WO2019241526A1 - Dosages génétiques et sérologiques pour un appariement amélioré donneur/receveur - Google Patents

Dosages génétiques et sérologiques pour un appariement amélioré donneur/receveur Download PDF

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WO2019241526A1
WO2019241526A1 PCT/US2019/036999 US2019036999W WO2019241526A1 WO 2019241526 A1 WO2019241526 A1 WO 2019241526A1 US 2019036999 W US2019036999 W US 2019036999W WO 2019241526 A1 WO2019241526 A1 WO 2019241526A1
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transplant
antibodies
subject
gcc2
rejection
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PCT/US2019/036999
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Krzysztof KIRYLUK
Ali GHARAVI
Nicholas STEERS
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The Trustees Of Columbia University In The City Of New York
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G01N2800/245—Transplantation related diseases, e.g. graft versus host disease

Definitions

  • the present invention relates to genetic and serological assays for donor/recipient matching before transplantation and for evaluating transplant rejection after transplantation.
  • Transplant and graft rejection is a life-threatening condition that occurs when the recipient’s immune system begins to attack the donor tissue. This can lead to many additional side effects and, without treatment, may lead to the loss of the transplanted organ.
  • CMR cell-mediated rejection
  • ABMR antibody-mediated rejection
  • HLA Human leukocyte antigen
  • the present disclosure provides for a method for identifying a transplant donor for a subject in need of a transplant, where the subject is homozygous for rs893403-G allele and/or is homozygous for CNVR915.1 -deletion.
  • the method may comprise the steps of: (a) obtaining at least one donor nucleic acid sample from at least one potential donor; (b) detecting in the at least one donor nucleic acid sample (i) the presence or absence of a G allele at single nucleotide polymorphism (SNP) rs893403, and/or (ii) the presence or absence of CNVR915.1 -deletion; and (c) identifying a transplant donor for the subject if the transplant donor is homozygous for rs893403-G allele and/or is homozygous for CNVR915.1 -deletion.
  • SNP single nucleotide polymorphism
  • the present disclosure provides for a method for identifying a transplant donor for a subject in need of a transplant, where the subject is homozygous for a variant or polymorphism in linkage disequilibrium with rs893403.
  • the present disclosure also provides for a method for identifying a transplant donor for a subject in need of a transplant, where the subject is homozygous for any of the following variants.
  • the method may comprise the steps of: (a) obtaining at least one donor nucleic acid sample from at least one potential donor; (b) detecting in the at least one donor nucleic acid sample the presence or absence of an allele of a variant/polymorphism (e.g., single nucleotide polymorphism (SNP)) as described herein; and (c) identifying a transplant donor for the subject if the transplant donor is homozygous for the allele of the variant/polymorphism.
  • a variant/polymorphism e.g., single nucleotide polymorphism (SNP)
  • the method may comprise the steps of: (a) obtaining at least one donor nucleic acid sample from at least one potential donor; (b) detecting in the at least one donor nucleic acid sample the presence or absence of an allele of a variant/polymorphism (e.g., single nucleotide polymorphism (SNP)) in linkage disequilibrium with rs893403; and (c) identifying a transplant donor for the subject if the transplant donor is homozygous for the allele of the
  • a variant/polymorphism e.g., single nucleotide polymorphism (SNP)
  • the present method may further comprise transplanting an organ, tissue or cell from the transplant donor to the subject.
  • the present disclosure also provides for a method for detecting transplant rejection in a subject who has received a transplant from a donor or assessing the subject’s risk of transplant rejection towards a transplant from a donor.
  • the method may comprise the steps of: (a) obtaining a recipient nucleic acid sample from the subject, and a donor nucleic acid sample from the donor; (b) detecting in the recipient nucleic acid sample and the donor nucleic acid sample (i) the presence or absence of a G allele at single nucleotide polymorphism (SNP) rs893403, or (ii) the presence or absence of CNVR915.1 -deletion; and (c) diagnosing that the subject has transplant rejection or an increased risk of transplant rejection, if (i) the subject is homozygous for rs893403-G allele, and the donor is not homozygous for rs893403-G allele; or (ii) the
  • subject/recipient is homozygous for CNVR915.1 -deletion, and the donor is not homozygous for CNVR915.1 -deletion.
  • the subject may be diagnosed to have transplant rejection or an increased risk of transplant rejection when the subject/recipient and donor have a LIMS 1 locus“High-Risk” or“Genomic Collision” genotype combination.
  • the present disclosure provides for a method for detecting transplant rejection in a subject who has received a transplant from a donor or assessing the subject’s risk of transplant rejection towards a transplant from a donor.
  • the method may comprise the steps of: (a) obtaining a recipient nucleic acid sample from the subject, and a donor nucleic acid sample from the donor; (b) detecting in the recipient nucleic acid sample and the donor nucleic acid sample the presence or absence of an allele of a variant/polymorphism (e.g., SNP) in linkage disequilibrium with rs893403 (or an allele of a variant/polymorphism (e.g., SNP) as described herein); and (c) diagnosing that the subject has transplant rejection or an increased risk of transplant rejection, if the subject/recipient is homozygous for the allele, and the donor is not homozygous for the allele.
  • a variant/polymorphism e.g., SNP
  • the present disclosure provides for a method for treating a subject with transplant rejection or an increased risk of transplant rejection where the subject has received a transplant from a donor.
  • the method may comprise the steps of: (a) obtaining a recipient nucleic acid sample from the subject, and a donor nucleic acid sample from the donor; (b) detecting in the recipient nucleic acid sample and the donor nucleic acid sample (i) the presence or absence of a G allele at single nucleotide polymorphism (SNP) rs893403, or (ii) the presence or absence of CNVR915.1 -deletion; and (c) treating the subject for transplant rejection or an increased risk of transplant rejection, if (i) the subject/recipient is homozygous for rs893403-G allele, and the donor is not homozygous for rs893403-G allele; or (ii) the recipient is homozygous for
  • step (c) at least one immunosuppressant may be administered to the subject.
  • the subject is homozygous for rs893403-G allele
  • the donor is homozygous for rs893403-A allele or is rs893403-AG.
  • the subject is homozygous for CNVR915.1 -deletion
  • the donor is heterozygous for CNVR915.1 -deletion or lacks CNVR915.1 -deletion.
  • the subject may be treated for transplant rejection or an increased risk of transplant rejection, when the subject/recipient and donor have a LIMS 1 locus “High-Risk” or“Genomic Collision” genotype combination.
  • the present disclosure provides for a method for treating a subject with transplant rejection or an increased risk of transplant rejection where the subject has received a transplant from a donor.
  • the method may comprise the steps of: (a) obtaining a recipient nucleic acid sample from the subject, and a donor nucleic acid sample from the donor; (b) detecting in the recipient nucleic acid sample and the donor nucleic acid sample the presence or absence of an allele of a variant/polymorphism (e.g., SNP) in linkage disequilibrium with rs893403 (or an allele of a variant/polymorphism (e.g., SNP) as described herein); and (c) treating the subject for transplant rejection or an increased risk of transplant rejection, if the subject is homozygous for the allele, and the donor is not homozygous for the allele.
  • step (c) at least one
  • immunosuppressant may be administered to the subject.
  • LIMS 1 locus“High-Risk” or“Genomic Collision” genotype combination may be defined as:
  • recipient rs893403-GG and donor rs893403-AA or rs893403-AG, or
  • recipient CNVR915.1 -del/del and donor CNVR9l5.l-wt/wt or CNVR9l5.l-del/wt.
  • LIMS1 locus“Low-Risk” or“Non-collision” genotype combination may be defined as:
  • Non-limiting examples of variants/polymorphisms that may be used in the present methods include: rsl0084l99, rs427l73l, rs7596l99, rs2683806, rsl464406, rsl l l23694, rs2460947, rs2460944, rsl474220, rs2683798, rs2683797, rs2049l5l, rs2049l50, rs2l39807, rs25776l2, rs2577599, rs27l8764, rs27l8765, rs2l 18448, rs2l 18450, rs2577627, rsl0084l99, rs2718740, rsl978839, rsl469966, rs920264, rs2577588, rs27l8733, rsl30
  • the method may comprise the steps of: (a) obtaining a sample from the subject; (b) determining level of anti-LIMS 1 antibodies and/or anti-GCC2 antibodies in the sample; (c) comparing the level obtained in step (b) with the level of the anti-LIMS 1 antibodies and/or anti-GCC2 antibodies in a control sample; and (d) diagnosing that the subject has transplant rejection or an increased risk of transplant rejection, if the level of the anti-LIMS 1 antibodies and/or anti-GCC2 antibodies obtained in step (b) increases by at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 1 fold, at least or about 1.2 folds, at least or about 1.5 fold, at least or about 2 folds,
  • the present disclosure provides for a method for treating a subject with transplant rejection or an increased risk of transplant rejection, the method comprising the steps of: (a) obtaining a sample from the subject; (b) determining level of anti-LIMS 1 antibodies and/or anti- GCC2 antibodies in the sample; (c) comparing the level obtained in step (b) with the level of the anti-LIMS 1 antibodies and/or anti-GCC2 antibodies in a control sample; and (d) treating the subject for transplant rejection or an increased risk of transplant rejection, if the level of the anti-LIMS 1 antibodies and/or anti-GCC2 antibodies obtained in step (b) increases by at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 1 fold, at least or about 1.2 folds, at least or about 1.5 fold, at least or about 2 folds, at least or about 3 folds, or at least or
  • step (d) at least one immunosuppressant may be administered to the subject.
  • the method may comprise the steps of: (a) obtaining a sample from the subject; (b) determining level of LIMS 1 mRNA and/or GCC2 mRNA in the sample; (c) comparing the level obtained in step (b) with the level of the LIMS 1 mRNA and/or GCC2 mRNA in a control sample; and (d) diagnosing that the subject has transplant rejection or an increased risk of transplant rejection, if the level of the LIMS 1 mRNA and/or GCC2 mRNA obtained in step (b) decreases by at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 1 fold, at least or about 1.2 folds, at least or about 1.5 fold, at least or about 2 folds
  • the present disclosure provides for a method for treating a subject with transplant rejection or an increased risk of transplant rejection, the method comprising the steps of: (a) obtaining a sample from the subject; (b) determining level of LIMS 1 mRNA and/or GCC2 mRNA in the sample; (c) comparing the level obtained in step (b) with the level of the LIMS 1 mRNA and/or GCC2 mRNA in a control sample; and (d) treating the subject for transplant rejection or an increased risk of transplant rejection, if the level of the LIMS 1 mRNA and/or GCC2 mRNA obtained in step (b) decreases by at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 1 fold, at least or about 1.2 folds, at least or about 1.5 fold, at least or about 2 folds, at least or about 3 folds, or at least
  • immunosuppressant may be administered to the subject.
  • the level of the anti-LIMS 1 antibodies and/or anti-GCC2 antibodies is determined by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the sample may be a plasma, serum or blood sample.
  • the transplant may be a kidney transplant, a heart transplant, a lung transplant, a liver transplant, a pancreas transplant, a bone marrow transplant, a portion thereof, or a combination thereof.
  • the transplant may be a tissue transplant.
  • the control sample may be from a healthy subject or a plurality of healthy subjects.
