Methods for assessment and treatment of relapse of antibody-mediated allograft rejection

Abstract

Disclosed herein includes a method for treating antibody-mediated kidney allograft rejection, comprising treating a subject determined to suffer from antibody-mediated kidney allograft rejection more than 6 months after transplantation by administering an anti-CD38 antibody or antigen-binding fragment thereof (e.g., daratumumab, felzartamab, or isatuximab) to the subject; extracting cell-free DNA from a blood, plasma, serum or urine sample collected from the subject after conclusion of the treatment; quantifying an amount of donor-derived cell-free DNA, a percentage of donor-derived cell-free DNA out of total cell-free DNA, or both, in the extracted DNA; and retreating the subject with an anti-CD38 antibody or antigen-binding fragment thereof if the amount of donor-derived cell-free DNA, the percentage of donor-derived cell-free DNA out of total cell-free DNA, or both, exceed one or more threshold values.

Description

N.064.W0.01METHODS FOR ASSESSMENT AND TREATMENT OF RELAPSE OF ANTIBODY- MEDIATED ALLOGRAFT REJECTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 735,457, filed December 18, 2024, the contents of which are hereby incorporated by reference in its entirety.BACKGROUND

[0002] Antibody-mediated rejection (AB MR), including chronic active ABMR (cABMR), remains the leading cause of allograft loss in kidney transplant recipients. Daratumumab and felzartamab are humanized IgGl antibodies that target CD38 and show promise for the treatment of cABMR. See Kwun et al., Journal of the American Society of Nephrology 30(7): 1206 (2019); Spica et al.. Case Reports in Nephrology and Dialysis 9(3): 149 (2020); Siisal et al., SAGE Open Medical Case Reports 11:2050313X231211050 (2023); Doberer et al., Transplantation 105(2):451 (2021). However, transplant recipients successfully treated for an episode of ABMR may later relapse with ABMR, posing a renewed risk of allograft loss.

[0003] There remains a need for techniques and methods to noninvasively detect ABMR relapse and provide timely retreatment to transplant recipients having ABMR relapse.SUMMARY

[0004] In one aspect, the present disclosure provides a method for treating antibody-mediated kidney allograft rejection, comprising treating a subject determined to suffer from antibody- mediated kidney allograft rejection more than 6 months after transplantation by administering an anti-CD38 monoclonal antibody or an antigen-binding fragment thereof to the subject; extracting cell-free DNA from a blood, plasma, serum or urine sample collected from the subject after conclusion of the treatment; quantifying an amount of donor-derived cell-free DNA, a percentage of donor-derived cell-free DNA out of total cell-free DNA, or both, in the extracted DNA; and retreating the subject with the anti-CD38 monoclonal antibody or an antigen-binding fragment thereof if the amount of donor-derived cell-free DNA, the percentage of donor-derived cell-free DNA out of total cell-free DNA, or both, exceed one or more threshold values.4926-4669-9902.1N.064.W0.01

[0005] In some embodiments, the subject was determined to suffer from antibody-mediated kidney allograft rejection using the Banff 2019 Classification. In some embodiments, the subject was determined to suffer from antibody-mediated kidney allograft rejection by a histological biopsy of renal tissue.

[0006] In some embodiments, the subject was determined to suffer from antibody-mediated kidney allograft rejection by quantifying an amount of donor-derived cell-free DNA, a percentage of donor-derived cell-free DNA out of total cell-free DNA, or both, in a blood, plasma, serum or urine sample collected from the subject before the treatment.

[0007] In some embodiments, the subject was determined to suffer from antibody-mediated kidney allograft rejection by quantifying a percentage of donor-derived cell-free DNA out of total cell-free DNA of >1.0%, in a blood, plasma, serum or urine sample collected from the subject before the treatment. In some embodiments, the subject was determined to suffer from antibody-mediated kidney allograft rejection by quantifying an amount of donor-derived cell-free DNA of >78 cp / mL, in a blood, plasma, serum or urine sample collected from the subject before the treatment. In some embodiments, the subject was determined to suffer from antibody- mediated kidney allograft rejection by quantifying a percentage of donor-derived cell-free DNA out of total cell-free DNA of >1.0%, or an amount of donor-derived cell-free DNA of >78 cp / mL, in a blood, plasma, serum or urine sample collected from the subject before the treatment.

[0008] In some embodiments, administering the anti-CD38 monoclonal antibody or an antigenbinding fragment thereof comprises administering a dose of the anti-CD38 monoclonal antibody or an antigen-binding fragment thereof once per week for four weeks and at 4, 7, and 10 months after the first administration; or wherein administering the anti-CD38 monoclonal antibody or an antigen-binding fragment thereof comprises administering a dose of the anti-CD38 monoclonal antibody or an antigen-binding fragment thereof once per week for four weeks and at 3, 6, and 9 months after the first administration.

[0009] In some embodiments, the anti-CD38 monoclonal antibody or an antigen-binding fragment thereof is administered subcutaneously or intravenously. In some embodiments, the24926-4669-9902.1N.064.W0.01 anti-CD38 monoclonal antibody or an antigen-binding fragment thereof comprises daratumumab, felzartamab, or isatuximab.

[0010] In some embodiments, the blood, plasma, serum or urine sample is collected from the subject between 9 months and 15 months after conclusion of the treatment.

[0011] In some embodiments, the quantifying step comprises performing targeted multiplex amplification of the extracted DNA or its derivative to amplify 100 to 20,000 different polymorphic target loci together in the same reaction volume using 100 to 20,000 different target-specific primers, and performing high-throughput sequencing on the amplicons to generate sequence reads.

[0012] In some embodiments, the quantifying step comprises: a) adding a Tracer DNA to extracted DNA or its derivative to obtain a mixed composition; b) performing targeted multiplex amplification on the mixed composition comprising the extracted DNA or its derivative and the Tracer DNA at 100 to 20,000 different polymorphic target loci together in the same reaction volume using 100 to 20,000 different target-specific primers; c) sequencing the amplicons by high-throughput sequencing to generate sequence reads; and d) quantifying an amount of donor- derived cell-free DNA and an amount of total cell-free DNA from the sequence reads, wherein the amount of total cell-free DNA is quantified using sequence reads derived from the Tracer DNA.

[0013] In some embodiments, the method comprises retreating the subject with the anti-CD38 monoclonal antibody or an antigen-binding fragment thereof when the percentage of donor- derived cell-free DNA out of total cell-free DNA exceeds about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, or any percentage therebetween. In some embodiments, the method comprises retreating the subject with the anti-CD38 monoclonal antibody or an antigen-binding fragment thereof when the percentage of donor-derived cell-free DNA out of total cell-free DNA exceeds 0.50%, 0.51%. 0.52%, 0.53%. 0.54%, 0.55%, 0.56%, 0.57%. 0.58%, 0.59%, 0.60%, 0.61%. 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.70%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.80%, 0.81%, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%. 0.89%. 0.90%, 0.91%, 0.92%, 0.93%. 0.94%, 0.95%, 0.96%, 0.97%. 0.98%,34926-4669-9902.1N.064.W0.010.99%, 1.0%, 1.01 %, 1.02%, 1.03%, 1.04%, 1.05%, 1.06%, 1.07%, 1.08%, 1.09%, 1.10%, 1.11%, 1.12%, 1.13%, 1.14%, 1.15%, 1.16%, 1.17%, 1.18%, 1.19%, 1.20%, 1.21%, 1.22%,1.23%, 1.24%, 1.25%. 1.26%, 1.27%, 1.28%, 1.29%. 1.30%, 1.31%, 1.32%, 1.33%. 1.34%.1.35%, 1.36%, 1.37%, 1.38%, 1.39%, 1.40%, 1.41%, 1.42%, 1.43%, 1.44%, 1.45%, 1.46%,1.47%, 1.48%, 1.49%, 1.50%, or any percentage therebetween.

[0014] In some embodiments, the method comprises retreating the subject with the anti-CD38 monoclonal antibody or an antigen-binding fragment thereof when the amount of donor-derived cell-free DNA (e.g„ genomic copies / mL) exceeds about 10 cp / mL, about 20 cp / mL, about 30 cp / mL, about 40 cp / mL, about 50 cp / mL, about 60 cp / mL, about 70 cp / mL, about 80 cp / mL, about 90 cp / mL, about 100 cp / mL, about 110 cp / mL, about 120 cp / mL, about 130 cp / mL, about 140 cp / mL, about 150 cp / mL, or any amount therebetween. In some embodiment, the method comprises retreating the subject with the anti-CD38 monoclonal antibody or an antigen-binding fragment thereof when the amount of donor-derived cell-free DNA exceeds 50 cp / mL, 51 cp / mL, 52 cp / mL, 53 cp / mL, 54 cp / mL, 55 cp / mL, 56 cp / mL, 57 cp / mL, 58 cp / mL, 59 cp / mL, 60 cp / mL, 61 cp / mL, 62 cp / mL, 63 cp / mL, 64 cp / mL, 65 cp / mL, 66 cp / mL, 67 cp / mL, 68 cp / mL, 69 cp / mL, 70 cp / mL, 71 cp / mL, 72 cp / mL, 73 cp / mL, 74 cp / mL, 75 cp / mL, 76 cp / mL, 77 cp / mL, 78 cp / mL, 79 cp / mL, 80 cp / mL, 81 cp / mL, 82 cp / mL, 83 cp / mL, 84 cp / mL, 85 cp / mL, 86 cp / mL, 87 cp / mL, 88 cp / mL, 89 cp / mL, 90 cp / mL, 91 cp / mL, 92 cp / mL, 93 cp / mL, 94 cp / mL, 95 cp / mL, 96 cp / mL, 97 cp / mL, 98 cp / mL, 99 cp / mL, or 100 cp / mL, or any amount therebetween.

[0015] In some embodiments, the method comprises retreating the subject with the anti-CD38 monoclonal antibody or an antigen-binding fragment thereof when the percentage of donor- derived cell-free DNA out of total cell-free DNA, the amount of donor-derived cell-free DNA, or both, exceed one or more abovementioned thresholds.

[0016] In another aspect, the present disclosure provides a method for identifying a subject who is susceptible to relapse from antibody-mediated kidney allograft rejection, wherein the subject has received treatment with an anti-CD38 antibody or antigen-binding fragment thereof, comprising: extracting cell-free DNA from a blood, plasma, serum or urine sample collected from the subject after conclusion of the treatment; quantifying an amount of donor-derived cell-free DNA, a percentage of donor-derived cell-free DNA out of total cell-free DNA, or both, in the44926-4669-9902.1N.064.W0.01 extracted DNA; and identifying the subject as susceptible to relapse from antibody-mediated kidney allograft rejection if the amount of donor-derived cell-free DNA, the percentage of donor- derived cell-free DNA out of total cell-free DNA, or both, exceed one or more threshold values.

[0017] In some embodiments, the anti-CD38 antibody or antigen-binding fragment thereof is daratumumab, felzartamab, or isatuximab.

[0018] In some embodiments, the method comprises identifying the subject as susceptible to relapse from antibody-mediated kidney allograft rejection when the percentage of donor-derived cell-free DNA out of total cell-free DNA exceeds about 0.5%, about 0.6%, about 0.7%, about 0.8%. about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, or any percentage therebetween. In some embodiments, the method comprises identifying the subject as susceptible to relapse from antibody-mediated kidney allograft rejection when the percentage of donor-derived cell-free DNA out of total cell-free DNA exceeds 0.50%. 0.51%. 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59%, 0.60%, 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%. 0.67%, 0.68%, 0.69%, 0.70%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%. 0.76%, 0.77%, 0.78%, 0.79%, 0.80%, 0.81%, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.90%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, 1.0%, 1.0%, 1.01%, 1.02%, 1.03%, 1.04%, 1.05%, 1.06%, 1.07%, 1.08%, 1.09%, 1.10%, 1.11%. 1.12%, 1.13%, 1.14%, 1.15%, 1.16%, 1.17%, 1.18%, 1.19%, 1.20%, 1.21 %, 1.22%, 1.23%,1.24%, 1.25%, 1.26%, 1.27%, 1.28%, 1.29%, 1.30%, 1.31%, 1.32%, 1.33%, 1.34%, 1.35%,1.36%, 1.37%, 1.38%. 1.39%, 1.40%, 1.41%, 1.42%. 1.43%, 1.44%, 1.45%, 1.46%. 1.47%.1.48%, 1.49%, 1.50%, or any percentage therebetween. Can I - get

[0019] In some embodiments, the method comprises identifying the subject as susceptible to relapse from antibody-mediated kidney allograft rejection when the amount of donor-derived cell-free DNA (e.g., genomic copies / mL) exceeds about 10 cp / mL, about 20 cp / mL, about 30 cp / mL, about 40 cp / mL, about 50 cp / mL, about 60 cp / mL, about 70 cp / mL, about 80 cp / mL, about 90 cp / mL, about 100 cp / mL, about 110 cp / mL, about 120 cp / mL, about 130 cp / mL, about 140 cp / mL, about 150 cp / mL, or any amount therebetween. In some embodiment, the method comprises identifying the subject as susceptible to relapse from antibody-mediated kidney allograft rejection when the amount of donor-derived cell-free DNA exceeds 50 cp / mL, 5154926-4669-9902.1N.064.W0.01 cp / mL, 52 cp / mL, 53 cp / mL, 54 cp / mL, 55 cp / mL, 56 cp / mL, 57 cp / mL, 58 cp / mL, 59 cp / mL, 60 cp / mL, 61 cp / mL, 62 cp / mL, 63 cp / mL, 64 cp / mL, 65 cp / mL, 66 cp / mL, 67 cp / mL, 68 cp / mL, 69 cp / mL, 70 cp / mL, 71 cp / mL, 72 cp / mL, 73 cp / mL, 74 cp / mL, 75 cp / mL, 76 cp / mL, 77 cp / mL, 78 cp / mL, 79 cp / mL, 80 cp / mL, 81 cp / mL, 82 cp / mL, 83 cp / mL, 84 cp / mL, 85 cp / mL, 86 cp / mL, 87 cp / mL, 88 cp / mL, 89 cp / mL, 90 cp / mL, 91 cp / mL, 92 cp / mL, 93 cp / mL, 94 cp / mL, 95 cp / mL, 96 cp / mL, 97 cp / mL, 98 cp / mL, 99 cp / mL, or 100 cp / mL, or any amount therebetween.

