antibodies, associated with allograft rejection.

[0062] In another embodiment, the invention provides non-HLA antibodies that predict allograft rejection, including, but not limited to, tubulin, LPHN1, SNRPN, KRT18, KRT8, LGALS3, SNRPB2, DEXI, collagen II, PLA2R1, GSTT1, VCL, CSF2, LGALS8, STAT6, GAPDH, IL-8, SHC3, ENO1, AGRN, EMCN, and SSB. In some embodiments, one or any combination of two or more of the above-mentioned non-HLA antibodies are predictive as markers of allograft rejection. In some embodiments, a combination of non-HLA antibodies that are predictive of allograft rejection based on additional organ-specific analyses includes tubulin, LPHN1, SNRPN, KRT18, KRT8, DEXI, and GAPDH.In some embodiments, determining levels of one or any combination of two or more of the following non-HLA antibodies: tubulin, LPHN1, SNRPN, KRT18, KRT8, LGALS3, SNRPB2, DEXI, collagen II, PLA2R1, GSTT1, VCL, CSF2, LGALS8, STAT6, GAPDH, IL-8, SHC3, ENO1, AGRN, EMCN, and SSB is predictive of allograft rejection. In some embodiments, a combination of non-HLA antibodies predictive of allograft rejection, e.g., based on additional organ-specific analyses, includes tubulin, LPHN1, SNRPN, KRT18, KRT8, DEXI, and GAPDH.

[0063] In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, or 15 of the tubulin, LPHN1, SNRPN, KRT18, KRT8, LGALS3, SNRPB2, DEXI, collagen II, PLA2R1, GSTT1, VCL, CSF2, LGALS8, and STAT6 non-HLA antibodies are informative as independent markers of allograft rejection. In some embodiments, one, two, three, four, five, six, or seven additional non-HLA antibodies of GAPDH, IL-8, SHC3, ENO1, AGRN, EMCN, and SSB are informative as independent markers of allograft rejection. In some embodiments, determining levels of one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, or 15 of the tubulin, LPHN1, SNRPN, KRT18, KRT8, LGALS3, SNRPB2, DEXI, collagen II, PLA2R1, GSTT1, VCL, CSF2, LGALS8, and STAT6 non-HLA antibodies is predictive of allograft rejection.In some embodiments, determining levels of one, two, three, four, five, six, or seven of the GAPDH, IL-8, SHC3, ENO1, AGRN, EMCN, and SSB non-HLA antibodies is predictive of allograft rejection.

[0064] In some embodiments, determining the levels of one, two, three, four, five, six, seven, eight, nine, or ten of the tubulin, LPHN1, TG, GAPDH, FN1, NPHS1, VIM, myosin, VCL, and PECR non-HLA antibodies is predictive of cardiac allograft rejection. For example, determining the levels of TG and LPHN1 non-HLA antibodies is predictive of cardiac allograft rejection. In another example, determining the levels of tubulin, LPHN1, TG, and VCL non-HLA antibodies is predictive of cardiac allograft rejection. In another example, determining the levels of tubulin, LPHN1, and TG non-HLA antibodies is predictive of cardiac allograft rejection. In another example, determining the levels of DEXI, EMCN, SNRPN, LPHN1, and SSB non-HLA antibodies is predictive of cardiac allograft rejection.In another example, determining levels of KRT18, GAPDH, AGRN, ENO1, and EMCN non-HLA antibodies is predictive of non-rejection. In some embodiments, determining levels of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, or eighteen of the ENO1, AGRN, EMCN, SSB, actin, FLT3LG, PRKCH, IL-21, tubulin, LPHN1, TG, GAPDH, FN1, NPHS1, VIM, myosin, VCL, and PECR non-HLA antibodies is predictive of cardiac allograft rejection.

[0065] In some embodiments, determining levels of one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, or 15 of the DEXI, LGALS3, SNPRN, CSF2, IL-8, STAT6, LGALS8, KRT18, KRT8, GSTT1, LMNA, Collagen II, ATP5B, SNRPB2, and PLA2R1 non-HLA antibodies is predictive of pediatric renal allograft rejection.

[0066] For example, the determination of DEXI, LGALS3, SNPRN, CSF2, IL-8, STAT6, and LGALS8 non-HLA antibody levels is predictive of renal allograft rejection. In another example, the determination of DEXI, LGALS3, SNPRN, CSF2, STAT6, LGALS8, KRT18, KRT8, GSTT1, collagen II, SNRPB2, and PLA2R1 non-HLA antibody levels is predictive of renal allograft rejection. In another example, determination of the levels of tubulin, DEXI, LGALS3, SNPRN, KRT18, KRT8, GSTT1, collagen II, SNRPB2, and PLA2R1 non-HLA antibodies is predictive of renal allograft rejection.

[0067] In some embodiments, determining levels of one, two, or three of the tubulin, SHC3, and CXCL11 non-HLA antibodies is predictive of adult renal allograft rejection.

[0068] In certain embodiments, the invention provides a composition for determining the presence of one or more antibodies in a biological sample. In certain embodiments, the composition comprises a collection of solid-phase substrates coated with one or more homogeneous populations of binding agents. In certain embodiments, each of the homogeneous populations of binding agents specifically binds to a non-HLA antibody disclosed herein.

[0069] In some embodiments, the coating occurs by conjugation, i.e., the one or more homogeneous populations of binders are conjugated to the surface of the solid-phase substrates. In some embodiments, the conjugation is covalent. In some embodiments, the coating occurs by covalent bonding. An example of a covalent bond includes, but is not limited to, glutaraldehyde. In some embodiments, the coating occurs by covalent cross-linking. Examples of covalent bonds include, but are not limited to, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), H-benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), (1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride) (EDAC), and N-hydroxysuccinimide (NHS). In some embodiments, the coating is done by physical adsorption.In some embodiments, the coating is achieved by encapsulation. Examples of encapsulation include, but are not limited to, polymers. In some embodiments, the coating is achieved by affinity attachment. Examples of physical adsorption include, but are not limited to, biotin / streptavidin, histidine / nickel, and antibody / antigen. Methods for covalent bonding, covalent crosslinking, physical adsorption, encapsulation, and affinity bonding are generally known in the art and are encompassed by the present invention.

[0070] In some embodiments, the solid-phase substrates comprise particles, nanoparticles, beads, nanospheres, or microspheres. In some embodiments, the solid-phase substrates can be porous or non-porous. In some embodiments, the substrate can be array-based. In another embodiment, a solid-phase substrate of the present invention comprises a magnetic-based protein assay component. In other embodiments, the substrate can be organic or inorganic; it can be metal (e.g., copper or silver) or non-metal; it can be polymeric or non-polymeric; it can be conductive, semiconducting, or non-conducting (insulating); it can be reflective or non-reflective; etc.The substrate may comprise, for example, polyethylene, polytetrafluoroethylene, polystyrene, polyethylene terephthalate, polycarbonate, gold, silicon, silicon oxide, silicon oxynitride, indium, tantalum oxide, niobium oxide, titanium, titanium oxide, platinum, iridium, indium tin oxide, diamond or diamond-like layers, etc.

[0071] The substrates described above may be made of any suitable material, including, but not limited to, a material selected from the group consisting of metals, metal oxides, alloys, semiconductors, polymers (such as organic polymers in any suitable form, including woven, non-woven, molded, extruded, cast, etc.), silicon, silicon oxide, ceramics, glass, and composites thereof.

[0072] In certain embodiments, the solid phase substrates are labeled. In certain embodiments, the binders are labeled. In certain embodiments, the biological samples are labeled. In some embodiments, one or two of the above components are labeled. In some embodiments, all of the above components are labeled.

[0073] In one embodiment, the label is a fluorescent moiety. Fluorescent moieties or labels of interest include, but are not limited to, coumarin and its derivatives, e.g., 7-amino-4-methylcoumarin, aminocoumarin, Bodipy dyes such as Bodipy FL, Cascade Blue, fluorescein and its derivatives, e.g., fluorescein isothiocyanate, Oregon Green, rhodamine dyes, e.g., Texas Red, tetramethylrhodamine, eosins and erythrosins, cyanine dyes, e.g., Cy3 and Cy5, macrocyclic chelates of lanthanide ions, e.g., Quantum Dye™, fluorescent energy transfer dyes such as thiazole orange-ethidium heterodimer, TOTAB, etc. In one embodiment, the fluorescent label is a phycoerythrin (e.g., R-phycoerythrin (R-PE)). R-PE exhibits extremely bright red-orange fluorescence with high quantum yield. It is excited by laser lines from 488 to 561 nm, with extinction maxima at 496, 546, and 565 nm and a fluorescence emission peak at 578 nm.R-PE is a large molecule used for fluorescence-based detection, e.g., in flow cytometry, microarray assays, ELISAs, and other applications that require high sensitivity but not photostability.

[0074] Fluorescent dyes can be detected in droplets in real time with high resolution, and the availability of many fluorescent dyes with different excitation and emission wavelengths allows the monitoring of many labels in a single experiment. Sets of fluorescent dyes can be selected to allow the simultaneous detection of more than one dye in the same reaction. A set of dyes that can be detected simultaneously can include, but is not limited to, Cy3, Cy5, FAM, JOE, TAMRA, ROX, dR110, dR6G, dTAMRA, dROX, or any mixture thereof. Each of these dyes can be used individually or in any combination to realize any of the present embodiments. One dye can enable the detection of single molecules. A large number of fluorescent dyes have been synthesized and are commercially available in various formats.

[0075] In some embodiments, the label is an affinity tag. Common affinity tags are known in the art, e.g., biotin, histidine, glutathione S-transferase (GST), and maltose-binding protein (MBP). Antibodies and antigens can also be used as affinity tags. In a specific embodiment, the binding agent is labeled with an affinity tag, and this label is used to apply the binding agent to the solid substrate.

[0076] In some embodiments, the label is an isotopic moiety. The isotopic moiety comprises, for example, 32 P, 33 P, 35 S, 125 I and the like. In some embodiments, the solid-phase substrates are magnetically labeled. In some embodiments, the solid-phase substrates are labeled with one or more small molecules.

[0077] In some embodiments, each solid phase substrate is detectably distinguishable from other solid phase substrates within the composition. In some embodiments, the detectably distinguishable solid phase substrates are distinguishable by labeling.

[0078] In one case, a method for detecting biomarkers for solid organ transplant rejection in a patient sample is described.In some cases, the method comprises: (a) detecting a first transplant rejection biomarker in a sample obtained from the individual, wherein the first transplant rejection biomarker is an antibody directed against tubulin, and one or more additional transplant rejection biomarkers, wherein the additional transplant rejection biomarker is an antibody directed against an antigen selected from the group consisting of: LPHN1, SNRPN, KRT18, KRT8, LGALS3, SNRPB2, DEXI, collagen II, PLA2R1, GSTT1, VCL, CSF2, LGALS8, STAT6, GAPDH, IL-8, SHC3, ENO1, AGRN, EMCN, and SSB; (b) determining whether the amount of the transplant rejection biomarker is significantly different from the amount in a control sample; and (c) detecting transplant rejection biomarkers if the determination in (b) shows a significant difference in the patient sample compared to the control sample.In other embodiments, the first transplant rejection biomarker is an antibody directed against tubulin, and the additional transplant rejection biomarker is an antibody directed against an antigen selected from the group consisting of LPHN1, SNRPN, KRT18, KRT8, LGALS3, SNRPB2, DEXI, Collagen II, PLA2R1, GSTT1, VCL, CSF2, LGALS8, STAT6, GAPDH, IL-8, SHC3, ENO1, AGRN, EMCN, and SSB. In some embodiments, detecting further comprises detecting one or more additional transplant rejection biomarkers in the sample of an individual selected from the group consisting of antibodies directed against TG, PECR, NPHS1, FN1, myosin, VIM, ATP5B, LMNA, CXCL11, actin, FLT3LG, PRKCH, and IL-21.

[0079] In one case, a method for detecting biomarkers for solid organ transplant rejection in a sample from an individual is described.In some cases, the method comprises: (a) detecting a first transplant rejection biomarker in a sample obtained from the individual, wherein the first transplant rejection biomarker is an antibody directed against dexamethasone-induced transcript (DEXI), and one or more additional transplant rejection biomarkers, wherein the additional transplant rejection biomarker is an antibody directed against an antigen selected from the group consisting of CXC motif chemokine ligand 11 (CXCL11), cytokeratin 18 (KRT18), cytokeratin 8 (KRT8), TUBα1b: tubulin alpha 1 b (TUBA1B), and tubulin; (b) determining whether the amount of the transplant rejection biomarker is significantly different from the amount in a control sample; and (c) detecting transplant rejection biomarkers if the determination in (b) shows a significant difference in an individual's sample compared to the control sample.In other embodiments, the first transplant rejection biomarker is an antibody directed against tubulin, and the additional transplant rejection biomarker is an antibody directed against an antigen selected from the group consisting of CXCL11, DEXI, KRT18, and KRT8.

[0080] In some embodiments, the detection further comprises detecting one or more additional transplant rejection biomarkers in the individual's sample selected from the group consisting of antibodies directed against LPHN1, CSF2, STAT6, LGALS8, SHC3, GAPDH, GSTT1, and PLA2R1. In other embodiments, the detection further comprises detecting one or more additional transplant rejection biomarkers in the individual's sample selected from the group consisting of antibodies directed against LPHN1, CSF2, STAT6, LGALS8, SHC3, GAPDH, GSTT1, PLA2R1, IL-8, LGALS3, SNPRN, myosin, PECR, VIM, ATP5B, collagen II, LMNA, and SNRPB2.

[0081] In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the non-HLA antigen targets disclosed herein are informative as an independent marker for cardiac allograft rejection. In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the non-HLA antigen targets disclosed herein are predictive of cardiac allograft rejection.

[0082] In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten, 11, or 12 of the non-HLA antigen targets disclosed herein are informative as independent markers of renal allograft rejection. In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten, 11, or 12 of the non-HLA antigen targets disclosed herein are predictive of renal allograft rejection.

[0083] In one case, the procedure is performed with 8 or fewer transplant rejection markers. Optionally, the detection can be performed with up to 5, 10, 15, 20, 25, 30, or up to 35 transplant rejection markers. In some embodiments, the transplant rejection markers are selected exclusively from the group consisting of antibodies directed against DEXI, CXCL11, KRT18, KRT8, TUBA1B, tubulin, LPHN1, CSF2, STAT6, LGALS8, SHC3, GAPDH, GSTT1, PLA2R1, IL-8, LGALS3, SNPRN, myosin, PECR, VIM, ATP5B, collagen II, LMNA, SNRPB2, VCL, TG, FN1, ENO1, AGRN, EMCN, SSB, actin, FLT3LG, PRKCH and IL-21, and combinations of two or more of these markers.In some embodiments, the transplant rejection markers are selected exclusively from the group consisting of antibodies directed against DEXI, CXCL11, KRT18, KRT8, TUBA1 B, tubulin, LPHN1, CSF2, STAT6, LGALS8, SHC3, GAPDH, GSTT1, PLA2R1, IL-8, LGALS3, SNPRN, myosin, PECR, VIM, ATP5B, collagen II, LMNA, SNRPB2, and combinations of two or more of these markers. In other embodiments, the transplant rejection markers further include one or more additional markers beyond those listed herein, such as other markers of interest to the user.

