WO2017136844A1 - Methods for predicting risk of antibody-mediated rejection - Google Patents

Abstract

In various embodiments, the invention is directed to methods and assays for determining the relative likelihood of a subject developing antibody-mediated rejection (AMR). In various embodiments, the invention further teaches methods for treating a subject who is determined to be likely to develop AMR, before the subject develops clinical AMR.

Description

METHODS FOR PREDICTING RISK OF ANTIBODY-MEDIATED REJECTION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62/291,268 filed on February 4, 2016, the contents of which are incorporated by reference in their entirety.

FIELD OF INVENTION

[0002] The invention generally relates to the field of transplant immunology. BACKGROUND

[0003] Antibody-mediated rejection (ABMR) is a major obstacle for successful transplantation in HLA-sensitized (HS) patients and a leading cause of graft failure for kidney transplant patients. HS patients develop allo-antibodies due to previous sensitizing events such as previous transplant(s), blood transfusion and pregnancy. ABMR. is a direct consequence of donor specific HLA antibody (DSA) development.

[0004] Timely and accurate diagnosis is critical for the treatment of ABMR and prevention of allograft failure. In addition to renal dysfunction, usually evidenced by serum creatinine elevation and presence of DSA, the clinical diagnosis of ABMR also requires the presence of histopathoiogical findings in renal allograft tissue. These include evidence for microvascular inflammation and microcirculation injury. The latest Banff 2013 classification of ABMR also recognizes molecular diagnostic markers such as "increased expression of gene transcripts in the biopsy tissue indicative of endothelial injury, if thoroughly validated", as one of the criteria for diagnosis of both acute and chronic active ABMR. There is increasing interest in effort to develop and validate gene signatures identified from the expression levels of large number of gene transcripts, using biopsy tissue samples of renal grafts, for the purposes of both clinical diagnosis of ABMR and also elucidation of molecular biological mechanisms of ABMR. However, it is important to recognize that biopsies do have inherent risk and may not always be possible, especially in patients on anti-coagulation. Thus, there would be benefits to develop reliable assays using blood and urine, which are predictive of ABMR and possible resolution after therapy.

[0005] Exosomes are small membrane vesicles with diameter of 30 to 100 nm, and secreted by most cell types. Exosomes exist abundantly in various types of body fluid, such as saliva, urine and blood plasma. Exosomes contain proteins, micro RNA (miRN A) and messenger RNA (mRNA), which are protected by the exterior membrane structures and directly related to the original cells secreting exosomes. Exosome-related research using various types of body fluid has increased over the last decade in multiple diseases, including cancer and organ transplantation. Here, we investigated the utility of gene expression in plasma exosomes as a possible non-invasive diagnostic tool to identify ABMR development in HS kidney transplant patients. We extracted exosomes from the archived plasma of kidney transplant patients with or without graft rejection, and purified RNA inside exosomes. Quantitative polymerase chain reaction (qPCR) was performed for multiple candidate genes likely related to ABMR based on previous research performed by our group and others using renal cortex tissue, and mR A transcript levels of these genes were compared among patients with or without ABMR or CMR. There is a need in the art for improved methods for diagnosing, predicting and preventing AMR, especially at an early stage.

SUMMARY

[0006] Provided herein methods for diagnosing and/or determining the likelihood of antibody -mediated rejection (ABMR) in a subject in need thereof. The methods include obtaining a biological sample from a subject desiring determination of likelihood of antibody- mediated rejection; determining the amount of mRNA encoding each of gpl 30, SH2D1 B, TNFa and CCL4 in the biological sample; and determining that the subject has an increased likelihood of ABMR if the amount of the mRNA encoding each of gpl30, SH2D1B, TNFa and CCL4 is higher than a reference sample.

[0007] Also provided herein methods for diagnosing and/or determining the likelihood of antibody -mediated rejection (ABMR) in a subject in need thereof. The methods include obtaining a biological sample from a subject desiring determination of likelihood of antibody- mediated rejection; determining the amount of gpI 30, SH2D1 B, TNFa and CCL4 proteins in the biological sample; and determining that the subject has an increased likelihood of ABMR if the amount of the gp^'130, SH2D1B, TNFa and CCL4 proteins is higher than a reference sample.

[0008] Further provided herein are methods for diagnosing and/or determining the likelihood of antibody-mediated rejection (ABMR). The methods include obtaining a biological sample from a subject desiring determination of likelihood of antibody -mediated rejection; determining the amount of one or more mRNA encoding one or more proteins associated with ABMR in the biological sample; and determining that the subject has an increased likelihood of ABMR if the amount of the one or more mRNA encoding one or more proteins associated with ABMR is higher than a reference sample.

[0009] Also provided herein are methods for diagnosing and/or determining the likelihood of antibody-mediated rejection (ABMR). The methods include obtaining a biological sample from a subject desiring determination of likelihood of antibody -mediated rejection; determining the amount of one or more proteins associated with ABMR in the biological sample; and determining that the subject has an increased likelihood of ABMR if the amount of the one or more proteins associated with ABMR is higher than a reference sample.

[0010] In some embodiments, the sample is blood or plasma. In some embodiments, the sample is exosomes in blood or plasma.

[0011] In some embodiments, the subject has received an allograft. In exemplary embodiments, the allograft is selected from the group consisting of kidney, heart, liver, lung, islet, intestine and other solid organs. In one embodiment, the allograft is a kidney allograft.

[0012] In some embodiments, the one or more mRNA for which an amount is determined encodes a protein selected from the group consisting of: IL-6, IL-6R, GP130, IL-23, TGFb, IFNg, TNFa, CD160, CRTAM, CCL3, CCL4, XCL1 , EGR1 , EGR2, CAV1, DARC, SH2D1B, CXCLl l, FGGBP2, GNLY, PLalA, IL-10, IL-12, IL-17 and IL-21.

[0013] In some embodiments, the one or more proteins for which an amount is a protein is determined is selected from IL-6, IL-6R, GP130, IL-23, TGFb, IFNg, TNFa, CD 160, CRTAM, CCL3, CCL4, XCL1, EGR1, EGR2, CAV1, DARC, SH2DIB, CXCLl l, FGGBP2, GNLY, PLalA, IL-10, IL-12, IL-17, IL-21 or combinations thereof.

[0014] In some embodiments, the one or more mRNA for which an amount is determined encodes a protein selected from the group consisting of: GP130, TNFa, CD160, CCL4, CAV1 , DARC, and SH2D1 B.

[0015] In some embodiments, the protein for which the amount is determined is selected from GP130, TNFa, CD160, CCL4, CAVI, DARC, SH2D1B or combinations thereof.

[0016] In some embodiments, the subject is a human. [0017] Also provided herein is a method, comprising: determining that a subject has been diagnosed with AMR or is an increased likelihood of having or developing AMR according to the methods described herein; and selecting a therapy for AMR if presence or likely presence of AMR is determined. In some embodiments, the therapy includes performing one or more medical procedure on the subject in order to prevent the subject from developing clinical AMR associated with decreased graft function. In some embodiments, the one or more medical procedure includes plasma exchange. In some embodiments, the therapy is prescribing or administering one or more therapeutic agents, wherein one or more therapeutic agent is selected from the group consisting of: pulse steroids, immunosuppressive drugs, IVIG, anti-CD20 antibody, anti-complement agents, anti-C5 antibody, C I inhibitor, anti-IL-6 receptor antibody, tocilizumab, IgG-digesting enzyme, IdeS, cyclosporine A, tacrolimus, mycophenolate mofetii, steroid, sirolimus, everolimus, belatacept, induction daigs, alemtuzumab, anti-thymoglubulin, and anti-IL-2 receptor antibody

[0018] In some embodiments, the reference value is the mean or median expression level of the one or more mRNA in a population of subjects that do not have AMR, or don't have a high likelihood of developing AMR. In some embodiments, the reference value is the mean or median expression level of the one or more mRNA from the subject tested at an earlier time points, wherein the earlier time point is before the subject received an allograft or before likelihood of AMR is determined in the subject. In some embodiments, the reference value is the expression level of one or more of the mRNA in a subject who does not have clinical AMR or pre-clinical.

BRIEF DESCRIPTION OF FIGURES

[0019] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0020] Fig, 1 depicts in accordance with various embodiments of the invention, differential levels of mRNA transcripts of 9 genes in plasma exosomes among ABMR, cell-mediated rejection (CMR), desensitized (DES) Control, and non-DES control patient groups. Horizontal long and short lines indicate mean ± standard deviation (SD) of results of 18 ABMR, 8 CMR, 18 DES Control and 20 non-DES control patients, respectively. Statistical analysis results by MW test for each gene are indicated on each panel. ** p<0.01, * 0.01 p 0.05; # 0.05≤p<0.10 by MW test. [0021] Fig. 2 depicts in accordance with various embodiments of the invention, mRNA transcript levels of the other 12 genes in plasma exosomes from ABMR, CMR, DES Control, and non-DES control patient groups. Horizontal long and short lines indicate mean ± standard deviation (SD) of results of 18 ABMR, 8 CMR, 18 DES Control and 20 non-DES control patients, respectively. p>0. 10 by KW Test.

[0022] Fig. 3 depicts in accordance with various embodiments of the invention, differential gene score based on the mRNA transcript levels of 4 selected genes (gp!30, SH2D1B, TNFa and CCL.4) in plasma exosomes among ABMR, CMR, DES Control, and non-DES control patient groups. Horizontal long and short lines indicate mean ± standard deviation (SD) of results of 18 ABMR, 8 CMR, 18 DES Control and 20 non-DES control patients, respectively. p<0.05 by KW test comparing the 4 patient groups. Statistical analysis results by MW test: ** p 0 01. * 0.0 ! - p 0.05 by MW test.

[0023] Fig, 4A~Fig, 43B depict in accordance with various embodiments of the invention, DSA levels in ABMR and DES control patient groups and correlation between gene combination score and DSA score in ABMR patient group. Fig. 4A: Significantly higher DSA score in ABMR than DES control patient group as expected. * p<0.05 by MW test. Horizontal long and short lines indicate mean ± standard deviation (SD) of results of 15 ABMR (3 patients had no PRA/DSA data available within the selected study timeframe), 18 DES Control patients, respectively. Fig. 4B: Gene score vs. DSA score in ABMR patient group. R2, coefficient of determination.