[0020] In some embodiments, the method comprises identifying the subject as susceptible to relapse from antibody-mediated kidney allograft rejection when the percentage of donor-derived cell-free DNA out of total cell-free DNA, the amount of donor-derived cell-free DNA, or both, exceed one or more abovementioned thresholds.

[0021] In some embodiments, the kidney transplant is from a human.

[0022] In some embodiments, the kidney transplant is a xenotransplant, optionally wherein the kidney transplant from a pig.

[0023] In some embodiments, the method is performed without prior knowledge of donor or recipient genotypes.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 shows a timeline for daratumumab treatment and data collection.

[0025] FIG. 2 shows graphs quantifying molecular histology active antibody-mediated rejection score (FIG. 2A), plasma dd-cfDNA fraction (FIG 2B), estimated glomerular filtration rate (eGFR; mL / min / 1.73m2; FIG. 2C), and urine albumin to creatine ratio (mg / g; FIG. 2D), before and after daratumumab treatment.

[0026] FIG. 3 shows graphs of daratumumab treatments and dd-cfDNA fraction over time for two kidney transplant recipients suffering from chronic active antibody-mediated rejection relapse.DETAILED DESCRIPTION64926-4669-9902.1N.064.W0.01

[0027] The present disclosure is generally directed to improved tests for assessing allograft injury including antibody-mediated rejection (ABMR) and chronic active antibody-mediated rejection (cABMR) relapse in kidney transplant recipients. The improved tests described herein leverage doner-derived cell-free DNA (dd-cfDNA; e.g., Prospera™ test) to enhance the precision of detecting ABMR relapse.

[0028] WO 2021 / 243045 titled “Methods for detection of donor-derived cell-free DNA” and filed May 27, 2021 as PCT / US2021 / 034561, and WO 2020 / 010255 titled “Methods for detection of donor-derived cell-free DNA” and filed August 3, 2019 as PCT / US 2019 / 040603, are hereby incorporated by reference in their entirety.

[0029] Transplant Recipient and Kidney Transplant

[0030] In some embodiments of the methods described herein, the kidney transplant recipient is a human subject. The kidney transplant recipient may have received, for example, a single kidney transplantation. The kidney transplant recipient may have received, for example, a double kidney transplantation.

[0031] In some embodiments, the kidney transplant is from a human. In some embodiments, the kidney transplant has been treated or reconditioned by ex vivo kidney perfusion prior to the transplantation.

[0032] In some embodiments, the kidney transplant is a xenotransplant. In some embodiments, the kidney transplant is from a pig, a primate, a baboon, a cow, or a dog. In some embodiments, the kidney transplant is from a genetically engineered pig. In some embodiments, the genetically engineered pig has been engineered to knockout or inactivate Porcine endogenous retrovirus (PERVs). In some embodiments, the genetically engineered pig has been engineered to knockout or inactivate GGTA1, CM AH, 04GalNT2, and / or GHR. In some embodiments, the genetically engineered pig has been engineered to express DAF, TBM, EPCR, HOI, CD46, and / or CD47.

[0033] Extraction of cfDNA

[0034] In some embodiments, the methods described herein comprise collecting a liquid sample (e.g., blood, plasma, serum or urine) from a kidney transplant recipient, wherein the kidney74926-4669-9902.1N.064.W0.01 transplant recipient was determined to suffer from antibody-mediated kidney allograft rejection more than 6 months after transplantation, and wherein the kidney transplant recipient has already completed treatment with an anti-CD38 antibody or antigen-binding fragment thereof (e.g., daratumumab, felzartamab, or isatuximab). In some embodiments, the methods described herein comprise collecting a liquid sample from the kidney transplant recipient between about 9 months and about 24 months, or between about 12 months and about 18 months, or between about 9 months and about 15 months after conclusion of the treatment with the anti-CD38 antibody or antigen-binding fragment thereof (e.g.. daratumumab, felzartamab, or isatuximab). In some embodiments, the methods described herein comprise collecting a liquid sample from the kidney transplant recipient about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, or about 24 months, after conclusion of the treatment with the anti-CD38 antibody or antigen-binding fragment thereof (e.g.. daratumumab, felzartamab, or isatuximab).

[0035] In some embodiments, the methods described herein comprise extracting cell-free DNA from a liquid sample or portion thereof of a kidney transplant recipient, wherein the extracted cell-free DNA comprises a mixture of donor-derived cell-free DNA and recipient-derived cell- free DNA. Methods that are particularly useful in exemplary embodiments include methods for isolating circulating free DNA (cfDNA) from a liquid sample, and in illustrative embodiments from a blood, serum, plasma, or urine, and in further illustrative embodiments, a plasma sample.

[0036] In certain illustrative embodiments, isolation of cfDNA from a liquid (e.g., blood or blood derivative sample such as a plasma sample) can involve binding DNA molecules from a sample to a matrix and isolating the DNA molecules in the presence of a solvent. In some embodiments, the method further comprises incubating the biological sample comprising DNA molecules with a protease, prior to contacting the DNA molecules to the matrix. In some embodiments, the method can further include the steps of washing the matrix with a wash buffer to remove impurities, and optionally, drying the matrix. Enriched nucleic acid samples can be eluted from the matrix with an elution buffer.84926-4669-9902.1N.064.W0.01

[0037] Other methods for nucleic acid isolation, for example cfDNA isolation, and optional enrichment of certain cfDNA can include ion exchange columns, or microfluidic devices, such as solid phase isolation, based on DNA capture by immobilized beads or functionalized surface. Additional methods include liquid phase isolation, utilizing an electric field, or chemical reagents, instead of a functionalized surface. In illustrative embodiments herein, isolation of cfDNA from a patient sample is performed using a DNA isolation kit (e.g.. QIAamp Circulating Nucleic Acid kit (Qiagen)).

[0038] In some embodiments, cfDNA or their derivatives of certain sizes can be enriched before or after subjecting the cfDNA to methods herein. In some embodiments, size selection can be performed before the sequencing library preparation. In some embodiments, size selection can be performed after the sequencing library preparation and before sequencing. In some embodiments, size selection is performed on a sequencing-ready pool. Enriched cfDNA molecules can be, for example 50 to 1200 base pairs in length, 70 to 500 base pairs in length, 100 to 200 base pairs in length, or 130 to 170 base pairs in length. In some embodiments, the enriched cfDNA molecules are from 50 to 200 bp in length. In some embodiments, the enriched cfDNA molecules are between 60 and 200 bp in length, between 60 and 150 bp in length, or between 80 and 140 bp in length, in length before the enriched cfDNA molecules, or derivatives thereof, are ligated to adapters in methods herein. In some embodiments, the enriched cfDNA molecules are about or less than 200, 175, 166, 150, 100 bp in length before they are ligated to adapters. Such enrichment methods can be performed for example using the methods described in WO2018 / 156418 and WO2019161244, each of which is incorporated herein by reference in its entirety.

[0039] Preparation of Sequencing Library

[0040] In some embodiments, the method described herein comprises preparing a sequencing library from the extracted cell-free DNA or its derivative. The preparation of the sequencing library can comprise, for example, appending an adapter to the extracted cell-free DNA or its derivative. The preparation of the sequencing library can comprise, for example, performing a universal amplification on the extracted cell-free DNA or its derivative. The preparation of the sequencing library can comprise, for example, performing targeted enrichment on the extracted94926-4669-9902.1N.064.W0.01 cell-free DNA or its derivative to enrich a plurality of polymorphic target loci, preferably a plurality of SNP loci.

[0041] Library Preparation

[0042] In some embodiments, the method further comprises appending an adapter to the extracted cfDNA or derivative thereof and generating adapted DNA before performing targeted enrichment (e.g.. with a panel of oligonucleotide probes). In some embodiments, the adapter is a Y-adapter. In some embodiments, the adapter comprises a universal priming site.

[0043] In some embodiments, the adapter comprises a molecular barcode or index sequence, wherein sequence reads generated from the high-throughput sequencing can be grouped together using the molecular barcode or index sequence. In some embodiments, the adapters do not comprise a molecular barcode or index sequence, wherein sequence reads generated from the high-throughput sequencing can be grouped together using the fragment-end sequence of the extracted cell-free DNA or DNA derived therefrom. In some embodiments, the sequence reads that are grouped together using the molecular barcode or index sequence and / or the fragment-end sequence can be subject to error correction to correct sequencing errors and generate a consensus sequence.

[0044] In some embodiments, the method further comprises amplifying the adapted DNA using a primer that binds to the universal primer binding site and generating adapted-amplified DNA before performing targeted enrichment (e.g., with a panel of oligonucleotide probes). In some embodiments, the adapted-amplified DNA further comprises a sequencing adapter sequence or sequencing primer binding site for high-throughput sequencing. In some embodiments, the adapted-amplified DNA further comprises a sample barcode or index sequence, which allows multiplexed sequencing of pooled sequencing libraries (e.g., multiplexed sequencing of sequencing libraries generated from multiple samples).

[0045] Typically, methods herein include a step of appending nucleic acid adapters to extracted cfDNA molecules or to nucleic acid derivatives generated therefrom. For example, adapters may be appended on to the DNA molecules by ligation. The extracted cfDNA molecules in illustrative embodiments are extracted from a sample of a subject. In some embodiments,104926-4669-9902.1N.064.W0.01 appending nucleic acid adapters is performed after the extracted cfDNA molecules are fragmented to form fragmented DNA molecules. Typically, methods include exposing the extracted cfDNA molecules to one or more polymerases or kinases, such as Klenow Large Fragment Polymerase and T4 polynucleotide kinase (PNK), as well as a ligase, such as T4 ligase. In some embodiments, the extracted cfDNA molecules or the fragmented DNA molecules are exposed to one or more polymerases and / or kinases to generate the nucleic acid derivatives. In some embodiments, the method further comprises appending adapters to the nucleic acid derivatives generated therefrom. In some embodiments, the extracted cfDNA molecules are not fragmented prior to appending nucleic acid adapters thereto.

[0046] In some embodiments, adapters are ligated to the extracted cfDNA molecules. In some embodiments, before such ligation, the extracted cfDNA molecules can be modified to form sample nucleic acid derivatives, for example to make them more amenable to adapter ligation. For example, extracted cfDNA molecules can be blunt ended, nucleotides can be added to the extracted cfDNA molecules or blunted-ended derivative therefrom, and / or phosphate moieties can be added or removed from the ends of DNA molecules or derivatives thereof. In some embodiments, prior to ligation, the extracted cfDNA molecules may be blunt ended, and then a single adenosine base can be added to the 3’ end. In some embodiments, prior to ligation the DNA may be cleaved using a restriction enzyme or some other cleavage method. In some embodiments, during ligation the 3’ adenosine of the DNA fragments and the complementary 3’ thymidine overhang of an adapter can enhance ligation efficiency. In some embodiments, adapter ligation is performed using a T4 ligase.

