Assessing the state of transplant rejection by analysis of t cell receptor sublibrary diversity

Abstract

New methods for assessing the status of transplant rejection in transplant recipients, such as kidney transplant recipients, are disclosed. The diagnostic methods utilize the measurement of TCR subunit repertoire diversity to identify stable subjects, subjects undergoing cell-mediated rejection processes, and subjects undergoing antibody-mediated rejection processes. The proportion of unique TCR alpha and beta subunit sequences to the total unique TCR subunit sequences (total sequences of alpha, beta, delta, and gamma subunits) provides a diagnostic measure that can identify stable subjects, subjects undergoing cell-mediated rejection processes, and subjects undergoing antibody-mediated rejection processes. If antibody-mediated rejection or cell-mediated rejection is detected, treatment methods include administration of appropriate therapy.

Description

[0001] Cross Reference to Related Applications:

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 641,992, filed March 12, 2018, entitled "Kidney Transplant Rejection And T-Cell Receptor Subunit Repertoire," the contents of which are incorporated by reference into this application.

[0003] Statement as to Federally Sponsored Research or Development:

[0004] This application was made with government support under Grant No. U19 AI128913 awarded by the National Institutes of Health. The government has certain rights in the application. BACKGROUND

[0005] Transplant rejection is a serious problem affecting a substantial portion of kidney transplant recipients. There are two main forms of kidney rejection: antibody-mediated rejection (AMR) and cell-mediated rejection (CMR) mediated through T cells. Importantly, as is known in the art, these two forms of rejection have different prognoses and are treated by different methods. Thus, it would be advantageous to be able to identify when a kidney transplant recipient is experiencing rejection and to accurately diagnose which form of rejection a subject is suffering from. It would be more advantageous to identify patients at risk for or experiencing AMR or CMR through an easily obtained sample as opposed to an invasive transplant biopsy.

[0006] The high variability of BCRs and TCRs is possible due to the recombination process, enabling T cells to be activated by a diverse array of antigens. This recombination involves three different gene segment types: V (variable), D (diversity), and J (joining) segments, similar to the recombination of immunoglobulins. Additional diversity is created at the junctions of the segments during the recombination process. The TCR complex comprises multiple subunits, including heterodimers of alpha and beta subunits and heterodimers of delta and gamma subunits. Most of the clonal diversity and most of the heterodimers exist between the alpha and beta subunits, and it is generally known that the proportion of heterodimers changes during disease progression. As disclosed herein, the inventors of the present disclosure have determined that certain measures of TCR subunit diversity provide diagnostic features indicative of the state of transplant rejection. SUMMARY

[0007] The inventors of the present disclosure have developed a new method of assessing whether a transplant recipient, who is undergoing some form of immune-mediated rejection, is stable (not undergoing a rejection process). The method can also be used to determine whether an AMR or CMR process is active in a transplant recipient. The method is accomplished by analyzing the diversity of certain T cell receptor subunits. Advantageously, these T cell receptor subunit diversity measures can be assessed in peripheral blood samples, which enables the renal rejection status to be assessed conveniently and non-invasively.

[0008] The present invention is based on the discovery that the ratio of the abundance of alpha and beta T cell receptor subunit clonotypes to the total number of alpha, beta, gamma and delta T cell receptor subunit clonotypes is highly indicative of the renal transplant rejection status. The predicted ratio can be expressed as:

[0009] (Na + Np) : (Na + Np + Nd + Ny)

[0010] where Na is the number of unique alpha subunit sequences, Np is the number of unique beta subunit sequences, Nd is the number of unique delta subunit sequences, and Ny is the number of unique gamma subunit sequences. Thus, this diagnostic ratio represents the proportion of total subunit diversity (total unique clonotypes) that is occupied by alpha and beta subunit clonotype diversity.

[0011] In a first aspect, the scope of the present invention includes a new method of diagnosing an ongoing transplant rejection process in a subject by measuring the diversity of TCR subunits. The method of the present invention enables the determination of the rejection status, wherein the no rejection, AMR rejection and CMR rejection status can be determined. The method of the present invention can be applied to any transplant recipient, for example, a kidney transplant recipient.

