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WO2025096938A1 - Procédés de prédiction de rechute de post-transplantation de cellules souches hématopoïétiques et méthodes de traitement - Google Patents

Procédés de prédiction de rechute de post-transplantation de cellules souches hématopoïétiques et méthodes de traitement Download PDF

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WO2025096938A1
WO2025096938A1 PCT/US2024/054113 US2024054113W WO2025096938A1 WO 2025096938 A1 WO2025096938 A1 WO 2025096938A1 US 2024054113 W US2024054113 W US 2024054113W WO 2025096938 A1 WO2025096938 A1 WO 2025096938A1
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patient
tcr
bone marrow
diversity
relapse
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Mahasweta GOOPTU
Andrew A. LANE
Nurefsan E. SARIIPEK
Peter Van Galen
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Brigham and Womens Hospital Inc
Dana Farber Cancer Institute Inc
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Brigham and Womens Hospital Inc
Dana Farber Cancer Institute Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G01N2800/245—Transplantation related diseases, e.g. graft versus host disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/54—Determining the risk of relapse

Definitions

  • 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).
  • CNB immune checkpoint blockade
  • Stem-cell transplantation is a targeted, regenerative approach that goes beyond the scope of standard chemotherapy, radiation, and invasive surgeries.
  • AML Acute myeloid leukemia
  • HSCT allogeneic or donor hematopoietic stem cell transplantation
  • 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.
  • 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.
  • GVL Graft Versus Leukemia
  • 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
  • TCR diversity T cell receptor clonal diversity
  • 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.
  • 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.
  • 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 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 preemptive intervention.
  • HSCT 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).
  • 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).
  • 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.
  • FIG. 1 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.
  • UMAP Uniform Manifold Approximation and Projection
  • FIG. 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).
  • FIG. 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).
  • 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.
  • HSCT hematopoietic stem-cell transplantation
  • 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.
  • 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.
  • 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.
  • a gene signature may comprise a list of genes differentially expressed in a distinction of interest.
  • 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.
  • 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.
  • administer 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.
  • 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.
  • 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).
  • any of the elements or combinations of elements that are recited in this specification in open-ended language are considered to additionally be recited in closed-ended language (e.g., consist and derivatives thereof) and in partially 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.
  • Ranges provided herein are understood to be shorthand for all values within the range, including fractions/decimals.
  • 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.
  • the immune system is a vital component in preventing and eliminating cancer.
  • T cell receptor 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.
  • MHC major histocompatibility complex
  • 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.
  • HSCT hematopoietic stem-cell transplantation
  • 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.
  • TCR T cell receptor
  • low TCR diversity preferably around at least two months post-HSCT, indicates an increased likelihood that a patient will experience relapse.
  • 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.
  • 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.
  • Bone marrow isolation also typically favors higher cell yields compared to peripheral blood isolation.
  • the patient has or has had acute leukemia, bone marrow failure syndrome, or Myelodysplastic Syndrome (MDS).
  • MDS Myelodysplastic Syndrome
  • 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.
  • the patient received a conventional hematopoietic stemcell (HSC) transplant. That is, the transplant is not from an alternative donor source.
  • HSC transplant is not a transplant that includes a T-cell depleted transplant or a haplo- cord transplant.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • RNA is released through lysis using standard techniques.
  • the extracted RNA is then reversed and transcribed into complementary DNA (cDNA), which serves as the basis for subsequent amplification via polymerase chain reaction (PCR).
  • cDNA complementary DNA
  • PCR polymerase chain reaction
  • 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.
  • 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.
  • the patient is at high risk for relapse and the patient is identified for preemptive intervention.
  • 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).
  • 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.
  • 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.
  • TCR T-cell receptor
  • 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.
  • an Inverse Simpson’s Index is used as a quantitative indicator of TCR clonal diversity.
  • an inverse Simpson’s index of a pre-specified threshold indicates low TCR clonal diversity.
  • 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 immune signature associated with relapse (low TCR diversity) could prompt similar rapid taper of immunosuppression to prevent or delay relapse.
  • calcineurin inhibitors such as tacrolimus
  • Donor lymphocyte infusions 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.
  • 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).
  • TCR T Cell Receptor
  • 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 embodiment 2, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • UMAP Uniform Manifold Approximation and Projection
  • 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.
  • 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.
  • HSCT hematopoietic stem-cell transplantation
  • 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.
  • 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.
  • HSCT hematopoietic stem-cell transplantation
  • 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.
  • HSCT hematopoietic stem-cell transplantation
  • 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 TP53 mutation.
  • Embodiment 32 is the method of any preceding embodiment, wherein the patient has or has had TP53-mutated acute myeloid leukemia.
  • 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.
  • 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.
  • 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.
  • 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.
  • HSCT hematopoietic stem-cell transplantation
  • 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.
  • cDNA complementary DNA
  • 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.
  • 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).
  • 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).
  • T cell clonotype expansion was correlated with the expression of key T cell genes, including CD8A, CD8B, PR 1, NKG7, and GZMA.