[0047] In some embodiments, adapters containing one or more universal priming sequences are utilized in methods herein. In some embodiments, the adapters are Y adapters, for example in methods involving NGS sequencing. In some embodiments, the adapters each comprises a universal priming site. In some embodiments, the adapter may comprise a barcode or index sequence. Thus, multiple samples can be analyzed in the same sequencing reaction. The sample barcode or index sequence can be used to process data according to the sample from which the data was generated.114926-4669-9902.1N.064.W0.01

[0048] In some embodiments, the adapter may comprise a molecular barcode or index sequence. In some embodiments, the number of adapters having different molecular barcode or index sequences is between 10 to 1.000, and wherein the ratio of the total number of template nucleic acid or cfDNA molecules to the number of different molecular barcode or index sequences in the ligation reaction is at least 1,000:1. The number of different molecular barcode or index sequences in the ligation reaction, in certain embodiments, ranges fromlO to 50, 10 to 100, 50 to 200, 100 to 300, 200 to 500, 300 to 600, 500 to 700, 600 to 800 or 700 to 1,000. In some embodiments, there are at least 1. 10, 20, 30, 40, 50, or at least 100; 200, 300, 400, 500, 600, 700, 800, 900, or 1000 different molecular barcode or index sequences in the ligation reaction. In some embodiments, the ratio of the total number of template nucleic acid or cfDNA molecules to the number of different molecular barcode or index sequences in the ligation reaction is at least 10,000:1. In some embodiments, the ratio of the total number of template nucleic acid or cfDNA molecules to the number of different molecular barcode or index sequences in the ligation reaction ranges from 50,000: 1 to 50:1, from 25,000: 1 to 100:1. from 10.000:1 to 100:1, from 10:000:1 to 8,000:1 to 500:1, from 5,000:1 to 200:1, from 10,000:1 to 50:1. In some embodiments, the methods disclosed herein result in at least 100; 200; 500; 750; 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000 different molecular barcode or index sequences to each one template nucleic acid or cfDNA molecules.

[0049] Exemplary library preparation protocols are provided in Abbosh et al., Nature 545:446- 451 (2017); and Sigdel et al., J. Clin. Med. 8(1): 19 (2019), each of which is incorporated herein by reference in its entirety.

[0050] Targeted Enrichment

[0051] In some embodiments, the method described herein comprises performing targeted enrichment on the extracted cell-free DNA or DNA derived therefrom to enrich a plurality of polymorphic loci (e.g., SNP loci), wherein at least some of said polymorphic loci can distinguish donor-derived cfDNA (dd-cfDNA) from recipient-derived cfDNA (rd-cfDNA). In some embodiments, the percentage of dd-cfDNA out of total cfDNA in the plasma fraction of the blood sample of the transplant recipient can be estimated using sequence reads at the plurality of enriched polymorphic or SNP loci.124926-4669-9902.1N.064.W0.01

[0052] The plurality of polymorphic or SNP loci being enriched can comprise, for example, at least 100 polymorphic or SNP loci, at least 200 polymorphic or SNP loci, at least 500 polymorphic or SNP loci, at least 1,000 polymorphic or SNP loci, at least 2,000 polymorphic or SNP loci, between 100 and 20,000 polymorphic or SNP loci, between 100 and 500 polymorphic or SNP loci, between 500 and 2,000 polymorphic or SNP loci, or between 2,000 and 20,000 polymorphic or SNP loci. Exemplary lists of polymorphic or SNP loci include those targeted by an exemplary 1,200-plex primer library, an exemplary 2,686-plex primer library, and an exemplary 10,984-plex primer library disclosed in U.S. Pat. No. 9,677,118 including sequence listing thereof, which is incorporated herein by reference in its entirety.

[0053] In some embodiments, the targeted enrichment enriches a plurality of polymorphic or SNP loci each having a minor allele frequency (MAF) of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10%. At an average MAF of 5%, 1,000 polymorphic or SNP loci may translate to 50 informative SNPs.

[0054] In some embodiments, the targeted enrichment comprises preforming targeted multiplex amplification to enrich the polymorphic loci. In some embodiments, the targeted enrichment comprises preforming targeted probe capture to enrich the polymorphic loci. In some embodiments, the targeted enrichment comprises preforming linked target capture to enrich the polymorphic loci. Exemplary target enrichment protocols based on multiplex PCR amplification are provided in Zimmermann et al., Prenat. Diagn. 32:1233-1241 (2012); and Sigdel et al., J. Clin. Med. 8(1): 19 (2019), each of which is incorporated herein by reference in its entirety.

[0055] Targeted amplification

[0056] In some embodiments, the targeted enrichment technique can involve targeted multiplex amplification (e.g., PCR or isothermal amplification). Methods in some aspects herein include performing one or, in some embodiments, two or more amplifications. Such amplifications in certain illustrative embodiments include at least one targeted amplification wherein at least one primer and in certain embodiments both primers of a primer pair, one or more primer pairs, or a set of primer pairs used for the amplification are each designed to bind to a specific nucleic acid sequence at or near, typically within, a genomic region of interest comprising a polymorphic134926-4669-9902.1N.064.W0.01 target loci (i.e. are target- specific primers) to generate target amplicons. Tn some embodiments, methods herein include one or more universal amplifications.

[0057] A number of amplification technologies can be used with methods herein. For example, such amplification can be an isothermal amplification (e.g., recombinase polymerase amplification (RPA) (Kersting et al. 2014 Microchim Acta 181 (13-14), 1715-1723, (incorporated by reference in its entirety)), a ligase-based amplification, PCR, or a combination thereof (e.g., ligation-mediated PCR). In some illustrative embodiments, the targeted amplification is a targeted PCR(s) that is performed using a PCR reaction mixture that includes one primer pair, or in illustrative embodiments a set of primer pairs, and at least a portion of the library of DNA molecules comprising the extracted cfDNA or DNA derived therefrom (e.g., adapted DNA, adapted-amplified DNA).

[0058] Typically, at least one primer of a primer pair used for targeted amplification herein, is a target- specific primer designed to bind to a specific nucleic acid sequence in or near, typically within, a genomic region of interest comprising the polymorphic target loci. A target- specific primer can be designed to bind to any sequence within or near a polymorphic target loci for amplification of the polymorphic target loci. One of the advantages of the methods described herein is increased flexibility in primer / probe design for targeted amplification or enrichment. In some embodiments, one primer of the one or more primer pairs or the set of primer pairs in the reaction mixture used for a targeted amplification is a universal primer and binds to a primer binding site on an adapter. Thus, for example, in such embodiments a universal primer that binds an adapter primer binding site can be used for an amplification reaction along with a targetspecific primer that binds a primer binding site on a sample DNA region.

[0059] Target-specific primers typically define the ends of target amplicons, which typically encompass at least a portion of the polymorphic target loci. In some embodiments, a PCR can be performed using two target-specific primers. The target amplicon in such embodiment would extend from the sample DNA region bound by target- specific primer on a 5’ end to the sample DNA region bound by primer on the 3’ end. In some embodiments, a PCR can be performed using a universal primer and a target-specific primer. The target amplicon in such embodiment144926-4669-9902.1N.064.W0.01 would extend from the sample DNA region bound by target-specific primer on a 3’ end of one strand to the end of the sample DNA fragment on the 5’ end of that strand.

[0060] In some methods herein, a universal amplification of the library of DNA molecules comprising the extracted cfDNA or DNA derived therefrom can be performed before the targeted amplification. Such universal amplification can be performed for example using a universal primer pair that binds primer binding sites in the adapter. Thus, in some embodiments, the methods herein include performing a universal PCR using the adapted DNA molecules, and a universal PCR primer pair comprising primers designed to bind universal primer binding sequences on the adapters, before performing one or more targeted PCRs.

[0061] The one or more primer pairs in illustrative embodiments is a set of primer pairs. In some embodiments, the set of primer pairs is a set of between 20 and 100,000, between 50 and 50,000, between 100 and 20,000, between 100 and 500. between 500 and 2,000, or between 2,000 and 20,000 primer pairs.

[0062] In some embodiments, at least one of the primer pairs comprises a universal primer and a target- specific primer. In some embodiments, at least one of the primer pairs comprises two target- specific primers. In some embodiments, at least one of the primers comprises a sequencing tag. In some embodiments, at least one of the primers comprises a sample index. In some embodiments, at least one of the primers comprises biotin modification. In some embodiments, performing a PCR further comprises using primers comprising a sequencing tag. In some embodiments, performing a PCR further comprises using primers comprising a sample index. In some embodiments, the primers of the primer pairs are probe-dependent primers and the amplification is a target capture polymerase chain reaction.

[0063] In some embodiments, one or both of the primer binding sites of a primer pair can include at least a portion of one of the adapter sequences. In some embodiments, one of the primer binding sites of a primer pair can include at least a portion of the adapter sequences. In some embodiments, both of the primer binding sites of a primer pair can include at least a portion of the adapter sequences. In some embodiments, neither of the primer binding sites of a primer pair include any of the adapter sequences.154926-4669-9902.1N.064.W0.01

[0064] Methods as described herein, in some embodiments, can include multiple amplification cycles (e.g., multiple PCR temperature cycles), and in some embodiments can include several sequential PCR reactions performed during the same set of temperature cycles. In some embodiments, amplification cycles can include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cycles. In some embodiments, amplification cycles can include at least 7, 8, 9, or 10 cycles. In illustrative embodiments, amplification cycles can include at least 11, 12, 13, 14, 15, 16, or 17 cycles.

[0065] Typically, in embodiments described herein, PCR amplification is performed by adding a PCR reaction mixture to the DNA template followed by addition of a polymerase enzyme, and then amplified through multiple amplification cycles. In some embodiments, the PCR reaction mixture contains one or more primer pairs, deoxynucleotides (dNTPs), PCR reaction buffer, and deionized water. In some embodiments, the dNTPs can comprise a mixture of dATP, dCTP, dGTP and dTTP. In some embodiments, the final concentration of each dNTP in the reaction mixture can range from 0.05 mM to 0.5 mM dNTPs, for example, from 0.05 mM to 0.5 mM, 0.05 mM to 0.1 mM, 0.05 mM to 0.15 mM, 0.05 to 0.2 mM, 0.05 mM to 0.25 mM, 0.05 mM to 0.3 mM, 0.05 mM to 0.35 mM, 0.05 to 0.4 mM 0.05 mM to 0.45 mM, from .1 mM to 0.5 mM, 0.15 mM to 0.5 mM, or 0.2 mM to 0.5 mM, 0.25 to 0.5 mM, 0.3 mM to 0.5 mM, 0.35 mM to 0.5 mM, 0.4 mM 0.5 mM, or from 0.45 mM to 0.5 mM. In illustrative embodiments, the final concentration of each dNTP in the reaction mixture is between 0.15 mM and 0.25 mM. In some embodiments, the final concentration of each dNTP in the reaction mixture is 0.2 mM.

[0066] PCR buffer solution creates a suitable environment for the polymerase chain reaction and can contain many different components, including magnesium chloride (MgC12), potassium chloride (K.C1), dimethyl sulfoxide (DMSO), and glycerin or bovine serum albumin (BSA). In some embodiments, the concentration of KC1 can be between 25 and 50 mM, between 25 and 75 mM, between 25 and 100 mM, between 30 and 100 mM, between 50 and 100 mM, or between 70 and 100 mM. In some embodiments, the MgC12 concentration can be in the range of 0.5 mM to 5 mM, between 0.5 mM to 4.5 mM, 0.5 to 4.0 mM, 0.5 to 3.5 mM, 0.5 to 3.0 mM, 0.5 to 2.5 mM, 0.5 to 2.0 mM, 0.5 to 1.5 mM, 1.0 to 5 mM, 1.5 to 5mM, 2.0 to 5 mM, 2.5 to 5 mM, 3.0 to 5 mM, 3.5 to 5 mM, 4.0 to 5 mM, or 4.5 to 5 mM. In some embodiments, the concentration of MgC12 is 2.0 mM.164926-4669-9902.1N.064.W0.01

[0067] In illustrative embodiments, the buffer solution is a Q5® Reaction Buffer (B9027S, New England Biolabs, Inc.). In some embodiments, the reaction buffer is Standard Taq Reaction Buffer (B9014S, New England Biolabs, Inc). In some embodiments, the reaction buffer is a Standard Taq (Mg-free) Reaction Buffer (B9015S, New England Biolabs, Inc.).

[0068] In some embodiments, a DNA polymerase is used to produce DNA amplicons using DNA as a template. In some embodiments, the polymerase is a Q5® DNA Polymerase, such as Q5® High-Fidelity DNA Polymerase (M0491S, New England BioLabs, Inc.) or Q5® Hot Start High-Fidelity DNA Polymerase (M0493S, New England BioLabs, Inc.). Q5® High-Fidelity DNA polymerase is a high-fidelity, thermostable, DNA polymerase with 3'^ 5 ' exonuclease activity, fused to a processivity-enhancing Sso7d domain. Q5® High-Fidelity DNA polymerase lacks 5'— ► 3 'exonuclease activity and strand displacement activity.