DETAILED DESCRIPTION OF THE INVENTION

[0024] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, 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. Allen et ai., Remington: The Science and Practice of Pharmacy 22nd ed., Pharmaceutical Press (September 15, 2012); Hornyak et a!., Introduction to Nanoscience and Nanotechnologv, CRC Press (2008); Singleton and Sainsbury, Dictionaiy of Microbiology and Molecular Biology 3rd ed., revised ed., J. Wiley & Sons (New York, NY 2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Staicture 7th ed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionaiy of DNA and Genome Technology 3rd ed., Wiley -Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2nd ed., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013); Kohler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur, J. Immunol. 1976 Jul, 6(7):51 I -9; Queen and Selick, Humanized immunoglobulins, U. S. Patent No. 5,585,089 (1996 Dec); and Riechmann et a!., Reshaping human antibodies for therapy, Nature 1988 Mar 24, 332(6162):323-7.

[0025] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below,

[0026] Some abbreviations used herein are defined as follows: ABMR, antibody-mediated rejection; ABOI, ABO incompatible; ADCC, antibody-dependent cellular cytotoxicity; CAVI, caveolin 1; CCL3, chemokine (C-C motif) ligand 3; CCL4, chemokine (C-C motif) ligand 4; CCL4L1, chemokine (C-C motif) ligand 4-like 1; CCL4L2, chemokine (C-C motif) ligand 4-like 2; CD 160, CD (cluster of differentiation) 160 molecule; CMR, cell-mediated rejection; CRTAM, cytotoxic and regulatory T cell molecule; Ct cycle threshold; DARC, atypical chemokine receptor 1 (duffy blood group); DES, desensitized; DSA, donor specific antibody; EDTA, ethylenediaminetetraacetic acid, EGR1 , early growth response I ; EGR2, early growth response 2 (Krox-20 homolog, Drosophila); FGFBP2, fibroblast growth factor binding protein 2; GAPDH, glyceraldehyde 3 -phosphate dehydrogenase; GNLY, granulysin; gpl30, glycoprotein 130 (interleukin 6 signal -transducing beta-receptor); HLA, human leukocyte antigen; HS, HLA-sensitized; IFNy, interferon gamma; EL- 10, interleukin 10; IL~ 12a, interleukin 12 alpha subunit p35; IL-23a, interleukin 23 alpha subunit pl9; DL-6, interleukin 6; IL-6Ra, interleukin 6 alpha-receptor; IVIG, intravenous immunoglobulin; KW test, Kmskal-Wallis H test; miRNA, micro-ribonucleic acid, MW test, Mann-Whitney U test; PRA, panel reactive antibody; qPCR, quantitative polymerase chain reaction; RQ, relative quantity; SFI, standard fluorescence intensities; SH2D1B, SH2 domain containing IB; TGFpi, transforming growth factor, beta 1; TNFa, tumor necrosis factor alpha; XCL1, chemokine (C motif) ligand 1 ; XCL2, chemokine (C motif) ligand 2. [0027] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.).

[0028] "Administering" and/or "administer" as used herein refer to any route for delivering a pharmaceutical composition to a patient. Routes of delivery may include non-invasive peroral (through the mouth), topical (skin), transmucosal (nasal, buccal /sublingual, vaginal, ocular and rectal) and inhalation routes, as well as parenteral routes, and other methods known in the art. Parenteral refers to a route of delivery that is generally associated with injection, including intraorbital, infusion, intraarterial, intracarotid, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.

[0029] As used herein, "AMR" and "ABMR" are used interchangeably and refer to antibody mediated rejection.

[0030] "Beneficial results" may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a patient developing the disease condition and prolonging a patient's life or life expectancy. In some embodiments, the disease condition is AMR. In some embodiments, the disease condition is clinically detectable AMR (also referred to herein as "clinical AMR"), which can include AMR in which organ dysfunction is detectable.

[0031] The term "effective amount" as used herein refers to the amount of a pharmaceutical composition comprising one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof, to decrease at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The phrase "therapeutically effective amount" as used herein means a sufficient amount of the composition to treat a disorder, at a reasonable benefit/risk ratio applicable to any medical treatment,

[0032] A therapeutically or prophylactically significant reduction in a symptom is, e.g. at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150% or more in a measured parameter as compared to a control or non-treated subject or the state of the subject prior to administering the peptides described herein. Measured or measurable parameters include clinically detectable markers of disease, for example, elevated or depressed levels of a biological marker, as well as parameters related to a clinically accepted scale of symptoms or markers for AMR. It will be understood, however, that the total daily usage of the compositions and formulations as disclosed herein will be decided by the attending physician within the scope of sound medical judgment. The exact amount required will vary depending on factors such as the type of disease being treated, gender, age, and weight of the subject.

[0033] As used herein, "subject" or "individual" or "animal" or "patient" or "mammal" means any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves, felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes, rodents such as mice, rats, hamsters and guinea pigs; and so on. In certain embodiments, the mammal is a human subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.

[0034] As used herein, the terms "treat," "treatment," "treating," or "amelioration" when used in reference to a disease, disorder or medical condition, refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, reverse, alleviate, ameliorate, inhibit, lessen, slow down or stop the progression or severity of a symptom or condition. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease-state is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Also, "treatment" may mean to pursue or obtain beneficial results, or lower the chances of the individual developing the condition even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition as well as those prone to have the condition or those in whom the condition is to be prevented.

[0035] "Significantly higher" as used herein relating to reference amounts refers to a statistically significant amount higher than the reference amount.

[0036] By way of additional background, it was determined that: 1) AMR-associated n RNA is upregulated much earlier than the time when organ dysfunction (e.g., kidney dysfunction) is observed (clinical AMR), 2) AMR-associated mRNA are included in exosomes circulating in blood, and 3) the levels of expression of the genes resulting in said mRNA ("AMR- associated genes") are associated with rejection status. After a series of experiments, it was determined that: 1) AMR-associated genes can be detected in the exosome by monitoring corresponding mRNA, 2) AMR-associated genes are upregulated in exosomes obtained from subjects with AMR compared to what is found in cell-mediated rejection (CMR) or no rejection, and 3) elevation of the expression of AMR-associated genes can be observed much earlier than the clinical AMR in subjects, indicating a utility of detection of the expression of AMR-associated genes in the exosome to predict and/or prevent AMR. Accordingly, the invention is based, at least in part, on these findings. In various embodiments described herein, provided herein are assays and methods for determining whether or not a subject who has received an allograft has a relatively high probability of developing clinical AMR. mRNA as bio-markers for diagnosing AMR

[0037] Provided herein is a method for diagnosing AMR or determining the likelihood of antibody-mediated rejection (AMR). The methods include obtaining a biological sample from a subject desiring determination of likelihood of antibody -mediated rejection; determining the level of one or more mRNA encoding one or more proteins associated with ABMR in the biological sample; and determining that the subject has an increased likelihood of ABMR if the level of the one or more mRNA encoding one or more proteins associated with ABMR is higher than a reference sample. In some embodiments, the subject has undergone an allograft transplant. In various embodiments, the biological sample includes one or more exosomes.

[0038] Provided herein is a method for diagnosing AMR or determining the likelihood of antibody -mediated rejection (AMR). The methods include obtaining a biological sample from a subject desiring determination of likelihood of antibody -mediated rejection; determining the presence or absence of one or more mRNA encoding one or more proteins associated with ABMR in the biological sample; and determining that the subject has an increased likelihood of ABMR if the level of the one or more mRNA encoding one or more proteins associated with ABMR is present in the biological sample from the subject. In some embodiments, the subject has undergone an allograft transplant. In various embodiments, the biological sample includes one or more exosomes.

[0039] Also provided herein is a method for diagnosing AMR or determining the likelihood of antibody-mediated rejection (AMR). The methods include detecting the level of one or more mRNA encoding one or more proteins associated with ABMR in a biological sample from a subject, wherein an increase in the level of the one or more mRNA encoding one or more proteins associated with ABMR relative to reference value indicates increased likelihood of ABMR. In some embodiments, the subject has undergone an allograft transplant. In various embodiments, the biological sample includes one or more exosomes. In some embodiments, detecting the level of one or more mRNA includes obtaining the results of analysis of the level of the one or more mRNA. In some embodiments, detecting the level of one or more mRNA includes requesting the results of analysis of the level of the one or more mRNA.

[0040] Also provided herein is a method for diagnosing AMR or determining the likelihood of antibody-mediated rejection (AMR). The methods include detecting the presence or absence of one or more mRNA encoding one or more proteins associated with ABMR in a biological sample from a subject, wherein the presence of the one or more mRNA encoding one or more proteins associated with ABMR indicates increased likelihood of ABMR. In some embodiments, the subject has undergone an allograft transplant. In various embodiments, the biological sample includes one or more exosomes. In some embodiments, detecting the presence or absence of one or more mRNA includes obtaining the results of analysis of the presence or absence of the one or more mRNA, In some embodiments, detecting the level of one or more mR A includes requesting the results of analysis of the presence or absence of the one or more mRN A.

[0041] In various embodiments, the invention provides a method for determining whether or not a subject who has received an allograft has a relatively high probability of developing AMR. In some embodiments, the method includes (1) obtaining a biological sample from the subject, wherein the biological sample includes one or more exosomes; (2) determining the amount of one or more mRNA encoding a protein associated with AMR in the biological sample; (3) determining that the subject has a relatively high probability of developing AMR, if the amount of one or more mRNA encoding a protein associated with AMR is significantly higher than a reference sample, or (4) determining that the subject does not have a relatively high probability of developing AMR, if the amount of one or more mRNA encoding a protein associated with AMR is not significantly higher than a reference sample.

[0042] Also provided herein are methods for determining efficacy of treatment for AMR in subjects in need thereof. The method include obtaining a sample from a subject desiring determination of efficacy of treatment for AMR., determining the presence or absence of one or more mRNA encoding one or more proteins associated with ABMR in the biological sample: and determining that the treatment is effective if the one or more mRNA encoding one or more proteins associated with ABMR is absent in the biological sample from the subject. In some embodiments, the subject has undergone an allograft transplant. In various embodiments, the biological sample includes one or more exosomes.

[0043] Further provided herein are methods for determining efficacy of treatment for AMR in subjects in need thereof. The method include obtaining a sample from a subject desiring determination of efficacy of treatment for AMR, determining the levels of one or more mRNA encoding one or more proteins associated with ABMR in the biological sample; and determining that the treatment is effective if the level of one or more mRNA encoding one or more proteins associated with ABMR is lower in the biological sample from the subject relative to a reference value. In some embodiments, the subject has undergone an allograft transplant. In various embodiments, the biological sample includes one or more exosomes,

[0044] Also provided herein is a method for determining efficacy of treatment for AMR in subjects in need thereof. The methods include detecting the level of one or more mRNA encoding one or more proteins associated with ABMR in a biological sample from a subject, wherein a decrease in the level of the one or more mRNA encoding one or more proteins associated with ABMR relative to reference value indicates that the treatment for AMR is effective. In some embodiments, the subject has undergone an allograft transplant. In various embodiments, the biological sample includes one or more exosomes. In some embodiments, detecting the level of one or more mRNA includes obtaining the results of analysis of the level of the one or more mRNA. In some embodiments, detecting the level of one or more mRNA includes requesting the results of analysis of the level of the one or more mRNA.

