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WO2025174664A1 - Marqueurs d'antigènes mineurs d'histocompatibilité associés à un effet de la greffe contre la leucémie et leurs utilisations - Google Patents

Marqueurs d'antigènes mineurs d'histocompatibilité associés à un effet de la greffe contre la leucémie et leurs utilisations

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Publication number
WO2025174664A1
WO2025174664A1 PCT/US2025/014985 US2025014985W WO2025174664A1 WO 2025174664 A1 WO2025174664 A1 WO 2025174664A1 US 2025014985 W US2025014985 W US 2025014985W WO 2025174664 A1 WO2025174664 A1 WO 2025174664A1
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snp
hla
donor
recipient
snps
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Inventor
Catherine J. Wu
Nicoletta CIERI
Gad Getz
Donald Arthur STEWART
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General Hospital Corp
Dana Farber Cancer Institute Inc
Broad Institute Inc
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General Hospital Corp
Dana Farber Cancer Institute Inc
Broad Institute Inc
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Publication of WO2025174664A1 publication Critical patent/WO2025174664A1/fr
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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
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4201—Neoantigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/50—Cellular immunotherapy characterised by the use of allogeneic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • 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

Definitions

  • T cell alloreactivity against minor histocompatibility antigens mHAgs
  • D-R donor-recipient
  • SNPs genetic polymorphisms
  • allo-HSCT allogeneic hematopoietic stem cell transplantation
  • determining the presence or absence of one or more SNPs from a panel of SNPs in the donor determining the presence or absence of one or more SNPs from the panel of SNPs in the recipient; and identifying one or more mismatches between the recipient and the donor, wherein a mismatch includes the presence in the recipient and the absence in the donor of a given SNP from the panel of SNPs; and selecting as the transplant donor the donor including one or more mismatches between the recipient and the donor; wherein GvHD-free and relapse-free survival (GRFS) is improved in the recipient after receipt of donor tissue from the donor; and wherein the panel of SNPs includes the TICRR p.R287C SNR; the CNTLN p.T695I SNP; the ZNF792 p.Rl 10Q SNP; the LY9 p.M240V SNP; the CTLA4 p.T17A SNP; the WDR49
  • KIAA1549 p.P436A SNP the LILRA4 p.P27L SNP; the NAALADL2 p.G68S SNP; the RGPD2 p.S177L SNP; the RTP4 p.T131M SNP; the S100Z p.E23A SNP; the SIGLEC6 p.P246S SNP; the THEMIS p.I551V SNP; the TTC30A p.P279H SNP; and/or the ZNF286B p.P466S SNP.
  • the one or more of the SNPs from the panel of SNPs includes the APOBEC3F p.A108S SNP and/or the MCPH1 p.R256I SNP.
  • the SNP information for the donor and/or the recipient is obtained by targeted next generation sequencing (NGS), whole exome sequencing (WES), whole genomic sequencing (WGS), and/or SNP array-based genotyping.
  • NGS next generation sequencing
  • WES whole exome sequencing
  • WGS whole genomic sequencing
  • SNP array-based genotyping SNP array-based genotyping
  • the donor tissue includes HLA-matched allogeneic hematopoietic stem cells.
  • compositions including one or more graft versus leukemia minor histocompatibility antigen (GvL mHAg)-specific antigenic epitopes and a pharmaceutical composition, wherein a GvL mHAg-specific antigenic epitope consists of a peptide of about 8-11 amino acids encoded by a single nucleotide polymorphism (SNP) selected from the group consisting of the TICRR p.R287C SNP; the CNTLN p.T695I SNP; the ZNF792 p.Rl 10Q SNP; the LY9 p.M240V SNP; the CTLA4 p.T17A SNP; the WDR49 p.L476P SNP; the HELB p.L191P SNP; the PRDM15 p.R88fs SNP; the PRTN3 p.V78I SNP; the C15orf40 p.C25R SNP; the MCPH1 p
  • SNP
  • KIAA1549 p.P436A SNP the LILRA4 p.P27L SNP; the NAALADL2 p.G68S SNP; the RGPD2 p.S177L SNP; the RTP4 p.T131M SNP; the S1OOZ p.E23A SNP; the SIGLEC6 p.P246S SNP; the THEMIS p.I551V SNP; the TTC30A p.P279H SNP; and/or the ZNF286B p.P466S SNP in a sample from the transplant donor and/or the transplant recipient.