[0069] In some embodiments, the polymerase is a T4 DNA polymerase (M0203S. New England BioLabs, Inc.). T4 DNA Polymerase catalyzes the synthesis of DNA in the 5'— ► 3' direction and requires the presence of template and primer. This enzyme has a 3'— > 5' exonuclease activity which is much more active than that found in DNA Polymerase I. T4 DNA polymerase lacks 5'^- 3' exonuclease activity and strand displacement activity.

[0070] In some embodiments of any of the aspects herein, the length of the primers can be between 10 to 100 nucleotides, such as between 10 to 75 nucleotides, 10 to 40 nucleotides, 10 to 35 nucleotides. 10 to 30 nucleotides, 10 to 20 nucleotides, 15 to 100 nucleotides. 20 to 100 nucleotides, from 25 to 100 nucleotides, from 30 to 100 nucleotides from 35 to 100 nucleotides, from 40 to 100 nucleotides, from 45 to 100 nucleotides, from 50 to 100 nucleotides, from 55 to 100 nucleotides, from 60 to 100 nucleotides, from 65 to 100 nucleotides, from 70 to 100 nucleotides, or from 75 to 100 nucleotides. In some embodiments, the range of the length of the primers is between 5 to 50 nucleotides, such as 5 to 40 nucleotides, 5 to 20 nucleotides, or 5 to 10 nucleotides. In some embodiments, the primers are between 5 and 50 bp in length, between 10 and 40 bp in length, between 15 and 30 bp in length, between 15 and 25 bp in length, between 20 and 40 bp in length, between 25 and 50 bp in length, or between 30 and 50 bp in length. In some embodiments, the primers are between 25 and 100 bp in length, between 35 and 100 bp in174926-4669-9902.1N.064.W0.01 length, between 45 and 100 bp in length, between 55 and 100 bp in length, between 65 and 100 bp in length, or between 75 and 100 bp in length.

[0071] In some embodiments of any of the aspects or embodiments herein, the number of primer pairs can range from 20 to 100,000 primer pairs that each bind to one or more primer binding sequences. In some embodiments, the primer pairs are a part of a set of primer pairs. In some embodiments, the set of primers range from 50 to 50,000, from 100 to 20,000, from 100 to 500, from 500 to 2,000, or from 2,000 to 20,000 primer pairs. In some embodiments, the number of primer pairs can range from 10 to 10,000, 10 to 1,000, 10 to 100, 10 to 50, 10 to 40, 10 to 30, 15 to 30, or 15 to 25 primer pairs.

[0072] In some embodiments, PCR is used to generate short amplicons. The fragment sizes of dd-cfDNA may be distributed in approximately a Gaussian fashion with a mean of 160 bp, a standard deviation of 15 bp, a minimum size of about 100 bp, and a maximum size of about 220 bp. Because cfDNA fragments are short, the likelihood of both primer sites being present the likelihood of a fragment of length L comprising both the forward and reverse primers sites is the ratio of the length of the amplicon to the length of the fragment. Under ideal conditions, assays in which the amplicon is 45, 50, 55, 60, 65, or 70 bp will successfully amplify from 72%, 69%, 66%, 63%, 59%, or 56%, respectively, of available template fragment molecules. Thus, in some embodiments target amplicons generated in method herein are between 40 and 100, 40 and 75, or 45 and 70 bp in length. The amplicon length is the distance between the 5 -prime ends of the forward and reverse priming sites. In an embodiment, a substantial fraction of the amplicons are between 25 on the low end of the range, and 100 bp, 90 bp, 80 bp, 70 bp, 65 bp, 60 bp, 55 bp, 50 bp, or 45 bp on the high end of the range.

[0073] In some embodiments, targeted amplification is performed using direct multiplexed PCR, sequential PCR, nested PCR, doubly nested PCR, one-and-a-half sided nested PCR, fully nested PCR, one sided fully nested PCR, one-sided nested PCR, hemi-nested PCR, hemi-nested PCR, triply hemi-nested PCR, semi-nested PCR, one sided semi-nested PCR, reverse semi-nested PCR method, or one-sided PCR, which are described in U.S. Publication No 2012 / 0270212, U.S. Publication No. 2013 / 0123120, and U.S. Publication No. 2015 / 0322507, each of which is incorporated herein by reference in their entirety.184926-4669-9902.1N.064.W0.01

[0074] In some embodiments, the method of amplifying target loci in a nucleic acid sample involves (i) contacting the nucleic acid sample with a library of primers that simultaneously hybridize to at least 100; 200; 500; 1,000; 2.000; 5,000; 10,000; 20,000; 50,000; or 100.000 different target loci to produce a single reaction mixture; and (ii) subjecting the reaction mixture to primer extension reaction conditions (such as PCR conditions) to produce amplified products that include target amplicons. In some embodiments, at least 50, 60, 70, 80, 90, 95, 96, 97, 98. 99, or 99.5% of the targeted loci are amplified. In various embodiments, less than 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1, or 0.05% of the amplified products are primer dimers. In some embodiments, the primers are in solution (such as being dissolved in the liquid phase rather than in a solid phase). In some embodiments, the primers are in solution and are not immobilized on a solid support. In some embodiments, the primers are not part of a microarray.

[0075] In certain embodiments, the multiplex amplification reaction is performed under limiting primer conditions for at least 1 / 2 of the reactions. In some embodiments, limiting primer concentrations are used in 1 / 10, 1 / 5, 1 / 4, 1 / 3, 1 / 2, or all of the reactions of the multiplex reaction. Provided herein are factors to consider in achieving limiting primer conditions in an amplification reaction such as PCR.

[0076] In certain embodiments, the multiplex amplification reaction can include, for example, between 50 and 50,000 multiplex reactions. In certain embodiments, the following ranges of multiplex reactions are performed: between 100, 200, 250, 500, 1000, 2500, 5000, 10,000, 20,000, 25000. 50000 on the low end of the range and between 200. 250, 500, 1000. 2500, 5000, 10,000, 20,000, 25000, 50000, and 100,000 on the high end of the range.

[0077] In an embodiment, a multiplex PCR assay is designed to amplify potentially heterozygous SNP or other polymorphic or non-polymorphic loci on one or more chromosomes and these assays are used in a single reaction to amplify DNA. The number of PCR assays may be between 50 and 200 PCR assays, between 200 and 1,000 PCR assays, between 1,000 and 5,000 PCR assays, or between 5,000 and 20,000 PCR assays (50 to 200-plex, 200 to 1,000-plex, 1,000 to 5,000-plex, 5,000 to 20,000-plex, more than 20,000-plex respectively). In an embodiment, a multiplex pool of at least 10,000 PCR assays (10,000-plex) are designed to amplify potentially heterozygous SNP loci a single reaction to amplify cfDNA obtained from a194926-4669-9902.1N.064.W0.01 blood, plasma, serum, or urine sample. The SNP frequencies of each locus may be determined by clonal or some other method of sequencing of the amplicons. In another embodiment the original cfDNA samples is split into two samples and parallel 5,000-plex assays are performed. In another embodiment the original cfDNA samples is split into n samples and parallel (~10,000 / n)- plex assays are performed where n is between 2 and 12, or between 12 and 24, or between 24 and 48, or between 48 and 96.

[0078] In an embodiment, a method disclosed herein uses highly efficient highly multiplexed targeted PCR to amplify DNA followed by high throughput sequencing to determine the allele frequencies at each target locus. One technique that allows highly multiplexed targeted PCR to perform in a highly efficient manner involves designing primers that are unlikely to hybridize with one another. The PCR probes, typically referred to as primers, are selected by creating a thermodynamic model of potentially adverse interactions between at least 100, at least 200, at least 500. at least 1,000. at least 2,000. at least 5,000. at least 10,000, at least 20,000, or at least 50,000 potential primer pairs, or unintended interactions between primers and sample DNA, and then using the model to eliminate designs that are incompatible with other the designs in the pool. Another technique that allows highly multiplexed targeted PCR to perform in a highly efficient manner is using a partial or full nesting approach to the targeted PCR. Using one or a combination of these approaches allows multiplexing of at least 100, at least 200, at least 500, at least 1 ,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, or at least 50,000 primers in a single pool with the resulting amplified DNA comprising a majority of DNA molecules that, when sequenced, will map to targeted loci. Using one or a combination of these approaches allows multiplexing of a large number of primers in a single pool with the resulting amplified DNA comprising greater than 50%, greater than 80%, greater than 90%, greater than 95%, greater than 98%, or greater than 99% DNA molecules that map to targeted loci.

[0079] Bioinformatics methods for analyzing the sequence data obtained from multiplex PCR are described in U.S. Publication No 2012 / 0270212, U.S. Publication No. 2013 / 0123120, and U.S. Publication No. 2015 / 0322507, each of which is incorporated herein by reference in its entirety.

[0080] Hybrid Capture204926-4669-9902.1N.064.W0.01

[0081] In some embodiments, the targeted enrichment technique can involve fragment capture by hybridization (i.e., hybrid capture). Although any hybrid capture method can be used to perform methods herein that include a targeted enrichment step, in some embodiments, a method of the present disclosure may involve using any of the hybrid capture methods disclosed herein to enrich cfDNA having one or more polymorphic target loci. In some embodiments described herein, the targeted enrichment steps can be performed after cfDNA molecules are extracted. In some embodiments described herein, the targeted enrichment steps can be performed after appending adapters to the extracted cfDNA molecules. In some embodiments described herein, the targeted enrichment steps can be performed after universal PCR amplification of the adapted DNA.

[0082] In capture by hybridization, hybrid capture oligonucleotide probes complementary to one or both strands of specific target DNA sequences, or DNA derived therefrom in a sample, are utilized, i.e., the probes may be strand specific. The specific target DNA sequence in illustrative embodiments overlaps with or is found within a target region of a sample DNA molecule such as a cfDNA. Thus, hybrid capture probes when used in methods herein can be designed to bind to a DNA molecule that contains at least one target region or a portion thereof. In some embodiments, the hybrid capture probes can be designed to bind to a target DNA sequence within or overlapping a target region. In other examples, the hybrid capture probes can be designed to bind to a common region that is flanking but not overlapping the target region and that can be a common region that was added to some, most, almost all or all of the DNA in a sample, or added to all amplicons using a common sequence on at least one primer of a primer pair. In illustrative embodiments, a hybrid capture probe or set thereof, are designed to bind to a target DNA sequence within target region, or set of target regions, respectively.

[0083] Hybrid capture probes may be added to a prepared sample and hybridized through a denature-reannealing process to form duplexes of exogenous-endogenous fragments (e.g.. hybrid capture probes bound to sample DNA molecules, or DNA derived therefrom). These duplexes may then be physically separated from the sample by various means. In some embodiments, once the hybrid capture probes are removed, the sample DNA molecules, or DNA derived therefrom can be amplified. Some ways to physically remove the hybrid capture probes are by covalently bonding the hybrid capture probes to a solid support, for example a magnetic bead, or a chip.214926-4669-9902.1N.064.W0.01Another way to physically remove the hybrid capture probes is by covalently bonding them to a molecular moiety with a strong affinity for another molecular moiety. An example of such a molecular pair is biotin and streptavidin, such as is used in SURE SELECT (Agilent). Thus, hybrid capture probes, for example that bind to a target DNA sequence within or overlapping a target region of a DNA molecule obtained or derived from a sample, can be covalently attached to a biotin molecule, and after hybridization with sample DNA or DNA derived therefrom, a solid support with streptavidin affixed can be used to pull down the biotinylated hybrid capture probes, which are hybridized to DNA molecules obtained or derived from a sample that include a target region that includes the target DNA sequence recognized by the hybrid capture probes. Thus, in some embodiments, the hybrid capture probes are immobilized, directly or indirectly to a solid support, hr some embodiments, the hybrid capture probes include a binding partner, for example biotin.

[0084] In some embodiments of any of the aspects herein, the hybrid capture probes can be a part of a set of at least two hybrid capture probes. In some embodiments, the set includes at least one hybrid capture probe for each polymorphic target loci. In some embodiments, the set includes two or more hybrid capture probes for each polymorphic target loci. In some embodiments, the set includes three or more hybrid capture probes for each polymorphic target loci. In some embodiments, the set includes four or more hybrid capture probes for each polymorphic target loci.