  • the control sample may be from a subject who has received a transplant without rejection or from a plurality of subjects who have received a transplant without rejection.
  • the transplant rejection may comprise acute cellular rejection (ACR) and/or antibody- mediated rejection (ABMR).
  • ACR acute cellular rejection
  • ABMR antibody- mediated rejection
  • the transplant rejection may be hyperacute rejection.
  • the transplant rejection may be acute rejection.
  • the transplant rejection may be chronic transplant rejection.
  • the subject is human.
  • the subject’s existing immunosuppressive regimen may be modified or maintained.
  • Figures 1A-1C Discovery Phase.
  • Figure 1A shows our strategy for selecting high- priority deletions for tagging and typing in the discovery cohort.
  • a total of 44 of 50 deletions were successfully tagged and genotyped in the discovery cohort; 6 of 50 deletion-tagging single nucleotide polymorphisms (SNPs) were either monomorphic or failed our genotype quality- control analysis.
  • Annotations were based on the human reference genome hgl8 (accessed in July 2010).
  • Copy-number polymorphisms (CNPs) were common copy-number variants (CNVs with an allele frequency of >1%).
  • MAF denotes minor allele frequency.
  • Figure 1B shows the probability-probability plot for the genetic screen for rejection in the discovery cohort of 705 recipients under a recessive model.
  • the circles represent P values for 44 successfully typed common deletions; the horizontal dashed lines represent significance thresholds of 0.05
  • Figures 2A-2D Effects of rs893403 on the Rejection-free Allograft Survival in Study Cohorts.
  • Figure 2A shows the results in the discovery phase (involving 705 kidney transplant recipients [the Columbia cohort] who had either a nonrisk genotype [upper] or a risk genotype [lower]). Tick marks indicate censored data.
  • Figure 2B shows the results in the replication phase, which involved a stratified analysis of three other cohorts (Belfast, TransplantFines, and Torino) that included a total of 2004 donor-recipient pairs. The P values correspond to the minimally adjusted model, with adjustment for cohort only (if applicable).
  • Figure 2C shows the results in all the cohorts combined, which involved a stratified analysis of the four cohorts (i.e., 2709 kidney transplants [in 705 recipients from the discovery cohort and 2004 donor-recipient pairs from the replication cohorts]).
  • Figure 2D shows the estimated hazard ratios (with 95%
  • Figure 3A shows the change in intensity (x axis) as compared with the -log P value (y axis) for the top-ranking proteins on the basis of the mean signal intensity in a protein array; the change is calculated as a ratio of the mean normalized intensity in the high-risk rejection group to the mean normalized intensity of all other groups (termed“fold change”).
  • the findings suggest the presence of anti-LIMSl reactivity in high-risk recipients with rejection.
  • Figure 3C shows the results of anti- LIMS 1 total IgG seroreactivity studies with the use of an enzyme-linked Figure 2
  • OD optical density
  • FIG. 3D through 3G show the anti-LIMSl reactivity of IgGl, IgG2, IgG3, and IgG4 subclasses, respectively; the results show predominant IgG2 and IgG3 responses.
  • An asterisk indicates a P value of less than 0.001 and a dagger a P value of less than 0.01 for the comparisons of the group of recipients with a high-risk genotype who had rejection as compared with all other groups.
  • Figures 4A-4D show the anti-LIMSl reactivity of IgGl, IgG2, IgG3, and IgG4 subclasses, respectively; the results show predominant IgG2 and IgG3 responses.
  • An asterisk indicates a P value of less than 0.001 and a dagger a P value of less than 0.01 for the comparisons of the group of recipients with a high-risk genotype who had rejection as compared with all other groups.
  • Figures 4A-4D show the anti-LIMSl reactivity of
  • ROC Receiver Operating Characteristic
  • the present disclosure provides a new genetic marker for donor/recipient matching that can decrease the risk of transplant rejection.
  • Genomic collision at a novel histocompatibility locus encoding LIMS 1 antigen is associated with a higher risk of transplant rejection and production of anti-LIMSl antibodies.
  • the risk of rejection may be modified by genetic matching based on rs893403 genotype. This technology can serve as a diagnostic tool for better selection of transplant donor tissues/organs and to determine risk factors for patients who have already received transplants.
  • a sample e.g., a plasma or serum sample
  • the levels of anti-LIMS 1 antibodies and/or anti-GCC2 antibodies in the sample can be used for assessing the onset or severity of transplant rejection, or as an indicator of the efficacy of a therapeutic intervention for treating transplant rejection.
  • transplant rejection may be diagnosed or predicted, and then the subject may be treated.
  • the therapeutic intervention may be continued when it is effective, or altered if ineffective or insufficient.
  • the method may also identify a transplant recipient at risk for transplant rejection or delayed graft function.
  • the methods of the present disclosure can impact the way transplant recipients are treated (before, during, and/or after a transplantation procedure). For example, patients identified as having a high risk of transplant rejection can be treated more aggressively with, for example, immunosuppressants or other therapeutic agents. Patients identified as low risk may be treated less aggressively (e.g., with minimal or no
  • the present methods can predict or diagnose transplant rejection in a subject before transplantation or who has received a transplant.
  • the present method can be used to identify individuals at high risk of rejection by performing a composite genetic test of donors and recipients (e.g., LIMS1 genetic mismatch), and/or performing a serological test that detects anti-LIMS 1 antibodies and/or anti-GCC2 antibodies.
  • the method can be non-invasive, and lead to early clinical detection of rejection events.
  • the present method can also provide pre-emptive donor-recipient matching based on the composite genetic tests of recipients and their prospective donors.
  • the recipient s inheritance of variants that disrupt genes expressed in the kidney predisposes to allosensitization and rejection.
  • CNVs large copy number variants
  • CNVs large copy number variants
  • donor-recipient genomic incompatibility at this locus is associated with the production of anti-LIMS 1 antibodies and increased risk of kidney allograft rejection.
  • LIMS1 antigen (encoded within the locus marked by rs893403) is a viable target for determining histocompatibility between the donor and recipient.
  • mismatched LIMS1 genotype has an about 63% increased risk of rejection.
  • the present method may be used for genetic screen for donor/recipient matching, and for detecting risk of rejection or assessing rejection after transplantation.
  • the present disclosure provides for a minimally invasive technique to identify signs of rejection, or risk of rejection in transplant recipients.
  • the method can guide patient-specific, anti-rejection medicine regimens, and provide additional targets for antibody depletion therapies.
  • similar genetic incompatibilities can arise as a result of donor- recipient differences in variants that alter protein immunogenicity, by changing either protein structure, localization, or expression.
  • an allograft recipient is homozygous for a common deletion polymorphism and receives an allograft (e.g., a kidney allograft) from a donor that caries at least one normal allele.
  • the product of the gene(s) affected by these polymorphisms can become targets of the recipient’s immune system when expressed in an allograft.
  • genomic incompatibilities between a donor and recipient may lead to alvantsitization against novel antigens.
  • recessive inheritance of gene- disrupting variants may represent a risk factor for allograft rejection.
  • donor-recipient genomic incompatibility at the LIMS 1 locus is associated with the production of anti-LIMS 1 antibodies and increased risk of allograft rejection (e.g., kidney allograft rejection).
  • genetic matching is defined by both a transplant recipient and donor being homozygous for a gene-intersecting deletion.
  • “Genomic collision” may be defined as a specific donor-recipient genotype combination in which a recipient homozygous for a gene-intersecting deletion receives a transplant from a non-homozygous donor.
  • “genomic collision” (high-risk) donor/recipient genotype combination is defined as recipient homozygosity for the rs893403-G allele in the absence of donor homozygosity for the same allele.
  • “Collision risk” may be defined by recipient homozygosity in the absence of donor homozygosity.
  • the present method comprises a composite donor-recipient genetic test.
  • DNA samples are obtained from both the recipient and the potential or actual donor.
  • the genotype at either rs893403 or CNVR915.1, or any proxies for these loci (such as variants which are in linkage disequilibrium with these loci) is determined in both donor and recipient DNA using any available DNA typing or sequencing technology.
  • The“genomic collision” (high-risk) donor-recipient genotype combination may be determined as recipient homozygosity for the rs893403-G allele (or CNVR915.1 -deletion or any other polymorphism in linkage disequilibrium with the rs893403-G allele) in the absence of donor homozygosity for the same allele(s).
  • LIMS 1 locus“High-Risk” or“Genomic Collision” genotype combination may be defined as:
  • recipient rs893403-GG and donor rs893403-AA or rs893403-AG, or
  • recipient CNVR915.1 -del/del and donor CNVR9l5.l-wt/wt or CNVR9l5.l-del/wt.
  • LIMS1 locus“Low-Risk” or“Non-collision” genotype combination may be defined as:
  • donor CNVR915.1 -del/del regardless of the recipient genotype.
  • CNVR915.1 -del refers to the deletion allele.
  • CNVR9l5.l-wt refers to the wildtype allele (without the deletion).
  • any other polymorphism in linkage disequilibrium with rs893403 may be used to replace rs893403 and CNVR915.1.
  • variants/polymorphisms that may be used in the present methods include: rsl0084l99, rs427l73l, rs7596l99, rs2683806, rsl464406, rsl 1123694, rs2460947, rs2460944, rsl474220, rs2683798, rs2683797, rs2049l5l, rs2049l50, rs2l39807, rs25776l2, rs2577599, rs27l8764, rs27l8765, rs2l 18448, rs2l 18450, rs2577627, rsl0084l99, rs27l8740, rsl9788
  • a recipient will receive organ or tissue transplant from a donor where matched,“low-risk”, or“non-collision” recipient-donor genotype combination is established.
  • the present method comprises assaying the presence or level of alloantibodies.
  • the presence or level of alloantibodies may be assayed using protein arrays, ELISA, and/or western blots.
  • anti-LIMS 1 antibodies (and/or anti-GCC2 antibodies) serological test may be used to detect circulating anti-LIMS 1 antibodies (and/or anti-GCC2 antibodies) in sera of kidney transplant recipients. Positive test indicates the presence of anti-LIMS 1 (and/or anti- GCC2) humoral response that is associated with allograft rejection risk (e.g., kidney allograft rejection risk).
  • allograft rejection risk e.g., kidney allograft rejection risk
  • any suitable assay may be used to assay the presence or level of alloantibodies (e.g., anti- LIMS 1 antibodies and/or anti-GCC2 antibodies), including, but not limited to, ELISA, immunodiffusion, immunoblotting techniques, immunofluorescence, immunohistochemistry, immunocytochemistry, immunoprecipitation, heamagglutination, enzyme immunoassays, ELISpot, flow cytometry and flow cytometry for multiplex bead-based assays such as Luminex- type assays.
  • the present disclosure provides for a method for detecting transplant rejection in a subject who has received a transplant from a donor, or a method for assessing the subject’s risk of transplant rejection towards an allograft from a donor.
  • the method contains the following steps: (a) obtaining a sample (e.g., a plasma or serum sample, or other samples as discussed herein) from the subject; (b) assaying the level of anti-LIMS 1 antibodies and/or anti-GCC2 antibodies in the sample; and (c) comparing the level obtained in step (b) with the level of the anti-LIMS 1 antibodies and/or anti- GCC2 antibodies in a control sample.