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Abstract

L'invention concerne des procédés de prédiction de rechute chez un patient ayant subi une post-transplantation de cellules souches hématopoïétiques et des méthodes de traitement, les méthodes comprenant l'obtention d'un échantillon de moelle osseuse à partir de la post-transplantation du patient; et la détermination de la diversité clonale de TCR d'une population de lymphocytes T à l'intérieur de l'échantillon de moelle osseuse; la diversité clonale de TCR indiquant le risque de rechute.
PCT/US2024/054113 2023-11-01 2024-11-01 Procédés de prédiction de rechute de post-transplantation de cellules souches hématopoïétiques et méthodes de traitement Pending WO2025096938A1 (fr)

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Citations (3)

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US20150167084A1 (en) * 2012-07-03 2015-06-18 Sloan Kettering Institute For Cancer Research Quantitative Assessment of Human T-Cell Repertoire Recovery After Allogeneic Hematopoietic Stem Cell Transplantation
US11180813B2 (en) * 2012-10-01 2021-11-23 Adaptive Biotechnologies Corporation Immunocompetence assessment by adaptive immune receptor diversity and clonality characterization
WO2022192699A1 (fr) * 2021-03-12 2022-09-15 The Board Of Regents Of The University Of Texas System Méthodes de reconstitution de sélection de lymphocytes t et leurs utilisations

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Publication number Priority date Publication date Assignee Title
US20150167084A1 (en) * 2012-07-03 2015-06-18 Sloan Kettering Institute For Cancer Research Quantitative Assessment of Human T-Cell Repertoire Recovery After Allogeneic Hematopoietic Stem Cell Transplantation
US11180813B2 (en) * 2012-10-01 2021-11-23 Adaptive Biotechnologies Corporation Immunocompetence assessment by adaptive immune receptor diversity and clonality characterization
WO2022192699A1 (fr) * 2021-03-12 2022-09-15 The Board Of Regents Of The University Of Texas System Méthodes de reconstitution de sélection de lymphocytes t et leurs utilisations

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NOVIELLO MADDALENA, MANFREDI FRANCESCO, RUGGIERO ELIANA, PERINI TOMMASO, OLIVEIRA GIACOMO, CORTESI FILIPPO, DE SIMONE PANTALEO, TO: "Bone marrow central memory and memory stem T-cell exhaustion in AML patients relapsing after HSCT", NATURE COMMUNICATIONS, vol. 10, no. 1, 1 January 2019 (2019-01-01), UK, pages 1 - 15, XP093311964, ISSN: 2041-1723, DOI: 10.1038/s41467-019-08871-1 *
NUREFSAN SARIIPEK , SA NA KSENIIA R, CUTLER COREY, HO VINCENT T, KORETH JOHN, LINDSLEY COLEMAN, LUSKIN MARLISE R, GARCIA JACQUELI: "Post-Transplant T Cell Clonotype Diversity Is Associated with Survival in Patients with TP53-Mutated Acute Myeloid Leukemia", BLOOD, vol. 142, 2 November 2023 (2023-11-02), pages 2176 - 2178, XP093311966, DOI: 10.1182/blood-2023-181863 *

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