[0012] In a second aspect, the scope of the present invention includes a new method of diagnosing an ongoing transplant rejection process in a subject by measuring the expression level of TCR subunits. For example, by comparison to the subunit expression levels determined for a stable subject, the measure of subunit expression levels can be used to determine the rejection status, wherein the no rejection, AMR rejection and CMR rejection status can be assessed. The method of the present invention can be applied to any transplant recipient, for example, a kidney transplant recipient.

[0013] In a third aspect, the scope of the present invention includes a new method of treating an ongoing rejection in a transplant recipient, wherein the transplant rejection status is determined and, following such determination, the subject is administered an appropriate treatment. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 . Figure 1 The subunit arrangement of the T cell receptor complex is described.

[0015] Figure 2 . Figure 2The sequence analysis workflow performed on samples from kidney recipients in Example 1 is described.

[0016] Figure 3A and 3B . Figure 3A is a box plot describing total reads, while Figure 3B is a box plot describing total clonotypes detected in the sample pool of kidney recipients representing stable subjects, subjects undergoing an AMR process, and subjects undergoing a CMR process.

[0017] Figure 4 . Figure 4 is a box plot describing the percentage of total reads for the alpha and beta subunits in kidney recipients.

[0018] Figure 5A , 5B , 5C, and 5D. Figure 5A , Figure 5B , Figure 5C and Figure 5D TCR subunit expression levels in samples from kidney recipients, including stable (STA), AMR, and CMR rejection status, are described. Figure 5A Expression of the TCR-alpha subunit is described. Figure 5A Expression of the TCR-alpha subunit is described. Figure 5B Expression of the TCR-beta subunit is described. Figure 5C Expression of the TCR-gamma subunit is described. Figure 5D Expression of the TCR-delta subunit is described. DETAILED DESCRIPTION

[0019] The various applications disclosed herein relate to assessing the rejection status of a transplant. The rejection status reflects whether there is a substantial, ongoing rejection process against the transplant or whether the transplant is stable. The rejection status also distinguishes between AMR and CMR rejection processes, which can be treated differently.

[0020] In a main embodiment, the transplant is a kidney transplant. However, the general methods of the application are applicable to diagnosing and treating rejection processes for other transplant types. The transplant can include any selected transplant type, for example, a type selected from the group consisting of organs, tissues, cells, hearts, lungs, livers, skin, corneas, intestines, pancreases, limbs, fingers, bones, ligaments, cartilage, and tendons. Reference to a transplant herein will include whole organs and portions thereof.

[0021] The transplant recipient subject can be a human, for example, the recipient can be a human patient receiving a transplant (e.g., a kidney transplant). In alternative embodiments, the subject can include a non-human animal, for example, a veterinary patient or a test animal. For convenience, the description provided herein is directed to human subjects. It will be appreciated that one skilled in the art can apply the methods and compositions described herein to non-human animals by utilizing TCR repertoire analysis methods appropriate for the chosen non-human animal species.

[0022] Several embodiments of the present application relate to assessing the rejection status of a transplant recipient. In some cases, the rejection status is "no rejection," i.e., the absence of a substantial immune response against the transplant.

[0023] In some cases, the rejection status is the occurrence of antibody-mediated rejection, which is any measure of antibody-mediated response against the transplant, antibody-mediated transplant failure (e.g., mediated by donor-specific antibodies (DSA) that bind to antigens present in the transplant (e.g., on the endothelium of the transplant)), and / or antibody-mediated injury and / or rejection.

[0024] In some cases, the rejection status is the occurrence of cell-mediated rejection, which is any measure of T cell-mediated response against the transplant, any injury or failure of the transplant mediated by T cells activated against the transplanted tissue, the presence of activated T cells against antigens of the transplant, and / or cell-mediated rejection.

