Methods of predicting relapse post hematopoietic stem-cell transplantation and methods of treatment

Abstract

Methods of predicting relapse in a patient post HSCT, and methods of treatment, the methods including obtaining a bone marrow sample from the patient post- transplantation; and determining TCR clonal diversity of a population of T cells within the bone marrow sample; wherein TCR clonal diversity is indicative of risk of relapse.

Description

METHODS OF PREDICTING RELAPSE POST HEMATOPOIETIC STEM-CELL TRANSPLANTATION AND METHODS OF TREATMENTCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 546,865, filed on November 1, 2023, the disclosure of which is incorporated by reference herein in its entirety.BACKGROUND

[0003] Cancer treatment protocols involve, for example, chemotherapy and radiotherapy, both of which suppress tumors by directly killing malignant cells, as well as more recently, immune checkpoint blockade (ICB)-based therapy, which rescues the antitumor activity of T cells through the targeted blockade of checkpoints (i.e., membrane proteins). Stem-cell transplantation is a targeted, regenerative approach that goes beyond the scope of standard chemotherapy, radiation, and invasive surgeries.

[0004] Acute myeloid leukemia (AML) is an aggressive hematologic malignancy affecting both children and adults. Allogeneic or donor hematopoietic stem cell transplantation (HSCT) is the only curative treatment; however, the likelihood of disease relapse after transplant can be as high as 50% resulting in dismal long-term survival. The first six months post-transplant are important for reconstitution of a healthy blood system and also the time period when relapse is most likely to occur. Hence, detecting early prognostic signs of relapse is important but thus far has remained challenging. Stratifying AML patients at risk of relapse early after HSCT could provide valuable information by nominating patients for additional treatment to prevent relapse.

[0005] In stem-cell therapies for leukemia involving hematopoietic stem-cell transplantation, Graft Versus Leukemia (GVL) occurs when the donor T-lymphocytes in the graft recognize antigens on the leukemic blast cell as foreign and initiate immune-mediated clearanceof the malignant cell. GVL constitutes an important part of the antileukemic effect of transplantation.

[0006] Certain patients have genetic mutations that allow for the evasion of GVL, evidenced by the increased risk of relapse in these patients. For example, TP53 mutations, which are associated with unfavorable outcomes in various cancer types, confer poor prognosis in acute myeloid leukemia and other hematologic malignancies. Even with HSCT, the risk of relapse approximates 80%, with dismal long-term survival in these patients

[0007] It is an unmet need in the field of transplantation to develop better tools for early prediction of relapse post-transplantation. The current guidelines for HSCT monitoring include regular complete blood count (CBC) checkups, measurement of chimerism studies, and proceeding with a bone marrow biopsy if any anomalies arise in the counts. This approach, however, often detects relapses too late, leaving physicians with limited treatment options.SUMMARY

[0008] There are provided methods of predicting relapse in patients post-HSCT. Such methods involve evaluating a population of T cells present in bone marrow of the patients, and are based on the discovery that T cell receptor (TCR) clonal diversity (i.e., TCR diversity) is an early prognostic indicator for relapse post-HSCT, particularly for AML. Specifically, low TCR diversity in the early post-HSCT period indicates an increased likelihood that a patient, particularly an AML patient, will experience relapse.

[0009] One such method of predicting relapse in a patient post HSCT includes: obtaining a bone marrow sample from the patient post-transplantation; and determining TCR clonal diversity of a population of T cells within the bone marrow sample; wherein TCR clonal diversity is indicative of risk of relapse.

[0010] Another method of predicting relapse in a patient post HSCT includes: obtaining a bone marrow sample from the patient post-transplantation; and determining TCR clonal diversity of a population of T cells within the bone marrow sample; wherein TCR clonal diversity is indicative of risk of relapse, and wherein the patient received a conventional hematopoietic stemcell transplant.

[0011] Another method of predicting relapse in a patient post HSCT includes: obtaining a bone marrow sample from the patient at 3-5 months post-transplantation; and determining TCRclonal diversity of a population of T cells within the bone marrow sample; wherein if the TCR clonal diversity is low, the patient is at high risk for relapse and the patient is identified for preemptive intervention.

[0012] There are also provided methods of treatment. That is, a method of treating a patientHSCT is provided that includes predicting relapse in the patient comprising one of the methods described herein; and administering a treatment to the patient (preferably, treating the patient with a pre-emptive intervention).

[0013] Another method of treating a patient post HSCT includes: predicting relapse in the patient comprising: obtaining a bone marrow sample from the patient at 3-5 months posttransplantation; and determining TCR clonal diversity of a population of T cells within the bone marrow sample; wherein if the TCR clonal diversity is low, the patient is at high risk for relapse and the patient is identified for pre-emptive intervention; and administering a treatment to the patient (preferably, treating the patient with a pre-emptive intervention).