[0085] In some embodiments of any of the aspects herein, the hybrid capture probes can have a length in the range of 30 bases to 170 bases, 30 bases to 160 bases, 30 bases to 150 bases, 30 bases to 140 bases, 30 bases to 130 bases, 30 bases to 120 bases, 30 bases to 110 bases, 30 bases to 100 bases. 30 bases to 90 bases, 30 bases to 80 bases, 30 bases to 70 bases, 30 bases to 60 bases, 30 bases to 50 bases, 40 bases to 160 bases, 40 bases to 150 bases, 40 bases to 140 bases, 40 bases to 130 bases, 40 bases to 120 bases, 40 bases to 110 bases, 40 bases to 100 bases, 40 bases to 90 bases, 40 bases to 80 bases, 40 bases to 70 bases, 40 bases to 60 bases, 50 bases to 150 bases, 50 bases to 140 bases, 50 bases to 130 bases, 50 bases to 120 bases, 50 bases to 110 bases. 50 bases to 100 bases, 50 bases to 90 bases, 50 bases to 80 bases, 50 bases to 70 bases. 60 bases to 140 bases, 60 bases to 130 bases, 60 bases to 120 bases, 60 bases to 110 bases, 60 bases to 100 bases, 60 bases to 90 bases, 60 bases to 80 bases, 70 bases to 130 bases, 70 bases to 120224926-4669-9902.1N.064.W0.01 bases, 70 bases to 110 bases, 70 bases to 100 bases, 70 bases to 90 bases, 80 bases to 120 bases, 80 bases to 110 bases, 80 bases to 100 bases, 90 bases to 120 bases, 90 bases to 110 bases, 100 bases to 165 bases, 100 bases to 150 bases, 100 bases to 140 bases, 100 bases to 130 bases, 100 bases to 120 bases, 110 bases to 150 bases, 110 bases to 140 bases, 110 bases to 130 bases, 120 bases to 150 bases, or 130 bases to 160 bases.

[0086] In some embodiments, the targeted probe capture is performed using tiling probes providing at least 2X, at least 3X, or at least 4X tiled coverage of each polymorphic target loci. In some embodiments, the hybrid capture panel was designed as a 4X tiling probe set (e.g„ 4 baits per base with ~90 bp overlap for ~120bp probes). In some embodiments, the hybrid capture panel was designed as a 2X tiling probe set (2 baits per base with ~60 bp overlap for ~120bp probes).

[0087] Linked Target Capture using Probe-Dependent Primers

[0088] In some embodiments, the targeted enrichment technique can involve probe-dependent primers. Probe-dependent primers (PDPs) have been disclosed (Pel, et al. “Rapid and highly- specific generation of targeted DNA sequencing libraries enabled by linking capture probes with universal primers” PLoS ONE 13(12):e0208283 (2018); WO 2017168332A1 “Linked duplex target capture”, which are hereby incorporated by reference in their entirety). Such embodiments can be considered linked target capture (LTC) methods. Briefly, in an LTC method, a targetspecific probe is linked to a universal primer. The target specific probe is designed to hybridize to a target of interest such as one of the polymorphic target loci. The universal primer linked to the probe is designed to hybridize to a universal priming site in the adapters that have been ligated to the cell-free DNA. The binding of the probe to the target brings the linked universal primer into proximity with the universal priming site and in fact, the ability of the universal primer to bind to the universal priming site and be extended depends on the probe binding to its target. Results to-date have shown that the universal primers do not hybridize to adapters attached to fragments that do not include the target. The linked target capture is highly target specific and primer extension depends on successful probe binding. For that reason, the universal primers linked to the probes are "probe-dependent primers". LTC may be performed using only one PDP, e.g., for a linear amplification, but preferably uses paired forward and reverse PDPs (as234926-4669-9902.1N.064.W0.01 shown in Fig. 1 (b) of Pel 2018 as target-capture PCR 1 ) to amplify the fragment exponentially. The bound probe does not interfere with primer extension to copy the entire fragment (including the distal adapter) when a strand-displacing polymerase is used. It is noted that the cell-free DNA fragment is copied by primers extended from within the ligated adapters, so the entirety of the fragment is copied into amplicons. Due to the probe, LTC gives the target specificity of conventional PCR with gene-specific primers while due to the priming sites in the adapters, the entire fragment is amplified. The resulting amplification products will include a copy of the entire target-containing fragment with adapters at both ends. Preferably, PDPs are designed to incorporate non-extendable capture probes linked 5’ to 5’ with a primer. Multiple linker types are possible as discussed below. Typically, probes of PDPs can be between 30 to 70 nucleotides in length, and include or comprise a 3’ inverted dT base or 3’ C3 spacer to inhibit polymerase extension. In some embodiments, probes are designed to cover the desired region with overlap between forward and reverse probes. In some embodiments, the probes are between 20 and 100 nucleotides in length. In some embodiments, the size of the probe can be between 20 and 40 nucleotides, between 30 and 50 nucleotides, between 40 and 60, between 50 and 70 between 60 and 80, between 70 and 90, 80 and 100, 90 and 110, 100 and 120 nucleotides in length. In some embodiments, at least one of the probes of a PDP pair comprises a sample index.

[0089] In PDPs, forward and reverse probes can be designed to bind to nucleic acid sequences within or near a polymorphic target loci on a sample DNA molecule to enrich nucleic acid molecules comprising the polymorphic target loci of interest or copies thereof. Typically, the primer portion of a PDP is a universal primer designed to bind to a universal primer site on the appended adapter. In some embodiments, the PDP is designed with a sequencer binding sequence, such as an Illumina flow cell binding sequence, incorporated therein. In some embodiments, the sequencer flow cell binding sequence is between the probe and universal primer, and adjacent to the primer. Linked primers of the invention may also include sequencing tags to ensure that all cluster reads originate from the same linked template molecule. The lengths of the primers can be extended or shortened at the 5' end or the 3' end to produce primers with desired melting temperatures. Also, the annealing position of each primer pair can be designed such that the sequence and length of the primer pairs yield the desired melting temperature. In illustrative embodiments, the primer is a low melting temperature universal primer complementary to a portion of the ligated adapter.244926-4669-9902.1N.064.W0.01

[0090] The primer can be tailed or untailed depending on the specific requirements. In some embodiments, the universal primer comprises an A tail. The length of the primers of the PDP can range from 5 to 40 nucleotides in length. In certain embodiments, the PDP primers are between 10 and 25 nucleotides long. In embodiments, the primers of the PDP can range from 5 to 15 nucleotides, from 10 to 25 nucleotides, from 15 to 35 nucleotides, or from 25 to 40 nucleotides in length.

[0091] Typically, probe dependent primers comprise a linker between the probe and the primer. Probe and primer portions of the PDP are typically linked by a polyethylene glycol derivative, an oligosaccharide, a lipid, a hydrocarbon, a polymer, or a protein. In some embodiments, the linker is a PEG molecule, or derivative thereof. In some embodiments, the linker is an oligosaccharide. In some embodiments, the linker is a lipid. In some embodiments, the linker is a hydrocarbon. In some embodiments, the linker is a polymer. In some embodiments, the linker is a protein, or portion thereof. Linkers based on click chemistry is described in WO2017 / 168332A1, which is incorporated herein by reference in its entirety.

[0092] Sequencing to Generate Sequence Reads

[0093] In some embodiments, the method described herein comprises performing sequencing on the sequencing library to generate sequence reads, and using the sequence reads to quantify an amount of donor-derived cell-free DNA and / or a percentage of donor-derived cell-free DNA out of total cell-free DNA.

[0094] In some embodiments, the sequencing is next-generation sequencing or high-throughput sequencing. In some embodiments, the sequencing is untargeted shotgun sequencing (e.g., whole-genome sequencing). In some embodiments, the sequencing is targeted sequencing performed on enriched DNA (e.g., enriched at a plurality of polymorphic loci that can distinguish between the lung transplant and the transplant recipient) or DNA derived thereof.

[0095] DNA sequencing techniques, particularly high throughput next- generation sequencing techniques (often referred to as massively parallel sequencing techniques) such as those employed NOVASEQ (ILLUMINA), MISEQ (ILLUMINA), HISEQ (ILLUMINA), ION TORRENT (LIFE TECHNOLOGIES), GENOME ANALYZER ILX (ILLUMINA), GS254926-4669-9902.1N.064.W0.01FLEX+(ROCHE 454) etc., can be used for determining the sequences of the enriched DNA or DNA derived thereof to elucidate the sequence of the original cfDNA. High throughput genetic sequencers are amenable to the use of barcoding (i.e., sample tagging with distinctive nucleic acid sequences) so as to identify specific samples from individuals thereby permitting the simultaneous analysis of multiple samples in a single run of the DNA sequencer. Methods as described herein that utilize NGS detection, in some embodiments can have an average or median depth of read of at least 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000, 150,000, or 200,000.

[0096] Methods herein can include analyzing data obtained from next- generation sequencing techniques. In some embodiments of methods herein, the enriched DNA or DNA derived thereof can be subjected to sequencing using next-generation sequencing techniques. For a skilled artisan, algorithm design tools are available that can be used and / or adapted to analyze the sequencing data. In addition, those skilled in the art can determine appropriate parameters for measuring alignment to a consensus sequence and / or to a known target region sequence, including any algorithms needed to achieve maximal alignment over the length of the sequences being compared.

[0097] Sequence reads can be demultiplexed using an in-house tool and mapped using the Burrows-Wheeler alignment software, Bwa mem function (BWA, Burrows-Wheeler Alignment Software (see Li H. and Durbin R. (2010) Fast and accurate long-read alignment with Burrows- Wheeler Transform. Bioinformatics.) in single end or paired end mode to a version of reference genome. The reference genome used can be hgl9 or hg38. Amplification statistics QC can be performed by analyzing one or more of, but not limiting to, total reads, number of mapped reads, number of mapped reads on target, and number of reads counted.

[0098] Methods herein can include a background error model that can be constructed using normal, healthy, or non-diseased liquid samples, in illustrative embodiments, normal, healthy, or non-diseased plasma samples, which are sequenced on the same sequencing run to account for run-specific artifacts. In some embodiments, 5, 10, 15, 20, 25, 30, 40, 50. 100, 150, 200, 250. or more than 250 normal, healthy, or non-diseased liquid samples, in illustrative embodiments, plasma samples can be analyzed on the same sequencing run. The number of samples that can be264926-4669-9902.1N.064.W0.01 sequenced on the same sequencing run can be in the range of 5 to 500, 5 to 400, 5 to 300, 5 to 250, 20 to 250, 30 to 250, 50 to 250, 75 to 250, 100 to 250, 50 to 500, or 100 to 500. Sample barcodes are used in illustrative embodiments. In some illustrative embodiments, 20, 25, 40, or 50 normal samples (e.g., plasma samples) can be analyzed on the same sequencing run. Outlier samples can be iteratively removed from the model to account for noise and contamination. In some embodiments, samples with a Z score of greater than 5, 6, 7. 8, 9. or 10 are removed from the data analysis. For each base substitution of every genomic loci, the DOR weighted mean and standard deviation of the error can be calculated.

[0099] Methods herein can include calculating percent identity that can be calculated by determining the number of matched positions in aligned DNA sequences, dividing the number of matched positions by the total number of aligned DNA sequences, and multiplying by 100. A matched position refers to a position in which identical nucleotides occur at the same position in aligned DNA sequences. The percent identity over a particular length can be determined by counting the number of matched positions over that length and dividing that number by the length followed by multiplying the resulting value by 100. A non-limiting example for calculating the percent identity, can be, if (i) a 500-nucleotide DNA target sequence is compared to a subject DNA sequence, (ii) an alignment program presents 200 nucleotides from the target DNA sequence aligned with a region of the subject DNA sequence where the first and last nucleotides of that 200-nucleotide region are matches, and (iii) the number of matches over those 200 aligned nucleotides is 180, then the 500-nucleotide nucleic acid target sequence contains a length of 200 and a sequence identity over that length of 90 percent (i.e„ 180, 200x100=90).