[0025] Various embodiments of the present application include the assessment of T cell subunit repertoire diversity, particularly the number of different clonotypes for each subunit. These values are used in novel diagnostic ratios that are highly correlated with transplant rejection status.

[0026] The general method of the present application includes a method of assessing the transplant rejection status of a transplant recipient subject, comprising the steps of

[0027] obtaining a sample from the subject;

[0028] assessing the subject's (Na+Np):(Na+Np+Nd+Ny) ratio from the sample; and

[0029] determining the transplant rejection status of the subject by comparing the observed (Na+Np):(Na+Np+Nd+Ny) ratio to statistically relevant ratios indicative of normal, CMR, or AMR status.

[0030] The various methods disclosed herein are directed to assessing factors in a sample. The sample type selected can include any biological material containing T lymphocytes. Peripheral blood is a particularly convenient source that is readily extracted. Other sources of T cells include, for example, serum, tissue fluid, skin, buccal swab, saliva, urine, or tissue samples, such as tissue obtained by biopsy.

[0031] T cells can be obtained from a sample by methods known in the art, such as by fluorescence activated cell sorting, magnetic cell sorting, leucapheresis, or density gradient centrifugation. As known in the art, cells obtained by these methods can be further subdivided into functionally or developmentally distinct subpopulations.

[0032] The methods of the present application include measuring TCR receptor diversity for each of the alpha, beta, delta, and gamma subunits, i.e., the number of unique clonotypes for each subunit type. In the practice of the present application, any suitable method that provides a measure of subunit diversity can be employed.

[0033] From the isolated T cells, genetic material reflecting the diversity of subunit sequences can be obtained. Various methods known in the art utilize genomic DNA or RNA. DNA provides better stability and can enable more accurate quantification of low abundance TCR clonotypes. RNA (e.g., mRNA) reflects the actual TCRs in the cell and also provides information about the expression level of the genes selected for analysis.

[0034] The selected genetic material can be analyzed by various methods known in the art to assess the clonotypes present for each subunit type. The practitioner can select from a variety of diversity assessment tools, including single cell and bulk methods. Any TCR gene sequence reflecting subunit diversity can be selected, including analysis of the CDR3 region, for which a variety of protocols are known. Analysis of CDR1 and CDR2 sequences can also be performed. The practitioner can select from any number of established library preparation methods, such as multiplex PCR, targeted enrichment, and rapid amplification of 5' complementary DNA ends (5' RACE). Sequencing of the selected material can be performed using any sequencing platform known in the art, such as the Illumina (TM), Ion Torrent (TM), and Roche 454 (TM) platforms. Exemplary methods include, for example, DNA barcoding, exhaustive sequencing, 5' RACE or other unbiased sequencing techniques, spectratyping, non-parametric abundance estimators, parametric estimators, Chao 1 estimator, Chao 1-bc estimator, Chao 2 estimator, abundance-based coverage estimators, incidence-based coverage estimators, f-Poisson abundance model, and dilution curve.

[0035] In one embodiment, sequence diversity is assessed by RNA-Seq whole transcriptome shotgun sequencing, for example, as described in D. Bolotin et al. MiXCR: software for comprehensive adaptive immunity profiling, Nature methods, 2015.

[0036] Through the selected sequencing and analysis protocols, the number of clonotypes for each of the a, b, d, and g subunits is determined. Surprisingly, the inventors of the present disclosure have found that the state of rejection is highly correlated with the following ratios:

[0037] (Na+ Np) : (Na+ Np+ Nd+ Ng)

[0038] where Na is the number of unique a subunit sequences, Np is the number of unique b subunit sequences, Nd is the number of unique d subunit sequences, Ng is the number of unique g subunit sequences; and where the ratio can be expressed as a percentage value.

[0039] In particular, the inventors of the present disclosure have determined that a relatively lower value of the diagnostic ratio is indicative of a stable state, i.e., no substantial rejection process; an intermediate value of the diagnostic ratio is indicative of a CMR process in progress, while a relatively higher value of the diagnostic ratio is indicative of an AMR process in progress.