  • the kit consists of each of the TICRR p.R287C SNP; the CNTLN p.T695I SNP; the ZNF792 p.Rl 10Q SNP; the LY9 p.M240V SNP; the CTLA4 p.T17A SNP; the WDR49 p.L476P SNP; the HELB p.L191P SNP; the PRDM15 p.R88fs SNP; the PRTN3 p.V78I SNP; the C15orf40 p.C25R SNP; the MCPH1 p.A761V SNP; the MEFV p.D424E SNP; the CR1 p.T1408M SNP; the LILRB1 p.L68P SNP; the ARL11 p.C148R SNP; the ATXN3 p.V33M SNP; the EXO1 p.E589K SNP; the LILRB4
  • the kit consists of each of the TICRR p.R287C SNP; the CNTLN p.T695I SNP; the ZNF792 p.Rl 10Q SNP; the LY9 p.M240V SNP; the CTLA4 p.T17A SNP; the WDR49 p.L476P SNP; the HELB p.L191P SNP; the PRDM15 p.R88fs SNP; the PRTN3 p.V78I SNP; the C15orf40 p.C25R SNP; the MCPH1 p.A761V SNP; the MEFV p.D424E SNP; the CR1 p.T1408M SNP; the LILRB1 p.L68P SNP; the ARL11 p.C148R SNP; the ATXN3 p.V33M SNP; the EXO1 p.E589K SNP; the LILRB4
  • isolated refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
  • FIGS. lA and lB Building a pipeline for systematic mHAg discovery.
  • FIG. lA is an overview of the analysis workflow for prediction of minor histocompatibility antigens (mHAgs).
  • WES whole exome sequencing
  • SNPs single-nucleotide polymorphisms
  • FIG. IB tissue-specificity filters
  • IB presents the GvL filter to identify genes with preferential expression in acute myeloid leukemia (AML): a single-cell based molecular classifier (van Galen et al., Cell 176, 1265-1281 el 224 (2019) was applied to the Beat AML dataset (Tyner et al., Nature 562, 526-531 (2016)) to define distinct AML expression clusters (ECs). AML genes with evidence of expression at the RNA or protein level in the GTEx repository of human adult healthy tissues were excluded, to define a list of 259 candidate genes with preferential expression in AML (heatmap).
  • AML acute myeloid leukemia
  • FIGS. 2A-2L Predicted GvL mHAgs as targets for leukemia immunotherapy.
  • FIG. 2A is a heatmap showing the 86 recurrent polymorphisms in the subgroup of GRFS patients (i.e., patients with long-term survival in the absence of both relapse and acute/chronic GvHD requiring systemic treatment).
  • FIG. 2B shows the number of recurrent GvL SNPs for GRFS patients (GRFSyes, pink) versus all the other patients (GRFSno, grey); p ⁇ 0.0001, Mann Whitney test.
  • FIG. 2D is Hazard Ratios (HR) from multivariate Cox proportional hazards regression modeling of variables influencing post-transplant outcomes. Statistically significant p values are in bold.
  • FIG. 2G is the cumulative incidence (CI) of relapse stratifying the overall HP-MRD cohort based on the median number of recurrent GRFS mHAgs.
  • FIG. 21 is the mass spectrum of a detected HLA-B4403 -presented GRFS mHAg derived from APOBEC3F in the SET2 cell line. Red, blue, and green peaks represent y-, b-, and internal ions, respectively, confirming the peptide sequence.