[0100] In some embodiments, the uniformity in DOR can be measured using standard methods such as. but not limiting to, DOR slope, normalized median depth of read (nmDOR), or breadth of read (BOR). DOR slope represents the slope of the line in the linear portion of a list of loci sorted in descending DOR order. Closer to zero is better, as it represents a flat line. In some embodiments, the uniformity in DOR can be measured using the percent of reads in the 90th- 95th percentile. For this measurement, the loci are sorted in descending DOR order. In illustrative embodiments, a DOR distribution using the 90th-95th percentile contains 5 percent of reads. The reads of all loci between the 90th percentile and 95th percentile can be counted and divided by the total reads for all loci.274926-4669-9902.1N.064.W0.01

[0101] In some embodiments, the magnitude of the DOR slope can be less than 0.005, 0.001 , 0.0005, 0.0001, 0.00005, 0.00001, 0.000005, or 0.000001. The magnitude of the DOR slope can be between 0 and 0.005. such as 0.000001 to 0.005. such as between 0.000005 to 0.00001, 0.00001 to 0.00005, 0.00005 to 0.0001, 0.0001 to 0.0005, 0.0005 to 0.001, or 0.001 to 0.005. The percent of reads in the 90th-95th percentile can be between 0.2 and 9 percent, such as between 0.2 to 8 percent, 0.2 to 7 percent, 0.2 to 6 percent. 0.4 to 9 percent, 0.4 to 8 percent, 0.4 to 7 percent, 0.4 to 6 percent, 1 to 9 percent, 1 to 8 percent, 1 to 7 percent, 1 to 6 percent, 2 to 9 percent, 2 to 8 percent, 2 to 7 percent, 2 to 6 percent, 3 to 9 percent, 3 to 8 percent. 3 to 7 percent, 3 to 6 percent, 0.2 to 1.0 percent, 1 to 2 percent, 2 to 3 percent, 2 to 4 percent, 3 to 4 percent, 4 to 5 percent, 5 to 6 percent, or 6 to 8 percent, or 7 to 9 percent. In some embodiments of methods herein, the method can produce a composition comprising at least 100 different amplicons (e.g., at least 300, 500, 750, 1,000, 2,000, 5,000, 7,500, 10,000, 15,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 non-identical amplicons) with the magnitude of the DOR slope in any of the ranges herein, or with a percent of reads in the 90th-95th percentile in any of the ranges herein. In some embodiments, different amplicons can range in between 100 to 500,000, 100 to 400,000, 100 to 300,000, 100 to 200,000. 100 to 100,000. 100 to 75,000, 100 to 50,000, 100 to 40,000, 100 to 30,000, 100 to 25,000, 100 to 20,000, or 100 to 15,000 non-identical amplicons.

[0102] In some embodiments, the high-throughput sequencing is performed with a median depth of read of at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20.000, at least 50,000. at least 100.000, about 100-10,000, about 200- 10,000, about 500-10,000, or about 1,000-100,000 per polymorphic locus.

[0103] The use of sequence reads generated by sequencing to quantify an amount of donor- derived cell-free DNA and / or a percentage of donor-derived cell-free DNA out of total cell-free DNA are described in Sigdel et al., J. Clin. Med. 8(1): 19 (2019); W02020 / 010255;WO2021 / 243045; and WO2022 / 182878, each of which is incorporated herein by reference in its entirety.

[0104] In some embodiments, the method comprises identifying the subject as susceptible to relapse from antibody-mediated kidney allograft rejection when the percentage of donor-derived284926-4669-9902.1N.064.W0.01 cell-free DNA out of total cell-free DNA, the amount of donor-derived cell-free DNA, or both, exceed one or more thresholds. See e.g., Sigdel et al., J. Clin. Med. 8(1): 19 (2019); Halloran et al., Transplantation 106(12):2435-2442 (2022), each of which is incorporated herein by reference in its entirety.

[0105] In some embodiments, the method comprises identifying the subject as susceptible to relapse from antibody-mediated kidney allograft rejection when the percentage of donor-derived cell-free DNA out of total cell-free DNA exceeds about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, or any percentage therebetween. In some embodiments, the method comprises identifying the subject as susceptible to relapse from antibody-mediated kidney allograft rejection when the percentage of donor-derived cell-free DNA out of total cell-free DNA exceeds 0.25%, 0.26%. 0.27%. 0.28%,. 0.28%. 0.30%. 0.31%. 0.32%. 0.33%. 0.34%. 0.35%. 0.36%. 0.37%. 0.38%. 0.39%, 0.40%, 0.41%. 0.42%, 0.43%, 0.44%, 0.45%, 0.46%..0.47%, 0.48%, 0.49%, 0.50%, 0.51%. 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%,0.59%, 0.60%, 0.61%. 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.70%,0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.80%, 0.81%, 0.82%,0.83%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.90%, 0.91%, 0.92%, 0.93%, 0.94%,0.95%, 0.96%, 0.97%. 0.98%, 0.99%, 1.0%. 1.01%, 1.02%, 1.03%, 1.04%. 1.05%, 1.06%, 1.07%, 1.08%, 1.09%, 1.10%, 1.11 %, 1.12%, 1.13%, 1.14%, 1.15%, 1.16%, 1.17%, 1.18%,1.19%, 1.20%, 1.21%, 1.22%, 1.23%, 1.24%, 1.25%, 1.26%, 1.27%, 1.28%, 1.29%, 1.30%,1.31%, 1.32%, 1.33%. 1.34%, 1.35%, 1.36%, 1.37%. 1.38%, 1.39%, 1.40%, 1.41%. 1.42%.1.43%, 1.44%, 1.45%, 1.46%, 1.47%, 1.48%, 1.49%, 1.50%, or any percentage therebetween.

[0106] In some embodiments, the method comprises identifying the subject as susceptible to relapse from antibody-mediated kidney allograft rejection when the amount of donor-derived cell-free DNA (e.g., genomic copies / mL) exceeds about 10 cp / mL, about 20 cp / mL, about 30 cp / mL, about 40 cp / mL, about 50 cp / mL, about 60 cp / mL, about 70 cp / mL, about 80 cp / mL, about 90 cp / mL, about 100 cp / mL, about 110 cp / mL, about 120 cp / mL, about 130 cp / mL, about 140 cp / mL, about 150 cp / mL, or any amount therebetween. In some embodiment, the method comprises identifying the subject as susceptible to relapse from antibody-mediated kidney allograft rejection when the amount of donor-derived cell-free DNA exceeds 50 cp / mL, 51294926-4669-9902.1N.064.W0.01 cp / mL, 52 cp / mL, 53 cp / mL, 54 cp / mL, 55 cp / mL, 56 cp / mL, 57 cp / mL, 58 cp / mL, 59 cp / mL, 60 cp / mL, 61 cp / mL, 62 cp / mL, 63 cp / mL, 64 cp / mL, 65 cp / mL, 66 cp / mL, 67 cp / mL, 68 cp / mL, 69 cp / mL, 70 cp / mL, 71 cp / mL, 72 cp / mL, 73 cp / mL, 74 cp / mL, 75 cp / mL, 76 cp / mL, 77 cp / mL, 78 cp / mL, 79 cp / mL, 80 cp / mL, 81 cp / mL, 82 cp / mL, 83 cp / mL, 84 cp / mL, 85 cp / mL, 86 cp / mL, 87 cp / mL, 88 cp / mL, 89 cp / mL, 90 cp / mL, 91 cp / mL, 92 cp / mL, 93 cp / mL, 94 cp / mL, 95 cp / mL, 96 cp / mL, 97 cp / mL, 98 cp / mL, 99 cp / mL, or 100 cp / mL, or any amount therebetween.

[0107] Tracer DNA

[0108] Use of Tracer DNA to improve quantification of the amount of total cell-free DNA is described in WO2021 / 243045 and WO2022 / 182878, each of which is incorporated herein by reference in its entirety. Tracer DNA (or internal calibration DNA) refers to a composition of DNA for which one or more of the following features are known - length, sequence, nucleotide composition, quantity, or biological origin. The Tracer DNA can be added to a biological sample derived from a human subject to help estimate the amount of total cfDNA in said sample.

[0109] In some embodiments, the method further comprises adding a Tracer DNA to extracted DNA or its derivative to obtain a mixed composition. In some embodiments, the Tracer DNA comprises synthetic double-stranded DNA molecules. In some embodiments, the Tracer DNA comprises DNA molecules of non-human origin.

[0110] In some embodiments, the Tracer DNA comprises DNA molecules having a length of about 50-500 bp, or about 75-300 bp, or about 100-250 bp, or about 125-200 bp, or about 125 bp, or about 160 bp, or about 200 bp. or about 500-1,000 bp.

[0111] In some embodiments, the Tracer DNA comprises DNA molecules having the same or substantially the same length, such as a DNA molecule having a length of about 125 bp. or about 160 bp, or about 200 bp. In some embodiments, the Tracer DNA comprises DNA molecules having different lengths, such as a first DNA molecule having a length of about 125 bp, a second DNA molecule having a length of about 160 bp, and a third DNA molecule having a length of about 200 bp. In some embodiments, the DNA molecules having different lengths are used to determine size distribution of the cell-free DNA in the sample304926-4669-9902.1N.064.W0.01

[0112] In some embodiments, the Tracer DNA comprises a target sequence, wherein the target sequence comprises a barcode positioned between a pair of primer binding sites capable of binding to a pair of primers. In some embodiments, at least part of the Tracer DNA is designed based on an endogenous human SNP locus, by replacing an endogenous sequence containing the SNP locus with the barcode. During the multiplex PCR target enrichment step, the primer pair targeting the SNP locus can also amplify the portion of Tracer DNA containing the barcode.

[0113] In some embodiments, the barcode is an arbitrary barcode. In some embodiments, the barcode comprises reverse complement of a corresponding endogenous genome sequence capable of being amplified by the same primer pair.

[0114] In some embodiments, the target sequence within the Tracer DNA is flanked on one or both sides by endogenous genome sequences. In some embodiments, the target sequence within the Tracer DNA is flanked on one or both sides by non-endogenous sequences.

[0115] In some embodiments, the Tracer DNA comprises a plurality of target sequences. In some embodiments, the Tracer DNA comprises a first target sequence comprising a first barcode positioned between a first pair of primer binding sites capable of binding to a first pair of primers, and a second barcode positioned between a second pair of primer binding sites capable of binding to a second pair of primers. In some embodiments, the first and / or second target sequence is designed based on one or more endogenous human SNP loci, by replacing an endogenous sequence containing a SNP locus with a barcode. In some embodiments, the first and / or second barcode is an arbitrary barcode. In some embodiments, the first and / or second barcode comprises reverse complement of a corresponding endogenous genome sequence capable of being amplified by the first or second primer pair. In some embodiments, the first and / or second target sequence within the Tracer DNA is flanked on one or both sides by endogenous genome sequences. In some embodiments, the first and / or second target sequence within the Tracer DNA is flanked on one or both sides by non-endogenous sequences.

[0116] In some embodiments, the Tracer DNA comprises DNA molecules having the same or substantially the same sequence. In some embodiments, the Tracer DNA comprises DNA molecules having different sequences.314926-4669-9902.1N.064.W0.01

[0117] In some embodiments, the Tracer DNA comprises a first DNA comprising a first target sequence and a second DNA comprising a second target sequence. In some embodiments, the first target sequence and second target sequence have different barcodes positioned between the same primer binding sites. In some embodiments, the first target sequence and second target sequence have different barcodes positioned between the same primer binding sites, wherein the different barcodes have the same or substantially the same lengths. In some embodiments, the first target sequence and second target sequence have different barcodes positioned between the same primer binding sites, wherein the different barcodes have different lengths. In some embodiments, the first target sequence and second target sequence are designed based on different endogenous human SNP loci, and hence comprise different primer binding sites. In some embodiments, the amount of first DNA and the amount of the second DNA are the same or substantially the same in the Tracer DNA. In some embodiments, the amount of first DNA and the amount of the second DNA are different in the Tracer DNA.

[0118] Determining Amount of Total cfDNA using Tracer DNA

[0119] In certain embodiments, the Tracer DNA can be used to improve accuracy and precision of the method described herein, help quantify over a wider input range, assess efficiency of different steps at different size ranges, and / or calculate fragment size-distribution of input material.

[0120] Some embodiments of the present invention relate to a method of quantifying the amount of total cell-free DNA in a biological sample, comprising: a) adding a Tracer DNA to extracted DNA or its derivative to obtain a mixed composition; b) performing targeted multiplex amplification on the mixed composition comprising the extracted DNA or its derivative and the Tracer DNA at 100 to 20,000 different polymorphic target loci together in the same reaction volume using 100 to 20,000 different target-specific primers; c) sequencing the amplicons by high-throughput sequencing to generate sequence reads; and d) quantifying an amount of donor- derived cell-free DNA and an amount of total cell-free DNA from the sequence reads, wherein the amount of total cell-free DNA is quantified using sequence reads derived from the Tracer DNA.324926-4669-9902.1N.064.W0.01

[0121] In some embodiments, the method comprises adding the Tracer DNA to a whole blood sample before plasma extraction. In some embodiments, the method comprises adding the Tracer DNA to a plasma sample after plasma extraction and before isolation of the cell-free DNA. In some embodiments, the method comprises adding the Tracer DNA to a composition comprising the isolated cell-free DNA. In some embodiments, the method comprises ligating adaptors to the isolated cell-free DNA to obtain a composition comprising adaptor-ligated DNA, and adding the Tracer DNA to the composition comprising adaptor-ligated DNA.