  • a sample e.g., a plasma or serum sample, or other samples as discussed herein
  • the subject is determined (or diagnosed to undergo transplant rejection, or diagnosed) to have an increased risk of transplant rejection, if the level of anti-LIMS 1 antibodies and/or anti-GCC2 antibodies obtained in step (b) increases by at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 1 fold, at least or about 1.2 folds, at least or about 1.5 fold, at least or about 2 folds, at least or about 3 folds, or at least or about 4 folds, compared to its level in the control sample.
  • the present methods may treat a subject with transplant rejection or an increased risk of transplant rejection.
  • the subject When diagnosed with transplant rejection, the subject may be treated with at least one immunosuppressant.
  • the subject when transplant rejection is predicted (or when an increased risk of transplant rejection is diagnosed), the subject may be treated with at least one immunosuppressant.
  • the method contains the following steps: (a) obtaining a sample (e.g., a plasma or serum sample, or other samples as discussed herein) from the subject; (b) assaying the level of anti-LIMS 1 antibodies and/or anti-GCC2 antibodies in the sample; (c) comparing the level obtained in step (b) with the level of the anti-LIMS 1 antibodies and/or anti- GCC2 antibodies in a control sample; and (d) treating the subject for transplant rejection or an increased risk of transplant rejection, if the level of anti-LIMS 1 antibodies and/or anti-GCC2 antibodies obtained in step (b) increases at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 1 fold, at least or about 1.2 folds, at least or about 1.5 fold, at least or about 2 folds, at least or about 3 folds, or at least or
  • the alloantibodies may be any isotype, including IgG (e.g., IgGl, IgG2, IgG3, IgG4), IgM, IgA (IgAl, IgA2), IgD and/or IgE.
  • the antibodies are IgG2 and/or IgG3.
  • proteins/polypeptides there may be a number of different isoforms for each of these proteins/polypeptides discussed in this disclosure, provided herein are the general accession numbers, NCBI Reference Sequence (RefSeq) accession numbers, GenBank accession numbers, and/or UniProt numbers to provide relevant sequences.
  • the proteins/polypeptides may also comprise other sequences.
  • the level of the alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) in the sample may increase or decrease by about 1% to about 100%, about 5% to about 90%, about 10% to about 80%, about 5% to about 70%, about 5% to about 60%, about 10% to about 50%, about 15% to about 40%, about 5% to about 20%, about 1% to about 20%, about 10% to about 30%, at least or about 5%, at least or about 10%, at least or about 15%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 100%, about 10% to about 90%, about 12.5% to about 80%, about 20% to about 70%, about 25% to about 60%, or about 25% to about 50%, about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, at least or about
  • the level of the LIMS 1 mRNA and/or GCC2 mRNA in the sample may decrease or increase by about 1% to about 100%, about 5% to about 90%, about 10% to about 80%, about 5% to about 70%, about 5% to about 60%, about 10% to about 50%, about 15% to about 40%, about 5% to about 20%, about 1% to about 20%, about 10% to about 30%, at least or about 5%, at least or about 10%, at least or about 15%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 100%, about 10% to about 90%, about 12.5% to about 80%, about 20% to about 70%, about 25% to about 60%, or about 25% to about 50%, about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, at least or about 1.1 fold, at least or about 1.2 fold, at
  • the control sample may be from a patient who has received a transplant without rejection or a plurality of patients who have received a transplant without rejection.
  • the control sample may be from a healthy subject or a plurality of healthy subjects.
  • the samples may include, but are not limited to, serum, plasma, blood, whole blood and derivatives thereof, cardiac tissue, bone marrow, urine, cerebrospinal fluid (CSF), myocardium, endothelium, skin, hair, hair follicles, saliva, oral mucus, vaginal mucus, sweat, tears, epithelial tissues, semen, seminal plasma, prostatic fluid, excreta, ascites, lymph, bile, kidney, and other tissues, organs, as well as other samples or biopsies.
  • the biological sample is plasma or serum.
  • the level or amount of alloantibodies in a subject’s sample can be compared to a reference level or amount of the alloantibodies present in a control sample.
  • the control sample may be from a patient or patients without undergoing transplant rejection, or a healthy subject or subjects.
  • a control sample is taken from a patient prior to transplant or treatment with a therapeutic intervention, or a sample taken from an untreated patient.
  • a control sample is from transplant recipients without transplant rejection.
  • Reference levels for a polypeptide can be determined by determining the level of a polypeptide in a sufficiently large number of samples obtained from normal, healthy control subjects to obtain a pre-determined reference or threshold value.
  • a reference level can also be determined by determining the level of the polypeptide in a sample from a patient prior to transplant.
  • Reference (or calibrator) level information and methods for determining reference levels can be obtained from publicly available databases, as well as other sources.
  • the transplant may be an allograft or a xenograft.
  • An allograft is a transplant of an organ, tissue, bodily fluid or cell from one individual to a genetically non-identical individual of the same species.
  • a xenograft is a transplant of an organ, tissue, bodily fluid or cell from a different species.
  • the transplant maybe any organ or tissue transplant, including, but not limited to, a kidney transplant, a heart transplant, a liver transplant, a pancreas transplant, a lung transplant, an intestine transplant, a skin transplant, a bone marrow transplant, a small bowel transplant, a trachea transplant, a cornea transplant, a limb transplant, and a combination thereof.
  • organ or tissue transplant including, but not limited to, a kidney transplant, a heart transplant, a liver transplant, a pancreas transplant, a lung transplant, an intestine transplant, a skin transplant, a bone marrow transplant, a small bowel transplant, a trachea transplant, a cornea transplant, a limb transplant, and a combination thereof.
  • the present methods may diagnose or predict any type of transplant rejection, including, but not limited to, hyperacute rejection, acute rejection, and/or chronic rejection.
  • the present methods may determine/detect the presence, type and/or severity of the transplant rejection.
  • the method may contain the following steps: (a) obtaining a first sample from the patient before initiation of the therapy (or at a first time point after initiation of the therapy); (b) assaying the levels of alloantibodies (e.g., anti-LIMS 1 antibodies and/or anti-GCC2 antibodies) in the first sample; (c) obtaining a second sample from the patient after initiation of the therapy (or at a second time point after initiation of the therapy); (d) assaying the levels of the alloantibodies (e.g., anti-LIMS 1 antibodies and/or anti-GCC2 antibodies) in the second sample; (e) comparing the levels of step (b) with the levels of step (d).
  • alloantibodies e.g., anti-LIMS 1 antibodies and/or anti-GCC2 antibodies
  • step (d) decreases (e.g., by about 1% to about 100%, about 5% to about 90%, about 10% to about 80%, about 5% to about 70%, about 5% to about 60%, about 10% to about 50%, about 15% to about 40%, about 5% to about 20%, about 1% to about 20%, about 10% to about 30%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 10% to about 90%, about 12.5% to about 80%, about 20% to about 70%, about 25% to about 60%, or about 25% to about 50%, about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, at least or about 1.1 fold, at least or about 1.2 fold, at least or about 1.3 fold, at least or about 1.4 fold, at least or about 1.5 fold
  • the present disclosure provides for a method for assessing efficacy of an
  • the method may contain the following steps: (a) obtaining a first sample from the patient before initiation of the therapy (or at a first time point after initiation of the therapy); (b) assaying the levels of LIMS1 mRNA and/or GCC2 mRNA in the first sample; (c) obtaining a second sample from the patient after initiation of the therapy (or at a second time point after initiation of the therapy); (d) assaying the levels of the LIMS 1 mRNA and/or GCC2 mRNA in the second sample; (e) comparing the levels of step (b) with the levels of step (d).
  • step (d) increases (e.g., by about 1% to about 100%, about 5% to about 90%, about 10% to about 80%, about 5% to about 70%, about 5% to about 60%, about 10% to about 50%, about 15% to about 40%, about 5% to about 20%, about 1% to about 20%, about 10% to about 30%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 10% to about 90%, about 12.5% to about 80%, about 20% to about 70%, about 25% to about 60%, or about 25% to about 50%, about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, at least or about 1.1 fold, at least or about 1.2 fold, at least or about 1.3 fold, at least or about 1.4 fold, at least or about 1.5 fold, at least or about 1.6 fold, at least or about
  • An effective therapy may be continued, or discontinued if the patient’ s condition has improved and is no longer in need of treatment.
  • An ineffective treatment may be altered or modified, or replaced with other treatment.
  • the present methods can include the steps of measuring the level of alloantibodies (e.g., anti-LIMS 1 antibodies and/or anti-GCC2 antibodies) in a sample from a patient receiving a therapeutic intervention, and comparing the measured level to a reference level or the level of alloantibodies (e.g., anti-LIMS 1 antibodies and/or anti-GCC2 antibodies) in a control sample.
  • the measured level of the alloantibodies e.g., anti-LIMS 1 antibodies and/or anti-GCC2 antibodies
  • the measured level of the alloantibodies is indicative of the therapeutic efficacy of the therapeutic intervention.
  • therapy may be continued or altered, e.g., by change of dose or dosing frequency, or by addition of other active agents, or change of therapeutic regimen altogether.
  • the present invention also encompasses a method of predicting or assessing the level of severity of transplant rejection in a patient.
  • the method comprises measuring the level of alloantibodies (e.g., anti-LIMS 1 antibodies and/or anti-GCC2 antibodies) in a biological sample from a patient; and comparing the measured level to a reference level or the level of the alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) in a control sample, wherein the measured level of the alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) is indicative of the level of severity of transplant rejection in the patient.
  • an increase or decrease (as described herein) in the level of the alloantibodies is indicative of the level of severity of transplant rejection in the patient.
  • kits containing a reagent for measuring the level of alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) in a biological sample, instructions for measuring alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies), and instructions for evaluating or monitoring transplant rejection in a patient based on the level of the alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies).
  • alloantibodies e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies
  • instructions for measuring alloantibodies e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies
  • instructions for evaluating or monitoring transplant rejection in a patient based on the level of the alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies).
  • the present disclosure also provides for a kit containing a reagent for measuring the mRNA level (e.g., LIMS 1 mRNA and/or GCC2 mRNA) in a biological sample, instructions for measuring mRNA level (e.g., LIMS1 mRNA and/or GCC2 mRNA), and instructions for evaluating or monitoring transplant rejection in a patient based on the level of the mRNA (e.g., LIMS1 mRNA and/or GCC2 mRNA).
  • a reagent for measuring the mRNA level e.g., LIMS 1 mRNA and/or GCC2 mRNA
  • instructions for measuring mRNA level e.g., LIMS1 mRNA and/or GCC2 mRNA
  • instructions for evaluating or monitoring transplant rejection in a patient based on the level of the mRNA (e.g., LIMS1 mRNA and/or GCC2 mRNA).
  • kits for assessing or predicting the severity or progression of transplant rejection in a subject may comprise a reagent for measuring alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) in a biological sample, and instructions for assessing severity or progression of transplant rejection based on the level of the alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies).
  • alloantibodies e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies
  • the kit may comprise a reagent for measuring mRNA level (e.g., LIMS1 mRNA and/or GCC2 mRNA) in a biological sample, and instructions for assessing severity or progression of transplant rejection based on the mRNA level (e.g., LIMS1 mRNA and/or GCC2 mRNA).