[0084] Furthermore, a method for testing a combination of markers in a sample of a biological fluid obtained from a human subject is described, the method comprising conducting an immunoassay by contacting the sample with the solid support of a kit or composition as described herein. Examples of immunoassays include, but are not limited to, an enzyme-linked immunosorbent assay (ELISA) and a bead-based, particle-based, or other multiplex assay.

[0085] In some cases, the sample is plasma or serum. In some cases, the method further comprises contacting the sample with the conjugates of the kit and testing the reaction of the conjugates with the sample. In some cases, the method further comprises contacting the antigen standards with the solid support and the conjugates and testing the relative levels of transplant rejection biomarkers in the sample relative to the antigen standards.

[0086] Furthermore, a method for determining transplant rejection biomarkers in a serum or plasma sample is described.In some embodiments, the method comprises: (a) providing a binding agent that specifically binds to an antibody directed against DEXI and one or more binding agents that specifically bind to an antibody directed against a single antigen selected from the group consisting of CXCL11, KRT18, KRT8, TUBA1 B, and tubulin; (b) providing a microtiter plate coated with the binding agents; (c) adding the serum or plasma to the microtiter plate; (d) providing alkaline phosphatase-antibody conjugates reactive with transplant rejection biomarkers on the microtiter plate; (e) providing p-nitrophenyl phosphate on the microtiter plate; and (f) examining the response occurring as a result of steps (a) to (e) relative to a standard curve to determine the levels of transplant rejection biomarkers in the sample.

[0087] In some embodiments, the detection further comprises detecting one or more additional transplant rejection biomarkers in the patient sample selected from the group consisting of antibodies directed against LPHN1, CSF2, STAT6, LGALS8, SHC3, GAPDH, GSTT1, PLA2R1, IL-8, LGALS3, SNPRN, myosin, PECR, VIM, ATP5B, collagen II, LMNA, SNRPB2, VCL, TG, FN1, ENO1, AGRN, EMCN, SSB, actin, FLT3LG, PRKCH, and IL-21. In some embodiments, the detection further comprises detecting one or more additional transplant rejection biomarkers in the patient sample selected from the group consisting of antibodies directed against LPHN1, CSF2, STAT6, LGALS8, SHC3, GAPDH, GSTT1, and PLA2R1.In some embodiments, the detection further comprises detecting one or more additional transplant rejection biomarkers in the patient sample selected from the group consisting of antibodies directed against LPHN1, CSF2, STAT6, LGALS8, SHC3, GAPDH, GSTT1, PLA2R1, IL-8, LGALS3, SNPRN, myosin, PECR, VIM, ATP5B, collagen II, LMNA, and SNRPB2.

[0088] Other groupings of transplant rejection biomarkers can be identified by reference to the figures and tables herein. For example, a selection of biomarkers for use together includes one or more members of the Fig. 5 or Fig. 6 as "Group I", "Group II" and / or "Group III" and / or in Table 4 and the grouping of one or more representatives of a group. In another example, a selection comprises one or more representatives of different groups of the markers identified in Fig. 5 or Fig.6 and Table 4. A representative grouping of biomarkers for use together may include one or more members of any of the columns presented in Table 3 and / or Table 5, thereby tailoring the grouping to the detection of cardiac transplant rejection, adult renal transplant rejection, or pediatric renal transplant rejection. In another example, a "core" biomarker (e.g., tubulin) associated with rejection in various organ systems and populations is selected and combined with one or more biomarkers associated with each of the columns listed in Table 3 and / or Table 5.Other criteria for selecting biomarkers for joint use include whether the biomarkers are predictive in the CART (classification and regression tree analysis) analysis, whether the biomarkers are ranked independently in analyses that show they are independent predictors of rejection, whether the markers are correlated in the correlation matrix (see . Fig. 2 and Fig. 3) are grouped together (or independently, as in VCL), whether the biomarkers are significantly associated with the time to first rejection (see Fig.1), whether the biomarkers are newly described here (Table 1 or Table 6) or whether they have previously been associated with transplant rejection, but are not limited thereto. Various other combinations of biomarker groupings are also conceivable. Representative examples include, but are not limited to, a selected single biomarker, such as an antibody directed against tubulin or DEXI or EMCN or LGALS3 or SNPRN or LPHN1 or SSB or TG or CXCL11 or KRT8 or KRT18 as suggested single examples, in combination with one, two, three, four, five or more additional biomarkers; selected subsets of markers grouped together here (e.g., as grouped in Groups I, II, III, or IV or in any of the tables or figures herein); selected combinations comprising one or more representative markers within such subsets.

[0089] In some embodiments, the above steps are performed for 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, or all 31 of the markers listed in Table 1. In one embodiment, the set of markers consists of 8 or fewer of the markers listed in Table 1. In another embodiment, the set of markers consists of 6 or fewer of the markers listed in Table 1. In yet another embodiment, the set of markers consists of 4 or fewer of the markers listed in Table 1. Representative groupings of transplant rejection biomarkers include antibodies against DEXI, KRT8, and KRT18; CXC11, and Tubulin / TUBA1 B; DEXI, KRT8, KRT18, CXC11, and Tubulin / TUBA1 B; DEXI, SNPRN, Smith antigen, LGALS3, ANXA2R, Jo-1, CCP, and TG; DEXI, LPHN1, CSF2, LGALS8, STAT6, and SHC3. Other groupings include tubulin, LPHN1, SNRPN, KRT18, KRT8, DEXI, and GAPDH, as well as various groupings found in the Fig. 5 and Fig. 6 and in the various exemplary embodiments listed herein.

[0090] In one instance, the present disclosure describes a method for determining the presence of one or more of the non-HLA antibodies disclosed herein in a biological sample obtained from an individual, e.g., an individual who has received or will receive an organ transplant. Using the methods described herein, one could screen for or monitor antibodies to non-HLA antigens in individuals with heart and / or kidney allografts and thus assess their risk of developing rejection.

[0091] The first step of the methods generally involves contacting a biological sample obtained from an individual with the composition disclosed herein.

[0092] Contacting the biological sample with the composition is generally accomplished by adding the composition to the sample, or vice versa, and incubating the mixture for a period of time long enough for the composition to specifically bind to the target antibodies present in the sample. Effective or optimal conditions can be determined using methods known in the art.

[0093] Any suitable protocol for obtaining such biological samples may be used, and suitable protocols are well known in the art. When obtaining a sample from an individual (e.g., a blood sample), the amount may vary depending on the size of the individual and the condition being studied. In some aspects, up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 ml of a sample is obtained. In some aspects, about 1-50, 2-40, 3-30, or 4-20 ml of a sample is obtained. In some aspects, more than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mL of a sample is obtained. In some aspects, up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 µL of a sample is obtained. In some aspects, about 1-50, 2-40, 3-30, or 4-20 µL of a sample is obtained.In some aspects, more than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 µI of a sample are obtained. In some aspects, less than about 1 pg, 5 pg, 10 pg, 20 pg, 30 pg, 40 pg, 50 pg, 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 100 ng, 200 ng, 500 ng, 1 µg, 5 µg, 10 µg, 20 µg, 30 µg, 40 µg, 50 µg, 100 µg, 200 µg, 500 µg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 200 mg, 500 mg, 1 gram, 5 grams, 10 grams, 20 grams, 50 grams, 100 grams, or more of the sample is extracted for analysis. In some aspects, approximately 1-5 pg, 5-10 pg, 10-100 pg, 100 pg-1 ng, 1-5 ng, 5-10 ng, 10-100 ng, or 100 ng-1 µg of the sample is extracted for analysis. In some aspects, approximately 1 mg of the sample is extracted for analysis.

[0094] Multiple biological samples can be collected at once. One or more biological samples can be collected from an individual at any time, including before transplantation, at the time of transplantation, or at any time after transplantation.

[0095] After contacting a biological sample with a composition described herein, a signal is generated from the contacting step, which can be detected by any suitable method known in the art. Exemplary methods include visual detection, fluorescence detection (e.g., fluorescence microscopy), scintillation counting, surface plasmon resonance, ellipsometry, atomic force microscopy, surface acoustic wave detection, autoradiography, and chemiluminescence. As one of skill in the art will appreciate, the choice of detection method depends on the particular labeling agent used.

[0096] In some cases, detection is achieved by measuring fluorescence intensity. Such methods are well known in the art. For example, with Luminex's xMAP technology (Luminex Corp., Austin, TX), up to 500 immunoassays in various combinations can be performed in a single reaction in a standard 96-well microplate. In some embodiments, detection is achieved by immunological analysis. The "Examples" section contains exemplary embodiments of the effective or optimal conditions for the methods described herein.

[0097] In some embodiments, detection is performed with labeled secondary anti-human antibodies. Labeled secondary anti-human antibodies are used in the art and are available from various suppliers. In some embodiments, the secondary anti-human antibodies are labeled with enzyme conjugates. Non-limiting examples include alkaline phosphatase (AP) or horseradish peroxidase (HRP). In some embodiments, the secondary anti-human antibodies are labeled with fluorescent conjugates. Non-limiting examples are fluorescein (FITC), tetramethylrhodamine (TRITC), Alexa Fluor, phycoerythrin, etc. In some embodiments, the secondary anti-human antibodies are labeled with biotin. Specific embodiments of labeled secondary anti-human antibodies are provided in the "Examples" section.

[0098] In certain cases, the present disclosure provides methods for diagnosing transplant rejection in an individual who has undergone a heart or kidney transplant. In another embodiment, the present disclosure describes a method for predicting the likelihood of transplant rejection in an individual in need of a heart or kidney transplant.

[0099] In certain cases, the methods provided herein comprise measuring the binding of the one or more homogeneous populations of binding agents to the one or more antibodies that may be present in the sample. In some cases, the methods comprise measuring the levels of the one or more antibodies in the sample. In some embodiments, elevated levels of the one or more antibodies, compared to reference levels, indicate that the individual has a likelihood of developing transplant rejection following a heart or kidney transplant.

[0100] In some embodiments, whether the individual will experience a rejection reaction (e.g., acute rejection) is determined based on the presence of one or more of the biomarkers disclosed herein. In some embodiments, the presence of one or more biomarkers includes one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, or 18 of the non-HLA antibodies disclosed herein at levels above the reference values ​​that are informative as independent markers of allograft rejection.

[0101] The accuracy of the diagnostic and / or prognostic methods can be measured by how close a measured or calculated value is to its actual value. For example, the accuracy of the methods provided herein can be measured by the proportion of correctly predicted rejection or non-rejection. In some embodiments of the present disclosure, the accuracy of the methods described herein is the number of non-rejection individuals for whom the methods described herein are not predicted to have rejection divided by the total number of individuals who actually have no rejection. In other embodiments, the accuracy of the methods described herein is the number of individuals for whom the methods described herein predict rejection divided by the total number of individuals who actually have rejection.In some embodiments, the methods described herein include assessing (e.g., predicting, diagnosing, identifying, etc.) a rejection reaction with an accuracy of about 60-100%. In some embodiments, the accuracy is about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, but not more than 100%. In some embodiments, the accuracy is about 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-100%, but not more than 100%. In some embodiments, the accuracy is about 90%. In some embodiments, the accuracy is about 87%. In some embodiments, the accuracy is about 86%. In some embodiments, the accuracy is about 80%. In some embodiments, the accuracy is about 70%.In some embodiments, the accuracy is about 60%.

[0102] The specificity of diagnostic and / or prognostic methods may be a measure of the proportion of individuals who are actually negative for a disease and are correctly identified by the model as negative for that disease. The specificity of a model may be equal to the number of true negatives divided by the sum of true negatives and false positives. In other words, the specificity of a model may be the probability of a negative test result when the individual is actually negative for the disease. In some cases, the specificity of the methods described herein is the number of non-rejection individuals predicted not to be rejected by the methods described herein divided by the total number of individuals predicted not to be rejected using the methods described herein.In some cases, the comparison step of the methods described herein includes the evaluation (e.g., prediction, diagnosis, identification, etc.) of a rejection reaction with a specificity of approximately 60-100%. In some embodiments, the specificity is about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, but not more than 100%. In some embodiments, the specificity is about 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-100%, but not more than 100%. In some embodiments, the specificity is about 90%. In some embodiments, the specificity is about 80%. In some embodiments, the specificity is about 70%. In some embodiments, the specificity is about 66.67%. In some embodiments, the specificity is about 60%.

[0103] The sensitivity of diagnostic and / or prognostic methods can be a measure of the proportion of individuals who are actually positive for a disease and are correctly identified by the model as being positive for the disease. The sensitivity of a model can be equal to the number of true positives divided by the sum of true positives and false negatives. In other words, the sensitivity of a model can be the probability of a positive test result when the individual is actually positive for the disease. In some cases, the sensitivity of the methods described here is the number of individuals with rejection for whom the methods described here predict rejection divided by the total number of individuals for whom rejection is predicted using the methods described here. In some cases, the comparison step of the methods described herein includes the evaluation (e.g.prediction, diagnosis, identification, etc.) of a rejection reaction with a sensitivity of about 70-100%. In some embodiments, the sensitivity is about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, but not more than 100%. In some embodiments, the sensitivity is about 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-100%, but not more than 100%. In some embodiments, the sensitivity is about 70%. In some embodiments, the sensitivity is about 92%. In some embodiments, the sensitivity is about 99%.

[0104] The present disclosure further encompasses methods of treating an individual in need of treatment for transplant rejection after receiving a heart or kidney transplant. In some cases, the number of non-HLA antibodies disclosed herein with altered levels in a sample obtained from an individual, e.g., elevated or present compared to reference levels, can inform the treatment method. In some embodiments, detection of an elevated level of one or more of the non-HLA antibodies described herein compared to reference levels indicates that the individual is in need of treatment.

[0105] In certain cases, if one or more of the non-HLA antibodies disclosed herein are detected in a biological sample, standard treatment procedures may be used to remove the antibodies. For example, the treating physician may administer, perform, or request plasmapheresis to the individual. Plasmapheresis is a known method by which harmful antibodies can be selectively removed from the individual's bloodstream. In certain cases, the method comprises contacting a biological sample obtained from the individual with the composition disclosed herein, measuring the level of the one or more antibodies in the sample, and administering an anti-graft rejection treatment to the individual if elevated levels of the one or more antibodies disclosed herein are present compared to reference levels of the one or more antibodies.