  • FIG. 2J is a schematic of the 1000 Genomes allo-HCT simulation: the 1000 Genomes repository was mined to identify individuals identical at HLA class I alleles (with tolerance for 1 mismatch) across different ethnicities (EUR: pink; EAS: yellow; SAS: green; AFR: teal; AMR: purple) to model population coverage for immunotherapeutic strategies exploiting GRFS mHAgs. DRP: donor-recipient pair.
  • FIG. 2K for each GRES SNP, the allelic frequency in each population is reported in the top graph, with the black bar indicating the allelic frequency in the overall simulation cohort. The blue shade represents the AF range with the highest probability of D-R mismatch.
  • FIG. 3 GRFS mHAgs.
  • the histogram bars denote the number of HLA alleles predicted to present an epitope encompassing the SNP.
  • CR complete remission
  • PBSC peripheral blood stem cells
  • BM bone marrow.
  • PTCy posttransplant cyclophosphamide; n.a.: not available.
  • mHAgs Minor histocompatibility antigens
  • HLA molecules can cause immune responses involved in graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) effects after allogeneic hematopoietic cell transplantation (alloHCT).
  • GvHD graft-versus-host disease
  • GvL graft-versus-leukemia
  • alloHCT allogeneic hematopoietic cell transplantation
  • the pathogenesis of GvHD the most detrimental immune-related complication after allogeneic hematopoietic cell transplantation, is attributed to a donor-derived immune response directed against mHAgs either broadly expressed across tissues or expressed specifically in GvHD-affected tissues.
  • an analytic framework to systematically identify autosomal encoded mHAgs associated with GvL clinical outcomes.
  • This analytic framework shown schematically in FIGS. 1 A and IB and described in more detail in the examples section included herewith, is based on the integration of polymorphism detection by whole exome sequencing of germline DNA from donor-recipient (D-R) pairs together with organ-specific transcriptional- and proteome-level expression.
  • D-R donor-recipient
  • Application of this analytic framework to a cohort of 220 HLA- matched allo-HSCT D-R pairs identified GvL targets for the prevention or treatment of posttransplant disease recurrence.
  • the analytic framework described herein identifies single nucleotide polymorphisms (SNPs) that produce amino acid coding differences between recipients and donors and are associated with GvL outcomes.
  • SNPs single nucleotide polymorphisms
  • GvL graft-versus- leukemia
  • HSCT allogeneic hematopoietic stem cell transplantation
  • T lymphocytes donor T cells
  • GvL might develop after recognizing tumor-specific or recipient-specific alloantigens. It can lead to remission or immune control of hematologic malignancies.
  • dbSNP a SNP database maintained by the National Center for Biotechnology Information (NCBI) (available on the worldwide web at ncbi.nlm.nih.gov/snp/). As of June 8, 2015, dbSNP listed 149,735,377 SNPs for the human genome.
  • NCBI National Center for Biotechnology Information
  • GRFS graft-versus-host disease-free, relapse-free survival
  • HCT allogeneic hematopoietic stem cell transplantation
  • Such methods include: determining the presence or absence of one or more SNPs from a panel of SNPs in the donor; determining the presence or absence of one or more SNPs from the panel of SNPs in the recipient; and identifying one or more mismatches between the recipient and the donor, wherein a mismatch comprises the presence in the recipient and the absence in the donor of a given SNP from the panel of SNPs; and selecting as the transplant donor the donor comprising one or more mismatches between the recipient and the donor; wherein GvHD-free and relapse-free survival (GRFS) is improved in the recipient after receipt of donor tissue from the donor; and wherein the panel of SNPs includes the TICRR p.R287C SNP; the CNTLN p.T695I SNP; the ZNF792 p.Rl 10Q SNP; the LY9 P.M240V SNP; the CTLA4 p.T17A SNP; the WDR49 p.L476P SNP; the HE
  • the panel of SNPs includes, but is not limited to, any one, any two, any three, any four, any five, any six, any seven, any eight, any nine, any ten, about any fifteen, about any twenty, about any twenty-five, about any thirty, about any thirty-five, about any forty, about any forty-five, about any fifty, about any fifty-five, about any sixty, about any sixty -five, about any seventy, about and seventy -five, about any eighty, about any eight-five, or all eighty-seven of these SNPs, or any range thereof.