[0045] Further provided herein is a method for determining efficacy of treatment for AMR in subjects in need thereof. The methods include detecting the presence or absence of one or more mRNA encoding one or more proteins associated with ABMR in a biological sample from a subject, wherein the absence of the one or more mRNA encoding one or more proteins associated with ABMR indicates that the treatment for AMR is effective. In some embodiments, the subject has undergone an allograft transplant. In various embodiments, the biological sample includes one or more exosomes. In some embodiments, detecting the presence or absence of one or more mRNA includes obtaining the results of analysis of the presence or absence of the one or more mRNA. In some embodiments, detecting the presence or absence of one or more mRNA includes requesting the results of analysis of the presence or absence of the one or more mRNA.

[0046] In some embodiments of the methods described herein, the subject does not have clinically evident ABMR. In some embodiments, the subject has no evidence of reduced graft function at the time of testing.

[0047] In some embodiments, the allograft is of a type that may include, but is not limited to, kidney, heart, liver, lung, islet, intestine and other solid organs. In some embodiments, the allograft is a kidney allograft.

[0048] In some embodiments, the one or more mRNA for which the level and/or presence or absence is determined, encodes a protein associated with AMR that include but are not limited to IL-6, IL-6R, GP130, IL-23, ΤΟΡβ, ΙΡΝγ, TNFa, CD 160, CRTAM, CCL3, CCL4, XCL1, EGR1, EGR2, CAVl, DARC, SH2D1B, CXCL1 1, FGGBP2, GNLY, PLal A, IL-IQ, IL-12, IL- 1 7, IL-21 or combinations thereof.

[0049] In some embodiments, the one or more mRNA for which the level and/or presence or absence is determined, encodes a protein associated with AMR that include but are not limited to CCL3, CCL4, CD 160, CRTAM, EGR1, EGR2, IFNy, XCL1, CAVl, DARC, FGFBP2, G LY, SH2D1B, IL-ό, IL-6Ra, gpl30, IL-10, IL-12a, IL-23a, TGFpl, TNFa or combinations thereof.

[0050] In some embodiments, one or more mRNA for which the level and/or presence or absence is determined, encodes a protein associated with AMR that include but are not limited to GP130, TNFa, CD160, CCL-4, CAVl, DARC, and SFI2D1B.

[0051] In some embodiments, the mRNA for which the level and/or presence or absence is determined, encodes a protein associated with AMR that include but are not limited to gp!30, SH2D1B, TNFa and CCL4,

[0052] In some embodiments, one or more mRNA for which the level and/or presence or absence is determined, encodes a protein associated with AMR that include but are not limited to gpl30, SH2D B, TNFa, CCL4 or combinations thereof.

[0053] In some embodiments, the amount of one or more of the mRNA that encodes a protein associated with AMR (for example, mRNAs set forth herein) could be measured in addition to, or instead of, any of those mRNA specifically listed herein, by analyzing biological samples containing exosomes, as described herein. In some embodiments an elevated amount of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, and 25 or more types of mRNA that encodes a protein associated with AMR, as determined by the exosome analysis described herein, is indicative of a relatively high probability of an allograft recipient developing AMR.

Methods for detecting presence and/or level of mRNA associated with AMR

[0054] In some embodiments, the amount of mRNA encoding a particular protein (e.g., an AMR-associated protein), and therefore the expression level of each corresponding gene (e.g., AMR-associated gene), is determined by extracting total RNA from a sample containing exosomes, followed by reverse transcription, pre-amplification and then quantitative PCR. In some embodiments, expression of each gene is then analyzed by- determining fold change (FC) relative to a reference RNA after normalizing with a housekeeping gene. In some embodiments, the housekeeping gene is GAPDH. However, one of skill in the art would readily appreciate that alternative housekeeping genes could be used for the same purpose. [0055] Methods of "quantitative" amplification are well known to those of skill in the art. Merely by way of example, quantitative PGR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PGR reaction. Quantitative PGR can also be done using an external control such as a reference RNA wherein the results are expressed as the relative quantitative value against the reference RNA. Detailed protocols for quantitative PGR are provided in Innis, et al . (1990) PGR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N. Y.). The known nucleic acid sequence for the genes is sufficient to enable one of skill in the art to routinely select primers to amplify any portion of the gene. Fluorogenic quantitative PGR may also be used in the methods of the invention. In fiuorogenic quantitative PGR, quantitation is based on amount of fluorescence signals, e.g., TaqMan and sybr green.

[0056] Other amplification methods may include, but are not limited to, ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren, et al. (1988) Science 241 : 1077, and Barringer et al. (1990) Gene 89: 17), transcription amplification (Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication (Guateili, et al. (1990) Proc, Nat. Acad, Sci , USA 87: 1874), dot PGR, and linker adapter PGR, etc,

[0057] In some embodiments, statistical analysis is used to determine the differences in the mRNA expression levels, for example between the subject and the reference value. In on embodiment, a two-tailed student t-test with unequal variation may be used to measure the differences between the subject's expression of one or more genes described herein and a normal biological sample (e.g., a biological sample from an individual who does not have AMR, or does not have a relatively high probability of developing AMR), or the subject's own biological sample from an earlier time point prior to receiving an allograft, or a reference, generate by computer algorithm or otherwise, pooling many control samples of individuals who do not have AMR, or a high likelihood of developing AMR. In some embodiments, a significant difference may be determined where the p value is equal to or less than 0.05.

[0058] In some embodiments, non-parametric Kruskal-Wallis H Test (KW test) statistical method may be used to determine the differences in the proteins and/or mRNA expression level between the subject and the reference value, for example, as described in Kruskai; Wallis (1952). "Use of ranks in one-criterion variance analysis". Journal of the American SSttaattiissttiiccaall AAssssoocciiaattiioonn.. 4477 ((226600)):: 558833--662211;; CCoorrddeerr,, GGrreeggoorryy WW..;; FFoorreemmaann,, DDaallee II.. ((22000099)).. NNoonnppaarraammeettrriicc SSttaattiissttiiccss ffoorr NNoonn--SSttaattiissttiicciiaannss.. HHoobbookkeenn:: JJoohhnn WWiilleeyy && SSoonnss.. pppp.. 9999--110055.. IISSBBNN 99778800447700445544661199;; SSiieeggeell;; CCaasstteellllaann ((11998888)).. NNoonnppaarraammeettrriicc SSttaattiissttiiccss ffoorr tthhee BBeehhaavviioorraall SScciieenncceess ((SSeeccoonndd cc ll.. )).. NNeeww YYoorrkk:: MMccGGrraaww--HHiillll.. IISSBBNN 00007700557733557733..22))..

[[00005599]] IInn ssoommee eemmbbooddiimmeennttss,, nnoonn--ppaarraammeettrriicc MMaannnn--WWhhiittnneeyy UU tteesstt ((MMWW tteesstt)) ssttaattiissttiiccaall mmeetthhoodd mmaayy bbee uusseedd ttoo ddeetteerrmmiinnee t thhee ddiiffffeerreenncceess iinn tthhee pprrootteeiinn aandd//oorr mmRRNNAA eexxpprreessssiioonn lleevveell bbeettwweeeenn tthhee ssuubbjjeecctt aanndd tthhee rreeffeerreennccee vvaalluuee,, ffoorr eexxaammppllee,, aass ddeessccrriibbeedd iinn MMaannnn,, HHeennrryy BB..;; WWhhiittnneeyy,, DDoonnaalldd RR.. ((11994477)).. ""OOnn aa TTeesstt ooff WWhheetthheerr oonnee ooff TTwwoo RRaannddoomm VVaaririaabblleess iiss SSttoocchhaassttiiccaallllyy LLaarrggeerr tthhaann tthhee OOtthheerr"".. AAnnnnaallss ooff MMaatthheemmaattiiccaall SSttaattiissttiiccss.. 1188 ((11)):: 5500--6600;; FFaayy,, MMiicchhaaeell PP..;; PPrroosscchhaann,, MMiicchhaaeell AA.. ((22001100)).. ""WWiillccooxxoonn--MMaannnn--WWhhiittnneeyy oorr tt--tteesstt?? OOnn aassssuummppttiioonnss ffoorr hhyyppootthheessiiss tteessttss aanndd mmuullttiippllee iinntteerrpprreettaattiioonnss ooff ddeecciissiioonn rruulleess"";; SSttaattiissttiiccss SSuurrvveeyyss.. 44:: 11--3399.. ((PPMMIIDD 2200441144447722))..

[0060] Provided herein is a method for diagnosing AMR. or determining the likelihood of antibody -mediated rejection (AMR). The methods include obtaining a biological sample from a subject desiring determination of likelihood of antibody -mediated rejection; determining the level of one or more proteins associated with ABMR in the biological sample; and determining that the subject has an increased likelihood of ABMR if the level of the one or more proteins associated with ABMR is higher than a reference sample. In some embodiments, the subject has undergone an allograft transplant. In various embodiments, the biological sample includes one or more exosomes.

[0061] Provided herein is a method for diagnosing AMR or determining the likelihood of antibody -mediated rejection (AMR). The methods include obtaining a biological sample from a subject desiring determination of likelihood of antibody -mediated rejection; determining the presence or absence of one or more proteins associated with ABMR in the biological sample, and determining that the subject has an increased likelihood of ABMR if the level of the one or more proteins associated with ABMR is present in the biological sample from the subject. In some embodiments, the subject has undergone an allograft transplant. In various embodiments, the biological sample includes one or more exosomes.

[0062] Also provided herein is a method for diagnosing AMR or determining the likelihood of antibody-mediated rejection (AMR). The methods include detecting the level of one or more proteins associated with ABMR in a biological sample from a subject, wherein an increase in the level of the one or more proteins associated with ABMR relative to reference value indicates increased likelihood of ABMR. In some embodiments, the subject has undergone an allograft transplant. In various embodiments, the biological sample includes one or more exosomes. In some embodiments, detecting the level of one or more proteins includes obtaining the results of analysis of the level of the one or more proteins. In some embodiments, detecting the level of the one or more proteins includes requesting the results of analysis of the level of the one or more proteins.