  • mRNA level e.g., LIMS1 mRNA and/or GCC2 mRNA
  • LIM zinc finger domain containing 1 is a protein that in humans is encoded by the LIMS1 gene.
  • the NCBI Reference Sequence (RefSeq) accession numbers for human LIMS 1 mRNA may include NM_00l 193482, NM_00l 193483, NM_00l 193484, NM_00l 193485 and NM_00l 193488.
  • the NCBI Reference Sequence (RefSeq) accession numbers for human LIMS1 protein may include NP 001180411, NP_00l 180412, NP_00l 180413, NP_00l 180414, and NP_00l 180417.
  • the NCBI Reference Sequence (RefSeq) accession numbers for murine LIMS1 mRNA may include NM_00l 193303, NMJ326148, NM_20l242, NM 001346676 NM_00l359l 15.
  • the NCBI Reference Sequence (RefSeq) accession numbers for murine LIMS1 protein may include NP_00l 180232, NPJ301333605, NPJ380424, NP_957694 and NPJ301346044.
  • SNPs single-nucleotide polymorphisms
  • LIMS1 chromosome 2ql2.3 in the intronic portion of LIMS1.
  • This gene encodes LIM zinc finger domain containing- 1, a protein involved in cell adhesion and integrin signaling through its interaction with integrin-linked kinase found in focal adhesion plaques.
  • CNVR915.1 was originally annotated to intersect LOC100288532, a pseudogene in hgl8 that was removed in subsequent releases of the human genome.
  • GCC2 a peripheral membrane protein of unclear function.
  • GCC2 protein is expressed in the cytoplasm of kidney proximal tubule cells.
  • Genotyping of polymorphic variants can be carried out using any suitable methodology known in the art.
  • Genotyping single nucleotide polymorphisms include ligation detection reaction (Day et ah, Genomics 29, 152 62 (1995)), mass spectrometry, such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), single nucleotide primer extension and DNA chips or microarrays (see review by Schafer et ah, Nature Biotechnology, Vol 16, pp33-39 (1998)).
  • the use of DNA chips or microarrays may enable simultaneous genotyping at many different polymorphic loci in a single individual or the simultaneous genotyping of a single polymorphic locus in multiple individuals. SNPs may also be scored by DNA sequencing.
  • SNPs are commonly scored using PCR-based techniques, such as PCR-SSP using allele- specific primers (Bunce et al., Tissue Antigens, 1995; 50: 23-31).
  • This method generally involves performing DNA amplification reactions using genomic DNA as the template and two different primer pairs, the first primer pair comprising an allele- specific primer which under appropriate conditions is capable of hybridizing selectively to the wild type allele and a non allele-specific primer which binds to a complementary sequence elsewhere within the gene in question, the second primer pair comprising an allele- specific primer which under appropriate conditions is capable of hybridizing selectively to the variant allele and the same non allele- specific primer.
  • Further suitable techniques for scoring SNPs include PCR ELISA and denaturing high performance liquid chromatography (DHPLC).
  • genotyping can be carried out by performing PCR using non-allele specific primers spanning the polymorphic site and digesting the resultant PCR product using the appropriate restriction enzyme (also known as PCR-RFLP). Restriction fragment length polymorphisms, including those resulting from the presence of a single nucleotide polymorphism, may be scored by digesting genomic DNA with an appropriate enzyme then performing a Southern blot using a labelled probe corresponding to the polymorphic region (Molecular Cloning: A Laboratory Manual, Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
  • Genotyping of any given polymorphic variant may comprise screening for the presence or absence in the genome of the subject of both the normal or wild type allele and the variant or mutant allele, or may comprise screening for the presence or absence of either individual allele, it generally being possible to draw conclusions about the genotype of an individual at a polymorphic locus having two alternative allelic forms just by screening for one or other of the specific alleles.
  • genotyping of multiple polymorphisms in a single subject sample may be carried out simultaneously, for example with the use of a microarray or "gene chip".
  • Multiple should be taken to mean two or more, three or more, four or more, five or more, six or more etc.
  • Genotyping may be carried out in vitro, and can be performed on an isolated sample containing genomic DNA prepared from a suitable sample obtained from the subject under test.
  • genomic DNA is prepared from a sample of whole blood or tissue, or any suitable sample as described herein, according to standard procedures which are well known in the art. If genomic sequence data for the individual under test in the region containing the SNP is available, for example in a genomic sequence database as a result of a prior genomic sequencing exercise, then genotyping of the SNP may be accomplished by searching the available sequence data.
  • the presence of the variant may be inferred by evaluating the mRNA expression pattern using any suitable technique.
  • the presence of the variant may be inferred by evaluating the sequence, structure or properties of the protein using any convenient technique.
  • the level of the DNA or RNA (e.g., mRNA) molecules may be determined/detected using routine methods known to those of ordinary skill in the art.
  • the level of the nucleic acid molecule may be determined/detected by nucleic acid hybridization using a nucleic acid probe, or by nucleic acid amplification using one or more nucleic acid primers.
  • Nucleic acid hybridization can be performed using Southern blots, Northern blots, nucleic acid microarrays, etc.
  • the DNA in a sample may be evaluated by a Southern blot.
  • a Northern blot may be used to detect an mRNA.
  • mRNA is isolated from a given sample, and then electrophoresed to separate the mRNA species. The mRNA is transferred from the gel to a solid support. Labeled probes are used to identify or quantity the nucleic acids.
  • labeled nucleic acids are used to detect hybridization.
  • Complementary nucleic acids may be labeled by any one of several methods typically used to detect the presence of hybridized polynucleotides. One method of detection is the use of autoradiography.
  • Other labels include ligands that bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand.
  • Nucleic acid microarray technology which is also known as DNA chip technology, gene chip technology, and solid-phase nucleic acid array technology, may be based on, but not limited to, obtaining an array of identified nucleic acid probes on a fixed substrate, labeling target molecules with reporter molecules (e.g., radioactive, chemiluminescent, or fluorescent tags such as fluorescein, Cye3-dUTP, or Cye5-dUTP, etc.), hybridizing target nucleic acids to the probes, and evaluating target-probe hybridization.
  • reporter molecules e.g., radioactive, chemiluminescent, or fluorescent tags such as fluorescein, Cye3-dUTP, or Cye5-dUTP, etc.
  • the sensitivity of the assays may be enhanced through use of a nucleic acid
  • amplification system that multiplies the target nucleic acid being detected.
  • Nucleic acid amplification assays include, but are not limited to, the polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, quantitative RT-PCR, etc.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • real-time RT-PCR real-time RT-PCR
  • quantitative RT-PCR quantitative RT-PCR
  • Measuring or detecting the amount or level of mRNA in a sample can be performed in any manner known to one skilled in the art and such techniques for measuring or detecting the level of an mRNA are well known and can be readily employed.
  • a variety of methods for detecting mRNAs have been described and may include, Northern blotting, microarrays, real time PCR, RT-PCR, targeted RT-PCR, in situ hybridization, deep- sequencing, single-molecule direct RNA sequencing (RNAseq), bioluminescent methods, bioluminescent protein reassembly, BRET (bioluminescence resonance energy transfer)-based methods, fluorescence correlation spectroscopy and surface-enhanced Raman spectroscopy (Cissell, K. A. and Deo, S. K. (2009) Anal. Bioanal. Chem., 394:1109-1116).
  • the methods of the present invention may include the step of reverse transcribing RNA when assaying the level or amount of an mRNA.
  • the present application measures the level of anti-LIMS 1 antibodies and/or anti-GCC2 antibodies in a biological sample.
  • the sample is a body fluid.
  • the body fluid can include, but are not limited to, serum, plasma, blood, whole blood and derivatives thereof, urine, tears, saliva, sweat, cerebrospinal fluid (CSF), oral mucus, vaginal mucus, seminal plasma, semen, prostatic fluid, excreta, ascites, lymph, bile, and amniotic fluid.
  • the biological sample is plasma or serum.
  • samples can include, but are not limited to, kidney tissue, cardiac tissue, bone marrow, myocardium, endothelium, skin, hair, hair follicles, epithelial tissues, as well as other samples or biopsies.
  • the biological sample is kidney tissue.
  • the sample may be obtained at any time point after the transplant procedure, such as about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours, about 15 hours, about 18 hours, about 20 hours, about 22 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 3 years, about 5 years or longer following the transplantation procedure.
  • the time point may also be earlier or later.
  • the level or amount of anti-LIMS 1 antibodies and/or anti-GCC2 antibodies in a patient sample can be compared to a reference level or amount of the anti-LIMS 1 antibodies present in a control sample.
  • the control sample may be from a patient who has received a transplant without rejection or a plurality of patients who have received a transplant without rejection.
  • the control sample may be from a healthy subject or a plurality of healthy subjects.
  • a control sample is taken from a patient prior to treatment with a therapeutic intervention or a sample taken from an untreated patient (e.g., a patient who has not received a transplant and/or an immunosuppressant therapy).
  • Reference levels for anti-LIMS 1 antibodies and/or anti-GCC2 antibodies can be determined by determining the level of anti-LIMS 1 antibodies and/or anti- GCC2 antibodies in a sufficiently large number of samples obtained from a patient or patients who have received a transplant without transplant rejection, or normal, healthy control subjects to obtain a pre-determined reference or threshold value.
  • a reference level can also be determined by determining the level of anti-LIMS 1 antibodies and/or anti-GCC2 antibodies in a sample from a patient prior to treatment with the therapeutic intervention.
  • Reference (or calibrator) level information and methods for determining reference levels can be obtained from publicly available databases, as well as other sources. (See, e.g., Bunk, D. M. (2007) Clin. Biochem. Rev., 28(4): 131-137; and Remington: The Science and Practice of Pharmacy, Twenty First Edition (2005)).
  • the level of anti-LIMS 1 antibodies and/or anti-GCC2 antibodies can be detected and/or quantified by any of a number of methods well known to those of skill in the art.
  • the anti- LIMS 1 antibodies and/or anti-GCC2 antibodies may be detected by, for example, mass spectrometry (e.g., LC-MS/MS) and Western blot.
  • the methods may include various immunoassays such as enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay (LFIA), immunohistochemistry, antibody sandwich capture assay, immunofluorescent assay, Western blot, enzyme-linked immunospot assay (EliSpot assay), precipitation reactions (in a fluid or gel), immunodiffusion, Immunoelectrophoresis, radioimmunoassay (RIA), competitive binding protein assays, chemiluminescent assays, and the like.
  • ELISA enzyme-linked immunosorbent assay
  • LFIA lateral flow immunoassay
  • immunohistochemistry immunohistochemistry
  • antibody sandwich capture assay immunofluorescent assay
  • Western blot Western blot
  • enzyme-linked immunospot assay EliSpot assay
  • precipitation reactions in a fluid or gel
  • Immunoelectrophoresis radioimmunoassay
  • RIA radioimmunoassay
  • competitive binding protein assays
  • analytic biochemical methods such as electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, liquid chromatography-tandem mass spectrometry, and the like.