[0106] In certain embodiments, treatment protocols for AMR utilize permutations of a multi-pronged approach, including, but not limited to: (1) suppression of the T cell-dependent antibody response, (2) removal of reactive antibodies, (3) blockade of residual alloantibodies, and (4) depletion of naive and memory B cells. In certain cases, the treatment regimen consists of the use of plasmapheresis. In certain embodiments, the treatment regimen consists of administration of rituximab. In certain embodiments, the treatment regimen includes, but is not limited to, administration of at least one proteasome inhibitor, such as bortezomib. In certain embodiments, the treatment regimen consists of administration of mycophenolate mofetil. Other treatment methods are known in the art; see, for example, Levine MH, et al., Treatment options and strategies for antibody mediated rejection after renal transplantation. Semin Immunol. 2012 Apr;24(2):136-42, which is incorporated by reference.

[0107] In certain embodiments, the therapeutic agents are selected from tacrolimus, mycophenolate mofetil, and everolimus, with or without corticosteroids. In certain embodiments, the therapeutic agents are tacrolimus, mycophenolate mofetil, and corticosteroids. In certain embodiments, the therapeutic agents are tacrolimus, everolimus, and corticosteroids.

[0108] In certain embodiments, the treatment further includes a steroid. In some embodiments, the steroids include, but are not limited to, corticosteroids (e.g., glucocorticoids and mineralocorticoids). In some embodiments, the corticosteroid is selected from prednisone (Deltasone, Orasone), budesonide (Entocort EC), and prednisolone (Millipred). In some embodiments, steroids are used to reduce inflammation and reduce immune system activity. In certain embodiments, the treatment does not include a steroid.

[0109] In certain cases, the method comprises administering to the individual a therapeutically effective amount of one or more therapeutic agents. In some embodiments, the therapeutic agent is a Janus kinase inhibitor (e.g., tofacitinib (Xeljanz)). In some embodiments, the therapeutic agent is a calcineurin inhibitor (e.g., cyclosporine (Neoral, Sandimmune, SangCya) or tacrolimus (Astagraf XL, Envarsus XR, Prograf)). In some embodiments, the therapeutic agent is an mTOR inhibitor (e.g., sirolimus (Rapamune) or everolimus (Afinitor, Zortress)). In some embodiments, the therapeutic agent is an IMDH inhibitor (e.g., azathioprine (Azasan, Imuran), leflunomide (Arava), or mycophenolate (CellCept, Myfortic)). In some embodiments, the therapeutic agent is a biologic (e.g.,, Abatacept (Orencia), Adalimumab (Humira), Anakinra (Kineret), Certolizumab (Cimzia), Etanercept (Enbrel), Golimumab (Simponi), Infliximab (Remicade), Ixekizumab (Taltz), Natalizumab (Tysabri), Rituximab (Rituxan), Secukinumab (Cosentyx), Tocilizumab (Actemra), Ustekinumab (Stelara), Vedolizumab (Entyvio)), or Belatacept (Nulojix). In some embodiments, the therapeutic agent is a monoclonal antibody (e.g., Basiliximab (Simulect) or Daclizumab (Zinbryta)). In certain embodiments, the treatment comprises one or more therapeutic agents selected from those mentioned above.

[0110] In certain embodiments, the treatment further includes a steroid. In some embodiments, the steroids include, but are not limited to, corticosteroids (e.g., glucocorticoids and mineralocorticoids). In some embodiments, the corticosteroid is selected from prednisone (Deltasone, Orasone), budesonide (Entocort EC), and prednisolone (Millipred). In some cases, steroids are used to reduce inflammation and reduce immune system activity. In certain embodiments, the treatment does not include a steroid.

[0111] In certain embodiments, the therapeutic agents are selected from tacrolimus, mycophenolate mofetil, and everolimus, with or without corticosteroids. In certain embodiments, the therapeutic agents are tacrolimus, mycophenolate mofetil, and corticosteroids. In certain embodiments, the therapeutic agents are tacrolimus, everolimus, and corticosteroids.

[0112] In another embodiment, the present disclosure provides a kit. Such a kit is a packaged combination comprising the following basic elements: (a) a composition comprising a collection of solid phase substrates coated with one or more homogeneous populations of binding agents, each homogeneous population of binding agents specifically binding to an antibody directed against a single antigen selected from the group consisting of (DEXI), CXC motif chemokine ligand 11 (CXCL11), cytokeratin 18 (KRT18), cytokeratin 8 (KRT8), tubulin, including tubulin alpha 1 b (also referred to as TUBa1b or TUBA1 B), latrophilin 1 (LPHN1), colony stimulating factor 2 (CSF2), signal transducer and activator of transcription 6 (STAT6), lectin galactoside-binding soluble 3 (“lectin galactoside-binding soluble 3”;LGALS3), SHC adaptor protein 3 (SHC3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutathione S-transferase theta-1 (GSTT1), phospholipase A2 receptor 1 (PLA2R1), interleukin 8 (IL-8), lectin galactoside-binding soluble 8 (LGALS8), small nuclear ribonucleoprotein polypeptide N (SNPRN), myosin, peroxisomal trans-2-enoyl-CoA reductase (PECR), vimentin (VIM), ATP synthase H+ transporting mitochondrial F1 complex beta polypeptide (ATP5B), collagen II, prelamin A / C (LMNA), small nuclear ribonucleoprotein polypeptide B (SNRPB2), Fibronectin 1 (FN1) and vinculin (VCL) and (b) reagents for detecting the binding of the one or more homogeneous populations of binders to the antibodies.;

[0113] In other embodiments, each homogeneous population of binding agents specifically binds to an antibody directed against a single antigen selected from the group consisting of tubulin, LPHN1, SNRPN, KRT18, KRT8, LGALS3, SNRPB2, DEXI, collagen II, PLA2R1, GSTT1, VCL, CSF2, LGALS8, STAT6, GAPDH, IL-8, SHC3, ENO1, AGRN, EMCN, SSB, TG, PECR, NPHS1, FN1, myosin, VIM, ATP5B, LMNA, CXCL11, actin, FLT3LG, PRKCH, and IL-21. In certain embodiments, the kits also include instructions for use.

[0114] In another embodiment, the kit may comprise one or more reference samples. Reference samples may be any biological samples as used herein. In another embodiment, the kit may comprise one or more reference levels of the non-HLA antibodies disclosed herein.

[0115] All patents and publications mentioned in this specification represent the level of knowledge of the person skilled in the art to which the disclosure relates. All patents and publications cited herein are incorporated by reference to the same extent as if each individual publication were expressly and individually indicated as being incorporated by reference in its entirety. Exemplary embodiments

[0116] The following are examples of the embodiments described herein: Embodiment 1: A composition comprising a collection of solid phase substrates coated with one or more homogeneous populations of binding agents, wherein each homogeneous population of binding agents specifically binds to an antibody directed against a single antigen selected from the group consisting of: dexamethasone-induced transcript (DEXI), CXC motif chemokine ligand 11 (CXCL11), cytokeratin 18 (KRT18), cytokeratin 8 (KRT8), tubulin, including tubulin alpha 1 b (also referred to as TUBα1 b or TUBA1 B), latrophilin 1 (LPHN1), colony stimulating factor 2 (CSF2), signal transducer and activator of transcription 6 (STAT6), lectin galactoside-binding soluble 3 (“lectin galactoside-binding soluble 3”;LGALS3), SHC adaptor protein 3 (SHC3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutathione S-transferase theta-1 (GSTT1), phospholipase A2 receptor 1 (PLA2R1), interleukin 8 (IL-8), lectin galactoside-binding soluble 8 (LGALS8), small nuclear ribonucleoprotein polypeptide N (SNPRN), myosin, peroxisomal trans-2-enoyl-CoA reductase (PECR), vimentin (VIM), ATP synthase H+ transporting mitochondrial F1 complex beta polypeptide (ATP5B), collagen II, prelamin A / C (LMNA), small nuclear ribonucleoprotein polypeptide B (SNRPB2), fibronectin 1 (FN1), nephrosis 1, congenital Finnish type nephrosis 1 (NPHS1), thyroglobulin (TG) and vinculin (VCL).; Embodiment 2: The composition of claim 1, wherein the collection of solid phase substrates further comprises one or more additional homogeneous populations of binding agents, each additional homogeneous population of binding agents specifically binding to an antibody directed against a single additional antigen selected from the group consisting of: alpha-enolase (ENO1), agrin (AGRN), endomucin (EMCN), Sjogren's syndrome antigen B (SSB), actin, fms-related tyrosine kinase 3 ligand (FLT3LG), protein kinase C eta (PRKCH), and interleukin 21 (IL-21). Embodiment 3: Composition according to embodiment 1 or 2, wherein the solid phase substrates are porous or non-porous. Embodiment 4: The composition of embodiment 2 or 3, wherein the solid phase substrates comprise particles, nanoparticles, beads, nanospheres or microspheres. Embodiment 5: Composition according to 4, wherein the beads are polystyrene beads. Embodiment 6: The composition of any preceding embodiment, wherein the collection of solid phase substrates comprises a microarray. Embodiment 7: Composition according to any one of the preceding embodiments, wherein the solid phase substrates are fluorescently labeled, magnetically labeled or radioactively labeled. Embodiment 8: Composition according to any one of the preceding embodiments, wherein the solid phase substrates are labeled with a small molecule. Embodiment 9: Composition according to any one of the preceding embodiments, wherein the one or more homogeneous populations of binders are conjugated to the surface of the solid phase substrates. Embodiment 10: The composition of embodiment 9, wherein the conjugation is covalent. Embodiment 11: A composition according to any one of the preceding embodiments, wherein the one or more homogeneous populations of binders are bound by affinity to the surface of the solid phase substrates. Embodiment 12: Composition according to any one of the preceding embodiments, wherein the binder is a polypeptide. Embodiment 13: Composition according to any one of the preceding embodiments, wherein the solid phase substrates are coated with at least three different homogeneous populations of binders that bind to at least three different antigens. Embodiment 14: The composition of embodiment 13, wherein the substrates are coated with a plurality of different homogeneous populations of binders that bind to the antibodies consisting of antibodies against tubulin, LPHN1, SNRPN, KRT18, KRT8, LGALS3, SNRPB2, DEXI, collagen II, PLA2R1, GSTT1, VCL, CSF2, LGALS8, and STAT6. Embodiment 15: The composition of embodiment 13, wherein the substrates are coated with a plurality of different homogeneous populations of binders that bind to the antibodies consisting of antibodies against tubulin, LPHN1, TG, GAPDH, FN1, NPHS1, VIM, myosin, VCL, and PECR. Embodiment 16: The composition of embodiment 15, wherein the substrates are further coated with a plurality of different homogeneous populations of binding agents that bind to the antibodies consisting of antibodies against ENO1, AGRN, EMCN, SSB, actin, FLT3LG, PRKCH, and IL-21. Embodiment 17: The composition of embodiment 13, wherein the substrates are coated with a plurality of different homogeneous populations of binding agents that bind to the antibodies consisting of antibodies against DEXI, LGALS3, SNPRN, CSF2, IL-8, STAT6, LGALS8, KRT18, KRT8, GSTT1, LMNA, Collagen II, ATP5B, SNRPB2, and PLA2R1. Embodiment 18: The composition of embodiment 13, wherein the substrates are coated with multiple different homogeneous populations of binders that bind to the antibodies consisting of antibodies against tubulin, SHC3, and CXCL11. Embodiment 19: The composition of embodiment 13, wherein the substrates are coated with a plurality of different homogeneous populations of binding agents that bind to the antibodies consisting of antibodies against DEXI, EMCN, SNRPN, LPHN1, and SSB. Embodiment 20: The composition of embodiment 13, wherein the substrates are coated with a plurality of different homogeneous populations of binding agents that bind to the antibodies consisting of antibodies against KRT18, GAPDH, AGRN, ENO1, and EMCN. Embodiment 21: The composition of embodiment 13, wherein the substrates are coated with a plurality of different homogeneous populations of binders that bind to the antibodies consisting of antibodies against tubulin, LPHN1, SNRPN, KRT18, KRT8, LGALS3, SNRPB2, DEXI, collagen II, GAPDH, ENO1, AGRN, EMCN, SSB, PLA2R1, GSTT1, VCL, CSF2, LGALS8, STAT6, IL-8, and SHC3. Embodiment 22: The composition of embodiment 1, wherein the single antigen is selected from the group consisting of: LPHN1, TG, DEXI, CSF2, IL-8, LGALS3, SNPRN, STAT6, SHC3 and LGALS8. Embodiment 23: The composition of embodiment 2, wherein the single additional antigen is selected from the group consisting of: EMCN and SSB. Embodiment 24: Composition according to any one of the preceding embodiments, wherein at least one solid phase substrate is detectably distinguishable from at least one other solid phase substrate. Embodiment 25: A kit comprising: the composition of any one of embodiments 1-24, and reagents for detecting binding of the one or more homogeneous populations of binding agents to the antibodies. Embodiment 26: The kit of embodiment 25, further comprising one or more reference samples. EXAMPLES

[0117] The following examples are provided for illustrative purposes. They are intended to show certain aspects and embodiments of the present disclosure, but are not intended to limit the disclosure in any way. Example 1. Non-HLA antibodies associated with heart and kidney allograft rejection

[0118] There is growing evidence that the development of antibodies to non-HLA antigens after transplantation is associated with rejection and reduced long-term graft survival. This example describes the use of sera from kidney and heart transplant recipients obtained before and after transplantation to create a panel of non-HLA antigen targets that can be used to identify transplant recipients with circulating non-HLA antibodies that may indicate the risk of graft damage and graft loss.

[0119] Non-HLA antibodies reactive with endothelial cells were identified by testing sera from heart and kidney transplant recipients diagnosed with acute rejection without detectable circulating donor-specific HLA antibodies for binding to human primary arterial endothelial cells in a flow-through crossmatch assay (Zhang and Reed, Transplantation 2005). Sera positive in the endothelial cell crossmatch assay were tested untreated and / or after absorption and elution from aortic endothelial cells on protoarrays containing >9000 full-length human protein antigens. Non-HLA antibody targets associated with the aortic endothelial cell plasma membrane and autoantigens were classified using a bioinformatics and gene ontology approach. These studies led to the identification of 31 non-HLA antigen targets (Table 1).These and other non-HLA antigens were conjugated to polystyrene beads to develop a multiplex bead array for further high-throughput testing in cardiac, pediatric, and adult kidney transplant recipients (Table 1).