[0063] Also provided herein is a method for diagnosing AMR or determining the likelihood of antibody-mediated rejection (AMR). The methods include detecting the presence or absence of one or more proteins associated with ABMR in a biological sample from a subject, wherein the presence of the one or more proteins associated with ABMR indicates increased likelihood of ABMR. In some embodiments, the subject has undergone an allograft transplant. In various embodiments, the biological sample includes one or more exosomes. In some embodiments, detecting the presence or absence of one or more proteins includes obtaining the results of analysis of the presence or absence of the one or more proteins. In some embodiments, detecting the presence or absence of the one or more proteins includes requesting the results of analysis of the presence or absence of the one or more proteins.

[0064] In various embodiments, the invention provides a method for determining whether or not a subject who has received an allograft has a relatively high probability of developing AMR. In some embodiments, the method includes (1) obtaining a biological sample from the subject, wherein the biological sample includes one or more exosomes; (2) determining the amount of one or more proteins associated with AMR in the biological sample; (3) determining that the subject has a relatively high probability of developing AMR, if the amount of one or more proteins associated with AMR is significantly higher than a reference sample, or (4) determining that the subject does not have a relatively high probability of developing AMR, if the amount of one or more proteins associated with AMR is not significantly higher than a reference sample.

[0065] Also provided herein are methods for determining efficacy of treatment for AMR in subjects in need thereof. The method includes obtaining a sample from a subject desiring determination of efficacy of treatment for AMR; determining the presence or absence of one or more proteins associated with ABMR in the biological sample; and determining that the treatment is effective if the one or more proteins associated with ABMR is absent in the biological sample from the subject. In some embodiments, the subject has undergone an allograft transplant. In various embodiments, the biological sample includes one or more exosomes.

[0066] Further provided herein are methods for determining efficacy of treatment for AMR in subjects in need thereof. The method include obtaining a sample from a subject desiring determination of efficacy of treatment for AMR, determining the levels of one or more proteins associated with ABMR in the biological sample; and determining that the treatment is effective if the level of one or more proteins associated with ABMR is lower in the biological sample from the subject relative to a reference value. In some embodiments, the subject has undergone an allograft transplant. In various embodiments, the biological sample includes one or more exosomes.

[0067] Also provided herein is a method for determining efficacy of treatment for AMR in subjects in need thereof. The methods include detecting the level of one or more proteins associated with ABMR in a biological sample from a subject, wherein a decrease in the level of the one or more proteins associated with ABMR relative to reference value indicates that the treatment for AMR is effective. In some embodiments, the subject has undergone an allograft transplant. In various embodiments, the biological sample includes one or more exosomes. In some embodiments, detecting the level of one or more proteins includes obtaining the results of analysis of the level of the one or more proteins. In some embodiments, detecting the level of the one or more proteins includes requesting the results of analysis of the level of the one or more proteins.

[0068] Further provided herein is a method for determining efficacy of treatment for AMR in subjects in need thereof. The methods include detecting the presence or absence of one or more proteins associated with ABMR in a biological sample from a subject, wherein the absence of the one or more proteins associated with ABMR relative to a reference value indicates that the treatment for AMR is effective. In some embodiments, the subject has undergone an allograft transplant. In various embodiments, the biological sample includes one or more exosomes. In some embodiments, detecting the presence or absence of one or more proteins includes obtaining the results of analysis of the presence or absence of the one or more proteins. In some embodiments, detecting the presence or absence of the one or more proteins includes requesting the results of analysis of the presence or absence of the one or more proteins.

[0069] In some embodiments of the methods described herein, the subject does not have clinically evident ABMR. In some embodiments, the subject has no evidence of reduced graft function at the time of testing.

[0070] In some embodiments, the allograft is of a type that may include, but is not limited to, kidney, heart, liver, lung, islet, intestine and other solid organs. In some embodiments, the allograft is a kidney allograft.

[0071] In some embodiments, the one or more proteins of which the level and/or presence or absence is determined include but are not limited to any one or more of IL-6, IL-6R, GP130, IL-23, TGF , IFNy, TNFa, CD160, CRTAM, CCL3, CCL4, XCL1, EGRl, EGR2, CAV1, DARC, SH2D1B, CXCL11, FGGBP2, GNLY, PLalA, IL-10, IL-12, IL-17, IL-21 or combinations thereof.

[0072] In some embodiments, the one or more proteins of which the level and/or presence or absence is determined include but are not limited to any one or more of CCL3, CCL4, CD 160, CRTAM, EGRl, EGR2, IFNy, XCL1 , CAV1, DARC, FGFBP2, GNLY, SH2D1B, IL-6, IL-6Ra, gpl30, IL-10, IL-12a, IL-23a, TGFpi, TNFa or combinations thereof.

[0073] In some embodiments, the one or more proteins of which the level and/or presence or absence is determined include GP130, TNFa, CD 160, CCL4, CAV1 , DARC, and SH2D1B.

[0074] In some embodiments, the one or more proteins of which the level and/or presence or absence is determined include any one or more of GP130, TNFa, CD 160, CCL4, CAV1, DARC, and SH2D1B or combinations thereof.

[0075] In some embodiments, the protein of which the level and/or presence or absence is determined include gpl30, SH2D1B, TNFa and CCL4.

[0076] In some embodiments, the protein of which the level and/or presence or absence is determined include any one or more of gpl30, SH2D1B, TNFa and CCL4 or combinations thereof. [0077] In some embodiments, one or more proteins of which the level and/or presence or absence is determined include any one or more of gpl30, SH2D1B, TNFot, CCL4 or combinations thereof.

[0078] In some embodiments, one or more proteins of which the level and/or presence or absence is determined include gpl30, SH2D1B, TNFot and CCL4.

[0079] In some embodiments, the amount of the one or more protein associated with AMR (for example, proteins set forth herein) can be measured in addition to, or instead of, any of those proteins specifically listed herein, by analyzing biological samples containing exosomes, as described herein. In some embodiments an elevated amount of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 1 1 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, and 25 or more protein associated with AMR as determined by the exosome analysis described herein, is indicative of a relatively high probability of an allograft recipient developing AMR.

Methods for detecting presence and/or level of proteins associated with AMR

[0080] Methods for detecting the levels of protein expression include any methods known in the art. For example, protein levels can be measured indirectly using DNA or mRNA arrays. Alternatively, protein levels can be measured directly by measuring the level of protein synthesis or measuring protein concentration.

[0081] DNA and mRNA arrays (microarrays) comprise a series of microscopic spots of DNA or RNA oligonucleotides, each with a unique sequence of nucleotides that are able to bind complementary nucleic acid molecules. In this way the oligonucleotides are used as probes to which only the correct target sequence will hybridize under high-stringency conditions. In embodiments of the present invention, the target sequence is either the coding DNA sequence or unique section thereof, corresponding to the protein whose expression is being detected, or the target sequence is the transcribed mRNA sequence, or unique section thereof, corresponding to the protein whose expression is being detected.

[0082] Directly measuring protein expression and identifying the proteins being expressed in a given sample can be done by any one of a number of methods known in the art. For example, antibodies specific to the proteins set forth herein may be used to detect and/or determine the level of the proteins using for example, Western Blot analysis or immunoprecipitation.

[0083] In some embodiments, 2-dimensional polyacrylamide gel electrophoresis (ZD- PAGE) has traditionally been the tool of choice to resolve complex protein mixtures and to detect differences in protein expression patterns. Differentially expressed proteins observed between samples from subjects and reference samples are separate by 2D-PAGE and detected by protein staining and differential pattern analysis. Alternatively, 2- dimensional difference gel electrophoresis (2D-DIGE) can be used, in which different protein samples are labeled with fluorescent dyes prior to 2D electrophoresis. After the electrophoresis has taken place, the gel is scanned with the excitation wavelength of each dye one after the other. This technique is particularly useful in detecting changes in protein abundance, for example when comparing a sample from samples from subjects and reference samples.

[0084] Commonly, proteins subjected to electrophoresis are also further characterized by mass spectrometry methods. Such mass spectrometry methods can include matrix- assisted laser desorption/ionization time-of-flight (MALDI-TOF).

[0085] MALDI-TOF is an ionization technique that allows the analysis of biomolecuies (such as proteins, peptides and sugars), which tend to be fragile and fragment when ionized by more conventional ionization methods. Ionization is triggered by a laser beam (for example, a nitrogen laser) and a matrix is used to protect the biomolecule from being destroyed by direct laser beam exposure and to facilitate vaporization and ionization. The sample is mixed with the matrix molecule in solution and small amounts of the mixture are deposited on a surface and allowed to dry. The sample and matrix co-crystallize as the solvent evaporates.

[0086] Protein rnicroarrays can also be used to directly detect protein expression. These are similar to DNA and mRNA rnicroarrays in that they comprise capture molecules fixed to a solid surface. Capture molecules are most commonly antibodies specific to the proteins being detected, although antigens can be used where antibodies are being detected in serum. Further capture molecules include proteins, aptamers, nucleic acids, receptors and enzymes, which might be preferable if commercial antibodies are not available for the protein being detected. Capture molecules for use on the protein arrays can be externally synthesized, purified and attached to the array. [0087] Alternatively, they can be synthesized in-situ and be directly attached to the array. The capture molecules can be synthesized through biosynthesis, cell-free DNA expression or chemical synthesis. In-situ synthesis is possible with the latter two. There is therefore provided a protein microarray comprising capture molecules (such as antibodies) specific for each of the biomarkers being quantified immobilized on a solid support. In one embodiment of the invention, the microarray comprises capture molecules specific for each of IL-6, IL-6R, GP130, IL-23, ΤίίΡβ, IFNy, TNFa, CD160, CRTAM, CCL3, CCL4, XCLl , EGRl , EGR2, CAVl , DARC, SH2D1.B, CXCLl l , FGGBP2, GNLY, PLalA, IL-10, IL-12, IL-17, IL-21 or combinations thereof. In one embodiment of the invention, the microarray comprises capture molecules specific for any one or more of IL-6, IL-6R, GP130, IL-23, TGF , IFNy, TNFa, CD160, CRTAM, CCL3, CCL4, XCLl, EGRl, EGR2, CAVl, DARC, SH2D 1B, CXCLl , FGGBP2, GNLY, PLal A, IL-10, IL-12, IL-17, IL-21 or combinations thereof.

[0088] Once captured on a microarray, detection methods can be any of those known in the art. For example, fluorescence detection can be employed. It is safe, sensitive and can have a high resolution. Other detection methods include other optical methods (for example colorimetric analysis, chemiluminescence, label free Surface Piasmon Resonance analysis, microscopy, reflectance etc.), mass spectrometry, electrochemical methods (for example voltametry and amperometry methods) and radio frequency methods (for example multipolar resonance spectroscopy).