[0122] In some embodiments, the method further comprises adding a second Tracer DNA before the targeted amplification. In some embodiments, the method further comprises adding a second Tracer DNA after the targeted amplification.

[0123] In some embodiments, the amount of total cfDNA in the sample is estimated using the NOR of the Tracer DNA (identifiable by the barcode), the NOR of sample DNA, and the known amount of the Tracer DNA added to the plasma sample. In some embodiments, the ratio between the NOR of the Tracer DNA and the NOR of sample DNA is used to quantify the amount of total cell-free DNA. In some embodiments, the ratio between the NOR of the barcode and the NOR of the corresponding endogenous genome sequence is used to quantify the amount of total cell-free DNA. In some embodiments, this information along with the plasma volume can also be used to calculate the amount of cfDNA per volume of plasma. In some embodiments, these can be multiplied by the percentage of donor DNA to calculate the total donor cfDNA and the donor cfDNA per volume of plasma.

[0124] Adjusting Threshold for Calling Transplant Rejection using Amount of cfDNA

[0125] Some embodiments use either a fixed threshold of the percentage of dd-cfDNA out of total cfDNA or the amount of dd-cfDNA, or a dynamic threshold that is not fixed but adjusted or scaled according to the amount of total cfDNA as noted herein. See W02020 / 010255, which is incorporated herein by reference in its entirety. The way that this is determined can be based on using a training data set to build an algorithm to maximize performance. It may also take into account other data such as patient weight, age, or other clinical factors.334926-4669-9902.1N.064.W0.01

[0126] In some embodiments, the method further comprises determining the occurrence, recurrence, relapse, or likely relapse of transplant rejection using the amount of donor-derived cell-free DNA. In some embodiments, the amount of donor-derived cell-free DNA is compared to a cutoff threshold value to determine the occurrence or likely occurrence of ABMR relapse, wherein the cutoff threshold value is adjusted or scaled according to the amount of total cell-free DNA. In some embodiments, the cutoff threshold value is a function of the number of reads of the donor-derived cell-free DNA.

[0127] In some embodiments, the method comprises applying a scaled or dynamic threshold metric that takes into account the amount of total cfDNA in the samples to more accurately assess transplant rejection. In some embodiments, the method further comprises flagging the sample if the amount of total cell-free DNA is above a pre-determined value. In some embodiments, the method further comprises flagging the sample if the amount of total cell-free DNA is below a pre-determined value.

[0128] Treatment of ABMR Relapse Patients

[0129] In some embodiments, the method described herein further comprises administering a treatment to a kidney transplant recipient suffering from or susceptible to ABMR relapse. In some embodiments, the treatment comprises administering the anti-CD38 antibody or antigenbinding fragment thereof (e.g., daratumumab, felzartamab, or isatuximab) subcutaneously to a kidney transplant recipient suffering from or susceptible to ABMR relapse. In some embodiments, the treatment comprises administering the anti-CD38 antibody or antigen-binding fragment thereof (e.g., daratumumab, felzartamab, or isatuximab) intravenously to a kidney transplant recipient suffering from or susceptible to ABMR relapse. In some embodiments, the treatment comprises administering daratumumab subcutaneously at a dose of 1800 mg to a kidney transplant recipient suffering from or susceptible to ABMR relapse. In some embodiments, the treatment comprises administering a dose of daratumumab to the ABMR relapse patient once per week for four weeks to a kidney transplant recipient suffering from or susceptible to ABMR relapse. In some embodiments, the treatment comprises administering a dose of the anti-CD38 antibody or antigen-binding fragment thereof (e.g., daratumumab,344926-4669-9902.1N.064.W0.01 felzartamab, or isatuximab) after identifying the subject as susceptible to relapse from antibody- mediated kidney allograft rejection.

[0130] In some embodiments, the method comprises retreating the AB MR relapse patient with the anti-CD38 antibody or antigen-binding fragment thereof (e.g., daratumumab, felzartamab, or isatuximab) when the percentage of donor-derived cell-free DNA out of total cell-free DNA, the amount of donor-derived cell-free DNA, or both, exceed one or more thresholds.

[0131] In some embodiments, the method comprises retreating the AB MR relapse patient with the anti-CD38 antibody or antigen-binding fragment thereof (e.g., daratumumab, felzartamab, or isatuximab) when the percentage of donor-derived cell-free DNA out of total cell-free DNA exceeds about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, or any percentage therebetween. In some embodiments, the method comprises retreating the cABMR relapse patient with the anti-CD38 antibody or antigen-binding fragment thereof (e.g., daratumumab, felzartamab, or isatuximab) when the percentage of donor-derived cell-free DNA out of total cell-free DNA exceeds 0.25%, 0.26%. 0.27%. 0.28%,. 0.28%. 0.30%. 0.31%. 0.32%. 0.33%. 0.34%. 0.35%. 0.36%. 0.37%. 0.38%. 0.39%, 0.40%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%., 0.47%, 0.48%, 0.49%, 0.50%, 0.51%. 0.52%, 0.53%, 0.54%, 0.55%. 0.56%, 0.57%, 0.58%, 0.59%, 0.60%, 0.61 %, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.70%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.80%, 0.81%, 0.82%, 0.83%, 0.84%. 0.85%, 0.86%, 0.87%, 0.88%. 0.89%, 0.90%, 0.91%, 0.92%. 0.93%.0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, 1.0%, 1.01%, 1.02%, 1.03%, 1.04%, 1.05%, 1.06%, 1.07%, 1.08%, 1.09%, 1.10%, 1.11%, 1.12%, 1.13%, 1.14%, 1.15%, 1.16%, 1.17%. 1.18%, 1.19%. 1.20%. 1.21%, 1.22%, 1.23%. 1.24%. 1.25%, 1.26%, 1.27%, 1.28%. 1.29%, 1.30%, 1.31%, 1.32%, 1.33%, 1.34%, 1.35%, 1.36%, 1.37%, 1.38%, 1.39%, 1.40%, 1.41%, 1.42%, 1.43%, 1.44%, 1.45%, 1.46%, 1.47%, 1.48%, 1.49%, 1.50%, or any percentage therebetween.

[0132] In some embodiments, the method comprises retreating the AB MR relapse patient with the anti-CD38 antibody or antigen-binding fragment thereof (e.g., daratumumab, felzartamab, or isatuximab) when the amount of donor-derived cell-free DNA (e.g., genomic copies / mL)354926-4669-9902.1N.064.W0.01 exceeds about 10 cp / mL, about 20 cp / mL, about 30 cp / mL, about 40 cp / mL, about 50 cp / mL, about 60 cp / mL, about 70 cp / mL, about 80 cp / mL, about 90 cp / mL, about 100 cp / mL, about 110 cp / mL, about 120 cp / mL, about 130 cp / mL, about 140 cp / mL, about 150 cp / mL, or any amount therebetween. In some embodiment, the method comprises retreating the AB MR relapse patient with the anti-CD38 antibody or antigen-binding fragment thereof (e.g., daratumumab, felzartamab, or isatuximab) when the amount of donor-derived cell-free DNA exceeds 50 cp / mL, 51 cp / mL, 52 cp / mL, 53 cp / mL, 54 cp / mL, 55 cp / mL, 56 cp / mL, 57 cp / mL, 58 cp / mL, 59 cp / mL,60 cp / mL, 61 cp / mL, 62 cp / mL, 63 cp / mL, 64 cp / mL, 65 cp / mL, 66 cp / mL, 67 cp / mL, 68 cp / mL,69 cp / mL, 70 cp / mL, 71 cp / mL, 72 cp / mL, 73 cp / mL, 74 cp / mL, 75 cp / mL, 76 cp / mL, 77 cp / mL,78 cp / mL, 79 cp / mL, 80 cp / mL, 81 cp / mL, 82 cp / mL, 83 cp / mL, 84 cp / mL, 85 cp / mL, 86 cp / mL,87 cp / mL, 88 cp / mL, 89 cp / mL, 90 cp / mL, 91 cp / mL, 92 cp / mL, 93 cp / mL, 94 cp / mL, 95 cp / mL,96 cp / mL, 97 cp / mL, 98 cp / mL, 99 cp / mL, or 100 cp / mL, or any amount therebetween.

[0133] Working Example

[0134] Patients and Methods

[0135] Sixteen kidney transplant recipients with biopsy-confirmed chronic AB MR were treated with daratumumab. All patients were aged >18 years and were transplanted more than 180 days prior to biopsy confirmation of chronic ABMR by histology using Banff 2019 Classification and the Molecular Microscope Diagnostic System (MMDx-Kidney, ThermoFisher).

[0136] In all cases, 30 step sections were cut and stained for PAS, H&E, silver and Masson trichrome for light microscopy. C4d staining was performed using either immunofluorescence or immunohistochemistry, with appropriate controls. After biopsy confirmation of cABMR status (index biopsy), patients were treated with subcutaneous daratumumab (Darzalex™, distributed by Janssen Phamaceuticals) at a flat dose of 1800 mg administered weekly for 4 weeks, and at months 4, 7 and 10 for a total of 7 injections (FIG. 1). The first injection of daratumumab was given over at least 15 minutes after premedication with oral dexamethasone 8 mg, chlorpheniramine 4 mg and paracetamol 1000 mg. Post- administration, the patients were observed for 30-60 minutes in the clinic. Subsequent administrations did not require premedications or observation. The patients were given symptomatic standby medications for fever, nausea and diarrhoea to be taken as required after daratumumab administration. All patients were364926-4669-9902.1N.064.W0.01 maintained on their baseline immunosuppression of tacrolimus, mycophenolate sodium with or without corticosteroids. Blood tacrolimus levels were optimized to a trough level of 5-7 ng / mL. Mycophenolate sodium doses were maintained at 500 mg twice per day. The prednisolone dose was maintained, ranging from 5 - 7.5 mg / day. No high dose corticosteroids, rituximab, plasma exchanges or intravenous immunoglobulins was given. The patients were given prophylaxis against Pneumocystis jerovecii and monitored for cytomegalovirus and BK virus infections.

[0137] Serum creatinine and eGFR (CKD-EPI 2021) levels, urine albumin / creatinine ratio, donor specific antibody, DSA (Luminex Single Antigen Bead assays, One Lambda, West Hills, CA) and donor-derived cell-free DNA, dd-cfDNA (the ProsperaTM Test, Natera Inc, San Carlos, CA) were measured prior to commencement of treatment and repeated at the end of treatment. The post-treatment (PT) biopsies were performed at the end of treatment and not later than 3 months after the completion of treatment. Donor specific antibody was interpreted as positive using an MFI cutoff of 500.

[0138] Results

[0139] Sixteen KTRs with a confirmed diagnosis of cABMR were treated with a 10-month course of subcutaneous daratumumab. The cohort was 75% male and 100% Asian with a median age of 50 years (IQR: 41-59). The median time from transplantation to diagnosis of chronic active-mediated transplant was 9.1 years (IQR: 5.4-11.4 years). 3 patients had a third transplant and 2 patients had a second transplant. The cohort had a median 5 HLA mismatches (IQR: 4-6), with de novo donor specific antibody present in 6 of 16 patients [37%].

[0140] Prior to treatment, all 16 patients were confirmed to have cABMR based on histological biopsy with a median microvascular inflammation (MVI) score (g+ptc) of 4 (IQR: 2.5-4.5), and molecular microscope diagnostic assessment also showed that all patients had either moderate or severe antibody-mediated rejection, with a median antibody-mediated rejection score of 0.78 (IQR: 0.62-0.85) (Table 1). The pretreatment biomarker assessment showed a median donor- derived cell-free DNA fraction of 2.58% (IQR:1.671-5.76%), with 14 of 16 patients (87%) with donor-derived cell-free DNA of >1.0%, the standard clinical threshold for considering a patient as “at high risk for rejection.” The remaining 2 patients had cell-free donor derived DNA test374926-4669-9902.1N.064.W0.01 results >0.8%. Median eGFR was 46 (IQR:32-63) mL / min / 1 ,73m2 and median urine albumin / creatinine was 47 (IQR: 8-130) mg / g. 13 of 16 patients [81%] were positive for DSA.