[0154] Table 1. Non-HLA antibodies associated with heart and kidney allograft rejection # Marking ID antigen Gene designation 1 Heart 1 DEXI Dexamethasone-induced transcript 2 Heart 2 EMCN Endomucin 3 Heart 3 SNRPN Small nuclear ribonucleoprotein polypeptide N (Smith antigen nuclear sequence) 4 Heart 4 NTRK1 Neurotrophic tyrosine kinase receptor type 1 5 Heart 5 ROR1 Receptor tyrosine kinase-like orphan receptor 1 6 Heart 6 LGALS3 Lectin, galactoside binding, soluble, 3 7 Heart 7 ANXA2R Annexin A2 receptor 8 Heart 8 IYD Iodotyrosine diiodinase 9 Heart 9 EMCN Endomucin 10 Herzl 10 FGFR1 Fibroblast growth factor receptor 1 11 Heart 11 HARS Jo-1 12 Heart 12 SPN Sialophorin, CD43 13 Heart 13 SSB Sjogren syndrome antigen B (autoantigen La) 14 Heart 14 IL-8 Interleukin 8, CXCL8 15 Hez 15 CCP cyclic citrullinated peptide 16 Heart 16 VEGFA vascular endothelial growth factor A 17 Heart 17 DQA1*01:02 HLA-DQA1 18 Heart 18 LPHN1 Latrophilin 1 19 Heart 19 TG Thyroglobulin 20 Kidneys 1 FGF2 Fibroblast growth factor receptor 2 21 Kidneys 2 CSF2 Colony stimulating factor 2 22 Kidneys 3 NGF Nerve growth factor 23 Kidneys 4 LGALS8 Lectin, galactoside binding, soluble, 8 24 Kidneys 5 LGALS1 Lectin, galactoside binding, soluble, 1 25 Kidneys 6 MAPK1 Mitogen-activated protein kinase 1 26 Kidneys 7 SDF1B CXC motif chemokine ligand 12 27 Kidneys 8 VWF Von Willebrand factor 28 Kidneys 9 STAT6 Signal transducer and activator of transcription 6 29 Kidneys 10 SHC3 SHC adapter protein 3 30 Kidneys 11 GNG5 G protein subunit gamma 5 31 Kidneys 12 IL18R1 Interleukin-18 receptor 1

[0120] Cohort for the detection of heart transplant rejection. The heart transplant detection cohort was determined among 12 heart allograft recipients transplanted at UCLA between 2001 and 2005 who tested positive for endothelial cell (EC) antibodies and negative for HLA-DSA and MICA antibodies using endothelial cell flow crossmatch (ECXM) testing (Zhang Transplantation 2005). Six ECXM and HLA-DSA patients without rejection were included as controls. Patients were HLA-typed using the LABType SSO DNA typing method (One Lambda, Canoga Park, CA) according to the manufacturer's specifications. MICA antibodies and HLA class I and class II antibodies were determined using a single antigen Luminex assay (One Lambda, Canoga Park, CA).Acute cellular rejection (ACR) and AMR were diagnosed by endomyocardial biopsy (EMB) according to the International Society for Heart and Lung Transplantation (ISHLT) criteria and as previously reported (Zhang Transplantation 2005, Stewart J Heart and Lung Transplant, 005). The average time for serum sample collection from the day of transplantation was 67 days and from the day of rejection + biopsy was 14 days.

[0121] Kidney transplant rejection detection cohort. The kidney transplant detection cohort (n=16) was identified from kidney allograft recipients transplanted at UCLA between 2010 and 2015 who were diagnosed with rejection and tested positive for endothelial cell antibodies using ECXM (Zhang Transplantation 2005). Pre-transplant serum from 3 of the 16 patients served as a negative control. Patient sera were tested untreated and after adsorption and elution from endothelial cells. The average time for serum sample collection from the day of transplantation was 21 days, and from the day of rejection + biopsy, 23 days.

[0122] Validation cohort of the single-center heart transplant study. The single-center study cohort consisted of 63 heart transplant recipients transplanted at UCLA between 2009 and 2016. Serum samples were collected at the time of EMB. Rejection (n=42, ACR >1R) was assessed according to the 1990 ISHLT revision of the working formula for heart transplant rejection. The median and interquartile range of days from serum collection to EMB were 0 for both rejection and non-rejection samples (p<0.26). After transplantation, patients maintained triple drug immunosuppression (tacrolimus, mycophenolate mofetil, and corticosteroids).

[0123] Pediatric Kidney Transplant Validation Cohort. Eighty-three pediatric kidney transplant patients transplanted at UCLA were enrolled in this study from August 2005 to November 2014. Twenty-one patients were excluded from this analysis because more than one study sample was missing at the listed time points. The remaining 62 patients were enrolled in this retrospective study. This study was approved by the UCLA Institutional Review Board (#11-002375) and complies with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards, as well as the tenets of the Istanbul Declaration. Informed consent from parental guardians was obtained for all patients. Immunosuppression regimens included induction with ATG for PRA ≥30%, delayed graft function or rapid steroid withdrawal protocol, or an anti-CD25 monoclonal antibody for PRA <30%.Maintenance immunosuppression consisted of steroid-free or steroid-based immunosuppression, a calcineurin inhibitor, and an antimetabolite. Acute rejection and chronic rejection were treated according to previously described protocols (Pearl, Pediatr Nephrol, 2016). Patients underwent protocol biopsies at 6, 12, and 24 months post-transplantation or when clinically indicated. Biopsy samples were evaluated based on the 2013 Banff criteria (Haas, Am J Transplant, 2014). Blood samples were collected pre-transplantation, as well as at 6, 12, and 24 months post-transplantation and during episodes of renal allograft rejection.

[0124] Validation cohort for adult kidney transplants. The cohort consisted of 163 kidney transplant recipients transplanted at UCLA between 2006 and 2012. Biopsy samples were evaluated based on the 2013 Banff criteria (Haas, Am J Transplant, 2014). Sera were collected at the time of biopsy (+ / - 6 weeks). After transplantation, patients received triple immunosuppression (tacrolimus, mycophenolate mofetil, and corticosteroids). Induction was primarily performed with solumedrol and basiliximab or anti-thymocyte globulin. IVIG is used to augment immunosuppression at the time of transplantation in patients with DSA diagnosed within one year before transplantation (current). In patients with a history of DSA, the use of IVIG at the time of transplantation is at the discretion of the treating nephrologist.This study was approved by the UCLA Institutional Review Board (#16-000786).

[0125] HLA typing and HLA-DSA testing. All patients were HLA-typed using the LABType SSO DNA typing method (One Lambda, Canoga Park, CA) according to the manufacturer's specifications. MICA antibodies and HLA class I and class II antibodies were identified using a single antigen Luminex assay (One Lambda, Canoga Park, CA).

[0126] Kidney transplant rejection. ACR and AMR were diagnosed by renal biopsy according to the Banff criteria (Haas M et al., AJT 2014).

[0127] Protoarray assay for the detection of non-HLA antigens. Serum antibodies were profiled using a human protein microarray as previously described. Briefly, the InVitrogen Human ProtoArray v5.0, which contains over 9,000 proteins from a baculovirus-based expression system, was blocked with blocking buffer for 1 hour and then incubated for 90 minutes with 5 µI of serum diluted 1:150 in PBST buffer. Slides were washed four times with 5 ml of fresh PBST buffer for 10 minutes each and probed for 90 minutes with goat anti-human Alexa Fluor 647 IgG secondary antibodies (Molecular Probes, Eugene, OR). After a second wash with PBST buffer, the slides were dried and scanned using a GenePix 4100A fluorescence microarray scanner and GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA). The protein arrays included 12 rejection+ / ECXM+ sera and 6 rejection- / ECXM- sera, which served as technical controls.Array normalization and analysis were performed using Prospector 2.0 software (Life Technologies, Grand Island, NY) as previously described. A gene ontology analysis of the 366 antigens was performed using the DAVID Gene Ontology Analysis Software (available online at david.ncifcrf.gov) to identify biological enrichment themes, as previously described.

[0128] Development of the multiplex non-HLA antigen panel for the detection of non-HLA antibodies. The newly described non-HLA antigen targets were discovered using the selected detection cohorts described above and in Table 1. Genetic ontological analyses and tissue expression data were used to select the most biologically relevant non-HLA target molecules for inclusion in the non-HLA panel. The non-HLA multiplex single-antigen bead panel was manufactured by Immucor, Inc. (Peachtree Corners, GA), and reagents for screening sera from selected cohorts of heart, adult kidney, and pediatric kidney transplant recipients were provided by Immucor, Inc. Briefly, 40 µl of antigen-coated beads were incubated with 10 µl of patient serum for 30 minutes.Unbound serum was removed by washing, and the beads were stained with 50 µl of a conjugate containing phycoerythrin (PE)-conjugated goat anti-human IgG, which was diluted 1:10 in wash buffer and incubated for 30 minutes in the dark on a rocking platform. Results were examined by reading the median fluorescence intensity (MFI) of IgG binding on the Luminex 100 analyzer (Luminex, Austin, TX). To determine a positive threshold for the Luminex assay for non-HLA antibodies, non-HLA antibodies were examined in sera isolated from 44 healthy individuals. The median MFI of antibody reactivity to the 18 antigens significantly associated with cardiac allograft rejection was 510 MFI (range: 67–8367 MFI, SD=900). Based on previous experience with Luminex-based solid-phase antibody detection methods, a positive threshold of MFI >1000 was chosen.

[0129] Statistics. The odds ratios associating non-HLA antibodies with allograft rejection were determined to be significantly greater than 1 using the two-sided Fisher's exact test (p<0.1; StataCorp. 2015. Stata Statistical Software: Release 14. College Station, TX: StataCorp LP). The threshold for significance of p<0.1 was set according to standard practice for evidence-based analyses (Fan, L. et al., Am J Transplant 2011), and the default alpha value (p-value) of 0.1 in STATA was used to prevent exclusion of antibodies that might interact with each other. Confidence intervals (95%) were constructed assuming that the standard error estimates were asymptotically independent and normal.

[0130] In cluster analysis, the arrangement of non-HLA markers, the size of the circles, and the representation of the inner boxes to identify clusters are determined according to the numerical value of the pairwise correlation coefficient. The analysis was performed using hierarchical clustering with full linkage and Euclidean distance in the R Corrplot application (Simko, T. wa V. 2017).

[0131] To perform the classification and regression tree (CART) analysis, the rpart function in the R library (R software package version 3.4.0 (available online at www.r-project.org / )) was used. To avoid overfitting and select a parsimonious set of predictor variables, the maxdepth (the maximum depth of any node of the final tree, with the root node counted as depth 0) was set to 3 and the minsplit (the minimum number of observations that must be present in a node for a split to be attempted) was set to 5. All other computer software parameters were set to their default values.

[0132] Development of a non-HLA panel for cardiac transplants: We hypothesize that antibodies targeting non-HLA antigens expressed by the allograft endothelium could be identified by protein microarray analysis using sera from cardiac allograft recipients with biopsy-proven rejection. Sera from twelve cardiac transplant recipients without HLA or MICA DSA but with biopsy-proven rejection and positive ECXM reactivity (median of 117 MCS; range: 62–529 MCS) were assembled as a detection cohort. Detection cohort sera were collected an average of 14 days after EMB (range: 0–76 days). EMB were obtained an average of 67 days after transplantation (range: 20–222 days). An additional six sera that were not associated with rejection and that were ECXM-negative and MICA DSA-negative were used as controls.The 18 sera (12 rejection+ / ECXM+ sera and 6 rejection- / ECXM-control sera) were hybridized to protein microarrays containing 9,000 full-length human proteins. Bioinformatic analysis of the protein microarrays identified 366 rejection-associated antigens with significantly increased fluorescence intensity (>1.5-fold; p<0.05), indicating positive antibody reactivity in sera isolated from rejection+ / ECXM+ patients compared to rejection- / ECXM controls. Subsequently, an ontological gene analysis of the 366 non-HLA antigens was performed to identify those biologically most relevant with respect to known functional properties. From this analysis, 22 plasma membrane antigens and 10 known autoantigens were selected because they are likely target antigens expressed on the endothelial cell surface and may contribute to a positive ECXM.Of these antigens, 19 could be expressed and conjugated to Luminex polystyrene beads for further downstream high-throughput testing (Table 1). Other non-HLA antigens (9 heart, 30 kidney, 8 lung, and 1 liver antigen) (Table 4) were selected to create a multiplex panel of 67 non-HLA antigens.

[0133] Development of a non-HLA panel for kidney transplants: Sera from 16 kidney transplant recipients without HLA or MICA DSA, but with biopsy-proven rejection and a positive ECXM, were tested. Three additional sera collected pre-transplant from 3 / 16 recipients served as negative controls. The sera were tested untreated in parallel and after adsorption and elution for endothelial cells. The non-adsorbed and eluted sera were tested on protein microarrays, resulting in the identification of 1252 antigenic targets (>1.5-fold increase, p<0.05) compared to the pre-transplant sera. Prospector analysis identified antibodies present in both the untreated and eluted rejection sera and reacting with 386 different proteins. Of these, 251 were excluded from the analysis because they were also present in the pre-transplant sera.This resulted in 135 unique proteins as potential candidates for the creation of a non-HLA panel. Following gene ontology and frequency analysis, 12 of the 135 antigen targets were selected based on their expression profile and functional properties and added to the 67 antigens used in the cardiac multiplex bead array to create the renal multiplex bead array.

[0134] Identification of non-HLA antibodies associated with allograft rejection. Multiplex bead array assays were used to examine serum samples from 34 non-rejection patients and 33 with rejection transplanted at UCLA between 2009 and 2016 to determine whether they developed non-HLA antibodies. A higher percentage of 10 non-HLA antibodies (tubulin, LPHN1, thyroglobulin (TG), GAPDH, FN1, NPHS1, VIM, myosin, VCL, PECR) was observed in heart transplant recipients with rejection compared to serum samples from non-rejection patients (Table 2). LPHN1 and thyroglobulin (TG) are newly described here.

[0170] Table 2. Non-HLA Abs associated with rejection in the UCLA cohort of adult cardiac patients Non-HLA-Ab No rejection (n=34) Rejection (n=33) Exact Fisher p-value Tubulin 23,53% 36,36% 0,189 LPHN1 17,65% 33,33% 0,116 TG 5,88% 12,12% 0,322 GAPDH 0 9,09% 0,114 FN1 0 9,09% 0,114 NPHS1 2,94% 12,12% 0,169 VIM 2,94% 9,09% 0,295 Myosin 5,88% 12,12% 0,322 VCL 5,88% 15,15% 0,201 PECR 5,88% 15,15% 0,201

[0135] Identification of non-HLA antibodies associated with kidney allograft rejection. Serum samples from pediatric kidney transplant recipients (samples: n=95 without rejection and n=34 rejection) and adult kidney transplant recipients (samples: n=90 without rejection and n=70 rejection) were analyzed using the multiplex bead array. Patients in the rejection group experienced at least one histologically proven rejection episode during the study period. Samples from the pediatric and adult kidney cohorts were analyzed independently ( Fig. 1A and B). In these independent analyses, 15 and 3 antibodies against non-HLA antigens, respectively, were identified as significantly associated with rejection (p<0.01 and a risk ratio >1) ( Fig.1). In the pediatric cohort, antibodies against 15 non-HLA antigens (y-axis) were identified as significantly associated with the time to first kidney rejection with an odds ratio >1 (x-axis) in pediatric kidney transplant recipients ( Fig. 1A). Seven of these, DEXI, CSF2, IL-8, LGALS3, SNPRN, STAT6, and LGALS8, are newly described here as significantly associated with kidney allograft rejection. The bars represent the 95% confidence interval (CI). Antibodies against three additional non-HLA antigens (tubulin, CXCL11, SHC3) were significantly associated with kidney allograft rejection in adult kidney transplant recipients ( Fig. 1B). SHC3 was newly found to be significantly associated with kidney transplant rejection.