[0040] With this teaching, one skilled in the art can determine the ratios associated with stable, CMR, and AMR states of rejection for any type of transplant and target population. This is obtained for a selected transplant type, a selected recipient population, and a selected diversity assessment method, by analyzing samples from a pool of appropriately matched subjects (e.g., subjects receiving the same type of transplant, subjects with similar health and demographic factors, etc.) representing stable, AMR, CMR, and optionally mixed AMR / CMR states of rejection. For example, the threshold values can be determined by using a historical prospective study, a retrospective cohort study, or similar analysis.

[0041] From these measurements, a statistically validated threshold value for each rejection state category can be calculated. The statistically validated threshold value is a value or range of values of the diagnostic ratio that can be used to classify a subject according to the state of rejection. The statistical value of (Na+ Np) : (Na+ Np+ Nd+ Ng) for a target population can be used to develop a classifier or other predictive model. Subsequently, the resulting classifier can be used to assess the state of rejection of any individual in a population by measuring (Na+ Np) : (Na+ Np+ Nd+ Ng) in a sample from the individual.

[0042] Such a threshold or cutoff value can be selected by methods generally known in the art to provide a selected sensitivity and / or specificity of the rejection state assessment, for example, a sensitivity and / or specificity of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%. The designation of a rejection state can be selected at any selected level of confidence, for example, a probability of a particular rejection state greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In one embodiment, a rejection state comprises a probability score, i.e., a measure of the likelihood that a subject is in a particular rejection state.

[0043] Thresholds of the present application can be determined using any statistical method known in the art and suitable for associating a diagnostic ratio with a rejection state. Exemplary methods of generating thresholds include logistic regression, linear regression methods, machine learning classifiers such as random forests, support vector machines, and deep learning and neural network methods.

[0044] In one embodiment, the rejection state of a kidney transplant recipient can be assessed. In one embodiment, a stable kidney rejection state is determined for subjects with a diagnostic ratio value within 3% standard deviation of 91%. In one embodiment, a CMR rejection state is determined for subjects with a diagnostic ratio value within 2% standard deviation of 95%. In one embodiment, an AMR rejection state is determined for subjects with a diagnostic ratio value within 2% standard deviation of 97% or greater. Subjects with a diagnostic ratio value between selected thresholds for CMR and AMR states (e.g., within a range of 94% to 99%) can optionally be determined to have a mixed AMR / CMR rejection state. For example, in one embodiment, a stable state is determined for subjects with a (Nα+Nβ):(Nα+Nβ+Nδ+Nγ) ratio below 94%; a CMR rejection state is determined for subjects with a (Nα+Nβ):(Nα+Nβ+Nδ+Nγ) ratio between 94% and 97%; and an AMR rejection state is determined for subjects with a (Nα+Nβ):(Nα+Nβ+Nδ+Nγ) ratio greater than 97%.

[0045] While the present disclosure relates to a diagnostic ratio of (Nα+Nβ):(Nα+Nβ+Nδ+Nγ), it will be appreciated that the methods disclosed herein can be practiced using any diagnostically equivalent or highly correlated measure.

[0046] The diagnostic method can be performed as a routine post-transplant monitoring protocol performed on a regular basis. Early onset of rejection symptoms exhibited at the T cell receptor level can provide a means for early detection. In some embodiments, the (Nα+Nβ):(Nδ+Nγ) ratio assessment can be applied as a diagnostic tool when used on a subject exhibiting potential rejection symptoms. For example, in the case of kidney rejection, symptoms that can indicate the need for a diagnostic test can include fever, fatigue, pain and tenderness in the transplant area, decreased urine output, or elevated biomarkers of graft injury (such as creatinine).

[0047] Rejection status assessment by TCR subunit expression.