[0014] The above summary is not intended to describe each disclosed embodiment or every implementation. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples may be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. Thus, the scope of the present disclosure should not be limited to the specific illustrative embodiments described herein, but rather extends at least to the embodiments described by the language of the claims, and the equivalents of those embodiments. Any of the elements that are positively recited in this specification as alternatives may be explicitly included in the claims or excluded from the claims, in any combination as desired. Although various theories and possible mechanisms may have been discussed herein, in no event should such discussions serve to limit the claimable subject matter.BRIEF DESCRIPTION OF THE FIGURES

[0015] Embodiments will be described, by way of example, with reference to the following drawings. It is to be noted, however, that the drawings illustrate only selected embodiments and are therefore not to be considered limiting of scope. Additional and equally effective embodiments and applications of the present disclosure exist.

[0016] FIG. 1 shows a Swimmer Plot of the clinical course of eight patients assigned to cohorts 1 and 2, including time of HSCT, relapse, and death. Samples were taken before HSCT (Pre), during remission (Reml and Rem 2), and after relapse (Rell). Samples were analyzed with single-cell RNA and TCR sequencing. The gray box indicates the time frame at which differences in T cell CD4 / CD8 ratio and TCR diversity were found.

[0017] Figure 2A. A Uniform Manifold Approximation and Projection (UMAP) shows dimensionality reduction of all T cells in cohorts. T cells are shaded according to cell type annotation. Treg: T regulatory cell, Mem: memory, Term Exh: terminally exhausted, NKT: NKT cells, yb: yb T lymphocytes, NKT: natural killer T lymphocytes.

[0018] Figure 2B. A UMAP shows the same T cells from FIG. 2 A (sub sampled to 513 cells per sample), shaded and connected by clonotype (i.e., cells with the same TCR sequence). For Cohort 1, the top 172 cells (ranked by clone size) are shaded, comprising 56 clonotypes (more diverse / smaller clone size).

[0019] Figure 2C. A UMAP shows the same T cells from FIG. 2A (subsampled to 513 cells per sample), shaded and connected by clonotype (i.e., cells with the same TCR sequence). For Cohort 2, the top 172 cells (ranked by clone size) are shaded, comprising 5 clonotypes (less diverse / larger clone size).DETAILED DESCRIPTIONDefinitions

[0020] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs and as commonly used in the art to which this application belongs. In case of conflict, the present specification, including definitions, will control.

[0021] Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. See, e g., www.informatics.jax.org / mgihome / nomen / gene.shtml and www.genenames.org / . Purification techniques are performed according to manufacturer’s specifications, or as describedherein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0022] Herein, “patient” refers to any human being having a hematologic malignancy or having had a hematologic malignancy prior to hematopoietic stem-cell transplantation (HSCT) and / or being treated for or having been treated for a hematologic malignancy.

[0023] Herein, “predicting” relapse includes predicting the likelihood of the recurrence of a hematologic malignancy post HSCT. Evidence of such relapse includes, for example, morphologic relapse and / or molecular relapse, which are well-defined in the practice of transplantation.

[0024] Herein, “conventional transplant” refers to an allogeneic hematopoietic stem-cell transplant from which T cells have not been depleted and / or a transplant that is not derived from cord blood. That is, the conventional hematopoietic stem-cell transplant referenced herein is not the T-cell depleted or haplo-cord (alternative) transplant used in Yew et al., Bone Marrow Transplantation (2015), 50, 1227-1234.

[0025] Herein, a “signature” encompasses genes whose expression profile or whose occurrence is associated with a specific cell type, subtype, or cell state of a specific cell type or subtype within a population of monocytes. Often in the art, the terms “signature,” “expression profile,” and “expression program” are used interchangeably. A “gene signature” as used herein, may refer to any set of up- and / or down-regulated genes that are representative of a cell type or subtype. A gene signature as used herein, may also refer to any set of up- and / or down-regulated genes between different cells or cell (sub)populations derived from a gene-expression profile. For example, a gene signature may comprise a list of genes differentially expressed in a distinction of interest.

[0026] Herein, it will be understood by one of skill in the art that “treating” encompasses enhancing treatment, or improving treatment efficacy. Treatment may include inhibition of tumor regression as well as inhibition of tumor growth, metastasis, or tumor cell proliferation, or inhibition or reduction of otherwise deleterious effects associated with a tumor. Efficacy of treatment may be determined by any known method for diagnosing or treating the particular cancer. It will be appreciated that, although not precluded, “treating” does not require that the symptoms associated with the condition being treated are completely eliminated. As used herein“treatment” or “treating,” includes any beneficial or desirable effect on the symptoms or pathology of the condition being treated and may include even minimal reductions in one or more measurable markers of the conditions.

[0027] The term “administer,” “administering,” or “administration” refers to the act of the attending physician or caregiver, prescribing the agent for administration and thereby causing the application of an agent to a patient, through ingestion, inhalation, infusion, injection, or any other means, whether self-administered or administered by a clinician or other qualified care giver.