[0136] Table 3 shows a list of non-HLA antibodies associated with rejection after adult heart transplantation (first column), pediatric kidney transplantation (second column), and adult kidney transplantation (third column). The same targets are present in the Fig. 1-3, 5 and 6, with indicators for those that are predictive in the CART analysis, are independently sorted, are newly described and form clusters in the correlation matrix. Table 3. Non-HLA Abs associated with rejection in the UCLA Adult Cardiac Cohort, the UCLA Pediatric Renal Cohort, and the UCLA Adult Renal Cohort. Heart, adult Kidneys, pediatric Kidneys, adult Tubulin DEXI Tubulin LPHN1 LGALS3 SHC3 TG SNPRN CXCL11 GAPDH CSF2 FN1 IL-8 NPHS1 STAT6 VIM LGALS8 Myosin KRT18 VCL KRT8 PECR GSTT1 LMNA Collagen II ATP5B SNRPB2 PLA2R1

[0137] A correlation matrix analysis of non-HLA antibodies associated with rejection after adult heart transplantation was performed ( Fig.2A). Non-HLA antibodies associated with cardiac allograft rejection in adults can be selectively grouped into four groups, one of which forms an independent cluster (VCL). (Non-HLA antibodies associated with pediatric renal allograft rejection can be selectively grouped into six groups ( Fig. 2B), four of which form independent clusters (PLAR1, CSF2, GSTT1, and LGALS8). Seven of the non-HLA antibodies were newly identified in this study as being associated with renal allograft rejection (CSF2, SNPRN, LGALS8, STAT6, IL-8, DEXI, and LGALS3). The non-HLA antibodies found to be significantly associated with transplant rejection in the pediatric kidney transplant cohort were extrapolated to the adult kidney transplant cohort to determine a similar correlation ( Fig.2C). The antigens that are independently correlated during a rejection episode are similar in pediatric and adult kidney transplant patients with rejection (adult kidneys: CSF2, GSTT1).

[0138] The pediatric and adult cohorts were then combined in an analysis to determine how antibodies specific for the panel of 18 non-HLA targets are sorted independently upon rejection ( Fig. 3, n=104). In the rejection samples, the antibodies cluster into 9 clusters, with four of the 18 forming independent clusters (PLA2R, STAT6, GSTT1, and CSF2). Three of these four (STAT6, GSTT1, and CSF2) were newly identified within the context of the proteomics-based detection.

[0139] To identify non-HLA antibodies capable of distinguishing rejection from non-rejection, a classification and regression tree analysis (CART) was performed. Fig. 4 shows the CART analysis of sera from adult heart (n=67 sera, Fig. 4A) and pediatric kidney allotransplantation patients (n=129 sera, Fig.4B). CART analysis of sera isolated from adult cardiac allotransplantation patients (n=67 sera) with an MFI cutpoint of 1000 was used for the analysis. The root node, LPHN1, containing all 67 sera, of which 49% are rejection samples, splits into subordinate nodes at an MFI<1000. As the algorithm proceeds to the terminal nodes, 65% of the rejection samples are correctly identified (far right, dark boxes). The scale bar indicates the association with rejection, with the lighter boxes of the terminal nodes correlating with non-rejection. Eight non-HLA antibodies (SNPRN, KRT18, LGALS3, KRT8, SNRPB2, DEXI, PLA2R1, Collagen II, and GSTT1) were informative predictors of renal allograft rejection. The root node SNPRN, which contains all 129 sera, of which 26% are rejection samples, divides into subordinate nodes at an MFI <1000.As the algorithm proceeds to the terminal nodes, 56% of the non-rejection samples are correctly identified (far left, darker boxes). The scale bar indicates association with rejection, with the lighter boxes of the terminal nodes correlating with non-rejection. PLA2R1 and GSTT1 were also found to independently cluster in the rejection samples, and three of the eight (SNPRN, LGALS3, and DEXI) were newly identified here.

[0140] Non-HLA antibodies significantly associated with kidney and heart allograft rejection are described below. Using a protein microarray containing 9000 full-length proteins, followed by gene ontology analysis and the development of a high-throughput multiplex bead array, 10 non-HLA antibodies were associated with heart allograft rejection (Table 2) and 18 non-HLA antibodies were found to be significantly associated with pediatric and adult kidney allograft rejection ( Fig. 1, Table 3). Two of these non-HLA antigens are newly described here as being associated with transplant rejection (LPHN1 and TG). Nine of these renal targets, DEXI, SHC3, SNPRN, LGALS3, CSF2, LGALS8, and STAT6, are newly described here as being associated with transplant rejection ( Fig. 1, Table 3). The non-HLA antibodies are shown in Table 3 and Fig.5. Some markers are found in multiple organs and are predictive in the CART analysis. Other markers are non-HLA antibodies that sort independently. Still other markers include non-HLA antibodies that sort together in the correlation matrix and are independent of other markers. The correlation matrix analysis identified a group of non-HLA antibodies that can be used independently to predict rejection.