  • HPLC high-performance liquid chromatography
  • TLC thin layer chromatography
  • hyperdiffusion chromatography liquid chromatography-tandem mass spectrometry, and the like.
  • the level of anti-LIMS 1 antibodies may be detected by using molecules (e.g., polypeptides, etc.) that bind to the anti-LIMS 1 antibodies and/or anti-GCC2 antibodies.
  • the binding polypeptide may be the LIMS1 protein (e.g., recombinant LIMS1 protein), or may be an antibody or antibody fragment, such as an Fab, F(ab) 2 , F(ab’) 2 , Fd, or Fv fragment of an antibody.
  • antibodies can be used for this purpose, including, but not limited to, polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies (e.g., generated using transgenic mice, etc.), single chain antibodies (e.g., single chain Fv (scFv) antibodies), heavy chain antibodies and chimeric antibodies.
  • the antibodies can be from various species, such as rabbits, mice, rats, goats, chickens, guinea pigs, hamsters, horses, sheep, llamas etc.
  • ELISA is used to detect and/or quantify anti-LIMS 1 antibodies in a sample.
  • the ELISA can be any suitable methods, including, but not limited to, direct ELISA, sandwich ELISA, and competitive ELISA.
  • Presence or level of anti-LIMS 1 antibodies and/or anti-GCC2 antibodies can also be assayed using immunodiffusion, immunoblotting techniques, immunofluorescence,
  • Western blot is used to detect and quantify anti- LIMS 1 antibodies and/or anti-GCC2 antibodies in a sample.
  • the technique may comprise separating sample proteins by gel electrophoresis, transferring the separated proteins to a suitable solid support, and incubating the sample with the antibodies that specifically bind the anti- LIMS 1 antibodies and/or anti-GCC2 antibodies.
  • the disclosure further includes protein microarrays (including antibody arrays) for the analysis of levels of a plurality of alloantibodies (e.g., including LIMS1 antibodies and/or anti- GCC2 antibodies).
  • Protein microarray technology which is also known as protein chip technology and solid-phase protein array technology, is well known to those of ordinary skill in the art. Protein microarray may be based on, but not limited to, obtaining an array of identified peptides or proteins on a fixed substrate, binding target molecules or biological constituents to the peptides, and evaluating such binding. See, e.g., MacBeath et ah, Printing Proteins as Microarrays for High-Throughput Lunction Determination, Science 289(5485): 1760- 1763,
  • control peptide or protein molecules are attached to the substrate.
  • the polypeptides that may be used to assay the level of anti-LIMS 1 antibodies and/or anti-GCC2 antibodies may be derived also from sources other than antibody technology. Lor example, such binding agents can be provided by degenerate peptide libraries which can be readily prepared in solution, in immobilized form or as phage display libraries. Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptides and non-peptide synthetic moieties.
  • the anti-LIMS 1 antibodies and/or anti-GCC2 antibodies can be used to screen peptide libraries, including phage display libraries, to identify and select peptide binding partners of the anti-LIMS 1 antibodies and/or anti-GCC2 antibodies. Yeast two-hybrid screening methods also may be used to identify polypeptides that bind to the anti-LIMS l antibodies and/or anti-GCC2 antibodies.
  • a label can be any material having a detectable physical or chemical property.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Such labels may include, but are not limited to, a fluorescent label, a radiolabel, a chemiluminescent label, an enzyme, a metallic label, a bioluminescent label, a chromophore, biotin etc.
  • a fluorescently labeled or radiolabeled antibody that selectively binds to a polypeptide of the invention may be contacted with a tissue or cell to visualize the
  • a label may be a combination of the foregoing molecule types.
  • the level, amount, abundance or concentration of anti-LIMS 1 antibodies and/or anti- GCC2 antibodies may be measured.
  • the measurement result may be an absolute value or may be relative (e.g., relative to a reference protein or polypeptide, etc.)
  • a difference (increase or decrease) in the measured level of the anti- LIMS 1 antibodies and/or anti-GCC2 antibodies relative to the level of the anti-LIMS 1 antibodies and/or anti-GCC2 antibodies in the control sample e.g., a sample in at least one patient who has received a transplant without rejection, in the patient prior to treatment, at a different time point during treatment, or an untreated patient
  • a pre-determined reference value is indicative of the therapeutic efficacy of the therapeutic intervention (e.g., an immunosuppressant therapy).
  • a reduction or decrease in the measured level of the anti-LIMS 1 antibodies and/or anti-GCC2 antibodies relative to the level of the anti-LIMS 1 antibodies and/or anti-GCC2 antibodies in the control sample (e.g., a sample in the patient prior to treatment or an untreated patient) or pre-determined reference value can be indicative of the therapeutic efficacy of the therapeutic intervention.
  • the level of anti-LIMS l antibodies and/or anti-GCC2 antibodies is decreased when compared to the level in a control sample or pre-determined reference value in response to a therapeutic intervention, the decrease is indicative of therapeutic efficacy of the therapeutic intervention.
  • Transplant rejection The present method may be used to assess the transplant status or outcome, including, but not limited to, transplant rejection, transplant function (including delayed graft function), non rejection based allograft injury, transplant survival, chronic transplant injury, or titer
  • the non-rejection based allograft injury may include ischemic injury, virus infection, peri-operative ischemia, reperfusion injury, hypertension, physiological stress, injuries due to reactive oxygen species and/or injuries caused by pharmaceutical agents.
  • the transplant status or outcome may comprise vascular
  • the methods described herein are used for diagnosing or predicting transplant status or outcome (e.g., transplant rejection). In some embodiments, the methods described herein are used to detect and/or quantify alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) to determine whether a subject is undergoing transplant rejection. In some embodiments, the methods described herein are used to detect and/or quantify alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) for diagnosis or prediction of transplant rejection.
  • alloantibodies e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies
  • the methods described herein are used to detect and/or quantify alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) for determining an immunosuppressive regimen for a subject who has received a transplant. In some embodiments, the methods described herein are used to detect and/or quantify alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) to predict transplant survival in a subject that have received a transplant. The invention provides methods of diagnosing or predicting whether a transplant in a transplant recipient will survive or be lost.
  • alloantibodies e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies
  • the methods described herein are used to detect and/or quantify alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) to diagnose or predict the presence of long-term graft survival. In some embodiments, the methods described herein are used to detect and/or quantify alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) for diagnosis or prediction of non-rejection based transplant injury. The present methods may be used to diagnose graft-versus-host-disease (GVHD).
  • GVHD graft-versus-host-disease
  • the term "diagnose” or “diagnosis” of a transplant status or outcome includes predicting or diagnosing the transplant status or outcome, determining predisposition to a transplant status or outcome, monitoring treatment of transplant patient, diagnosing a therapeutic response of transplant patient, and prognosis of transplant status or outcome, transplant progression, and response to a particular treatment.
  • the transplant may be an allograft or a xenograft.
  • An allograft is a transplant of an organ, tissue, bodily fluid or cell from one individual to a genetically non-identical individual of the same species.
  • a xenograft is a transplant of an organ, tissue, bodily fluid or cell from a different species.
  • the transplant maybe any organ or tissue transplant, including, but not limited to, a heart transplant, a kidney transplant, a liver transplant, a pancreas transplant, a lung transplant, an intestine transplant, a skin transplant, a bone marrow transplant, a small bowel transplant, a trachea transplant, a cornea transplant, a limb transplant, and a combination thereof.
  • Transplant rejection includes a partial or complete immune response to a transplanted cell, tissue, organ, or the like on or in a recipient of said transplant due to an immune response to a transplant.
  • a transplant can be rejected through either a cell-mediated rejection (CMR) or antibody-mediated rejection (AMR).
  • CMR cell-mediated rejection
  • AMR antibody-mediated rejection
  • the rejection may be acute cellular rejection (ACR).
  • ACR acute cellular rejection
  • the rejection may be T-cell-mediated rejection.
  • the present methods may diagnose or predict any type of transplant rejection, including, but not limited to, hyperacute rejection, acute rejection, and/or chronic rejection.
  • Hyperacute rejection can occur within minutes or hours to days following transplantation and may be mediated by a complement response in recipients with pre-existing antibodies to the donor.
  • hyperacute rejection antibodies are observed in the transplant vasculature very soon after transplantation, possibly leading to clotting, ischemia, and eventual necrosis and death.
  • Acute rejection occurs days to months or even years following transplantation. It can include a T-cell mediated response and is identified based on presence of T-cell infiltration of the transplanted tissue, structural injury to the transplanted tissue, and injury to the vasculature of the transplanted tissue.
  • Chronic rejection occurs months to years following transplantation and is associated with chronic inflammatory and immune response against the transplanted tissue.
  • Chronic rejection may also include chronic allograft vasculopathy, which is associated with fibrosis of vasculature of the transplanted tissue.
  • U.S. Patent No. 8,637,038. Fibrosis is a common factor in chronic rejection of all types of organ transplants. Chronic rejection can typically be described by a range of specific disorders that are characteristic of the particular organ.
  • disorders include fibrotic atherosclerosis; in lung transplants, such disorders include fibroproliferative destruction of the airway (bronchiolitis obliterans); in kidney transplants, such disorders include obstructive nephropathy, nephrosclerorsis, tubulointerstitial nephropathy; and in liver transplants, such disorders include disappearing bile duct syndrome.
  • Chronic rejection can also be characterized by ischemic insult, denervation of the transplanted tissue, hyperlipidemia and hypertension associated with immunosuppressive drugs.
  • the invention provides methods of determining whether a patient or subject is displaying transplant tolerance.
  • transplant tolerance includes when the subject does not reject a graft organ, tissue or cell(s) that has been introduced into/onto the subject. In other words, the subject tolerates or maintains the organ, tissue or cell(s) that has been transplanted.
  • GVHD graft-versus-host-disease
  • GVHD immunocompromised subjects, who when transplanted, receive "passenger" lymphocytes in the transplanted stem cells or solid organ. These lymphocytes recognize the recipient's tissue as foreign. Thus, they attack and mount an inflammatory and destructive response in the recipient.
  • GVHD has a predilection for epithelial tissues, especially skin, liver, and mucosa of the gastrointestinal tract. GVHD subjects are immunocompromised due the fact that prior to transplant of the graft, the subject receives immunosuppressive therapy.
  • Certain embodiments of the invention provide methods of predicting transplant survival in a subject that has received a transplant.
  • the invention provides methods of diagnosing or predicting whether a transplant in a transplant patient or subject will survive or be lost.
  • the invention provides methods of diagnosing or predicting the presence of long term graft survival.
  • Long-term graft survival refers to graft survival for at least about 5 years beyond current sampling, despite the occurrence of one or more prior episodes of acute rejection.
  • transplant survival is determined for patients in which at least one episode of acute rejection has occurred. As such, these embodiments provide methods of determining or predicting transplant survival following acute rejection.
  • transplant rejection may be diagnosed or predicted (a risk of transplant rejection assessed), and then the subject may be treated with a therapy for the rejection, such as an immunosuppressant therapy.
  • a therapy for the rejection such as an immunosuppressant therapy.
  • An immunosuppressant also referred to as an immunosuppressive agent, can be any compound that decreases the function or activity of one or more aspects of the immune system, such as a component of the humoral or cellular immune system or the complement system.