[0028] In this application, terms such as “a,” “an,” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terms “a,” “an,” and “the” are used interchangeably with the term “at least one.” The phrases “at least one of’ and “comprises at least one of’ followed by a list refers to any one of the items in the list and any combination of two or more items in the list.

[0029] As used herein, the term “or” is generally employed in its usual sense including “and / or” unless the content clearly dictates otherwise. The term “and / or” means one or all of the listed elements or a combination of any two or more of the listed elements. Thus, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include “A and / or B and / or C” and to thus encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0030] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.

[0031] The words “preferred” and “preferably” refer to embodiments of the disclosure that may afford certain benefits, under certain circumstances. However, other claims may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred claims does not imply that other claims are not useful, and is not intended to exclude such other claims from the scope of the disclosure.

[0032] Herein, any of the elements or combinations of elements that are recited in this specification in open-ended language (e.g., comprise and derivatives thereof), are considered to additionally be recited in closed-ended language (e.g., consist and derivatives thereof) and inpartially closed-ended language (e.g., consist essentially, and derivatives thereof). That is, it is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also contemplated. In other words, if an embodiment comprises A, B and C, embodiments consisting essentially of A, B and C are also contemplated, as are embodiments consisting of A, B and C.

[0033] Groupings of alternative elements or embodiments disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found therein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0034] Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the invention. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0035] The term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” may be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. All numerical values provided herein are modified by the term about.

[0036] Ranges provided herein are understood to be shorthand for all values within the range, including fractions / decimals. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0037] The immune system is a vital component in preventing and eliminating cancer. Cytotoxic T cells (CTL) and natural killer cells (NK) have potent ability to kill tumor cells and numerous studies show that effector T cells at the tumor site predict favorable outcome across many cancers. The T cell receptor (TCR) is a molecule found on the surface of T lymphocytes (i.e., T cells) that is responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules on target cells. The TCR is a heterodimer composed of two different protein chains. In most T cells (about 95%), these two protein chains are termed the alpha (a) and beta (P) chains. The genetically programmed variability of TCRs underlies immune recognition of diverse antigens. The selection of antigen-specific T cells under different pressures — such as infections, vaccines, autoimmune diseases, allergy, and tumors — can dramatically alter the repertoire in individuals either transiently or permanently.

[0038] There are provided herein methods of predicting relapse in patients post hematopoietic stem-cell transplantation (HSCT). Such methods include: obtaining a bone marrow sample from the patient post-transplantation; and determining TCR clonal diversity of a population of T cells within the bone marrow sample; wherein TCR clonal diversity is indicative of risk of relapse.

[0039] Advantageously, the present methods provide an earlier indication that a patient is likely to experience relapse, even when the blood counts and vitals are stable. The methods are based on the discovery that T cell receptor (TCR) diversity is an early prognostic indicator for relapse post-HSCT, particularly for AML. Specifically, low TCR diversity, preferably around at least two months post-HSCT, indicates an increased likelihood that a patient will experience relapse. Given the uncertainty that physicians face about a patient’s outlook after HSCT, there is a pressing need for enhanced monitoring methods. Employing TCR sequencing in the early months following transplantation helps in identifying patients who would benefit from additional treatments, with the goal of preventing relapse and increasing long-term survival.

[0040] With respect to TCR repertoire diversity, in Yew et al. (Bone Marrow Transplantation (2015), 50, 1227-1234), there is a statement that “higher diversity early after transplant possibly implies lower risks of . . . relapse following the HSCT transplantation” (Abstract, page 1120); however, the data presented does not support this statement. In the carryover paragraph at pages 1229-1230, referring to Figures 4a and 4b, it is stated that “weexamined the correlation of the diversity index of TCR repertoire of samples collected at day 50, 75-100 or 1 year with relapse status. The TCR diversity in relapsed patients was not significantly different from that of non-relapsed patients . . . .” And, as noted in the first column at page 1230, the patients were separated into a non-GVHD group and a GVHD (graft-vs-host disease) group, with opposite results. Referring to Figures 4c and 4d, it is stated that “in the non-GVHD group, we found that the diversity index was significantly higher in non-relapsed patients compared to that in relapsed patients.” Referring to Figures 4e ad 4f, “relapsed patients in the GVHD group showed higher TCRA diversity . . . compared with the non-relapsed patients . . . while we only observed a similar trend in the case of TCRB for the relapsed vs non-relapsed patients.” This data does not support a broad conclusion that higher TCR diversity early after transplant implies a lower risk of relapse, or that a lower TCR diversity early after transplant indicates a higher risk of relapse.

[0041] Significantly, cancer patients tested by the methods described herein are typically in remission, so there are no (or very few) cancer cells to evaluate. Furthermore, bone marrow, rather than peripheral blood, is tested in the present methods. Bone marrow contains both hematopoietic and n on-hematopoietic cells (i.e., stromal cells), making it a prime site for cell isolation. Hematopoietic cells comprise blood ceils from either the lymphoid or myeloid lineage. Stromal cells (osteoblasts, endothelial cells, pericytes, etc.) are integral in maintaining the bone marrow niche required for hematopoiesis. Bone marrow isolation also typically favors higher cell yields compared to peripheral blood isolation.