[0048] In another aspect, expression of individual TCR subunit types can be used to assess rejection status. As shown in Figures 5C and 5D, individual subunit expression profiles correlate with rejection status. The general method includes a method of assessing the transplant rejection status of a transplant recipient subject, comprising the steps of: Figure 5A , 5B

[0049] Obtaining a sample from the subject;

[0050] Assessing the expression of one or more of TCR-a, TCR-β, TCR-δ, and / or TCR-γ subunits in the subject by the sample;

[0051] Determining whether the subject has CMR, has AMR, or is stable by comparing the observed expression value(s) of the selected TCR subunit(s) to values defining stable, AMR, or CMR rejection status of members of the selected population.

[0052] The measured expression level can be any measure of gene expression, for example, the abundance of transcripts encoding the selected TCR subunit type, the average copy number of the selected TCR subunit type, the abundance of translated or functional TCR subunits of the selected subunit type, or any other measure of expression known in the art.

[0053] In one embodiment, the assessment of rejection status can be determined by comparing the expression level of one or more selected recipient subunits to a threshold or range determined for similar stable subjects. An "increase" and a "decrease" in expression of the selected subunit refers to the expression level relative to that determined for stable subjects of the same population. By this method, the rejection status can be determined by any of the following:

[0054] An increase in TCR a subunit expression indicates AMR;

[0055] A decrease in TCR-β subunit expression indicates AMR;

[0056] A decrease in TCR γ subunit expression indicates AMR;​

[0057] Reduced TCR-δ subunit expression indicates AMR.

[0058] Reduced TCR-β subunit expression indicates CMR;

[0059] Reduced TCR-δ subunit expression indicates CMR;

[0060] Reduced TCR-γ subunit expression indicates CMR;

[0061] When TCR-α expression is normal (same as level in stable subjects) but elevated, and TCR-β, TCR-δ, and / or TCR-γ expression is reduced, CMR is indicated;

[0062] When TCR-α expression is elevated and TCR-β, TCR-δ, and / or TCR-γ expression is reduced, AMR is indicated;

[0063] When TCR-δ expression is between levels observed for stable subjects and AMR subjects, CMR is indicated; and

[0064] Stable rejection status is indicated when expression of TCR-β, TCR-δ, or TCRγ is not increased or reduced relative to expression levels determined for stable subjects.

[0065] Therapeutic methods. In another aspect, the scope of the present application includes methods of treating a subject who can have a rejection process, comprising:

[0066] Obtaining a sample from a subject who is a member of a selected population;

[0067] Evaluating the subject's (Nα+Nβ):(Nα+Nβ+Nδ+Nγ) ratio by the sample; and

[0068] Determining whether the subject has CMR, has AMR, or is stable by comparing the observed (Nα+Nβ):(Nα+Nβ+Nδ+Nγ) to the ratios defining stable, CMR, or AMR rejection status of members of the selected population; and

[0069] If the subject is determined to have CMR rejection, administering a treatment appropriate for CMR; if the subject is determined to have AMR, administering a treatment appropriate for AMR; and if the subject is determined to be stable, not administering a treatment.

[0070] In one embodiment, the subject is a kidney recipient.

[0071] In another embodiment, the scope of the present application includes methods of treating a subject who can have a rejection process, comprising:

[0072] Samples were obtained from subjects who were members of a selected group;

[0073] The expression of one or more TCR subunits in the subjects was assessed through samples;

[0074] Whether a subject has CMR, AMR, or is stable is determined by comparing the observed expression values ​​of one or more selected TCR subunits with values ​​that define the stable, AMR, or CMR rejection status of members of the selected population; and

[0075] If a subject is determined to have CMR rejection, treatment appropriate for CMR is administered; if a subject is determined to have AMR, treatment appropriate for AMR is administered; and if a subject is determined to be stable, no treatment is administered.

[0076] In one implementation, the expression of one or more TCR subunits includes the expression of TCR-α, TCR-β, TCR-δ, and TCR-γ. In one implementation, the subject is a kidney transplant recipient. In one implementation, a value defining stable rejection status is... Figure 5A , 5B The values ​​described in 5C and 5D.