  • identifying one or more mismatches between the recipient and the donor includes identifying about ten mismatches, about fifteen mismatches, about twenty mismatches, about twenty-five mismatches, about thirty mismatches, about thirty-five mismatches, about forty mismatches, about forty-five mismatches, about fifty mismatches, about fifty-five mismatches, about sixty mismatches, about sixty-five mismatches, about seventy mismatches, about seventy-five mismatches, about eighty mismatched, about eighty-five mismatches from the panel of SNPs, or any range thereof.
  • determining the presence or absence of one or more SNPs from a panel of SNPs in the donor and/or determining the presence or absence of one or more SNPs from the panel of SNPs in the recipient includes determining the presence or absence the APOBEC3F p.A108S SNP and/or the MCPH1 p.R256I SNP.
  • Methods are provided for inducing a GvL reaction in a HLA-matched transplant recipient receiving donor tissue from a donor.
  • Such methods include: determining the presence or absence of one or more SNPs from the panel of SNPs for the donor; determining the presence or absence of SNPs from the panel of SNPs for the recipient; identifying one or more mismatches between the recipient and the donor, wherein a mismatch comprises the presence in the recipient and the absence in the donor of a given SNP from the panel of SNPs; and transplanting the recipient with donor tissue from the donor when there is one or more mismatches between the recipient and the donor; wherein the panel of SNPs comprises the TICRR p.R287C SNP; the CNTLN p.T695I SNP; the ZNF792 p.Rl 10Q SNP; the LY9 p.M240V SNP; the CTLA4 p.T17A SNP; the WDR49 p.L476P SNP; the HE
  • the panel of SNPs includes, but is not limited to, any one, any two, any three, any four, any five, any six, any seven, any eight, any nine, any ten, about any fifteen, about any twenty, about any twenty-five, about any thirty, about any thirty-five, about any forty, about any forty-five, about any fifty, about any fifty-five, about any sixty, about any sixty-five, about any seventy, about and seventy -five, about any eighty, about any eight-five, or all eighty-seven of these SNPs, or any range thereof.
  • the donor and the recipient may be HLA-matched, wherein HLA stands for human leukocyte antigens.
  • HLA type of HLA-matched donor and recipient includes the HLA type HLA A0101, HLA A0201, HLA A0208, HLA A0301, HLA A0302, HLA Al 101, HLA A2301, HLA A2402, HLA A2403, HLA A2601, HLA A2902, HLA A3001, HLA A3101, HLA A3201, HLA A3301, HLA A6601, HLA A6801, HLA A6802, HLA A7401, HLA A8001, HLA B0702, HLA B0801, HLA B1302, HLA B1401, HLA B1402, HLA B1501, HLA B1503, HLA B1801, HLA B2705, HLA B3501, HLA B3502, HLA B3508, HLA B3701, HLA B3801, HLA B3901, HLA B4001, HLA B4001, HLA B400
  • SNP information for a panel of SNPs may be obtained for a donor and/or a recipient by any of a variety of methods.
  • SNPs are allelic germline variations
  • SNPs information for a donor and/or a recipient may be determined from a genomic DNA sample.
  • the panel of SNPs includes, but is not limited to, any one, about any two, about any three, about any four, about any five, about any ten, about any fifteen, about any twenty, about any twenty-five, about any thirty, about any thirty-five, about any forty, about any forty-five, about any fifty, about any fifty-five, about any sixty, about any sixty-five, about any seventy, about and seventy-five, about any eighty, about any eight-five, or all eighty-seven of these SNPs, or any range thereof.