[0042] In certain of the methods, the patient has or has had acute leukemia, bone marrow failure syndrome, or Myelodysplastic Syndrome (MDS). In certain of the methods, the patient has a TP53 mutation, and preferably has or has had TP53-mutated acute myeloid leukemia (AML). It is also believed that similar effect, if not the same effect, will occur for patients with TP53-wild- type AML.

[0043] In certain of the methods, the patient received a conventional hematopoietic stemcell (HSC) transplant. That is, the transplant is not from an alternative donor source. In certain methods, the HSC transplant is not a transplant that includes a T-cell depleted transplant or a haplo- cord transplant.

[0044] Early prediction of relapse is important. If treatment is started when there is a lower disease burden there may be better outcomes (e g., there may be a higher success rate and / or maybe more tolerable for a patient). Using a biomarker / immune signature as described herein to identify patients at high risk for relapse in the post-transplantation setting would allow risk-adapted treatments and focus interventions on the most appropriate patients while minimizing toxicity for those who do not need this intervention.

[0045] In certain of the methods, the bone marrow sample is obtained (e.g., isolated) at least 2 months, at least 2.5 months, or at least 3 months, post-transplantation. In certain methods, the bone marrow sample is obtained (e.g., isolated) up to 6 months, up to 5.5 months, or up to 5 months, post-transplantation. In certain embodiments, obtaining (e.g., isolating) a bone marrow sample from the patient occurs at 3-5 months post-transplantation. Obtaining a bone marrow sample from a patient uses standard techniques used in conventional HSCT.

[0046] In the methods, TCR clonal diversity of a population of T cells within a bone marrow sample is indicative of risk of relapse. TCR clonal diversity can be determined using conventional techniques involving sorting and / or isolating T cells using known systems and methods, sequencing using known systems and methods, and determining the TCR clonal diversity using known systems and methods.

[0047] T cell sorting / isolation typically involves the use of flow cytometry, although other techniques can be used if desired. Flow cytometry uses surface markers, such as CD3+, as canonical identifiers for T cells. Cells flow through a laser beam, and detectors capture light scatter and fluorescence emissions, facilitating the precise identification and sorting and / or isolating of specific cell types. Following this step, cells can be separated into distinct groups, such as T cells or viable mononuclear cells.

[0048] T cell receptor (TCR) diversity can be determined by constructing a library' of TCR sequences and sequencing. The library; of TCR sequences can be constructed using DNA or RNA, preferably RNA. In certain methods, evaluating the T cells to determine their T cell receptor (TCR) clonal diversity includes using single-cell RNA sequencing or bulk sequencing of the T cells to evaluate their gene expression profile. Also, known methods of deriving genetic information from RNA-sequencing libraries (e.g., a sequencing technique used in U.S. Pat. Pub. No. 2020 / 0248175 (van Galen et al.)) can be used in determining TCR clonal diversity.

[0049] More specifically, RNA is released through lysis using standard techniques. The extracted RNA is then reversed and transcribed into complementary DNA (cDNA), which servesas the basis for subsequent amplification via polymerase chain reaction (PCR). The amplification process typically focuses solely on the CDR3 region of the T cell receptors, targeting the variable regions of T Cell Receptors' alpha and beta chains in TCR sequencing. Amplified cDNA is transformed into a sequencing library and subsequently loaded onto a sequencing instrument, yielding detailed genetic and transcriptomic information for each individual cell.

[0050] A higher TCR clonal diversity correlates to a lower chance of the patient relapsing due to the immune system having a better chance of working. Thus, in certain methods, if the TCR clonal diversity is low, the patient is at high risk for relapse and the patient is identified for preemptive intervention.

[0051] In certain methods, the TCR clonal diversity is determined qualitatively via comparison with the TCR clonal diversity of a non-relapsed patient (i.e., a patient in remission). In certain methods, the qualitative determination is made by overlaying a UMAP of the TCR diversity from the patient with a UMAP of the TCR diversity from a control sample. A control sample can be obtained from one or more patients in remission post HSCT.

[0052] In certain methods, the TCR clonal diversity is quantitatively determined using a diversity index. A diversity index is a quantitative measure that reflects how many different types (such as species) there are in a dataset (a community), and that can simultaneously take into account the relations among the individuals distributed among those types, such as richness, divergence, or evenness. These indices are statistical representations of biodiversity in different aspects.