  • immunosuppressants include, (1) antimetabolites, such as purine synthesis inhibitors (such as inosine monophosphate dehydrogenase (IMPDH) inhibitors, e.g., azathioprine, mycophenolate, and mycophenolate mofetil), pyrimidine synthesis inhibitors (e.g., lefhmomide and terifhmomide), and antifolates (e.g., methotrexate); (2) calcineurin inhibitors, such as tacrolimus, cyclosporine A, pimecrolimus, and voclosporin; (3) TNF-alpha inhibitors, such as thalidomide and lenalidomide; (4) IL-l receptor antagonists, such as anakinra; (5) mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin (sirolimus), deforolimus, everolimus, temsirolimus, zotaroli
  • Non-limiting exemplary cellular targets and their respective inhibitor compounds include, but are not limited to, complement component 5 (e.g., eculizumab); tumor necrosis factors (TNFs) (e.g., infliximab, adalimumab, certolizumab pegol, afelimomab and golimumab); IL-5 (e.g., mepolizumab); IgE (e.g., omalizumab); BAYX (e.g., nerelimomab); interferon (e.g., faralimomab); IL-6 (e.g., elsilimomab); IL-12 and IL-13 (e.g., lebrikizumab and ustekinumab); CD3 (e.g., muromonab-CD3, otelixizumab, teplizumab, visilizumab); CD4 (e.g., clen
  • receptor/CD25 e.g., basiliximab, daclizumab, inolimomab.
  • the present disclosure provides for methods of evaluating and/or monitoring the efficacy of a therapeutic intervention (e.g., an immunosuppressant therapy) for treating transplant rejection.
  • a therapeutic intervention e.g., an immunosuppressant therapy
  • These methods can include the step of measuring the level of alloantibodies (e.g., anti- LIMS1 antibodies), or a panel of alloantibodies (e.g., anti-LIMSl antibodies), in a biological sample from a patient who has received a transplant.
  • the level of alloantibodies (e.g., anti-LIMSl antibodies) in the biological sample is compared to a reference level, or the level of the alloantibodies (e.g., anti-LIMSl antibodies) in a control sample.
  • the control sample may be taken from the patient at a different time point after transplantation, or from the patient before initiation of the therapeutic intervention (e.g., an immunosuppressant therapy), or from the patient at a different time point after initiation of the therapeutic intervention (e.g., an immunosuppressant therapy).
  • the measured level of the alloantibodies e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies
  • a decrease in the level of the alloantibodies is indicative of the therapeutic efficacy of the therapeutic intervention. In some cases, a decrease in the level of the
  • alloantibodies e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies
  • a change in the measured level of the alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) relative to a sample from the patient taken prior to treatment or earlier during the treatment regimen is indicative of the therapeutic efficacy of the therapeutic intervention.
  • the method comprises detecting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
  • alloantibodies e.g., including anti-LIMSl antibodies and/or anti-GCC2 antibodies
  • the patient sample may be classified as indicative of effective or non-effective intervention on the basis of a classifier algorithm. For example, samples may be classified on the basis of threshold values as described, or based upon mean and/or median alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) levels in one population or versus another (e.g., a population of healthy controls or a population of patients having received a transplant without rejection, or levels based on effective versus ineffective therapy).
  • classification schemes are known for classifying samples between two or more classes or groups, and these include, without limitation: Principal Components Analysis, Naive Bayes, Support Vector Machines, Nearest Neighbors, Decision Trees, Logistic, Artificial Neural Networks, Penalized Logistic Regression, and Rule-based schemes.
  • the predictions from multiple models can be combined to generate an overall prediction.
  • a classification algorithm or "class predictor" may be constructed to classify samples. The process for preparing a suitable class predictor (reviewed in Simon (2003) British Journal of Cancer (89) 1599-1604).
  • the present invention also provides methods for modifying a treatment regimen comprising detecting the level of alloantibodies (e.g., anti-LIMSl antibodies) in a biological sample from a patient receiving the therapeutic intervention and modifying the treatment regimen based on an increase or decrease in the level of the alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) in the biological sample.
  • alloantibodies e.g., anti-LIMSl antibodies
  • the methods for modifying the treatment regimen of a therapeutic intervention may comprise the steps of: (a) detecting the level of at least one alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) in a biological sample from a patient receiving the therapeutic intervention; and (b) modifying the treatment regimen based on an increase or decrease in the level of the at least one alloantibodies (e.g., anti-LIMSl antibodies) in the biological sample.
  • the method comprises detecting 2, 3, 4, 5, 6, 7, 8, 9, 10 or more alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) described herein.
  • the levels of less than 50, less than 30, or less than 20 alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) are detected.
  • Modifying the treatment regimen can include, but is not limited to, changing and/or modifying the type of therapeutic intervention, the dosage at which the therapeutic intervention is administered, the frequency of administration of the therapeutic intervention, the route of administration of the therapeutic intervention, as well as any other parameters that would be well known by a physician to change and/or modify.
  • one or more alloantibodies e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies
  • the therapeutic intervention is continued.
  • one or more alloantibodies e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies
  • do not decrease (or increase) during therapy or match reference levels the therapeutic intervention is modified.
  • the information regarding the increase or decrease in the level of at least one alloantibodies can be used to determine the treatment efficacy, as well as to tailor the treatment regimens of therapeutic interventions.
  • the present methods are used for the titration of a subject's immunosuppression. Additionally, the present method can be utilized to determine whether the response to drug therapy indicates resolution of rejection risk. It can also be used to test whether the reduction of drug therapy increases the risk of rejection and whether drug therapy, if discontinued, should be resumed. This helps avoiding over-medication and/or under-medication of a given patient and duration of treatment can be tailored to the needs of the patient.
  • the titration of immunosuppression can be after organ transplantation, or during a viral or bacterial infection. Further, the titration can be during a viral or bacterial infection after a subject has undergone organ transplantation.
  • the method can include monitoring the response of a subject to one or more immunosuppressive agents, the withdrawal of an immunosuppressive agent, an antiviral agent, or an anti-bacterial agent.
  • the disclosure further provides methods for developing personalized treatment plans for transplant recipients.
  • the methods can be carried out by, for example, carrying out any of the methods of alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) analysis described herein and, in consideration of the results obtained, designing a treatment plan for the patient whose transplant is assessed. If the levels of alloantibodies (e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies) indicate that the patient is at risk for an undesirable clinical outcome (e.g., transplant rejection, developing delayed graft function, or compromised graft function), the patient is a candidate for treatment with an effective amount of an immunosuppressant.
  • alloantibodies e.g., anti-LIMSl antibodies and/or anti-GCC2 antibodies
  • the patient may require a treatment regime that is more aggressive than a standard regime, or it may be determined that the patient is best suited for a standard regime. When so treated, one can treat or prevent transplant rejection (or, at least, prolong the time the transplanted organ functions adequately).
  • a different result i.e., a different level of alloantibodies (e.g., anti- LIMS 1 antibodies and/or anti-GCC2 antibodies) may indicate that the patient is not likely to experience an undesirable clinical outcome. In that event, the patient may avoid
  • the samples may be drawn before, during or after transplantation.
  • the samples may be drawn at different time points during transplantation, and/or be drawn at different time points after transplantation.
  • the sample When the sample is drawn after transplantation, it can be obtained from the subject at any point following transplantation.
  • the sample is obtained about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, at least 1, 2, 3, or 6 months following transplantation.
  • the sample is obtained least 1, 2, 3, 4, 6 or 8 weeks following transplantation.
  • the sample is obtained at least 1, 2, 3, 4, 5, 6, or 7 days following transplantation.
  • the sample is obtained at least 10 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 18 hours or 24 hours after transplantation. In other embodiments, the sample is obtained at least one week following transplantation.
  • one or more alloantibodies are measured between 1 and 8 weeks, between 2 and 7 weeks, at 1, 2, 3, 4, 5, 6, 7 or 8 weeks following transplantation.
  • kits containing a reagent or reagents for measuring anti-LIMS 1 antibodies and/or anti-GCC2 antibodies in a biological sample, instructions for measuring the anti-LIMS l antibodies and/or anti-GCC2 antibodies, and/or instructions for evaluating or monitoring transplant rejection in a patient based on the level of the anti-LIMS 1 antibodies and/or anti-GCC2 antibodies, and/or instructions for assessing an immunosuppressant therapy in a patient.
  • the present disclosure provides for a kit containing a reagent or reagents for measuring LIMS 1 mRNA level and/or GCC2 mRNA level in a biological sample, instructions for measuring the LIMS 1 mRNA level and/or GCC2 mRNA level, and/or instructions for evaluating or monitoring transplant rejection in a patient based on the LIMS 1 mRNA level and/or GCC2 mRNA level, and/or instructions for assessing an immunosuppressant therapy in a patient.
  • the kit comprises LIMS 1 protein (e.g., recombinant LIMS 1 protein) and/or GCC2 protein. In certain embodiments, the kit comprises primers and/or probe for genetic testing according to the present methods as described herein.
  • the kit contains a reagent for measuring anti-LIMS 1 antibodies and/or anti-GCC2 antibodies levels in a biological sample, instructions for measuring anti-LIMS 1 antibodies and/or anti- GCC2 antibodies levels, and instructions for evaluating or monitoring transplant rejection in a patient based on the anti-LIMS 1 antibodies and/or anti-GCC2 antibodies levels.
  • the kit contains a reagent for measuring LIMS 1 protein and/or GCC2 protein in a biological sample, instructions for measuring the LIMS 1 protein and/or GCC2 protein, and instructions for evaluating or monitoring transplant rejection in a patient based on the level of the LIMS 1 protein and/or GCC2 protein.
  • the kit may also be customized for determining the efficacy of therapy for transplant rejection.
  • kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed (e.g., sterile, pharmaceutically acceptable buffer and/or other diluents). However, various combinations of components may be comprised in a vial.
  • the kits of the present invention also will typically include a means for containing the nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.
  • the liquid solution may be an aqueous solution.
  • the components of the kit may also be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
  • kits may also include components that preserve or maintain the reagents or that protect against their degradation. Such components may be protease inhibitors or protect against proteases. Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution.
  • kits will also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented.
  • the study design had two stages.
  • the first stage involved a screen of 50 high-priority copy-number polymorphisms (see below) in kidney allograft recipients who had undergone transplantation at the Columbia University Irving Medical Center in New York. Signals that reached a P value of less than 0.05 were advanced to the second stage (the replication phase).
  • the replication phase involved genotyping of the top signals from the discovery phase in additional cohorts involving donor-recipient pairs.
  • the primary outcome in the genetic association study was the first rejection in a time-to-event analysis, defined as a rejection event (antibody-mediated rejection or T-cell-mediated rejection) that occurred between the date of transplantation and the date of allograft biopsy showing such an event.
  • the replication cohorts included the Harbor cohort (387 donor-recipient pairs; mean follow-up, 9.2 years; overall rejection rate, 24%), the TransplantLines Genetics cohort (833 donor-recipient pairs; mean follow-up, 6.6 years; overall rejection rate, 36%), and the Torino cohort (784 donor-recipient pairs; mean follow-up, 9.6 years; overall rejection rate,
  • genomic collision as a specific donor-recipient genotype combination in which a recipient who was homozygous for a deletion-tagging allele received a transplant from a nonhomozygous donor.