  • SNP information may be obtained, for example, by utilizing any of a variety of DNA sequencing approaches, including for example, targeted next generation sequencing (NGS), whole exome sequencing (WES), and whole genomic sequencing (WGS).
  • NGS next generation sequencing
  • WES whole exome sequencing
  • WGS whole genomic sequencing
  • NGS Next Generation Sequencing
  • SNP information may be obtained utilizing microarray-based SNP genotyping assays.
  • SNP information may be obtained by capillary electrophoresis, mass spectrometry, single-strand conformation polymorphism (SSCP), single base extension, electrochemical analysis, denaturating HPLC and gel electrophoresis, restriction fragment length polymorphism (RFLP), chip detection, various novel PCR or qPCR-based technologies, such as amplification refractory mutation system PCR (ARMS-PCR), and kompetitive allele-specific PCR (KASP), or hybridization analysis.
  • SSCP single-strand conformation polymorphism
  • RFLP restriction fragment length polymorphism
  • RFLP restriction fragment length polymorphism
  • chip detection various novel PCR or qPCR-based technologies, such as amplification refractory mutation system PCR (ARMS-PCR), and kompetitive allele-specific PCR (KASP), or hybridization analysis.
  • SNP information for a panel of SNPs for the donor and/or SNP information for a panel of SNPs for the recipient may have been previously determined and may be provided as a computer-readable medium having stored thereon computer-readable SNP information for the panel of SNPs.
  • donor and/or recipient refers to a human donor and/or recipient as well as a non-human mammalian subject.
  • examples herein concern humans and the language is primarily directed to human concerns, the concept is applicable to any mammal, and is useful in the fields of veterinary medicine, animal sciences, research laboratories and such.
  • the methods provided herein may be used in association with transplantation of donor tissue for the treatment of a malignant or a nonmalignant hematologic disease with indication to allogeneic hematopoietic cell transplantation.
  • Such disorders may include, but are not limited to, cancers such as, for example, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS).
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • any of a variety of donor tissues may be transplanted, including but not limited to, allogeneic hematopoietic stem cells.
  • Such stem cells may be obtained from the bone marrow or the bloodstream.
  • the donor tissue includes HLA-matched allogeneic hematopoietic stem cells.
  • compositions including one or more graft versus leukemia minor histocompatibility antigen (GvL mHAg)-specific antigenic epitopes, wherein a GvL mHAg- specific antigenic epitope consists of a peptide of about 8-11 amino acids in length encoded by a single nucleotide polymorphism (SNP) selected from the TICRR p.R287C SNP; the CNTLN p.T695I SNP; the ZNF792 p.Rl 10Q SNP; the LY9 p.M240V SNP; the CTLA4 p.T17A SNP; the WDR49 p.L476P SNP; the HELB p.L191P SNP; the PRDM15 p.R88fs SNP; the PRTN3 p.V78I SNP; the C15orf40 p.C25R SNP; the MCPH1 p.A761V SNP; the M
  • the composition may include one, two, three, four, five, about ten, about fifteen, about twenty, about twenty-five, about thirty, about thirty-five, about forty, about forty- five, about fifty, about fifty-five, about sixty, about sixty-five, about seventy, about seventy-five, about eighty, or about eight-five of these GvL mHAg-specific antigenic epitopes, or any range thereof.
  • a composition may further include a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. Such a carrier may be pyrogen free.
  • Such a composition may be used as an immunotherapeutic vaccine. Such a vaccine may further include an adjuvant.
  • Kits for use in selecting an HLA-matched transplant donor wherein GvHD-free and relapse-free survival (GRFS) is improved in the recipient after receipt of donor tissue from the donor are provided.
  • a kit is any manufacture (for example, a package or container) including at least one reagent for specifically detecting one or more of the SNPs described herein.
  • the kit may be promoted, distributed, or sold as a unit for performing the methods provided herein.