[0077] If CMR rejection is detected, appropriate treatments to alleviate CMR can be administered, such as corticosteroids and T-cell depletion agents. If AMR rejection is detected, appropriate treatments to treat AMR can be administered, such as plasma exchange, intravenous immunoglobulin administration, or B-cell depletion therapy.

[0078] In an alternative implementation, the treatment method may be used to distinguish normal subjects from subjects with any form of rejection, and if a subject is determined to be experiencing a rejection process, a treatment suitable for all forms of transplant rejection associated with the subject may be administered.

[0079] Example

[0080] Example 1. Development of a diagnostic ratio for kidney transplant recipients. Peripheral blood was collected from 37 human kidney transplant recipients. Twelve samples were obtained from stable recipients without rejection symptoms. Thirteen samples were obtained from recipients identified as undergoing the CMR process, prior to any additional immunosuppressive intervention. Twelve samples were obtained from recipients identified as undergoing the AMR process, prior to any additional immunosuppressive intervention. Sequence diversity of the TCRα, β, δ, and γ subunit CDR3 sequences was determined using RNA-Seq methods performed using the MixCR software (MiLaboratory LLC) workflow, such as... Figure 2For stable subjects, an average of 7,234 reads and 497 total clones (143 alpha, 164 beta, 8 delta, and 68 gamma) were observed. For CRM subjects, an average of 5,059 reads and 237 clones (63 alpha, 81 beta, 2 delta, and 34 gamma) were observed. For AMR subjects, an average of 8,090 reads and 286 total clones (75 alpha, 79 beta, 3 delta, and 26 gamma) were observed.

[0081] The average of the (Na+ Np) : (Na+ Np+ Nd+ Ny) diagnostic ratio was 91% for stable subjects, 95% for CMR subjects, and 97% for AMR subjects.

[0082] All patents, patent applications, and publications cited in this specification are incorporated by reference herein as if each individual patent application, or publication were specifically and individually indicated to be incorporated by reference. The disclosed embodiments are presented for purposes of illustration and not limitation. While the application has been described with reference to the described embodiments, those skilled in the art will understand that modifications can be made to the described embodiments without departing from the spirit and scope of the application.

Claims

1. Use of an agent that evaluates the (Nα+Nβ):(Nα+Nβ+Nδ+Nγ) ratio in a sample obtained from a transplant recipient subject, wherein Nα is the number of unique α subunit sequences, Nβ is the number of unique β subunit sequences, Nδ is the number of unique δ subunit sequences, Nγ is the number of unique γ subunit sequences, in the manufacture of a diagnostic agent for evaluating the status of rejection in said subject; wherein said diagnostic agent determines the transplant rejection status of said subject by comparing the observed (Nα+Nβ):(Nα+Nβ+Nδ+Nγ) ratio with status threshold values indicative of normal, cell-mediated rejection or antibody-mediated rejection; and wherein the relevant statistical thresholds are: a steady state is determined for subjects with a (Nα+Nβ):(Nα+Nβ+Nδ+Nγ) ratio lower than 94%; a cell-mediated rejection status is determined for subjects with a (Nα+Nβ):(Nα+Nβ+Nδ+Nγ) ratio between 94% and 97%; and an antibody-mediated rejection status is determined for subjects with a (Nα+Nβ):(Nα+Nβ+Nδ+Nγ) ratio greater than 97%.

2. Use according to claim 1, wherein said transplant recipient is a kidney transplant recipient.

3. Use according to claim 1, wherein said transplant recipient is a recipient of a graft selected from the group consisting of organs, tissues and cells.

4. Use according to claim 1, wherein said transplant recipient is a recipient of a graft selected from the group consisting of heart, lung, liver, skin, cornea, intestine, pancreas, limb, bone, ligament and tendon.

5. Use according to claim 1, wherein said transplant recipient is a recipient of a graft selected from the group consisting of fingers and cartilage.

6. Use according to claim 1, wherein said agent evaluates Nα, Nβ, Nδ and Nγ sequence diversity by RNA-Seq. ​

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