[0053] Many diversity indices used in assessing T-cell receptor (TCR) clonal diversity are mathematically derived from information theory, a concept widely employed in the field of ecology to quantify biodiversity in ecosystems. Within T-cell repertoires, diversity accounts for the clonal composition, including the number of unique TCR sequences, referred to as “richness,” as well as the distribution spectrum of these sequences, known as “relative abundance.” This diversity metric reflects the degree of uncertainty in sorting a TCR sequence from a repertoire and associating it with a specific T-cell clone (i.e., a unique TCR sequence). A variety of diversity indices may be used. One of the many of diversity indices, the Simpson’s Index (also referred to as the “Simpson Index”) or the Inverse Simpson’s Index (also referred to as the “Inverse Simpson Index) is particularly favored in the field of studying immune reconstruction, as it focuses on dominant TCR clones and potential clonal expansions within the T-cell population.

[0054] The Inverse Simpson’s Index can be calculated based on the following equation:wherein K is the total number of clonotypes, in is the number of sequences belonging to the i-th type and N is the total number of sequences for which clonotypes are determined. Statistical analysis of TCR sequences to determine a diversity index can be done using conventional algorithms.

[0055] Preferably, an Inverse Simpson’s Index is used as a quantitative indicator of TCR clonal diversity. In certain methods, an inverse Simpson’s index of a pre-specified threshold (e.g., 100 or less) indicates low TCR clonal diversity.

[0056] As noted above, early prediction of relapse is important and can lead to pre-emptive intervention. Thus, herein certain methods involve causing the patient to be treated with a preemptive intervention. The pre-emptive intervention may be selected from rapid taper of immunosuppression, donor lymphocyte infusion, therapy with one or more immune-boosting agents, such as hypomethylating agents, or using one or more targeted therapies.

[0057] More specifically, in the setting of early prediction of relapse after transplant, one might be able to: (1) reduce immunosuppression earlier than planned, leading to a better graft- versus-leukemia effect; (2) administer a donor lymphocyte infusion (DLI) to add additional graft- versus-leukemia T cells and other immune cells; (3) administer an immune-boosting therapy, like a T cell checkpoint blockade inhibitor (e.g., anti-CTLA4 antibody); and / or (4) start anti-AML chemotherapy, targeted therapy, or immunotherapy to intercept relapse before it becomes overt disease. The specific methods for implementing such pre-emptive interventions are well-known to those of skill in the art or in cancer treatment.

[0058] Rapid taper of immunosuppression: The graft-versus tumor (GVT) effect is thought to be mediated by donor T-lymphocytes. Post allogeneic transplantation, donor T-Lymphocytes are suppressed with calcineurin inhibitors such as tacrolimus and other agents to prevent graft- versus host disease while still maintaining a GVT effect. For patients where disease markers such as minimal residual disease are identified early after transplant, rapid taper of immunosuppression can activate the donor graft and amplify the GVT effect. In this case, identification of an immunesignature associated with relapse (low TCR diversity) could prompt similar rapid taper of immunosuppression to prevent or delay relapse. Rapid taper of immunosuppression has successfully been used to treat relapse post allotransplant in the reduced intensity setting (Kekre, N., et al. (2015), Haematologica 100, 1222-1227. 10.3324 / haematol.2015.129650).

[0059] Donor lymphocyte infusions (DLI): Unstimulated T-lymphocytes can be obtained from the donor and be given to the recipient when relapse occurs or sometimes to boost T-cell reconstitution. DLI has been shown to reverse T-cell exhaustion phenotypes which are associated with relapse. (Bachireddy, P. et al. (2014), Blood 123, 1412-1421. 10.1182 / blood-2013-08- 523001.) Reversal of TCR diversity with the use of DLI in chronic myeloid leukemia patients post allogeneic transplantation is known. Once a biomarker / immune signature that predicts relapse is identified (low TCR diversity), pre-emptive DLI could be used to re-invigorate T-cells and prevent or delay relapse.

[0060] Therapy with immune-boosting agents, such as hypomethylating agents, and targeted therapies: Maintenance therapy with hypomethylating agents in combination with TP53 targeted agents can reduce relapse rates in TP53-mutated acute myeloid leukemia and myelodysplastic syndrome from 80% historically to 38.3%. (Mishra, A. et al. (2022), JCO 40, 3985-3993. 10.1200 / JC0.22.00181.) Hypomethylating agents alone in an unselected population, however, has not been shown to improve survival. (Oran, B. et al., Blood Adv 4, 5580-5588. 10.1182 / bloodadvances.2020002544.)ILLUSTRATIVE EMBODIMENTS

[0061] Embodiment 1 is a method of treating a patient post hematopoietic stem-cell transplantation (HSCT), the method including: obtaining a bone marrow sample from the patient at least 2 months post-transplantation; determining T Cell Receptor (TCR) clonal diversity of a population of T cells within the bone marrow sample; wherein TCR clonal diversity is indicative of risk of relapse; and administering a treatment to the patient (preferably, treating the patient with a pre-emptive intervention).

[0062] Embodiment 2 is the method of embodiment 1, wherein the bone marrow sample is isolated at least 2.5 months post-transplantation. Embodiment 3 is the method of embodiment2, wherein the bone marrow sample is isolated at least 3 months post-transplantation. Embodiment 4 is the method of any preceding embodiment, wherein the bone marrow sample is isolated up to 6 months post-transplantation. Embodiment 5 is the method of any preceding embodiment, wherein the bone marrow sample is isolated up to 5 months post-transplantation. Embodiment 6 is the method of any preceding embodiment, wherein the sample includes CD3+ T cells.