[0141] Table 1 (Diagnostic Results):

[0142] After completion of treatment with SC daratumumab, patients showed stabilized renal function, with no transplant glomerulopathy evident, and with discrete improvement in certain biomarker scores (Table 1, FIG. 2). Histology indicated that 9 of 16 patients [56%] were no longer experiencing active-mediated antibody rejection with a microvascular inflammation score of 0-1 and 4 of 16 patients [25%] showed an amelioration of microvascular inflammation scores, with the median decreasing to 1 (IQR: 0-2) (Table 1). Molecular histology showed 8 patients with complete resolution of rejection, 3 with mild antibody-mediated rejection, and 4 with moderate antibody-mediated rejection, with antibody-mediated rejection scores dropping in 15 / 16 patients by a median drop of 74% (IQR: 45-86%), to a median of 0.17 (IQR: 0.11-0.44) (FIG. 2A). Following treatment, the median cell-free donor derived DNA decrease was 85%. and dropped by more than 50% in all 16 patients, and by more than 75% in 14 of 16 patients (FIG. 2B). Median donor-derived cell-free DNA was 0.33% (IQR: 0.25-0.61), with 12 of 16 patients below 0.5%, and 14 of 16 patients below 1.0%. Median eGFR did not change appreciably at 51 (IQR: 37-77) mL / min / 1.73m2while the median urine albumin / creatinine ratio dropped to 9 (IQR: 3-40) mg / g (FIGs. 2C-2D).384926-4669-9902.1N.064.W0.01

[0143] After daratumumab treatment was completed, observations of all patients were continued using serum creatinine and eGFR levels, urine albumin / creatinine ratio and donor-derived cell- free DNA. During this period, two patients were assumed to have a recurrence of their antibody- mediated rejection based on their rebound donor-derived cell-free DNA levels. One patient was a highly sensitized female with positive pre-transplant CDC and flow crossmatches who was transplanted 7 years previously. At 9-months post-treatment, she had a rebound of her donor- derived cell-free DNA level to her pre-treatment level. She was treated with a single dose of daratumumab. 6 months later when her donor-derived cell-free DNA level had risen, another dose of daratumumab was administered (FIG. 3, top). At 18-months post treatment her biopsy showed stabilization of microvascular inflammation and molecular antibody-mediated rejection scores. A second male patient also had rebound donor-derived cell-free DNA levels at 9-months post treatment and was retreated with a single dose of daratumumab (FIG. 3, bottom). At 15- months post-treatment his biopsy also showed stabilization of microvascular inflammation and molecular antibody-mediated rejection scores. Both patients showed significant reductions in their donor-derived cell-free DNA levels in tandem with their biopsy results.

[0144] cABMR Relapse was observed in 2 of the 4 patients who had relatively high donor- derived cell-free DNA levels at the end of treatment (>0.5%), and 1 of the 2 patients who had high donor-derived cell-free DNA levels at the end of treatment (>1%), whereas no patients with <0.5% donor-derived cell-free DNA levels experienced a cABMR relapse.

[0145] 12 of 16 patients (75%) showed significant improvement after a 10-month course of treatment, 8 of 16 patients had complete remission of antibody-mediated rejection as adjudicated by molecular microscopy results and another 4 patients had mild residual antibody-mediated rejection. The impact of daratumumab therapy on the severity of rejection across the full cohort was dramatic, with the median molecular antibody-mediated rejection score dropping by 74%, and the median microvascular inflammation score dropping from 4 to 1. The donor-derived cell- free DNA percentage reduction mirrored the antibody-mediated rejection score reduction, with a median decrease of 85%, and with all 16 patients showing a donor-derived cell-free DNA percentage decrease of >50% after daratumumab treatment. The post-treatment donor-derived cell-free DNA percentage dropped below the 1% threshold, which is used to indicate patients as “at high-risk for rejection”, in all but 2 patients, whose donor-derived cell-free DNA were 1.01%394926-4669-9902.1N.064.W0.01 and 1.1 1 %. Tn contrast, the traditional biomarkers were not as consistent, with serum creatinine, eGFR and urine albumin / creatinine values dropping in 11 of 16 patients, but rising in the remainder, reflecting their lack of sensitivity in predicting the underlying renal pathology.

[0146] Although daratumumab has a K light chain compared to felzartamab’s light chain, the results suggest that daratumumab’ s efficacy in treating antibody-mediated rejection are comparable with the results observed in the felzartamab trial, in which 82% of the patients experienced a complete remission. The felzartamab treated patients also showed reductions in the median microvascular inflammation and molecular antibody-mediated rejection scores and donor-derived cell-free DNA levels. See Osmanodja, et al., Transplant Int. 37:13213 (2024).

[0147] An aspect of the daratumumab treatment that made the regimen practical and convenient was the subcutaneous route of administration. There are also case reports on the use of intravenous daratumumab for the treatment of acute antibody-mediated rejection. In contrast to intravenous daratumumab, subcutaneous daratumumab is administered at a flat-dose of 1800 mg for patients with a body weight from 40 kg to 130 kg. The initial experience with daratumumab in a patient with acute antibody-mediated rejection demonstrated that weekly subcutaneous administration of daratumumab for 4 weeks dramatically reduced the donor-derived cell-free DNA to an exceptionally low level. Therefore, the frequency of subsequent administrations was empirically modified and reduced to quarterly and the treatment duration was limited to 10 months. The use of this regimen was adopted for the treatment of chronic antibody-mediated rejection to this present cohort of patients with remarkably positive results as disclosed herein.

[0148] Post-treatment dd-cfDNA levels were also associated with cABMR relapse. Among the patients who showed complete remission of antibody-mediated rejection after the initial course of daratumumab, 2 / 4 patients with dd-cfDNA >0.5% relapsed, whereas none of the patients with dd-cfDNA levels <0.5% relapsed, suggesting utility in monitoring and predicting rejection during anti-CD38 treatment. The 2 patients were biopsied after retreatment, and the results showed stability of antibody-mediated rejection in-line with the decrease of the donor-derived cell-free DNA levels. In contrast, serum creatinine and eGFR was unchanged post-treatment in both patients who relapsed, and urine albumin / creatinine decreased in one of the two patients, demonstrating poor ability of these biomarkers to predict underlying allograft pathology.404926-4669-9902.1N.064.W0.01

[0149] In the felzartamab study, rejection features had recurred in 33% of patients 6 months after showing remission of AB MR after treatment was complete. In our study 1 of the 8 patients with complete remission at the completion of treatment relapsed 12 months after completion of treatment. In the 2 patients that were retreated, antibody-mediated rejection appeared to be controlled. The precise proportion of patients who relapse following cABMR treatment is presently unknown.

[0150] For at least these reasons, dd-cfDNA provides a noninvasive way to monitor cABMR relapse after completion of treatment with daratumumab. Furthermore, dd-cfDNA was significantly more predictive of cABMR relapse than other biomarkers.* * * *414926-4669-9902.1

Claims

N.064.W0.01CLAIMSWHAT IS CLAIMED IS:

1. A method for treating antibody-mediated kidney allograft rejection, comprising: treating a subject determined to suffer from antibody-mediated kidney allograft rejection more than 6 months after transplantation by administering an anti-CD38 monoclonal antibody or an antigen-binding fragment thereof to the subject; extracting cell-free DNA from a blood, plasma, serum or urine sample collected from the subject after conclusion of the treatment; quantifying an amount of donor-derived cell-free DNA, a percentage of donor-derived cell-free DNA out of total cell-free DNA, or both, in the extracted DNA; and retreating the subject with the anti-CD38 monoclonal antibody or an antigen-binding fragment thereof if the amount of donor-derived cell-free DNA, the percentage of donor-derived cell-free DNA out of total cell-free DNA, or both, exceed one or more threshold values.

2. The method of claim 1, wherein the subject was determined to suffer from antibody-mediated kidney allograft rejection using the Banff 2019 Classification or by histological biopsy of renal tissue.

3. The method of claim 1 , wherein the subject was determined to suffer from antibody-mediated kidney allograft rejection by quantifying an amount of donor-derived cell-free DNA, a percentage of donor-derived cell-free DNA out of total cell-free DNA, or both, in a blood, plasma, serum or urine sample collected from the subject before the treatment.

4. The method of claim 3. wherein the subject was determined to suffer from antibody-mediated kidney allograft rejection by quantifying a percentage of donor-derived cell-free DNA out of total cell-free DNA of >1.0%, in a blood, plasma, serum or urine sample collected from the subject before the treatment.

5. The method of any of claims 1-4, wherein administering the anti-CD38 monoclonal antibody or an antigen-binding fragment thereof comprises administering a dose of the anti- CD38 monoclonal antibody or an antigen-binding fragment thereof once per week for four424926-4669-9902.1N.064.W0.01 weeks and at 4, 7, and 10 months after the first administration; or wherein administering the anti- CD38 monoclonal antibody or an antigen-binding fragment thereof comprises administering a dose of the anti-CD38 monoclonal antibody or an antigen-binding fragment thereof once per week for four weeks and at 3, 6, and 9 months after the first administration.

6. The method of any of claims 1-5, wherein the anti-CD38 monoclonal antibody or an antigenbinding fragment thereof is administered subcutaneously or intravenously.

7. The method of any of claims 1-6, wherein the anti-CD38 monoclonal antibody or an antigenbinding fragment thereof comprises daratumumab, felzartamab, or isatuximab.

8. The method of any of claims 1-7, wherein the blood, plasma, serum or urine sample is collected from the subject between 9 months and 15 months after conclusion of the treatment.

9. The method of any of claims 1-8, wherein the quantifying step comprises performing targeted multiplex amplification of the extracted DNA or its derivative to amplify 100 to 20,000 different polymorphic target loci together in the same reaction volume using 100 to 20,000 different target- specific primers, and performing high-throughput sequencing on the amplicons to generate sequence reads.

10. The method of any of claims 1-9, wherein the quantifying step comprises: a) adding a Tracer DNA to extracted DNA or its derivative to obtain a mixed composition; b) performing targeted multiplex amplification on the mixed composition comprising the extracted DNA or its derivative and the Tracer DNA to amplify 100 to 20,000 different polymorphic target loci together in the same reaction volume using 100 to 20,000 different target- specific primers; c) sequencing the amplicons by high-throughput sequencing to generate sequence reads; and434926-4669-9902.1N.064.W0.01 d) quantifying the amount of donor-derived cell-free DNA and the amount of total cell- free DNA from the sequence reads, wherein the amount of total cell-free DNA is quantified using sequence reads derived from the Tracer DNA.

11. The method of any of claims 1-10, wherein the method comprises retreating the subject with the anti-CD38 monoclonal antibody or antigen-binding fragment thereof when the percentage of donor-derived cell-free DNA out of total cell-free DNA exceeds 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%.

12. A method for identifying a subject who is susceptible to relapse from antibody-mediated kidney allograft rejection, wherein the subject has received treatment with an anti-CD38 monoclonal antibody or antigen-binding fragment thereof, comprising: extracting cell-free DNA from a blood, plasma, serum or urine sample collected from the subject after conclusion of the treatment: quantifying an amount of donor-derived cell-free DNA, a percentage of donor-derived cell-free DNA out of total cell-free DNA, or both, in the extracted DNA; and identifying the subject as susceptible to relapse from antibody-mediated kidney allograft rejection if the amount of donor-derived cell-free DNA, the percentage of donor-derived cell-free DNA out of total cell-free DNA, or both, exceed one or more threshold values.

13. The method of claim 12, wherein the anti-CD38 monoclonal antibody or antigen-binding fragment thereof is daratumumab, felzartamab, or isatuximab.

14. The method of any of claims 12-13, wherein the method comprises identifying the subject as susceptible to relapse from antibody-mediated kidney allograft rejection when the percentage of donor-derived cell-free DNA out of total cell-free DNA exceeds 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%.

15. The method of any of claims 12-14, wherein the blood, plasma, serum or urine sample is collected from the subject between 9 months and 15 months after conclusion of the treatment.444926-4669-9902.1N.064.W0.0116. The method of any of claims 12-15, wherein the quantifying step comprises performing targeted multiplex amplification of the extracted DNA or its derivative to amplify 100 to 20,000 different polymorphic target loci together in the same reaction volume using 100 to 20,000 different target-specific primers, and performing high-throughput sequencing on the amplicons to generate sequence reads.

17. The method of any of claims 12-16, wherein the quantifying step comprises: a) adding a Tracer DNA to extracted DNA or its derivative to obtain a mixed composition; b) performing targeted multiplex amplification on the mixed composition comprising the extracted DNA or its derivative and the Tracer DNA at 100 to 20,000 different polymorphic target loci together in the same reaction volume using 100 to 20,000 different target-specific primers; c) sequencing the amplicons by high-throughput sequencing to generate sequence reads; and d) quantifying an amount of donor-derived cell-free DNA and an amount of total cell-free DNA from the sequence reads, wherein the amount of total cell-free DNA is quantified using sequence reads derived from the Tracer DNA.

18. The method of any of claims 12-17, wherein the kidney transplant is from a human.

19. The method of any of claims 12-17, wherein the kidney transplant is a xenotransplant, optionally wherein the kidney transplant from a pig.

20. The method of any of claims 12-19, wherein the method is performed without prior knowledge of donor or recipient genotypes.454926-4669-9902.1

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