[0089] Additional methods of determining protein concentration include mass spectrometry and/or liquid chromatography, such as LC-MS, UPLC, or a tandem UPLC-MS/MS sy stem.

Systems for diagnosing AMR

[0090] Also provided herein is a system comprising an isolated biological sample from a subject desiring a determinations of the likelihood of AMR, and an assay for detecting a presence or absence of one or more mRNA encoding a protein associated with AMR, wherein the one or more mRNA encodes anv one or more of IL-6, IL-6R, GP130, IL-23. TGF , IFNy, TNFa, CD160, CRTAM, CCL3, CCL4, XCLl, EGRl, EGR2, CAVl, DARC, SH2D1B, CXCLl , FGGBP2, GNLY, PLal A, IL-10, IL-12, IL-17, IL-21 or combinations thereof, to diagnose or determine the likelihood of AMR in the subject. [0091] Also provided herein is a system comprising an isolated biological sample from a subject desiring a determinations of the likelihood of AMR, and an assay for detecting the level of one or more mRNA encoding a protein associated with AMR, wherein the one or more mRNA encodes any one or more of IL-6, IL-6R, GP130, IL-23, TGFp, IFNy, TNFa, CD160, CRTAM, CCL3, CCL4, XCL1, EGR1 , EGR2, CAV1, DARC, SH2D1B, CXCLl l, FGGBP2, ONLY, PLal A, IL-1Q, IL-12, IL-17, IL-21 or combinations thereof, to diagnose or determine the likelihood of AMR in the subject.

[0092] Also provided herein is a system comprising an isolated biological sample from a subject desiring a determinations of the likelihood of AMR, and an assay for detecting a presence or absence of one or more proteins associated with AMR, wherein the one or more proteins includes but is not limited to IL-6, IL-6R, GP130, IL-23, TGFp, IFNy, TNFa, CD160, CRTAM, CCL3, CCL4, XCL1, EGR1, EGR2, CAV1, DARC, SH2D1 B, CXCLl l, FGGBP2, GNLY, PLal A, IL-10, IL-12, IL-17, IL-21 or combinations thereof, to diagnose or determine the likelihood of AMR in the subject.

[0093] Also provided herein is a system comprising an isolated biological sample from a subject desiring a determinations of the likelihood of AMR, and an assay for detecting the level of one or more proteins associated with AMR, wherein the one or more proteins includes but is not limited IL-6, IL-6R, GP130, IL-23, TGF , IFNy, TNFa, CD 160, CRTAM, CCL3, CCL4, XCLl, EGR1, EGR2, CAV1, DARC, SH2D1B, CXCLl l, FGGBP2, GNLY, PLal A, IL-10, IL-12, IL-17, IL-21 or combinations thereof, to diagnose or determine the likelihood of AMR in the subject.

Selecting therapies to treat AMR

[0094] Various embodiments provide a method of selecting a therapy for AMR for a subject in need thereof. In various embodiments, the method includes detecting the presence of one or more mRNA encoding a protein associated with AMR, wherein the one or more mRNA encodes any one or more of IL-6, IL-6R, GP130, IL-23, TGF , IFNy, TNFa, CD160, CRTAM, CCL3, CCL4, XCLl, EGR1, EGR2, CAV1, DARC, SH2D1B, CXCLl l, FGGBP2, GNLY, PLal A, IL-10, IL-12, IL-17, IL-21 or combinations thereof in a subject desiring determination of likelihood of AMR; and selecting a therapy to treat AMR.

[0095] Various embodiments provide for a method of selecting a therapy for AMR for a subject in need thereof. In various embodiments, the method includes detecting the level of one or more mR A encoding a protein associated with AMR, wherein the one or more mRNA encodes any one or more of IL-6, IL-6R, GP130, IL-23, ΤΟΡβ, IFNy, TNFa, CD 160, CRTAM, CCL3, CCL4, XCL1, EGR1, EGR2, CAV1, DARC, SH2D1B, CXCLl l, FGGBP2, GNLY, PLalA, IL-10, IL-12, IL-17, IL-21 or combinations thereof in a subject desiring determination of likelihood of AMR; and selecting a therapy to treat AMR.

[0096] Various embodiments provide a method of selecting a therapy for AMR for a subject in need thereof. In various embodiments, the method includes detecting the presence of one proteins associated with AMR, wherein the one or more proteins includes but is not limited to IL-6, IL-6R, GP130, IL-23, ΤίίΡβ, IFNy, TNFa, CD160, CR TAM, CCL3, CCL4, XCL1, EGR1, EGR2, CAV1, DARC, SH2D1 B, CXCLl l, FGGBP2, GNLY, PLal A, IL-10, IL-12, IL-17, IL-21 or combinations thereof in a subject desiring determination of likelihood of AMR; and selecting a therapy to treat AMR.

[0097] Various embodiments provide for a method of selecting a therapy for AMR for a subject in need thereof. In various embodiments, the method includes detecting the level of protein associated with AMR, wherein the one or more proteins includes but is not limited to IL-6, IL-6R, GP130, IL-23, TGF , IFNy, TNFa, CD160, CRTAM, CCL3, CCL4, XCL1, EGR1, EGR2, CAVl , DARC, SH2D1B, CXCLl l, FGGBP2, GNLY, PLalA, IL-10, IL-12, IL-17, IL-21 or combinations thereof in a subject desiring determination of likelihood of AMR; and selecting a therapy to treat AMR.

[0098] Selecting a therapy as used herein, includes but is not limited to selecting, choosing, prescribing, advising, recommending, instructing, or counseling the subject with respect to the treatment.

[0099] In various embodiments, the method further comprises administering the therapy to treat AMR. In various embodiments, the therapy is a therapy as described herein. In various embodiments, the therapy is an available therapy in the prior art,

[0100] In various embodiments, detecting the presence or level of mRNA encoding a protein associated with AMR including mRNA encoding any one or more of IL-6, IL-6R, GP130, IL-23, ΤΓτΡβ, IFNy, TNFa, CD 160, CRTAM, CCL3, CCL4, XCL1, EGR1, EGR2, CAVl, DARC, SH2D1 B, CXCLl l, FGGBP2, GNLY, PLalA, IL-10, IL-12, IL-17, IL-21 or combinations thereof can be performed as described by the methods or systems of the present invention [0101] In various embodiments, detecting the presence or level of any one or more of IL-6, IL-6R, GP130, 11.-23 , ΤΟΡβ, IFNy, TNFa, CD 160, CRTAM, CCL3, CCL4, XCLl, EGRl, EGR2, CAV1, DARC, SH2D1B, CXCLl l, FGGBP2, GNLY, PLalA, IL-10, IL-12, IL-17, IL-21 or combinations thereof can be performed as described by the methods or systems of the present invention.

[0102] In some embodiments, the subject can be a subject presenting no symptoms of AMR or a subject presenting one or more symptoms of AMR, for example, as discussed herein.

[0103] In further embodiments, the above diagnosis may be used to direct the treatment for the subject. In one embodiment, a subject with the likely presence of AMR may be treated with one or more therapies for AMR. One of ordinary skill in the art will be able to select an available treatment for AMR based on the diagnosis of AMR.

[0104] In some embodiments, the therapies include one or more therapeutic substances and/or performing one or more medical procedures on the subject, if it determined that the subject has an increased likelihood of developing AMR. In some embodiments, one or more therapeutic substance and/or treatment protocol administered to the subject determined to have an increased likelihood of developing AMR, or suspected of AMR, may include, but is in no way limited to pulse steroids, IVIG, anti-CD20 antibody such as rituximab, plasma exchange, anti-complement agents such as anti-C5 antibody (e.g. eculizumab) and CI inhibitor (e.g. Berinert), anti-IL-6 receptor antibody such as tocilizumab, IgG-digesting enzyme (e.g. IdeS), In some embodiments, treatment may include, but is in no way limited to, adjustment of dose or type (stronger immunosuppression) of regular maintenance immunosuppressive drugs (e.g., cyclosporine A, tacrolimus, mycophenolate mofetil, steroid, sirolimus, everolimus, belatacept), induction drugs (e.g., alemtuzumab, anti-thymoglubulin, anti-IL-2 receptor antibody) and/or other immunosuppressive drugs. In some embodiments, the treatment further includes monitoring anti-HLA, especially donor specific anti-HLA antibody levels, in order to adjust treatment as necessary.

Biological Samples

[0105] Examples of biological samples include but are not limited to body fluids, whole blood, plasma, pulmonary secretions, intestinal fluids or aspirate, stomach fluids or aspirate, serum, cerebral spinal fluid (CSF), urine, sweat, saliva, tears, breast aspirate, prostate fluid, seminal fluid, cervical scraping, amniotic fluid, intraocular fluid, mucous, and stool. In particular embodiments of the present invention, the biological sample is whole blood, blood plasma, blood serum, or pulmonary secretions. In various embodiments, the biological sample is whole blood. In various embodiments, the biological sample is serum. In various embodiments, the biological sample is plasma. In some embodiments, the sample comprises one or more exosomes.

[0106] In some embodiments, the sample for determining the presence or level of any one or more of IL-6, IL-6R, GP130, IL-23, ΤΟΡβ, IFNy, TNFa, CD160, CRTAM, CCL3, CCL4, XCL1, EGR1, EGR2, CAV1, DARC, SH2D1B, CXCL1 1, FGGBP2, ONLY, PLalA, IL-10, IL-12, IL-17, IL-21 or combinations thereof is plasma. In some embodiments, the sample for determining the presence or level of any one or more of IL-6, IL-6R, GP130, IL-23, ΤΟΡβ, IFNy, TNFa, CD160, CRTAM, CCL3, CCL4, XCL1, EGR1, EGR2, CAVI, DARC, SH2DIB, CXCL1 1, FGGBP2, GNLY, PLalA, IL-10, IL-12, IL-17, IL-21 or combinations thereof is a sample comprising exosomes.

Reference Values

[0107] In various embodiments of the methods and assays described herein, the reference value is based on the expression level of one or more genes that encode for a protein selected from IL-6, IL-6R, GP130, IL-23, ΤίίΡβ, IFNy, TNFa, O) i 60, CRTAM, CCL.3, CCL4, XCL1, EGR1, EGR2, CAVI, DARC, SH2D1.B, CXCL1 1 , FGGBP2, GNLY, PLal A, IL-10, IL-12, IL-17 and IL-21. In some embodiments, the reference value is the mean or median expression level of any of the aforementioned genes in a population of subjects that do not have AMR, or don't have a high likelihood of developing AMR. In other embodiments, the reference value is the mean or median expression level of any of the aforementioned genes from the tested subject at earlier time points, such as, but in no way limited to, before the subject received an allograft, or before AMR could result in the subject. In some embodiments, the reference value is the expression level of any of the genes described herein from the tested subject at a single time point prior to the subject receiving an allograft, or shortly after the subject has received an allograft, but before the subject is likely to exhibit signs of rejection. In some embodiments, the reference value is the expression level of one or more of the aforementioned genes in a subject who does not have AMR at any stage (e.g. clinical AMR or pre-clinical AMR (before organ dysfunction and other symptoms are clinically detectable). When the results of the mRNA analyses described herein are considered in light of the reference value (determined by any means described herein - including in the examples), it can be determined whether or not an allograft recipient has an increased likelihood or a relatively high probability of developing AMR.