[0178] Table 4 # Antigen Gene designation function reference 1 DEXI Dexamethasone-induced transcript This gene encodes a member of the Ras superfamily of small GTPases and is induced by dexamethasone. The encoded protein is an activator of G protein signaling and acts as a direct nucleotide exchange factor for Gi-Go proteins. This protein interacts with the neuronal nitric oxide adaptor protein CAPON and a nuclear adaptor protein FE65, which interacts with the amyloid precursor protein of Alzheimer's disease. This gene may play a role in dexamethasone-induced changes in cell morphology, cell growth, and cell-extracellular matrix interactions. Epigenetic inactivation of this gene is closely associated with dexamethasone resistance in multiple myeloma cells. Alternatively spliced ​​transcript variants encoding different isoforms have been found for this gene. Small GTPase. Negatively regulates the transcriptional regulatory activity of the APBB1 / FE65-APP complex. 2 EMCN Endomucin EMCN is a mucin-like sialoglycoprotein that impairs the assembly of focal adhesion complexes and inhibits the interaction between cells and the extracellular matrix (Kinoshita et al., 2001). 3 SNRPN Small nuclear ribonucleoprotein polypeptide N (Smith antigen nuclear sequence) The protein encoded by this gene is a polypeptide of a small nuclear ribonucleoprotein complex and belongs to the snRNP SMB / SMN family. The protein plays a role in pre-mRNA processing, possibly in tissue-specific alternative splicing events. Although individual snRNPs are thought to recognize specific nucleic acid sequences through RNA-RNA base pairing, the specific role of this family member is unknown. The protein arises from a bicistronic transcript that also encodes a protein called the SNRPN upstream reading frame (SNURF). Several transcription initiation sites have been identified, and extensive alternative splicing occurs in the 5' untranslated region. Additional splice variants have been described, but the sequences of the complete transcripts have not been determined. The 5' UTR of this gene has been identified as an imprinting center.Alternative splicing or a deletion caused by a translocation event in this patemally expressed region is responsible for Angelman syndrome or Prader-Willi syndrome due to failure of the parental imprinting switch. 4 NTRK1 Neurotrophic tyrosine kinase receptor type 1 This gene encodes a member of the neurotrophic tyrosine kinase receptor (NTKR) family. This kinase is a membrane-bound receptor that, upon binding to a neurotrophin, phosphorylates itself and members of the MAPK signaling pathway. The presence of this kinase leads to cell differentiation and may play a role in the specification of sensory neuron subtypes. Mutations in this gene have been associated with congenital insensitivity to pain, anhidrosis, self-mutilating behavior, mental retardation, and cancer. Alternative transcriptional splice variants of this gene have been found, but only three have been characterized to date. Receptor tyrosine kinase involved in the development and maturation of the central and peripheral nervous system by regulating the proliferation, differentiation, and survival of sympathetic and neurons. 5 ROR1 Receptor tyrosine kinase-like orphan receptor 1 A receptor tyrosine kinase-like orphan receptor that modulates neurite outgrowth in the central nervous system. The encoded protein is a glycosylated type I membrane protein belonging to the ROR subfamily of cell surface receptors. It is a pseudokinase that lacks catalytic activity and can interact with the noncanonical Wnt signaling pathway. This gene is highly expressed during early embryonic development but is expressed at very low levels in adult tissues. Increased expression of this gene is associated with B-cell chronic lymphocytic leukemia. Alternative splicing generates multiple transcript variants encoding different isoforms. A tyrosine-protein kinase receptor whose role is still unclear. A receptor tyrosine kinase whose mutation leads to skeletal diseases in mice. It may be a biomarker for B-cell lymphocytic leukemia and kidney cancer. 6 LGALS3 Lectin, galactoside-binding, soluble, 3 Galactose-specific lectin that binds IgE, chemoattractant activity. This protein plays a role in numerous cellular functions, including apoptosis, innate immunity, cell adhesion, and T cell regulation. The protein exhibits antimicrobial activity against bacteria and fungi. A member of the galectin family of carbohydrate-binding proteins, members of this protein family have an affinity for beta-galactosides. The encoded protein is characterized by an N-terminal proline-rich tandem repeat domain and a single C-terminal carbohydrate recognition domain. This protein can self-associate through the N-terminal domain, allowing it to bind to multivalent saccharide ligands. This protein localizes to the extracellular matrix, cytoplasm, and nucleus. This protein plays a role in numerous cellular functions including apoptosis, innate immunity, cell adhesion and T cell regulation.The protein exhibits antimicrobial activity against bacteria and fungi. Alternative splicing leads to multiple transcript variants. 7 ANXA2R Annexin A2 receptor Can act as a receptor for annexin II on bone marrow stromal cells to induce osteoclast formation. "Annexin A2 (A2) is a profibrinolytic receptor that binds both plasminogen and its activator, tissue plasminogen activator (tPA), acts as a cofactor for plasmin formation, and localizes fibrinolytic activity to the surface of ECs. A2 is found on the surface membrane of ECs and monocytes and also on the brush border membrane of placental syncytiotrophoblasts, all of which are recognized targets of pathogenic antiphospholipid antibodies. Several lines of evidence suggest that A2 functions as a tPA-dependent cofactor for plasmin formation at the cell surface in vivo." (Romay-Penabad 2009 Blood) 8 IYD Iodotyrosine diiodinase This gene encodes an enzyme that catalyzes the oxidative NADPH-dependent deiodination of mono- and diiodotyrosine, the halogenated byproducts of thyroid hormone production. The N-terminus of the protein acts as a membrane anchor. Mutations in this gene lead to congenital hypothyroidism due to dyshormonogenesis type 4, also known as deiodinase deficiency, iodotyrosine dehalogenase deficiency, or thyroid hormoneogenesis type 4. Alternative splicing results in multiple transcript variants. 9 EMCN Endomucin EMCN is a mucin-like sialoglycoprotein that impairs the assembly of focal adhesion complexes and inhibits the interaction between cells and the extracellular matrix (Kinoshita et al., 2001). 10 FGFR1 Fibroblast growth factor receptor 1 Tyrosine-protein kinase, which acts as a cell-surface receptor for fibroblast growth factors and plays an essential role in the regulation of embryonic development, cell proliferation, differentiation, and migration. The protein encoded by this gene belongs to the fibroblast growth factor receptor (FGFR) family, whose amino acid sequence is highly conserved among members and throughout evolution. FGFR family members differ from each other in their ligand affinity and tissue distribution. A representative full-length protein consists of an extracellular region composed of three immunoglobulin-like domains, a single hydrophobic membrane-spanning segment, and a cytoplasmic tyrosine kinase domain.The extracellular portion of the protein interacts with fibroblast growth factors, initiating a cascade of downstream signals that ultimately influence mitogenesis and differentiation. This particular family member binds both acidic and basic fibroblast growth factors and is involved in limb induction. Mutations in this gene have been associated with Pfeiffer syndrome, Jackson-Weiss syndrome, Antley-Bixler syndrome, osteoglophonous dysplasia, and autosomal dominant Kallmann syndrome 2. Chromosomal aberrations involving this gene have been implicated in myeloproliferative stem cell disorder and stem cell leukemia-lymphoma syndrome. Alternately spliced ​​variants encoding different protein isoforms have been described; however, not all variants are fully characterized. 11 HARS Jo-1 Aminoacyl-tRNA synthetases are a class of enzymes that load tRNAs with the corresponding amino acids. This enzyme is responsible for the synthesis of histidyl transfer RNA, which is essential for the incorporation of histidine into proteins. It is a common target of autoantibodies in human 12 SPN Sialophorin, CD43 The protein encoded by this gene is an important sialoglycoprotein found on the surface of thymocytes, T lymphocytes, monocytes, granulocytes, and some B lymphocytes. It may be part of a physiological ligand-receptor complex involved in T cell activation. During T cell activation, this protein is actively removed from the T cell-antigen-presenting cell (APC) contact site, suggesting a negative regulatory role in the adaptive immune response. It plays a role in the physicochemical properties of the T cell surface and in lectin binding. 13 SSB Sjogren syndrome antigen B (autoantigen La) The protein encoded by this gene is involved in various aspects of RNA metabolism, including binding and protecting the poly(U) termini of nascent RNA polymerase III transcripts from exonuclease digestion, processing of the 5' and 3' ends of pre-tRNA precursors, acting as an RNA chaperone, and binding viral RNAs associated with the hepatitis C virus. Autoantibodies that react with this protein are found in the sera of patients with Sjogren's syndrome and systemic lupus erythematosus. Alternative use of the promoter results in two different transcript variants encoding the same protein. 14 IL-8 Interleukin 8, CXCL8 A member of the CXC chemokine family. This chemokine is one of the most important mediators of the inflammatory response. This chemokine is secreted by various cell types. It acts as a chemoattractant and is also a potent angiogenic factor. It is a chemotactic factor that attracts neutrophils, basophils, and T cells, but not monocytes. It is also involved in neutrophil activation. 15 CCP cyclic citrullinated peptide Cross-sectional studies have shown that anticyclic citrullinated peptide (anti-CCP) antibodies are present in approximately 1% of the population. 1,2 Their presence has been associated with a high risk of later development of rheumatoid arthritis (RA). 3,4 However, anti-CCP antibodies can be detected more than 10 years before disease onset. 5 The risk of RA progression in anti-CCP-positive individuals from the general population has been estimated at 5% over a 5-year period, 1 meaning this test is unlikely to be of much use as a screening tool. However, retrospective studies have shown that the predictive value of CCP testing is much higher in the years immediately before diagnosis, with a positive predictive value (ppv) of 85% found within 1.5 years of symptom onset. 16 VEGFA vascular endothelial growth factor A Growth factor active in angiogenesis, vasculogenesis, and endothelial cell growth. Induces endothelial cell proliferation, promotes cell migration, inhibits apoptosis, and induces blood vessel permeabilization. Binds to the receptors FLT1 / VEGFR1 and KDR / VEGFR2, heparan sulfate, and heparin. NRP1 / Neuropilin-1 binds the isoforms VEGF-165 and VEGF-145. Isoform 17 DQA1*01:02 HLA-DQA1 HLA-DQA1 belongs to the alpha chain paralogs of HLA class II. The class II molecule is a heterodimer consisting of an alpha (DQA) and a beta (DQB) chain, both anchored in the membrane. It plays a central role in the immune system by presenting peptides derived from extracellular proteins. Class II molecules are expressed in antigen-presenting cells (APC: B lymphocytes, dendritic cells, macrophages). Within the DQ molecule, both the alpha and beta chains contain the polymorphisms that specify peptide binding specificities, resulting in up to four different molecules. 18 LPHN1 Latrophilin 1 A member of the latrophilin subfamily of G protein-coupled receptors (GPCRs). Latrophilins may play a role in both cell adhesion and signal transduction. 19 TG Thyroglobulin It is a glycoprotein homodimer produced primarily by the thyroid gland. It serves as a substrate for the synthesis of thyroxine and triiodothyronine, as well as for the storage of the inactive forms of thyroid hormone and iodine. 20 FGF2 Fibroblast growth factor receptor 2 Members of the FGF family bind heparin and exhibit broad mitogenic and angiogenic effects. This protein is implicated in various biological processes, including limb and nervous system development, wound healing, and tumor growth. The mRNA for this gene contains multiple polyadenylation sites and is alternatively translated from non-AUG (CUG) and AUG initiation codons, resulting in five distinct isoforms with distinct properties. 21 CSF2 Colony stimulating factor 2 Cytokine that controls the production, differentiation and function of granulocytes and macrophages. 22 NGF Nerve growth factor This gene is a member of the NGF-beta family and encodes a secreted protein that undergoes homodimerization and is incorporated into a larger complex. This protein has a growth-promoting effect, and the complex is involved in growth regulation. 23 LGALS8 Lectin, galactoside-binding, soluble, 8 Galectins are beta-galactoside-binding animal lectins with conserved carbohydrate recognition domains. Galectins play a role in development, differentiation, cell-cell adhesion, cell-matrix interaction, growth regulation, apoptosis, and RNA splicing. This gene is frequently expressed in tumor tissues and appears to be involved in integrin-like cell interactions. Alternative spliced ​​transcript variants encoding different isoforms have been identified. 2 LGALS1 Lectin, galactoside-binding, soluble, 1 Galectins are a family of beta-galactoside-binding proteins involved in the regulation of cell-cell and cell-matrix interactions. This gene product can act as an autocrine negative growth factor that regulates cell proliferation. 25 MAPK1 Mitogen-activated protein kinase 1 MAP kinases, also known as extracellular signal-regulated kinases (ERKs), act as an integration point for numerous biochemical signals and are involved in a variety of cellular processes such as proliferation, differentiation, transcriptional regulation, and development. Activation of this kinase requires its phosphorylation by upstream kinases. 6 SDF1 B CXC motif chemokine ligand 12 CXCL12, ligand for the G protein-coupled receptor, chemokine (CXC motif) receptor 4, and plays a role in many different cellular functions, including embryogenesis, immune surveillance, inflammatory response, tissue homeostasis, and tumor growth and metastasis. 7 VWF Von Willebrand factor A glycoprotein involved in hemostasis. The encoded preprotein undergoes proteolytic processing after assembly into large multimeric complexes. 28 STAT6 Signal transducer and activator of transcription 6 A member of the STAT family of transcription factors. In response to cytokines and growth factors, members of the STAT family are phosphorylated by receptor-associated kinases. 2 SHC3 SHC adapter protein 3 Related pathways include the immune system and GPCR signaling. GO annotations for this gene include protein kinase binding and receptor tyrosine kinase binding. 30 GNG5 G protein subunit gamma 5 G proteins are trimeric (alpha-beta-gamma) membrane-associated proteins that regulate the flow of information from cell-surface receptors to a variety of internal metabolic effectors. The interaction of a G protein with its activated receptor promotes the exchange of GTP for GDP bound to the alpha subunit. 31 IL18R1 Interleukin-18 receptor 1 The protein encoded by this gene is a cytokine receptor belonging to the interleukin-1 receptor family. 3 2 IL-21 Interleukin 21 The encoded protein plays a role in both innate and adaptive immune responses by inducing the differentiation, proliferation, and activity of multiple target cells such as macrophages, natural killer cells, B cells, and cytotoxic T cells. Cytokine with immunoregulatory effects. May promote the transition between innate and adaptive immunity. Induces the production of IgG(1) and IgG(3) in B cells (by similarity). May play a role in the proliferation and maturation of natural killer (NK) cells in synergy with IL15. May regulate the proliferation of mature B and T cells in response to activating stimuli. In synergy with IL15 and IL18, it stimulates the production of interferon gamma in T cells and NK cells. May inhibit the activation and maturation of dendritic cells (DCs) during the T cell-mediated immune response. Sigdel, JASN, 2012 33 VIM Vimentin A member of the intermediate filament family. Intermediate filaments, along with microtubules and actin microfilaments, form the cytoskeleton. It is responsible for maintaining cell shape, cytoplasmic integrity, and stabilizing cytoskeletal interactions. It is also involved in the immune response and controls the transport of low-density lipoprotein (LDL)-derived cholesterol from a lysosome to the site of esterification. It functions as an organizer of several important proteins involved in binding, migration, and cell signaling. Its associated pathways include ERK signaling. Jurcevic, Transplantation, 2001; Besarani, Transplantation, 2014 3 TUBA1B Tubulin, Alpha 1 b Tubulin is the main component of microtubules. It binds two moles of GTP, one to an exchangeable site on the beta chain and one to a non-exchangeable site on the alpha chain. *Microtubules are cylindrical tubes 20–25 nm in diameter. They are composed of protofilaments, which in turn are composed of alpha and beta tubulin polymers. Each microtubule is polarized, with the alpha subunits exposed at one end (-) and the beta subunits (+) at the other. Microtubules serve as a scaffold that determines cell shape and forms the backbone upon which cellular organelles and vesicles move, a process that requires motor proteins. The main microtubule motor proteins are kinesin, which generally moves toward the (+) end of the microtubule, and dynein, which generally moves toward the (-) end. Microtubules also form the spindle fibers for the separation of chromosomes during mitosis. Hoffman, Autoimmunity, 2016; Bilalic, J Proteom Res, 2010 39 Tubulin Tubulin Tubulin is the main component of microtubules. It binds two moles of GTP, one to an exchangeable site on the beta chain and one to a non-exchangeable site on the alpha chain. *Microtubules are cylindrical tubes 20–25 nm in diameter. They are composed of protofilaments, which in turn are composed of alpha and beta tubulin polymers. Each microtubule is polarized, with the alpha subunits exposed at one end (-) and the beta subunits (+) at the other. Microtubules serve as a scaffold that determines cell shape and forms the backbone upon which cellular organelles and vesicles move, a process that requires motor proteins. The main microtubule motor proteins are kinesin, which generally moves toward the (+) end of the microtubule, and dynein, which generally moves toward the (-) end. Microtubules also form the spindle fibers for the separation of chromosomes during mitosis. Hoffman, Autoimmunity, 2016; Bilalic, J Proteom Res, 2010 36 APOA1 Apolipoprotein A-I This gene encodes apolipoprotein AI, the major protein component of high-density lipoprotein (HDL) in plasma. The protein promotes cholesterol efflux from tissues for excretion in the liver and is a cofactor for lecithin-cholesterol acyltransferase (LCAT), which is responsible for the formation of most cholesterol esters in plasma. This gene is closely linked to two other apolipoprotein genes on chromosome 11. Defects in this gene have been associated with HDL deficiency, including Tangier disease, and with systemic non-neuropathic amyloidosis. Ziegler, Transplantation, 2011 37 ICAM1 Intracellular adhesion molecule 1 This gene encodes a cell surface glycoprotein typically expressed on endothelial cells and cells of the immune system. It binds to CD11a / CD18 or CD11b / CD18 integrins and is also used as a receptor by rhinoviruses. Lawson, Transplantation, 2005 38 Myosin Myosin Muscle myosin is a hexameric protein containing two heavy chain subunits, two alkaline light chain subunits, and two regulatory light chain subunits. This gene encodes the beta heavy (or slow chain) subunit of cardiac myosin. It is predominantly expressed in the normal human heart chamber. It is also expressed in skeletal muscle tissues rich in type I slow-twitch muscle fibers. Alterations in the relative abundance of this protein and the alpha heavy (or fast) subunit of cardiac myosin correlate with cardiac contraction velocity. Its expression is also altered in thyroid hormone deficiency and hemodynamic overload.Mutations in this gene are associated with familial hypertrophic cardiomyopathy, myosin storage myopathy, dilated cardiomyopathy, and early-onset distal Laing myopathy. Kalache, Journal of Immunology, 2011; Nath, Hum Immunol, 2010 39 KRT8 Cytokeratin 8 This gene is a member of the type II keratin family located on the long arm of chromosome 12. Type I and type II keratins heteropolymerize to form intermediate-sized filaments in the cytoplasm of epithelial cells. The product of this gene typically dimerizes with keratin 18 to form an intermediate filament in simple monolayer epithelial cells. This protein plays a role in maintaining cellular structural integrity and is also involved in signal transduction and cellular differentiation. Mutations in this gene lead to cryptogenic cirrhosis. Alternately spliced ​​transcript variants have been identified for this gene. Alvarez-Marquez, Hum Immunol, 2008 40 KRT18 Cytokeratin 18 Alvarez-Marquez, Hum Immunol, 2008 41 HSPB1 Heat shock protein beta-1 The protein encoded by this gene is induced by