[0063] Embodiment 7 is the method of any preceding embodiment, wherein if the TCR clonal diversity is low, the patient is at high risk for relapse. Embodiment 8 is the method of any preceding embodiment, wherein the treatment includes a pre-emptive intervention. Embodiment 9 is the method of embodiment 8, wherein the pre-emptive intervention is selected from rapid taper of immunosuppression, donor lymphocyte infusion, therapy with one or more immune-boosting agents, and one or more targeted therapies.

[0064] Embodiment 10 is the method of any preceding embodiment, wherein the patient received a conventional hematopoietic stem-cell transplant. Embodiment 11 is the method of embodiment 10, wherein the conventional hematopoietic stem-cell transplant is not a T-cell depleted transplant or a haplo-cord transplant.

[0065] Embodiment 12 is the method of any preceding embodiment, wherein the patient has or has had acute leukemia, bone marrow failure syndrome, or Myelodysplastic Syndrome (MDS). Embodiment 13 is the method of any preceding embodiment, wherein the patient has a TP53 mutation. Embodiment 14 is the method of embodiment 13, wherein the patient has or has had TP53-mutated acute myeloid leukemia.

[0066] Embodiment 15 is the method of any preceding embodiment, wherein the TCR clonal diversity is determined qualitatively via comparison with the TCR clonal diversity of a nonrelapsed patient. Embodiment 16 is the method of embodiment 15, wherein the qualitative determination is made by overlaying a Uniform Manifold Approximation and Projection (UMAP) of the TCR diversity from the patient with a UMAP of the TCR diversity from a control sample. Embodiment 17 is the method of any preceding embodiment wherein the TCR clonal diversity is determined quantitatively using a diversity index. Embodiment 18 is the method of embodiment 17, wherein an Inverse Simpson’s Index is used as a quantitative indicator of TCR clonal diversity. Embodiment 19 is the method of any preceding embodiment, wherein evaluating the T cells to determine their T cell receptor (TCR) clonal diversity includes single-cell RNA sequencing.Embodiment 20 is the method of embodiment 19, wherein single-cell RNA sequencing includes TCR sequencing.

[0067] Embodiment 21 is a method of predicting relapse in a patient post hematopoietic stem-cell transplantation (HSCT), the method including: obtaining a bone marrow sample from the patient at least 2 months post-transplantation; and determining TCR clonal diversity of a population of T cells within the bone marrow sample; wherein TCR clonal diversity is indicative of risk of relapse.

[0068] Embodiment 22 is the method of any preceding embodiment, wherein the bone marrow sample is isolated at least 2.5 months post-transplantation. Embodiment 23 is the method of embodiment 22, wherein the bone marrow sample is isolated at least 3 months posttransplantation. Embodiment 24 is the method of any preceding embodiment, wherein the bone marrow sample is isolated up to 6 months post-transplantation. Embodiment 25 is the method of embodiment 24, wherein the bone marrow sample is isolated up to 5.5 months post-transplantation. Embodiment 26 is the method of embodiment 25, wherein the bone marrow sample is isolated up to 5 months post-transplantation.

[0069] Embodiment 27 is a method of predicting relapse in a patient post hematopoietic stem-cell transplantation (HSCT), the method including: obtaining a bone marrow sample from the patient at 3-5 months post-transplantation; and determining TCR clonal diversity of a population of T cells within the bone marrow sample; wherein if the TCR clonal diversity is low, the patient is at high risk for relapse and the patient is identified for pre-emptive intervention.

[0070] Embodiment 28 is a method of predicting relapse in a patient post hematopoietic stem-cell transplantation (HSCT), the method including: obtaining a bone marrow sample from the patient post-transplantation; and determining TCR clonal diversity of a population of T cells within the bone marrow sample; wherein TCR clonal diversity is indicative of risk of relapse, and wherein the patient received a conventional hematopoietic stem-cell transplant.

[0071] Embodiment 29 is the method of embodiment 28, wherein the conventional hematopoietic stem-cell transplant is not a T-cell depleted transplant or a haplo-cord transplant. Embodiment 30 is the method of any of the preceding embodiments, wherein the patient has or has had acute leukemia, bone marrow failure syndrome, or Myelodysplastic Syndrome (MDS). Embodiment 31 is the method of any preceding embodiment, wherein the patient has a TP53mutation. Embodiment 32 is the method of any preceding embodiment, wherein the patient has or has had TP53-mutated acute myeloid leukemia.