  • a kit may include a plurality of probes capable of binding to and/or identifying one or more of the TICRR p.R287C SNP; the CNTLN p.T695I SNP; the ZNF792 p.Rl 10Q SNP; the LY9 p.M240V SNP; the CTLA4 p.T17A SNP; the WDR49 p.L476P SNP; the HELB p.L191P SNP; the PRDM15 p.R88fs SNP; the PRTN3 p.V78I SNP; the C15orf40 p.C25R SNP; the MCPH1 p.A761V SNP; the MEFV p.D424E SNP; the CR1 p.T1408M SNP; the LILRB1 p.L68P SNP; the ARL11 p.C148R SNP; the ATXN3 p.V33M SNP; the EX01 p
  • the kit may include probes capable of binding to and/or identifying any one, about any two, about any three, about any four, about any five, about any ten, about any fifteen, about any twenty, about any twenty-five, about any thirty, about any thirty-five, about any forty, about any forty-five, about any fifty, about any fifty-five, about any sixty, about any sixty-five, about any seventy, about and seventy-five, about any eighty, about any eight-five, or all eighty-seven of these SNPs, or any range thereof, in a sample from the transplant donor and/or the transplant recipient.
  • a kit may include a plurality of primers for selectively amplifying one or more of the TICRR p.R287C SNP; the CNTLN p.T695I SNP; the ZNF792 p.Rl 10Q SNP; the LY9 p.M240V SNP; the CTLA4 p.T17A SNP; the WDR49 p.L476P SNP; the HELB p.L191P SNP; the PRDM15 p.R88fs SNP; the PRTN3 p.V78I SNP; the C15orf40 p.C25R SNP; the MCPH1 p.A761 V SNP; the MEFV p.D424E SNP; the CR1 p.T1408M SNP; the LILRB1 p.L68P SNP; the ARL11 p.C148R SNP; the ATXN3 p.V33M SNP; the EX01 p.E5
  • mHAgs are sensed as foreign by donor T cells and are expected to be highly immunogenic due to the lack of central tolerance against them.
  • mHAgs are inherited as germline traits encoded by polymorphic genes rather than presenting as somatic events, hence they are not tumor-specific antigens per se.
  • the pathogenesis of graft-versus-host disease (GvHD) the most detrimental immune-related complication after allo-HCT, can be thus attributed to a donor-derived immune response directed against mHAgs either broadly expressed across tissues or expressed specifically in GvHD-affected tissues.
  • Peptides were pulsed on CD3-depleted PBMCs, and cocultured with autologous female-derived T cells. Cells were similarly restimulated after 7 days; on day 14, they were screened for antigen specificity by dextramer staining.
  • GvL filter While the analysis focused on AML/MDS, the scalable design of the GvL filter enables incorporating expression profiles from other hematological malignancies with indication to allo-HCT but only a paucity of immunotherapy targets (such as T-ALL or PTCLs). Systematic identification of GvL mHAgs may lead to their effective use to prevent or treat posttransplant relapse in an analogous fashion as has been tested for tumor neoantigens.
  • matched donor and recipient DNA was analyzed, as well as PBMCs in some cases, collected from all adult patients who underwent first T-replete allo-HSCT from a matched related donor (MRD) between January 1st, 2013 and December 31st, 2020.
  • MRD matched related donor
  • HP -MRD cohort we analyzed matched donor and recipient DNA collected from 58 adult patients who underwent first T-replete MRD allo-HCT for myeloid disease (AML/MDS) between January 1, 2011, and April 30, 2021, at Hospital de la Princesa, Madrid. Patients were considered in complete remission if disease activity could not be documented by BM evaluation. All other patients not falling within this definition were categorized as having active disease.
  • the ELN and R-IPSS scores were consolidated in a single variable, termed ‘overall risk score’ and comprising 3 categories: i) favorable, including ELN favorable and R-IPSS very low/low; ii) intermediate, encompassing ELN intermediate and R- IPSS intermediate; and iii) adverse, including ELN adverse and R-IPSS high/very high.