environmental stress and developmental changes. The encoded protein is involved in stress resistance and actin organization and is translocated from the cytoplasm to the nucleus upon stress induction. Defects in this gene are a cause of Charcot-Marie-Tooth disease type 2F (CMT2F) and distal hereditary motor neuropathy. Pockley, Transplantation, 2001 42 FLRT2 Transmembrane protein with leucine-rich repeats FLRT2 This gene encodes a member of the fibronectin leucine-rich transmembrane protein (FLRT) family. Members of the FLRT family may play a role in cell adhesion and / or receptor signaling. Their protein structures resemble small leucine-rich proteoglycans found in the extracellular matrix. Shirai, Arthritis Res Ther, 2012 43 Collagen VI Collagen VI The proteins of the extracellular matrix share a triple-helical domain as a common structural element. Collagen VI is an important structural component of microfibrils. The basic structural unit of collagen VI is a heterotrimer of the alpha1(VI), alpha2(VI), and alpha3(VI) chains. Nath, Transplantation, 2011 44 Collagen I Collagen I Type I is a fibril-forming collagen found in most connective tissues and abundant in bone, cornea, dermis, and tendons. Mutations in this gene are associated with osteogenesis imperfecta types I-IV, Ehlers-Danlos syndrome type VIIA, Ehlers-Danlos syndrome classic type, Caffey disease, and idiopathic osteoporosis. Nath, Transplantation, 2011 4E Collagen II Collagen II A fibrillar collagen found in cartilage and the vitreous humor of the eye. Mutations in this gene are associated with achondrogenesis, chondrodysplasia, early-onset familial osteoarthritis, SED congenita, Langer-Saldino achondrogenesis, Kniest dysplasia, Stickler syndrome type I, and Strudwick spondyloepimetaphyseal dysplasia. Nath, Transplantation, 2011 46 Collagen III Collagen III a fibrillar collagen found in extensible connective tissues such as the skin, lungs, uterus, intestines, and the vascular system, often in association with type I collagen. Mutations in this gene are associated with Ehlers-Danlos syndrome type IV and with aortic and arterial aneurysms. Nath, Transplantation, 2011 47 Collagen IV Collagen IV Type IV collagen, the main structural component of basement membranes, is a multimeric protein composed of three alpha subunits. These subunits are encoded by six different genes, alpha 1 through alpha 6, each of which can form a triple helix structure with two other subunits to create type IV collagen. This gene encodes alpha 3. In Goodpasture syndrome, autoantibodies bind to the collagen molecules in the basement membranes of the alveoli and glomeruli. Nath, Transplantation, 2011 48 Collagen V Collagen V This gene encodes an alpha chain for one of the low-abundance fibrillar collagens. Fibrillar collagen molecules are trimers that can consist of one or more types of alpha chains. Type V collagen is found in tissues containing type I collagen and appears to regulate the assembly of heterotypic fibers composed of both type I and type V collagen. Nath, Transplantation, 2011; Burlingham, J Clin Invest, 2007 49 GSTT1 Glutathione S-transferase theta-1 The protein encoded by this gene, glutathione S-transferase (GST) theta 1 (GSTT1), is a member of a superfamily of proteins that catalyze the conjugation of reduced glutathione to a variety of electrophilic and hydrophobic compounds. Human GSTs can be divided into five major classes: alpha, mu, pi, theta, and zeta. The theta class includes GSTT1 and GSTT2. GSTT1 and GSTT2 share 55% amino acid sequence identity and may play an important role in human carcinogenesis. The GSTT1 and GSTT2 genes have a similar structure, consisting of five exons with identical exon / intron boundaries. The GSTT1 gene is haplotype-specific and absent in 38% of the population. Alternative splicing of this gene results in multiple transcript variants. Two related pseudogenes, also located on chromosome 22, have been identified. Aguilera, Clin Exp Immunol, 2001; Aguilera, Transplant Proc, 2005 50 ENO1 Alpha-enolase A multifunctional enzyme that, in addition to its role in glycolysis, is involved in various processes such as growth control, hypoxia tolerance, and allergic reactions. It stimulates the production of immunoglobulins. Murtas, Clin J Am Soc Nephrol, 2012 51 Actin Actin Alvarez-Marquez, Hum Immunol, 2008 521 GAPDH Glyceraldehyde-3-phosphate dehydrogenase It has both glyceraldehyde-3-phosphate dehydrogenase and nitrosylase activities, thus playing a role in glycolysis and nuclear functions, respectively. It participates in nuclear processes such as transcription, RNA transport, DNA replication, and apoptosis. Its nuclear functions are likely due to its nitrosylase activity, which mediates cysteine ​​S-nitrosylation of nuclear target proteins such as SIRT1, HDAC2, and PRKDC. It modulates cytoskeletal organization and assembly. It facilitates CHP1-dependent microtubule-membrane associations through its ability to stimulate CHP1 binding to microtubules (by similarity). Buse, Electropheresis, 2008 53 LMNA Prelamin-A / C Lamins are components of the nuclear lamina, a fibrous layer on the nucleoplasmic side of the inner nuclear membrane that is thought to provide a scaffold for the nuclear envelope and may also interact with chromatin. Cortes, Transplant Proc, 2009; Bilalic, J Proteome Res, 2010 54 VCL Vinculin Actin filament (F-actin)-binding protein involved in cell-matrix adhesion and cell-cell adhesion. Wilke, Heart, 1995 51 NCL Nucleolin The major nucleolar protein of growing eukaryotic cells. It is associated with intranucleolar chromatin and preribosomal particles. It induces chromatin decondensation by binding to histone H1. Qin, Transplantation, 2011 56 PRKCH Protein kinase C, eta Calcium-independent protein kinase C activity, which is involved in the regulation of cell differentiation into keratinocytes and pre-B cell receptors, mediates the regulation of epithelial tight junction integrity. Sutherland, Kidney International, 2009 57 PRKCZ Protein kinase C, zeta A member of the PKC family of serine / threonine kinases involved in a variety of cellular processes such as proliferation, differentiation, and secretion. In contrast to the classical PKC isoenzymes, which are calcium-dependent, PKC zeta exhibits kinase activity that is independent of calcium and diacylglycerol, but not phosphatidylserine. Furthermore, it is insensitive to typical PKC inhibitors and cannot be activated by phorbol esters. Unlike the classical PKC isoenzymes, it possesses only a single zinc-finger module. These structural and biochemical properties indicate that the zeta subspecies is related to, but distinct from, other PKC isoenzymes. Alternative splicing results in multiple transcript variants encoding different isoforms. Sutherland, Kidney International, 2009 58 PLA2R1 Phospholipase A2 receptor 1, 180kDa This gene represents a phospholipase A2 receptor. The encoded protein likely exists in both a transmembrane and a soluble form. The transmembrane receptor may play a role in the clearance of phospholipase A2, thereby inhibiting its action. Polymorphisms at this locus have been associated with susceptibility to idiopathic membranous nephropathy. Alternately spliced ​​transcript variants encoding different isoforms have been identified. Kanigicherla, Kidney Int, 2013 59 FN1 Fibronectin 1 This gene encodes fibronectin, a glycoprotein present in a soluble dimeric form in plasma and in a dimeric or multimeric form on the cell surface and in the extracellular matrix. Fibronectin is involved in cell adhesion and migration processes such as embryogenesis, wound healing, blood coagulation, host defense, and metastasis. The gene has three regions that can be alternatively spliced, resulting in 20 different transcript variants. However, the full-length nature of some variants has not been determined. Angaswamy, Am J Transplant, 2014 50 PECR Peroxisomal trans-2-enoyl-CoA reductase PECR (peroxisomal trans-2-enoyl-CoA reductase) is a protein-coding gene. Related pathways include peroxisome and mitochondrial LC fatty acid beta-oxidation. GO annotations associated with this gene include receptor binding and trans-2-enoyl-CoA reductase (NADPH) activity. An important paralog of this gene is DECR2. Dinavahi, J Am Soc Nephrol, 2011 61 HSPG2, perlecan Heparan sulfate proteoglycan 2 This gene encodes the perlecan protein, which consists of a core protein to which three long chains of glycosaminoglycans (heparan sulfate or chondroitin sulfate) are attached. The perlecan protein is a large multidomain proteoglycan that binds to and cross-links many components of the extracellular matrix and cell surface molecules. This protein has been shown to interact with laminin, prolargin, collagen type IV, FGFBP1, FBLN2, FGF7, and transthyretin, among others, and it plays an essential role in numerous biological activities. Perlecan is a key component of the extracellular matrix of vessels, where it contributes to the maintenance of endothelial barrier function. It is a potent inhibitor of smooth muscle cell proliferation and thus presumably contributes to the maintenance of vascular homeostasis. It can also inhibit the activity of growth factors (e.g.,FGF2) and thus stimulate the growth and regeneration of endothelial cells. It is a major component of basement membranes, where it is involved in the stabilization of other molecules, as well as in glomerular permeability to macromolecules and cell adhesion. Mutations in this gene cause Schwartz-Jampel syndrome type 1, Silverman-Handmaker type of dyssegmental dysplasia, and tardive dyskinesia. Alternative splicing of this gene results in several transcript variants. Joosten, Am J Transplant, 2005 62 AGRN Agrin This gene encodes one of several proteins critical for neuromuscular junction (NMJ) development, as determined in mouse knockout studies. The encoded protein contains multiple laminin G, Kazal-type serine protease inhibitor, and epidermal growth factor domains. Additional posttranslational modifications occur to add glycosaminoglycans and disulfide bonds. A mutation in this gene was found in a family with congenital myasthenic syndrome affecting the limb-girdle muscles. Alternative splicing results in multiple transcript variants encoding different isoforms. Joosten, Am J Transplant, 2005 63 ATP5B ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide This gene encodes a subunit of mitochondrial ATP synthase. Mitochondrial ATP synthase catalyzes ATP synthesis by utilizing an electrochemical gradient of protons across the inner membrane during oxidative phosphorylation. ATP synthase consists of two interconnected multisubunit complexes: the soluble catalytic core, F1, and the membrane-spanning component, Fo, which comprises the proton channel. The catalytic part of mitochondrial ATP synthase consists of five different subunits (alpha, beta, gamma, delta, and epsilon), assembled in a stoichiometry of three alpha, three beta, and a single representative of the other three. The proton channel consists of three major subunits (a, b, and c). This gene encodes the beta subunit of the catalytic core. Hsu, Arthritis Rheum, 2006 64 NPHS1 Nephrosis 1, congenital, Finnish type (nephrin) This gene encodes a member of the immunoglobulin family of cell adhesion molecules that function in the glomerular filtration barrier in the kidney. The gene is primarily expressed in kidney tissues, and the protein is a type 1 transmembrane protein found at the slit diaphragm of glomerular podocytes. The slit diaphragm is thought to act as an ultrafilter to exclude albumin and other macromolecules from the plasma during urine formation. Mutations in this gene lead to Finnish type 1 congenital nephrosis, characterized by severe proteinuria and loss of the slit diaphragm and foot processes. Kuusniemi, Transplantation, 2007 65 APOL2 Apolipoprotein L, 2 This gene is a member of the apolipoprotein L gene family. The encoded protein is located in the cytoplasm, where it can influence the movement of lipids or facilitate the binding of lipids to organelles. Two transcript variants have been identified for this gene, each encoding the same protein. Delville, Sci Transl Med, 2014 66 CD40 CD40 molecule, member of the TNF receptor superfamily 5 This gene is a member of the TNF receptor superfamily. The encoded protein is a receptor on antigen-presenting cells of the immune system and is essential for mediating a variety of immune and inflammatory responses, including T cell-dependent immunoglobulin class switching, the development of memory B cells, and the formation of germinal centers. The AT-hook transcription factor AKNA is reported to coordinately regulate the expression of this receptor and its ligand, which may be important for homotypic cell interactions. The adaptor protein TNFR2 interacts with this receptor and serves as a mediator of signal transduction. The interaction between this receptor and its ligand is necessary for amyloid-beta-induced microglial activation and is therefore considered an early event in the pathogenesis of Alzheimer's disease.Mutations affecting this gene are the cause of autosomal recessive hyper-IgM immunodeficiency type 3 (HIGM3). Several alternatively spliced ​​transcript variants of this gene, encoding different isoforms, have been reported. Delville, Sci Transl Med, 2014 67 CGB5 Chorionic gonadotropin, beta polypeptide 5 This gene is a member of the beta-chain glycoprotein hormone family and encodes the beta-5 subunit of chorionic gonadotropin (CG). Glycoprotein hormones are heterodimers consisting of a common alpha subunit and a unique beta subunit, which confers biological specificity. CG is produced by the trophoblastic cells of the placenta and stimulates the ovaries to synthesize the steroids essential for maintaining pregnancy. The beta subunit of CG is encoded by six genes arranged in tandem and inverted pairs on chromosome 19q13.3, adjacent to the gene for the beta subunit of luteinizing hormone. Delville, Sci Transl Med, 2014 68 FAS Fas - cell surface death receptor The protein encoded by this gene is a member of the TNF receptor superfamily. This receptor contains a death domain. It has been shown to play a central role in the physiological regulation of programmed cell death and is implicated in the pathogenesis of various malignancies and immune system disorders. The interaction of this receptor with its ligand enables the formation of a death-inducing signaling complex, which includes Fas-associated death domain protein (FADD), caspase 8, and caspase 10. Autoproteolytic processing of the caspases in this complex triggers a downstream caspase cascade, leading to apoptosis. This receptor has also been shown to activate NF-kappaB, MAPK3 / ERK1, and MAPK8 / JNK and is involved in the transduction of proliferation signals in normal diploid fibroblasts and T cells.Several alternatively spliced ​​transcript variants have been described, some of which are candidates for nonsense-mediated mRNA decay (NMD). Isoforms lacking the transmembrane domain may negatively regulate apoptosis mediated by the full-length isoform. Delville, Sci Transl Med, 2014 69 P2RY11 purinergic receptor P2Y, G-protein coupled, 11 The product of this gene belongs to the family of G protein-coupled receptors. This family includes several receptor subtypes with different pharmacological selectivity, which in some cases overlaps, for various adenosine and uridine nucleotides. This receptor is coupled to the stimulation of the phosphoinositide and adenyl cyclase pathways and behaves like a selective purinoreceptor. Naturally occurring read-through transcripts have been found that arise from intergenic splicing between this gene and an immediately upstream gene (PPAN, which encodes the Peter Pan homolog). The PPAN-P2RY11 read-through mRNA is ubiquitously expressed and encodes a fusion protein identical to each individual gene product. Delville, Sci Transl Med, 2014 70 PTPRO Tyrosine-protein phosphatase receptor type U This gene encodes a member of the R3 subtype family of receptor-type protein tyrosine phosphatases. These proteins are localized to the apical surface of polarized cells and may have tissue-specific functions by activating Src family kinases. This gene contains two distinct promoters, and alternatively spliced ​​transcript variants encoding multiple isoforms have been observed. The encoded proteins may have multiple isoform-specific and tissue-specific functions, including regulation of osteoclast production and activity, inhibition of cell proliferation, and facilitation of apoptosis. This gene is a candidate tumor suppressor, and decreased expression of this gene has been observed in various cancers. Delville, Sci Transl Med, 2014 71 SNRPB2 Small nuclear ribonucleoprotein polypeptide B The protein encoded by this gene associates with stem-loop IV of the U2 small nuclear ribonucleoprotein (U2 snRNP) in the presence of snRNP-A'. The encoded protein may play a role in pre-mRNA spiking. Autoantibodies from patients with systemic lupus erythematosus frequently recognize epitopes on the encoded protein. Two transcript variants have been identified for this gene, encoding the same protein. Delville, Sci Transl Med, 2014 72 CXCL11 Chemokine (CX-C motif) ligand 11 Chemokines are a group of small (approximately 8 to 14 kDa), mostly basic, structurally related molecules that regulate the cellular trafficking of various types of leukocytes through interactions with a subset of G protein-coupled 7-transmembrane receptors. Chemokines also play a fundamental role in the development, homeostasis, and function of the immune system and have effects on cells of the central nervous system and on endothelial cells involved in angiogenesis or angiostasis. Chemokines are divided into two major subfamilies, CXC and CC. This antimicrobial gene is a CXC member of the chemokine superfamily. The protein it encodes induces a chemotactic response in activated T cells and is the dominant ligand for CXC receptor 3. The gene encoding this protein contains four exons and at least three polyadenylation signals, which may reflect cell-specific regulation of expression.IFN-gamma is a potent inducer of transcription of this gene. Two transcript variants encoding different isoforms have been identified for this gene. Sigdel, J Am Soc Nephrol, 2012 73 GDNF Neurotrophic factor from glial cells This gene encodes a highly conserved neurotrophic factor. The recombinant form of this protein has been shown to promote the survival and differentiation of dopaminergic neurons in culture and can prevent axotomy-induced apoptosis of motor neurons. The encoded protein is processed into a mature secreted form, which exists as a homodimer. The mature form of the protein is a ligand for the product of the proto-oncogene RET (rearranged during transfection). Several transcript variants encoding different isoforms have been identified for this gene. Mutations in this gene can be associated with Hirschsprung's disease. Sigdel, J Am Soc Nephrol, 2012 74 IFNG Interferon Gamma This gene encodes a member of the type II interferon family. The encoded protein is a soluble cytokine with antiviral, immunoregulatory, and antitumor properties and is a potent activator of macrophages. Mutations in this gene are associated with aplastic anemia. Sigdel, J Am Soc Nephrol, 2012 75 CXCL9 CXC motif chemokine 9 This antimicrobial gene encodes a protein thought to be involved in T cell trafficking. The encoded protein binds to the CXC motif chemokine 3 and is a chemoattractant for lymphocytes but not for neutrophils. Sigdel, J Am Soc Nephrol, 2012 76 EDIL3 EGF-like repeats and discoidin I-like domains 3 The protein encoded by this gene is an integrin ligand. It plays an important role in mediating angiogenesis and may be important for the remodeling and development of vascular walls. It also influences the behavior of endothelial cells. Jackson, J Am Soc Nephrol, 2015 77 CLOSELY Endoglin This gene encodes a homodimeric transmembrane protein, a major glycoprotein of the vascular endothelium. This protein is a component of the transforming growth factor-beta receptor complex and binds with high affinity to peptides beta1 and beta3. Mutations in this gene cause hereditary hemorrhagic telangiectasia, also known as Osler-Rendu-Weber syndrome 1, an autosomal dominant multisystem vascular dysplasia. This gene may also be involved in preeclampsia and various cancers. Alternately spliced ​​transcript variants encoding different isoforms have been identified for this gene. Jackson, J Am Soc Nephrol, 2015 78 CAM4 intercellular adhesion molecule 4 (Landsteiner-Wiener blood group) This gene encodes the Landsteiner-Wiener (LW) blood group antigen(s), which belongs to the immunoglobulin (Ig) superfamily and shares similarity with the intercellular adhesion molecule (ICAM) protein family. This ICAM protein contains two C2-type Ig-like domains and binds to the leukocyte adhesion protein LFA-1. The molecular basis of the LW(A) / LW(B) blood group antigens is a single aa variation at position 100; Gln-100=LW(A) and Arg-100=LW(B). Alternative splicing results in multiple transcript variants encoding different isoforms. Jackson, J Am Soc Nephrol, 2015 79 FLT3LG fms-related tyrosine kinase 3 ligand Dendritic cells (DCs) are the crucial link between innate and adaptive immunity by recognizing pathogens and inducing pathogen-specific immune responses. FLT3LG regulates DC development and is particularly important for plasmacytoid DCs and CD8 (see MIM 186910)-positive classical DCs and their CD103 (ITGAE; MIM 604682)-positive tissue counterparts. Jackson, J Am Soc Nephrol, 2015 Example 2. Validation of non-HLA antibodies in association with cardiac allograft rejection