[0072] Embodiment 33 is the method of any preceding embodiment wherein the TCR clonal diversity is determined qualitatively via comparison with the TCR clonal diversity of nonrelapsed patient. Embodiment 34 is the method of embodiment 10, wherein the qualitative determination is made by overlaying a UMAP of the TCR diversity from the patient with a UMAP of the TCR diversity from a control sample. Embodiment 35 is the method of any preceding embodiment wherein the TCR clonal diversity is determine quantitatively using a diversity index. Embodiment 36 is the method of embodiment 35, wherein an Inverse Simpson Index is used as a quantitative indicator of TCR clonal diversity.

[0073] Embodiment 37 is the method of any preceding embodiment, wherein evaluating the T cells to determine their T cell receptor (TCR) clonal diversity includes single-cell RNA sequencing.

[0074] Embodiment 38 is the method of any of any preceding embodiment, further including causing the patient to be treated with a pre-emptive intervention. Embodiment 39 is the method of embodiment 38, wherein the pre-emptive intervention is selected from rapid taper of immunosuppression, donor lymphocyte infusion, therapy with one or more immune-boosting agents, and one or more targeted therapies.

[0075] Embodiment 40 is a method of treating a patient post hematopoietic stem-cell transplantation (HSCT), the method including: predicting relapse in the patient including any of the methods of any preceding embodiment: and treating the patient with a pre-emptive intervention. Embodiment 41 is a method of treating a patient post hematopoietic stem-cell transplantation (HSCT), the method including: predicting relapse in the patient including: obtaining a bone marrow sample from the patient at 3-5 months post-transplantation; and determining TCR clonal diversity of a population of T cells within the bone marrow sample; wherein if the TCR clonal diversity is low, the patient is at high risk for relapse and the patient is identified for pre-emptive intervention; and treating the patient with a pre-emptive intervention. Embodiment 42 is the method of embodiment 40 or 41, wherein the pre-emptive intervention is selected from rapid taper of immunosuppression, donor lymphocyte infusion, therapy with one or more immune-boosting agents, and one or more targeted therapies.EXAMPLES

[0076] The examples of this section are merely for illustrative purposes and are not meant to be overly limiting on the scope of the appended claims. Notwithstanding that the numerical ranges and parameters setting forth the broad scope are approximations, the numerical values set forth in the specific examples are reported as precisely as possible.Methods

[0077] Twenty-six (26) longitudinal bone marrow aspirates and 1 peripheral blood sample were collected from 12 patients with TP 53 -mutated AML who underwent HSCT. All patients received HLA-matched T-cell replete HSCT in morphologic remission, with standard tacrolimus / methotrexate-based prophylaxis and peripheral blood stem cells. The patients were categorized into two cohorts based on their treatment response: long-term remission (>3.5 years, cohort 1, n=4); or relapse (cohort 2, n=8). Cell isolation involved gentle thawing of frozen bone marrow samples to preserve cell viability. Subsequently, the thawed cells were thoroughly washed and resuspended in a suitable buffer or stained to enable their identification during cytometry analysis. Specifically, flow cytometry was used based on identifying the surface marker CD3+. Following this, cells were separated into distinct groups, such as T cells and viable mononuclear cells. Paired 5’ single-cell RNA and T cell receptor (TCR) sequencing was then performed on sorted viable mononuclear cells from each sample and CD3+ T cells from selected samples using lOx Genomics procedures. Specifically, RNA was released through lysis using standard techniques and the extracted RN A was then reversed and transcribed into complementary DNA (cDNA), which served as the basis for subsequent amplification via polymerase chain reaction (PCR). The amplification process focused solely on the CDR3 region of the T cell receptors, targeting the variable regions of T Cell Receptors’ alpha and beta chains in TCR sequencing. Amplified cDNA was transformed into a sequencing library and subsequently loaded onto a sequencing instrument, yielding detailed genetic and transcriptomic information for each individual cell. In total, 44 samples (27 mononuclear, 17 CD3+), were analyzed resulting in 250,640 high-quality single-cell transcriptomes, including 137,649 T cells and NK cells. Data analysis was performed using Cell Ranger Multi (lOx Genomics) and R 4.3 with the tidyverse 2.0, Seurat 5.1, and scRepertoire 1.1 package.Results

[0078] The initial analysis was focused on the 2-6-month post-HSCT period that is critical for immune reconstitution. This included 8 samples from 7 patients in the 2 cohorts (1 : remission, 2: relapse, gray box in FIG. 1). Of these 7 patients, 4 were in the relapse cohort, with these relapses occurring >4 months post-HSCT. No significant difference in T-cell chimerism was seen between these 7 patients in this 2-6-month period. Based on canonical gene expression signatures, cell types were annotated and T cells selected for further evaluation (FIG. 2A-C). First, it was discovered that the CD4 / CD8 ratio differed between the cohorts, indicating a relative CD8+ T cell expansion in the relapse cohort (cohort 1 : 1.69, cohort 2: 0.55, / J<0 0001 ). Second, lower TCR diversity was discovered in the relapse cohort (Inverse Simpson Index, cohort 1 : 234.6, cohort 2: 83.0, <0.0001). This difference remained significant when subsampling the same number of T cells per patient and when restricting all samples to the 3 to 4-month timepoint. Considering the disparity in CD8+ T cell numbers between the cohorts, repertoire diversity was also analyzed, specifically in CD8+ T cells, and it was found that the observed significance persisted. To further investigate TCR diversity, T cell clonotypes (cells that share the same TCR sequence) were overlaid in a UMAP visualization, which highlighted increased hyperexpanded CD8+ T cells in the relapse cohort (FIG. 2C). Finally, we greater clonotype expansion was correlated with the expression of key T cell genes, including CD8A, CD8B, PR 1, NKG7, and GZMA. These results suggest that cytotoxic CD8+ T cells link lower TCR diversity to the likelihood of disease relapse.Conclusion