  • genome wide significance as an alpha level of 5.0xl0 -8 , as generally accepted for genetic association studies.
  • Stage 1 (the discovery phase) involved a genome-wide screen of 50 high priority common copy number polymorphisms (CNPs) in 705 kidney allograft recipients transplanted at Columbia University Irving Medical Center (CUIMC); signals reaching nominal P-value ⁇ 0.05 were advanced to the replication phase.
  • CNPs common copy number polymorphisms
  • CUIMC Columbia University Irving Medical Center
  • Clinical Outcomes and Statistical Methods The primary outcome was time-to-first-rejection, defined from the date of first transplant to the date of first biopsy demonstrating a rejection event, including both antibody mediated rejections (ABMR) and T-cell mediated rejections (TCMR).
  • ABMR antibody mediated rejections
  • TCMR T-cell mediated rejections
  • Kaplan-Meier survival analysis for univariate analysis
  • Cox proportional hazards model for multivariate analysis
  • Multivariate modeling of clinical covariates was done using variables that were nominally associated with outcome on univariate analysis ( P ⁇ 0.05). Such covariates were then subjected to stepwise selection using a BIC-guided selection.
  • the multivariate model was used to test recipient’s genotype coded under a recessive model, with risk genotype defined by
  • the risk genotype (“collision genotype”) was defined by recipient homozygosity in the absence of donor homozygosity for the deletion-tagging allele. Under this coding, any DR pair with a donor genotype homozygous for the deletion-tagging allele was coded as non-risk regardless of the paired recipient genotype, including when recipient genotype was not available. Similar to recipient-only analyses, the“collision genotype” was used as one of the predictors in the multivariate Cox proportional hazards model to derive adjusted effect estimates and
  • Stage 1 ( Discovery ) Methods The clinical characteristics of the CUIMC discovery cohort are summarized in Table 3.
  • the association screen was performed using a tag-SNP approach based on the filtering strategy depicted in Figure 1.
  • 50 high priority candidate SNPs were genotyped in a cohort of 705 kidney transplant recipients recruited by the Columbia Chronic Kidney Disease (CKD) Bio-bank; genomic DNA was extracted from whole blood (QuickGene-6lOL, Kurabo) and individuals SNPs were typed using KASP ( Kompetitive Alelle Specific PCR) assay by LGC Genomics.
  • CKD Columbia Chronic Kidney Disease
  • KASP Kompetitive Alelle Specific PCR
  • Strict genotype quality control (QC) analysis was performed, including per-SNP and per- individual genotyping rates >95% and elimination of SNPs deviating from Hardy- Weinberg equilibrium within each ethnic group. In total, 44 SNPs passed all QC filters ( Figure 1). To test for effects of deletion homozygosity, we used a time-to-event survival analysis under a recessive model with and without adjustment for relevant clinical covariates. We applied a Bonferroni- corrected alpha to declare statistical significance.
  • the clinical characteristics of the replication cohorts are presented in Table 5.
  • Targeted genotyping was performed using KASP assay (TransplantLines cohort), Sequenom iPLEX MassARRAY® (Belfast cohort) and by direct Sanger sequencing (Torino cohort).
  • the QC assessment included genotyping rate >95% in the entire cohort and passing Hardy- Weinberg equilibrium test (P>0.05) in both donor and recipient groups separately.
  • genotype frequencies of rs893403 were nearly identical in donors and recipients within each European cohort, and comparable across cohorts, assuring against potential genotyping errors or bias.
  • the statistical models applied to the replication cohorts were the same as in the discovery analysis.
  • the combined statistical analyses across replication cohorts were stratified by cohort membership. Because in our replication cohorts the majority of rejection events occurred within the first year post-transplant, we also compared alternative statistical models that do not rely on the assumption of proportional hazards. This included non-parametric (log-rank) and logistic-regression-based tests, as summarized in Table 8.
  • the deleted sequence was next interrogated for various regulatory motifs and features using UCSC Genome Browser 47 and a variety of other data types, including Human mRNAs 48 , Human ESTs 49 , predicted Retroposed Genes (UCSC Genes V5), C/D and H/ACA Box snoRNAs, scaRNAs, and microRNAs from snoRNABase 50 and miRBase 51 , tRNA Genes predicted by using tRNAscan-SE v. l.23 52 ; TargetScan miRNA Regulatory Sites 53 ; repeat sequences by
  • PCR-based Deletion Confirmation The CNVR915.1 deletion was confirmed by genomic DNA quantitative PCR in participant samples with rs893043-AG and GG genotypes and primer walking PCR method was adapted to confirm deletion boundaries based on the presence of a PCR product between close primer pairs. The below primer pair detected a single band PCR product and Sanger sequencing of this amplicon using the same primer pair identified the deletion breakpoints:
  • FUN-LDA Latent Dirichlet Allocation
  • LDA Latent Dirichlet Allocation
  • rs893403 we detected no predicted functional variants within the CNVR915.1 deletion region.
  • rsl0084l99 we detected several variants with high probability of being functional, including rsl0084l99 with high scores across nearly all 127 tissues and types (avg. posterior FUN-LDA probability 1.0).
  • GTEx Genotype-Tissue Expression
  • transcript quantification was performed with Affymetrix 2.1 ST arrays; after normalization of expression levels across genes using robust multi-array average (RMA) 61 and derivation of PEER factors as previously described 62, 63 , rs893403 was tested for eQTL effects with transcripts within a l-Mb window using linear regression under additive genotype coding with adjustments for age, sex, PEER factors and the first 4 principal components of ancestry.
  • RMA multi-array average
  • Standard pre-processing was applied to the microarrays to account for technical variability and protein prospector was used to normalize the samples using a linear model and calculate the M- scores.
  • Z-scores were calculated as the number of standard deviations of the signal derived from the signal of the mean and a Z score >2.5 was considered positive, on the condition this was concurrent with both protein spots on the microarray.
  • LIMS 1 protein (ab 116807) was diluted to 2 pg/l lml in carbonate bicarbonate buffer and coated on an Immulon H2 plate overnight at 4°C. Plates were washed x3 with 200 pl of washing buffer (lxPBS, 0.05% tween) and blocked for two hours with blocking buffer (lxPBS, 0.05% tween, 1% fish gelatin). Plates were washed x3 with 200 m ⁇ of washing buffer, serum was diluted 1:1000 in washing buffer, 100 m ⁇ was added per well and incubated for two hours at room temperature. Plates were washed x7 with 200 m ⁇ of washing buffer, before the addition of the detection antibodies diluted according to
  • TMB peroxidase substrate and Peroxidase substrate Sol B were mixed at a 1:1 ratio at room temperature and 100 m ⁇ was added to the plate, and the reaction was stopped by adding 100 m ⁇ of 2M H2S04, left on the bench for 20 minutes before reading at 450 nm.
  • anti-biotin-HRP antibody abl922l
  • lxPBS 0.05% tween
  • TMB peroxidase substrate and Peroxidase substrate Sol B were mixed at a 1:1 ratio and 100 m ⁇ was added to the plate at room temperature, and the reaction was stopped by adding 100 m ⁇ of 2M H 2 S0 4 , left on the bench for 20 minutes before reading at 450 nm.
  • Our second control was serum taken from normal healthy controls which were non-reactive to LIMS 1. These samples were used as normalization controls between the plates. The reactivity of individual serum samples was measured as a fold-change in OD compared to the average for the normalization controls ( Figure 3).
  • Tissue antibody staining was performed with the use of mouse monoclonal (IgGl) to human LIMS1 (LSBio LS-C169391) as well as rabbit polyclonal IgG antibody to human GCC2 (Genetex GTX51372) on paraffin-embedded tissues with the use of heat-induced antigen retrieval.
  • the following human tissues were sectioned and examined: kidney, liver, heart, lung, pancreas, and skin.
  • RNAscope® in situ Hybridization was performed using the
  • Flow cytometry Cells were stained using standard flow cytometry protocols. Briefly, for the staining of LIMS1, cells were resuspended in flow buffer (1 x PBS, 2 % FBS and blocked using Human TruStain FcX (BioLegend), before incubation with the anti-LIMSl antibody (LSBio LSC169391, dilution 1:200, manufacturer’s recommendation), and incubated for 40 minutes at 4°C. Cells were washed twice in flow buffer, and incubated with anti-mouse IgGl (Alexa Fluor 488, BioLegend) for 30 minutes at 4°C. Cells were washed twice with flow buffer and fixed using 1% formaldehyde in PBS. Cells were analyzed on an LSR II flow cytometer (BD
  • Cells were washed x3 in PBS, followed by 20 min incubation in PBS, 0.1% Triton xlOO, and washed x3 in PBS. Cells were initially incubated with anti-LIMSl antibody (1:200 dilution in PBS, 1% BSA, manufacturer’s instructions) for 60 minutes, and washed x2 in PBS. Cells were incubated with anti-mouse IgGl (Alexa Fluor 488) and DAPI for 45 minutes, before washed 4 times in PBS. Cells were attached to a slide using vector shield (Vector Labs) and stored at 4oC. Images were taken using Nikon Al confocal microscope at 600x, images were processed using ImageJ (NIH).
  • Cells were stained with phalloidin (Alexa Fluor 594, Invitrogen) and DAPI (BioLegend) for 45 minutes at room temperature, before washed 4 times in PBS. Cells were attached to a slide using vector shield (Vector Labs) and stored at 4oC. Images were taken on an Olympus 1X73 microscope at 600x, images were processed using ImageJ (NIH).
  • phalloidin Alexa Fluor 594, Invitrogen
  • DAPI BioLegend
  • Cytotoxicity Assay Cell cytotoxicity was measured using the LDH-Cytotoxicity Assay (Abeam) following manufacturer’s instructions. Briefly, HEK-293 cells and HRCE cells and were treated with anti-LIMS 1 antibody (3 pg/ml) or a control antibody (mouse IgG, 3 pg/ml) overnight. Both antibodies were filter washed using sterile PBS before use on the cells. Cell supernatants were removed from the culture plate, centrifuges at 250g to remove cellular debris and used in the LDH-Cytotoxicity Assay. The OD was measured using a Bio-Tek Powerwave XS reader, absorbance of samples was measured at 490 nm and the reference wave length was 630 nm.
  • the primary outcome in genetic association studies was time-to-first-rejection, defined from the date of transplant to the date of allograft biopsy demonstrating a rejection event, including both Antibody Mediated Rejections (ABMR) and/or Acute Cellular Rejections (ACR).
  • ABMR Antibody Mediated Rejections
  • ACR Acute Cellular Rejections
  • the significance of this association surpassed our prespecified Bonferroni- adjusted threshold (alpha level of l.lxlO -3 ).
  • genomic- collision model was superior to recipient-only recessive or additive models (Table 7).
  • the top SNP rs893403, resides on chromosome 2ql2.3 in the intronic portion of LIMS1. This gene encodes a protein that is involved in cell adhesion and integrin signaling found in focal adhesion plaques.
  • the risk allele, rs893403-G is frequent in persons of European and African ancestry but absent in persons of East Asian ancestry and tags a common l.5-kb deletion
  • CNVR915.1 was originally annotated to intersect LOC100288532, a gene in the human reference genome hgl8 that was removed in subsequent releases of the human genome.