[0141] Additional analyses of the non-HLA antigens described above were performed as described in CL Butler et al., Am J Transplant. 2020;00:1-13. In the study of adult cardiac allograft recipients (samples: n = 477 without rejection; n = 69 with rejection), 18 non-HLA antibodies associated with rejection were identified (P < .1), including four newly identified non-HLA antigen targets (DEXI, EMCN, LPHN1, and SSB). CART analysis showed that 5 / 18 non-HLA antibodies discriminated between rejection and non-rejection. Antibodies against 4 / 18 non-HLA antigens synergize with HLA donor-specific antibodies and significantly increase the probability of rejection (P < .1). The non-HLA panel was validated using an independent adult heart transplant cohort (n = 21 no rejection; n = 42 rejection, >1R) with an area under the curve of 0.87 (P < .05) at 92.86% sensitivity and 66.67% specificity.The results confirm that multiplex bead array assessment of non-HLA antibodies identifies heart transplant recipients at risk of rejection.

[0142] Table 5 lists the targets identified by the additional analysis, confirming the value of these targets previously identified in Table 4. Table 6 summarizes the various targets identified in both kidney and heart transplant studies. Fig. 6 provides an extended summary of the Fig. 5 and presents the results of the additional analysis.

[0182] Table 5. Targets identified and grouped by additional analysis Target group Predictive Independently sorted Newly identified ENO1 1 / 2 X X AGRN 1 / 2 X X EMCN 1 / 2 X X X SSB 2 X X Actin 3 FLT3LG 3 PRKCH 3 IL-21 3 Group 1 = Core + Predictive in the CART analysis Group 2 = Independently sorting + redescribed by UCLA Group 3 = Joint clustering in the correlation matrix

[0183] Table 6. All identified targets Additional analysis Independent analysis Heart, adult Heart, adult Kidneys, pediatric Kidneys, adult ENO1* , ** Tubulin DEXI* Tubulin AGRN* , ** LPHN1 * LGALS3* SHC3*** EMCN* , ** TG*** SNPRN* CXCL11*** SSB** GAPDH*** CSF2** Actin*** FN1*** IL-8*** FLT3LG*** NPHS1 *** STAT6** PRKCH*** VIM*** LGALS8** IL-21*** Myosin*** KRT18* VCL** KRT8* PECR*** GSTT1 * , ** LMNA*** Collagen II* ATP5B*** SNRPB2* PLA2R1* , **

[0143] Italics means newly identified.

[0144] Bold print indicates the nucleus (of various organs). *Means that the CART analysis is predictive. **Means they are sorted independently. ***Means they form common clusters in the correlation matrix. References Fan, L. et al. Neutralizing IL-17 prevents obliterative bronchiolitis in murine orthotopic lung transplantation. Am J Transplant 11, 911-22 (2011). Haas, M., et al. Banff meeting report writing (2014). “Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions.” Am J Transplant 14(2): 272-283. Huang da, W., BT Sherman and RA Lempicki (2009). "Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists". Nucleic Acids Res 37(1):1-13. Michaels, P. J., et al. (2003). „Humoral rejection in cardiac transplantation: risk factors, hemodynamic consequences and relationship to transplant coronary artery disease“. J Heart Lung Transplant 22(1): 58-69. Pearl, M. H., et al. (2016). „Bortezomib may stabilize pediatric renal transplant recipients with antibody-mediated rejection.“ Pediatr Nephrol 31(8): 1341-1348. Simko, T. w. a. V. (2017). „R package „corrplot“: Visualization of a Correlation Matrix (Version 0.84)." Smith, N. R. D. a. H. (1998). „Applied Regression Analysis“. Stewart, S., et al. (2005), „Revision oft he 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection“. J Heart Lung Transplant 24(11): 1710-1720. Zhang, Q., et al. (2005). „Development of posttransplant antidonor HLA antibodies is associated with acute humoral rejection and early graft dysfunction“, Transplantation 79(5): 591-598. Zhang, Q. and E. F. Reed (2016). "The importance of non-HLA antibodies in transplantation". Nat Rev Nephrol 12(8): 484-495. Figure 1 (continued) Figure 2 (continued) Figure 3 (continued) Figure 5 (continued) Figure 6 (continued) QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] DK 104687

[0002] Cited non-patent literature

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[0011] Li et al., Am J Transplant., 2012, 12(10):2710-8

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[0011] Chaudhuri et al., Pediatric Transplantation., 2012, 16(5):E183-7

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[0011] Molecular Cloning: A Laboratory Manual“, zweite Auflage (Sambrook et al., 1989

[0059] Current Protocols in Molecular Biology“ (Ausubel et al., Hrsg., 1987, regelmäßige Aktualisierungen

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[0059] Singleton et al., Dictionary of Microbiology and Molecular Biology, 2. Auflage, J.Wiley & Sons (New York, N.Y. 1994

[0059] Levine MH, et al., Treatment options and strategies for antibody mediated rejection after renal transplantation. Semin Immunol. 2012 Apr;24(2):136-42

[0106] Zhang und Reed, Transplantation 2005

[0119] Zhang Transplantation 2005, Stewart J Heart and Lung Transplant, 005

[0120] Pearl, Pediatr Nephrol, 2016

[0123] Haas, Am J Transplant, 2014 [0123, 0124] Haas M et al., AJT 2014

[0126] p<0,1; StataCorp. 2015. Stata Statistical Software: Release 14. College Station, TX: StataCorp LP

[0129] Fan, L. et al. Am J Transplant 2011

[0129] Simko, T. and V. 2017

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[0140] Bilalic, J Proteom Res, 2010

[0140] Ziegler, Transplantation, 2011

[0140] Kalache, Zeitschrift für Immunologie, 2011

[0140] Nath, Hum Immunol, 2010

[0140] Alvarez-Marquez, Hum Immunol, 2008

[0140] Shirai, Arthritis Res Ther, 2012

[0140] Nath, Transplantation, 2011

[0140] Burlingham, J Clin Invest, 2007

[0140] Aguilera, Clin Exp Immunol, 2001

[0140] Aguilera, Transplant Proc, 2005

[0140] Murtas, Clin J Am Soc Nephrol, 2012

[0140] Cortes, Transplant Proc, 2009

[0140] Bilalic, J Proteome Res, 2010

[0140] Angaswamy, Am J Transplant, 2014

[0140] Dinavahi, J Am Soc Nephrol, 2011

[0140] Joosten, Am J Transplant, 2005

[0140] Hsu, Arthritis Rheum, 2006

[0140] Kuusniemi, Transplantation, 2007

[0140] Delville, Sci Transl Med, 2014

[0140] Sigdel, J Am Soc Nephrol, 2012

[0140] Jackson, J Am Soc Nephrol, 2015

[0140] C.L. Butler et al., Am J Transplant. 2020;00:1-13

[0141] Fan, L. et al. Neutralizing IL-17 prevents obliterative bronchiolitis in murine orthotopic lung transplantation. Am J Transplant 11, 911-22 (2011

[0144] Haas, M., et al. Banff meeting report writing (2014). „Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions.“ Am J Transplant 14(2): 272-283

[0144] Huang da, W., B. T. Sherman und R. A. Lempicki (2009). "Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists ". Nucleic Acids Res 37(1): 1-13

[0144] Michaels, P. J., et al. (2003). „Humoral rejection in cardiac transplantation: risk factors, hemodynamic consequences and relationship to transplant coronary artery disease“. J Heart Lung Transplant 22(1): 58-69

[0144] Pearl, M. H., et al. (2016). „Bortezomib may stabilize pediatric renal transplant recipients with antibody-mediated rejection.“ Pediatr Nephrol 31(8): 1341-1348

[0144] Simko, T. w. a. V. (2017). „R package „corrplot“: Visualization of a Correlation Matrix (Version 0.84

[0144] Smith, N. R. D. a. H. (1998). „Applied Regression Analysis

[0144] Stewart, S., et al. (2005), „Revision oft he 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection“. J Heart Lung Transplant 24(11): 1710-1720

[0144] Zhang, Q., et al. (2005). „Development of posttransplant antidonor HLA antibodies is associated with acute humoral rejection and early graft dysfunction“, Transplantation 79(5): 591-598

[0144] Zhang, Q. und E. F. Reed (2016). „The importance of non-HLA antibodies in transplantation“. Nat Rev Nephrol 12(8): 484-495

[0144]

Claims

[1] A composition comprising a collection of solid phase substrates coated with one or more homogeneous populations of binding agents, each homogeneous population of binding agents specifically binding to an antibody directed against a single antigen selected from the group consisting of: dexamethasone-induced transcript (DEXI), CXC motif chemokine ligand 11 (CXCL11), cytokeratin 18 (KRT18), cytokeratin 8 (KRT8), tubulin, including tubulin alpha 1 b (also referred to as TUBa1b or TUBA1B), latrophilin 1 (LPHN1), colony stimulating factor 2 (CSF2), signal transducer and activator of transcription 6 (STAT6), lectin galactoside-binding soluble 3 (lectin galactoside-binding soluble 3“;LGALS3), SHC adaptor protein 3 (SHC3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutathione S-transferase theta-1 (GSTT1), phospholipase A2 receptor 1 (PLA2R1), interleukin 8 (IL-8), lectin galactoside-binding soluble 8 (LGALS8), small nuclear ribonucleoprotein polypeptide N (SNPRN), myosin, peroxisomal trans-2-enoyl-CoA reductase (PECR), vimentin (VIM), ATP synthase H+ transporting mitochondrial F1 complex beta polypeptide (ATP5B), collagen II, prelamin A / C (LMNA), small nuclear ribonucleoprotein polypeptide B (SNRPB2), fibronectin 1 (FN1), nephrosis 1, congenital Finnish type nephrosis 1 (NPHS1), thyroglobulin (TG) and vinculin (VCL).; [2] The composition of claim 1, wherein the collection of solid phase substrates further comprises one or more additional homogeneous populations of binding agents, each additional homogeneous population of binding agents specifically binding to an antibody directed against a single additional antigen selected from the group consisting of: alpha-enolase (ENO1), agrin (AGRN), endomucin (EMCN), Sjogren's syndrome antigen B (SSB), actin, fms-related tyrosine kinase 3 ligand (FLT3LG), protein kinase C eta (PRKCH), and interleukin 21 (IL-21). [3] The composition of claim 1 or 2, wherein the solid phase substrates are porous or non-porous. [4] The composition of claim 2 or 3, wherein the solid phase substrates comprise particles, nanoparticles, beads, nanospheres or microspheres. [5] The composition of claim 4, wherein the beads are polystyrene beads. [6] A composition according to any one of the preceding claims, wherein the collection of solid phase substrates comprises a microarray. [7] A composition according to any one of the preceding claims, wherein the solid phase substrates are fluorescently labelled, magnetically labelled or radioactively labelled. [8] A composition according to any one of the preceding claims, wherein the solid phase substrates are labelled with a small molecule. [9] A composition according to any one of the preceding claims, wherein the one or more homogeneous populations of binders are conjugated to the surface of the solid phase substrates. [10] The composition of claim 9, wherein the conjugation is covalent. [11] A composition according to any one of the preceding claims, wherein the one or more homogeneous populations of binders are bound by affinity to the surface of the solid phase substrates. [12] A composition according to any one of the preceding claims, wherein the binder is a polypeptide. [13] A composition according to any one of the preceding claims, wherein the solid phase substrates are coated with at least three different homogeneous populations of binding agents that bind to at least three different antigens. [14] The composition of claim 13, wherein the substrates are coated with a plurality of different homogeneous populations of binders that bind to the antibodies consisting of antibodies against tubulin, LPHN1, SNRPN, KRT18, KRT8, LGALS3, SNRPB2, DEXI, collagen II, PLA2R1, GSTT1, VCL, CSF2, LGALS8 and STAT6. [15] The composition of claim 13, wherein the substrates are coated with a plurality of different homogeneous populations of binders that bind to the antibodies consisting of antibodies against tubulin, LPHN1, TG, GAPDH, FN1, NPHS1, VIM, myosin, VCL and PECR. [16] The composition of claim 15, wherein the substrates are further coated with a plurality of different homogeneous populations of binders that bind to the antibodies consisting of antibodies against ENO1, AGRN, EMCN, SSB, actin, FLT3LG, PRKCH and IL-21. [17] The composition of claim 13, wherein the substrates are coated with a plurality of different homogeneous populations of binders that bind to the antibodies consisting of antibodies against DEXI, LGALS3, SNPRN, CSF2, IL-8, STAT6, LGALS8, KRT18, KRT8, GSTT1, LMNA, collagen II, ATP5B, SNRPB2 and PLA2R1. [18] The composition of claim 13, wherein the substrates are coated with a plurality of different homogeneous populations of binders that bind to the antibodies consisting of antibodies against tubulin, SHC3 and CXCL11. [19] The composition of claim 13, wherein the substrates are coated with a plurality of different homogeneous populations of binding agents that bind to the antibodies consisting of antibodies against DEXI, EMCN, SNRPN, LPHN1 and SSB. [20] The composition of claim 13, wherein the substrates are coated with a plurality of different homogeneous populations of binders that bind to the antibodies consisting of antibodies against KRT18, GAPDH, AGRN, ENO1 and EMCN. [21] The composition of claim 13, wherein the substrates are coated with a plurality of different homogeneous populations of binding agents that bind to the antibodies consisting of antibodies against tubulin, LPHN1, SNRPN, KRT18, KRT8, LGALS3, SNRPB2, DEXI, collagen II, GAPDH, ENO1, AGRN, EMCN, SSB, PLA2R1, GSTT1, VCL, CSF2, LGALS8, STAT6, IL-8 and SHC3. [22] The composition of claim 1, wherein the single antigen is selected from the group consisting of: LPHN1, TG, DEXI, CSF2, IL-8, LGALS3, SNPRN, STAT6, SHC3 and LGALS8. [23] The composition of claim 2, wherein the single additional antigen is selected from the group consisting of: EMCN and SSB. [24] A composition according to any one of the preceding claims, wherein at least one solid phase substrate is detectably distinguishable from at least one other solid phase substrate. [25] A therapeutic agent for use in the treatment of an individual requiring treatment for transplant rejection after receiving a heart or kidney transplant, wherein the individual has elevated levels of one or more antibodies in a biological sample from the individual compared to reference levels, and wherein the levels were determined by contacting a biological sample obtained from the individual with a composition according to any one of claims 1-24. [26] Therapeutic agent for use in the treatment according to claim 25, wherein the therapeutic agent comprises one or more of a Januskinase inhibitor (e.g. Tofacitinib (Xeljanz)), a calcineurin inhibitor (e.g. Cyclosin Sandimmune, SangCya) or Tacrolimus (Astagraf XL, Envarsus XR, Prograf)), an mTOR-Inhibitor (zB Sirolimus (Rapamune) or Everolimus (Afinitor, Zortress)), an IMDH-Inhibitor (zB Azathioprine (Azasan, Imuran), Lefluvade (Cell Arcophenol), Myfortic)), einem Biologikum (zB, Abatacept (Orencia), Adalimumab (Humira), Anakinra (Kineret), Certolizumab (Cimzia), Etanercept (Enbrel), Golimumab (Simponi), Infliximab (Remicade), Ixekizumab (Taltz), Natalizumab (Txitusa), Rituximab (Ritux), Ricum (Cosentyx), Tocilizumab (Actemra), Ustekinumab (Stelara), Vedolizumab (Entyvio)) or Belatacept (Nulojix) or a monoclonal antibody (zB Basiliximab (Simulect) or Daclizumab (Zinbryta)). [27] Kit comprising: a) the composition according to any one of claims 1-24, and b) Reagents for detecting the binding of the one or more homogeneous populations of binding agents to the antibodies. [28] A kit according to claim 27, further comprising one or more reference samples.

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