[0079] By performing deep analysis of single-cell gene expression linked to TCR clonotypes, insights into the biology of hematopoietic reconstitution after HSCT of patients with 77<53-mutated AML were discovered. Despite having analyzed a limited number of samples, patients who remained in long-term remission have significantly higher CD8+ TCR diversity between 2-6 months post-HSCT, while those who relapsed between 5-18 months post-HSCT showed lower TCR diversity and hyperexpanded CD8+ clones at the same timepoints. This work suggests that early post-HSCT TCR diversity could be a biomarker associated with GVL.

[0080] The complete disclosures of the patents, patent documents, and publications cited herein are incorporated by reference in their entirety as if each were individually incorporated. To the extent that there is any conflict or discrepancy between this specification as written and thedisclosure in any document that is incorporated by reference herein, this specification as written will control. Various modifications and alterations to this disclosure will become apparent to those skilled in the art without departing from the scope and spirit of this disclosure. It should be understood that this disclosure is not intended to be unduly limited by the illustrative embodiments and examples set forth herein and that such examples and embodiments are presented by way of example only with the scope of the disclosure intended to be limited only by the claims set forth herein as follows.

Claims

Claims:

1. A method of treating a patient post hematopoietic stem-cell transplantation (HSCT), the method comprising: obtaining a bone marrow sample from the patient at least 2 months posttransplantation; determining T Cell Receptor (TCR) clonal diversity of a population of T cells within the bone marrow sample; wherein TCR clonal diversity is indicative of risk of relapse; and administering a treatment to the patient.

2. The method of claim 1, wherein the bone marrow sample is isolated up to 6 months posttransplantation.

3. The method of claim 1, wherein the bone marrow sample comprises CD3+ T cells.

4. The method of claim 1, wherein if the TCR clonal diversity is low, the patient is at high risk for relapse.

5. The method of claim 1, wherein administering a treatment comprises treating the patient with a pre-emptive intervention.

6. The method of claim 5, wherein the pre-emptive intervention is selected from rapid taper of immunosuppression, donor lymphocyte infusion, therapy with one or more immune-boosting agents, and one or more targeted therapies.

7. A method of predicting relapse in a patient post hematopoietic stem-cell transplantation (HSCT), the method comprising: obtaining a bone marrow sample from the patient at least 2 months posttransplantation; anddetermining TCR clonal diversity of a population of T cells within the bone marrow sample; wherein TCR clonal diversity is indicative of risk of relapse.

8. The method of claim 7, wherein the bone marrow sample is isolated at least 2.5 months post-transplantation.

9. The method of claim 8, wherein the bone marrow sample is isolated up to 5 months posttransplantation.

10. A method of predicting relapse in a patient post hematopoietic stem-cell transplantation (HSCT), the method comprising: obtaining a bone marrow sample from the patient post-transplantation; and determining TCR clonal diversity of a population of T cells within the bone marrow sample; wherein TCR clonal diversity is indicative of risk of relapse, and wherein the patient received a conventional hematopoietic stem-cell transplant.

11. The method of claim 10, wherein the conventional hematopoietic stem-cell transplant is not a T-cell depleted transplant or a haplo-cord transplant.

12. The method of claim 10, wherein the patient has a TP53 mutation.

13. The method of claim 10, wherein the TCR clonal diversity is determined qualitatively via comparison with the TCR clonal diversity of non-relapsed patient.

14. The method of claim 13, wherein the qualitative determination is made by overlaying aUMAP of the TCR diversity from the patient with a UMAP of the TCR diversity from a control sample.

15. The method of claim 10, wherein the TCR clonal diversity is determine quantitatively using a diversity index.

16. The method of claim 15, wherein an Inverse Simpson Index is used as a quantitative indicator of TCR clonal diversity.

17. The method of claim 10, wherein determining TCR clonal diversity comprises single-cell RNA sequencing.

18. The method of claim 10, further comprising causing the patient to be treated with a preemptive intervention.

19. The method of claim 18, wherein the pre-emptive intervention is selected from rapid taper of immunosuppression, donor lymphocyte infusion, therapy with one or more immune- boosting agents, and one or more targeted therapies.

20. The method of claim 10, wherein the bone marrow sample is isolated at least 2.5 months and up to 5 months post-transplantation.

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