[0108] In various embodiments, the expression level, as assessed by exosome mRNA analysis described herein, of one or more of 11. ··(:·. IL-6R, GP130, IL-23, TGFb, ΪΡΝγ, TNFa, CD160, CRTAM, CCL3, CCL4, XCLl, EGRl, EGR2, CAVl, DARC, SH2D1 B, CXCL1 1, FGGBP2, GNLY, PLalA, IL-10, IL-12, IL-17 and 11.-2 1 in the subject with a relatively high probability of developing AMR, compared to the reference value, is increased by at least or about 1-5%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% or more. In various embodiments, the expression level, as assessed by exosome mRNA analysis described herein, of one or more of IL-6, IL-6R, GP 130, IL-23, TGFb, IFNy, TNFa, CD160, CRTAM, CCL3, CCL4, XCLl, EGRl, EGR2, CAVl, DARC, SH2D1B, CXCLl l, FGGBP2, GNLY, PLalA, IL- 10, IL-12, IL- 17 and IL-21 in the subject with a relatively high probability of developing AMR, compared to the reference value, is increased by at least or about 1-fold, 2-fold, 3 -fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80- fold, 85-fold, 90-fold, 95-fold, 100-fold or more.

[0109] In determining efficacy, the expression level as assessed by exosome mRNA and/or protein analysis described herein, of one or more of IL-6, IL-6R, GP 130, IL-23, TGFb, IFNg, TNFa, CD 160, CRTAM, CCL3, CCL4, XCLl , EGRl, EGR2, CAVl , DARC, SH2D1 B, CXCL l l, FGGBP2, GNLY, PLal A, IL-10, IL-12, IL-17 and IL-21 in the subject in whom the treatment for AMR is effective, is decreased relative to the reference value, by at least or about 1-5%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% or more. In determining efficacy, the expression level as assessed by exosome mRNA and/or protein analysis described herein, of one or more of IL-6, IL-6R, GP 130, IL- 23, TGFb, IFNg, TNFa, CD 1 60, CRTAM, CCL.3, CCL4, XCLl, EGRl, EGR2, CAVl, DARC, SH2D1B, CXCLl l, FGGBP2, GNLY, PLalA, IL-10, IL- 12, IL-17 and IL-21 in the subject in whom the treatment for AMR is effective, is decreased relative to the reference value, by at least or about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25- fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold or more. EXAMPLES

[0110] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Example 1

[0111] Total R A was extracted from exosomes purified from 226 EDTA-piasma samples from 51 patients (7 AMR, 6 CMR, 38 no rejection Control [18 HLA-sensitized (HS)/20 non- HS]) for reverse transcription, pre-amplifi cation and then qPCR. By way of background, whole blood can be collected in various tubes that contain different types of anti-coagulant, such as no-anti-coagulant to obtain serum, EDTA [Ethylenediaminetetraacetic acid] to obtain plasma, heparin to obtain heparinized plasma, ACD (acid citrate dextrose) to obtain ACD- plasma. As used herein, the term "EDTA-plasma" means EDTA-anti-coagulated plasma. The results of each gene were presented as fold change (FC) relative to a reference RNA after normalizing with a housekeeping gene, GAPDH. The FC was then normalized with the average FC of ail samples in the non-HS Control group for each gene (nFC). The average nFC (anFC) of multiple samples of each patient at or prior to diagnosis of rejection and the anFC from the 1st year post-transplant of each patient in the Control group (transplant recipients without rejection during the 1st year post-transplant) were used for comparison among all groups. A gene score (GS) was calculated by averaging selected 7 genes anFC for each patient for further comparison.

[0112] Table 1

[0113] Among 23 AMR-associated genes selected, 7 genes (GP130, TNF , CD 160, CCL4, CAV1, DARC, SH2D1B) showed increase of anFC in the AMR compared to other groups. The GS of these 7 genes was significantly higher in the AMR than other groups, and also significantly higher in HS Control than CMR and non-HS Control. HS Control patients with relatively higher GS tended to have donor-specific antibodies during the period.

[0114] Significant changes in AMR-associated gene expression were detected in exosomes from blood plasma and correlated with AMR status. This result indicates the utility of exosome mRNA measurement to predict a relative risk for AMR,

[0115] Total RNA was extracted from exosomes purified from 200 μΐ of archived EDTA- plasma samples using ExoRNeasy Serum/Plasma Midi Kit (Qiagen), and eluted in 15 μΐ of deionized-distilled H20. The next step (reverse transcription) was performed right after the RNA samples were prepared,

[0116] All eluted total RNA (typically 14-15μ1 although 13.2μ1 is the recommended volume) was submitted for reverse transcription (High-Capacity cDNA Reverse Transcription Kit, Life Technologies). The 20μ1 of cDNA obtained can be kept at -80°C.

[0117] 3.75μ1 of the cDNA was pre-amplified at the final reaction volume of 15μ1 with primers of the 24 genes including a housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using TaqMan Pre Amp Master Mix (Life Technologies) at 14 amplification cycles following the protocols recommended by the vendor. The 15μ1 of pre- amp product was then diluted into 300μ1 with TE (lOmM Tris-HCl pH 8.0, lmM EDTA) and kept at ~80°C. For the reference sample, 20ng of cDNA was added into the pre-amp reaction.

[0118] ΙΟμΙ of diluted pre-amp product was submitted for qPCR of each specific gene and GAPDH at the reaction volume of 25 μΐ. Thus, theoretically approximately 150 genes including GAPDH per sample can be tested for RT-qPCR using 200μ1 of plasma at the maximum,

[0119] The result (Ct: threshold cycle) of each gene was first normalized by the Ct of GAPDH, This normalization is for variation of amounts of RNA used for RT-PCR among samples.

[0120] The fold change (FC) of each gene vs. that in the reference RNA was calculated by comparing GAPDH-normalized Ct of each gene with that in the reference RNA. This normalization is for variation among qPCR runs. [0121] The FC of each gene was further normalized with the same gene's average FC of all samples in the non-FIS Control group (nFC). This normalization is for vari ation of FC among genes.

[0122] In some analysis where multiple nFC results are available per patient, the average of nFC (an !·^'(^' ) was used.

0123] Table 2

Example 2

[0124] Study Subjects: This study was approved by the Institutional Review Board at Cedars- Sinai Medical Center (IRB numbers Pro00021002, Pro00034039). 152 archived EDTA- plasma samples obtained from 64 kidney transplant recipients who were transplanted between August 2008 and July 2014 were used for this study. Among these 64 patients, 12 and 6 developed biopsy-proven ABMR and ABMR/CMR, respectively (ABMR group), 8 had CMR (CMR group) and 38 no rejection (control groups). ABMR and CMR were diagnosed based on the Banff 2013 and Banff 1997 classification, respectively. ABMR was developed at median 4.0 months post-transplant (9 days to 6.5 years) and CMR at 3.8 months (1.3 to 6.5 months).

[0125] All patients in the ABMR group except one were HLA-sensitized prior to transplant (HLA class I antibody | Ab ): 75% ± 38%, HLA class II Ab: 57% ± 35%) and only one patient in the ABMR group received an ABO incompatible (ABOI) transplant. Sixteen of the 17 HS patients received desensitization therapy with IVIG+rituximab with or without plasma exchange prior to transplant, and the remaining I US patient did not. The one non-HS ABMR patient developed de novo HLA class II Ab (>60%) including de novo DSA at rejection, 6.5 years post-transplant. No patient in the CMR group was HLA-sensitized (HLA class I Ab: 4% ± 1 1%, HLA class II Ab: 3% ± 9%), therefore none received desensitization pre- transplant. There are 2 non-rejection control groups; DES (desensitized) and non-DES control groups. All 18 patients in the DES control group (HLA class I Ab: 51% ± 43%, HLA class II Ab: 25% ± 32%) received pre-transplant desensitization due to HLA-sensitized status (15 patients), ABOI transplant without HLA-sensitization (2 patients), or both (1 patient). None of the 20 patients in the non-DES group was HLA-sensitized (HLA class I Ab: 4% ± 8%, HLA classll Ab: 0% ± 2%) or ABOI transplant, therefore none was desensitized pre- transplant.

[0126] The desensitization protocols used for ABO compatible transplant in HS and ABOI transplant in non-HS patients have been reported (Kahwaji, J., et al., Rituximab: An emerging therapeutic agent for kidney transplantation. Transplant Research and Risk Management, 2009. 1 : p. 1 5-29). Briefly, a standard protocol for HLA-desensitization consisted of 2 doses of IVIG (2g/kg) one month apart with one dose of rituximab (Ig) two weeks after the first IVIG dose. The protocol for ABOI transplant consisted of one dose of rituximab (Ig) two weeks prior to initiation of 5 sessions of plasma exchange followed by one dose of IVIG (2g/kg). The combination of both protocols was used for HS patients who received an ABOI transplant. If a negative or acceptable crossmatch was achieved and/or the anti-blood group titer became <1 :8 after desensitization, the patient was transplanted (Vo, A.A., et al., Rittiximah and intravenous immune globulin for desensitization during renal transplantation. N Engl J Med, 2008. 359(3): p, 242-51; Vo, A. A., et al., Benefits of rituximab combined with intravenous immunoglobulin for desensitization in kidney transplant recipients. Transplantation, 2014. 98(3): p. 312-9).

[0127] All but one patient received induction therapy. Most patients in the ABMR and DES control groups received lymphocyte depleting agents (alemtuzumab or anti -thymocyte globulin) except two patients in the DES control group receiving anti-IL-2 receptor antibody (daclizumab). The majority of patients in the CMR and non-DES groups received anti-IL-2 receptor antibody (basiliximab or daclizumab) except 3 patients in the CMR group and 6 patients in the non-DES control groups who received lymphocyte depleting agents (alemtuzumab or anti-thymocyte globulin). Maintenance immunosuppression consisted of calcineurin inhibitor (tacrolimus or cyclosporine A), mycofenolate mofetil and steroids. The target levels were dependent on the type of induction as reported elsewhere (Barbosa, D., et al., Polyomavirus BK viremia in kidney transplant recipients after desensitization with WIG ami maximal). Transplantation, 2014. 97(7): p. 755-61).