  • Clinical diagnosis and grading of acute GvHD were annotated according to consensus criteria (Przepiorka et al., 1995, Bone Marrow Transplant, 15:825-828; and Glucksberg et al., 1974, Transplantation, 18:295-304).
  • Chronic GvHD diagnosis and grading were based on the National Institutes of Health consensus criteria (Pavletic et al., 2010, Biol Blood Marrow Transplant, 16:871-890).
  • clusters of hematopoietic origin were identified using standard lineage markers (PTPRC, CD3E, MS4A1, CD79B, KLRB KLRG1, LYZ, CD68, CD14, KIT) and excluded from further analysis (with the only exceptions of Langerhans cells in skin and Kupffer cells in liver).
  • Clustering was repeated after the removal of immune cells, and non-immune cell types were manually annotated using the same set of lineage-defining genes used in the original publications.
  • gene expression profiles were compiled, upon exclusion of non-coding genes as well as HLA genes. Genes with a sum count >5 counts per million (CPM) were retained to create the final list of genes to be used for the GvHD filter.
  • Pseudo-bulk expression of AIRE, HLA-A, HLA-B and HLA-C within the 3 clusters was calculated using the ‘ AggregateExpressiori function in Seurat and the resulting expression levels were normalized on the number of cells present in each cluster.
  • Input fdes for the pipeline were donor and recipient exomes in the form of BAM/CRAM files aligned to the hg!9 reference genome, that were processed using Deep Variant (Poplin et al., 2018, Nat Biotechnol', 36:983-987) version 1.1.0 and Funcotator (part of the GATK package, v4.2.6.1) to define germline variants (including SNPs, indels and frameshifts) and proceed to their annotation, respectively.
  • Deep Variant Praplin et al., 2018, Nat Biotechnol', 36:983-987
  • Funcotator part of the GATK package, v4.2.6.1
  • the ‘GvHD filter’ has been already described in the previous section.
  • the GvL filter features 2 main components: the ‘AML filter’ (to define genes expressed by malignant myeloid cells) and the ‘Hematopoietic filter’ (to define genes expressed across the different hematopoietic lineages).
  • the list of GTEx tissues varies to include either female reproductive organs (for male patients) or male reproductive organs (for female patients); for both female and male patients, whole blood, spleen, and lung are excluded given the inherent large proportion of hematopoietic cells present.
  • the resulting list of genes is then subjected to a second filtering round using the GTEx proteomics dataset (Jiang et al., 2020, Cell,' 183:269-283 e219), with all genes with a tissue specificity score (TS) >2 in any GTEx tissue being removed; in instances when protein data was missing, we applied a second RNA-Seq filtering step, using a cutoff of log2(TPM)>5 in any GTEx tissue, a threshold shown to reliably correspond to protein expression (Jiang et al., 2020, Cell,' 183:269-283 e219).
  • TS tissue specificity score
  • AML cell lines The following AML cell lines were purchased from DSMZ: Mono- MAC6, MUTZ-3, OCI-AML3 and SET2; MOLM-13 were kindly gifted by the Genovese Lab (DFCI). All cell lines are part of the LL-100 panel (Quentmeier et al., 2019, Sci Rep 9:8218), i.e., profiled at the genomic and transcriptomic level with publicly available results. For each cell line, paired WES and RNA-Seq data were downloaded from ENA (accession numbers: PRJEB30297 and PRJEB30312, respectively).
  • RNA-Seq data was used to evaluate the expression levels of the genes harboring the GvL SNPs and to infer HLA class I typing with OptiType (Szolek et al., 2014, Bioinformatics, 30:3310-3316). Up to 50 million or 0.2 g of each AML cell line were immunoprecipitated, as previously described (Sarkizova et al., 2020, Nat Biotechnol, 38: 199- 209).