  • Our deletion breakpoint mapping and detailed functional annotations of the region indicated that the rs893403-G risk allele was associated with lower messenger RNA (mRNA) expression of LIMS1 and GCC2, the neighboring gene, across multiple GTEx project tissues than was the alternative allele.
  • mRNA messenger RNA
  • rs893403 has a direction-consistent cis-eQTL effect on the LIMS1 mRNA level in the kidney tubulointerstitium. (Details are provided in Table 9.)
  • LIMS1 was strongly expressed in human kidneys and other commonly transplanted organ tissues, such as heart and lung. Within the kidney, LIMS 1 staining is strongest in the distal nephron, including the basolateral surface of distal tubules in the cortex and medulla, the medullary thick ascending limb of the loop of Henle, and medullary collecting ducts. The proximal-to-distal gradient of LIMS 1 expression was consistent with human kidney single nuclear RNA sequencing data and was confirmed by means of RNAscope in situ hybridization.
  • cell-surface LIMS 1 was induced by hypoxia in HEK293 cell lines, a finding consistent with previously reported hypoxia- induced LIMS1 gene expression in cultured endothelial cells. 25
  • GCC2 was detected predominantly in the cytoplasmic compartment of proximal tubules (most strongly in S3) and in kidney vascular smooth muscle. (Details are provided in Table 11.)
  • ProtoArray protein arrays To obtain an unbiased characterization of alloantibody response in kidney recipients with a high-risk genotype, we used ProtoArray protein arrays to screen serum specimens that had been obtained from recipients. These protein arrays capture the human proteome with 9375 immobilized recombinant human proteins. We tested serum specimens that had been obtained from 16 recipients, including 8 persons with rejection (4 recipients with a high-risk genotype and 4 with a low-risk genotype) and 8 controls who had not had rejection. The seroreactivity to proteins was detected with antihuman IgG as an increased intensity normalized to control protein gradients printed on each array.
  • the LIMS 1 protein ranked l4th among the 9375 proteins (0.l5th percentile) on the array according to the mean intensity in the group of recipients with a high-risk genotype who had allograft rejection.
  • No other proteins that were encoded within a l-Mb window of rs893403 showed significant seroreactivity, although the GCC2 protein was not captured on protein arrays.
  • IgG subclass analysis showed that the anti-LIMSl response was predominantly of IgG2 and IgG3 subtype, but weaker IgG4 reactivity was also detected (Figure 3D-3G, and Figure 4).
  • 29 recipients 9%) tested positive by either IgG2 or IgG3 subtype.
  • IgG2 and IgG3 comprise only a small percentage of total IgG (up to 30% and 8%, respectively), and there were no detectable differences between groups in levels of IgGl, the main IgG subclass, which explains why total IgG had overall lower reactivity than these subtypes.
  • kidney tissue eQTL data is not available in Genotype-Tissue Expression (GTEx) project, data supported strong effect of rs893403 on the mRNA levels of LIMS1 and nearby GCC2 gene across multiple tissues. In each case, the risk allele was associated with lower mRNA levels for both genes.
  • GTEx Genotype-Tissue Expression
  • our annotations suggest that rs893403 or another variant in LD, such as rsl0084l99, is associated with gene expression of LIMS1 in human kidney tubules and other organs.
  • MFI Mean Florescent Intensity
  • genomic-collision scenario in which an allograft recipient was homozygous for a deletion polymorphism and received a kidney allograft from a donor who had at least one normal allele.
  • genomic collision at chromosome 2ql2.3 led to a risk of rejection that was nearly 60% higher than the risk among donor-recipient pairs with noncollision genotypes.
  • the risk associated with the collision genotype is equivalent to a mismatch of three of six HLA alleles, which is both clinically significant and potentially modifiable by genetic testing and matching.
  • the genomic collision at chromosome 2ql2.3 would be expected to occur in approximately 12 to 15% of transplants from unrelated donors among persons of European and African ancestry but would be very rare among persons of East Asian ancestry.
  • the collision genotype was associated with the presence of anti-LIMS 1 antibodies.
  • the risk genotype was associated with a lower kidney mRNA level of LIMS1 and that LIMS 1 protein was induced on the cell surface under hypoxic conditions.
  • the recessive model potentially supports a loss-of-function effect, and our data point to LIMS1 as the most likely culprit gene, but the precise causal variant underlying this locus is still unclear.
  • rs893403 also regulates mRNA expression of GCC2, encoding a protein of unclear function.
  • the GCC2 protein is expressed in proximal tubule cells. Further genotype- specific analysis of expression patterns of genes within the LIMS1 locus may be useful, especially in the context of hypoxic injury and other forms of injury.
  • the immune system of a donor who was homozygous for a gene-disrupting deletion may recognize epitopes that are encoded by that gene in the tissues of a recipient, leading to graft-versus-host disease. 22
  • a similar mechanism may also apply to other types of variants that were not examined in this study, such as loss-of-function variants, variants altering the expression of immunogenic proteins, or missense variants that create new immunogenic epitopes.
  • a population-based sequencing study has shown that a large proportion of persons are natural“human gene knockouts” (i.e., have two copies of loss-of-function variants in the same gene) for a number of nonessential genes. 34 Given our hypothesis, we speculate that such persons may be at risk for rejection if they receive an allograft expressing an intact protein.
  • SNV Single Nucleotide Variant
  • SNP Single Nucleotide Polymorphism
  • Copy Number Variant is a phenomenon in which a segment of the genome is either missing (deletion) or is repeated (duplication); the number of copies (segment repeats) can also vary between individuals.
  • CNP Copy Number Polymorphism
  • Deletion Polymorphism refers to a deletion type of CNP; i.e. common deletion of a genomic segment with population frequency greater than 1%.
  • Expression Quantitative Trait Locus a genetic variant that is associated with mRNA expression levels; cis-eQTLs (or local eQTLs) are genetic variants associated with transcript levels of nearby genes; trans-eQTLs (or distant eQTLs) are associated with transcript levels of distant genes (e.g. located on a different chromosome).
  • Table 1 Cox Proportional-Hazards Association Analysis of LIMS1 Collision Genotype with Allograft Rejection in a Time-to-Event Analysis, According to Cohort.
  • the power for the discovery phase was calculated for a range of expected effect sizes (HR 1.50-2.00) and MAFs (from 10% to n
  • This test can be used to detect circulating anti-LIMS 1 antibodies in sera of kidney transplant recipients. Positive test indicates the presence of anti-LIMS 1 humoral response that is associated with kidney allograft rejection risk.
  • Recombinant LIMS1 protein (Abeam abl 16807), Immulon H-2B ELISA plates, Anti-human IgG antibody-HRP (Abeam ab97l60), Anti-human IgGl antibody-Biotin (Sigma B6775), Anti human IgG2 antibody-Biotin (Sigma B3398), Anti-human IgG3 antibody-Biotin (Sigma B3523), Anti-human IgG4 antibody-Biotin (Sigma B3648), Anti-LIMSl antibody-HRP (LSBio
  • 8a Dilute serum to 1:1000 with the blocking buffer, and add 100 pl of the diluted serum to each well. This is done in duplicates or triplicates. Cover plate and incubate at room temperature for 2 hours.
  • 8b Using the mouse derived LIMS1 antibody, generate serial dilutions of this control antibody starting at 1:2000 (through 1: 128,000) leaving the last well blank (no antibody). Add 100 pl of the diluted control antibody to each well, cover plate and incubate at room temperature for 2 hours.
  • 8c Use serum from a minimum of 5 healthy controls, diluted to 1: 1000 with blocking buffer, and add 100 pl of the diluted serum to each well. This is done in duplicates or triplicates. Cover plate and incubate at room temperature for 2 hours.
  • 9 Wash plates ten times (10 x) with 250 m ⁇ of wash buffer (1 x PBS, 0.05% tween), ensure all the liquid is removed from each of the wells.
  • a positive result is calculated by the OD of the sample that is above the average OD of the healthy control serum plus 3 times the standard deviation.
  • Dragun D Miiller DN, Hinrich Brasen J, et al. Angiotensin II type l-receptor activating antibodies in renal-allograft rejection. N Engl J Med 2005; 352: 558-69.
  • Non-HLA antibodies angiotensin II type 1 receptor (anti- AT 1R) and endothelin-l type A receptor (anti-ETAR) are associated with renal allograft injury and graft loss.
  • TransplantLines a prospective cohort study and biobank of solid organ transplant recipients.
  • Genomes Project C Abecasis GR, Auton A, et al. An integrated map of genetic variation from 1,092 human genomes. Nature 2012;491:56-65.
  • SEQ ID NO. 1 (Y: C or T)

Abstract

La présente invention concerne un marqueur génétique pour l'appariement donneur/receveur qui peut diminuer le risque de rejet de greffe. Une collision génomique au niveau d'un nouveau locus d'histocompatibilité codant l'antigène LIMS 1 est associée à un risque plus élevé de rejet de greffe et de production d'anticorps anti-LIMS 1.
PCT/US2019/036999 2018-06-14 2019-06-13 Dosages génétiques et sérologiques pour un appariement amélioré donneur/receveur WO2019241526A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109811061A (zh) * 2019-02-20 2019-05-28 新疆农业大学 Coil基因特异snp标记、和田乔达红羊产羔数性状的检测方法及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150337361A1 (en) * 2009-11-06 2015-11-26 The Board Of Trustees Of The Leland Stanford Junior University Non-Invasive Diagnosis of Graft Rejection in Organ Transplant Patients
WO2017079736A1 (fr) * 2015-11-06 2017-05-11 The Trustees Of Columbia University In The City Of New York Profilage de protéines exosomales pour la détection du rejet de greffe cardiaque

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150337361A1 (en) * 2009-11-06 2015-11-26 The Board Of Trustees Of The Leland Stanford Junior University Non-Invasive Diagnosis of Graft Rejection in Organ Transplant Patients
WO2017079736A1 (fr) * 2015-11-06 2017-05-11 The Trustees Of Columbia University In The City Of New York Profilage de protéines exosomales pour la détection du rejet de greffe cardiaque

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GOH, SK ET AL.: "Probe-Free Digital PCR Quantitative Methodology to Measure Donor-Specific Cell -Free DNA after Solid-Organ Transplantation", CLINICAL CHEMISTRY, vol. 63, no. 3, 18 January 2017 (2017-01-18), pages 742 - 750, XP009502842, DOI: 10.1373/clinchem.2016.264838 *
STEERS, NJ ET AL.: "Genomic Mismatch at LIMS1 Locus and Kidney Allograft Rejection", THE NEW ENGLAND JOURNAL OF MEDICINE, vol. 380, no. 20, 16 May 2019 (2019-05-16), pages 1918 - 1928, XP055664330 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109811061A (zh) * 2019-02-20 2019-05-28 新疆农业大学 Coil基因特异snp标记、和田乔达红羊产羔数性状的检测方法及其应用
CN109811061B (zh) * 2019-02-20 2023-05-09 新疆农业大学 Coil基因特异snp标记、和田乔达红羊产羔数性状的检测方法及其应用

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