[0128] All patients received anti-viral prophylaxis consisting of valganciclovir or aciclovir for 6 months post-transplant depending on a risk for viral infection and viral-PCR monitoring as previously reported (Kahwaji, J., et al., Infectious complications in kidney-transplant recipients desensitized with rituximab and intravenous immunoglobulin. Clin J Am Soc Nephrol, 2011. 6(12): p. 2894-900). ABMR was treated with pulse steroids, IVIG and rituximab with or without plasma exchange. CMR was treated with pulse steroids. Refractory or Banff 2 rejection was treated with anti -thymocyte globulin.

[0129] Candidate Genes: We selected 21 candidate genes for this study (Table 3). Among them, CCL3, CCL4, CD160, CRT AM, EGR1, EGR2, IFNy, XCL1 are ADCC-activated genes identified by our previous study (Suvioiahti, E., et al., Genes associated with antibody- dependent cell activation are overexpressed in renal biopsies from patients with antibody - mediated rejection. Transpl Immunol, 2015. 32(1): p. 9-17). ADCC is an important mechanism of allograft injury which leads to ABMR (Hirohashi, T., et al., Complement independent antibody-mediated endarteritis and transplant arteriopathy i mice. Am J Transplant, 2010. 10(3): p. 510-7; Hirohashi, T., et al., A novel pathway of chronic allograft rejection mediated by NK cells and alloantibody. Am J Transplant, 2012. 12(2): p. 313-21; Lee, C.Y., et al., The involvement of FcR mechanisms in antibody-mediated rejection. Transplantation, 2007. 84(10): p. 1324-34). CAV1, DARC, FGFBP2, GNLY, SH2D1B are ABMR-associated genes as identified in previous study of biopsy samples from renal transplant recipients (Venner, J.M., et al., The molecular landscape of antibody-mediated kidney transplant rejection: evidence for NK involvement through CD 16a Fc receptors. Am J Transplant, 201 5. 15(5): p. 1336-48; Suvioiahti, E., et al., Genes associated with antibody- dependent cell activation are overexpressed in renal biopsies from patients with antibody- mediated rejection. Transpl Immunol, 2015. 32(1): p. 9-17; Dominy, K.M., et al., Use of Quantitative Real Time Polymerase Chain Reactio to Assess Gene Transcripts Associated With Antibody-Mediated Rejection of Kidney Transplants. Transplantation, 2015. 99(9): p. 1981-8). IL-6, IL6Ra and gpl30 (IL-6 β receptor) are selected due to the important role of IL-6/TL-6R signaling in mediating ABMR and DSA development. IL-10, IL-12a, IL-23a, TGFpl, TNFa are selected due to their important roles in all types of inflammatory or antiinflammatory immune events. [0130] Panel reactive antibody (PRA), anti-HLA antibody specificity and DSA score: PRA and antibody specificity assays were performed at Cedars-Sinai Medical Center HLA Laboratory using the methods previously described (Reinsmoen, N.L., et al., Acceptable donor-specific antibody levels allowing for successful deceased and living donor kidney transplantation after desensitization therapy. Transplantation, 2008. 86(6): p. 820-5). Briefly, HLA antibodies were detected by either flow quick screen or single antigen Luminex bead assay (Luminex, Austin, TX). The binding levels of HLA-specific antibodies were determined by multi-analyte bead assay performed on Luminex platform. The single antigen Luminex bead assay was standardized with Quantiplex beads (One Lambda/Thermo Fisher Scientific, Canoga Park, CA). Final specificity was analyzed through HLA Visual 2.2 software (One Lambda/Thermo Fisher Scientific). DSA scores were calculated based on standard fluorescence intensities (SFI) of DSAs as previously described (Vo, A. A., et al., A Phase I/II Trial of the Interletikin-6 Receptor-Specific Humanized Monoclonal (Tocilizumab) + Intravenous Immunoglobulin in Difficult to Desensitize Patients. Transplantation, 2015. 99(11): p. 2356-63); score=10 for SFI>200,000, score=5 for 100,000<SFI<200,000, score=2 for 0<SFI<100,000, score^;=0 for SFI=0. If DSA against multiple donor HLA antigens were detected in the same patient then the sum of each DSA score were used as the final DSA score for that patient.

[0131] Plasma samples selection and exosome RNA extraction: For patients in the ABMR and CMR groups, all the archived EDTA-plasma samples from each patient, which were collected within 1 month prior to diagnosis of rejection by biopsy, were included in this study. The mean sample number per patient included in the ABMR and CMR groups were 1.4 ± 0.6 (range 1-3 samples) and 1.9 ± 0.6 (range 1-3), respectively. For most patients in the DES and non-DES control groups, 3 plasma samples per patient were included in this study, which were generally collected over the time course of 1 to!2 months post-transplant (2.9 ± 0.4 samples per patient, range 2-4). 200μ1 of each selected plasma sample was submitted for exosome RNA extraction with exoRNeasy Serum/Plasma Midi Kit (Qiagen, Hilden, Germany) following the standard protocols.

[0132] Reverse transcription, pre-amplificaiion and. qPCR: cDNA was synthesized from total exosome RNA using High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosy stems/Thermo Fisher Scientific, Foster City, CA), and then cDNA was pre- amplified with the pooled TaqMan^ Gene Expression Assays (Applied Biosystems/Thermo Fisher Scientific) of all the genes we studied in addition to the reference gene of GAPDH with Human GAPD Endogenous Control (Applied Biosystems/Thermo Fisher Scientific), following the standard protocols with TaqMan^~' PreAmp Master Mix (Applied Biosystems/Thermo Fisher Scientific). The pre-ampiification product (from exosome RNA in approximately 1.25μ1 of original plasma for each gene) was then submitted for qPCR for each of 21 genes using TaqMan^® Gene Expression Assay (Table 3) as well as GAPDH, and TaqMan® Gene Expression Master Mix (Applied Biosystems/Thermo Fisher Scientific) on 7500 Real-Time PCR system (Applied Biosystems/Thermo Fisher Scientific). Total RNA prepared from a normal healthy individual ' s peripheral blood mononuclear cel ls stimulated with phorboi 12-myristate 13 -acetate and ionomycin was included at each qPCR run in the entire study and used as the reference RNA. Because of the high sequence similarities between XCLl and XCL2 (98%), and between CCL4, CCL4L1 and CCL4L2 (96% between CCL4 and CCL4L1, 100% between CCL4L1 and CCL4L2), the selected assays for XCL l and CCL4 will detect two and three transcripts, respectively. The expression level of each gene was first normalized to GAPDH using ACt (cycle threshold) method for each RNA sample respectively, and then presented as relative quantity (RQ) to the expression level of the same gene in the reference RNA sample (also normalized to GAPDH) by calculating A Ct.

[0133] Table 3. Candidate Genes and Taqman* Assays

related gene

[0134] Data analysis: Since the number of plasma samples included in the study varied for each individual patient, we first calculated the average RQ of each gene for each individual patient. For patients who had only one plasma sample included in this study, the RQ result from that single plasma/exosome RNA sample was used as the average RQ of that gene for this patient. If a patient had more than one plasma sample (collected at various time points) included in this study, we calculated the average RQ of each gene by averaging the RQ results from all plasma/exosome RNA samples. The average RQ results of each gene for each patient were then used in the following analysis. Non-parametric Kruskal -Walls s H Test (KW test) was first performed to identify genes whose mRNA transcript levels (average RQ) exhibited significant difference among the 4 study groups of ABMR, CMR, DES control and non-DES control. If the p-value was less than 0.10 by KW test, non-parametric Mann- Whitney U test (MW test) was performed to make pairwise comparison within these 4 study groups. To calculate the gene combination score, genes showing significant elevation in ABMR patients were first selected based on the KW and MW test analysis. In order for each selected gene to make equal contribution to the final gene combination score, we used the normalized average RQ of each selected gene. The initial average RQ of each gene for each patient was normalized to the overall average RQ of the same gene among all 64 patients from ail 4 study groups of ABMR, CMR, DES control and non-DES control. The gene combination score for each patient was then obtained by calculating the average of normalized average RQ values from 4 selected genes. Further statistical analysis of gene combination scores was then conducted by KW and MW tests to identify the group(s) significantly different among the 4 study groups. Statistical analysis was done by Prism 6,0 (GraphPad Software, La Jolia, CA). The p value <0.05 was considered statistically significant.

Results

[0135] Among the 21 candidate genes, we identified 9 genes whose mRNA transcript levels in plasma exosomes varied significantly or near significance when all 4 study groups were compared (p<0.05 for 8 genes and p 0 07 for SH2D1 B by KW test) (Fig. 1) (Table 4). These 9 genes include the IL-6 signaling-related gene (gp^'130), ABMR-associated genes identified from renal biopsies (CAV1, DARC, SH2D1B), ADCC-associated genes (CCL4 and EGR1), and cytokine/inflammation-related genes (TNFa, 11.-- 10. lL-23a). Among the 9 genes, the mRNA transcript levels of 6 genes (gp l 30, CCL4, TNFa, CAV1, DARC and SH2D1 B) exhibited significant elevation in the ABMR patient group compared with either some or all of the other 3 patient groups (p<0.05 for pairwise comparison by MW test), while the mRNA transcript levels of IL-10 and IL-23a in plasma exosome appeared to decrease in the ABMR patient group compared with some of the other 3 patient groups (p<0.05 for pairwise comparison by MW test). The remaining 12 candidate genes showed no statistically significant variation of mRNA transcript levels (p>0.10 by KW test) (Fig. 2). Among the 6 genes whose mRNA transcript level in plasma exosome exhibited significant elevation in the ABMR patient group, only gpl30 gene showed significant elevation of mRNA transcripts in the ABMR patient group compared with all the other 3 study groups, while the other 5 genes showed significant elevation of mRNA transcripts in ABMR patient group above some but not all of the other three patient groups.

[0136] Table 4. Summary of Statistical Analysis Results of All 21 Candidate Genes

[0137] We next determined if gene scores combining multiple genes would better differentiate patients with ABMR from those with CMR and controls. We found that the gene score combining 4 genes (gp l 30, SH2D1B, TNFa and CCL4) was more robust in predicting risk for ABMR compared to controls. In addition, it was also significantly lower in the CMR patients compared to ABMR and controls (Fig. 3).