  • PBMCs for immunogenicity testing were isolated by Ficoll- Paque PLUS (Fisher Scientific) density gradient centrifugation from peripheral blood samples of healthy donors. DNA was extracted with the DNA mini kit (Qiagen) following manufacturer’s instructions and used for HLA typing (through the Brigham and Hospital HLA typing lab), and for SRY PCR to select those from female donors with appropriate HLA restrictions. SRY PCR primers were used, following the protocol described by Cui et al. (Cui et al., 1994, Lancet, 343: 79-82).
  • T cells were co-incubated with autologous EBV-LCL pulsed with the appropriate peptides for 6 hours, and then labeled with the IFNy secretion Assay detection kit in PE (Miltenyi Biotech) following manufacturer’s instructions.
  • CD8a BV785, clone RPA-T8, Biolegend
  • CD4 PerCP-Cy5.5, clone RPA-T4, Biolegend
  • Zombie Violet vitality dye, Biolegend
  • CD19 APC-Fire750, clone HIB19, Biolegend
  • CD14 APC-Fire750, clone M5E2, Biolegend
  • Data were acquired on a high throughput sampler (HTS)-equipped Fortessa cytometer (BD Biosciences) and analyzed using Flowjo vl0.8 software (BD Biosciences).
  • HTS high throughput sampler
  • NRM non-relapse mortality
  • relapse acute GvHD
  • chronic GvHD chronic GvHD
  • mHAg load i.e., overall acute and chronic, as well as organ-specific GvHD
  • initial evaluation of deciles of mHAg load was used to guide subsequent analyses and define stratification criteria (mHAg load median vs. analysis of individual SNPs).
  • the log-rank test and the Gray test were used for group comparison of OS and cumulative incidence of relapse and GvHD, respectively.
  • the risk factor analysis on acute GvHD-specific hazard was firstly assessed by the Cox proportional-hazards model, and hazard ratio (HR) with the associate 95% confidence interval were calculated for each variable.
  • Forest plots were performed by the forest model function in the ‘forestmodel’ (version 0.6.2) R package.

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Abstract

L'invention concerne des antigènes mineurs d'histocompatibilité (mHAgs) associés à des résultats cliniques d'effet de la greffe contre la leucémie (GvL) identifiés par des polymorphismes mononucléotidiques (SNP), et leurs utilisations.
PCT/US2025/014985 2024-02-16 2025-02-07 Marqueurs d'antigènes mineurs d'histocompatibilité associés à un effet de la greffe contre la leucémie et leurs utilisations Pending WO2025174664A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150278434A1 (en) * 2012-11-08 2015-10-01 Umc Utrecht Holding B.V. Method for prediction of an immune response against mismatched human leukocyte antigens
US20190359681A1 (en) * 2015-02-09 2019-11-28 Université de Montréal Novel minor histocompatibility antigens and uses thereof
US20200224271A1 (en) * 2017-07-14 2020-07-16 The Regents Of The University Of California Novel Methods of Predicting Transplant Rejection Risk
US20220228212A1 (en) * 2013-10-15 2022-07-21 Illumina, Inc. Major histocompatibility complex single nucleotide polymorphisms
WO2022235577A2 (fr) * 2021-05-03 2022-11-10 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Récepteurs des lymphocytes t (tcr) ciblant l'antigène d'histocompatibilité mineure ha-1

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150278434A1 (en) * 2012-11-08 2015-10-01 Umc Utrecht Holding B.V. Method for prediction of an immune response against mismatched human leukocyte antigens
US20220228212A1 (en) * 2013-10-15 2022-07-21 Illumina, Inc. Major histocompatibility complex single nucleotide polymorphisms
US20190359681A1 (en) * 2015-02-09 2019-11-28 Université de Montréal Novel minor histocompatibility antigens and uses thereof
US20200224271A1 (en) * 2017-07-14 2020-07-16 The Regents Of The University Of California Novel Methods of Predicting Transplant Rejection Risk
WO2022235577A2 (fr) * 2021-05-03 2022-11-10 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Récepteurs des lymphocytes t (tcr) ciblant l'antigène d'histocompatibilité mineure ha-1

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