[0138] Since DSA is a major factor contributing to the development of ABMR, we next examined the correlation between the gene score and DSA levels. As shown in Fig, 4A, the ABMR patients had significantly higher DSA score than the patients in the DES control group. However, the correlation between the gene combination score and DSA score in ABMR patients was not high (R²=0.0396), although more than half of the ABMR patients showed a gene score >1 .3 (Fig, 4B).

[0139] ABMR remains one of the major obstacles to the long-tenn survival of allografts in kidney transplant patients. The importance of timely and accurate diagnosis of ABMR cannot be underestimated for the wellbeing of kidney transplant patients, particularly those with HLA sensitization. Renal biopsy remains the gold standard for diagnosis of allograft rejection including ABMR in kidney transplant patients. However, attempts to identify plasma or urine markers that would accurately predict risk for ABMR are desirable. Noninvasive tools capable of evaluating the overall physiologic and immunologic conditions of transplant patients including subclinical allograft rejection would be excellent alternative or supplement to traditional renal biopsy. Using peripheral blood or urine from kidney transplant patients, previous studies have attempted to establish and validate various types of molecular signatures based on proteins, metabolites and also mRNA transcript levels of multiple genes to predict the immune injuries to allograft and/or allograft rejection. For most of this research on mRNA transcripts and gene signatures, intracellular total mRNA transcripts extracted from intact cells were investigated; therefore, the requirements on sample freshness and storage are relatively strict. In comparison, exosome studies utilizing various body fluids (including blood plasma) may have less stringent requirements, particularly on sample freshness. Due to the exterior lipid membrane, the inner content of exosome is well-protected, therefore it is possible to detect and quantify multiple biologies such as proteins and RNA which are prone to quick degradation or denaturing once leaving the in vivo cellular environment. A previous study has confirmed that exosomes provide very strong protection to the mRNA transcripts inside and found that using the same exosome RNA extraction kit as we use in our study, high quality RNA preparations containing intact, full-length mRNA transcripts can be achieved from blood plasma samples including patient plasma sample biobanked over a decade. Likewise, we found excellent stability of mRN A transcripts for both the reference gene (GAPDH) and tested candidate genes in the exosomes of archived plasma samples in our study, many of which had been frozen for over 5 years, when compared to freshly collected plasma samples from healthy individuals.

[0140] Studies assessing mRNA transcript profiles of multiple genes in exosomes have been conducted for various diseases of the urinary system, including allograft rejection in kidney transplant patients. These studies all investigated exosomes from relatively large amount of urine samples per patient (usually 20ml or more). So far, we have not found research studies reported on assessing mRNA transcripts profile related to allograft rejection in kidney transplant patients using blood plasma exosomes. Since we have a sizable archive of plasma samples from kidney transplant patients, we selected 21 candidate genes for this study, which included genes whose mRNA transcript levels were elevated in the renal biopsies of kidney- transplant patients with ABMR as previously described, and other genes related to general inflammatory and/or IL-6 signaling events, the significant changes of whose expression very likely also contribute to the pathogenesis of ABMR. [0141] Among the 21 candidate genes, we identified multiple genes whose mRNA transcript levels exhibited significant changes (mostly elevation) in the plasma exosomes of ABMR patients compared with the other groups of kidney transplant patients. These genes include IL-6 β receptor of gp!30, chemokine/cytokine genes like CCL4, T F , IL-10 and IL-23a, and also other ABMR-associated genes, SH2D1 B, CA.V1 , DARC, as identified in previous published study of renal allograft biopsies. Analysis of our plasma exosome samples detected almost no IL-6 mRNA and the levels of IL-6Ra were similar in all groups. However, there were consistently higher levels of gpl 30 mRNA transcripts in the plasma exosomes of ABMR patients compared to other groups.

[0142] In addition to gpl30, we also found significant change of mRNA transcript levels for several other genes in the plasma exosomes of ABMR patients, including CCL4, TNFa, SH2D1B, CAVT, DARC, IL-10 and IL-23a, compared with some of the other three patient groups. These candidate genes were initially selected according to various criteria. TNFa and IL-10 were selected because of their well-known critical function in inflammatory conditions. IL-23/TL~17 immune axis is the most critical for the differentiation and activation of T helper 17 cells which are involved in the pathogenesis of various types of autoimmunity as well as the development of ABMR in transplant patients. CCL4, SH2D1B, CAV1 and DARC were selected because they are ABMR-associated genes identified by published studies using renal allograft biopsies of kidney transplant patients. Previous studies also reported that these 4 genes were either involved in the important function of various immune cells which may contribute to, or directly involved in the pathogenesis of ABMR in transplant patients. We found elevated levels of CAV1 and DARC mRNA transcripts in the plasma exosomes of ABMR patients compared with CMR and non-DES control patients, but not so compared with the DES-control patients, even though previous study using renal allograft biopsies identified both of them as highly ABMR-associated genes. However, it should be noted that DARC was also previously reported to be DSA-selective transcript in kidney transplant patients, which is consistent with our finding since a number of patients in the DES control patient group indeed had DSA detected (Fig. 4A).

[0143] Since ABMR is a highly complex immunologic and pathologic event, we generated a gene score combining the transcript levels of multiple genes (gpl 30, SH2D1 B, TNF and CCL4) which was significantly higher in the ABMR group and significantly lower in the CMR group compared with the control groups. But we did not find any significant correlation between our gene score and the DSA levels in these patients. DSA is a major risk factor associated with the development of ABMR in kidney transplant patients. High DSA levels (usually SFI>150,000), are associated with a risk for ABMR, while lower levels of DSA (SFI< 150,000) do not necessarily correlate with the development of ABMR. In fact, within the ABMR group of patients included in our study, there are also several who did not have detectable DSA when the biopsy diagnosis of ABMR was made. Since the development of ABMR involves multiple immunologic events such as 1L-6 signaling, NK cell function, inflammation and immune homeostasis, and chemokine/chemotaxis more than just generation of DSA, measuring a comprehensive biomarker with components from various immune functions such as our gene score might be more accurate to predict ABMR than measuring DSA alone,

[0144] Overall our current study confirmed the feasibility of quantifying the mRNA transcript levels of various genes in plasma exosome of kidney transplant patients. Using our platform, differential mRNA transcriptions of multiple genes were detected in plasma exosomes among ABMR, CMR and control patients, suggesting that exosome mRNA contains information related to events occurred at allograft and/or overall medical and immunologic conditions of kidney transplant patients. Elevated mRNA level of gpl 30 in plasma exosomes of ABMR patients suggests the involvement of IL-6/gpl30 signaling in the development of ABMR in some HLA-sensitized patients. More studies are warranted using larger number of samples and covering more comprehensive set of genes, in order to develop a plasma exosome mRNA-based diagnostic tool that can be used to predict allograft rejection for kidney transplant patients.

[0145] The various methods and techniques described above provide a number of ways to carry out the invention. Of course, it is to be understood that not necessarily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein.

[0146] A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by inclusion of one, another, or several advantageous features. Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein.

[0147] Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments. Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the application extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof. In some embodiments, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment of the application (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range.

[0148] Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and ail examples, or exemplar}' language (for example, "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application. Preferred embodiments of this application are described herein, including the best mode known to the inventors for carrying out the application. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context,

[0149] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and/or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and/or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and/or the use of the term in the present document shall prevail. In closing, it is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the application. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.

Claims

What is claimed is:

1. A method for determining the likelihood of antibody-mediated rejection (ABMR), comprising:

obtaining a biological sample from a subject desiring determination of likelihood of antibody-mediated rej ection;

determining the amount of mRNA encoding each of gpl30, SH2D1B, TNFoc and

CCL4 in the biological sample; and

determining that the subject has an increased likelihood of ABMR if the amount of the mRNA. encoding each of gp O, SH2D1 B, TNFoc and CCL4 is higher than a reference sample,

2. A method for determining the likelihood of antibody-mediated rejection (ABMR), comprising:

obtaining a biological sample from a subject desiring determination of likelihood of antibody-mediated rej ection;

determining the amount of one or more mRNA encoding one or more proteins associated with ABMR in the biological sample;

determining that the subject has an increased likelihood of ABMR if the amount of the one or more mRNA encoding one or more proteins associated with ABMR is higher than a reference sample.

3. The method of claim J or 2, wherein the sample blood or plasma.

4. The method of claim J or 2, wherein the sample is exosomes in blood or plasma.

5. The method of claim J or 2, wherein the subject has received an allograft,

6. The method of claim 5, wherein the allograft is selected from the group consisting of kidney, heart, liver, lung, islet, intestine and other solid organs.

7. The method of claim 5, wherein the allograft is a kidney allograft.

8. The method of claim 2, wherein one or more mR A for which an amount is determined encodes a protein selected from the group consisting of: IL-6, IL-6R, GP130, IL-23, TGFb, IFNg, TNFa, CD 160, CRT AM, CCL3, CCL4, XCL1, EGR1, EGR2, CAV1 , DARC, SH2D1B, CXCL11, FGGBP2, GNLY, PLalA, Π .- ! ().. IL-12, IL-17 and IL-21 .

9. The method of claim 2, wherein one or more mRNA for which an amount is determined encodes a protein selected from the group consisting of: GP130, TNFa, CD 160, CCL4, CAV1, DA C. and SH2D1B.

10. The method of any of claims 1-9, wherein the subject is a human.

1 1. The method of claims 1 or 2, wherein the reference value is the mean or median expression level of the one more more mRNA in a population of subjects that do not have AMR, or don't have a high likelihood of developing AMR.

12. The method of claims 1 or 2, wherein the reference value is the mean or median expression level of the one or more mRNA from the subject tested at an earlier time points, wherein the earlier time point is before the subject received an allograft or before likelihood of AMR is determined in the subject.

13. The method of claims 1 or 2, wherein the reference value is the expression level of one or more of the mRNA in a subject who does not have clinical AMR or preclinical.

14. A method, comprising:

determining that a subject has a relatively high probability of developing AMR according to the method of any of claims 1 -10; and

administering or modifying the dose of one or more therapeutic agent to the subject and/or performing one or more medical procedure on the subject in order to prevent the subject from developing clinical AMR. associated with decreased graft function. 5. The method of claim 11, wherein one or more therapeutic agent is selected from the group consisting of: pulse steroids, immunosuppressive drugs, IVIG, anti-CD20 antibody, anti-complement agents, anti-C5 antibody, C I inhibitor, anti-IL-6 receptor antibody, tociiizumab, IgG-digesting enzyme, IdeS, cyciosporine A, tacrolimus, mycophenolate mofetil, steroid, sirolimus, everolimus, belatacept, induction drugs, alemtuzumab, anti-thymogiubulin, anti-IL-2 receptor antibody.

16. The method of claim 7, wherein one or more medical procedure includes plasma exchange.